synthesized at John Hopkins University in 1877, and i t was f ir s t used in c lin ic a l ...... (1977) and Grisham et al_ (1978). Michalapolous e t al_ ..... membrane which forms the base of a reaction chamber into which small volumes (approx.
THE USE OF ISOLATED HEPATOCYTES TO STUDY THE TOXIC EFFECTS OF CHEMICALS
by
Jennie Gwynn, B .S c ., M .S c ., M.P.S.
A th esis submitted in accordance w ith the requirements o f the U n iv e rs ity o f Surrey fo r the degree o f Doctor o f Philosophy.
Department o f Biochem istry, U n iv e rs ity o f S urrey, G u ild fo rd , Surrey.
November 1981.
ProQuest N um ber: 10798499
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uest ProQuest 10798499 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346
To Jim and Dorothy
ACKNOWLEDGEMENTS The completion o f th is th esis would not have been achieved w ith o u t the dedicated supervision o f Professor James W. B rid g e s .. There are many o th e rs , too numerous to m ention, who have co n trib u ted by t h e ir p ra c tic a l a ss istan c e, exp ert advice and encouragement.
I would lik e to thank p a r t ic u la r ly Dr. J e ffre y F ry , Dr. Carol Jones and Dr. P h ilip W iebkin, w ith whom I worked so c lo s e ly , f o r passing on t h e ir valuab le experience in the use o f is o la te d hepatocytes. I would also l ik e to thank Dr. Tim Fennel, Dr. Richard Hinton and Dr. Diane Benford fo r t h e ir assistance in some o f the s p e c ia lis e d techniques used here.
I am also extrem ely g ra te fu l to my frie n d s and colleagues a t the D .H .S .S . and Smith K line and French whose continued encouragement has helped me complete th is m anuscript.
In p a r tic u la r I would
lik e to mention Dr. Susan Sullman, Dr. P eter Johnson and Dr. Paul Sharpe.
*
F in a lly I am indebted to Mrs Jan Hamilton whom w ith extreme care and unending patience typed th is manuscript Council fo r fin a n c ia l support.
and to the Medical Research
ABSTRACT
Some c h a ra c te ris tic s o f r a t hepatocytes, is o la te d by a non-perfusion technique, have been examined in order to v a lid a te t h e i r use f o r the in v e s tig a tio n o f chem ically induced t o x i c it y .
Untreated hepatocytes synthesized p r o te in , RNA and GSH and m etabolised exogenous su b s tra te s .
GSH was depleted during is o la tio n but a c tiv e
resynthesis was evid en t on incubation in complete c u ltu re medium. The le v e ls o f GSH, ATP and c y c lic AMP were s im ila r to those reported in the l i t e r a t u r e fo r hepatocytes is o la te d by p e rfu s io n .
Hence the
hepatocytes were shown to possess a number o f in vivo c h a r a c te r is tic s . Although th ere was an in h e re n t v a r i a b i l i t y between in d iv id u a l preparations c h a ra cterise d by the v a r i a b i l i t y in , and la c k o f c o r r e la tio n between, the ra te o f p ro te in syn th e s is , c e ll y ie ld and v i a b i l i t y , the e f f e c t o f a number o f xenobiotics were in v e s tig a te d .
Paracetamol induced a rap id f a l l
in ATP, dose-dependent in h ib it io n
o f p r o te in , RNA s y n th e s is , and the metabolism o f 7-EC, d e p le tio n o f GSH and covalent binding o f drug re la te d m a te ria l to p ro te in . Exposure o f c u ltu red hepatocytes to 40mM paracetamol f o r < 4 hrs produced re v e rs ib le in h ib it io n o f p ro te in s yn th e sis , but c e ll death was the r e s u lt o f a longer exposure.
S a fro le and phenobarbitone also induced a rap id f a l l
in ATP le v e ls
follow ed by dose-dependent in h ib it io n o f p ro te in and RNA syn th e sis . This was, th e r e fo r e , a common sequence o f events in response to a to x ic in s u lt but was u n lik e ly to be the d ir e c t cause o f c e ll death.
The a c t i v it i e s o f 7-EC O -deeth ylase, NADPHg diaphorase, SDH and G6PDH were a l l increased in c u ltu res tre a te d w ith 2mM phenobarbitone (a concentration which did not cause c e ll d e a th ).
Paracetamol also caused
increases in NADPH£ diaphorase a c t i v i t y .
The e ffe c ts o f these xenobiotics in hepatocytes were compared and discussed.
I t was concluded th a t the cause o f c e ll death'was not
id e n t if ie d and th a t i t was u n lik e ly to be due to one f a c to r alone but to
a combination o f fa c to rs dependent on the le v e l and d u ration o f to x ic in s u lt and a decreased a b i l i t y o f the c e ll to r e p a ir damage. With f u r th e r refinem ents cu ltu re d hepatocytes should provide a useful model f o r the in v e s tig a tio n o f mechanisms o f chem ically induced t o x i c it y .
CONTENTS CHAPTER 1:
INTRODUCTION
1.1
GENERAL BACKGROUND
1.2
INTRODUCTION TO TOXICOLOGY
1 .3
THE STRUCTURE OF THE LIVER
1 .4
MECHANISMS OF TOXIC LIVER INJURY
1.5
PROTEIN BIOSYNTHESIS AND THE MODE OF ACTION OF SOME INHIBITORS
1.6
ACTIVE METABOLITES AND TOXICITY
1 .7
RELATIONSHIP OF ENZYME INDUCTION AND INHIBITION TO TOXICITY
1 .8
PARACETAMOL HEPATOTOXICITY
CHAPTER 2:
ISOLATED HEPATOCYTES
2.1
INTRODUCTION
2 .2
ISOLATION TECHNIQUES
2 .3
BIOCHEMICAL AND MORPHOLOGICAL CHARACTERISTICS OF ISOLATED HEPATOCYTES
2 .4
ISOLATED HEPATOCYTES IN CULTURE-
2 .5
DRUG METABOLISM IN HEPATOCYTE SUSPENSIONS AND CULTURES
2 .6
MAINTENANCE OF CYTOCHROME P-450 IN PRIMARY MONOLAYER CULTURES OF RAT HEPATOCYTES
2 .7
THE USE OF ISOLATED HEPATOCYTES IN TOXICOLOGY
2 .8
AIMS .
CHAPTER 3: 3.1
3.2
MATERIALS AND METHODS
MATERIALS 3 .1 .1
Animals
3 .1 .2
Apparatus
3 .1 .3
Chemicals
HEPATOCYTE ISOLATION 3 .2 .1
The enzymic is o la tio n o f a d u lt r a t hepatocytes
3 .2 .2
The enzymic is o la tio n o f a d u lt hamster hepatocytes
Page 3 .3
CRITERIA USED TO ASSESS CELL VIABILITY
58
3 .3 .1
Dye exclusion t e s t
58
3 .3 .2
Lactate dehydrogenase assay fo r v i a b i l i t y
59
3 .3 .3
Measurement o f the ra te o f oxygen uptake in is o la te d hepatocytes
3 .3 .4
6p
Measurement o f 7-ethoxycoumarin O-deethylase a c t i v it y in c e ll suspensions
3 .4
61
PROTEIN SYNTHESIS IN ISOLATED HEPATOCYTES 3 .4 .1
Measurement o f
r i4
64
CJ -Leucine uptake in to
is o la te d hepatocytes
64
3 .4 .2
P re lim in a ry in v e s tig a tio n s
65
3 .4 .3
The e f f e c t o f xenobiotics on [ ^ c ] -Leucine uptake by is o la te d hepatocytes
3 .4 .4
The e f f e c t o f paracetamol on
65 L^CJ -Leucine
uptake in hepatocytes is o la te d from ra ts p re tre a te d w ith phenobarbitone 3 .4 .5
E ffe c t o f paracetamol on p ro te in degradation
3 .4 .6
E ffe c t o f paracetamol on the uptake o f
[*c]
( in r e la tio n to incubation tim e)
66
3 .4 .7
E ffe c t o f paracetamol on p ro te in sec retio n
67
3 .4 .8
To determine whether the e f f e c t on p ro te in
3 .4 .9
67
E ffe c t o f paracetamol on p ro te in synthesis in vivo in the r a t
67
TO DETERMINE WHETHER PARACETAMOL HAS ASPECIFIC EFFECT ON EXPORT PROTEINS 3 .5 .1
3 .5 .2
3 .5 .3
69
Prep aration o f cyanogen bromide a c tiv a te d Sepharose conjugates f 14 n P rep aration o f [ CJ - la b e lle d p ro te in from
69
is o la te d r a t hepatocytes
71
A f f i n i t y experiments w ith a c tiv a te d Sepharose conjugate
3.6
66
-Leucine in to is o la te d hepatocytes
synthesis is r e v e rs ib le
3.5
65
71
THE EFFECT OF XENOBIOTICS ON RNA SYNTHESIS 3 .6 .1
Measurement o f
r i4'
is o la te d hepatocytes
74
-U rid in e uptake in to 74
Page 3 .7
COVALENT BINDING 3 .7 .1
Measurement o f covalent binding o f paracetamol m etabolites to c e llu la r p ro te in
3 .8
3 .9
75
75
GLUTATHIONE IN ISOLATED HEPATOCYTES
76
3 .8 .1
76
G lu tath io n e assay
ASSAY OF ADENOSINE TRIPHOSPHATE IN ISOLATED HEPATOCYTES
77
3.10 ASSAY OF CYCLIC AMP IN ISOLATED HEPATOCYTES
78
3.11 THE PREPARATION OF MICROSOMES FROM ISOLATED HEPATOCYTES
79
3 .1 1 .1
Sodium dodecyl sulphate-polyacrylam ide gel e le c tro p h o re s is
79
3 .1 1 .2
NADH - cytochrome C reductase assay
81
3 .1 1 .3
Assay o f 7-ethoxycoumarin O -d e eth yla tio n in microsomes
3.12 PRIMARY MONOLAYER CULTURES OF ADULT RAT HEPATOCYTES
82 84
3 .1 2 .1
Hepatocyte a rc h ite c tu re
84
3 .1 2 .2
The e f f e c t o f xenobiotics on c e ll attachm ent
86
3 .1 2 .3
Assay fo r glutam ic o x a lo a c e tic transaminase
86
3 .1 2 .4
Enzyme cytochem istry
86
3 .1 2 .5
P ro tein synthesis in prim ary cu ltu re s o f a d u lt r a t hepatocytes
3 .1 2 .6
7-Ethoxycoumarin O -deethylase a c t i v i t y in cu ltu re s
CHAPTER 4:
88
RESULTS
88
89
4.1
INTRODUCTION
90
4 .2
VIABILITY ASSESSMENT
90
4 .2 .1
Trypan blue exclusion
90
4 .2 .2
Lactate dehydrogenase release
96
4 .2 .3
Comparison o f trypan blue exclusion and la c ta te dehydrogenase release
4 .3
4 .2 .4
C e ll re s p ira tio n
184
4 .2 .5
7-Ethoxycoumarin O-deethylase a c t i v it y
189
THE EFFECT OF EXOGENOUS CHEMICALS ON PROTEIN SYNTHESIS IN ISOLATED HEPATOCYTES
4 .4
99
113
DETERMINATION OF THE SPECIFICITY OF THE ACTION OF PARACETAMOL ON PROTEIN SYNTHESIS 4 .4 .1
132
The p reparation o f microsomesfrom is o la te d hepatocytes
132
Page 4 .5
THE EFFECT OF PARACETAMOL AND OTHER XENOBIOTICS ON RNA SYNTHESIS
138
4 .6
COVALENT BINDING
,
143
4 .7
EFFECTSON REDUCED GLUTATHIONE
145
4 .8
EFFECTS ON ADENOSINE TRIPHOSPHATE
151
4 .9
EFFECTS ON CYCLIC ADENOSINE MONOPHOSPHATE
153
4.1 0 INVESTIGATIONS WITH PRIMARY MONOLAYER CULTURES OF ADULT RAT HEPATOCYTES 4 .1 0 .1
155
The e ffe c ts o f xenobiotics on p ro te in synthesis in c u ltu re s
4 .1 0 .2
157
Comparison o f the e ffe c ts o f paracetamol and carbon te tr a c h lo rid e on cu ltu res using cytochemical and o th e r methods
4 .1 0 .3
164
E ffe c t o f cycloheximide on the m etabolic a c t i v it y o f prim ary cu ltu re s o f r a t hepatocytes
4 .1 0 .4
172
Comparison o f the inducing c a p a b ilitie s o f fo u r b a rb itu ra te s in prim ary monolayer cu ltu re s o f a d u lt r a t hepatocytes
CHAPTER 5:
DISCUSSION
5.1
VIABILITY CHARACTERISTICS OFISOLATED HEPATOCYTES
5 .2
ISOLATED RAT HEPATOCYTES INPRIMARYMAINTENANCECULTURE 5 .2 .1
182
190 191 201
The p ro p e rtie s o f cu ltu re s compared to suspensions o f is o la te d hepatocytes
5 .2 .2
Mixed fu n c tio n oxidase a c t i v it y in primary maintenance cu ltu re s o f a d u lt r a t hepatocytes
5 .2 .3
203
Comparison o f the inducing c a p a b ilitie s o f 0
5 .2 *4
some b a rb itu ra te s
205
The a p p lic a tio n o f q u a n tita tiv e cytochem istry to prim ary cu ltu re s
5 .3
201
208
THE EFFECTS OF XENOBIOTICS ON THE VIABILITY CHARACTERISTICS OF ISOLATED HEPATOCYTES
211
5 .3 .1
Paracetamol
211
5 .3 .2
A n ilin e analogues
219
5 .3 .3
S a fro le
220
5 .3 .4
Phenobarbitone
224
5 .3 .5
Carbon te tr a c h lo rid e
226
5 .3 .6
A summary o f some postulated mechanisms o f t o x ic it y
228
Page CHAPTER 6: 6.1
CONCLUSIONS
232
THE VALUE OF ISOLATED HEPATOCYTES FOR STUDYING THE TOXIC EFFECTS OF CHEMICALS
REFERENCES
233
237
\
ABBREVIATIONS
ADP
adenosine diphosphate
AMP
adenosine monophosphate
ATP
adenosine tripho sphate
BSP
sulphobromophthalein
DNA
deoxyribonucleic acid
7-EC
7-ethoxycoumarin
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
GDH
glycero l-3-p h o sp h ate dehydrogenase
GOT
g lu ta m ic -o x a lo a c e tic transaminase
G6P
D -g lucose-6-phosphate
G6PDH
glucose-6-phosphate dehydrogenase
GPT
g lu tam ic-p yru v ic transaminase
GSH
g lu ta th io n e
LDH
la c ta te dehydrogenase
NAD
nicotinam ide adenine d in u c le o tid e
NADH
nicotinam ide adenine d in u c le o tid e , reduced
NADP
nicotinam ide adenine d in u c le o tid e phosphate
NADPH
nicotinam ide adenine d in u c le o tid e phosphate, reduced
PAPS
3-phospoadenosine 5 ' -phosphosulphate
PBS'A1
phosphate b u ffered s a lin e
PGK
3-phospoglycerate kinase
PMS
phenazine methosulphate
PVA
p o lyvin yl alcohol
RNA
rib o n u c le ic acid
SDH
succinate dehydrogenase
TCA
t r ic h lo r o a c e tic acid
TIM
triosephosphate isomerase
TXT 00
t r i t o n X-100
UDP
u rid in e diphosphate
UDPG
u ri di n e -5 1-d i phosphoglucose
UTP
u rid in e tripho sphate
CHAPTER
1
I N T R O D U C T I O N
INTRODUCTION
1.1
GENERAL BACKGROUND
The ev o lu tio n o f s o c ie ty to the s o p h is tic a tio n s o f modern day l i f e has been made possible by the
numerous
o f science since the dawn o f mankind.
d isco veries in the f i e l d Development o f fu e ls and
machinery has f a c i l i t a t e d the foundation o f an in d u s t r ia lly based s o c iety in which l i f e
expectancy has been s u b s ta n tia lly increased.
The population explosion has n ecessitated the processing o f foods to reduce wastage and to enable more mouths to be fe d .
The in v e n tio n
o f the steam and combustion engines has improved means o f tra n s p o rta tio n and communication.
In more rece n t years the advancement o f medical
science has provided many new drugs to combat disease in humans and anim als.
S c ie n tis ts have, th e r e fo r e , provided much to improve the q u a lity o f life .
However, they also r e a lis e th a t they have introduced hazards,
many o f which are s t i l l unrecognised.
For- example, the in d u s tr ia l
re v o lu tio n began the la rg e scale p o llu tio n o f the environm ent.
Food
processing requires the use o f unnatural a d d itiv e s to s t a b i li s e , colour and fla v o u r products to improve t h e ir q u a lity , appearance storage l i f e .
and
I t is now recognised th a t many o f these agents can
cause damage to humans and animals e s p e c ia lly i f the d u ratio n o f exposure covers a l if e t im e .
In the race to provide new and more
e f fe c tiv e drugs mistakes have been made.
The a p p a llin g e ffe c ts o f
th a lid o m id e, which caused congenital d e fo rm itie s in c h ild re n born to mothers who had taken the drug during pregnancy, hastened the advancement o f the science o f to x ic o lo g y in the United Kingdom.
1 .2
INTRODUCTION TO TOXICOLOGY
The fundamental basis o f to xico lo g y was recognised many c e n tu ries ago by Paracelsus (1493-1541) who observed th a t "A ll thin gs are poisons, fo r th ere is nothing w ithout poisonous q u a lit ie s .
I t is
only dose which makes a th in g a poison" (c ite d by S ig e r is t , 19 5 8 ). Since the end o f-th e nineteenth century th ere has been a vast increase in the number and a v a i l a b i l i t y o f e f fe c tiv e and p o t e n t ia lly to x ic drugs.
Advances in organic chem istry have enabled the synthesis o f numerous new th e ra p e u tic agents, many by m anipulation o f e x is tin g s tru c tu re s . Increased usage o f drugs has in e v ita b ly led to the observation o f more and v a rie d to x ic re a c tio n s .
These reac tio n s can m anifest themselves in
a number o f ways which may be categorised as fo llo w s :
dose r e la te d ,
id io s y n c r a tic , a l l e r g i c , to le r a te d , physical dependency, c a rc in o g en ic, mutagenic and te ra to g e n ic .
D o se-related t o x ic it y may be e xe m p lified by the actio n o f the coumarin an ticoagu lants which are used to prolong the c lo t tin g tim e o f blood.
Excessive dosing w il l lead to a tendency to bleed due to
the prolonged c lo t tin g tim e.
Id io s yn crac y, which is defined as a c h a r a c te r is tic d is tin g u is h in g an in d iv id u a l, may be demonstrated by the d if f e r in g responses to is o n ia z id which is used in the chemotherapy o f tu b e rc u lo s is .
The
major route o f is o n ia z id metabolism is v ia N -a c e ty la tio n (Jenne, 1965) catalysed by an ac e ty l tra n s fe ra s e enzyme.
Epidem iological stu d ies
have found groups o f people who are slow a c e ty la to rs (La Du, 1972). A good example o f th is is found in Eskimos who th e re fo re d e to x ify the drug less q u ic k ly so th a t i t can accumulate to to x ic le v e ls w ith in the body.
Drug a lle r g y is an adverse re a c tio n which is the r e s u lt o f previous s e n s itis a tio n to the same drug, o r a c lo s e ly re la te d one.
The most
dangerous o f these types o f adverse reactio n s is the onset o f anaphylaxis which can lead to death in a very sho rt tim e .
One w e ll
known drug which w il l cause th is type o f re a c tio n in some p a tie n ts is p e n ic illin
(Schwartz and Vaughan, 1963).
Repeated dosing o f drugs can cause two undesired side e f f e c t s .
The
f i r s t o f these is to le ra n ce which is a decreased responsiveness to the actio n o f a drug.
This type o f e f f e c t is demonstrated by
sympathomimetic amines w hich,w ith repeated dosing, e l i c i t s m a lle r and sm aller card io va scu lar e f fe c ts .
A c la s s ic example o f th is is the
experiments done w ith ephedrine in the dog (Chen and Meek, 192 6).
The second undesired e f f e c t o f repeated dosing is th a t o f physical dependence where the presence o f the drug becomes necessary f o r the normal fu n ctio n in g o f the body.
The w ithdrawal symptoms s u ffe re d by
a person addicted to morphine are a s t r ik in g example o f physical dependence (Andrews and Himmelsbach, 1944).
Carcinogenesis is the development o f cancer which is the m alignant unrestrain ed p r o lif e r a t io n o f somatic c e l l s .
Chemical carcinogenesis
was discovered in man long before i t could be demonstrated in anim als. In 1775
cancer o f the scrotum was recognised as an occupational
disease o f chimney sweeps
and i t was the prolonged exposure to soot
th a t was presumed to be the cause (W o lf, 1952).
I t has since been
re a lis e d th a t the agents involved are the p o ly c y c lic hydrocarbons. Much research is now dedicated to e lu c id a tin g the mechanisms by which cancer is produced.
Any stim ulus which causes a permanent change in the genotype which could cause adverse e ffe c ts in fu tu re generations- is c la s s if ie d as a mutagen.
One o f the best examples o f th is is r a d ia tio n .
Although
the serious im p lic a tio n s o f exposure to mutagens are understood the methods fo r assessing whether o r not a chemical is mutagenic in vivo are s till
in t h e ir r e la t iv e in fa n c y .
The c la s s ic example o f a teratogen is thalidom ide which is known to cause ab n o rm alities in fo e ta l development (W illia m s , 196 3). Teratogens may a ct in a number o f ways, many o f which may not be apparent due to the e a r ly abo rtion o f the fo e tu s .
The discovery o f
the dreadful e ffe c ts o f the drug thalidom ide shocked the world and has caused governments to la y down s t r i c t le g is la tio n concerning the production and m arketing o f new drugs.
This has led to e xten s ive
te s tin g o f drugs in animals to determine any adverse e ffe c ts to enable the s a fe ty o f a drug in humans to be more a c c u ra te ly p re d ic te d .
This b r i e f summary demonstrates the broad im p lic a tio n s o f the study o f to xico lo g y
and suggests some o f the many problems in p re d ic tin g
whether o r not a drug o r chemical exposed to humans o r animals w i l l be s a fe .
A fu r th e r r e a lis a tio n is th a t i t is not only im portant to know
i f an agent is t o x ic , but also how i t causes t o x i c it y .
Approaches to the study o f mechanisms o f t o x ic it y have been many and v a rie d .
One o f the main areas is the in v e s tig a tio n o f e ffe c ts o f
drugs on the l i v e r .
When a drug or chemical is exposed to the body,
whether o r a l l y , in je c te d , inhaled or app lied t o p ic a lly once i t enters the blood stream i t very ra p id ly reaches the l i v e r . o f the l i v e r is the breakdown o f exogenous compounds
One main fu n c tio n .
by metabolism to more w ater so lu b le conjugates which may then be excreted ,
m ainly by the kidney.
The l i v e r is th e re fo re subjected to
p o t e n t ia lly to x ic chem icals, many o f which m anifest t h e i r symptoms w ith in the l i v e r , although others w il l be observed in o th er organs o f the body.
The l i v e r as a m etabolising organ may also produce
to x ic m etabolites from r e l a t i v e l y innocuous parent compounds.
The
importance o f a c tiv e m etab o lites in r e la tio n to t o x i c it y w il l be discussed l a t e r .
The in v e s tig a tio n o f the mechanisms o f t o x ic it y o f chemicals in the l i v e r is th e re fo re o f g re a t importance and w i l l h o p e fu lly give a g re a te r understanding o f the o v e ra ll t o x ic it y o f a compound. I t may also help to .provide in fo rm ation fo r the production o f less hazardous chemicals and drugs in the fu tu re .
1.3
THE STRUCTURE OF THE LIVER
The s tru c tu re o f the l i v e r has been c la s s ic a lly described in terms o f the hexagonal lo b u le (see Fig. 1 ).
This fu n c tio n a l u n it contains
the term inal hepatic venule a t i t s c e n tre , w ith the p o rta l space, hepatic a r t e r io le and b ile duct s itu a te d a t the periphery o f the lo b u le . According to th is c o n fig u ra tio n path o lo g ical lesions o f the l i v e r have been c la s s if ie d as c e n tr ilo b u la r , midzonal o r p e r ip o r t a l.
More recent evidence has in d ic a te d th a t th is is not a tru e d e s c rip tio n o f the fu n c tio n a l u n it o f the l i v e r (Rappaport, 196 9).
If
coloured g e la tin is in je c te d in to the p o rta l vein or hep atic a r te r y i t becomes apparent th a t the term inal a ffe r e n t vessels supply blood to only sections o f the hepatic lo b u le .
These sectors have been demonstrated
to l i e around the term in al p o rta l branches and extend from the c e n tra l vein o f one hexagon to the c e n tra l vein o f an adjacent hexagon.
These
Figure 1.
PS
o o
Q
O
ps
C^>
oOr
PS
o
THV c e n tr ilo b u la r zone
midzonal
O PS
o
Oh
PS
p e rip o rta l zone
O
PS
Schematic re p res en tatio n o f the c la s s ic hexagonal hep atic lo b u le (from P la a , 1975). PS is the p o rta l space, con sisting o f a branch o f the p o rta l v e in , a h ep atic a r t e r io l e , and a b ile duct; (c e n tra l v e in ).
THV is the term inal h ep atic venule
fu n c tio n a l u n its have been defined by Rappaport and co-workers as l i v e r a c in i (Rappaport, 1969) (see Fig . 2 ) .
The simple acinus
consists o f a small parenchymal mass which is ir r e g u la r in s ize and shape, arranged around an axis co n sistin g o f a term in al p o rta lven ule, an hepatic a r t e r i o l e , a b ile d u c tu le , lymph vessels and nerves.
This acinus lie s between two o r more term inal h ep atic venules
(c e n tra l vein s) w ith which i t s vascular and b i l i a r y axis i n t e r d ig i t a t e . The hep atic c e lls o f a simple a c in i are in c e l l u la r and sinuso idal contact w ith the c e lls o f adjacent o r overlapping a c in i.
However,
the c e lls o f one p a r tic u la r acinus are p r e f e r e n t ia lly supplied by t h e i r parent vessels.
This concept has been developed to show th a t
th ere are c ir c u la to r y zones w ith in each acinus which can be d ivid ed in to th ree depending on t h e ir distance from the term in al vascular branch.
I t has been demonstrated th a t th ree o r more sim ple a c in i
can c o n s titu te a complex acinus.
This u n it consists o f th ree simple
u n its and a sleeve o f parenchyma around the preterm inal a ffe r e n t Vessels, the lymph vessels and the nerves th a t e v e n tu a lly give r is e to the term inal a x ia l vessels o f the simple a c in i.
I t has been demonstrated th a t th ere is a lack o f u n ifo rm ity o f fu n ctio n o f the l i v e r c e lls (Chayen, 1974).
The a c t i v it y o f the
re s p ira to ry enzymes has been found to be highest in the zone c lo s e s t to the term inal a ffe r e n t v e s s e l, whereas the most d is ta n t zone has a high le v e l o f microsomal enzymes.
The c la s s ic a l d e s c rip tio n s
o f f o c a l, m idzonal, p e rip o rta l and c e n tr ilo b u la r lesio n s can be explained by Rappaportfs th e o rie s .
C e n trilo b u la r n e c ro s is , a le s io n
caused by carbon te tr a c h lo rid e (Recknagel, 196 7), occurs in the region most d is ta n t from the term inal p o rta l venule (zone 3 ) .
Midzonal
n ec ro sis , s im ila r to th a t caused by b e ry lliu m (W itschi and A ld rid g e , 1967), would occur in the area corresponding to the a c in a r zone c lo s e s t to the term inal a ffe r e n t vessel (zone 1 ) .
A c la s s ic example
o f a compound producing p e rip o rta l necrosis is a l ly ! alcohol ( R o u ille r , 1964).
M orphologically chem ically-induced l i v e r in ju r y can m anifest i t s e l f in d if f e r e n t ways. o f lip id s
The acute e ffe c ts can co n sist o f accumulation
( f a t t y l i v e r ) and the appearance o f degenerative processes
leading to c e ll, death (n e c ro s is ).
The n e c ro tic process can a f f e c t
Figure 2.
THV
PS
'
6
-
CD THV o
Schematic rep res en tatio n o f a simple hep atic acinus (Rappaport, 1969) PS and THV are as in F i g . l . 1, 2 and 3 represent the various zones d ra in in g o f f the term inal a ffe r e n t vessel.
small groups o f is o la te d parenchymal c e lls
(fo c a l n e c ro s is ),
groups o f c e lls located in zones (c e n t r ilo b u la r , m idzonal, o r p e rip o rta l necro sis) o r v i r t u a l l y a l l o f the c e lls w ith in a hep atic lo b u le (massive n e c ro s is ).
Chemical-induced l i v e r in ju r y re s u ltin g from chronic exposure can produce marked a lte r a tio n s o f the e n tir e l i v e r s tru c tu re g iv in g r is e to the degenerative and p r o lif e r a t iv e changes observed in the d if f e r e n t forms o f c ir r h o s is .
N eo p lastic changes may be another end
point o f chemical l i v e r in ju r y .
1 .4
MECHANISMS OF TOXIC LIVER INJURY
The study o f pathology deals m ainly w ith the s tru c tu re d response to d isease.
The t o x ic it y o f any compound is, however, the sum t o t a l o f
i t s in te ra c tio n s w ith c e l l u la r c o n s titu en ts to produce chemical a lte ra tio n s and the response o f the c e ll to those a b e rra tio n s .
I t is
th e re fo re im portant to determine the biochemical le sio n which occurs w ith in the c e l l .
The concept o f the biochemical le s io n was form ulated
by Peters (1 9 6 3 ), who to g eth er w ith others have la id the s o lid modern foundation fo r biochemical pathology.
Two approaches have developed w ith in the f i e l d o f to x ic o lo g y : (a )
the response o f c e lls or organs to in te rfe re n c e w ith selected m etabolic processes, i . e . the response to known biochemical lesions - ATP d e fic ie n c y , in h ib it io n o f p ro te in s yn th e sis , RNA and DNA syn thesis.
(b)
the biochemical mechanisms underlying the in d u ctio n o f d is c re te and s e le c tiv e b io lo g ic a l responses to some to x ic agents such as selected experim ental disease, e .g . carbon te tr a c h lo r id e , galactosamine and e th io n in e .
A wide v a r ie ty o f to x ic chem icals, viruses
and o th e r hazards in the
c e lls environment such as anoxia, are known to induce h ep atic c e ll death I t has also become c le a r th a t p e rs is te n t o r repeated episodes o f l i v e r c e ll necrosis seems to be the basis fo r the chronic progression o f l i v e r disease to c ir r h o s is .
Magee
(1966) made the fo llo w in g observations on the approach to
to x ic
l i v e r c e ll necrosis based on research c a rrie d out up to 1966.
I t would appear th a t the mechanisms o f c e ll death are q u ite s p e c ific and studies have shown th a t one can in t e r f e r e w ith most o f the basic p ro p e rtie s o f the c e ll w ith o u t inducing c e ll death.
C e ll death is
not a degenerative phenomenon in which the e s s e n tia l le s io n is some in h ib it in g e f fe c t
on one o f the e s s e n tia l m etabolic processes in the
c e ll.
i t simply the consequence o f slowing down the c e lls
N e ith e r is
m etabolic a c t i v it y as a r e s u lt o f the in te rfe re n c e w ith one o r more o f i t s major m etabolic fu n c tio n s .
Hence any biochemical change in
l i v e r c e lls in response to agents th a t do not produce necrosis must be by i t s e l f in s u f f ic ie n t to produce c e ll death and n ecro sis.
There have been th ree major hypotheses as to the prim ary ta r g e t f o r the m olecular mechanisms th a t produce c e ll death.
H is t o r ic a lly the f i r s t o f
these was the m itochondrial hypothesis o f C h ris tie and Judah (1954) based on experiments w ith carbon te tr a c h lo r id e which was shown to cause severe m itochondrial damage.
This involved the uncoupling o f
o x id a tiv e phosphorylation, loss o f re s p ira to ry control and the a c tiv a tio n o f ATPase.
I t is now known, however, th a t th is is not cau sative o f
c e ll death as the c e lls have already shown d is ru p tio n o f i n t r a c e ll u l a r membranes, in h ib it io n o f p ro te in and RNA syn th e sis , and an increase in c e l l u la r le v e ls o f t r ig ly c e r id e .
The h e p a to to x ic ity o f carbon
te tr a c h lo r id e was e x te n s iv e ly reviewed by Recknagel (1967) and Plaa and W itschi (1 9 7 6 ).
M itochondrial in ju r y is th e re fo re simply a
m an ife sta tio n o f the general m etabolic d e te r io r a tio n o f a dying c e l l .
The second of the major hypotheses was the lysosomal theory suggested by de Duve a f t e r his discovery o f lysosomes in 1955.
He im p lied th a t
lysosomes could be p o te n tia l su ic id e bags, knowing th a t they contain soluble hydrolases, w ith an acid pH optimum, surrounded by a lip o p ro te in membrane
preventing t h e i r escape.
De Duve (1963)
suggested th a t the normal segregation o f the lysosomal enzymes might have played an e s s e n tia l ro le in keeping the l y t i c processes o f the c e ll lo c a lis e d and
th e re fo re
p ro te c tin g ag ain st a u to ly s is .
This
theory raised a major question as to whether the release o f lysosomal enzymes causes c e ll death, o r only occurs when the c e ll is dead o r
dying?
Or one could ask whether c e ll death is simply au to ly s is ?
H o w ever,it has been shown th a t acute l i v e r c e ll in ju r y by carbon te tr a c h lo r id e (D ia n z a n i, 196 3), thioacetam ide ( S la te r e t a J , 1 9 6 5 ), dim ethylnitrosam ine (Rees, 1962) and b e ry lliu m s a lts (W itschi and A ld rid g e , 1967) gave no evidence im p lic a tin g lysosomal d is ru p tio n as a fa c to r in the cause o f c e ll death.
Lysosomes in fa c t appeared to
be r e l a t i v e l y s ta b le p a r tic le s during the development o f c e ll death. The changes in the d is tr ib u tio n o f the lysosomal hydrolases between the p a r tic u la te and soluble phases o f the c e ll th a t occur la t e a f t e r the a d m in is tra tio n o f hepatotoxins probably only r e f le c ts a scavanging ro le f o r the lysosomes.
The t h ir d theory in d ic a te s the plasma membrane as the prim ary ta r g e t in the pathogenesis o f c e ll death.
A very e a rly e f f e c t o f carbon
te tr a c h lo rid e t o x ic it y is a d e s tru c tiv e a tta c k on the membrane components o f the c e l l .
This is thought to in vo lve hom olytic cleavage
o f a carb o n -ch lo rid e bond follow ed by fre e ra d ic a l a tta c k w ith p e ro x id a tiv e decomposition o f the membrane s tru c tu re d lip id s (Recknagel, 1967).
W ithin the f i r s t hour o f exposure changes w ith in
the endoplasmic reticu lu m are evid en t
and the calcium content o f the
c e ll is increased w ith in twenty fo u r hours.
Associated w ith the
raised le v e ls o f calcium are o th e r e le c t r o ly te d isturbances, s w e llin g o f the l i v e r c e l l s , w ith i n t r a c e ll u l a r enzymes appearing in the plasma.
These changes suggest a severe breakdown o f the s e le c tiv e
p e rm e a b ility o f the c e ll plasma membrane.
Electron m icroscopic
studies o f l i v e r c e lls poisoned w ith dim ethylnitrosam ine (Emmelot and B e n e d e tti, 1960) and thioacetam ide (Rees e t al_, 1961) also demonstrated a lte r a tio n s in the membranes o f the endoplasmic re tic u lu m .
Plasma
membranes were found to be abnormal as e a rly as th ree hours a f t e r the ad m in is tra tio n o f dim eth yln itrosam ine.
I t has been known f o r many years th a t calcium accumulates in n e c ro tic tis s u e .
Judah e t al (1964) suggested th a t the s p e c ific fu n c tio n a l
consequence o f d ir e c t membrane in ju r y is an increase in calcium ion concentration w ith in the c e ll
and th a t the calcium ion is the common
agent m ediating to x ic l i v e r c e ll death.
This view is supported by
re s u lts demonstrating the p ro te c tiv e e f f e c t o f promethazine and r e la te d compounds on the t o x ic it y o f carbon te tr a c h lo r id e (Rees and S p ecto r, 1961).
A ll o f the above observations have the same problem to fa c e .
How
does one decide i f any one given s p e c ific disturbance is ca u s a lly re la te d to c e ll death, o r sim ply a m a n ife sta tio n o f the fa c t th a t the c e ll is ir r e v e r s ib ly in ju r e d , dead o r dyin g , as th is im p lic a te s th a t a l l metabolism and c e l l u la r functio ns are going to be perturbed and e v e n tu a lly cease?
Research in to the mechanisms o f hep atic necrosis has centred around a few model compounds in clu d in g carbon te tr a c h lo r id e , galactosamine and e th io n in e . compound in
By understanding the mechanisms o f t o x ic it y o f one
i t s m inutest d e ta il i t is hoped th a t the mechanism o f
actio n o f o th er compounds might
be more e a s ily understood.
The h e p a to to x ic ity o f e th io n in e has been b r i e f l y reviewed by Farber (1 9 7 1 ).
I t is known to produce a v a r ie ty o f p ath o lo g ical changes in
animals in c lu d in g f a t t y l i v e r and l i v e r cancer (S te k o l, 1963 and F arb e r, 1967).
This methionine analogue has a t le a s t fo u r m etabolic
e ffe c ts on the l.iv e r: (1 )
rapid in ductio n o f ATP d e p le tio n (S h u ll, 1962).
(2 )
e th y la tio n o f RNA, o f h ig h ly s e le c tiv e p ro te in s , e s p e c ia lly nuclear p ro te in s , and o f several o th e r nuclear p ro te in s .
(3 )
in h ib it io n o f m ethylation o f RNA, and o th e r components u t i l i z i n g m ethionine as donor.
(4 )
in co rp o ratio n in to p ro te in in place o f m ethionine.
The evidence stro n g ly suggests th a t most o f the acute h ep atic e ffe c ts o f eth io n in e are due predominantly o r e x c lu s iv e ly to th e d e fic ie n c y o f ATP.
These in clu d e f a t t y l i v e r (F a rb e r, 19 6 7 ), in h ib it io n o f p ro te in
synthesis (V illa -T r e v in o e t al_, 19 6 3 ), a number o f u ltr a s tr u c t u r a l a lte r a tio n s to cytoplasm ic org an elles o f the l i v e r c e ll and nucleolus and in h ib itio n o f RNA synthesis ( V illa -T r e v in o e t a l , 1966).
I t has
been suggested th a t the r a t io o f ATP to ADP to AMP is probably more im portant to c e llu la r fu n ctio n than the absolute value o f any s in g le adenine n u cleo tid e (A tkin son, 1966).
The in h ib it io n o f p ro te in synthesis
and RNA synthesis c lo s e ly p a r a lle l the decrease in ATP le v e ls although the mechanism by which th is occurs is not known.
E thionine also causes
a s t r ik in g loss o f l i v e r potassium w ith a corresponding increase in
sodium le v e ls (Judah e t al,, 1966 and C h ris tie and Judah, 1968) but no apparent e f f e c t on h ep atic BSP uptake, conjugation w ith GSH, o r ex c re tio n as fr e e o r conjugated BSP in the b ile (Schenker and Combes, 1967).
The e ffe c ts o f galactosamine on the l i v e r have been reviewed by Farber and E l-M o fty (1 9 7 5 ).
Figure 3 shows the proposed pathway
leading to c e ll death as caused by galactosam ine.
I t s major i n i t i a l
e f f e c t is to d ep lete the u rid in e nucleo tides by i t s metabolism to UDP hexosamine which traps the u rid in e n u c le o tid e s .
This leads to
re v e rs ib le plasma membrane in ju r y and increased in t r a c e ll u l a r calcium . At higher dose le v e ls o f galactosamine the plasma membrane becomes ir r e v e r s ib ly damaged, follow ed by a marked increase in i n t r a c e ll u l a r calcium and c e ll death.
The experiments described above w ith e th io n in e and galactosamine demonstrate th a t the d e p le tio n o f ATP and UTP re s p e c tiv e ly do not cause c e llu la r necrosis on t h e ir own, as these changes may be r e v e r s ib le .
P ro tein s yn th e sis , and to a le s s e r e x te n t, RNA synthesis are in h ib ite d by a wide range o f to x ic agents.
A lte ra tio n s in the s u b c e llu la r
org an isa tio n and fu n ctio n o f the r a t l i v e r parenchyma have been a ttr ib u te d to th is in h ib it io n (Trump and Ericsson, 196 5).
I t has been
d i f f i c u l t to c o r r e la te s tru c tu ra l changes to a s p e c ific biochemical le s io n , as the m a jo rity o f compounds have many biochemical e ffe c ts in a d d itio n to the in h ib it io n o f p ro te in syn th e sis . in te r e s t
I t is also o f
th a t marked in h ib it io n o f p ro te in synthesis by cyclohexim ide
in ra ts f o r many hours leads to only a few c e l l u la r changes in s e le c te d organs and i t s e ffe c ts are r e v e rs ib le (H a rris e t al_, 1968).
The in h ib it io n o f p ro te in synthesis also appears to be associated w ith several types o f f a t t y l i v e r , e .g . carbon te tr a c h lo r id e , e th io n in e , and puromycin (F a rb e r, 1967;
and Lombardi, 1965 and 1966).
The
l i v e r plays a major ro le in the conversion o f f a t t y acids from the blood to trig ly c e r id e s which are put back as plasma lip o p ro te in s in to the blood.
I f p ro te in synthesis is in h ib it e d , the supply o f e s s e n tia l
p ro tein is h alted causing an accumulation o f t r ig ly c e r id e w ith in the
Figure 3.
NORMAL LIVER CELL
galactosamine
1.
v u rid in e nucleotides
2.
UDP - hexoses
3.
UDP - hexosamines
R eversible plasma membrane in ju r y
| In tra c e llu la r^ c a lc iu m ^ ? Ir r e v e r s ib le plasma membrane in ju r y
Markedly increased in t r a c e ll u l a r calcium
CELL DEATH
T e n ta tiv e scheme f o r the sequence o f events in the development o f l i v e r c e ll necrosis induced by galactosamine (F arb er and E l-M o fty , 1975)
liv e r .
T h is , however, does not always occur, f o r cycloheximide
(V erbin ert al_, 1969) and e th io n in e (Schlunk e t al_, 1968) both in h ib it p ro te in synthesis w ith o u t an accompanying increase in t r ig ly c e r id e le v e ls .
The mechanisms must, th e r e fo r e , be more complex perhaps
in v o lv in g changes in polysomes.
Carbon te tr a c h lo rid e causes disaggre
gation o f polysomes to ribosomal monomers and sub units, whereas w ith cycloheximide and w ith e th io n in e ( in the male r a t ) th is does not occur. The bulk
o f t r ig ly c e r id e is synthesized in the endoplasmic re tic u lu m .
I t may, th e r e fo r e , fo llo w th a t in ta c t polysomes c lo s e ly associated w ith the endoplasmic reticu lu m may allo w a re g u la to ry control o f t r ig ly c e r id e syn th esis.
The main conclusion is th a t in h ib it io n
o f p ro te in synthesis alone is probably not the cause o f c e ll death.
Attempts to associate c e ll death w ith in h ib it io n o f a s p e c ific re a c tio n have also im p lic a te d RNA syn th esis. problems a ris e to those c ite d above.
S im ila r in te r p r e ta tio n
For example,
a -a m a n itin , which
induces severe lesions in several organs o f experim ental anim als, in h ib it s RNA polymerase (T ata and W id n e ll, 1966).
I t causes c e ll
death in the l i v e r and kidney o f the mouse, but i t s e ffe c ts are r e v e rs ib le in r a t l i v e r .
1.5
PROTEIN BIOSYNTHESIS AND THE MODE OF ACTION OF SOME INHIBITORS
Proteins are synthesized by both e u k aryo tic and p ro k a ry o tic c e lls . Since th is th esis is concerned w ith the e f f e c t o f chemicals in e u k aryo tic organisms the emphasis o f th is b r i e f summary is on e u k a ry o tic p ro te in synthesis although much o f the work to e lu c id a te the pathways has been c a rrie d out in p ro k a ry o tic organisms l ik e E. c o l i .
The pathways
a re , however, e s s e n tia lly s im ila r in both types o f c e l l .
The synthesis o f p roteins takes place on the surface o f the ribosomes which in the eu karyo tic c e ll occur e ith e r fre e in the cytoplasm bound to the endoplasmic re tic u lu m . ric h in ribosomes.
or
Furthermore the m itochondria are
The types o f p ro te in synthesized a t these d i f f e r e n t
s ite s vary in t h e ir fu n c tio n s .
The f i r s t stage o f p ro te in synthesis req u ires the a c tiv a tio n o f amino a c id s , the subunits o f p ro te in s , to t h e i r corresponding ami noacyl-tRNAs. This stage req u ires ATP and Mg++ and the ami noacyl-tRNA synthetase s p e c ific f o r the amino acid undergoing a c tiv a tio n .
The ami noacyl-tRNA
synthetases are h ig h ly s p e c ific fo r both the amino acid and i t s corresponding tRNA.
Errors can occur a t th is a c tiv a tio n stage since c e rta in amino acid analogues such as p -flu o ro p h e n y la la n in e , an analogue o f p h e n ylala n in e , and e th io n in e and n o r-le u c in e , analogues o f methionine may be accepted by the ami noacyl-tRNA synthetase and hence be incorporated in to p ro tein s in the p o s itio n s norm ally occupied by m ethionine or p h e n ylala n in e .
The second stage o f p ro te in synthesis involves the i n i t i a t i o n o f the polypeptide chain which occurs on the ribosomes.
Ribosomes co n s is t o f
RNA and p ro tein s and can d is s o c ia te in to two fu n c tio n a l su b units.
In
extram itochondrial p ro te in synthesis the i n i t i a t i n g amino acid is m ethionine in the form o f a s p e c ific methionyl-tRNA which responds to the i n i t i a t i n g AUG codon in the mRNA.
The form ation o f th is i n i t i a t i o n
complex .on the 40S subunit o.f the ribosome also req u ires i n i t i a t i o n fa c to rs and GTP. su b unit.
The i n i t i a t i o n complex then combines w ith the 60S
The methionyl-tRNA is positio n ed a t the i n i t i a t i o n codon
AUG so th a t the ribosomes s t a r t tra n s la tio n a t the c o rre c t p o in t on the mRNA.
The t h ir d stage o f p ro te in synthesis is the elongation o f the p o lypeptide chain which occurs in th ree major steps.
The f i r s t o f these is the b indi
o f the incoming ami noacyl-tRNA to the ami noacyl s it e on the 80S ribosome f a c i l i t a t e d by the binding o f an elongation fa c to r and GTP to the ami noacyl-tRNA.
This step can be in h ib ite d by c e rta in a n t ib io t ic s
p a r t ic u la r ly the te tr a c y lin e s .
The second step in elongation concerns the form ation o f the pep tide bond between the methionyl-tRNA and the ami noacyl-tRNA which is catalysed by p ep tid yl tra n s fe ra s e and re s u lts in the displacem ent o f tRNA from the pep tidyl-tR N A a t the p ep tid yl s i t e .
The a n t ib io t ic puromycin can i n h ib it p ro te in synthesis a t th is stage by becoming bound to the ami noacyl s it e re s u ltin g in the form ation o f a covalent p ep tid yl puromycin d e r iv a tiv e .
Since th is d e r iv a tiv e is not
recognised by the mRNA elongation is h a lte d and p ro te in synthesis in h ib it e d .
The t h ir d step in elo n g atio n o f the polypeptide chain is tra n s lo c a tio n . The ribosome now moves to the next codon on the mRNA and s h if t s the newly formed pep tidyl-tR N A from the aminoacyl to the p e p tid y l s it e on the ribosome le a v in g the aminoacyl s it e fre e to accept another aminoacyl-tRNA.
This step req u ires a s p e c ific elo n g atio n f a c to r as w e ll
as GTP and Mg++.
In eu k a ry o tic c e lls th is elongation fa c to r (EF2) can be
in a c tiv a te d by d ip h th e ria to x in re s u ltin g in an in h ib it io n o f tra n s lo c a tio n .
The f in a l stages o f p ro te in synthesis in vo lve the te rm in a tio n o f the polypeptide chain form ation which is s ig n a lle d by a sp e cia l te rm in a tio n codon on the mRNA.
Releasing fa c to rs then promote the re le a s e o f the
completed polypeptide chain from the ribosome.
The completed p o lypeptide
chain may then undergo covalent m o d ific a tio n to y ie ld i t s b io lo g ic a lly a c tiv e form.
Some o th e r in h ib ito r s o f p ro te in synthesis include chloramphenicol which blocks the p e p tid y l tr a n s fe r re a c tio n in m itochondrial p ro te in syn th e s is .
Cycloheximide blocks peptide bond form ation in extra m ito c h o n d ria l
p ro te in syn th esis.
Both chloramphenicol and cycloheximide bind to the
la rg e ribosomal subunits o f d isso c iated ribosomes.
Streptom ycin on the o th e r hand binds to the small ribosomal su b unit o f p ro k a ry o tic c e lls causing in h ib it io n o f p ro te in synthesis and m isreading o f the gen etic code on the mRNA.
Other aminoglycoside a n t ib io t ic s l ik e
the neomycins and kanomycins a c t in a s im ila r fa s h io n .
The a lk a lo id
emetine in h ib it s the binding o f ami noacyl-tRNAs.
Abrin and
r i c i n , which are to x ic p la n t p ro te in s , in h ib it p ro te in synthesis in eu k a ry o tic c e lls by in a c tiv a tin g the 60S subunit o f the ribosome and thus blocking e lo n g a tio n .
1.6
ACTIVE METABOLITES AND TOXICITY
One o f the main functio ns o f the l i v e r is to m etabolise drugs and fo re ig n compounds to more p o la r compounds to f a c i l i t a t e t h e i r e x c re tio n .
The main route o f e lim in a tio n is v ia the kidney, e x c re tio n
v ia the b i l i a r y system, the in te s tin e and the lungs being secondary to th is ro u te .
During re c e n t years research in to drug metabolism has
demonstrated th a t many fo re ig n compounds are m etabolised to p otent a lk y la t in g , a r y la tin g o r fr e e ra d ic a l in term ed iates which are in f a c t more to x ic than t h e i r parent compounds. . These re a c tiv e in te rm ed iates are very sh o rt liv e d and many e x e rt t h e i r to x ic ac tio n by binding ir r e v e r s ib ly to tis s u e macromolecules.
The lesio n s caused by th is
type o f re a c tio n are norm ally located in the p a rt o f the m etabolising tis s u e which contains the enzyme pathways responsible f o r t h e i r m etabolic a c t iv a t io n .
Many o f the i n i t i a l concepts o f m etabolic
a c tiv a tio n were developed during studies o f chemical carcinogenesis ( M i l l e r and M i l l e r , 1947, 1951 and 196 6).
I t is now known th a t s im ila r
re ac tio n s occur w ith some drugs a t high dosage le v e ls ( G i l l e t t e e t a l , 1974, M itc h e ll and J o llo w , 1975).
The re a c tiv e m etab o lites can lead to
various t o x ic it ie s in clu d in g c e l l u la r n ec ro sis , c a n c e r,.m u ta tio n s , immunological re a c tio n s , blood dyscrasias and fo e ta l damage.
A few
examples o f these types o f agents are given in Table 1, some w i l l be discussed in more d e t a i l.
The h e p a to to x ic ity o f carbon te tr a c h lo r id e has been e x te n s iv e ly in v e s tig a te d (Recknagel, 1967).
I t was not u n t il 1965-1966 th a t
evidence th a t the metabolism o f carbon te tr a c h lo r id e was e s s e n tia l f o r i t s t o x ic it y was published (Goshal and Recknagel, 1965; S la t e r , 1966; McLean and McLean, 196 6).
I t was demonstrated
in v it r o th a t the carbon
te tr a c h lo rid e s tim u la tio n o f l i p i d p ero x id a tio n in l i v e r microsomes is dependent on a supply o f NADPH, suggesting the involvem ent o f the NADPH-cytochrome P450 complex.
This was supported by the f a c t th a t
phenobarbital inductio n o f the complex g r e a tly increased the t o x i c it y o f carbon te tr a c h lo rid e (McLean and McLean, 196 6).
I t has since been
suggested th a t the most l i k e l y re a c tiv e product o f metabolism is the tric h lo ro m e th y l r a d ic a l, CCl^.
Gordis (1969) showed th a t the C C 1 g ro u p
attaches c o v a le n tly to lip id s and p ro te in s , the p a tte rn produced by la b e lle d CCl^ in vivo being s im ila r to th a t obtained a f t e r producing
Table 1.
Toxic Agent
CC14 CBrCl3 Halothane
References
)
S la te r (1972)
J
Burdino e t al (1973)
Koch e t a l (1974)
Wood e t a l (1976) Dim ethylnitrosam ine
j Magee e t al (1976) ( Gravela e t a l (1974)
T ric h lo ro e th y le n e
V inyl C hloride
Van Duuren and Banerjee (1976)
j B o lt
sLsl (1976)
( Kappus e i a l (1976)
Carbon D isulphide
Paracetamol
De M a tte is and Seawright (1976)
j Oollow e t a l (1973) ( Labadarios e t a l ( 1 9 /7 ).
A fla to x in
Patterson (1978)
N -acetylam inofluorene
M il le r (1970)
S u b stitu ted azobenzenes
Kadlabar
Is o n ia z id
Nelson e t a l (1976)
e t al (1976)
/ Nakatsugawa and Dahm (1967) Parathion
< Ptashne e t a j (1971)
Phenyl th io u rea
Poore and Neale (1972) j De M a tte is and Seawright (1976)
a -n ap h th ylis o th io c yan ate
1
Examples o f to x ic agents th a t re q u ire m etabolic a c tiv a tio n in the l i v e r in order to e x e rt t h e ir f u l l p o te n tia l as hepatotoxins ( S la t e r , 1978).
the CCl^ ra d ic a l in v it r o in the presence o f l i v e r microsomes.
Studies
by Uehleke e t . a l (1973) and Recknagel . e t . a l (1976) have shown th a t c o valen t binding by CCl^ occurs a n a e ro b ic a lly as w ell as a e r o b ic a lly , but l i p i d p ero xid atio n (which is a consequence o f CCl^ production) only occurs in the presence o f oxygen.
Associated w ith the l i p i d p ero x id a tio n
is the lib e r a tio n o f b io lo g ic a lly r e a c tiv e breakdown products, some o f which cause increases in c a p illa r y p e rm e a b ility and p la t e le t aggregation (Ugazio jet ert, 1976).
I t can be seen from the above
e ffe c ts th a t the m etabolic a c tiv a tio n o f carbon te tr a c h lo r id e to the tric h lo ro m e th y l ra d ic a l is v i t a l to the production o f i t s hep ato toxic e f fe c ts .
F u rth er work by S la te r e t a l (1980) has i d e n t if ie d , using techniques in clu d in g pulse ra d io ly s is and e le c tro n spin resonance, a more re a c tiv e fre e r a d ic a l, C C l^ * , derived from carbon te tr a c h lo r id e a f t e r i t s in te ra c tio n w ith a c tiv e oxygen.
The amount o f 0 ^ present
a t the s it e o f C C l^ -a c tiv a tio n may a f f e c t the r e a c t iv it y o f the fre e ra d ic a l products and may, th e r e fo r e , have a q u a lit a t iv e and q u a n tita tiv e a f f e c t on the c e l l u la r le s io n produced by CCl^.
The halogenated hydrocarbon bromobenzene, which is known to be hepatotoxic has been shown to be converted to a chem ically re a c tiv e m e ta b o lite , bromobenzene 3 ,4 epoxide, by a cytochrome P450 mediated metabolism (Brodie e t al_, 1971: 1973).
Jollow e t a l , 1974a:
and Zampaglione,
About 70% o f th is epoxide is converted to a g lu ta th io n e
conjugate by the g lu ta th io n e tra n s fe ra s e s in the l i v e r .
The g lu ta th io n e
conjugate is then converted to a m ercapturic acid which is excreted v ia the kidney.
I t has been demonstrated th a t c e n tr ilo b u la r necrosis
o f the l i v e r does not occur u n t il the dose o f bromobenzene is high enough to d ep lete the le v e l o f g lu ta th io n e below a th resh o ld l e v e l. When the g lu ta th io n e le v e ls are reduced the epoxide m e ta b o lite c o v a le n tly binds to tis s u e macromolecules causing tis s u e le s io n s . The re la tio n s h ip o f g lu ta th io n e to h e p a to to x ic ity has been demonstrated fo r o th e r hepatotoxins in clu d in g paracetamol which w i l l be discussed in g re a te r d e ta il in a l a t e r s e c tio n .
1 .7
RELATIONSHIP OF ENZYME INDUCTION AND INHIBITION TO TOXICITY
The previous section demonstrated the importance o f the cytochrome P450 mediated enzyme system in the metabolism o f drugs and fo re ig n compounds by the l i v e r .
I t is now known th a t the a c t i v i t y o f
th is system can be induced iji vivo by a number o f agents such as phenobarbitone (Conney, 1967;
G oldstein et_ al_, 1974).
Other known
inducers include the carcinogenic hydrocarbon 3,4,benzyp yrene, some o f the methylenedioxyphenyV compounds, eth y l alcohol and the s te ro id hormones.
I f t o x ic it y is metabolism m ediated, in d u ctio n
o f the drug m etab o lizin g enzyme system w i l l increase t o x i c it y . This has been made use o f in in v e s tig a tio n s o f the t o x i c it y o f drugs and chemicals in experim ental co n d itio n s .
I t is also o f
g re a t importance in the c lin ic a l s itu a tio n , e s p e c ia lly i f polypharmacy is being p ra c tis e d .
The c o -a d m in is tra tio n o f
b a rb itu ra te s may enhance o r dim inish the t o x i c it y o f o th e r drugs, o r o f fo re ig n chemicals present in the body from o th er sources.
It
has also become w id ely recognised th a t th ere are inducers o f drug m etabolizing enzymes present in the environment and d i e t .
These must
be taken in to co n sid eratio n when designing experiments w ith anim als, as any v a r ia tio n in conditions o f the d ie t o r environment may produce d if f e r e n t re s u lts from those expected, e s p e c ia lly when in v e s tig a tin g e ffe c ts r e la te d to metabolism.
In h ib it io n o f drug metabolism is also o f g re a t importance.
A
to x ic response w i l l be reduced i f t o x i c it y is m e ta b o lite m ediated, o r increased i f the parent molecule is the to x ic agent.
SKF 525A,
o r /?-diethylam i noethyl diphenyl propyl a c e ta te , is a p o ten t in h ib it o r o f cytochrome P450 mediated drug metabolism w ith no pharm acological a c tio n o f i t s own but w ith wide uses in medical research. a -N a p h th y l isocyanate and c o b a lt c h lo rid e are also used as experim ental in h ib it o r s .
Use o f in h ib ito r s o f drug metabolism has been made in the c lin ic a l s itu a tio n .
D is u lfira m produces very unpleasant side e ffe c ts
in c lu d in g flu s h in g , dyspnoea, nausea, vom iting and hypotension, when i t is ingested concommitantly w ith a lc o h o l.
I t has, th e r e fo r e ,
become a useful agent in the treatm ent o f alcoholism .
I t s a c tio n
is thought to be due to the accumulation o f acetaldehyde (H ald e t
a l , 1948) caused by the in h ib it o r y e f f e c t o f d is u lfir a m on aldehyde dehydrogenase, an enzyme which norm ally oxid ises acetaldehyde to a c e tic a c id .
In h ib ito r s o f the monoamine oxidase enzyme system have been used in the treatm en t o f severe depression, f o r example tranylcyprom ine and p a rg y lin e .
A side e f f e c t o f these drugs is caused by in g e s tin g
tyram in e , a monoamine present in a number o f food s tu ffs l ik e cheese and marmite.
Tyramine would norm ally be ra p id ly o xid ised
by the monoamine oxidases, but i f the l a t t e r are in h ib ite d by compounds l i k e tranylacyprom ine the tyram ine w i l l accumulate and may cause serious lesio n s due to c ard io va scu lar e ffe c ts (G oldberg, 196 4).
Another useful in h ib it o r o f drug metabolism is a llo p u r in o l, an in h ib it o r o f xanthine oxidase which c a ta ly s e s -th e o x id a tio n o f xanthine to u r ic a c id .
In gout u ric acid production is exc e s s iv e ly
high and a llo p u rin o l has proved to be o f g re a t value in a lle v ia t in g the s u ffe rin g caused by th is disease.
1 .8
PARACETAMOL HEPATOTOXICITY
Paracetamol was used as the main model hepatotoxin in the research c a rrie d out f o r th is th e s is .
A f u l l e r review o f the l i t e r a t u r e
r e l a t i ng to paracetamol h ep ato to x ici ty w i11, th e r e fo r e , be gi ven h e re .
Paracetamol (acetaminophen, N -a c e ty l-p ara-am in o -p h en o l) is a commonly used analgesic w ith a n tip y r e tic p ro p e rtie s .
M .I.M .S .
(th e
monthly index o f medical s p e c ia lit ie s , 1979) l i s t s f o r t y - f i v e p rep aratio n s con taining paracetamol which are in re g u la r use w ith in the United Kingdom. . Over two hundred p rep aratio n s are l is t e d by Ameer (1977) as being used in the United S ta te s .
This drug was
synthesized a t John Hopkins U n iv e rs ity in 1877, and i t was f i r s t used in c lin ic a l medicine by von Mehring in 1893 (Spooner and Harvey, 1976).
I t s use in medicine did not become exten sive u n t il
i t was recognised as the p rin c ip a l a c tiv e m e tab o lite o f a c e ta n ilid e and phenacetin by Brodie and Axelrod in 1949 (1948 , 194 9).
Paracetamol
is now used r e g u la r ly in the treatm ent o f co ld s, coughs and m ild p a in , and i t s use in preference to a s p ir in , which is known to cause b le e d in g , is encouraged (Madan, 1976).
In a double b lin d t r i a l
c a rrie d out by M ie lk e _ e t a l (1976) a s p ir in was shown to have e ffe c ts on bleeding tim e , p l a t e le t aggregation and p l a t e le t fa c to r 3 re le a s e , whereas paracetamol did not demonstrate these e ffe c ts .
Paracetamol is very safe when ingested a t the normal th e ra p e u tic dose, u s u a lly one gram taken up to fo u r times d a ily .
Large
overdoses, however, are now known to produce hep atic n ec ro sis . This was f i r s t reported to occur in ra ts by Boyd and Bereczky (1 9 6 6 ), follow ed by a re p o rt o f i t s occurrence in man by Davidson and Eastham (1 9 6 6 ).
F a ta l hepatic necrosis has been demonstrated in
ra ts and mice (M itc h e ll e t a l , 1973a), hamsters (Davis e t a l , 1 9 7 4 ), dogs (Gazzard j i t a l , 1975) and man (P re s c o tt e t a l , 19 7 1 ). The evidence suggests th a t the minimum le th a l dose in a d u lt humans is about 25g (Proudfoot and W rig h t, 1 9 7 0 ), although P re s c o tt and W right (1973) reported a f a t a l case a f t e r in g e s tio n o f only 18g. One o f the main problems associated w ith paracetamol is i t s usage in s e l f poisoning.
In 1973 alone about 5000 cases w e re 're p o rte d in
B r ita in w ith about f i f t y deaths (McLean, 197 4).
The normal blood
le v e ls o f paracetamol a f t e r a 500-600 mg dose are between 5 and 20 ju g/ml in humans, to x ic e ffe c ts are not e vid en t u n t il i t reaches 300 ju g/ml (Madan, 1977).
Dixon e t al_ (1975) c a rrie d out a d e ta ile d a n a ly s is o f the c e l l u la r events leading to necrosis using l i g h t - and e le c tro n -m ic ro s c o p ic , and histochem ical studies in ra ts s a c r ific e d a t in te rv a ls o f up to f o r t y e ig h t hours fo llo w in g overdosage w ith paracetamol (3g per k g ).
They
found glycogen d e p le tio n , loss o f ribosomes and cytoplasm ic m a trix sw e llin g commencing between th ree and s ix hours a f t e r a d m in is tra tio n o f a dose which caused fra n k c o a g u lative necrosis between tw elve and tw e n ty -fo u r hours in c e n tr ilo b u la r hepatocytes.
Midzonal c e lls
showed more prominent aqueous s w e llin g , w ith v e s ic u la tio n o f the endoplasmic reticu lu m and some hydropic va c u o !a tio n .
There was an
e a r ly tra n s ie n t r is e in succinate dehydrogenase a c t i v i t y fo llo w ed by a progressive reduction between s ix and tw elve hours which appeared to p a r a lle l sw ellin g and d is ru p tio n o f m itochondria c e lls .
in degenerating
There appeared to be no e a r ly involvem ent o f the lysosomes,
acid phosphatase a c t i v i t y being reduced only in c e lls alread y n e c ro tic .
Between tw e n ty -fo u r and f o r t y - e ig h t hours macrophages
accumulated in the n e c ro tic zones where the phagocytosis o f c e l l u la r deb ris was accompanied by a prominent m ito tic a c t i v it y in. su rv iv in g hepatocytes.
B u tta r e t al_ (1976) in v e s tig a te d the e ffe c ts o f s u b -le th a l, but to x ic doses o f paracetam ol, in ra ts using biochemical parameters as in d ices o f h e p a to to x ic ity .
They found dose-dependent e le v a tio n s in a c t i v i t i e s
o f the serum enzymes g lu ta m ic -o x a lo a c e tic transam inase, g lu ta m ic pyruvic transaminase and s o rb ito l dehydrogenase, to g e th e r w ith an increase in h e p atic t r ig ly c e r id e le v e ls , fo llo w in g s in g le doses o f 0 .5 and lg per kilogram bodyweight.
Maximal damage occurred w ith in
tw elve to eighteen hours, h ep atic fu n c tio n , in terms o f serum enzymes and h ep atic t r ig ly c e r id e re tu rn in g to norm al, between fo r ty -e ig h t, and seventy-two hours.
They also found an increase in hexo b arb ital induced
sleeping time in d ic a tin g th a t paracetamol in h ib it s the microsomal enzyme a c t i v it y o f the l i v e r .
T h o rg eirsso n -et aJ_ (1976) have in v e s tig a te d s im ila r parameters in mice using doses o f 400 mg/kg.
They found decreases in microsomal
p ro te in con centration w ith in th ree hours o f paracetamol a d m in is tra tio n , reductions in cytochromes P450 and b^ occurring a f t e r nine hours, but no e f f e c t on t r ig ly c e r id e le v e ls .
There was also a 60% reduction in
in vivo p ro te in synthesis measured b y [3H)-Leucine in c o rp o ra tio n in to to ta l l i v e r p ro te in s .
The LD5q o f paracetamol in ra ts was found to be 5 .2g /kg (McLean and Day, 1975).
This value was a ffe c te d by d ie t w ith low p ro te in and y e a s t
d ie ts reducing the LD^q .
In a chronic study where ra ts were given
200mg/kg paracetamol f o r 200 days no adverse e ffe c ts were found (Thomas e t ail^, 1977).
I t has also been demonstrated to have no
te ra to g e n ic e ffe c ts in ra ts (Lubway and B u rriss G a r r e tt, 1 9 7 7 ), o r mice (Lambert and Thorgeirsson, 1976).
The c lin ic a l s ig n ific a n c e o f paracetamol t o x ic it y in man has generated a l o t o f research in to the mechanism by which paracetamol m anifests i t s t o x i c it y .
Much o f th is work has been c a rrie d out a t the N atio n al
In s t it u t e o f Health (Bethseda, U .S .)*
M itc h e ll e t al_ (1973a) demonstrated
t h a t p retreatm ent o f ra ts and mice w ith phenobarbitone o r 3-methyl cholanthrene, inducers o f drug metabolism, increased the disappearance o f paracetamol from the plasma and tissu es o f the animals and increased the s e v e r ity o f hep atic n ec ro sis .
Conversely p retreatm ent w ith
in h ib ito r s o f drug metabolism, c o b a lt c h lo rid e and piperonyl buto xid e, in h ib ite d the metabolism and disapperance o f paracetamol from the tis s u e s , y e t protected ag a in s t necro sis.
On the basis o f these
observations they postu lated th a t paracetam ol-induced h e p a to to x ic ity is mediated by a to x ic m e ta b o lite o f paracetam ol.
Covalent binding o f ra d io la b e lle d paracetamol to mouse l i v e r p ro te in was found to be dose-dependent (Jo llo w e t a l , 19 7 3 ), as was h ep atic nec ro sis .
Pretreatm ent w ith inducers o r in h ib ito r s o f drug metabolism
were found to have e ffe c ts on the degree o f covalent binding which c o rre la te d w ith e ffe c ts on n ec ro sis .
I t was, th e r e fo r e , suggested
th a t the h ep atic necrosis may be caused by the co valen t binding o f a chem ically r e a c tiv e m e ta b o lite o f paracetamol to v i t a l h ep atic macromolecules. F u rth e r studies by P o tte r e t a l (1973) on the binding 3 o f H-paracetamol to h ep atic microsomes in v it r o demonstrated th a t the binding was mediated by a cytochrome P450 dependent mixed fu n c tio n oxidase.
The treatm ents which a lte re d the t o x i c it y o f paracetamol
in vivo s im ila r ly a ffe c te d th e e x te n t o f binding in v i t r o in d ic a tin g th a t
in v i tr o binding was a v a lid index o f paracetamol h e p a to to x ic ity .
P o tte r j i t a l (1973) also in v e s tig a te d the binding o f 2-a c e ty la m in o flu o re n e demonstrating th a t i t s binding was also dependent on cytochrome P450.
The t o x ic it y o f 2 -a c e ty l am inofluorene is thought to
r e s u lt from i t s conversion to an N-hydroxy d e r iv a tiv e (Grantham e t a l , 1965).
The s im ila r it ie s between the two compounds suggested th a t the
hepatotoxic m e ta b o lite o f paracetamol m ight also be an N-hydroxy d e r iv a tiv e .
The a r y la tio n by th e hepatotoxic m e ta b o lite o f paracetamol was found to be prevented in v it r o by the a d d itio n o f e le c t r o p h ilic sulphydryl compounds l ik e g lu ta th io n e and cys te in e (P o tte r e t a l , 197 3). M itc h e ll e t a j (1973b) then demonstrated th a t g lu ta th io n e also protected a g a in s t h e p a to to x ic ity in vivo in mice.
/
Pretreatm ent o f
mice w ith d ie th y l m aleate, which depletes g lu ta th io n e , p o te n tia te d paracetam ol-induced n ec ro sis .
Paracetamol caused a dose dependent
d e p le tio n o f g lu ta th io n e , w ith co v ale n t binding o f the to x ic in te rm e d ia te to the tis s u e macromolecules not occurring u n t il the g lu ta th io n e had been depleted by more than 70%.
Davis e t a l (1974) in v e s tig a te d the hepatotoxic e ffe c ts o f paracetamol in d if f e r e n t animal species fin d in g th a t the mouse and hamster were more su s cep tib le to h e p a to to x ic ity than the r a t and guinea p ig . The s e v e rity o f damage was c o rre la te d to the r a te o f form ation o f the a r y la tin g m e ta b o lite by the hep atic microsomal enzymes, and to the r a te o f d e p le tio n o f g lu ta th io n e .
The N -h yd ro xylatio n o f
2-a c e ty la m in o flu o re n e , also an N -a c e ty la ry la m in e , occurs to a g re a te r e x te n t in those species more s u s cep tib le to paracetam ol-induced h e p a to to x ic ity , f u r th e r supporting th e theory th a t the to x ic m e ta b o lite o f paracetamol re s u lts from N -h y d ro xylatio n .
I t is known th a t d ru g -g lu ta th io n e conjugates are f u r th e r m etabolised j n vivo and appear in th e u rin e as m ercapturic acids- (Boyland, 197 1). In v e s tig a tio n s by Jollow e t a] (1974b) demonstrated th a t the amount o f paracetamol m etabolised to a m ercapturic acid was g re a te r in the species more s u s cep tib le to t o x i c it y .
When the dose o f paracetamol reached
hepatotoxic le v e ls the fr a c tio n o f the dose excreted as the m ercapturic acid decreased m arkedly, supporting
the evidence th a t
g lu ta th io n e is depleted in paracetamol h e p a to to x ic ity .
They also
suggested th a t the normal m etabolic in te rm e d ia te which conjugates w ith g lu ta th io n e in the m ercapturic acid pathway could also be the e le c t r o p h ilic m e ta b o lite , which in the absence o f g lu ta th io n e a r y la te s hep atic macromolecules and causes c e ll death.
F u rth er studies were c a rrie d out in hamsters, a species very s u s cep tib le to paracetamol t o x i c i t y , which are known to N -hyd ro xylate o th er N -acetylarylam ines more r a p id ly than o th er species (P o tte r e t a l , 1974).
3-M ethylcholanthrene p retreatm ent increased the s e v e r ity o f the
damage whereas phenobarbitone did n o t.
F u rth e r studies on the metabolism
o f o th er N -acetylarylam ines demonstrated th a t 3-m ethylcholanthrene induced N -h y d ro x y la tio n , whereas phenobarbitone in hamsters increased the g lu cu ro n id atio n o f the parahydroxyl group o f paracetamol which, is
a d e to x ify in g re a c tio n r e s u ltin g in a f a s t e r clearance o f paracetamol from the tis s u e s .
Figure 4 shows the p ostulated m etabolic pathway f o r paracetamol (M itc h e ll e t al_, 1976a) which suggests th a t the to x ic pathway is v ia an N-hydroxy
d e r iv a tiv e o f paracetamol to an imidoquinone, which then
ir r e v e r s ib ly binds to the c e l l u la r macromolecules, leading to necrosis and death o f the l i v e r c e lls .
The g lu ta th io n e conjugate is excreted
as a m ercapturic acid which has been id e n t if ie d in the u rin e o f animals and man (M itc h e lle t al_, 1974).
On the basis o f th is observation th a t
metabolism o f paracetamol in humans also involves g lu ta th io n e much research is now being d ire c te d to fin d in g an a n tid o te to paracetamol overdosage.
M itc h e ll e t al_ (1974) suggested cysteam ine, a sulphydryl
group con taining compound
which p rotected ag a in s t the to x ic e ffe c ts o f
paracetamol in anim als, supported by Gazzard e t a l (1 9 7 4 ).
Boyland and
Chasseaud (1969) give fu r th e r support to the ro le played by g lu ta th io n e in m ercapturic acid b io s y n th e s is .
S im ila r ly th ere is much evidence to
support the ro le o f the cytochrome P450 mixed fu n c tio n oxidases in the form ation o f a c tiv e m etab o lites (Sims and G rover, 1974; .1964;
J e rin a and Daly
and Nebert e t a l , 1975).
-
In fu r th e r studies by Davis e t al_ (1976) on species v a r ia tio n i t was p o stu lated th a t the d if f e r e n t s u s c e p t ib ilit ie s o f animals to paracetamol t o x ic it y could be due to d if f e r in g c a p a c itie s to synthesize g lu ta th io n e , o r to form glucuronide o r sulphate conjugates.
They found
th a t the sulphate conjugation o f paracetamol was a s a tu ra b le process which co u ld , th e r e fo r e , lead to a g re a te r form ation o f to x ic m e ta b o lite . S la t te r y and Levy (1977) have suggested the use o f in o rg an ic sulphate as a p ro te c tiv e agent ag ain st t o x ic it y by i t s a b i l i t y to increase the sulphate conjugating c a p a c ity , having demonstrated a reduction o f t o x ic it y in mice a f t e r tr e a tin g w ith sodium su lp h ate.
F u rth er work is s t i l l being c a rrie d out to determine the i d e n t it y o f the to x ic m etabolic in te rm e d ia te , and to develop the id e a l therapy in overdosage o f paracetam ol, as w ell as continuing studies to develop animal models o f t o x i c it y .
A lready some doubt has been c a s t,
on the actual mechanisms already postulated', by Labadarios e t al_, (1977)
Figure 4.
NHCOCH.
NHCOCH.
sulphate
NHCOCH.
OH P-450 "
glucuronide Mixed fu n ctio n
H0-N-C0CHol
► Postulated to x ic in te rm ed iates NCOCH
g lu ta th io n e
N u c le o p h ilic c e ll macromolecules
HNCOCH
HNCOCH
g lu ta th io n e
MERCATPTURIC ACID
c e ll macromolecules
CELL DEATH
Postulated m etabolic pathways o f paracetamol (M itc h e ll e t a l , 1976a)
They demonstrated th a t a sulphydryl type compound, a-m ercap to p ro p io n y lg lycin e* reduced necrosis but did not a f fe c t the degree o f covalent binding in mice. They concluded th a t the binding o f the re a c tiv e m e tab o lite o f paracetamol to hepatocyte macromolecules need not lead to hep atic necrosis provided th a t sulphydryl groups are present in the l i v e r to prevent the d e le te rio u s e ffe c ts o f such b in ding.
More re c e n tly N-hydroxyparacetamol has been synthesized by Gemborys e t^ I
(1 9 7 8 ),
This postulated m e ta b o lite o f paracetam ol,
a t a p h y sio lo g ical pH in v i t r o , is f a r more s ta b le than p re v io u s ly suggested
w ith a h a l f - l i f e o f s lig h t ly less than 20 m inutes.
N-hydroxyparacetamol has been shown to dep lete g lu ta th io n e and cause h e p a to to x ic ity (Healey e t a l , 1978).
McMurtry e t al (1978) have demonstrated
th a t the kidney t o x ic it y which can also be* produced by paracetamol is probably due to a m e ta b o lite produced in s itu since p retreatm en t w ith 3-m ethylcholanthrene increased l i v e r but not kidney t o x i c it y .
I t could be expected th a t i f the N-hydroxy m e tab o lite was responsible f o r t o x ic it y produ ctio n .in creased production in the l i v e r would also cause increased kidney t o x ic it y since the h a l f - l i f e is r e l a t i v e l y long. This however is not the case.
These re s u lts have th e re fo re questioned
the p o s tu la te th a t N-hydroxyparacetamol is the to x ic m e ta b o lite ( G i l l e t t e , 1981).
De V ries (1981) has suggested th a t the hep ato toxic a c tio n o f paracetamol is dependent upon o x id a tio n to a to x ic m e ta b o lite v ia another pathway. He postulated th a t o x id a tio n o f paracetamol or tra n s fe r o f e le c tro n s to a semiquinone, which is more re a c tiv e than the quinoneim ine, is t h e o r e t ic a lly possible and can also e x p la in the conjugation o f g lu ta th io n e :
NCOCH.
HNCOCH
HNCOCH.
OH
HNCOCH.
quinoneimi ne
semiquinone
paracetamol
HNCOCH.
HNCOCH-
HNCOCH GS'
0
•SG
V!
OH
*
g lu ta th io n e conjugate
semiquinone
The h e p a to to x ic ity o f paracetamol may th e re fo re be due to in a c tiv a tio n o f in t r a c e ll u l a r p ro tein s v ia the re a c tiv e semiquinone o r a c tiv e oxygen species.
The to x ic m etab o lite o f paracetamol s t i l l
remains u n id e n tifie d .
Furthermore
the complete m echanistic pathway involved in the production o f the to x ic m e ta b o lite o f paracetamol has not been com pletely e lu c id a te d .
C H A P T E R
I S O L A T E D
2
H E P A T O C Y T E S
ISOLATED HEPATOCYTES 2.1
INTRODUCTION
The use o f is o la te d c e ll systems has become widespread in recent years due to the development o f r e lia b le is o la tio n techniques.
This
area o f research has become im portant f o r two main reasons.
F i r s t l y the increasin g use o f animals in the te s tin g o f drugs and chemicals has led to c r itic is m from the general p u b lic who fe e l th a t the use o f larg e numbers o f animals is not only unnecessary, but also c r u e l.
Recently a number o f pressure groups have been formed
to voice these fe e lin g s more f o r c e f u lly .
Research la b o ra to rie s and
pharmaceutical companies have become the ta rg e ts o f a n t i-v iv is e c t io n groups who w il l do t h e i r utmost to stop any animals being used in experim ents.
Although researchers and in d u s t r ia lis t s are sym pathetic
towards these views they have to comply w ith the increasing demands o f re g u la to ry agenc.ies to provide evidence fo r the s a fe ty o f t h e i r products from animal te s ts .
Many s c ie n t is ts , however, are becoming aware o f
the need to develop a lte r n a tiv e methods f o r the s a fe ty screening o f drugs and chemicals which w il l u t i l i s e less anim als. .Hence much research is now d ire c te d towards developing in v it r o screening te s ts fo r p re d ic tin g the s a fe ty o f chem icals.
One area in which some progress
has been made is the te s tin g o f chemicals fo r mutagenic p o t e n t ia l.
A
number o f te s ts have been designed which u t i l i s e b a c te r ia , y e a s ts , drosophila
f l i e s o r c u ltu red mammalian c e lls ra th e r than whole anim als.
The use o f these types o f te s ts is becoming ro u tin e among chemical and pharmaceutical companies although many o f the te s ts have given equivocal re s u lts in some cases which are d i f f i c u l t to in t e r p r e t .
There is
th e re fo re a need to develop and v a lid a te in v i t r o te s ts which w i l l enable drugs and chemicals to be te s te d f o r p o te n tia l t o x i c it y .
Is o la te d
hepatocytes provide one possible a lte r n a tiv e which uses small numbers o f anim als.
I f these types o f te s ts can be developed the number o f chronic
t o x ic it y te s ts which a t the present tim e have to be c a rrie d out on animals may be reduced, to g eth er w ith the vast fin a n c ia l investments necessary to develop new and s a fe r products.
This la s t p o in t is
becoming a major fa c to r in lim it in g o r ig in a l research aimed a t fin d in g new drugs.
Secondly the recent advances in biomedical research have demonstrated th a t c e llu la r functio ns and the mechanisms o f actio n o f to x ic agents are very complex.
Is o la te d c e lls provide an experim ental tool
whereby mechanisms may be in v e s tig a te d in a system removed from the many influences involved in whole animal experim entation in clu d in g pharm acokinetics, environmental and hormonal c o n tro ls .
The l i v e r is a major organ w ith in the body involved in endogenous and exogenous metabolism and i t is the p rin c ip a l ta r g e t organ f o r the to x ic actio n o f many chem icals.
I t is th e re fo re p a r t ic u la r ly re le v a n t to
use i t in an is o la te d s itu a tio n .
The f a c t th a t i t is e a s ily removed
from the body and i t s te x tu re renders i t s u ita b le fo r the m anipulations required fo r the is o la tio n o f separate c e lls is a g re a t advantage.
The l i v e r contains a number o f d if f e r e n t c e ll types which f a l l w ith in two main classe s, parenchymal and non-parenchyma1.
Parenchymal c e lls
are now known to have d if f e r e n t functio ns dependent on t h e i r lo c a tio n w ith in the l i v e r acinus.
I t is th e re fo re im portant th a t the y ie ld
o f c e lls is o la te d from a l i v e r should be high enough to provide a sample containing re p re s e n ta tiv e numbers o f the d if f e r e n t c e ll types.
2 .2
ISOLATION TECHNIQUES
The techniques i n i t i a l l y employed fo r is o la tin g l i v e r c e lls used chemical and mechanical means.
Although the c e ll y ie ld s were o fte n
high the v i a b i l i t y was in v a r ia b ly very low, the m a jo rity o f the c e lls having damaged plasma membranes.
The many studies reported using these
c e lls were, th e r e fo r e , inadequate as they were not re p re s e n ta tiv e o f in vivo fu n c tio n s .
Many o f these studies have been reviewed by
S chreib er and S chreib er (1 9 7 3 ).
In 1967 Howard
et _al_ introduced a technique in v o lv in g d ig e s tio n o f the
l i v e r w ith a m ixture o f enzymes, collagenase and hyaluron idase. l i v e r was i n i t i a l l y perfused w ith the enzymes in a Ca
4* +
The
- fre e s o lu tio n
in the c o ld , th is was follow ed by s lic in g the tis s u e and in cu b atin g the s lic e s w ith the enzymes a t 37°C.
This method produced c e lls w ith a
more acceptable v i a b i l i t y but the y ie ld was s t i l l
r e l a t i v e l y low.
The
major breakthrough came when Berry and Friend (1969) f i r s t described a technique f o r the perfusion o f the l i v e r w ith collagenase under p h ysio lo g ical conditions .which re s u lte d in high y ie ld s o f in t a c t c e lls . This technique is r e l a t i v e l y d i f f i c u l t to c a rry o u t, and numerous m o d ificatio n s have been introduced by d if f e r e n t research groups.
This
has re s u lte d in a number o f problems associated w ith the s ta n d a rd is a tio n o f the technique, and comparisons o f re s u lts between d if f e r e n t la b o ra to rie s .
Fry_et_al (1976) introduced a fu r th e r s ig n ific a n t m o d ific a tio n in the technique which removed the p e rfu sio n .
T h e ir technique in vo lves the
enzymic d ig estio n o f l i v e r s lic e s w ith collagenase and hyaluron idase. This is the method th a t was used fo r the research presented here and i t w il l be described in f u l l in the methods s e c tio n .
This method has a
number o f advantages over the o ther a v a ila b le techniques, the main one being th a t i t does not re q u ire the s o p h is tic a te d su rg ic a l operations necessary to set up a l i v e r p e rfu s io n , which means th a t the operators do not re q u ire a Home O ffic e animal lic e n c e .
The method may also be
app lied to pieces o f l i v e r tis s u e in c lu d in g biopsy samples from humans and la r g e r mammals, which may enable more d ir e c t comparisons to be made between animals and humans.
The c e ll y ie ld by th is method is lower than
th a t obtained by perfusion but the v i a b i l i t y is high and the numbers are
adequate f o r a wide range o f experim ents.
The technique employed
p r e f e r e n t ia lly is o la te s the parenchymal c e lls o f the l i v e r , which have been shown to have in ta c t plasma membranes, and c a rry out a number o f biochemical functio ns which re q u ire the presence o f in t a c t and fu n c tio n a l hormone rece p to r s ite s and a f u l l statu s o f endogenous cofactors (F ry _ e t _aT, 1976).
C e lls have been is o la te d from a number
o f d if f e r e n t species by th is technique, and a method has also been developed to c u ltu re the hepatocytes in prim ary monolayers.
2 .3 BIOCHEMICAL AND MORPHOLOGICAL CHARACTERISTICS OF ISOLATED HEPATOCYTES A larg e number o f studies are documented concerning the fu n c tio n a l and morphological c h a ra c te r is tic s o f is o la te d hepatocytes using the many v a ria tio n s o f the is o la tio n methods. recent studies w il l be given here. reviews a v a ila b le (S eglen, 1976c;
A sho rt review o f the more There are a number o f more comprehensi
Jeejeebhoy and P h il li p s , 1976;
Fry
and B ridges, 1979)
P r io r to using is o la te d hepatocytes i t must be demonstrated th a t the c e lls are in ta c t and fu n c tio n a lly v ia b le .
The most w id e ly used method
f o r determ ining v i a b i l i t y is dependent on the a b i l i t y o f c e lls w ith an in ta c t plasma membrane to exclude dyes l ik e trypan b lu e , n ig ro s in and eosin (P a u l, 1970).
This technique is quick and simple and i's
considered by many to be the most r e l ia b le , however some workers have questioned i t s accuracy and suggested o th e r methods.
Expression o f the
c e l l 's m etabolic c a p a c ity , e .g . i t s a b i l i t y to synthesize glycogen o r r e s p ir e , have been suggested as a lte r n a tiv e s .
These methods, however,
are not com pletely r e lia b le as th ere is an i n t r i n s i c v a r i a b i l i t y in the c e lls c a p acity to carry out various m etabolic fu n c tio n s .
Damaged c e lls
w il l continue to re s p ire due to the presence o f in ta c t m itoch ondria, s im ila r ly o th er c e llu la r functio ns c o n tro lle d by s u b c e llu la r o rg a n e lle s may continue to be a c tiv e .
Baur e t al_ (1975) compared a number o f
d if f e r e n t methods f o r e v a lu a tin g c e ll v i a b i l i t y , suggesting th a t the measurement o f c e llu la r potassium le v e ls and membrane p o te n tia l may be more s u ita b le c r i t e r i a .
The loss and uptake o f c e l l u la r potassium
i s , however, a re v e rs ib le process and may not be a tru e in d ic a to r o f c e llu la r damage.
The leakage o f soluble enzymes has also been u t il is e d
and is probably e q u iva len t to the trypan blue te s t in assessing ir r e v e r s ib le membrane damage.
I f i t is accepted th a t the l a t t e r t e s t
is only a measure o f gross s tru c tu ra l in t e g r it y and not n e c e s s a rily the c e lls tru e v i a b i l i t y , i t is probably the most r e lia b le parameter fo r assessing the success o f a c e ll is o la tio n procedure.
The s u rv iv a l
o f the c e ll a f t e r th is i n i t i a l trauma is dependent on many fa c to rs in c lu d in g the incubation media and c o n d itio n s .
Is o la te d parenchymal l i v e r c e lls are sp h erical in shape w ith the whole o f t h e ir surface covered w ith m ic r o v illi
(S eglen, 1 9 7 6 c ).
The
in te rn a l s tru c tu re is the same as th a t o f the organised tis s u e (Howard
et_ al_, 1967;
Berry and F rie n d , 1969;
Moldeus e t a l , 1974).
S p ecialised membrane regions l i k e b ile c a n a lic u li do not appear to be present on the surface o f the c e l l , although these may reappear when the c e lls are c u ltu re d .
Many fa cets o f carbohydrate metabolism have been in v e s tig a te d in is o la te d r a t hepatocytes, w ith re s u lts suggesting th a t they c lo s e ly resemble the in ta c t perfused l i v e r .
C e lls is o la te d from fed ra ts
r e ta in glycogen, and synthesize glucose which is released in to the surrounding medium, whereas c e lls is o la te d from starved ra ts con tain no glycogen and can only synthesize glucose in the presence o f added substrates l ik e la c ta te and pyruvate (G arrison and Haynes, 1973; Geelen e t al_, 1977;
S tory e t al_, 1976;
M u llh o fe r
et al_, 19 7 7 ).
Gluconeogenesis is stim u lated by hormones and c y c lic nucleo tides (G arrison and Haynes, 1973;
P ilk is e t al_, 1975).
F u rth er work on the
hormonal con trol o f gluconeogenesis has been reported by Byus e t al (1 9 7 6 ), Riou e t al_ (1 9 7 6 ), A rinze (1977) and Siess _et al_ (1977 and 197 8). More s p e c ific studies on the le v e ls o f c y c lic AMP in r a t hepatocytes and i t s re la tio n s h ip to gluconeogenesis have also been reported (Moxley and A lle n , 1975;
Birnbaum and F a in , 1977;
Chan and Exton, 1977).
Hems e t
Fain and Shepherd, 1977;
(1978) found th a t glycogen breakdown
stim u lated by hormones is dependent on the presence o f e x t r a c e llu la r
++
Ca
.
D ioch loroacetate has also been shown to i n h ib it gluconeogenesis
in is o la te d hepatocytes (S ta c p o o le ,1977;
Demaugre_et_al, 1978).
Walker (1977a) found th a t is o la te d hepatocytes are in flu en c ed by d iu rn a l rhythms, t h e ir a b i l i t y to synthesize glycogen being a ffe c te d by the tim e o f day a t which they are prepared.
Is o la te d r a t hepatocytes w il l synthesize f a t t y acids and g ly c e r o lip id s . S o le r-A rg ila g a e t al_ (1978) have demonstrated th a t calcium plays a ro le in f a t t y acid syn th e s is , a d e p le tio n reducing the basal r a te o f t r ia c y l g ly c e ro l synthesis
and dim inishing the e ffe c ts o f glucagon
and the c y c lic n u c le o tid e s .
A lte ra tio n s in the d ie ta r y status o f
animals a f f e c t the synthesis o f g ly c e r o lip id s , these being r e fle c te d by is o la te d r a t hepatocytes (Groener and van Golde, 1977).
The same
authors have also in v e s tig a te d the ro le played by exogenous and
•
endogenously synthesized f a t t y acids in g ly c e r o lip id synthesis (Groener and van Golde, 197 8).
Akesson e t al_ (1976) in v e s tig a te d the
metabolism o f s p e c ific monoacyl phospholipids fin d in g th a t the monoacyl phospholipid acyl tra n s fe ra s e reac tio n s operating a t p o s itio n s one o r two y ie ld d if f e r e n t satu rated acyl chain p r o file s in p h o sp atidylethanolam ine and pho sphatid ylcholine o f a s p e c ific u n s a tu ra tio n . This is o f importance because many membrane functions are re la te d to the physical s ta te o f membrane lip i d s . synthesize c h o les tero l
Is o la te d r a t hepatocytes w i l l
(Gibbons and P u llin g e r , 1977).
F a tty acid
synthesis is s ig n if ic a n t ly in h ib ite d by very low d en sity lip o p ro te in s whereas c h o les tero l synthesis is not (Lakshmanan e t al_, 197 7).
Panek
e t a l (1977) reported th a t both f a t t y acid and c h o les tero l synthesis were in h ib ite d by 5 -(te tr a d e c y lo x y )-2 -fu r o ic a c id , a l i p i d low ering agent.
Hormones also play an im portant ro le in f a t t y acid and
g ly c e r o lip id synthesis in the l i v e r .
Geelen e t al_ (1978) in v e s tig a te d
the in flu e n c e o f glucagon and in s u lin on the synthesis o f g ly c e r o lip id s , and Ochs and H a rris (1978) have studied the r e la tio n s h ip between g ly c o ly s is , lip o g e n e s is , gluconeogenesis and pyruvate kinase a c t i v i t y in is o la te d r a t hepatocytes.
Urea is synthesized by the l i v e r from a s p a rta te and ammonia.
Rognstad
(1977) has shown th a t production o f ammonia by the p ro lin e n u c le o tid e cycle is not a major pathway because hadacidin which in h ib it s th a t pathway does not i n h ib it urea synthesis in r a t l i v e r c e l l s .
L -N o rv a lin e
and L -le u c in e however do i n h ib it urea synthesis w ith t h e i r main s it e o f actio n a t o rn ith in e transcarbam ylase.
Hensgens e t al_ (1978) suggest
th a t the synthesis o f urea from p ro lin e is p r im a rily lim ite d by the ra te o f form ation o f ammonia from glutam ate v ia glutam ate dehydrogenase. F u rth er studies on ureogenesis in is o la te d r a t hepatocytes have been reported by M e ije r e t al_ (1 9 7 8 ), Siess e t al_ (1977) and Triebw asser and Freedland (1 9 7 7 ).
Is o la te d r a t hepatocytes w il l synthesize and secrete p ro tein s (S eglen, 1976 a and b ) , synthesis being measured by the in c o rp o ra tio n o f r a d io a c t iv it y from a c e llu la r p ro te in s .
la b e lle d amino acid m ixture in to the
The in co rp o ratio n ra te v a rie s depending on the
concentration o f the c e lls in suspension and the e x t r a c e llu la r amino acid c o n c en tratio n .
Hepatic p ro te in synthesis is su b jec t to
re g u la tio n by amino a c id s , e xe m p lified by the e f f e c t o f a la n in e which when added to the e x t r a c e llu la r medium stim u lates syn th e sis .
The
substrates la c ta te and pyruvate also s tim u la te s y n th e s is , t h e i r e ffe c ts being abolished by ami n o acetate, an in h ib it o r o f glutam ic transam inases, in d ic a tin g the presence o f a p o s itiv e con trol o f p ro te in synthesis by a transam ination-dependent agent (Seglen and Solheim , 1978a).
Seglen
has also in v e s tig a te d the nature o f the precursor pools fo r p ro te in synthesis using
- V a lin e , an amino acid which is not m etabolised
to any s ig n ific a n t e x te n t by the l i v e r .
The re s u lts suggested th a t
both the i n t r a c e ll u l a r and e x t r a c e llu la r v a lin e pools can d ir e c t ly provide precursors f o r p ro te in syn th e s is , the r e la t iv e c o n trib u tio n from each pool being p ro p o rtio n al to the r e la t iv e v a lin e co n centration in th a t pool (Seglen and Solheim , 1978b).
The e ffe c ts o f b io re g u la to rs
and hormones on p ro te in synthesis and amino acid uptake have been in v e s tig a te d by Donner e t al_ (1978) and Jeejeebhoy e t aT (1 9 7 7 ). Bezooijen
Van
(1977) found th a t the synthesis o f to ta l p ro tein s was
increased in o ld e r ra ts aged between.24 and 36 months.
Ricca e t al
(1978) however reported an age re la te d decrease in p ro te in synthesis in r a t hepatocytes.
The e x t r a c e llu la r media and amino acids used by
these two groups d if f e r e d , however, which could ex p la in t h e i r d if f e r in g r e s u lts .
The normal process o f p ro te in degradation also occurs sim ultaneously w ith p ro te in synthesis in is o la te d hepatocytes (S eglen, 1977a). F u rth er studies in to the control o f p ro te in degradation c a rrie d out by Hopgood e £ al_(1977b) demonstrated th a t degradation o f p r e la b e lle d p ro te in was in h ib ite d by in s u lin and c e rta in amino a c id s , ammonia, proteinase in h ib ito r s and by in h ib ito r s o f c e ll-e n e rg y produ ction.
B ile acids are also synthesized by is o la te d r a t hepatocytes (Yousef e t a l , 1978) a t a r a t e , and w ith a com position, s im ila r to th a t in v iv o .
The uptake o f the b ile acid ta u ro ch o late by is o la te d hepatocytes is in h ib ite d by c h o le s ta tic s te ro id hormones (Schwarz e t _al_, 1977) and ta u ro lith o c h o la te (Schwenk _et _al_, 1977).
The re s p ira to ry
a c t i v it i e s o f is o la te d hepatocytes have been in v e s tig a te d , showing th a t th e re is no a g e -re la te d d e c lin e in the fu n c tio n a l in t e g r it y o f the m itochondria (Brouwer _et _al_, 1977).
F u rth er studies on
c y to s o lic phosphorylation s tates have been c a rrie d out by van der Meer_et_al (1 9 7 8 ).
Evidence also s tro n g ly suggests th a t energy
dependent, c a r r ie r m ediated, a c tiv e tra n s p o rt systems are e x is te n t in is o la te d r a t hepatocytes.
This is supported by the work on
a -am in o iso b u tyric acid (Chen and Lee, 197 7), ouabain (Eaton and Klaassen, 19 7 8 ), ATP (Akerboom e t a l , 1977) and amino acids (Le Cam and F reychet, 1977 a and b;
2 .4
Edmondson _et ^1_, 1977).
ISOLATED HEPATOCYTES IN CULTURE
The c u ltu rin g o f is o la te d r a t hepatocytes involves a number o f te c h n ic a l problems, w ith the r e s u lt th a t r e l a t i v e l y less studies have been c a rrie d out in prim ary monolayer c u ltu re s than w ith suspensions o f fre s h ly is o la te d c e lls .
The method o f c e ll is o la tio n introduced by
Fry £ t aT (1976) may be c a rrie d out under s t e r i l e con ditio ns which • has enabled the successful c u ltu rin g o f a d u lt r a t l i v e r c e lls .
One
o f the major problems associated w ith c e ll c u ltu re s is the loss o f c e rta in s p e c ia lis e d c e l l u la r functio ns as the tim e in c u ltu re in crease s, w ith the r e s u lt th a t the c e lls may e v e n tu a lly bear no re la tio n s h ip to the tis s u e from which they were o r ig in a lly d e riv e d . This is e s p e c ia lly the case w ith c e ll lin e s which are composed o f u n d iffe re n tia te d e p it h e lia l c e lls .
A number o f workers have, th e r e fo r e ,
in v e s tig a te d the fu n c tio n a l aspects o f prim ary monolayer c u ltu re s o f a d u lt r a t hepatocytes in some d e t a i l.
Functional studies c a rrie d out by B is s e ll e t aj_ (1973) demonstrated th a t c e lls derived from regen erating a d u lt l i v e r synthesized p ro te in s . They also produced glucose from la c ta te and pyruvate in d ic a tin g th a t gluconeogenesis was o p e ra tio n a l to g eth er w ith an in te g ra te d in te r a c tio n between the m itochondrial and cytoplasm ic compartments o f the c e l l . The response o f the c e lls to in s u lin and glucagon v e r if ie d the presence o f in ta c t receptors f o r those hormones as w e ll as dem onstrating the
i
in t e g r it y o f the adenyl cyclase system.
The s tru c tu re o f the smooth
endoplasmic reticu lu m was re ta in e d and the le v e ls o f ATP returned to p h y sio lo g ical le v e ls w ith in a few hours o f in cu b a tio n .
F urther
studies were c a rrie d out on glucose metabolism (B is s e ll e t al_, 1 9 7 8 ), showing th a t prim ary c u ltu re s u t ilis e d r e l a t i v e l y small amounts o f glucose l ik e the in ta c t l i v e r , although on extended c u ltu re th is p a tte rn changed to th a t observed in c e ll lin e s where the u t i l i s a t i o n o f glucose is much g re a te r.
Studies c a rrie d out by Tanaka et _al_ (1 9 7 8 ), using a d u lt c e lls from normal r a t l i v e r , demonstrated th a t some o f the functio ns lo s t during the c e ll is o la tio n were recovered during c u ltu r in g .
These included the
in ductio n o f ty ro s in e transaminase by dexamethasone, reaggregated polysomes, and the s tim u la tio n o f p ro te in synthesis by in s u lin and dexamethasone which was in h ib ite d by glucagon.
They also demonstrated
th a t the c e lls secreted proteins in to the medium, album in, t r a n s f e r r in , fib rin o g e n and lip o p ro te in s being s p e c if ic a lly id e n t if ie d .
W illiam s and co-workers (Laishes and W illia m s , 1976; 1977;
W illiam s e t a l ,
W illiam s _et ^al., 1978) have c a rrie d out a system atic exam ination
o f the conditions a ffe c tin g the primary c e ll cu ltu re s o f a d u lt r a t hepatocytes fin d in g th a t a medium supplemented w ith dexamethasone increased the s u rv iv a l tim e in c u ltu r e .
Goldfarb
et _al_ (1978)
provided fu r th e r support fo r the recovery o f the a b i l i t y to synthesize lip id s and glycogen, and an improvement in morphology o f is o la te d a d u lt r a t hepatocytes in primary monolayer c u ltu r e .
F urther studies on gluconeogenesis have been c a rrie d out by O liv e r J2t_ a l (1 9 7 8 ), Walker (1977b) and K le tz ie n _et _al (1 9 7 6 ). (1977)
P a riza e t al
found th a t the inductio n o f amino acid tra n s p o rt by epinephrine
is independent o f c y c lic AMP le v e ls in prim ary c u ltu re s , and Hopgood e t_ a l (1977b) found c e lls in primary c u ltu re were more s e n s itiv e to the e ffe c ts o f in s u lin and glycogen on p ro te in degradation than hepatocytes in suspension.
Bellemann et abusing the is o la tio n method o f Fry_et_al
(1976) found
th a t the .prim ary monolayer c u ltu res r e ta in t h e ir m etabolic and enzymic
c h a ra c te ris tic s f o r several days (Gebhardt e t a l , 1978).
They also
found th a t in c u ltu re s derived from normal and allo xan d ia b e tic ra ts in s u lin stim u lated glycogen synthesis in both, and p ro te in synthesis more e f f e c t iv e ly in normal c e lls (Bellemann
2 .5
et al_, 1977a).
DRUG METABOLISM IN HEPATOCYTE SUSPENSIONS AND CULTURES
The metabolism o f xen o biotics in c e ll suspensions and c e ll c u ltu re s has been w ell reviewed (Bridges and F ry , 1976; Orrenius
_et_al_, 1977).
Fry and B ridges, 1977b;
The g e n e ra lly accepted method f o r the
in v e s tig a tio n o f drug metabolism in v it r o has been the use o f microsomes.
There a r e , however, a number o f reasons why s u b c e llu la r
fra c tio n s are probably not re p re s e n ta tiv e o f in vivo metabolism. Tissue homogenization may lib e r a t e lysosomal enzymes, and i t removes the com partm entalisation o f the enzymes and membrane components w ith in the c e l l .
Hence c e l l u la r metabolism which req u ires the in te g r a tio n o f
d if f e r e n t c e l l u la r components w il l not be seen in c e l l u la r fr a c tio n s . I t is also necessary to add u n n a tu ra lly high le v e ls o f c o -fa c to rs to enable metabolism to proceed.
Before metabolism commences the drug o r
chemical must e n te r the c e l l , the mechanisms c o n tr o llin g th is may a f fe c t m etabolism, and th is w i l l not be assessed in c e ll fr a c tio n s .
The perfused l i v e r and l i v e r s lic e s have also been employed in studies o f drug metabolism but th ere are problems in h e re n t in the use o f these techniques.
The perfused l i v e r , although a good system, is d i f f i c u l t
and expensive to s e t up, and the number o f te s ts th a t can be run on one l i v e r is lim ite d .
The main problems associated w ith l i v e r s lic e s
are f i r s t l y the o u ter layers o f the s lic e s contain damaged c e lls as a r e s u lt o f the s lic in g procedure.
Secondly the uptake o f xen o b io tics
in s lic e s is c o n tro lle d by passive d iffu s io n w ith the r e s u lt th a t little
i f any w i l l reach the cen tre o f the s l i c e , th is also a p p lie s
to the n u trie n ts added to the medium.
The c e lls in the cen tre o f the
s lic e s w i l l become n e c ro tic q u ite r a p id ly re s u ltin g in a p rep a ra tio n th a t is not homogeneous.
Is o la te d hepatocytes in suspension and c u ltu re .d o not s u ffe r these many drawbacks, and as techniques are now a v a ila b le f o r the p re p a ra tio n o f v ia b le and fu n c tio n a l c e lls they have proved to be very useful in the study o f drug metabolism.
A number o f drug o xid atio n s dependent on
cytochrome P-450 have been in v e s tig a te d in is o la te d hepatocytes and a number o f these are lis t e d in Table 1.
Moldeus e t a l (1978a) used is o la te d hepatocytes to in v e s tig a te the in te ra c tio n between ethanol and the metabolism o f fo re ig n compounds. They s p e c if ic a lly in v e s tig a te d the e ffe c ts o f ethanol on g lu cu ro n id atio n o f a number o f substrates in clu d in g harm ol, 2-n a p h th o l, 4 -m e th y lu m b e lliferone and phenolphthalein fin d in g th a t i t was in h ib ite d by up to 50% by low concentrations o f ethanol (lOmM).
F u rth er in v e s tig a tio n s were
made in to the e f f e c t o f ethanol on the i n t r a c e ll u l a r NADH/NAD+ r a t io and the
synthesis o f UDP glucuronic a c id , and the e f f e c t o f 4 -m eth yl-
pyrazole an in h ib it o r o f alcohol dehydrogenase dependent ethanol o x id a tio n .
This led the authors to suggest th a t the in h ib it o r y
e f f e c t o f ethanol on glu cu ro n id atio n is due to a decreased synthesis o f UDP glucuronic acid caused by an increased NADH/NAD
r a t io re s u ltin g
from the alcohol dehydrogenase dependent o x id a tio n o f e th a n o l.
The same group (N o rlin g e t a l , 1978) in v e s tig a te d the uptake o f glucuronides in to is o la te d hepatocytes and t h e ir e ffe c ts on the glucu ro n id atio n o f model s u b strates .
The re a c tio n sequence involved in the metabolism o f paracetamol v ia cytochrome P-450 o x id a tio n to th e form ation o f the m ercapturic acid d e r iv a tiv e has been d e lin e a te d in is o la te d r a t l i v e r and kidney c e lls by Moldeus e t al (1978 b ).
O xidation and g lu ta th io n e conjugation were
catalysed p rim a rily by the l i v e r c e l l s , w h ile conversion o f the g lu ta th io n e conjugate to the m ercapturic acid d e r iv a tiv e was catalysed p r im a r ily , by the kidney c e lls .
Moldeus (1978) performed a d e ta ile d ana lys is o f the form ation o f the d if f e r e n t m etabolites from paracetamol in hepatocytes is o la te d from mouse and r a t .
The hepatocytes from both species catalysed the
form ation o f g lucuronide, s u lp h a te, g lu ta th io n e and cystein e conjugates o f paracetam ol.
Sulphation had a higher a f f i n i t y fo r paracetamol than
g lucuronidation in both the r a t and mouse, whereas g lu cu ro n id atio n had a higher c a p a c ity .
The maximal ra te o f glucu ro n id atio n was s im ila r in
hepatocytes from both species but th a t o f sulphation was s e v e r a l-fo ld less in the mouse.
The ra te o f g lu ta th io n e conjugate form ation was found
to be d ir e c t ly c o rre la te d to the loss o f in t r a c e ll u l a r g lu ta th io n e , th is
T a b le 1.
Drug M e ta b o lis m in I s o l a t e d R a t H e p a to c y te s
Drug studied
Reference
A lp ren e lo l
Orrenius
Aminopyrene
Weigl
Antipyrene
Aarbakke e_t
Ethanol
Crow e t al_ (1977)
Harmol
Andersson e t al_ (1978)
B a rb itu ra te s
Yih and Rossum (1977)
Biphenyl
Wiebkin e t j * l (1976 and 1978b)
Benzoic Acid
Kao
Ethoxycoumarin
Fry e t al (1978)
4-Methyl Um bel!iferone
F ry_et_al (1978)
Benzo( a )pyrene
Jones _et_a] (1978)
Diphenylhydantoin
Inaba_et_al (1975)
4 -an d ro s ten e-3 , 17-dione
Stenberg _et _al (1978)
17 a -e th in y lo e s tr a d io l
Schwenk e t al (1978)
Oestrone sulphate
Schwenk e t al (1978)
Sulphadimidine and sulphanilam ide
Morland and Olsen (1977)
et_ ^1_ (1975)
et _al_ (1977) (1977)
et al_ (1978)
Burke and Orrenius
(1978)
E thoxyresorufin Burke and Hallman (1978) Phenacetin and N-Hydroxyphenacetin
McLean (1978)
Benzo( a )pyrene
Ashurst and Cohen (1980)
Paracetamol
Moldeus e t al_ (1980)
P yrid in e and s u b s titu te d p yrid in es
Blaauboer and Paine (1979)
Carbon te tr a c h lo rid e
Anundi e t al_ (1979)
ra te however was f a r less in the r a t than the mouse.
The mouse is a
species more su s cep tib le to the to x ic e ffe c ts o f paracetam ol.
This
was supported here by the progressive loss o f plasma membrane in t e g r it y o f the mouse hepatocytes a f t e r 90 minutes o f incubation w ith 2mM paracetamol which did not occur in the r a t hepatocytes incubated in the presence o f lOmM paracetamol f o r f iv e hours.
F u rth er in v e s tig a tio n s by Moldeus e t £l_ (1980) have shown th a t chronic a d m in is tra tio n o f ethanol to ra ts f o r 6 -8 weeks caused an e le v a tio n in the con centration o f cytochrome P-450 in is o la te d r a t hepatocytes. These hepatocytes showed enhanced ra te s o f o x id a tio n and UDP-glucuronic acid conjugation o f paracetam ol, w ith the binding a f f i n i t y o f paracetamol to cytochrome P-450 being increased from 2.8mM“ ^ in con trol hepatocytes to 0.75mM“ ^ in e th a n o l-tre a te d r a t hepatocytes.
There have only been a lim ite d number o f studies in prim ary c u ltu re s o f a d u lt r a t hepatocytes.
This is m ainly due to the i n a b i l i t y o f the
e a r l i e r techniques to produce cu ltu re s in which the c e lls m aintained ph ysio lo g ical le v e ls o f cytochrome P-450.
2.6
MAINTENANCE OF CYTOCHROME P-450 IN PRIMARY MONOLAYER CULTURES
OF RAT HEPATOCYTES I t has been w ell documented th a t during the f i r s t 24 hours, o f c u ltu re the con centration o f cytochrome P-450 f a l l s markedly in prim ary maintenance c u ltu res o f a d u lt r a t hepatocytes (G uzelian and B is s e ll, 1976;
Guzel ian
et al_, 1977).
A f u r th e r study by the same group
(G uzelian and Barw ick, 1979) presented supplementary evidence th a t t h is phenomenon occurred but was prevented by incubating c u ltu re s w ith in h ib ito r s o f p ro te in synthesis such as cyclohexim ide,puroinycin, actinonycin D, o r aza s e rin e .
I t was p o stu lated since cyclohexim ide
had no e f f e c t on the degredation o f t o ta l p ro te in and i t s e f f e c t appeared to be due to a decreased breakdown o f haem, th a t the s e le c tiv e degradation o f cytochrome P-450 in c e ll c u ltu re may re q u ire p ro te in syn th esis.
E a r lie r studies had suggested th a t the reduction in cytochrome P-450 could be ca u s a lly re la te d to an increase in haem oxygenase a c t i v i t y
(Maines and Kappas, 1977).
This hypothesis was in v e s tig a te d by Paine
and Legg (1978) using c u ltu red a d u lt r a t hepatocytes.
When c e lls were
cu ltu re d in W illia m 's medium E supplemented w ith 5% fo e ta l c a l f serum they obtained re s u lts s im ila r to those o f Guzelian and Barwick (1 9 7 9 ). A loss in concentration o f cytochrome P-450 was associated w ith a r is e in haem oxygenase a c t i v i t y , although i t was demonstrated by G uzelian.and Barwick in t h e ir study th a t the r is e in haem oxygenase occurred a f t e r the decrease in cytochrome P-450 became marked suggesting th a t th ere is not a d ir e c t l in k .
Decad e t a ! (1977) reported th a t le v e ls o f
cytochrome P-450 could be m aintained in c u ltu re d hepatocytes i f the c u ltu re media was supplemented w ith pharmacological le v e ls o f hormones. Paine and Legg (1978) u t ilis e d media supplemented w ith hormones and measured cytochrome P-450 con centration and haem oxygenase a c t i v i t y a f t e r varying the is o la tio n and c u ltu rin g co n d itio n s .
They found th a t
i f the l i v e r p erfu sate was supplemented w ith hormones in .a d d itio n to the c u ltu re media and the p la s t ic p e tr i dishes were coated w ith collagen the le v e ls o f cytochrome P-450 a f t e r 24 hours o f c u ltu re o f 121pmole/mg p ro te in were s im ila r to in vivo le v e ls and the a c t i v i t y o f haem oxygenase was high.
I t would appear th e re fo re th a t high le v e ls o f
cytochrome P-450 can be m aintained even when high le v e ls o f haem oxygenase were p resen t.
Paine ejfc
(1 979a) have also shown th a t the sole a d d itio n o f h ig h ,
unphysiological concentrations o f nicotinam ide (25mM) to c u ltu re media m aintained cytochrome P-450 le v e ls .
These le v e ls o f cytochrome P-450
were fu r th e r increased to those s im ila r to whole l i v e r values in vivo i f ImM nicotinam ide was added to the p erfu sate f o r the is o la tio n o f hepatocytes.
In a subsequent study (Paine e t al_, 1979b) hepatocytes
cu ltu re d f o r 24 hours were shown to lose 60% o f t h e ir NADH content as w ell as a marked reduction in cytochrome P-450 co n c e n tra tio n .
A d d itio n
o f 2mM nicotinam ide to the c u ltu re medium caused a marked increase in NADH le v e ls a f t e r 24 hours o f c u ltu r e , as did 25mM n ico tin a m id e.
Since
cytochrome P-450 concentration can only be m aintained w ith the high le v e l o f nicotinam ide i t was suggested th a t th e re is not a causal re la tio n s h ip between the content o f NADH and the maintenance o f cytochrome P-450 in c u ltu re s .
This was fu r th e r confirmed by the fin d in g
th a t isonicotinam ide was tw ice as e f f i c i e n t as nicotinam ide a t m ain ta in in g cytochrome P-450 but had no e f fe c t on NADH le v e ls .
Paine e t al_ have also demonstrated th a t c u ltu re o f hepatocytes in a simple balanced s a lt s o lu tio n prevents the loss o f cytochrome P-450 (Paine and Hockin, 1980) suggesting th a t a component(s) o f the c u ltu re media is responsible f o r the loss o f cytochrome P-450.
In v e s tig a tio n o f
the e ffe c ts o f d if f e r e n t c u ltu re media on cytochrome P-450 content o f hepatocytes a f t e r 24 hours o f c u ltu re in d ic a te d th a t cy s tin e and cysteine were the components o f media responsible fo r the decreased le v e ls .
They also found th a t incubation in media w ith o u t c ys tin e
or c y s te in e , but w ith added 5-amino la e v u lin ic acid m aintained even higher le v e ls o f cytochrome P-450.
I t is o f in te r e s t to note here th a t
cysteine has been shown to be a c y to to x ic c o n s titu e n t o f c u ltu re media by Nishiuch
et e\_ (1 9 7 6 ).
Added pyruvate had a p ro te c tiv e e f f e c t
probably due to complex form ation w ith cysteine in the media since preincub ation o f media a t 37°C was shown to have a dose-dependent e f f e c t on the s t a b ilis a t io n o f cysteine reducing the con centration o f o x id is a b le c y s te in e .
L e ib o v itz L-15 medium, used in the studies reported in the p rese n t work contains cystein e a t a con centration o f 120mg/l and pyruvate (550mg/l sodium s a l t ) but no c y s tin e .
Paine and Hockin (1980)
compared the e f f e c t o f L I 5 w ith th a t o f o th e r media (Waymouth's MB752/1: cysteine-81 mg/1, c y s tin e 18m g/l; c y s tin e -2 3 .7 0 , pyruvate-25.O m g/1;
W illia m 's medium E: c y s te in e -5 2 .0 5 m g /l, RPMI 1640: c y s tin e -5 9 .1 5 m g /l) and found
th a t the le v e l o f cytochrome P-450 a f t e r 24 hours in c u ltu re f e l l between 26 and 39% o f the i n i t i a l
to
le v e l w ith a l l fo u r media.
P ain e's group have in v e s tig a te d the s t r u c t u r e - a c t iv it y r e la tio n s h ip f o r the s u b s titu te d p y rid in es in r e la tio n to t h e ir a b i l i t y to m aintain cytochrome P-450 ( V i l l a
et ail_, 1980) to g eth er w ith t h e i r e f f e c t on
p ro te in synthesis in h ib it io n compared to th a t o f cyclohexim ide to t e s t the hypothesis o f Guzelian and Barwick (1 9 7 9 ).
They found th a t p y rid in e
i t s e l f was able to m aintain high le v e ls o f cytochrome P-450 as were the 3 - a c e ty l- and 3 -a m in o -p yrid in es .
These re s u lts provided f u r th e r support
fo r t h e i r e a r l i e r fin d in g s f o r the e ffe c ts o f nicotinam ide and is o n ico tin a m id e, although the amide group o f nicotinam ide was found to be im portant as th io n ico tin am id e was not e f f e c t iv e .
I t was also
demonstrated th a t both nicotinam ide and isonicotinam ide in h ib ite d p ro te in
synthesis by 48 and 60% re s p e c tiv e ly a f t e r 24 hours in c u ltu r e . Cycloheximide a t a con centration o f between 0 .5 and 1 . 0 /zM produced s im ila r e ffe c ts on in h ib it io n o f p ro te in synthesis but the le v e ls o f cytochrome P-450 were low er.
P y rid in e , however, was found to m aintain
high le v e ls o f cytochrome P-450 w ith only minimal in h ib it io n o f p ro te in syn th esis.
These re s u lts do not th e re fo re support the hypothesis o f
Guzelian and Barwick (1979) th a t the in h ib it io n o f p ro te in synthesis is the prim ary mechanism underlying the maintenance o f cytochrome P-450 in hepatocyte c u ltu r e .
Paine e t al_ (1980) have fu r th e r weakened th is hypothesis by dem onstrating th a t the a b i l i t y o f p yrid in es to m aintain cytochrome P-450 in hepatocyte c u ltu re is h ig h ly c o rre la te d w ith t h e i r a b i l i t y to bind to cytochrome P -450, suggesting th a t lig a n d form ation prevents i t s a cc ele rate d tu rn o ver in l i v e r c e ll c u ltu r e .
Furthermore metyrapone m aintains cytochrome P-450
le v e ls due p a r t i a l l y to preventing degradation o f the cytochrome (a p roperty o f lig a n d forming compounds) but m ainly by i t s e f f e c t o f increasing' the synthesis (Paine and V i l l a , 1980).
Increased synthesis
o f cytochrome P-450 was known to occur since the s p e c ific a c t i v i t y o f la b e lle d cytochrome P-450 in the cu ltu re s was reduced (anim als were in je c te d w ith la b e lle d la e v u lin ic acid p r io r to the is o la tio n o f hep ato cytes).
This increased synthesis by metyrapone was not com pletely
prevented by concomitant incubation w ith 5
cycloheximide which
in h ib ite d p ro te in synthesis by 95%.
I t may th e re fo re be concluded from the present evidence th a t the loss o f cytochrome P-450 in cu ltu re s is due to im paired synthesis and enhanced degradation.
2 .7
THE USE OF ISOLATED HEPATOCYTES IN TOXICOLOGY
Is o la te d hepatocytes in suspension and c u ltu re m aintain many biochemical functions w ith a c t i v it i e s s im ila r to those in v iv o .
They, th e r e fo r e ,
lend themselves to the study o f the mechanisms o f to x ic a c tio n o f drugs and chem icals.
W ithin the la s t few years a number o f stu d ies have been
c a rrie d o u t, some o f which w i l l be reported here.
This review w i l l be
lim ite d to is o la te d hepatocytes in suspension and prim ary c u lt u r e , f o r
more general studies on in v it r o t o x i c it y eva lu a tio n the reviews o f T a r d if f (1 9 7 8 ), Nardone (1 9 7 7 ), Bridges (1976) and Worden (1974) are re le v a n t.
The main endpoint o f t o x ic it y is the loss o f c e ll v i a b i l i t y . Zawydiwski and Duncan (1978). described a method f o r measuring spontaneous [5^Cr] release as an in d ic a to r o f membrane damage.
The
leakage o f c e l l u la r enzymes in to the e x t r a c e llu la r medium has been used by Zimmerman
et al_ (1974) to demonstrate the h e p a to to x ic ity o f
erythronjycin and chlorprom azine, glutam ic o x a lo a c e tic transaminase being used as the enzyme.
S im ila r studies were c a rrie d out to in v e s tig a te
the t o x i c it y o f the t r i c y c l i c a n ti depressants (Abernathy e t a l , 1975) and the th io x a n th in e n e u ro le p tic s (Abernathy and Zimmerman, 197 5).
Orrenius
et al_ (1976) in v e s tig a te d the e ffe c ts o f anoxia on is o la te d
hepatocytes.
I t caused an increase in plasma membrane p e rm e a b ility
demonstrated by a decrease in trypan blue exclusion frequency, leakage o f c y to s o lic la c ta te dehydrogenase, and an increase in s u c c in a te stim u lated oxygen uptake.
These changes appeared to be preceded by
a marked decrease in c e llu la r ATP c o n c en tratio n .
S im ila r e ffe c ts to
those caused by anoxia were observed i f the c e lls were incubated w ith an uncoupler o f o x id a tiv e pho sphorylation.
A number o f studies have been c a rrie d out in is o la te d hepatocytes to in v e s tig a te the e f f e c t o f xenobiotics on the m etabolic processes o f the l i v e r .
S tory and Freedland (1978) found th a t feeding DDT to starved
ra ts in h ib ite d gluconeogenesis from la c ta te a t the step between pyruvate and phosphoenol pyruvate in the is o la te d hepatocytes. D-Galactosamine in h ib it s glycogenolysis by causing a decrease in the content o f hepatocytes (Sternmann e t a l , 1978).
Mapes (1977) concluded
th a t the in h ib it io n o f lipogenesis by halothane is probably caused by in h ib it io n o f the o x id a tio n o f NADH.
Gluud and Dich (1977) observed
during studies on the ro le o f c y c lic AMP in l i v e r p ro te in metabolism th a t the xanthines am inophylline, c a ffe in e and th e o p h y llin e , in h ib it e d the in co rp o ratio n o f
[ ^ c ] -V a lin e in to the p roteins o f is o la te d
hepatocytes and the se c re tio n o f album in.
P ro te in s e c re tio n is also
in h ib ite d by c o lc h ic in e , ammonia and anoxia (Seglen and R e ith , 197 7).
A s e rie s o f studies have been c a rrie d out on the to x in p h a llo id in which is obtained from Amanita p h a llo id e s .
P h a llo id in is to x ic v ia
i t s d ir e c t actio n on the plasma membrane o f is o la te d hepatocytes (Frim m er, 19 7 7 ), and a t very low concentrations (20 M g/m l) i t
in h ib it s
amino acid in c o rp o ratio n in to p ro te in s (G ravela and P o li, 1977). Trypsin delays or prevents the plasma membrane response o f hepatocytes to p h a llo id in (Frimmer
et al_, 1977a) as do temperatures below 21°C,
which suggests th a t the f l u i d i t y o f the membrane lip id s might con trol an e a r ly step o f the p h a llo id in response (Frimmer and Rufeger, 1977). The uptake o f p h a llo id in by hepatocytes is not by simple d iffu s io n but probably v ia the pathway o f p h y sio lo g ical s u b s tra te .
System atic
screening has shown th a t the b i le acids i n h ib it the response o f is o la te d hepatocytes to p h a llo id in in a dose dependent manner (Frimmer e t a l , 1977b).
F urther studies in d ic a te th a t p h a llo id in induces an accumulation
o f tr ig ly c e r id e s w ith in the l i v e r c e ll probably by im p airin g p ro te in synthesis and se c re tio n (G ra v e la e t a l , 1977).
The p ero x id a tio n o f polyunsaturated lip id s is associated w ith the h e p a to to x ic ity o f several chem icals, e s p e c ia lly carbon te tr a c h lo r id e . Hogberg and co-workers have u t ilis e d is o la te d hepatocytes fo r the in v e s tig a tio n o f fa c to rs which may c o n trib u te to the p ro te c tio n o f l iv e
et al_, 1975a, b and c ) . These fa c to rs include the low permeation ra te of iro n in to the c e l l , the c e lls from l i p i d p ero x id a tio n (Hogberg
in a c tiv a tio n o f iro n by i t s complexing to a p o fe r r e tin , the metabolism o f l i p i d peroxides by g lu ta th io n e peroxidase, and the o x id a tio n o f malonaldehyde.
.
Weddle e t a l (1976) studied the e f f e c t o f carbon
te tr a c h lo rid e on l i p i d p ero x id a tio n in is o la te d hepatocytes fin d in g th a t i t increased.
Is o la te d hepatocytes from ra ts having consumed
alcohol fo r several months, produce malondialdehyde - a measure o f l i p i d p e ro x id a tio n , in s ig n if ic a n t ly higher amounts than c e lls from untreated anim als, w ith o u t the accumulation o f tr ig ly c e r id e s
(Remmer
e t a l , 1977).
F u rth er studies on the e ffe c ts o f carbon te tr a c h lo rid e in is o la te d hepatocytes have been c a rrie d out by P o li e t a ! (1978 and 19 7 9 ). They also demonstrated th a t l ip i d p ero x id a tio n is stim u la te d in is o la te d r a t hepatocytes which is dependent on the metabolism o f carbon te tr a c h lo rid e by cytochrome P-450.
I t was also shown to i n h i b i t
p ro te in synthesis and p ro te in and lip o p ro te in s e c retio n and induce the
accumulation o f f a t w ith in the c e lls .
The fre e ra d ic a l scavanger
promethazine was found to i n h ib it malondialdehyde production due to carbon te tr a c h lo rid e but not to in flu e n c e i t s e ffe c ts on reducing the cytochrome P-450 content o f is o la te d hepatocytes.
Lindstrom e£ al_ (1978) demonstrated th a t is o la te d hepatocytes from p h e n o b a rb ita l-tre a te d ra ts showed an increased r a te o f b io a c tiv a tio n o f carbon te tr a c h lo r id e producing a more marked e f f e c t on the re le a s e o f la c t a te dehydrogenase from these c e lls than on c e lls from u n treated r a ts .
This e f f e c t was more prominant than the e f f e c t o f phenobarbital
pretreatm ent on malondialdehyde production,
D epletion o f the
g lu ta th io n e le v e ls o f hepatocytes by d ie th y l maleate was, however, associated w ith increased malondialdehyde form ation.
Studies by Stacey and P r ie s t ly (1 9 7 8 a ), using a number o f agents shown to be h ep ato p ro te ctive in o th e r experim ental models o f carbon te tr a c h lo r id e induced t o x ic it y includin g- SKF-525A, prom ethazine, g lu ta th io n e and c y s te in e , demonstrated th a t they were in e f fe c tiv e in is o la te d r a t hepatocytes in reducing the t o x ic it y o f carbon te tr a c h lo r id e .
F u rth e r
studies (Stacey and P r ie s t ly , 1978b) suggested th a t l i p i d p e ro x id a tio n is not a r e q u is ite step in carbon te tr a c h lo r id e induced t o x i c it y in is o la te d hepatocytes.
Brom otrichloromethane, another halogenated hydrocarbon, has been used by K o ste r-A lb rec h t e t al (1978) to in v e s tig a te l i p i d p e ro x id a tio n and c e ll damage in is o la te d r a t hepatocytes.
I t produces s im ila r
a lte r a tio n s in l i v e r c e lls to carbon te tr a c h lo rid e but w ith in a lower con centration range.
Bromobenzene is another compound which is m etabolised by the cytochrome P-450 system to produce an a c tiv e m e tab o lite which is to x ic to the l i v e r . The conjugation o f the a c tiv e m e ta b o lite w ith g lu ta th io n e appears to be a major p ro te c tiv e mechanism in preventing the h e p a to to x ic ity o f bromobenzene Zampaglione e t al_ (1 9 7 3 ).
Thor £ t al_ (1978a, 1978b and 1979)
have used is o la te d r a t hepatocytes to in v e s tig a te the metabolism and t o x ic it y o f bromobenzene.
They found th a t the i n t r a c e ll u l a r le v e l o f
g lu ta th io n e in the hepatocytes is o f major importance in r e la t io n to the
c y to to x ic ity o f bromobenzene and th a t hepatocytes incubated in a complete medium are protected ag a in s t t o x ic it y by t h e i r a b i l i t y to resynthesize g lu ta th io n e .
Pretreatm ent o f ra ts w ith d ie th y l m aleate
p o te n tia te d the hepatotoxic e f f e c t whereas the presence o f c y s te in e , methionine and N -a c e ty lc y s te in e in the incubation medium p ro tected against t o x i c it y .
D epletion o f g lu ta th io n e leads to covalent binding o f r e a c tiv e m etabolites to tis s u e macromolecules.
Dent and Sun (1980) have
developed a model fo r studying the s p e c if ic it y o f co valent binding in is o la te d hepatocytes.
Using th is model they in v e s tig a te d the e ffe c ts
o f bromobenzene fin d in g th a t binding occurs f i r s t w ith macromolecules in the 20-40 ,00 0 dal ton range.
The c e ll appears to be able to
recognise these macromolecules and by some mechanism degrades them. When the ra ts are p re tre a te d w ith phenobarbitone the ra te a t which the binding occurs is g re a te r than w ith untreated hepatocytes re s u ltin g in an accumulation o f the high m olecular w eight fr a c tio n since the r a te o f form ation appears to exceed th a t o f degredation.-
This method may
prove to be useful in id e n tify in g the nature o f the macromolecules w ith which covalent adducts have formed.
G lutathio ne plays a valuab le ro le in the c e ll in p ro te c tin g a g a in s t the t o x ic it y o f endogenous chem icals.
A number o f studies have been c a rrie d
out to in v e s tig a te the le v e l and tu rn o ver o f th is t h io l in h ep ato cytes, and the e f f e c t o f is o la tio n techniques and incubation media. Orrenius
Reed and
(1977) demonstrated th a t hepatocytes fre s h ly is o la te d from
ra ts tre a te d w ith d ie th y l m aleate perform a net biosynthesis o f g lu ta th io n e a t a ra te s im ila r to th a t observed i_n vivo o f about 2 jum oles/hr/g wet w t. of liv e r .
Vina e t al_ (1978) however found th a t the le v e l o f g lu ta th io n e
in is o la te d hepatocytes f e l l during in c u b a tio n .
Hogberg and K ris to fe rs o n
(1977 and 1 9 7 8 ), who had also experienced loss o f g lu ta th io n e le v e ls during in c u b a tio n , found th a t by decreasing the time o f is o la tio n procedure and by supplementing the medium w ith amino acids and serum the le v e ls o f g lu ta th io n e in fre s h ly is o la te d hepatocytes were increased and a net ra te o f biosynthesis occurred th e r e a f te r .
The c h a ra c te ris tic s o f prim ary hepatocyte c u ltu re s are less w e ll defined than those o f is o la te d hepatocytes in suspension w ith the r e s u lt th a t only a few studies have been c a rrie d out so f a r .
However,
the in d ic a tio n s are th a t th is system w il l be u t ilis e d f a r more in the fu tu r e .
Most studies to date have been c a rrie d out in c e ll lin e s ,
the reviews mentioned p re vio u s ly may be supplemented here by those o f Desi e t
(1977) and Grisham
et al_ (1 9 7 8 ).
Michalapolous e t al_ (1976)
provide data w ith in t h e ir review to support the use o f prim ary h ep atic c e ll c u ltu re s in the in v e s tig a tio n o f hepatocarcinogenesis by ^chemicals. Huberman and Jones (1980) have used l i v e r c e ll c u ltu re s in mutagenesis s tu d ie s .
Two recent reviews provide more in fo rm atio n on the use o f
prim ary l i v e r c e ll c u ltu re s in assessing the t o x ic it y o f chemicals (F ry and B ridges, 1979;
Grisham, 1979).
W illiam s (1977) described a system where ten carcinogens produced unscheduled DNA synthesis in prim ary c u ltu re s , suggesting i t as a possible p re d ic to r o f p o te n tia l carcinogens.
Fry and Bridges (1977a)
describe another system f o r assessing the c y to to x ic ity o f chemicals which are metabolism m ediated.
They took advantage o f the f a c t th a t
prim ary c u ltu re s o f hepatocytes become overgrown w ith fib r o b la s ts a f t e r th ree or fo u r days, by using the hepatocytes as the m etabolising component and the fib r o b la s ts as the te s tin g component.
Using th is
system-cyclophosphamide was shown to be f a r more to x ic to the fib r o b la s ts in the presence o f hepatocytes, th is being reduced i f the hepatocytes are tre a te d w ith SKF-525A, an in h ib it o r o f drug metabolism.
This
f ib r o b la s t suppression t e s t was used to te s t a w ider range o f chemicals w ith promising re s u lts by Wiebkin e t al_ (1978a).
The leakage o f c e llu la r enzymes has again been used as a measure o f t o x ic it y in cu ltu re d c e ll lin e s to in v e s tig a te the e ffe c ts o f carbon te tr a c h lo rid e (Watanabe e t al_, 1977) and some general an aesth etics (Goto e t aQ_, 1976).
This could also be ap p lied to prim ary monolayer
cu ltu re s o f hepatocytes.
There are s t i l l many problems to be solved, and a g re a te r s ta n d a rd is a tio n o f is o la tio n and c u ltu rin g techniques, f o r is o la te d hepato cytes, before b a tte r ie s o f sh o rt term te s ts can be introduced f o r the t o x i c it y screening o f new drugs and chem icals. h ig h lig h te d by the fo llo w in g examples.
A couple o f problems may be F i r s t l y by S eglen's study (1977b)
demonstrating th a t ammonia accumulating in the suspending c u ltu re medium in h ib ite d p ro te in deg rad atio n , and secondly the suggestion by Nishiuch e t al (1976) th a t c y s te in e , a component o f many tis s u e c u ltu re media, is h ig h ly to x ic to c u ltu re d c e lls .
2 .8
AIMS
The main aims o f the work presented in th is th e s is were to v a lid a te the use o f is o la te d hepatocytes, in suspension o r c u ltu r e , f o r the in v e s tig a tio n o f the t o x ic it y o f drugs and chemicals in the l i v e r . Some model h ep ato to xin s, "p rim a rily paracetam ol, were then in v e s tig a te d in these systems f o r t h e ir e ffe c ts on the hepatocytes w ith the aim of providing fu r th e r evidence f o r the mechanism o f actio n o f h ep ato to xin s.
C H A P T E R
M A T E R I A L S
AND
3
METHODS
3.1
3 .1 .1
MATERIALS
Animals
Male W is tar a lb in o weanling ra ts were obtained from the U n iv e r s ity o f Surrey breeding s to c k .
They were housed in the U n iv e rs ity o f Surrey
animal u n it in M a k ra lo n ® S te r o le t® .
cages on a bedding o f wood shavings o r
They had fre e access to w ater and a p e lle te d standard
la b o ra to ry d ie t ( S p ille r s No. 1 , S p ille r s , Croyden) and were m aintained under c o n tro lle d environmental c o n d itio n s.
Male Golden Syrian weanling hamsters were obtained from s u ita b le s u p p lie rs , dependent on a v a i l a b i l i t y , and were housed and m aintained in a s im ila r manner to the r a ts .
3 .1 .2
Apparatus
The sonicators used were as fo llo w s :
Soniprobe (Dave Instrum ents L td ,
London) and sonic bath (K e rry , England).
Manufacturers fo r. o th e r
apparatus used are as s p e c ifie d in the Methods S ectio n .
3 .1 .3
Chemicals
Collagenase (typ e IV , or s p e c ific use re : C -213 9), hyaluronidase. (typ e I I ) ,
|8-glucuronidase (typ e H - l ) , sulphatase (typ e H - l ) ,
saccharo-1, 4 -la c to n e , e th y le n e g ly c o l-b is -( /3 -ami noethyl e t h e r ) - N ,N 't e t r a - a c e t ic a c id , paracetam ol, NAD, 2 , 4 -d in itro p h e n y l-h y d ra z in e , N-methylphenazonium m ethosulphate, NADPH, N -l-n a p h th yleth yle n ed iam in e d ih y d ro c h lo rid e , reduced g lu ta th io n e , G6PD, NADP, cyclohexim ide and n eo tetrazo liu m c h lo rid e were obtained from Sigma L td, London.
Pure
0 - glucuronidase ('K etodase1) was obtained from W illia m Warner and Co. L td , Hampshire.
7-Ethoxycoumarin was synthesized by the method o f U llr ic h and Weber (1 9 7 2 ).
A ll tis s u e c u ltu re media and supplements were obtained from GibcoB io c u lt L td , Scotland.
Sepharose 4B was obtained from Pharmicia Fine Chemicals, Uppsala, Sweden.
Phenobarbitone, am ylobarbitone, q u in a lb a rb ito n e and thiopentone (as the sodium s a lt ) were a l l obtained from May and Baker L td , Dagenham, Essex.
L— [ll-^ c ]-L e u c in e ( s p e c ific a c t i v it y 351mCi/mmol) ,
[ u - ^ c ] - U r id in e
(s p e c ific a c t i v it y 497mCi/mmol), £-Hydroxy [ r i n g - 3 - 5 - ^ 4c ] - a c e t a n i l i d e ( s p e c ific a c t i v it y 14.6mCi/mmol) and the c y c lic AMP assay k i t (Code TRK.432) were a l l obtained from The Radiochemical C en tre, Amersham, Buckinghamshire.
ATP and GOT assay k its were obtained from Boehringer Mannheim D iag n o stica .
A ll o th er chemicals used were o f the best reagent grade a v a ila b le .
METHODS
3 .2
HEPATOCYTE ISOLATION
3 .2 .1
The enzymic is o la tio n o f a d u lt r a t hepatocytes
A dult r a t hepatocytes were is o la te d by a co llagenase/hyaluronidase technique developed by Fry e t
(1976) from the o r ig in a l method o f
Howard e t £]_ (1 9 6 7 ).
Male W istar Albino r a t s , weighing between 60 and lOOg, were k i ll e d by c e rv ic a l d is lo c a tio n .
The abdomen was swabbed w ith 70% alcohol p r io r
to d is s e c tio n to expose the l i v e r .
Each o f the s ix lobes o f the l i v e r
++ ++ - and Mg - fre e
was removed s e p a ra te ly and placed in Dulbecco's Ca phosphate b u ffered s a lin e (P B S 'A ')(P a u l, 197 0).
Each lobe was c a r e fu lly
d rie d between two f i l t e r papers to remove any excess blood then placed on a clean f i l t e r paper supported by a square sheet o f glass which acted as an anchor f o r the lobes and s im p lifie d the c u ttin g .
The lobes were
cut w ith s in g le strokes o f a microtome blade (Swann-Morton PM40, ■ Swann-Morton, S h e ffie ld ) to obtain s lic e s o f approxim ately 0.5mm thickness which, were placed in PBS'A' .
I t is im portant to complete
th is process q u ic k ly so th a t the l i v e r is not allowed to d e te r io r a te . PBS'A'(10m l) co n taining about 3g o f tis s u e was placed in a 250ml con ical f la s k .
The fla s k was shaken f o r 10 minutes in a shaking w ater bath
(approxim ately 90 o s c illa tio n s per m inute) m aintained a t a tem perature o f 37°C.
’The supernatant was then removed c a r e fu lly and replaced w ith
fresh PBS'A'.
This washing procedure was repeated tw ice more.
A fte r
th ree washes w ith PBS'A' the supernatant was removed and replaced by 10ml o f PBS'A' con taining 0.5mM ethylene g ly c o l- b is - ( 0 -ami no eth yl e th e r) N ,N '- te t r a a c e tic acid (EGTA).
The s lic e s were incubated f o r a f u r th e r
10 m inutes, follow ed by a second s im ila r in c u b a tio n .
The supernatant
was removed and replaced by 10ml o f Hanks Mg++- fre e balanced s a l t s o lu tio n (B SS )(Paul, 1970) containing 5mM CaC^ w ith c o llag en a se/ hyaluronidase (0.05% /0.10% w/ v ) .
The fla s k was incubated f o r approxim ately
45 minutes a t 37°C in the shaking w ater bath.
The re s u ltin g supernatant
was f i l t e r e d through a la y e r o f b o u ltin g c lo th (150/xm pore s iz e ;
Henry
Simon L td , Cheadle Heath, Stockport) to remove any la rg e c e ll clumps and undigested m a te r ia l.
The f i l t r a t e was ce n trifu g e d a t 50g f o r one minute
in an MSE
bench c e n trifu g e , the supernatant was c a r e fu lly removed and
the p e lle t resuspended in
PBS'A1 (10m l) to wash the c e lls .
A fte r spinning
the c e lls to a p e lle t th is washing procedure was repeated again.
The
f in a l c e ll p e lle t was resuspended in 10ml L ie b o v itz L I5 medium con taining 10% v/v fo e ta l c a l f serum.
The c e ll v i a b i l i t y and y ie ld were determined
using the trypan blue exclusion method (see 3 . 3 . 1 ) .
A ll the so lu tio n s
used in the is o la tio n procedure contained phenol red as an in d ic a to r .
3 .2 .2
The enzymic is o la tio n o f a d u lt hamster hepatocytes
The method follow ed was s im ila r to th a t described in sec tio n 3 .2 .1 . Hamster l i v e r has only f iv e lobes and contains a g a ll b la d d e r, not present in r a t s , which must be c a r e fu lly removed p r io r to s lic in g the lobes.
The only m o d ific a tio n
to the above method ( 3 .2 .1 ) was th a t the
s lic e s were incubated f o r a s h o rte r period o f time (approx. 30 mins) w ith the collagenase/hyaluronidase s o lu tio n .
The reason f o r th is was
th a t the l i v e r tis s u e o f the hamster is less robust and d is so c iates more r e a d ily .
The f in a l c e ll p e lle t was s im ila r ly suspended in L I5 plus
10% fo e ta l
c a l f serum and
the c e ll v i a b i l i t y and y ie ld determined
the trypan
blue exclusion
method ( 3 . 3 . 1 ) .
by
3 .3
3 .3 .1
CRITERIA USED TO ASSESS CELL VIABILITY
Dye exclusion te s t
This t e s t was used to assess the y ie ld o f v ia b le c e lls d ir e c t ly a f t e r c e ll is o la t io n , and also as a measure o f changes in v i a b i l i t y w ith tim e and due to t o x i c it y .
The t e s t - i s based on the fa c t th a t v ia b le c e lls can exclude c e rta in p o la r dyes whereas n o n -viab le c e lls do n o t.
A c e ll suspension (0.2 5m l) was added to 0.1ml o f trypan blue s o lu tio n (0.4%) and mixed g e n tly w ith a Pasteur p ip e tte .
A cover s l ip was placed
over the g rid o f an improved Neubauer haemocytometer applying g e n tle pressure u n t il Newtons rings were v i s ib le .
The mixed c e ll suspension was
allowed to pass from a Pasteur p ip e tte under the cover s l i p .
The v ia b le
and n o n -viab le c e lls which s e ttle d w ith in the c e n tra l g rid were then counted.
The t o ta l c e ll y ie ld and v i a b i l i t y were c a lc u la te d as fo llo w s :
.
V ia b ility
=
X
x 100%
X + Y Total y ie ld o f v ia b le c e lls
=
X x
7
x 10
4
c e lls /m l
5 where X = Y =
number o f v ia b le c e lls number o f n o n -viab le c e lls
A fte r an i n i t i a l c e ll is o la tio n c a lc u la tio n o f the v i a b i l i t y enabled an assessment o f the q u a lity o f the is o la tio n procedure, c a lc u la tio n o f the y ie ld enabled the o r ig in a l c e ll suspension to be d ilu te d to contain the number o f c e lls required f o r exp erim en tatio n .
Experiments to assess t o x i c it y , using c e ll v i a b i l i t y as the c r it e r io n o f t o x i c i t y , were c a rrie d out using one ml samples o f c e ll suspension containing 10 in L I5 plus
c e lls /m l and te s t chemical a t the req u ired c o n c e n tra tio n , 10%
in 10ml conical assessed by
fo e ta l c a l f serum.
The incubations
fla s k s in a shaking incubator a t 37°C.
dye exclusion as q u ic k ly as possible a f t e r
removed from the in cu b a to r.
were c a rrie d out V i a b i l i t y was the samples were
3 .3 .2
Lactate Dehydrogenase assay fo r v i a b i l i t y
When hepatocytes are damaged i n t r a c e ll u l a r enzymes are released in to the e x t r a c e llu la r medium, th is f a c t was used to develop an assay whereby the amount o f la c ta te dehydrogenase (LDH) released from is o la te d hepatocytes could be used as a measure o f c e ll v i a b i l i t y .
The method used was a m o d ific a tio n o f the c o lo rim e tric method o f King (V a rle y , 1967) based on the form ation o f a coloured hydrazone, i . e . p y ru v a te -d in itro p h e n y l-h y d ra zo n e .
Reagents: (a )
Buffered s u b s tra te :
4g lith iu m la c ta te in 125ml 0 .1 M g ly c in e b u ffe r con taining 75ml 0.1N NaOH.
(b)
S o lu tio n o f nicotinam ide adenine d in u c le o tid e ;
a s o lu tio n o f
lOmg in 2ml d i s t i l l e d w ater was fre s h ly prepared. (c )
2 , 4 -d in itro p h e n y lh y d ra zin e reagent;
200mg o f the reagent was
dissolved in hot N HC1 and made up to a l i t r e w ith th is a c id . (d)
Sodium hydroxide:
0.4N .
Procedure: c
One ml samples o f is o la te d hepatocytes con taining 10
c e lls /m l and the
required concentrations o f te s t chemical were incubated in 10ml conical fla s k s a t 37°C.
A fte r the s p e c ifie d incubation tim e 0.5ml o f the c e ll
suspension was placed in an LP3 tube and the c e lls spun to a p e lle t by c e n trifu g in g a t 50g f o r one m inute.
The supernatant (O J rn l) was added to
0.75ml o f d i s t i l l e d w ater and used to determ ine the amount o f LDH released from the c e lls in to the e x t r a c e llu la r medium.
To the rem aining
supernatant and c e ll p e lle t was added 0.1ml 5% T rito n X-100 (aqueous), mixing w ell to lyse a l l the c e lls ;
0.1ml o f the l a t t e r was added to
0.75ml o f d i s t i l l e d w ater and used to determine the t o t a l amount o f LDH present in the c e lls and the medium.
Assay: 0.5ml b u ffered sub strate (a )
+ 50 At! sample j Add
0.1ml
mix and leave a t 37°C f o r 2 mins
NAD s o lu tio n |
Add
incubate a t 37°C f o r 15 mins
0.5ml colour reagent J
Add
5ml
(b)
0.4N NaOH
(c )
incubate a t 37°C f o r 15 mins (d)
I
mix and read absorbance a t 440nm
The standard curve was lin e a r f o r the concentrations o f LDH found a t a fi c e ll concentration o f 10 /m l. Total LDH was represented by the absorbance a t 440nm m u ltip lie d by a fa c to r o f 1.25 to allo w fo r d ilu t io n .
The LDH
released was c a lc u la te d by d iv id in g the absorbance o f the medium, con taining released LDH, by the absorbance o f the t o ta l LDH, expressed as a percentage.
A number o f experiments were c a rrie d out to determine the e ffe c ts o f d if f e r e n t chemicals on c e ll v i a b i l i t y , some w ith a simultaneous assessment o f the trypan blue exclusion conducted on the same c e ll sample.
3 .3 .3
Measurement o f the r a te o f oxygen uptake in is o la te d hepatocytes
The ra te o f c e l l u la r re s p ira tio n in is o la te d hepatocytes was determined using a C lark Oxygen E lectrode (Rank B ro s ., Bottisham , Cambridge).
This
oxygen ele c tro d e consists o f a platinum e le c tro d e covered by a T e f l o n ® membrane which forms the base o f a re a c tio n chamber in to which small volumes (approx. 3ml) o f re a c tio n medium may be placed.
The re a c tio n
chamber is sealed to exclude a i r except f o r a small hole through which a d d itio n s may be made to the re a c tio n medium using a s y rin g e .
A small
magnetic f le a is placed in the re a c tio n chamber so th a t the medium may be s t ir r e d a t a constant r a te .
The platinum e le c tro d e is p o la ris e d a t
-0 .6 V w ith respect to a s i l v e r - s i l v e r c h lo rid e reference e le c tro d e .
Under
( R}
these conditions the oxygen, which d iffu s e s through the T e f l o n ^ membrane, a t the surface o f the e le c tro d e produces a c u rre n t.
Any change in the
oxygen a c t i v it y causes a corresponding change in c u rren t which may be monitored on a c h a rt recorder (H e a th k it).
C a lib ra tio n o f the oxygen e le c tro d e : The e lectro d e was c a lib ra te d by the method o f Robinson and Cooper (1 9 7 0 ), a chemical method using N-methylphenazonium methosulphate (PMS) as a c a ta ly s t in stead o f is o la te d m itochondria.
L I 5 plus 10% fo e ta l c a l f serum (3m l) con taining 800 u n its o f c a talase and 2 0 jm.g PMS was placed in the re a c tio n chamber, which was surrounded by a w a te r-ja c k e t containing w ater a t 37°C.
The re a c tio n medium was
s t ir r e d fo r 20 minutes to allo w tem perature e q u ilib r a t io n , and a i r s a tu ra tio n .
The chamber was then sealed and 10 ,ul o f NADH ( 0 . 1 - 0 . 3/* mole)
added to the s t ir r e d medium by s y rin g e .
When the c u rre n t reached a
steady s ta te the a d d itio n o f NADH was repeated.
The mean scale
d e fle c tio n was c a lc u la te d f o r a known concentration o f NADH (c a lc u la te d s p e c tro p h o to m e tric a lly ), from these values the oxygen con centration o f the a i r saturated s o lu tio n may be c a lc u la te d , and hence the nmoles oxygen consumed per u n it o f d e fle c tio n f o r one ml o f re a c tio n medium determ ined.
Procedure: Is o la te d hepatocytes were incubated a t 37°C in a shaking w ater bath.
c
Twenty ml volumes con taining between 0 .5 and 2 .0 x 10
c e lls /m l and
t e s t chemical were used, incubating in 250ml conical fla s k s , to allo w 3ml samples to be removed a t time in te r v a ls f o r e stim a tio n o f the c e l l u la r re s p ira tio n r a t e .
The re a c tio n was monitored on the c h a rt reco rd er
u n t il a s tr a ig h t lin e response was recorded th a t was adequate to determ ine the ra te o f oxygen consumption.
3 :3 .4
Measurement o f 7 - ethoxycoumarin 0-d eeth ylase a c t i v i t y in c e ll
suspensions 7-Ethoxycoumarin 0-d eeth ylase a c t i v it y was used as a measure o f the m etabolic cap acity o f the c e lls w ith the aim o f fin d in g out whether th is could be used as a s e n s itiv e measure o f c e ll v i a b i l i t y .
Reagents: (a )
7-Ethoxycoumarin (7 -E C ); 35mM in dimethyl formamide
(b)
7-Hydroxycoumarin standard;
(c )
0.3M a ce ta te b u ffe r ;
(d)
0 -g lu c u ro n id ase/su lp h a tas e;
(DMF).
400 ^g/m l in DMF.
pH 4 .5 . d isso lve crude
0 -glucuronidase
co n taining sulphatase a c t i v it y (typ e H - l , Sigma) in ace ta te b u ffe r (c ) a t 5mg/ml. (e )
D ieth yl e th e r con taining 1.5% isoamyl a lc o h o l,
(f)
0.2M Glycine-NaOH b u ffe r pH 1 0 .4 .
(g)
Ketodase (pure 0-glucuronidase in a c e ta te b u f f e r ) .
(h)
Sulphatase;
(i)
Saccharo-1, 4 0 -la c to n e ; lOOmg in 2ml ac e ta te
100mg/6ml in ace ta te b u ffe r .
Procedure:
b u ffe r .
c
Samples o f c e ll suspension (1m l) con taining 10
c e lls /m l were placed in
10ml conical fla s k s con taining 2 /*1 o f 35mM 7-EC to give a f in a l concentration o f 70juM.
The incubation was continued f o r the req u ired
tim e , then the re a c tio n was stopped by p lacing the samples on ic e .
A
0.5ml a liq u o t o f the c e ll suspension was then added q u ic k ly to 4.5ml d i s t i l l e d w ater to lyse the c e lls and also d ilu t e the flu o re scen c e. S u ita b le blanks and standards were also run.
A number o f experiments
were c a rrie d out to determ ine the e f f e c t o f chemicals on the a c t i v i t y o f 7-ethoxycoumarin 0 -d e e th y la s e , some were c a rrie d out in the presence or absence o f added NADPH since i t is known th a t NADPH only r e a d ily p enetrates damaged plasma membranes.
Assay: One ml samples ( in t r i p l i c a t e ) o f the d ilu te d incubate were placed in 10ml S o v e ril tubes to which was added 0.2ml o f crude 0 -g lu c u ro n id a s e . The tubes were incubated overn igh t a t 37°C then e x tra c te d w ith 5ml e t h e r / alcohol f o r 10 m inutes. 2000g fo r 10 m inutes.
The phases were separated by c e n trifu g in g a t One ml samples o f the e th e r phase were back-
e x tra c te d in to glycine/NaOH b u ffe r f o r 10 m inutes.
The e th e r phase
was asp irated and the fluorescence present in the a lk a li phase was determined in a flu o rim e te r s e t a t A
370nm and A 450nm. ex em gave the t o ta l m etabolites produced by the c e ll suspension.
This
The products could be separated in to fre e and conjugated m etabolites by e x tra c tin g w ith e th e r p r io r to tr e a tin g w ith deconjugating enzymes. The fre e m etabolites would be extra c te d in to the e th e r phase and could be b ack-extracted in to g lycin e b u ffe r .
The conjugated m etabolites
could be fu r th e r separated in to glucuronide and sulphate by tr e a tin g samples s e p a ra te ly w ith ketodase (pure /3-glucuronidase) and sulphatase w ith added 1 ,4 saccharolactone.
3 .4
PROTEIN SYNTHESIS IN ISOLATED HEPATOCYTES
The in c o rp o ra tio n o f
^ c ]-L e u c in e in to t r ic h lo r a c e t ic acid in s o lu b le
p ro te in o f is o la te d hepatocytes v/as used as a measure o f p ro te in synthesis The method used was adopted from th a t o f Seglen (1976a).
P re lim in a ry
experiments were c a rrie d out to determ ine the id e a l c e ll con centration and incu b atio n times before the fo llo w in g procedure was adopted.
3 .4 .1
Measurement o f ft^ c ]-L e u c in e uptake in to is o la te d hepatocytes
One ml a liq u o ts o f a suspension o f is o la te d hepatocytes con taining 2 x 10^ c e l l s , 100 nCi L- [ u -^ c ] - L e u c in e (s p e c ific a c t i v it y 351 mCi/mmol) and t e s t chemical a t the required concentration were incubated in 10ml conical fla s k s in a shaking w ater bath a t 37°C.
A fte r the req u ired
incubation time the c e ll suspension was tra n s fe rre d to LP3 tubes and the c e lls spun g e n tly to a p e l l e t .
The supernatant was removed and
replaced by 1ml o f 10% t r ic h lo r o a c e tic acid (TCA) to p r e c ip ita te the p ro te in from the c e ll p e l l e t .
The p r e c ip ita te was washed th re e times
w ith 1% TCA to remove any resid u al la b e lle d amino acids not in co rp o rated in to the p ro te in s .
The f in a l p ro te in p e lle t was s o lu b ilis e d w ith one ml
o f 0.5N NaOH overn igh t a t 37°C.
P ro tein content was estim ated by the
method o f Lowry e t al (1951) using 0.1ml o f the s o lu b ilis e d sample. remaining 0.9ml o f sample was n e u tra lis e d w ith 0.1ml 4.5N HC1.
The
250 ^1
a liq u o ts o f the n e u tra lis e d sample were added to 4ml o f a s c i n t i l l a t i o n c o c k ta il composed o f:
Toluene, 65%
PP0, 1.5%;
Metapol HC100, 32.5%)
to g eth er w ith 250 jul o f w ate r.
The w ell mixed s c i n t i l l a t i o n c o c k ta il con tain in g the sample was l e f t overn igh t a t 0°C in the dark to reduce quenching then counted f o r a c t i v i t y using a H ew lett Packard T ric a rb s c i n t i l l a t i o n counter.
A quench curve was determined fo r the above p rep a ra tio n using carbon te tr a c h lo r id e as a quenching agent.
The fa c to rs f o r the equation o f
th is curve, obtained using a computer program, were programmed in to the s c i n t i l l a t i o n counter to enable decompositions per minute to be determined d ir e c t ly from the sample counts.
The [^ c ]-L e u c in e
incorporated in to the hepatocytes was expressed in terms o f dpm/mg o f c e l l u la r p ro te in , or as a percentage o f the c o n tro l.
3 .4 .2
P re lim in ary in v e s tig a tio n s
One ml samples o f is o la te d r a t hepatocytes containing 0 . 5 , 1 and 2 x 10 c e lls /m l were incubated f o r 30, 60 and 90 m inutes. tr ip lic a te .
Samples were run in
A fte r spinning the c e lls to a p e lle t the samples were
tre a te d as described in the method above ( 3 . 4 . 1 ) .
3 .4 .3
T14 The e f fe c t o f xen obiotics on L C -Leucine uptake by is o la te d
hepatocytes The e ffe c ts o f a number o f x e n o b io tic s , over a range o f c o n cen tratio n s , on the uptake o f in v e s tig a te d .
[^ c ]-L e u c in e by is o la te d r a t hepatocytes were fi Hepatocytes a t a concentration o f 2 x 10 c e lls /m l
were incubated f o r one hour, w ith t r i p l i c a t e samples f o r each te s t chemical con centration and c o n tro l.
Where the experiments were repeated
on more than one occasion means and standard d e v ia tio n were c a lc u la te d . The t e s t chemicals were dissolved d ir e c t ly in the medium i f p o s s ib le , or in DMF a t concentrations which enabled 10
m!
o r less o f DMF to
be added to a one ml sample o f suspended c e lls .
Samples were tre a te d
as described in 3 .4 .1 .
Experiments w ith hamster hepatocytes were .c a rrie d out according to the same procedure.
3 .4 .4
The e f f e c t o f paracetamol on [^ c ]- L e u c in e uptake in hepatocytes
is o la te d from ra ts p re tre a te d w ith phenobarbitone Rats were tre a te d w ith i . p . in je c tio n s o f phenobarbitone (70mg/kg) d a ily f o r th ree days p r io r to s a c r if ic in g the animals and is o la tin g hepatocytes U ntreated animals were m aintained under the same co n d itio n s .
A fte r
is o la tio n o f the c e lls cytochrome P450 was measured in both the p h e n ibarbitone p re tre a te d and the u n treated c e lls s p e c tr a lly by the method o f Omura and Sato (1964) to check th a t in ductio n o f the m etabolising enzymes had occurred.
[^ C -L eu cin e uptake was determined as p re v io u s ly
described ( 3 . 4 . 1 ) .
This experim ent was repeated on th ree occasions,
each sample being run in t r i p l i c a t e .
Means and standard d e v ia tio n s were
c a lc u la te d f o r each concentration o f paracetam ol.
3 .4 .5
E ffe c t o f paracetamol on p ro te in degradation
A fte r is o la tio n hepatocytes were d ilu te d to 10ml w ith L I5 medium p' us 10% fo e ta l c a lf serum to contain a f in a l con centration o f 100 nCi per m l.
-L eu cin e
This 10ml sample o f c e ll suspension was incubated f o r one hour in
a 100ml conical fla s k in a shaking w ater bath a t 37°C.
A fte r about 50
minutes incubation a sample o f c e ll suspension was taken to determ ine the c e ll v i a b i l i t y and number.
At one hour the c e lls were spun g e n tly to a
p e lle t and the supernatant removed.
The c e ll p e lle t was washed tw ice
c a r e fu lly w ith medium to remove re sid u al r a d io a c t iv it y and then made up
c
to a c e ll concentration o f 4 x 10 [ ^ c ] - L e u c in e .
c e lls /m l w ith medium not con tain in g
A liquots o f the c e ll suspension were then added to
medium co n taining paracetamol to give a f in a l c e ll con centration o f 2 x 10* c e lls /m l and paracetamol concentrations o f 0 , 0 .1 , 5 and 40mM in one ml d u p lic a te samples.
Two samples, w ith o u t paracetamol were processed
im m ediately to determ ine the la b el present in the c e l l u la r p ro te in a f t e r the one hour in c u b a tio n .
Control and tre a te d samples were then incubated
f o r a fu r th e r hour and processed as described p re vio u s ly ( 3 . 4 . 1 ) .
3 .4 .6
E ffe c t o f paracetamol on the uptake o f
14c
-Leucine in to
is o la te d hepatocytes ( in r e la tio n to incubation tim e) Is o la te d r a t hepatocytes were incubated as one ml samples co n tain in g 2 x 10^ c e lls a t 37°C in a shaking w ater b ath.
At 5 , 10, 15, 2 0 , 25,
30, 45 and 60 minutes two samples were removed the c e ll suspension placed in LP3 tubes and spun g e n tly to a p e l l e t .
The c e ll p e lle ts
were washed tw ice w ith u n la b e lle d medium and then the p ro te in was p re c ip ita te d w ith 6.5% TCA and the p ro te in p r e c ip ita te spun to a p e lle t , The supernatant was removed c a r e fu lly and a sample was counted to determine the TCA soluble r a d io a c t iv it y , the p ro te in p e l l e t was washed th ree times w ith 1% TCA and then s o lu b ilis e d w ith NaOH (o.5N ) and tre a te d as p revio u s ly described ( 3 . 4 . 1 ) .
This experim ent was c a rrie d out w ith co n tro l and samples tre a te d w ith 40mM paracetam ol.
3 .4 .7
E ffe c t o f paracetamol on p ro te in s e c retio n
Hepatocytes were is o la te d from two ra ts and the c e lls were pooled and made up to 20ml w ith medium con taining 100 nCi [ ^ c ] - L e u c in e /m l. The c e lls were incubated f o r 30 minutes in a 250ml conical fla s k a t 37°C in a shaking w ater bath then spun g e n tly to a p e l l e t and washed tw ice c a r e fu lly w ith medium.
The f in a l p e lle t was made up to 10ml
w ith medium and 2.5ml o f the suspension dispensed to fo u r 50ml conical fla s k s .
Medium (2 .5 m l) was added to two samples o f c e lls and 2.5ml
o f medium con taining 80mM paracetamol was added to the remaining two 50ml conical fla s k s .
The fo u r samples were incubated f o r one hour the
c e lls were then spun to a p e lle t and the supernatants removed and re ta in e d .
TCA (5ml o f 20%) was added to the supernatants to p r e c ip ita te
the p ro te in .
The p r e c ip ita te was then spun to a p e lle t and tre a te d as
alread y described ( 3 . 4 . 1 ) .
3 .4 .8
To determine whether the e f f e c t o f paracetamol on p ro te in
synthesis is re v e rs ib le Is o la te d r a t hepatocytes were incubated w ith o r w ith o u t 40mM paracetamol f o r one hour.
The c e lls were then washed c a r e fu lly and the media
replaced w ith L I5 + 10% serum con taining 100 nCi/ml [ ^ c ]- L e u c in e . Control and paracetamol tre a te d samples were removed a t 5 minute in t e r v a ls , f o r 30 m inutes, the c e lls spun to a p e lle t then tre a te d as p re v io u s ly described ( 3 . 4 . 1 ) .
3 .4 .9
E ffe c t o f paracetamol on p ro te in synthesis in vivo in the r a t
W is tar Albino ra ts weighing approxim ately 70g were in je c te d w ith 1000 mg/kg paracetamol in DMF con taining 2 w ith la b e l alone.
Ci
[^ c ]-L e u c in e o r DMF
The animals were observed f o r one hour and then k i l l e d
by c e rv ic a l d is lo c a tio n .
The liv e r s were removed im m ediately and placed
in 20ml 10% TCA, chopped f in e ly and homogenised w ith th re e strokes o f a P o tte r Eveljhem homogeniser.
A 5ml sample o f the homogenate was
placed in a S o v e ril tube and the p r e c ip ita te spun to a p e l l e t and washed tw ice w ith 1% TCA then once w ith 70% e th a n o l, tw ice w ith 96% ethanol and f i n a l l y tw ice w ith e th e r. s o lu b ilis e d in 5ml o f 0.5N NaOH.
The f in a l p ro te in p e l l e t was
Samples were taken to determ ine the
p ro te in concentration and the a c t i v it y o f [^ c ]-L e u c in e present in the p ro te in .
The experim ent was repeated on th re e occasions using th re e
t e s t animals in each case.
The animals were tre a te d and k i l l e d by
c e rv ic a l d is lo c a tio n in a staggered fashion a t 10 minute in te r v a ls to enable each animal to have received the la b e l f o r the same length o f time p r io r to s a c r if ic e .
V
3.5
TO DETERMINE WHETHER PARACETAMOL HAS A SPECIFIC EFFECT ON EXPORT
PROTEINS
3 .5 .1
P rep aration o f cyanogen bromide a c tiv a te d Sepharose conjugates
Sepharose 4B
(10m l) co n sistin g o f an equal volume o f gel and w a te r,
was washed w ith 100ml o f w ater through a s in te re d -g la s s fu n n e l.
The
Sepharose was placed in a small beaker w ith 10ml w ater and 20ml 2M Na2C0g and s t ir r e d in a fume cupboard.
Cyanogen bromide (1m l) in a c e t o n it r ile
was then added r a p id ly , the m ixture was s t ir r e d vig o ro u sly f o r 2 minutes and th e n .re tu rn e d to the s in te re d -g la s s funnel and washed w ith 200ml o f cold 0.1M NaHCOg pH 9 .5 follow ed by 200ml o f cold w ater and 200ml 0.1M NaHCOg pH 9 .0 .
A fte r the la s t wash the s lu r r y was f i l t e r e d to a m oist
compact cake
under vacuum.
(Three times th is q u a n tity was a c tu a lly
prepared, as
one batch , to produce s u f f ic ie n t a c tiv a te d Sepharose.)
Two th ird s o f the Sepharose was tra n s fe rre d to a b o ttle to which was added 17ml 0.1M NaHCO^ pH 9 .0 w ith 0.5M NaCl and 3ml o f r a t a n tis e ra ( a ) , the remaining t h ir d o f the Sepharose was tra n s fe rre d to another b o t t le to which was
added 8.5ml o f the same b u ffe r and 1.5ml o f non immune
ra b b it serum
(b ).
Coupling
o f the a c tiv a te d Sepharose w ith conjugates
was done o vern ig h t a t 4°C on a shaker.
Conjugates: (a )
An a n ti serum prepared ag ain st the serum o f laparotom ised r a t s .
in je c tio n schedule was th a t used by M ullock ejt al_ (1 9 7 4 ).
The
The a n ti serum
was shown by two dimensional crossed immune e le c tro p h o re s is (Axelson e t al_, 1 9 7 3 ), w ith a commercially a v a ila b le a n t i - ( r a t serum) in co rp o rated in to the upper g e l, to re a c t w ith a l l the major p ro tein s o f r a t serum. (b)
Non immune r a b b it serum.
In each case (a ) and ( b ) , a g lo b u lin fr a c tio n was prepared by p re c ip i-ta tio n w ith an equal volume o f ammonium su lp h a te.
The next day the two samples o f conjugated Sepharose were returned to separate s in te re d -g la s s funnels and drained q u ic k ly under vacuum.
The
fo llo w in g procedure is described f o r a 10ml q u a n tity o f conjugated Sepharose, the q u a n titie s were doubled f o r the r a t a n ti sera con jugate.
The s lu r r y was washed w ith 70ml o f coupling mix (0.1M NaHCOg, 0.5M NaCl, pH 9 .0 ) and allowed to stand in the cold f o r about 10 minutes w h ile IN! ethanolamine pH 9 .0 was prepared.
The Sepharose was tra n s fe rre d to
a glass beaker con taining 40ml o f the ethanolamine per 10ml o f Sepharose, and l e f t a t room tem perature f o r 2 hours.
The m ixture was then returned
to the funnel and washed a lte r n a te ly w ith 60ml portio ns o f coupling mix (pH 9 .0 ) and 0.1M ac e ta te b u ffe r pH 4 .0 co n taining 0.5M NaCl, s ta r tin g and fin is h in g w ith coupling m ix, the la s t p o rtio n o f each wash being drained g e n tly under vacuum;
th e re were 5 acid washes in a l l .
The
Sepharose was then placed in a graduated tube washing out the funnel w ith 0.15M NaCl (+ 15mM NaN^).
The Sepharose was allowed to s e t t l e .
Each conjugated sample was adjusted so th a t i t contained 50% Sepharose beads.
The conjugated Sepharose was then incubated w ith la b e lle d serum
to determine whether or not i t was a c tiv e as fo llo w s :
Sample 1.
0.3ml normal ra b b it conjugated Sepharose 0.1ml [ ^ c ] - f u c o s e la b e lle d r a t serum 0.6ml 0.15M NaCl
Sample 2.
0.3ml a n t i - r a t conjugated Sepharose 0.1ml
[^ c ]-fu c o s e la b e lle d r a t serum
0.6ml 0.15M NaCl
These two samples (1 and 2) were placed in LP3 tubes and shaken o v e rn ig h t a t 4°C.
The Sepharose was then spun to a p e lle t and washed tw ice w ith
0.15M NaCl.
Sodium hydroxide (one ml o f 0.5M) was then added to the
p e l l e t o f Sepharose beads and heated a t 80°C f o r 30 m inutes.
A liq u o ts
were then added to s c i n t i l l a n t (p re v io u s ly described in 3 .4 .1 ) and counted in a s c i n t i l l a t i o n counter.
M
- la b e lle d r a t serum:
A r a t was in je c te d in trav en o u s ly w ith c o lle c tio n o f serum.
[}^c] -fucose 135 minutes p r io r to
Analysis by crossed im m unoelectrophoresis, fo llow ed
by autoradiography demonstrated th a t the la b el was in co rp o rated in to a l l the major serum g ly c o p ro te in s .
3 .5 .2
P rep aration o f L ^ c j - la b e lle d p ro te in from is o la te d r a t
hepatocytes Is o la te d r a t hepatocytes were prepared as described p re vio u s ly ( 3 . 2 . 1 ) . The f in a l hepatocyte suspension was d ivid ed in to two portio ns (a ) and fi ( b ) , each containing 20 x 10 c e lls in 10ml.
Sample (a )
contained L I5 + 10'% fo e ta l c a l f serum w ith 2 ^ c i
Sample (b)
contained 40mM paracetamol in the medium w ith 2 u-Ci
L. C
Leucine
Leucine
Both samples were incubated a t 37°C in a shaking w ater bath f o r 2 hours. The c e lls were then spun to a p e lle t in S o v e ril tubes. medium was removed c a r e fu lly and stored fro z e n .
The incubation
Lubrol (5ml o f 0.5%)
was added to each c e ll p e lle t and mixed w ell to lyse the c e lls com pletely. This was checked m ic ro s c o p ic a lly .
The Lubrol samples co n tain in g the
c e llu la r m a te ria l were then c e n trifu g e d a t 40,000g in an MSE Superspeed
50
f o r 1£ hours to remove a l l the membraneous m a te r ia l. The supernatant was removed and stored fro zen u n t il re q u ire d , the p e lle ts were also r e ta in e d . A ll samples were stored a t -20°C u n t il re q u ire d .
3 .5 .3
A f f i n i t y experiments w ith a c tiv a te d Sepharose conjugate
The Lubrol e x tr a c ts , containing i n t r a c e ll u l a r p ro te in , and the in cu b a tio n media, con taining e x t r a c e llu la r p ro te in s , were thawed and 0.6ml a liq u o ts o f the samples were added in d u p lic a te to both a c tiv a te d Sepharose conjugates (0 .4 m l) s e p a ra te ly in LP3 tubes.
The samples were incubated
overn igh t a t 4°C on r o lle r s to continuously mix them.
A fte r in cu b a tio n
the samples were spun in a bench c e n trifu g e (2000g) to p e l l e t the Sepharose beads.
The supernatant was c a r e fu lly removed and re ta in e d .
The p e lle ts were washed th ree times w ith one ml 0 .1 5M NaCl and the washings pooled w ith the supernatant.
One ml o f 0.5N NaOH was added
to the f in a l p e l l e t o f Sepharose which was m inutes.
then heated a t 80°C f o r 30
A liquots were taken f o r counting r a d io a c t iv it y .
To the super
natant plus washings was added 3ml o f 10% TCA to p r e c ip ita te the p r o te in . The p r e c ip ita te was washed th ree times w ith 1% TCA then one ml o f 0.5N NaOH was added to the f in a l p ro te in p e lle t and l e f t o vern ig h t a t 37°C to s o lu b ilis e the p ro te in .
Samples were then taken f o r counting.
The
membrane p e lle t obtained a f t e r the p rep a ra tio n o f the Lubrol supernatant
Figure 1. Flow ch a rt fo r procedures used in experiments w ith a c tiv a te d Sepharose conjugates
• c e ll suspension incubate a t 37°C f o r 2 hours c e n trifu g e a t 2 0 0 g f o r one min. >£--------CELLS
a MEDIUM
add 5 ml Lubrol (mix w e ll) /c e n tr ifu g e 40,000rpm ( f o r 75 mins
supernatant
a . membranous p e lle t
incubate 0.6ml w ith 0.4ml o f conjugated CNBr Sepharose
spin down Sepharose and wash x 3 w ith NaCl
Sepharose beads (bound)
heat a t 90 C fo r 30 mins w ith 0.5N NaOH
supernatant (unbound) acid ppt p ro te in s (wash w e ll) p ro te in p r e c ip ita te
count r a d io a c t iv it y
s o lu b ilis e w ith 0.5N NaOH
count r a d io a c t iv it y
was also counted.
The procedure is presented as a flo w c h a rt
(F ig . 1) f o r c l a r i t y .
The d ilu t io n fa c to rs were c a lc u la te d .
c
as dpm per 10
c e lls .
R a d io a c tiv ity was expressed
3 .6
THE EFFECT OF XENOBIOTICS ON RNA SYNTHESIS ------------------------------------The in co rp o ratio n o f [ C l -U rid in e in to the rib o n u c le ic acids o f is o la te d r a t hepatocytes was used as a measure o f RNA s y n th e s is .
3 .6 .1
Measurement o f
-U rid in e uptake in to is o la te d hepatocytes
The p ro te in p r e c ip ita te obtained from is o la te d hepatocytes a f t e r treatm ent w ith TCA also contains RNA.
•
I t was found th a t f u r th e r
treatm en t o f the p r e c ip ita te by heating a t 90°C f o r 30 minutes fo llow ed by an e x tra c tio n w ith 70% alcohol to remove lip id s had no e f f e c t on - U r id in e . Leucine was, th e r e fo r e , follow ed f o r L ^ c J -U r id in e .
A p re lim in a ry experim ent was c a rrie d out to compare the e f f e c t o f d if f e r e n t c e ll concentrations on the ra te o f uptake.
Is o la te d r a t
hepatocytes a t concentrations o f 0 .5 , 1 .0 , 1.5 and 2 .0 x 10
c
c e lls /m l
were incubated w ith 100 nCi [ u - ^ c ] -U rid in e (s p e c ific a c t i v i t y 497 mCi/mmol) per ml f o r 30, 60 and 90 m inutes.
There was an i n i t i a l tim e lag but a f t e r
30 minutes the uptake was approxim ately lin e a r w ith tim e and c e ll co n cen tratio n .
I t was, th e r e fo r e , decided to m aintain a con cen tratio n
o f 2 x 1 0 6 c e lls /m l fo r incubations as th is gave a r e l a t i v e l y high a c tiv ity .
The e ffe c ts o f paracetam ol, phenobarbitone and s a fr o le a t d i f f e r e n t concentrations on the uptake o f
-U rid in e were in v e s tig a te d .
3 .7 3 .7 .1
COVALENT BINDING Measurement o f co valent binding o f paracetamol m etab o lites
to c e llu la r p ro te in Is o la te d r a t hepatocytes (2 .3 x 10
c e lls /m l) were incubated in L I5 +
10% fo e ta l c a l f serum con taining paracetamol a t a con centration o f 40 and 20mM w ith 0.422% £-hydroxy [ r i n g - 3 , 5 -
^ c ]]-a c e ta n ilid e
(s p e c ific a c t i v it y 14.6 mCi/mmol, 97jj^Ci/m g).
Tw enty-four samples
were prepared f o r in c u b a tio n , the la b e lle d paracetamol being added to the samples ju s t p r io r to commencing incubation a t 37°C.
Samples, in
d u p lic a te were removed from incubation a t 5 , 10, 20, 4 0 , and 60 minutes f o r both 20 and 40mM paracetam ol.
The c e lls were spun to a p e l l e t ,
the supernatant removed and one ml o f IN p e rc h lo ric acid added to p r e c ip ita te the p ro te in .
The p r e c ip ita te was washed f iv e times w ith
p e rc h lo ric acid to remove a l l the unbound la b e lle d paracetam ol.
The
p r e c ip ita te was s o lu b ilis e d in 0.5N NaOH and a liq u o ts were counted to determine the amount o f a c t i v i t y bound to the c e llu la r p r o te in . P ro te in was assayed by the method o f Lowry e t al_ (1 9 5 1 ).
The re s u lts
are expressed in nmoles o f t o ta l paracetamol bound per mg o f p r o te in , assuming th a t a s im ila r percentage binding o f unlabel led.paracetam ol (o r m e ta b o lite ) also occurred.
3 .8
GLUTATHIONE IN ISOLATED HEPATOCYTES
The g lu ta th io n e content o f is o la te d hepatocytes was assayed by a m o d ific a tio n o f the method o f S a v ille (1 9 5 8 ).
G lutathio ne was e x tra c te d by adding 6.5% TCA (1m l) to the c e ll p e lle t
c
(2 x 10
c e lls in one ml) to p r e c ip ita te the p ro te in .
A fte r mixing
w e ll the samples were spun a t 1500g f o r 15 minutes and 0.1ml o f the supernatant was used f o r the assay.
3 .8 .1
G lutathio ne assay
Reagents: (a )
0 . 0 0 1 M aqueous sodium n i t r i t e .
(b)
0.5% aqueous ammonium sulphonate.
(c )
Mix one volume o f a 1% aqueous s o lu tio n
o f m ercuric c h lo rid e w ith
4 volumes o f a 3.4% s o lu tio n o f sulphanilam ide (d)
in 0.4N
HC1.
0.1% s o lu tio n o f N -l-n ap h th yleth yle n ed iam in e d ih yd ro c h lo rid e in 0.4N HC1 fre s h ly prepared.
0.1ml o f sample in 6.5% TCA was added to 0.5ml o f s o lu tio n (a ) and l e f t f o r 5 m inutes. m inutes.
0.1ml o f (b) was added, mixed w ell and l e f t f o r two
One ml o f s o lu tio n (c ) was then added follow ed by 0.8ml
o f s o lu tio n ( d ) .
A fte r 10 minutes the absorbance was read a t 540nm.
S u ita b le co n tro ls and standards were always run.
At concentrations o f
up to IGQjig/ml o f g lu ta th io n e the standard curve was l in e a r , passing through zero .
3 .9
ASSAY OF ADENOSINE TRIPHOSPHATE IN ISOLATED HEPATOCYTES
Is o la te d hepatocytes (1m l) were incubated a t a c e ll con cen tratio n o f 0 .5 x 10^ c e lls /m l w ith 40mM paracetam ol, 20mM phenobarbitone, lOmM s a fr o le w ith con trol samples f o r th ree minutes a t 37°C.
The re a c tio n
was stopped by the a d d itio n o f 4ml cold IN p e rc h lo ric a c id .
A f t e r 10
minutes the p re c ip ita te d p ro te in was spun a t 1500g f o r .15 minutes to a p e lle t.
The supernatant was c a r e fu lly removed and n e u tra lis e d w ith the
required amount o f 5M K2 CO3 which p r e c ip ita te d the p e rc h lo ric a c id . A fte r 30 minutes the p e rc h lo ric acid p r e c ip ita te was spun to a p e lle t (1500g f o r 15 minutes) and the supernatant was used as q u ic k ly as possible to assay f o r ATP.
ATP was assayed using a Boehringer t e s t k i t based on the method o f Bucher (1 9 4 7 ).
The fo llo w in g reagents were used: (1 )
B u ffe r/G lycerate-3-p h o sp h ate containing tr ie th a n o l amine b u ffe r pH 7.6
0 .5 m o l/l
MgSO^
4mmol/l
glycerate-3-phosp hate
6 mmol/l
(2 )
NADH
(3 )
GAPDH/PGK/GDH/TIM
2.5m m ol/l > 560u/mol ;>450u/ml ;>800u/m l;>1000u/m l
Two ml o f s o lu tio n ( 1 ) , 0.2mls o f s o lu tio n (2 ) and the d e p ro te in is e d supernatant, were placed in a cuvette and mixed w e ll.
The absorbance
was read a t 340nm then 0.02ml o f suspension (3 ) was added.
A f t e r 10
minutes the absorbance was read a g a in , readings being repeated u n t il the re a c tio n stopped.
Using the conversion fa c to r provided, and allo w in g fo r a p p ro p ria te
c
d ilu t io n s , the concentration of.A TP /10 sample.
c e lls was c a lc u la te d f o r each
3.10
ASSAY OF CYCLIC AMP IN ISOLATED HEPATOCYTES
C y c lic AMP was assayed using a k i t supplied by the Radiochemicals C entre, Amersham (code TRK.432).
The assay is based on the com petition between
u n la b e lle d c y c lic AMP and a fix e d q u a n tity o f the t r it iu m la b e lle d compound fo r binding to a p ro te in which has a high s p e c if ic it y and a f f i n i t y fo r c y c lic AMP.
The amount o f la b e lle d p r o te in :c y c lic AMP
complex formed is in v e rs e ly r e la te d to the amount o f u n la b e lle d c y c lic AMP present in the assay sample.
Measurement o f the p ro te in bound
r a d io a c t iv it y enables the amount o f u n la b e lle d c y c lic AMP in the samples to be c a lc u la te d .
Separation o f the p ro te in bound c y c lic AMP from the unbound n u c leo tid e is achieved by adsorption o f the fre e n u c leo tid e on to coated ch a rc o a l, follow ed by c e n tr ifu g a tio n .
An a liq u o t o f the supernatant is then
removed f o r liq u id s c i n t i l l a t i o n counting.
The concentration o f
u n la b e lle d c y c lic AMP in the sample is then determined from a lin e a r standard curve.
Prep aration o f samples fo r the assay: fi Is o la te d r a t hepatocytes (2 x 10 c e lls /m l) were incubated w ith ^ c o n tro l medium, 40mM paracetam ol, 20mM phenobarbitone, and lOmM s a fr o le a t 37°C in a shaking w ater bath.
At in te rv a ls o f 5 , 10, 30 and 60 minutes
d u p lic a te samples were removed from the in cu b ato r and placed on ic e . The samples were tra n s fe rre d to LP3 tubes and the c e lls spun g e n tly to a p e lle t follow ed by removal o f the supernatant.
One ml o f cold 5%
TCA was added to the p e l l e t , mixed w ell and l e f t f o r 15 minutes a t 0°C. The samples were mixed again then the p r e c ip ita te was separated by spinning the samples o f 2000 rpm fo r 15 m inutes. c a r e fu lly removed and placed in S o v e ril tubes.
The supernatants were HC1 was added to bring
the f in a l con centration to 0.1N HC1 ( i . e . 0.1ml o f 1.1N HC1 to one ml o f sam ple).
TCA was e x tra c te d from the samples w ith 5ml w ater s a tu ra te d
d ie th y l e th e r.
The 10 minute e x tra c tio n s were repeated f iv e tim es.
A fte r the la s t e x tra c tio n any excess e th e r was removed by heatin g the samples in a w ater bath a t 70-90°C in a fume cupboard.
The samples were
then lyo p h ylised and re c o n s titu te d in a small volume o f a c e ta te b u ffe r (50mM, pH 6 .2 ) and a liq u o ts o f th is used in the c y c lic AMP assay.
3.1 1
THE PREPARATION OF MICROSOMES FROM ISOLATED RAT HEPATOCYTES
A number o f attempts were made to o btain a microsomal fr a c tio n from is o la te d hepatocytes w ith the aim o f carryin g out polyacrylam ide gel e lec tro p h o re s is on the fra c tio n in order to give fu r th e r in fo rm atio n concerning the e ffe c ts o f xenobiotics on p ro te in synthesis in hepatocytes. I t was r e a lis e d , however, th a t th is was te c h n ic a lly very d i f f i c u l t as the y ie ld s were f a r too small to enable fu r th e r fr a c tio n a tio n in to samples con taining s m alle r m olecular w eight ranges o f p ro te in s and d is tin c tio n between the separate p ro te in bands on the g e ls .
Some o f
the techniques attempted are described below.
Is o la te d hepatocytes were suspended in 1.15% KC1 pH 7 .4 and homogenisation was attempted using the fo llo w in g techniques:
P o tte r-E lv e h je m , a p o ly tro n ,
a Dounce homogeniser, sonic bath and a sonic probe.
Microscopy was used
i n i t i a l l y to determ ine whether or not the c e lls were d is ru p te d . homogenetes were then ce n trifu g e d a t 20,000g f o r 20 m inutes.
The
I f i t was
f e l t th a t the homogenisation had been successful the supernatant was fu r th e r c e n trifu g e d a t 1 0 0 , 0 0 0 g f o r one hour in an attem pt to obtain, a microsomal p e lle t which could be resuspended in a su cro se/tris/E D T A b u ffe r . The p ro te in con centration o f the microsomes were assayed by the method o f Lowry e t al_ (1 9 5 1 ).
The v i a b i l i t y o f some microsomal preparations was assessed by measuring the a c t i v it y o f
NADH cytochrome C reductase o r ethoxycoumarin 0 -d e e th y la s e
a c tiv ity .
Sodium dodecyl su lphate-polyacrylam ide gel e le c tro p h o re s is was used f o r a couple o f the microsomal preparations in an attem pt to separate the p ro te in s .
3 .1 1 .1
Sodium dodecyl su lphate-polyacrylam ide gel e le c tro p h o re s is
Sodium dodecyl sulphate (SDS)-polyacrylam ide gel e le c tro p h o re s is was used as a means o f sep arating microsomal p ro tein s fo llo w in g the s o lu b ilis a tio n o f microsomes in b u ffe r con taining SDS.
The method used
was th a t o f Laemmli (1970) which u t il is e d a discontinuous b u ffe r system.
Reagents: Gel stock:
30% ( V/^) Acrylamide con taining 0.8% ( w/v) NN1methylene bisacrylam ide (b is )
B u ffe r 1
: 1.5M T ris-H C l (pH 8 . 8 ) containing 0.4% ( w/v) SDS
B u ffe r 2
: 500mM T ris-H C l (pH 6 . 8 ) containing 0.4% (w>^) SDS
B u ffe r 3
: 25mM T ris-H C l (pH 8 .3 ) con taining 0.1% (W y^) SDS and 192mM g lycin e
B u ffe r 4
: 62.5mM T ris-H C l (pH 6 . 8 ) con taining 2.3% (w/v ) SDS, 15% ( v/v) g ly c e r o l, 5% ( v/ )
mercaptoethanol and 0.001% ( W A ) bromophenol
blue P ro p an -2-o l/a c e t ic a c id /w a te r (2 5 :1 0 :6 5 , by v o l) containing 0.05% (w/y ) Coomassie Blue G-250 P ro p an -2-o l/a c e t ic a c id /w a te r (1 0 :1 0 :8 0 , by v o l) .
Procedure: An a ir-c o o le d v e r t ic a l slab gel apparatus was used f o r these stu d ies and elec tro p h o re s is was always c a rrie d out in a cold-room a t 4°C.
A glass
cuvette was used to contain the g e l, which had f in a l dimensions o f 120 x 80mm x 1.5mm.
The glass cuvette was assembled by clamping perspex spacers between the glass p la te s to form the sides and bottom o f the c u v e tte .
The gaps
between the perspex and glass were sealed by running a warm Agar s o lu tio n (2% W /^) along the edges.
The glass cuvette was then clamped in a v e r t ic a l
p o s itio n .
The lower (s e p a ra tin g ) gel was prepared by mixing B u ffe r 1 (1 0 m l), gel stock (1 3 .4 m l), w ater (1 6 .6 m l), ammonium persulphate s o lu tio n ( fr e s h ly prepared, 10% ( W /v )» 240 (TEMED, 2 0 ^ 1 ) .
jj^I
) and N ,N ,N ! ,N '-te tra m e th y le th y le n e d ia m in e
The so lu tio n s were mixed thoroughly and p ip e tte d in to
the glass cuvette to a h eight o f about 70mm, using a Pasteur p ip e t te . A la y e r o f w ater (about 5mm) was c a r e fu lly introduced above the gel m ixture to ensure a com pletely f l a t in te rfa c e between the w ate r and the acrylam ide gel a f t e r i t had polym erised.
When p o lym erisation was
complete-, the w ater la y e r was removed and the upper (s ta c k in g ) g e l, co n sistin g o f a m ixture o f B u ffe r 2 (2 .5 m l) , gel stock ( 1 .0 m l) , w ater 6 .5 m l), ammonium persulphate s o lu tio n (10% (w/v ) , 6 ( ju.1) and TEMED (2 0 ju .l)
was added to the cuvette above the sep arating g e l.
A perspex comb
was introduced in to the top o f the cuvette to form the sample w e lls w ith in the stacking gel a f t e r polym erisation had occurred.
When the
stacking gel had s e t, the comb was c a r e fu lly removed and a small volume o f B u ffe r 3 introduced in to the top o f the cuvette to ensure sep aratio n o f the sample w e lls .
The perspex spacer was removed from the bottom o f
the cu vette before clamping the cuvette in to the e le c tro p h o re s is apparatus.
The upper and lower b u ffe r re s e rv o irs were f i l l e d to s u ita b le
le v e ls w ith e le c tro d e b u ffe r (B u ffe r 3 ) .
Microsomal suspensions were
d ilu te d w ith sample b u ffe r (B u ffe r 4) to a f in a l con centration o f 3mg microsomal p ro te in /m l.
The samples were placed in a b o ilin g w ater bath
(2 -3 min) p r io r to a p p lic a tio n to the stacking g e l. microsomal p ro te in app lied to the gel was 5 /x g .
The amount o f
E le c tro
phoresis was c a rrie d out using a constant c u rre n t o f 20mA in the stacking gel and th is was increased to 40mA when the bromophenol blue tra c k in g dye entered the sep aratin g g e l.
The e le c tro p h o re s is was continued u n t il the
tra c k in g dye was w ith in 5mm o f the end o f the g e l.
A fte r the e le c tro p h o re s is was com plete, the cuvette was removed from the apparatus and the two halves separated.
The stacking gel was cut .away
from the sep arating gel and the l a t t e r immersed o vern ig h t in a s o lu tio n which stained f o r p ro te in (p ro p a n -2 -o l/a c e t ic a c id /w a te r, 2 5 :1 0 :6 5 , by v o l, con taining 0.05% ( w/y) Coomassie Blue G -250).
D estaining o f the background
gel was achieved by immersion o f the gel in a m ixture o f propan- 2 - o l / a c e t i c a c id /w a te r (1 0 :1 0 :8 0 , by v o l) f o r a s u ita b le length o f tim e .
Gels were
stored in 3% ( w/v ) g lycero l a t 4°C.
3 .1 1 .2
NADH - cytochrome C reductase assay
NADH - cytochrome C reductase a c t i v it y was measured by m onitoring the ra te o f reduction o f exogenous cytochrome C a t 550nm (E= 2 1 ,000M"^cnf^) in a dual beam spectrophotometer (Cary)(Prough e t al_, 1976).
The re a c tio n
m ixture consisted o f 4 0 ^M cytochrome C, 200 jjlM NADH, IOOjjlM EDTA in 0.1M potassium phosphate b u ffe r pH 7 .7 contained in a 1ml cuvette (1cm l i g h t b a th ).
KCN (500^*.M) was added to the incubation to i n h ib it the
re o x id a tio n o f cytochrome C.
The re a c tio n tem perature was 25°C.
Reaction m ixture: I n i t i a l Cone.
F inal Volume in M ixture
NADH
2mM
0.1ml 0.1ml
KCN
5mM
0 . 1ml
EDTA
ImM
0 . 1 ml
0.1M
0.5ml
400
Cytochrome C
b u ffe r pH 7 .7
2 x 1 0 ^ c e lls /m l
c e lls /s u p e rn a ta n t
0 . 1ml
The a d d itio n o f
1 0 ^ 1 o f ImM Rotenone was used to determine i t s e f f e c t
on the a c t i v it y
o f NADH cytochrome reductase.
P ro te in content
o f the supernatant was assayed by the method o f Lowry
et
3 .1 1 .3
(1 9 5 1 ).
Assay o f 7-ethoxycoumarin 0 -d e e th y la tio n in microsomes
7-Ethoxycoumarin 0-d eeth ylase a c t i v it y was measure by determ ining the amount o f 7-hydroxycoumarin produced from exogenously added 7-ethoxycoumarin during a 10 minute incubation w ith microsomes.
The method was adapted from
th a t o f A it io (1 9 7 8 ).
Reaction m ixture: B u ffe r (0.1M T ris /H C l pH 7 .4 ) con taining 100^M 7-EC
0.35ml
G6 PD (lO u /m l)
0.35ml
Regenerating system: NADP
10yjumol/ml
J
G6 P
50^m ol/m l
I
MgCl 2
50jjimol/ml
j
0.05ml
Incubate fo r 2 minutes
Microsome (lOmg p ro te in /m l) in tris /H C l b u ffe r
Incubate f o r 10 minutes
0.05ml
The re a c tio n was stopped by adding 0.5ml o f 0 .3 1 M TCA.
A fte r
thorough mixing the pH o f the m ixture was brought to about pH 10 by adding 4ml o f 1.6M NaOH-glycine b u ffe r (pH 1 0 .3 ) .
The p ro te in
p r e c ip ita te was removed by c e n trifu g a tio n and the amount o f 7-hydroxycoumarin measured f lu o r im e t r ic a lly ( \
ca
370, X
S u ita b le blanks and standards were run sim ultaneously.
em
4 5 0 ).
3.12
PRIMARY MONOLAYER CULTURES OF ADULT RAT HEPATOCYTES
Hepatocytes were is o la te d from a d u lt r a t l i v e r according to the method p revio u s ly described w ith the fo llo w in g m o d ific a tio n s .
A ll the
procedures were c a rrie d out a s e p tic a lly in a lamina a i r flow cab in e t using s t e r i l e glassw are, so lu tio n s and media.
S t e r i le PBS'A 1 was
supplemented w ith gentam icin (7 6 ju j/m 1 ) aiid the co llag en ase/hyaluronidase s o lu tio n was f i l t e r s t e r ilis e d p r io r to use.
A fte r is o la tio n the f in a l
c e ll p e l l e t was resuspended in s t e r i l e L ie b o v itz ( L I5) medium supplemented w ith 1 0 % ( v/v ) fo e ta l c a l f serum, 1 0 % ( v/v ) try p to s e phosphate broth and ( V/ , ) p e n ic illin /s tr e p to m y c in (complete medium). A fte r determ ining the v g c e ll y ie ld and v i a b i l i t y the c e lls were d ilu te d to 10 c e lls /m l.
Hepatocyte cu ltu re s were prepared in Falcon fla s k s (sometimes w ith the p la s tic substratum coated, then a i r dryed w ith r a t t a i l
c o lla g e n ), o r
Leighton tubes con taining co llag en -co ated co v erslip s (10.5mm x 22mm). Suspension (2m l) was introduced in to the form er, one ml in to the l a t t e r . The cu ltu re s were incubated a t 37°C fo r 2 hours.
The medium co n tain in g
unattached c e lls was then c a r e fu lly removed and replaced w ith fre s h medium.
About 20 hours l a t e r the medium was again removed and the c e ll
monolayer was washed c a r e fu lly w ith PBS *A 1 then fresh medium was introd uced.
A fte r a fu r th e r incubation o f about 24 hours, during
which the c u ltu re s were able to recover from the trauma o f is o la tio n y e t contained only very small numbers o f f ib r o b la s ts , the c u ltu re s were ready to use to study the e ffe c ts o f xenobiotics on a number o f d i f f e r e n t param eters.
Xenobiotics were added to the cu ltu re s as a s o lu tio n , o r a
sonicated m ixture in complete medium, o r dissolved in DMF a t a con centration which enabled less than 10 jxl to be added per ml o f incubation medium.
3 .1 2 .1
Hepatocyte a rc h ite c tu re
Changes in hepatocyte a rc h ite c tu re were in v e s tig a te d a f t e r s ta in in g cu ltu re s w ith Giemsa, o r haematoxylin and eosin s ta in s .
(a )
Giemsa s ta in in g o f cu ltu red hepatocytes:
Reagents: 1.
PBS'A1
2.
Absolute methanol
3.
Giemsa s ta in - Difco 'Bacto 71'
4.
5.
B u ffers
(a )
M/lg Na2 HP04 -2H20
(b )
M/15 Na H2 P04 -2H20
Working b u f f e r : -
s o lu tio n
(a )
50ml
(b)
50ml
D is t il l e d w ater to 900ml.
Method: Cultures were mixed w ith PBS'A' then flooded w ith methanol and l e f t f o r one m inute.
They were drained and replaced w ith fresh methanol which
was l e f t f o r a fu r th e r 10 m inutes.
The methanol was discarded and the
c u ltu re flooded w ith the prepared Giemsa s ta in . was allowed
The u n d ilu te d s ta in
to remain in contact w ith the c e lls f o r one minute then
working b u ffe r
was added to d ilu t e the s ta in 1 in 5 and the p re p a ra tio n
l e f t fo r a fu r th e r two m inutes, follow ed by rap id rin s in g in working b u ffe r u n t il fre e o f excess s ta in .
A fte r a i r drying glass cover s lip s
were e ith e r immersed in xylene and mounted in DPX, o r f o r exam ination by microscopy and photography o f the c u ltu re s in p la s t ic fla s k s the working b u ffe r was added or the fla s k s simply stored a f t e r a i r d ry in g .
(b)
Haematoxylin and eosin s ta in in g o f c u ltu re d hepatocytes:
Cultures on cover s lip s from Leighton tubes were used in the fo llo w in g procedure:
1. .2 .
Rinse w ith PBS'A'. F ix in A c etic Alcohol Formalin f o r 5m inutes.
3.
Rinse w ith tap w a te r.
4.
S ta in w ith 50% v/v aqueous Haematoxylin (H a r r is ) f o r 2 m inutes.
5.
Rinse w ith tap w a te r.
6.
Immerse in 0.5% HC1 f o r 15 seconds.
7.
Immerse in 0.05% lith iu m carbonate f o r
8.
Rinse w ith 70% a lc o h o l.
9.
S ta in w ith Eosin f o r one m inute.
10.
Rinse tw ice in absolute a lc o h o l.
11.
C lear w ith xylene.
12.
Mount in DPX.
5 m inutes.
3 .1 2 .2
The e f f e c t o f xenobiotics on c e ll attachment
This was determined by counting the n u c lei o f the attached c e lls .
Method: The incubation was removed from the c u ltu re fla s k and replaced w ith 2ml o f 0.033% c ry s ta l v io le t in 0.1M c i t r i c a c id . a t 37°C f o r one hour.
The fla s k s were incubated
The c e lls were removed from the c u ltu re surface
by a g ita tin g them w ith a Pasteur p ip e tte . n uclei suspension was then
An a liq u o t o f the w e ll mixed
counted to determine th e number o f n uclei
using an improved Neubauer counting chamber.
3 .1 2 .3
Assay f o r glutam ic o x a lo a c e tic transaminase
Glutamic o x a lo a c e tic transaminase (GOT) was assayed Test Combination K it (C a t. No. 124478).
using aBoehringer
A fte r the ap p ro p riate treatm ent
the medium was removed from the c u ltu re fla s k s .
The monolayer c u ltu re
was washed tw ice w ith PBS'A1, the washings being pooled w ith the medium. Water (1m l) was then added to the c u ltu re s and the c e lls were detached from the c u ltu re surface using a "rubber policem an".
The c e ll suspension
was tra n s fe rre d to LP3 tubes, t o ta l ly s is o f the c e lls was ensured by fre e z in g and thawing th ree tim es. medium and the c e lls .
GOT a c t i v it y was measured .in both the
The amount o f GOT leaked from the c e lls was
c a lc u la te d from the t o ta l present in both the c e lls and the medium.
3 .1 2 .4
Enzyme cytochem istry
The fo llo w in g enzymes were monitored in cu ltu re d r a t hepatocytes using cytochemical methods based on those o f Chayen e t
(a )
(1 9 7 3 ).
Glucose- 6 -phosphate dehydrogenase:
Reaction medium:
0.05M g ly c y ly c in e b u ffe r pH 7.9 0.3%
neotetrazoliurn c h lo rid e
5.0%
p o lyp e p tid e.
-
Just before use 2.5mg/ml NADP (disodium s a lt ) ,, 1.5mg/ml G6 P (disodium s a lt ) and 0 . 2 mg/ml phenazine methosulphate were added, the f in a l s o lu tio n was bubbled w ith n itro g en and one ml introduced in to Leighton tubes.
(b)
NADPH^ - diaphorase:
To the re a c tio n medium (a ) was added 5mg/ml NADPf^, th is was then bubbled w ith n itrog en and one ml introduced in to Leighton tubes.
(c )
Succinate dehydrogenase:
Sodium succinate hexahydrate ( 6 8 mg) was added to 5ml o f the re a c tio n medium and 0.2mg/ml phenazine methosulphate.
The s o lu tio n was bubbled
w ith n itrog en and added to Leighton tubes as b efo re.
The re a c tio n time f o r a l l th ree enzymes was 45 m inutes.
The cover
s lip s were then removed from the Leighton tubes, a ir -d r y e d , and mounted in g ly c e rin j e l l y .
The colour reactio n s were q u a n tifie d using a
densitom eter (V ic k e rs , M85, Scanning and In te g ra tin g M ic ro d e n s ito m e te r).
These cytochemical techniques had been used p revio u s ly in our la b o ra to ry . I t was th e re fo re considered th a t they had been s u ita b ly v a lid a te d , fu r th e r supported by the work o f Chayen e t al_ (1 9 7 3 ).
P r io r to q u a n tify in g
the colour re ac tio n s f o r any experim ent the m icrodensitom eter was adjusted to give the optim al wavelength and spot s iz e . .The machine s e ttin g s were then unchanged f o r the assessment o f a l l s lid e s o f a s p e c ifie d colour re a c tio n w ith in each experim ent. terms o f a r b itr a r y machine u n its .
The re s u lts are expressed in
3 .1 2 .5
P r o t e in s y n th e s is i n p r im a ry c u l t u r e s o f a d u l t r a t h e p a to c y te s
The method used was e s s e n tia lly the same as th a t alread y described fo r hepatocyte suspensions.
Complete c u ltu re medium con taining lOOnCi [ ^ c ] -
Leucine per ml to g eth er w ith the required amount o f te s t compound was added to the cu ltu re s and incubated a t 37°C fo r one hour.
The medium
was then removed and the c e lls washed w ith PBS'A' to remove any re sid u al lab el not incorporated in to the c e lls .
PBS'A' ( lm l) was then added to
the c u ltu re s and the c e lls were detached from the c u ltu re surface w ith the aid o f a "rubber policem an".
The c e ll suspension was then removed
from the Falcon fla s k and placed in LP3 tubes and tre a te d as p re vio u s ly described ( 3 . 4 . 1 ) .
3 .1 2 .6
7-Ethoxycoumarin 0-d eethylase a c t i v it y in cu ltu re s
The method used was id e n tic a l to th a t described in 3 .3 .4 f o r hepatocyte suspensions except th a t 1 0 /x l o f 140mM 7-ethoxycoumarin was added to 20ml o f c u ltu re medium.
The cu ltu re s were incubated w ith a 4ml volume o f the
substrate/medium s o lu tio n .
A fte r deconjugation the samples were e x tra c te d
w ith 5ml hexane p r io r to the e th e r alcohol e x tra c tio n to remove any unreacted 7-ethoxycoum arin.
Samples could be stored f o r up to one week
a t -20°C w ith o u t Toss o f 7-hydroxycoumarin a c t i v i t y .
C H A P T E R
RESULTS
4
RESULTS
4.1
INTRODUCTION
Hepatocytes were is o la te d by the method o f Fry
et_ al_ (1976) from a d u lt
male W is tar Albino ra ts or Golden S yrian hamsters.
I t was assumed th a t
i f g re a te r than 80% o f the hepatocytes excluded trypan blue the p rep a ra tio n was v ia b le f o r the purposes o f the work described in th is th e s is .
This assumption was based on the work c a rrie d out by Fry e t al
(1976) w ith in the same la b o ra to ry .
These workers demonstrated
th a t is o la te d hepatocytes m etabolise fo re ig n compounds w ith o u t the a d d itio n o f exogenous cofactors,w h ich to g eth er w ith the a b i l i t y o f the c e lls to exclude trypan b lu e ,is in d ic a tiv e o f a fu n c tio n a l in t a c t plasma membrane (F ry e t al_, 1976;
Wiebkin
e t al_, 1976).
They also demonstrated
th a t endogenous glycogenolysis was s tim u lated by glucagen pro vid in g evidence f o r the existence o f fu n c tio n in g hormonal re ce p to r s ite s w ith in the is o la te d hepatocytes (F ry e t al_, 1976).
Figure 1 is a photograph o f a ty p ic a l sample taken from a p re p a ra tio n o f is o la te d .h e p a to c y te s .
Trypan blue was added and the sample viewed under
a l ig h t microscope a t x200 m a g n ific a tio n .
The c e lls coloured blue are
the non -viab le c e lls which have taken up the dye.
4 .2
VIABILITY ASSESSMENT
The i n i t i a l s e rie s o f experiments to be reported in v e s tig a te d the e f f e c t o f exogenously added chemicals on the v i a b i l i t y o f is o la te d hepatocytes. A number o f d if f e r e n t c r i t e r i a o f v i a b i l i t y were compared.
4 .2 .1
Trypan blue exclusion
The f i r s t c r ite r io n in v e s tig a te d was the a b i l i t y o f hepatocytes to exclude the dye trypan b lu e.
Tables la and lb give the re s u lts from two experim ents
which are re p re s e n ta tiv e o f a se rie s c a rrie d out to determ ine the e f f e c t o f a range o f concentrations o f paracetamol on the v i a b i l i t y o f is o la te d r a t hepatocytes.
Table la demonstrates th a t th ere was very l i t t l e
change in
v i a b i l i t y w ith time except a t the 40mM le v e l a t w h ic h ,a fte r 90 m inutes, the v i a b i l i t y f e l l to 46%.
Table lb shows the re s u lts o f a very s im ila r
experim ent where th ere were no dram atic changes in the v i a b i l i t y o f the hepatocytes as a r e s u lt o f treatm ent w ith paracetam ol.
V i a b i l i t y was
reduced only s l ig h t l y to 82% a f t e r 90 minutes treatm ent w ith 40mM paracetam ol.
In view o f the v a r i a b i l i t y experienced w ith the e f f e c t o f
paracetamol on is o la te d r a t hepatocytes, supported by e a r l i e r in v e s tig a tio n s (Gwynn, 1 9 7 6 ), the species was changed.
The hamster has been shown to
be more su s cep tib le to the e ffe c ts o f paracetamol in vivo (Davis e t a l , 1974) and has also been used as an in v it r o model fo r t o x ic it y (Smith and J o llo w , 1977).
The e f f e c t o f paracetamol was then in v e s tig a te d in hamster hepatocytes (F ig u re 2) using a s lig h t l y d if f e r e n t method.
The is o la te d c e lls were
incubated f o r two hours in medium con taining 25mM paracetam ol, the co n tro l was incubated under id e n tic a l conditions but w ith o u t paracetam ol.
A fte r
two hours the c e lls were spun to a p e l l e t and the medium was replaced w ith co n tro l medium w ith o u t paracetam ol.
The v i a b i l i t y o f the c e lls was
assessed by trypan blue exclusion a t two hours when the medium was changed and again a t in te rv a ls f o r another th re e hours.
The paracetamol was
found to be more to x ic to the hamster hepatocytes than i t was in the previous experiments w ith ra t. hepatocytes.
In f u r th e r experiments w ith hamster hepatocytes a range o f concentrations o f paracetamol was used.
I t is in te r e s tin g to note (see Figure 3) th a t
the lower concentrations were c o n s is te n tly more to x ic to the hepatocytes in the e a rly stages o f the tim e course than the higher co n c e n tra tio n s . Although th is phenomenon was reproduced in f iv e s im ila r experiments some doubt was cast as to the v a l i d it y o f the trypan blue exclusion t e s t as a c r ite r io n o f v i a b i l i t y assessment in hamster hepatocytes.
This was due
to the f a c t th a t w ith hamster hepatocytes the n o n -viab le c e lls formed la rg e aggregates during the incubations which meant th a t an accurate d eterm in atio n o f the v i a b i l i t y index was not p o s s ib le . was in v e s tig a te d .
The cause o f these aggregates
The a d d itio n o f DNAase had no e f f e c t on the la rg e
clumps o f c e lls in d ic a tin g th a t DNA was not responsible (Bellemann 1977b).
The a d d itio n o f p rotease,
and i t was,
th e r e fo r e , considered
et a l,
however, helped to disaggregate
the clumps
th a t p ro te in was being secreted
by
the c e l l s , o r being released when a c e ll was damaged, causing the c e lls to-clump to g e th e r.
These c e ll aggregates were f a r more apparent w ith the
hamster hepatocytes, where they were present both a f t e r tre a tm e n t w ith paracetamol
a t a l l concentrations
and in con trol preparations a f t e r twoto
three hours
o f in c u b a tio n , than w ith r a t hepatocytes.
F ig u re 1:
A ty p ic a l suspension o f is o la te d r a t hepatocytes stained w ith trypan blu e.
The dark blue stained c e lls are n o n -v ia b le .
(M a g n ific a tio n x200)
T a b le 1 E ffe c t o f paracetamol on the v i a b i l i t y o f is o la te d r a t hepatocytes using trypan blue exclusion as the c r it e r io n o f assessment.
(a ) Percentage v i a b i l i t y
Time Mins.
Control
O.lmM
5mM
40mM -
0
91
30
86
83
84
80
60
82
84
86
59
90
81
82
87
46
-
-
(b) Percentage v i a b i l i t y
Time Mins.
Control
lOmM
20mM
40mM
0
96
92
•91
93
30
92
87
89
91
60
90
85
90
86
90
88
90
91
82
These two ta b les give the re s u lts o f two experiments (ty p ic a l o f 6 ) demonstrating the e ffe c ts o f a range o f concentrations o f paracetam ol.
Figure 2 :
E ffe c t o f paracetamol on the v i a b i l i t y o f is o la te d hamster hepatocytes using trypan blue exclusion as the c r it e r io n o f assessment
100
•r—
'p“ >
c O) o S
ex.
Time (hours)
6
Is o la te d hepatocytes (10 /m l) were preincubated in the presence,® — • or absence,▼— ▼ , o f 25mM paracetamol f o r two hours, the medium was then changed f o r medium w ithout paracetamol (in d ic a te d by a rro w ).
Each
point is the mean o f three values and the experim ent is ty p ic a l o f two c a rrie d o u t.
Figure 3 :
E ffe c t o f a range o f concentrations o f paracetamol on c e ll v i a b i l i t y o f hamster hepatocytes assessed by trypan blue exclusion
100
60 .
2 Time (hours)
c Is o la te d hepatocytes (10 /m l) were preincubated w ith paracetamol f o r
two hours; *
v , c o n tro l; ▼— ▼ ,lmM; ■-------■ ,10mM; o— o,20mM.
The medium was then changed fo r control medium (in d ic a te d by a rro w ). Each p o in t is the mean o f th ree e s tim a tio n s . ty p ic a l o f f i v e .
This experim ent is one
3
4 .2 .2
Lactate dehydrogenase release
The second c r it e r io n in v e s tig a te d f o r v i a b i l i t y assessment was leakage o f the cytoplasm ic enzyme la c ta te dehydrogenase from is o la te d hepatocytes. Lysis o f the c e lls w ith T rito n X I00 enabled an e stim a tio n o f the to ta l in t r a c e ll u l a r la c ta te dehydrogenase.
The percentage o f the t o ta l c e l l u la r
la c ta te dehydrogenase released in to the e x t r a c e llu la r medium was used as the index o f v i a b i l i t y .
There was always some la c ta te dehydrogenase
d e te c ta b le in the e x t r a c e llu la r medium which was due to e it h e r the presence o f a small percentage o f n o n -v ia b le c e lls w ith in the suspension, or to the fo e ta l c a lf serum used in the medium which may have contained a small amount o f the enzyme.
Table 2 shows the e f f e c t o f a range o f concentrations o f paracetamol and a c e ta n ilid e on the release o f la c ta te dehydrogenase a f t e r one hour’ s in c u b a tio n .
There was a s lig h t increase in the amount o f la c ta te
dehydrogenase d e te c ta b le in the e x t r a c e llu la r medium a t concentrations g re a te r than lOmM but the re s u lts were not very marked in d ic a tin g th a t paracetamol and a c e ta n ilid e were not very to x ic to the plasma membranes, o f is o la te d r a t hepatocytes w ith in the conditions o f the experim ent.
Figure 4 dem onstrates, however, th a t s a fr o le had a much more marked e f f e c t on c e ll v i a b i l i t y .
At a con centration o f 0.5mM th ere was l i t t l e
e f f e c t , w ith l.OmM causing a s lig h t increase in the e x t r a c e llu la r la c ta te dehydrogenase.
At higher concentrations o f 5 and lOmM s a fr o le
caused a la rg e release o f la c ta te dehydrogenase dem onstrating th a t s a fro le is r e l a t i v e l y to x ic to the r a t hepatocytes a t h ig h er con centrations w ith in a very sho rt tim e o f in c u b a tio n .
Table 2 ;
E ffe c t o f paracetamol and a c e ta n ilid e on v i a b i l i t y o f is o la te d r a t hepatocytes using la c ta te dehydrogenase release as the c r it e r io n o f assessment
LDH released
{% o f to ta l c e llu la r LDH)
Concentration (mM)
Paracetamol
A c e ta n ilid e
0
23.5
22
2 .5
2 5 .0
21
5 .0
23.5
22
7.5
-
19
1 0 .0
25.0
29
15.0
-
23
2 0 .0
28.5
28
4 0 .0 '
32.0
-
c Hepatocytes a t 10 c e lls /m l were incubated f o r one hour w ith paracetamol o r a c e ta n ilid e a t various co n cen tratio n s.
Samples
o f c e lls were taken a t zero tim e to assess t o ta l c e llu la r LDH. (One experim ent ty p ic a l o f tw o .)
Figure 4 :
E ffe c t o f s a fro le on the v i a b i l i t y o f is o la te d r a t hepatocytes using la c ta te dehydrogenase release as the c r it e r io n o f assessment
100
80
o 4->
60
4-
O
*o a> in fO a) p— a>
40
O—
S-
zxz
a
20
0 10
20
30
40
50
Time (m inutes)
Hepatocytes were incubated a t 10 c e lls /m l as described in 'M ethods'. s*
v , C o n tro l;
■------- ■ , 0.5mM;
o------ o , l.OmM;*
▼— w , 5mM; □-------□ , lOmMs a fr o le . Each sample is the mean o f two e s tim a tio n s . experim ent ty p ic a l o f
two.
The re s u lts are from one
60
4 .2 .3
Comparison o f tr y p a n b lu e e x c lu s io n and l a c t a t e dehydrogenase
release Figures 5 t o 8 give the re s u lts o f a number o f experiments where the e ffe c ts on v i a b i l i t y in terms o f trypan blue exclusion and la c ta te dehydrogenase release were d ir e c t ly compared by measuring both parameters oh the same incubation sample.
Figure 5 demonstrates t h a t paracetam ol,
on th is occasion, e x h ib ite d a d ir e c t dose response e f f e c t in reducing the v i a b i l i t y o f is o la te d r a t hepatocytes.
The p r o f ile obtained f o r the
e f f e c t o f d if f e r e n t concentrations o f paracetamol on v i a b i l i t y measured in terms o f la c ta te dehydrogenase released from the c e lls was very s im ila r to th a t fo r trypan blue exclu sio n .
Figure 6 gives the re s u lts o f a comparison between the two c r i t e r i a o f plasma membrane in t e g r it y f o r hamster hepatocytes tre a te d w ith paracetam ol. The ImM paracetamol -dose was again more to x ic than th a t a t 10 o r 20mM in terms o f the trypan blue exclusion t e s t and th is was endorsed by the la c ta te dehydrogenase release measured on the same samples g iv in g support to the re s u lts alread y discussed from Figure 3.
Phenobarbitone was demonstrated to reduce the v i a b i l i t y o f is o la te d r a t hepatocytes by both c r i t e r i a a t a 20mM concentration (F ig u re 7 ) .
S a fro le (F ig u re 8 ) was also found to be very to x ic a t lOmM, by both c r i t e r i a supporting the re s u lts from Figure 4 ..
In th is experim ent the
v i a b i l i t y was zero according to the trypan blue exclusion method but only 75% according to la c ta te dehydrogenase released in to the e x t r a c e llu la r medium. This is probably due to some o f the la c t a te de hydrogenase remaining w ith in the n o n -viab le c e lls which were removed from the medium by c e n trifu g a tio n p r io r to assaying the la c ta te dehydrogenase.
These re s u lts suggest th a t th ere was l i t t l e c r i t e r i a o f v i a b i l i t y assessment.
d iffe re n c e between these two
Both parameters provided a measure o f
ir r e v e r s ib le plasma membrane damage.
For a ra p id assessment o f v i a b i l i t y
trypan blue exclusion was the method p re fe rre d .
F ig u r e 5 :
A com parison o f th e e f f e c t o f p a ra c e ta m o l on tr y p a n b lu e e x c lu s io n and th e r e l e a s e o f l a c t a t e dehydrogenase from i s o l a t e d r a t h e p a to c y te s
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Time (m inutes)
Experimental conditions were as described in 'M ethods'. [ 14C] -Leucine in co rp o ratio n was estim ated in t r i p l i c a t e f o r each time p o in t. O
O , C o n tro l; • ------ •
, paracetamol tre a te d .
30
Figure 28: ----------------
Time course o f the e ffe c ts o f paracetam ol, phenobarbitone T 14 1 and s a fro le on L CJ -Leucine in c o rp o ra tio n in to p ro te in s o f is o la te d r a t hepatocytes
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Time (m inutes)
Experimental conditions were as described in 'Methods1.
The
concentration o f chemicals chosen was th a t which gave approxim ately a 50% in h ib it io n a f t e r one hour. paracetam ol; •
, C o n tro l;
• , 5mM phenobarbitone;
Results are the mean o f d u p lic a te samples.
o-
lOmM
ImM s a fr o le .
60
4 .4
DETERMINATION OF THE SPEC IFIC ITY OF THE ACTION OF PARACETAMOL
ON PROTEIN SYNTHESIS In order to determine whether paracetamol had an e f f e c t on s p e c ific p ro tein s ra th e r than a general in h ib ito r y e f f e c t on p ro te in synthesis attempts were made to is o la te a microsomal fr a c tio n from is o la te d r a t hepatocytes w ith an aim to c arryin g out SDS polyacrylam ide gel e le c t r o pheresis.
4 .4 .1
The p rep a ra tio n o f microsomes from is o la te d r a t hepatocytes
A number o f d if f e r e n t techniques were in v e s tig a te d in c lu d in g homogenisation w ith a P o tte r-E lv e h je m , a Dounce homogeniser, a P o ly tro n , and two types o f so n ica to r - a sonic
probe and a sonic bath.
I t was found w ith both
the P o tter-E lveh jem and the Dounce homogeniser th a t even w ith a la rg e number o f strokes w ith the p e s tle the hepatocytes s t i l l in ta c t when examined m ic ro s c o p ic a lly .
remained la r g e ly
The Polytron was found to be more
successful in d is ru p tin g the hepatocytes th e re fo re a number o f experiments were conducted to determine the id e a l tim e f o r homogenisation.
A fte r the exposure o f each hepatocyte suspension to the P o lytron the a c t i v it y o f NADH cytochrome C reductase was measured in the homogenates. Table 3 gives the in d iv id u a l values f o r the a c t i v it i e s obtained from two experim ents.
In the f i r s t experim ent a c t i v it i e s were measured a f t e r 10,
30 and 60 second exposures in the presence and absence o f rotenone w ith
c
the re s u lts expressed in nm ole/min/10 c e lls .
In the second experim ent
a c t i v it i e s were also measured a f t e r a 5 second exposure, a c t i v i t i e s were expressed in nmole/min/mg p ro te in .
The to ta l enzyme a c t i v i t y a v a ila b le
w ith in the c e lls was obtained by tr e a tin g the c e lls w ith T rito n X I00 which t o t a l l y d isru p ts the c e lls and the in t r a c e ll u l a r o rg a n e lle s .
In
the f i r s t experim ent a 10 second exposure released the maximal a c t i v i t y from the c e lls -w ith g re a te r exposure times causing a decrease in a c t i v i t y . In the second experim ent maximal a c t i v i t y was released w ith in 5 seconds w ith a marked reduction in a c t i v it y by 10 seconds.
The decrease in
a c t i v it y was probably due to the severe shearing forces and heat produced by the P olytron causing den atu ratio n o f the enzyme.
I t was
apparent from the re s u lts th a t a very sh o rt period o f homogenisation tim e was required to d is ru p t the c e lls although the e f fe c tiv e tim e was v a r ia b le , as were the re s u lts w ith in each tim e p o in t f o r each separate
0 experim ent.
A la r g e r sample o f hepatocytes con taining 74 x 10 c e lls
in 5ml was homogenised f o r 10 seconds w ith the P o ly tro n .
The homogenate
was then ce n trifu g e d a t 10,000g fo r 20 minutes follow ed by 105,000g f o r 1 hour to is o la te the 105,000g p e l l e t .
At the end o f the second
c e n trifu g a tio n the p e l l e t was b a re ly v is ib le to the naked eye.
The
use o f the P o lytron was, th e r e fo r e , abandoned as u n s a tis fa c to ry .
Homogenisation w ith a sonic probe proved to be more e f fe c tiv e in terms o f d is ru p tin g the c e lls .
The probe th a t was a v a ila b le , however, had
in h e re n t problems due to i t s
la rg e s iz e and the n ec essity f o r a c o n tain er
f o r the suspension o f hepatocytes to have a diam eter o n ly s lig h t ly g re a te r than the probe i t s e l f .
These r e s t r ic tio n s meant th a t only very
small volumes could be sonicated a t one tim e , and there was also a p erce n ta g e.o f m a te ria l lo s t during s o n ic a tio n .
The most e f fe c tiv e method was found to be the sonic b ath.
Samples o f
is o la te d hepatocytes con taining 10 c e lls /m l in 1 0 ml o f b u ffe r were placed in a 10ml conical f la s k .
A range o f so n ica tio n times were t r i e d and 60
seconds.was found to be enough to d is ru p t the m a jo rity o f the c e lls when observed m ic ro s c o p ic a lly .
A 10ml suspension o f r a t hepatocytes was then
homogenised f o r 60 seconds follow ed by c e n trifu g a tio n a t 1 0 , 0 0 0 g and 105,000g to produce a small p e l l e t which was resuspended in tris/E T D A b u ffe r to give a concentration o f lOmg microsomal p ro te in per m l.
The
a c t i v it y o f 7-ethoxycoumarin 0-d eethylase was measured in th is microsomal p e lle t and found to be 10.25nmoles/10 min/0.05mg p ro te in .
This a c t i v i t y
was s im ila r to th a t found in a control microsomal p rep a ra tio n (12.05nm oles/ 10 min/0.05mg p ro te in ) assayed a t the same tim e.
The con trol microsomes
had been prepared from whole r a t l i v e r by someone experienced in the standard method.
Samples o f the microsomal f r a c t io n , derived from the c e lls tre a te d in the sonic b a th , were app lied to an SDS polyacrylam ide gel f o r e le c t r o phoresis to separate the proteins in to bands according to t h e i r re s p e c tiv e m olecular w eights.
Even though a small amount o f p ro te in (5 g per
a p p lic a tio n ) was app lied to the gel the r e s u ltin g sep aration was complex and d i f f i c u l t to in te r p r e t.
The fr a c tio n was not a pure microsomal
p e l l e t , and probably contained fragmented membranes derived from o th e r org an elles due to the n o n -s e le c tiv ity o f the s o n ic a tio n .
Hence f u r th e r
sep aration o f the p e l l e t would have been necessary to gain any more in fo rm a tio n .
T h is , however, would have proved d i f f i c u l t as the o r ig in a l
p e lle t obtained was so sm all.
An a lte r n a tiv e technique was considered instead o f the SDS polyacrylam ide gel electro p h o res is,w h ic h although less s p e c ific would d is tin g u is h between i n t r a c e ll u l a r p ro tein s produced f o r export and those produced f o r o th e r purposes to be u t ilis e d i n t r a c e ll u l a r ly .
This technique could p o t e n t ia lly
provide an answer to the question as to whether the e f f e c t o f paracetamol was s p e c ific f o r e ith e r o f the two major classes o f p ro te in o r p u re ly a n o n -s p e c ific a c tio n .
This a lte r n a tiv e technique involved conjugating a n t i - r a t serum to cyanogen bromide a c tiv a te d Sepharose.
The Sepharose conjugate was
incubated w ith samples derived from is o la te d hepatocytes which had been la b e lle d w ith [ ^ c ] -L eu cin e, w ith o r w ith o u t p r io r treatm ent w ith paracetam ol.
These samples were also incubated w ith a c tiv a te d sepharose
conjugated w ith a n t i- r a b b it serum which acted as a con trol by q u a n tify in g the amount o f n o n -s p e c ific b in d in g .
Table 4 gives the re s u lts obtained from th is experim ent.
Lysis o f the
washed c e ll samples w ith lu b ro l a f t e r incubation w ith [ ^ c ] released a l l the c e l l u la r p ro te in .
-Leucine
S c in t i ll a t i o n counting o f the
Sepharose beads a f t e r overn ig h t incubation w ith the c e ll samples demonstrated th a t paracetamol in h ib ite d the amount o f la b e lle d p r o te in , and hence p ro te in s y n th e s is , which was a v a ila b le to bind to the a n t i - r a t
c
serum conjugate from 412.4 to 39.15dpm/10 c e lls . export pro tein s present w ith in the c e lls .
This represents the
The la b e lle d p ro te in s in the
incubation not bound to the a n t i - r a t serum conjugate rep res en t the non export p ro tein s which were also in h ib ite d from 387.5 in the c o n tro l sample
c
to 21.25dpm/10 c e lls in the paracetamol tre a te d samples.
Hence i t would
appear th a t paracetamol in h ib it s the synthesis o f both groups o f p ro te in s to a s im ila r e x te n t.
A fte r the two hour incubation o f is o la te d hepatocytes w ith o r w ith o u t paracetam ol, the incubation medium was also re ta in e d .
The p ro te in present
in the medium, which represents the p ro te in exported during the two hour incubation was p re c ip ita te d from the medium and s o lu b ilis e d in NaOH.
These samples were also incubated w ith the a c tiv a te d Sepharose conjugates.
There was a g re a te r amount o f n o n -s p e c ific binding to the
a n ti (r a b b it serum) conjugate f o r these samples.
There was also some
la b e lle d p ro te in which did not bind to the a n ti (ra t-s e ru m ) conjugate which was probably derived from c e lls damaged o r dying during the two hour incubation period.-
Allowing f o r these fa c to rs i t would s t i l l
appear .
th a t is o la te d r a t hepatocytes produced export p ro tein s which they s e c re te d , e ith e r a c tiv e ly or p a s s iv e ly , and th a t the production o f these exp ort p ro tein s was in h ib ite d by paracetam ol.
Table 3 :
E ffe c t o f homogenisation tim e w ith a Polytron on the a c t i v it y o f NADH cytochrome C reductase
NADH cytochrome C reductase
Expt. 1
Homogenisation
Expt. 2
c
time (s e c .)
nmole/min / 1 0 c e lls
Total
nmole/min/mg p ro te in
Rotenone In s e n s itiv e
Total
5
-
-
5
-
-
73.1
10
6 8 .0
4 9 .0
57.2
10
63.0
5 2.0
3 6.2
30
71.0
4 7 .0
52.9
30
44.4
36.9
48.1
60
15.9
15.5
-
60
28.7
20.9
28.5
TX 100
58.0
49 .0
76.5
Treated c e lls
6 0 .0
5 0 .0
77.5
•
51.1
c Hepatocytes (2 x 10 /m l) in su cro se/tris/E D TA b u ffe r pH 7 .4 were exposed to d if f e r e n t times o f homogenisation using a P o ly tro n . NADH cyt.C reductase a c t i v it y was measured in the re s u ltin g suspensions. The re s u lts from two experiments are given here.
Treatment o f the
c e lls w ith TX 100 gave the to ta l enzyme a c t i v it y a v a ila b le w ith in the hepatocytes.
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E x p e rim e n ta l c o n d itio n s were as d e s c rib e d in 'M e th o d s '.
[ 14c ] - U r i d i n e i n c o r p o r a t i o n was e s tim a te d i n t r i p l i c a t e f o r each c o n c e n t r a t i o n .
20
F ig u r e 3 2 :
E f f e c t o f s a f r o l e on th e i n c o r p o r a t i o n o f
[ ^ c ] -U rid in e
i n t o th e r i b o n u c l e i c a c id s o f i s o l a t e d r a t h e p a to c y te s
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E x p e rim e n ta l c o n d it io n s w ere as d e s c r ib e d i n 'M e th o d s '.
[ 14c ] - U r i d i n e in c o r p o r a t i o n was e s tim a te d in t r i p l i c a t e a t each c o n c e n t r a t i o n .
10
4 .6
COVALENT BINDING '
w ith
l 14c j
samples.
Ibated w ith 20 and 40mM paracetamol spiked
-paracetam ol a t the same percentage concentration in a l l Samples o f hepatocytes in d u p lic a te were incubated f o r 5 , 10,
2 0 , 30, 40 and 60 m inutes.
On removal from the in cu b ato r th e c e lls were
spun to a p e l l e t and p e rc h lo ric acid was added to p r e c ip ita te the p ro te in . Repeated washing was c a rrie d out w ith the p e rc h lo ric acid to ensure removal o f a l l the unbound la b e l.
The f in a l p r e c ip ita te was then
s o lu b ilis e d in NaOH and the samples counted in a s c i n t i l l a t i o n co u n ter. The re s u lts (F ig u re 33) in d ic a te th a t the m a jo rity o f the binding occurred w ith in the f i r s t f iv e minutes o f in c u b a tio n .
There also appears to be
a t the dose le v e ls o f paracetamol used a d ir e c t c o r r e la tio n between the con centration o f paracetamol and the amount o f covalent b in d in g .
Figure 33 :
E ffe c t o f paracetamol concentration on the covalent binding o f
[ ^ c ] -paracetam ol to c e l l u la r p e rc h lo ric
a c id -in s o lu b le m a te ria l o f is o la te d r a t hepatocytes
40mM paracetamol
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[ ^ c ] ■ Leucine
i n c o r p o r a t i o n was e s tim a te d i n t r i p l i c a t e f o r each c o n c e n t r a t io n , a r e means t S .D . f o r two e x p e rim e n ts .
Values
Figure 40: — -----------
E ffe c t o f the tim e o f exposure to paracetamol on the H4 1 in c o rp o ratio n o f [ CJ -Leucine in to p ro te in s o f prim ary c u ltu re s o f a d u lt r a t hepatocytes
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4 .1 0 .3
E f f e c t o f c y c lo h e x im id e on th e m e t a b o lic a c t i v i t y o f p r im a ry
cu ltu res o f r a t hepatocytes The maintenance o f p h y sio lo g ical le v e ls o f cytochrome P-450 in c u ltu re s is a major problem associated w ith prim ary maintenance c u ltu re s (see 2 . 6 ) .
Guzelian and Barwick (1979) demonstrated th a t
the marked loss in le v e ls o f cytochrome P-450 occurring during the f i r s t 12 hours o f c u ltu re may be prevented by incubating cu ltu re s w ith in h ib ito r s o f p ro te in syn th esis.
The purpose o f the fo llo w in g
two experiments was to in v e s tig a te whether the a d d itio n o f cyclohexim ide to the incubation medium would m aintain the a c t i v i t y o f the mixed fu n c tio n oxidase system and to v e r if y the fin d in g s o f G uzelian and Barwick.
On the basis o f previous re s u lts (see F ig . 17) a con centration o f 10” ^M cycloheximide was chosen since i t in h ib ite d p ro te in synthesis in suspensions o f is o la te d hepatocytes by about 50%. A fu r th e r co n centration -5 o f 10 M cycloheximide was also employed. The cycloheximide was added to the c u ltu re s on day 0 when the c e lls were f i r s t seeded in to c u ltu re fla s k s and was present a t the same concentrations throughout the th ree day experim ent.
C ell number, in terms o f the number o f nuclei.,
and the a c t i v it y o f 7-ethoxycoumarin 0-d eeth ylase (an in d ic a to r o f the a c t i v i t y o f the microsomal mono-oxygenase system) were monitored d a ily fo r th ree days.
The ra te o f p ro te in sysnthesis was estim ated
a f t e r 24 hours in c u ltu r e .
Two experiments were c a rrie d out w ith the hepatocytes seeded in to Falcon fla s k s coated w ith collagen in the f i r s t but not the second. Some cu ltu re s were stain ed w ith Giemsa s ta in to i l l u s t r a t e the c e l l u la r morphology.
The morphology o f the c u ltu re s from the f i r s t experim ent
is described in the fo llo w in g s e c tio n .
Figures 44-50 are i ll u s t r a t i o n s
o f the c u ltu res from the second experim ent.
C e llu la r morphology: A fte r 24 hours in c u ltu re la rg e numbers o f c e lls were attached to the monolayer in the untreated c u ltu re s .
The c e lls were predominantly
e p it h e lia l in ch a ra cter w ith some re tic u lo e n d o th e lia l c e lls and fib r o b la s ts also p resent.
They appeared la r g e ly as c lu s te rs w ith
occasional s in g le c e lls .
The e p it h e lia l c e lls were spread out w ith
w e ll-d e fin e d morphology and i n t e r c e ll u l a r gaps w ith evidence o f occasional d u c t-lik e s tru c tu re s .
The c e l l u la r cytoplasm was g ra n u la r w ith
occasional vacuoles, the nuclei containing two to fo u r n u c le o li w ith b in u c le a te c e lls predom inating. were s t i l l
A fte r a fu r th e r day in c u ltu re there
la rg e numbers o f c e lls attached w ith the e p it h e lia l morphology
la r g e ly preserved in the centre o f the c e ll sheets but becoming less d is t in c t a t the p e rip h ery.
The plasma membranes were becoming less
d is t in c t due to the spreading o f the cytoplasm , re s u ltin g in less w e ll-d e fin e d i n t e r c e ll u l a r spaces, to g eth er w ith some misshapen n u c le i. F ib ro b la s ts and re tic u lo e n d o th e lia l c e lls were s t i l l apparent w ith the l a t t e r demonstrating phagocytic a c t i v i t y .
By day th ree the m orphological
degeneration was becoming more marked w ith some c e lls showing pseudopodia-like p ro je c tio n s w ith m ito tic fig u re s present in some f ib r o b la s ts .
By day fo u r the fib r o b la s ts were beginning to predominate
in the c u ltu re w ith the e p it h e lia l morphology degenerating fu r th e r w ith la rg e numbers o f vacuoles present in the cytoplasm o f the e p it h e lia l c e lls .
A fte r one day o f c u ltu re in the presence o f 10” ^M cycloheximide the monolayer had s im ila r it ie s to the untreated cu ltu re s although th ere appeared to be less c e lls attached and the c e ll s ize tended to be s m a lle r.
There were a number o f round c e lls w ith a la rg e nucleus
to cytoplasm r a t i o , the e p it h e lia l c e lls tended to contain more vacuoles, and th ere were also some n e c ro tic c e lls apparent.
By the
second day o f c u ltu re the vacuolation was more extensive w ith some c e lls containing la rg e p e rin u c le a r vacuoles.
Some o f the e p it h e lia l
c e lls m aintained t h e ir e p it h e lia l morphology b e tte r than those o f the co n tro ls and th ere were also fewer ir r e g u la r ly shaped n u c le i.
By
the t h ir d day o f c u ltu re about 50% o f the e p it h e lia l c e lls were showing extensive vacuolation w ith the remaining c e lls showing b iz a r r e n u clei and extensive spreading o f the cytoplasm such th a t the e p it h e lia l morphology observed on day one had la r g e ly disappeared.
Cycloheximide a t the higher concentration o f 10 on the c u ltu re s .
-5
M had a severe e f f e c t
Although there were some c e lls attached on day one
o f the c u ltu re they were mostly round, densely stain ed and n e c ro tic
w ith a high nucleus to cytoplasm r a t io .
The surface o f the c u ltu re
fla s k was l i t t e r e d w ith amorphous m a te ria l which was probably c e ll d e b ris .
By the second day o f c u ltu re th ere were even fewer c e lls
attached and a l l appeared n e c ro tic .
F ig u re 4 4 :
P rim a r y m o n o la y e r c u l t u r e o f a d u l t r a t h e p a to c y te s a f t e r 24 hours (d a y 1 ) , s t a in e d w it h Giemsa s t a i n (x 2 0 0 )
Figure 4 5 :
Primary monolayer c u ltu re o f a d u lt r a t hepatocytes a f t e r 48 hours (day 2 ) , stained w ith Giemsa s ta in (x200)
F ig u re 4 6 :
P rim a ry m o nolayer c u l t u r e o f a d u l t r a t h e p a to c y te s a f t e r 72 hours (d a y 3 ) , s t a in e d w it h Giemsa s t a i n (x 2 0 0 )
C i nnv»n
i aw. v- -A r*7/ •.
D»
r i imary monolayer c u ltu re o f a d u lt r a t hepatocytes tre a te d
w ith 10” M cycloheximide a f t e r 24 hours (day 1 ) , stained w ith Giemsa s ta in (x200)
F ig u re 4 8 :
P rim a r y m o nolayer c u l t u r e o f a d u l t r a t h e p a to c y te s t r e a t e d w i t h l C f 7M c y c lo h e x im id e a f t e r 48 hours (d ay 2 ) , s t a in e d w it h Giemsa s t a i n (x 2 0 0 )
Figure 4 9 :
Primary monolayer c u ltu re o f a d u lt r a t hepatocytes tre a te d w ith 10’ 7M cycloheximide a f t e r 72 hours (day 3 ) , stained w ith Giemsa s ta in (x200)
%
F ig u re 5 0 :
P rim a ry m o nolayer c u l t u r e o f a d u l t r a t h e p a to c y te s t r e a t e d w i t h 10’ ^M c y c lo h e x im id e a f t e r 24 hours (d a y 1 ) , s t a i n e d w i t h Giemsa s t a i n (x 2 0 0 )
i
* $
1
% %
«
% *
4
\ *
7-Ethoxycoumarin O -deethylase a c t i v i t y , p ro te in synthesis and c e ll number a f t e r treatm ent w ith cyclohexim ide: The above th ree parameters were also measured a f t e r treatm ent w ith -5 -7 cycloheximide a t 10 M and 10 M. In both experim ents, as p re v io u s ly -5 d escribed, 10 M cycloheximide was very to x ic to the c e lls causing a la rg e reduction in the c e ll numbers attached by day one o f the c u ltu r e .
A marked decrease in the ra te o f p ro te in synthesis was also
ev id e n t being 1.4% and 3.0% o f the co n tro l values fo r the two experiments re s p e c tiv e ly .
The re s u lts o f the f i r s t experim ent are d e ta ile d in Figure 51.
The r a te
o f p ro te in synthesis was reduced to 50.8% o f the con trol value by 1 (T 7M cycloheximide although the e f f e c t o f th is con centration on c e ll number did not d i f f e r from the co n tro ls over the th ree day p e rio d .
7-E thoxy-
coumarin 0 -d eeth ylase a c t i v it y was measured f o r fo u r hours in the cu ltu re s days one to th ree and expressed in terms o f the number o f nuclei present in the f la s k . 0 -d eeth ylase a c t i v i t y
A fte r one day in c u ltu re the 7-ethoxycoum arin
in the 1 0 " 7M cyclohexim ide tre a te d c u ltu re s was tw ice
th a t o f the con trol v a lu e .
This was fo llow ed by a near lin e a r red u ctio n
in a c t i v it y on days two and th ree o f the c u ltu r e .
In the co n tro l c u ltu re s
the a c t i v it y increased on the second day o f c u ltu re to tw ice the le v e l on day one.
This was follow ed by a marked f a l l
in a c t i v it y by day th re e .
In the second experim ent described in Figure 5 2 , 10~7m cyclohexim ide caused a reduction in p ro te in synthesis to 44.5% o f the co n tro l le v e l. At th is le v e l the cycloheximide did not appear to .b e to x ic to the c e lls compared to the co n tro ls over the th ree day period o f in c u b a tio n .
The
re s u lts in th is second experim ent fo r the a c t i v it y o f the 7-etho^ycoumarin 0 -d eeth ylase a c t i v it y
experim ent.
d if f e r e d , however, from those o f the previous
In each case the a c t i v it y in the co n tro ls was g re a te r than
th a t in the cycloheximide tre a te d c u ltu re s but the a c t i v it y increased in both from days one to two o f c u ltu re follow ed by a f a l l o f f in a c t i v i t y in both tre a te d and untreated by day th ree although the a c t i v i t y in the co n tro ls was more than double th a t in the cycloheximide tre a te d c u ltu re s on the t h ir d day o f c u ltu re .
F ig u re 5 1 :
The e f f e c t o f c y c lo h e x im id e on n u c le i number, and 7 -e th o x y c o u m a rin 0 - d e e t h y la s e a c t i v i t y in p r im a ry m o n o la y e r c u l t u r e o f a d u l t r a t h e p a to c y te s
to
rtJ
2
c
3
o
1
o , •T— c > '
• I—
10
4 ->
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S -
4 ->
E
>
03
4 -> Ll I
+ J CU
5
X i— >> 9 _c -£= 1 i o
0
2
3
Time in c u ltu re (days) C ultures were incubated w ith medium con taining 10"^M cyclohexim ide • (O O ), 10"5Mcycloheximide (□ ------ □) or co n tro l medium (v v ) . Nuclei counts and 7-ethoxycoumarin 0-d eethylase a c t i v it y were measured a t the time points in d ic a te d . A ll re s u lts were the mean o f th ree samples. (The nuclei count fo r day 0 was estim ated from the number o f c e lls seeded in to the fla s k s assuming a 30% binuclea.tion o f the c e l l s . )
Figure 5 2 :
Second experim ent to determine the e f f e c t o f cyclohexim ide on nuclei number, p ro te in synthesis and 7-ethoxycoumarin 0 -d eeth ylase a c t i v it y
in prim ary monolayer c u ltu re s o f
a d u lt r a t hepatocytes
JxZ CO
as
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lo X +->
C
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o
(U o
3
0
2
3
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SZ
100
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as O
E fO 3 O
CD
U
CO
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x
~o I
o
1
2
Time in c u ltu re (days) C ulture fla s k s were incubated w ith medium con taining 10 10"^M (□ ------ □) cycloheximide w ith co n tro ls ( v
v ).
M(o-
■O).
Nuclei counts and
7 -ethoxycoumarin 0 -deeth ylase a c t i v it y were measured a t the tim e p oints
in d ic a te d .
A ll re s u lts were the mean o f th ree samples.
(The n uclei count fo r Day 0 was estim ated from the number o f c e lls seeded in to the fla s k s assuming a 30% b in u c le a tio n o f the c e l l s . )
4 .1 0 .4
Comparison o f th e in d u c in g c a p a b i l i t i e s o f f o u r b a r b i t u r a t e s
in primary monolayer c u ltu res o f a d u lt r a t hepatocytes The experiments described in the previous section ( 4 .1 0 .4 ) demonstrated th a t the microsomal mono-oxygenase system was a c tiv e in prim ary c u ltu re s and th a t the le v e ls o f a c t i v it y may increase a f t e r the f i r s t day in c u ltu re even in con trol c u ltu re s .
C ultures should th e re fo re
serve as a s u ita b le model f o r in v e s tig a tin g the e ffe c ts o f inducing agents and studying the mechanisms o f in d u c tio n .
Furthermore by
comparing d if f e r e n t b a rb itu ra te s i t may be possible to id e n t if y s tru c tu re a c t i v it y re la tio n s h ip s r e q u is ite f o r in ductio n o f the microsomal mono-oxygenase system.
In the fo llo w in g experiments the e f f e c t o f fo u r b a rb itu ra te s were compared, each w ith a d if f e r in g d u ratio n o f a c t i v it y in v iv o , on the m etabolic a c t i v it y o f a d u lt r a t l i v e r c e lls in prim ary monolayer c u ltu re s using the a c t i v it y o f 7-ethoxycoumarin 0-d eeth ylase as a measure o f microsomal mono-oxygenase a c t i v i t y .
P re lim in a ry experiments were conducted to assess the t o x i c it y o f the •four b a r b itu r a te s , phenobarbitone, am ylobarbitone, q u in a lb a rb ito n e and thiopentone (as t h e ir sodium s a lts ) to the c u ltu re s .
They were
added to the c u ltu re s , a f t e r an i n i t i a l 24 hour period o f incu b atio n w ith con trol media to allow the c e lls to reco v er, on day one and were incubated continuously u n t il day fo u r when the nuclei were counted as a measure o f the number o f c e lls a tta c h e d -to the monolayer c u ltu re s . The re s u lts in Table 11 demonstrate th a t a l l fo u r o f the b a rb itu ra te s were extrem ely to x ic a t a con centration o f lOmM w ith no c e lls rem aining attach ed .
At ImM concentrations phenobarbitone was not to x ic whereas
the o th e r th ree b a rb itu ra te s were q u ite markedly to x ic .
There was
no evidence o f any o f the b a rb itu ra te s being to x ic a t IOOjjlM o r le s s .
Two experiments were then conducted to determine the co n cen tratio n fo r each b a rb itu ra te which would cause the optim al increase in enzyme a c t i v it y w ith minimal t o x i c it y . are given in Table 12.
The re s u lts o f these two experim ents
7-Ethoxycoumarin 0-d eethylase a c t i v i t y was
measured a f t e r the cu ltu re s had been exposed to the b a rb itu ra te s f o r 48 hours (day th r e e ).
The re s u lts were expressed as nmole o f
c 7 -ethoxycoumarin 0-deethylase a c t i v it y
incubation p erio d .
per 10
nuclei over a 4 hour
The optimal con centration o f phenobarbitone was
found to be ImM a f t e r the second experim ent.
Amylobarbitone and q u in a l-
barbitone were both found to induce maximal a c t i v it y a t O.lmM and thiopentone a t a concentration o f 0.2mM.
7-Ethoxycoumarin 0-d eeth ylase a c t i v it y was then measured days one to fo u r in c lu s iv e ly f o r am ylobarbitone and thiopentone a t t h e ir optim al concentrations and days one to fo u r and day seven f o r phenobarbitones and q u in a lb a rb ito n e . Figures 53 and 54.
The re s u lts o f these two experiments are given in In the untreated co n tro ls the a c t i v i t y on day
two increased to a t le a s t double the i n i t i a l a c t i v it y on day one and then tended to f a l l o f f .
Phenobarbitone caused a marked increase in
a c t i v it y which was approxim ately lin e a r from days one to fo u r and then f e l l d ra m a tic a lly by day seven although i t was s t i l l con trol values.
higher than the
Amylobarbitone and q u in a lb a rb ito n e had s im ila r
p r o file s o f a c t i v i t y , increasing ,to day two and then tending to plateau or decrease s l ig h t l y . i t s a c tio n .
Thiopentone, however, was delayed in
I t caused a marked increase in a c t i v it y by day fo u r but
the increases were small and increm ental days two and th re e .
A fu r th e r experim ent was c a rrie d out using the same optim al concentrations f o r the fo u r b a rb itu ra te s over a seven day c u ltu re period to t r y and confirm the re s u lts o f the previous experim ents.
The q u a lity o f the
cu ltu re s prepared f o r th is experim ent was not as good as the previous ones and the con trol cu ltu re s gave some spurious r e s u lts .
The re s u lts
fo r the b a rb itu ra te s (F ig . 55) in d ic a te th a t ImM phenobarbitone was again the most a c tiv e over the f i r s t fo u r days o f c u ltu r e , w ith the a c t i v it y increasin g s t i l l
fu r th e r by day seven.
The p r o f ile s f o r
amyl obarbi tone and q u in alb arb ito n e were again s im ila r although the increase in enzyme a c t i v it y was not as g re a t on day one f o r an\yl obarbi to n e. Thiopentone again had a delayed e f f e c t in inducing the a c t i v i t y o f the enzyme but by day seven there was a marked increase in a c t i v i t y .
The
nuclei counts, which were very low in many o f the con trol c u ltu r e s , were also very low in the am ylobarbitone and q u in a lb a rb ito n e c u ltu re s on day seven and th is may have a ffe c te d the day seven a c t i v i t y re s u lts fo r these two b a rb itu ra te s in th is experim ent.
Although th is l a t t e r
experiment was not e n t ir e ly successful i t did tend to confirm the previous re s u lts fo r the e ffe c ts o f the fo u r b a rb itu ra te s on microsomal mono-oxygenase a c t i v i t y .
T a b le 1 1 :
T o x i c i t y s tu d y w i t h f o u r b a r b i t u r a t e s on p r im a ry m o n o la y e r c u l t u r e s o f a d u l t r a t h e p a to c y te s
Concentration o f b a rb itu ra te Compound lOmM
ImM
18.4 x 104
Phenobarbitone
0
Thiopentone
0
Q uinalbarbitone
0
0.8
Amylobarbitone
0
IOOjiM
10/iM
2 4 .4 x 104
1 8 .8 x 1 0 4
0
30.1 x 104
3 0 .8 x 10 4
x 104
24.0 x 104
17.3 x 10 4
1.07 x 104
23.1 x 104
25.1 x 10 4
T o x ic ity was measured in terms o f the number o f nuclei present in each fla s k (each value is the mean o f three f la s k s ) . obtained on day 4 o f c u ltu r e . 29.4
The values were
The con trol values on days 1 and 4 were
x 104 and 18.9 x 104 re s p e c tiv e ly .
Table 12:
D eterm ination o f the optim al concentrations f o r fo u r b a rb itu ra te s fo r maximal in ductio n o f ethoxycoumarin 0 -d eeth ylase a c t i v it y
7-Ethoxycoumarin 0-d eethylase a c t i v it y (nmole/10^ n u c le i/4 h r )
Experiment 1
Experiment 2
Control (day 1)
30 .8
108.2
Control (day 3)
4 4 .7
4 6 .8
Phenobarbitone
Amylobarbitone
Q uinalbarbitone
Thiopentone
3mM
12.3
-
2mM
61.9
108.1
ImM
134.0
145.8 *
0.5mM
-
111.8
O.lmM
-
6 5 .9
0.6mM
3 .5
-
0.3mM
91 .2
-
0 . 2 mM
89.5
O.lmM
120.4
200.9 126.1 *
0.05mM
-
118.1
O.OlmM
-
128.6
0.3mM
2.1
0.2mM
75.2
O.lmM
118.2
-
85 .2 7 2 .8 *
0.05mM
-
70.1
O.OlmM
-
6 2 .3
0.3mM
24.6
-
0.2mM
217.2 *
-
O.lmM
159.2
-
A ll values above are the means o f th ree in d iv id u a l fla s k s .
Wider
con centration ranges were tested but the re s u lts are only given fo r those le v e ls which were not very to x ic to the c u ltu re s , i . e . nuclei count was not too low.
* These concentrations were regarded as being o p tim a l.
the
F ig u re 5 3 :
The e f f e c t o f p h e n o b a rb ito n e and q u i n a l b a r b i t o n e on 7 -e th o x y c o u m a rin 0 - d e e t h y la s e a c t i v i t y in p r im a ry m o nolayer c u l t u r e s o f a d u l t r a t h a p a to c y te s
200 £ +■>
160
cu $1 to sz n5 I— >> \ +■> CU CU 1
LxJ
0 1
2
3
4
5
6
7
Days in c u ltu re
Cultures were incubated w ith medium con taining ImM phenobarbitone (O
O ), O.lmM q u in alb arb ito n e (□ ------ □ ) , o r control medium ( v
v ).
7-Ethoxycoumarin 0-deethylase a c t i v it y was measured a t the tim e points in d ic a te d .
Each p o in t was the mean o f d u p lic a te samples.
F ig u r e 5 4 :
The e f f e c t o f a m y lo b a r b ito n e and th io p e n to n e on th e a c t i v i t y o f 7 -e th o x y c o u m a rin 0 - d e e t h y la s e a c t i v i t y in p r im a r y m o nolayer c u l t u r e s o f a d u l t r a t h e p a to c y te s
200 > 1 + J
•r—
>
•r—
+ ->
160
O n3 . S-
»
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•r—
+ J (U
O) r—
0 ) T3 1
O 3 £Z
O
sz •I—
O r—
\
s fCJ
CD T—
E
o
zs o o
1.20
80
E e
> >
X
o
sz
+J LU ■ 1
40
0 1
2
3
4
5
6
7
Days in c u ltu re
C ultures were incubated w ith medium con taining O.lmM am ylobarbitone (■ ------ ■ ) , 0.2mM thiopentone ( • ------ • ) , o r control medium ( v
v ).
7-Ethoxycoumarin 0-d eethylase a c t i v it y was measured a t the tim e points in d ic a te d .
Each p o in t was the mean o f d u p lic a te samples.
F ig u r e 5 5 :
The e f f e c t o f f o u r b a r b i t u r a t e s on 7 -e th o x y c o u m a rin 0 - d e e t h y l a s e a c t i v i t y in p r im a ry m o nolayer c u l t u r e s o f a d u l t r a t h e p a to c y te s
200
4-> O i.m ole/g l i v e r (1.6m g/g l i v e r ) in whole l i v e r p re p a ra tio n s .
The values obtained f o r the le v e ls o f g lu ta th io n e in Table 2 again in d ic a te some v a r i a b i l i t y between anim als.
The le v e l o f g lu ta th io n e
is a ffe c te d by the d ie ta r y status o f animals (McLean and Day, 1 9 7 5 ), i t also shows a marked d iu rn al v a r ia tio n (Isaacs and B in k le y , 1977) w ith the le v e l a t 6 am being about tw ice th a t a t 6 pm.
The values obtained f o r g lu ta th io n e in fre s h ly is o la te d hepatocytes
± 1 .1 2 /x g /lO ^ c e lls which compare w ell w ith those obtained by Thor e t al_ (1978) and Hogberg e t a\_ (1977) o f about 1 0 .1 3 /Jig/10^
were 7.5 2
c e lls .
Hogberg ejt aj_ (1977) -found th a t by using a more ra p id
perfusion technique the g lu ta th io n e le v e ls obtained in the fre s h ly is o la te d hepatocytes were h igher (1 3 .5 jJig/10 c e l l s ) .
The re s u lts obtained f o r the p reparations o f r a t hepatocytes used here in d ic a te th a t incubation in complete medium f a c i l i t a t e s the rap id resynthesis o f g lu ta th io n e .
This is in agreement w ith the
fin d in g s o f the K arolinska group but a t variance w ith the fin d in g s o f Vina e t a^ (1 9 7 8 ).
These v a ria tio n s in the g lu ta th io n e le v e ls reported
by d if f e r e n t groups may be an in d ic a tio n o f possib le d iffe re n c e s in v i a b i l i t y c h a ra c te r is tic s o f hepatocytes prepared by d if f e r e n t methods but are more l i k e l y a r e f le c tio n o f the v a ria tio n s in the incubation media used.
The L I5 medium used throughout the stu d ies
reported here contains s u f f ic ie n t amounts o f the amino a c id s , cysteine (120m g/l) and m ethionine (7 5 m g /l),w h ich are u t ilis e d by the c e ll to resynthesize g lu ta th io n e .
The K arolinska group also used
c u ltu re medium containing amino acids whereas Vina e t al_ used KrebsH e in s le t b u ffe r which had to be supplemented w ith m ethionine o r homocysteine to ju s t m aintain the le v e ls o f g lu ta th io n e during in cu b atio n s.
K reb s-H ein slet b u ffe r th e re fo re may not provide a
s u ita b le i n t r a c e ll u l a r environment f o r the synthesis o f g lu ta th io n e .
The re s u lts discussed here suggest th a t the q u a lity o f the is o la te d hepatocytes prepared by the s lic in g method o f Fry e t a]_ (1976) does not d i f f e r markedly from th a t o f hepatocytes prepared by perfu sio n o f in ta c t l i v e r .
They appear to c a rry out a number o f fu n ctio n s
which occur in vivo but w ith o u t the a d d itio n o f exogenous c o fa c to rs . There is however some v a r i a b i l i t y between in d iv id u a l p rep a ra tio n s o f hepatocytes in terms o f t h e ir y ie ld and v i a b i l i t y c h a ra c te r is tic s which may r e f le c t the status o f the animal from which they are derived and serves to emphasise th a t th ere is a considerable v a r ia tio n between d if f e r e n t animals o f the same species.
T h is -v a r ia tio n may be
a ffe c te d by age, d i e t , environmental conditions which may also cause stress e ffe c ts and the tim e o f day the animals are k i l l e d .
For the
experiments reported here these v a ria b le s were standardised as f a r as possible in th a t animals o f s im ila r ages were used throughout, they were fed a standard la b o ra to ry d ie t and were u s u a lly s a c r ific e d between 9am and 11am in the morning.
The time th a t the animals had
to a c c lim a tis e to the environment w ith in the housing f a c i l i t i e s however was v a ria b le and stress fa c to rs may also have v a rie d .
used It
should be possible to standardise most v a ria b le s w ith in one la b o ra to ry but between la b o ra to ry comparisons may be hindered however by the involvement o f d if f e r e n t v a ria b le s .
5 .2 5 .2 .1
ISOLATED RAT HEPATOCYTES IN PRIMARY MAINTENANCE CULTURE The p ro p e rtie s o f cu ltu re s compared to suspensions o f is o la te d
hepatocytes Suspensions o f is o la te d r a t hepatocytes can norm ally only be used f o r short term experiments o f a few hours.
A fte r th is time the v i a b i l i t y
o f the hepatocytes is reduced to below le v e ls a t which the c e lls are l i k e l y to fu n c tio n as they would under normal p h y sio lo g ical con d itio n s in v iv o .
The incubation method used here was to place one ml volumes o f c e ll suspension in to 10ml glass conical fla s k s in a shaking in c u b a to r.
As
the incubation time increased not only did the v i a b i l i t y o f u n treated c e lls tend to decrease but the c e lls also tended to aggregate in clumps and attach to the sides o f the f la s k , predom inantly around the meniscus o f the incubating medium.
This lo c a lis a tio n probably
c o n trib u tes to the reductions in v i a b i l i t y since the hepatocytes would not be homogeneously dispersed w ith in the medium hence the a v a i l a b i l i t y o f e s s e n tia l n u trie n ts from the medium would be hindered and oxygen supply no longer co n sis ten t w ith c e lls in the c en tre o f clumps p ossib ly being starved o f oxygen.
This became a serious
problem f o r incubations o f g re a te r than th ree hours.
A lte r n a tiv e incubation techniques have been used by o th e r research groups which may enable longer in cu b a tio n s.
For example the K arolinska
group (see Moldeus, 1978) incubate hepatocyte suspensions in round
bottomed fla s k s which are c o n tin u a lly ro ta te d .
This fa c to r may
c o n trib u te to the f a c t th a t they claim to achieve longer incubation tim es.
Other fa c to rs which may a f fe c t the v i a b i l i t y o f the
hepatocytes over long periods may be the is o la tio n procedure i t s e l f . I f th is is c a rrie d out under s t e r i l e conditions a suspension c u ltu re may be achieved.
Subtle d iffe re n c e s in the supplements added to
c u ltu re media, whose ro le is as y e t u n id e n tifie d , may a s s is t in m aintaining v i a b i l i t y o f suspensions.
Furtherm ore, d iffe re n c e s in
the apparatus such as the q u a lity o f the glass used may a f f e c t the c h a ra c te r is tic s o f a hepatocyte suspension.
The c h a r a c te r is tic property o f reaggregating and a tta c h in g to surfaces l ik e glass has been u t ilis e d in developing techniques f o r c u ltu rin g hepatocytes.
I f hepatocytes, is o la te d under a s e p tic
c o n d itio n s , are innoculated in to c u ltu re f la s k s , e .g . p la s t ic fla s k s , o r glass tre a te d w ith collagen, they w i l l atta ch to the surface o f the f la s k .
They w i l l then tend to reaggregate as i f to reg ain the
co n fig u ra tio n o f the tis s u e from which they were d e riv e d . they f la t t e n and spread to form a c o n flu en t monolayer.
With time
I t takes
about 24-48 hours fo r a c u ltu re to achieve a co n flu en t monolayer i f . . the n o n -viab le detached c e lls are removed a t in te r v a ls and the c u ltu re medium replaced thereby removing any to x ic l y t i c enzymes and o th e r m a tte r.
E p ith e lia l hepatocytes w il l not d iv id e to any
s ig n ific a n t e x te n t in such prim ary c u ltu re s but n o n -e p ith e lia l fib r o b la s ts w i l l .
A fte r about 5 days in c u ltu re fib r o b la s ts u s u a lly
begin to predominate.
I t th e re fo re fo llo w s th a t prim ary maintenance
c u ltu re s o f r a t hepatocytes may be used to in v e s tig a te the e ffe c ts o f chemicals f o r up to 3 to 5 days which is a marked improvement over hepatocyte suspensions.
This property o f cu ltu re s was considered to be p o t e n t ia lly useful f o r in v e s tig a tin g the longer term e ffe c ts o f paracetamol on r a t l i v e r in v i t r o .
I t has been w ell reported (see Chapter 1) th a t an acute
overdose o f paracetamol causes l i v e r damage in the r a t in v iv o .
The
onset o f th is l i v e r damage however is not apparent fo r some hours a f t e r the a d m in is tra tio n o f the to x ic dose.
I t was th e re fo re considered
re le v a n t to u t i l i s e cu ltu re s to in v e s tig a te in p a r t ic u la r the e ffe c ts
o f a la rg e dose o f paracetamol (40mM) on hepatocytes over a 48 hour period a f t e r exposure to paracetamol f o r between 2 and 8 hours.
In
the experiments c a rrie d out i t became apparent th a t treatm en t w ith 40mM paracetamol fo r more than 4 hours could cause ir r e v e r s ib le in h ib it io n o f p ro te in synthesis c e ll damage and.death, whereas a sh o rte r exposure time led to re v e rs ib le e ffe c ts on p ro te in syn th e sis . The e ffe c ts o f 2mM phenobarbitone on p ro te in synthesis were also found to be re v e rs ib le a f t e r up to 8 hours o f exposure to c u ltu re d hepatocytes w ith no evidence o f c e ll damage when compared to co n tro l c u ltu re s in terms o f c e ll attachm ent.
5 .2 .2
Mixed fu n ctio n oxidase a c t i v it y in prim ary maintenance
cu ltu re s o f a d u lt r a t hepatocytes Suspensions o f is o la te d r a t hepatocytes have been demonstrated to have the a b i l i t y to m etabolise fo re ig n compounds whose metabolism is mediated by the microsomal monooxygenase enzyme system.
Prim ary
monolayer cu ltu re s o f r a t hepatocytes have been reported to lose much o f th is a c t i v it y however during the f i r s t tw elve hours o f c u ltu re (see Chapter 2 ) .
Guzelian and Barwick (1979) reported th a t supplementing
c u ltu re medium w ith cycloheximide m aintained the le v e l o f cytochrome P-450 in primary maintenance c u ltu re s .
Two experiments were c a rrie d
out here in o rd er to t e s t the fin d in g s o f Guzelian and Barwick and furtherm ore to in d ic a te whether the c u ltu re system used here would m etabolise fo re ig n compounds v ia the microsomal monooxygenase pathways.
In our study cytochrome P-450 le v e ls were not measured,
instead the a c t i v i t y o f the microsomal enzyme ethoxycoumarin 0 -d e e th y la s e was assessed.
In the f i r s t o f these experiments cu ltu re s were m aintained on a su b s tra tio n o f collagen whereas in the second no collagen was used. The presence o f collagen did not appear to s ig n if ic a n t ly a f f e c t the attachment o f the c e lls to the p la s t ic c u ltu re s u rface .
The
morphological appearance o f the untreated con trol c u ltu re s , however, appeared to d e te r io r a te more ra p id ly in the presence o f collagen which is why i t was om itted in the.second experim ent.
In both experiments treatm en t o f cu ltu re s w ith 10 which in h ib ite d p ro te in synthesis by
-5
M cycloheximide
> 95% was markedly to x ic
causing a ra p id decrease in the number o f c e lls attached to the monolayer over the f i r s t 24 hours.
Treatment w ith cyclohexim ide (10”^M)
which in h ib ite d p ro te in synthesis by about 50% did not cause s ig n if ic a n t ly d if f e r e n t le v e ls o f c e ll attachm ent compared to control c u ltu re s .
The numbers o f c e lls attached f e l l by about 60% during
the th ree days o f c u ltu re in both the control and cycloheximide (1 0 ’ ^M) tre a te d c u ltu re s .
7-Ethoxycoumarin 0-d eethylase a c t i v i t y was present in both experiments although th ere was a marked d iffe re n c e in the le v e ls o f a c t i v i t y w ith about a 6 - f o ld g re a te r a c t i v i t y in the second experim ent.
I t is
u n lik e ly th a t the absence o f collagen would have co n trib u te d to th is d iffe re n c e in a c t i v i t y .
I t is more l i k e l y th a t the animals had
t h e ir mixed fu n c tio n oxidase a c t i v it y induced p r io r to the is o la tio n o f the hepatocytes f o r the second experim ent.
In both experiments
the le v e l o f a c t i v it y o f 7-ethoxycoumarin 0-d eeth ylase in the
c
co n tro ls doubled between days one and two to about 20 and 1 2 0 nmoles/ 1 0 n u c le i/4 hr which would not support the g e n e ra lly accepted fin d in g th a t the le v e ls o f microsomal monooxygenase a c t i v it y are reduced in c u ltu re s .
By day 3 the le v e ls o f a c t i v it y were reduced.
The fin d in g s from the two experiments f o r the e f f e c t o f 10’ ^M cycloheximide were not c o n s is te n t.
In the f i r s t experim ent the
le v e l o f a c t i v it y o f 7-ethoxycoumarin 0-d eeth ylase a c t i v i t y was doubled by the presence o f cycloheximide on day 1 fo llow ed by a lin e a r f a l l o f f during days 2 and 3 in c u ltu r e .
In the second experim ent,
however, the a c t i v it y on day 1 was 2 0 % less than in the con trol c u ltu re s .
The a c t i v it y increased on day 2 but not to the same e x te n t
as in the c o n tro ls . s till
By day 3 the a c t i v i t y had f a lle n again but was
less than th a t o f c o n tro ls .
Since
the re s u lts o f these two experiments were v a ria b le l i t t l e
may
be concluded in r e la tio n to the experiments o f Guzelian and Barwick (1 9 7 9 ).
I t may be suggested however th a t the c h a ra c te r is tic s o f the
cu ltu re s used here were d if f e r e n t to those o f Guzelian and Barwick
and o th er groups who have reported a marked loss o f cytochrome P-450 when hepatocytes are c u ltu re d .
The le v e l o f a c t i v it y o f the microsomal
monooxygenase enzymes o f the cu ltu re s used here was not very d if f e r e n t to the le v e ls seen in suspensions o f hepatocytes.
F u rth e r
more the a c t i v it y rose during c u ltu re which may be due to the presence o f an inducing agent in the fo e ta l c a l f serum.
I t th e re fo re
appears to be unnecessary to supplement the medium w ith an unnatural p h ysio lo g ical a d d itiv e l ik e cycloheximide to m aintain the le v e ls . In fa c t the re s u lts obtained w ith cycloheximide here do not tend to support the re s u lts o f Guzelian and Barwick.
I t may be th a t cyc lo
heximide is only e f fe c tiv e a t m ain tain in g le v e ls o f cytochrome P-450 in c e lls o f poor q u a lity .
5 .2 .3
Comparison o f the inducing c a p a b ilitie s o f some b a rb itu ra te s
The experiments c a rrie d out w ith cycloheximide in d ic a te th a t the prim ary maintenance cu ltu re s o f r a t hepatocytes used here con tain an a c tiv e microsomal monooxygenase system in v o lv in g cytochrome P-450. In i n i t i a l
cu ltu re s the le v e ls o f mixed fu n ctio n oxidase a c t i v it y
were r e l a t i v e l y low, although not very d if f e r e n t to those o f suspensions, but increased in co n tro ls during the f i r s t two days in c u ltu r e .
I t has
been demonstrated th a t phenobarbitone can induce the microsomal monooxygenase system in these c u ltu re s (F ry e t al_, 198 0);
benzanthracine
also induced microsomal monooxygenase a c t i v it y and the e f fe c ts o f the two inducers were a d d itiv e in d ic a tiv e o f the f a c t th a t two types o f haemoprotein were induced.
The b a rb itu ra te class o f drugs contains a number o f compounds w ith d if f e r in g d u ratio n o f actio n and physicochemical p ro p e rtie s .
The
a c t i v it y o f the microsomal monooxygenase system, as r e fle c te d by the a c t i v i t y o f 7-ethoxycoumarin 0 -d e e th y la s e , was used to compare the inducing c a p a c itie s o f fo u r d if f e r e n t b a rb itu ra te s .
Phenobarbitone, the c la s s ic b a rb itu ra te in d u ce r, is a l i p i d soluble long actin g b a rb itu ra te w ith a r e l a t i v e l y long in vivo h a lf l i f e . Amylobarbitone is an in te rm ed iate ac tin g and q u in a lb a rb ito n e a sh o rt actin g b a r b itu r a te .
Thiopentone is a h ig h ly l i p i d s o lu b le , very sh o rt
ac tin g b a rb itu ra te which is used as an a n a e s th e tic .
The stru c tu re s
f o r these b a rb itu ra te s are given in Figure 4.
P r io r to in v e s tig a tin g the inducing c a p a b ilitie s o f these fo u r b a rb itu ra te s t h e i r p o te n tia l t o x ic it y was in v e s tig a te d in c u ltu re s by m onitoring the e ffe c ts o f various concentrations o f each on the number o f c e ll nuclei attached to the surface o f c u ltu re s .
Pheno
barbitone was found to be the le a s t to x ic o f the fo u r b a rb itu ra te s . Once the approximate to x ic le v e ls o f the b a rb itu ra te s had been id e n t if ie d concentrations o f each were selected fo r the e ffe c ts on the a c t i v it y o f 7-ethoxycoumarin 0-d eeth ylase w ith the aim o f id e n tify in g the le v e ls which would achieve a maximal in d u ctio n o f a c t i v it y w ith minimal t o x ic it y over a two day c u ltu re p e rio d .
These
concentrations were id e n t if ie d as ImM, O.lmM, O.lmM and 0.2mM f o r phenobarbitone, am ylobarbitone, q u in a lb a rb ito n e and thiopentone r e s p e c tiv e ly .
The p r o f ile o f inductio n f o r these fo u r b a rb itu ra te s
was then in v e s tig a te d over a seven day period measuring the a c t i v i t y o f 7-ethoxycoumarin 0-d eethylase on days 1 , 2 , 3 and 4 , and in some cases day 7 (day 1 was 24 hours a f t e r the fla s k s were innoculated w ith hep ato cytes).
Phenobarbitone was found to be the most e f f e c t iv e inducer in th is system.
Of the fo u r b a rb itu ra te s studied phenobarbitone is the
le a s t l i p i d so lu b le and is m etabolised and e lim in a te d from the c e ll more s lo w ly .
I t is o f in te r e s t th a t Pelkonen and Karki (1973) found
an inverse r e la tio n s h ip in vivo between l i p i d s o l u b i li t y and enzyme inductio n f o r the b a rb itu ra te s .
The p r o file s o f inductio n f o r am ylobarbitone and q u in a lb a rb ito n e were s im ila r in th a t th ere was a marked increase in the a c t i v i t y o f 7 ethoxycoumarin during the f i r s t 24 hours o f exposure which then remained constant or f e l l
s l ig h t l y .
Both were f a r less e f f e c t iv e
than phenobarbitone which may be exp lained by the f a c t th a t they are both more l i p i d soluble and also- more ra p id ly m etabolised by the l i v e r . There is however a marked d iffe re n c e between the s tru c tu re s o f these two b a rb itu ra te s .
Q uinalbarbitone contains an a l l y l
side c h a in , not
present in any o f the o th er b a rb itu ra te s studied h ere, which may p o te n tia lly be metabolised to an a c tiv e m e ta b o lite , p o ssib ly an a l l y l i c
F ig u r e 4 :
S tru c tu re s o f b a rb itu ra te s
C2 h 5
C2H5 HN
H
ch2 ch2 ch(ch3 ).
H
Phenobarbitone
Amylobarbitone
ch2 c h = c h 2 chch2 ch2 ch3
C2H5 CHCH„CH,CH,
HN
I
CH.
CH.
O ^ N - ^ O H
H
Q uinalbarbitone
Thiopentone
2
c
3
epoxide which could lead to the d e s tru c tio n o f cytochrome P-450.
The
fa c t th a t the a c t i v i t y o f 7-ethoxycoumarin 0-deethylase was s t i l l r e l a t i v e l y high a f t e r 6 days o f treatm ent w ith q u in a lb a rb ito n e may suggest th a t another haemoprotein is induced o r th a t a lim ite d amount o f the a l l y l epoxide m e ta b o lite is formed re s u ltin g in less damage to cytochrome P-450.
Thiopentone, although h ig h ly l i p i d s o lu b le , did cause an in d u ctio n o f 7-ethoxycoumarin 0-d eeth ylase a c t i v it y but th is e f f e c t was delayed. A fte r th ree days the inductio n by thiopentone was q u ite marked. This delayed e f f e c t suggests th a t thiopentone i t s e l f was not resp o n sib le f o r the inducing a c t i v it y since i t is m etabolised very r a p id ly by the l i v e r .
One o f i t s m etabolites however is pentobarbitone and th is
or another m e tab o lite may be responsible f o r the e f f e c t o f thiopentone on the delayed increase in a c t i v i t y o f the microsomal monooxygenase system.
The e a r ly lag may be r e la te d to the production o f a c tiv e
sulphur ( S ') from thiopentone which would be to x ic to haemoproteins.
I t may be concluded from these p re lim in a ry in v e s tig a tio n s th a t the mixed fu n ctio n oxidase system may be induced in prim ary maintenance c u ltu re s o f a d u lt r a t hepatocytes and th is may prove to be a useful property fo r in v e s tig a tin g the mechanisms o f t o x ic it y o f o th e r fo re ig n compounds to the l i v e r in v i t r o .
T h is , however, may be dependent on
the nature o f haemoprotein induced (F ry eit aj_, 1980) and whether th is is s im ila r to the in vivo response o f the l i v e r .
F u rth e r in v e s tig a tio n s
would be required to c l a r i f y t h is .
5 .2 .4
The a p p lic a tio n o f q u a n tita tiv e cytochem istry to prim ary c u ltu re s
During the la s t few years much work has been dedicated to improving techniques f o r the histochem ical and cytochemical a n a ly s is o f enzymes in tis s u e s .
There are numerous advantages to being ab le to id e n t if y
the c e ll types o f a tis s u e where a p a r t ic u la r enzyme is lo cated and to q u a n tify the amounts o f enzyme in s itu ra th e r than use d isru p ted tis s u e s and hence only being able to determine o v e ra ll enzyme a c t i v i t i e s .
The
id e n t if ic a t io n o f tis s u e s ta b ilis e r s which are chem ically
in e r t (Altm an, 1980) has enabled the measurement o f enzymes w ith in unfixed tis s u e sections w ith o u t loss o f enzyme from i t s a c tiv e s ite in the tis s u e o r loss in i t s a c t i v i t y .
Primary c u ltu re s o f r a t hepatocytes can be m aintained on collagen coated glass covers!ips in Leighton tubes.
The r e s u lta n t c e ll
monolayer can be tre a te d l ik e an unfixed tis s u e section and th e re fo re provides a s u ita b le system fo r in v e s tig a tin g cytochem istry in an in v it r o s itu a tio n .
Methods have been developed fo r the cytochemical
id e n t if ic a t io n o f a number o f dehydrogenase enzymes (Chayen e t al_, 197 3). The dehydrogenases o x id is e t h e ir substrates by removing hydrogen and passing i t to a s u ita b le acceptor.
T etrazo liu m s a lts have been id e n t if ie d
as s u ita b le acceptors which when reduced y ie ld a coloured p r e c ip ita te o f t h e ir corresponding formazan.
This re a c tio n can be speeded up by
the a d d itio n o f a powerful hydrogen acceptor phenazine methosulphate which ra p id ly takes hydrogen from the donar and passes i t to the te tra z o liu m acceptor.
The e ffe c ts o f exogenous chemicals on the a c t i v it y o f th ree dehydrogenases were in v e s tig a te d :
succinate dehydrogenase (SDH), glucose 6 -phospate
dehydrogenase ( 6 6 PDH) and NADPH2 diaphorase.
SDH is a m itochondrial marker enzyme which plays an e s s e n tia l r o le in the t r ic a r b o x y lic acid cycle during the o x id a tio n o f succin ate to fum arate.
The hydrogen y ie ld e d from th is re a c tio n is norm ally u t il is e d
in the e le c tro n tra n s p o rt chain.
The use o f phenazine methosulphate
w i l l sho rt c i r c u i t the e le c tro n tra n s p o rt chain as a strong hydrogen acceptor.
6 6 PDH is a cytoplasm ic enzyme involved in the pentose phosphate
pathway which is a major pathway involved in the o x id a tio n o f glucose. This p a r tic u la r pathway produces pentose sugars which may be used to produce ribose and deoxyribose fo r t h e i r corresponding n u c le ic a c id s . G6 PDH requires NADP as a coenzyme and is th e re fo re a major source o f NADPH. This is o f considerable importance in a number o f e s s e n tia l m etabolic reactio n s in clu d in g the synthesis o f s te ro id s and f a t t y acids
and in the hyd ro xylatio n o f many l i p i d soluble compounds by cytochrome P-450.
NADPH2 diaphorase is a marker enzyme fo r the endoplasmic re tic u lu m , o r more s p e c if ic a lly f o r the microsomal re s p ira to ry system, which passes hydrogen from NADPH2 to re a c t w ith atmospheric oxygen by means o f cytochrome P-450.
I t s tru e p h ysio lo g ical ro le remains c o n tro v e rs ia l.
I t may be e q u iv a le n t to cytochrome P-450 reductase.
The a c t i v it i e s o f these th ree enzymes were q u a n tifie d in c u ltu re d hepatocytes using a Vickers scanning and in te g ra tin g m icrodensitom eter to measure the absorbance o f the p r e c ip ita te d formazan produced by the ap p ro p riate enzyme reac tio n s in the presence o f phenazine m ethosulphate, tis s u e s t a b i li s e r (PVA) and necessary c o fa c to rs .
Carbon te tr a c h lo rid e a very w id ely used model hepatotoxin was found to cause dose dependent reductions in c e ll number attached to c u ltu r e s , in h ib it io n o f p ro te in synthesis and increases in GOT leakage.
I t also
caused dose re la te d decreases in the a c t i v i t y o f G6 PDH a f t e r two and s ix hours o f in c u b a tio n , 1 0 , 0 0 0 ppm causing a ra p id f a l l
in a c t i v i t y
w ith in two hours such th a t i t was not d e tectab le a t 2 o r 6 hours. SDH a c t i v it y was decreased by lOOOppm carbon te tr a c h lo r id e by 20% w ith in two hours remaining a t th is le v e l a f t e r s ix hours.
At the
higher concentration o f 1 0 , 0 0 0 ppm however th ere was a continued f a l l in the a c t i v it y o f SDH between two and s ix hours to about 10% o f the con trol a c t i v i t y .
The a c t i v i t y o f NADPH2 diaphorase, however,
showed a biphasic response to exposure to carbon t e tr a c h lo r id e . At high concentrations (5 ,0 0 0 and 10,000ppm) i t s a c t i v i t y was reduced. At lower concentrations (1000 and 500ppm) however the a c t i v i t y o f NADPH2 diaphorase was increased.
This increase in a c t i v it y over
co n tro ls was m aintained f o r 24 hours although the peak a c t i v i t y was seen a t 8 hours.
The reductions in enzyme a c t i v i t y could be due to
in h ib it io n o r damage to the enzyme.
G6 PDH is a cytoplasm ic enzyme
which may e x p la in why the e ffe c ts o f carbon te tr a c h lo r id e were more marked on th is enzyme since th is enzyme would be more r e a d ily lo s t through damaged plasma membranes and also more a c c e s s ib le .
An attem p t
was made to determine whether the increase in NADPH2 diaphorase a c t i v it y was due to inductio n o r a c tiv a tio n by incubating in the presence o f cyclohexim ide.
This experim ent was not however successful
and fu r th e r in v e s tig a tio n would be needed to id e n t if y the mechanism by which th is increase in a c t i v i t y occurs.
The re s u lts obtained fo r lOmM paracetamol were very s im ila r to those obtained f o r lOOOppm carbon t e tr a c h lo r id e .
A fte r s ix hours o f
incubation th ere were s lig h t reductions in c e ll number and s lig h t increases in GOT leakage w ith a 40% in h ib it io n o f p ro te in s yn th e sis . Although the a c t i v it i e s o f SDH and G6 PDH were reduced the a c t i v i t y o f NADPH2 diaphorase increased in a lin e a r manner over 6 hours to about 2 0 0 % o f the control
Phenobarbitone dehydrogenases.
le v e l.
(2mM) on the o th e r hand produced increases in a l l th ree This e f f e c t was more marked and prolonged on the
a c t i v it i e s .of NADPH2 diaphorase and G6 PDH w ith the e f f e c t tending to increase over a 30 hour p erio d .
For SDH the e f f e c t o f phenobarbitone
was more marked w ith in the f i r s t two hours o f exposure.
The e f f e c t o f
phenobarbitone on the hepatocytes is th e re fo re d if f e r e n t to th a t o f paracetam ol.
5 .3
THE EFFECTS OF XEN0BI0TICS ON THE VIABILITY CHARACTERISTICS OF
ISOLATED HEPATOCYTES 5 .3 .1
Paracetamol
The main compound whose to x ic e ffe c ts were in v e s tig a te d in the studies reported here was paracetam ol.
I t is known to cause h e p a to to x ic ity
in animals and man when adm inistered a t high doses, v a s tly in excess o f th e ra p e u tic le v e ls .
The s u s c e p t ib ilit y o f d if f e r e n t animal species
to the hep ato toxic e ffe c ts o f paracetamol is however very v a r ia b le (see Chapter 1 ).
Paracetamol was found to be r e l a t i v e l y n o n -to xic to is o la te d r a t hepatocytes in terms o f i t s e f f e c t on c e ll v i a b i l i t y as measured by trypan blue exclusion or la c ta te dehydrogenase release, parameters which are l i k e l y to be in d ic a to rs o f ir r e v e r s ib le plasma membrane damage.
Incubations o f r a t hepatocytes w ith paracetamol a t concentrations o f up to 40mM f o r one hour caused l i t t l e
change in e ith e r parameter
when compared to those o f control incubations o f a s im ila r d u ra tio n . There was however some evidence to suggest th a t 40mM paracetamol did produce plasma membrane damage when the exposure time was increased to 90 minutes although th is was not a co n s is te n t fin d in g .
From the above data i t may th e re fo re be assumed t h a t , f o r the o th e r e ffe c ts in v e s tig a te d , the in t e g r it y o f the c e ll membrane was preserved a f t e r one hour o f incubation o f hepatocytes w ith concentrations o f up to 40mM paracetam ol.
I t is th e re fo re o f in te r e s t th a t paracetamol
was found to cause dose dependent reductions in both and
[ 14c] -U rid in e in c o rp o ra tio n .
[ ^ c ] -Leucine
P ro tein synthesis was in h ib ite d
by >75% w ith in 5 minutes o f exposure to high concentrations o f paracetamol (40mM).
This con centration caused a maximal in h ib it io n
throughout one hour o f incubation since the amount o f la b e l in co rp o rated in to p ro te in was not increased a f t e r 5 m inutes.
I f paracetamol (40mM)
was removed from the hepatocytes a f t e r 30 minutes o f incu b atio n the hepatocytes began to synthesize p r o te in , a l b e i t a t a slower r a te than c o n tro ls , in d ic a tin g th a t th is e f f e c t on p ro te in synthesis is re v e rs ib le i f the exposure time is r e l a t i v e l y s h o rt.
The in co rp o ratio n o f
[ ^ c ] -U rid in e was also m axim ally in h ib ite d
(95%) by 40mM paracetam ol, w ith a concentration o f about 5mM causing a 50% in h ib it io n (compared to ^9mM f o r 50% in h ib it io n o f p ro te in s y n th e s is ).
The e ffe c ts o f paracetamol a t e a r ly time po in ts and on
r e v e r s ib i li t y were not in v e s tig a te d fo r th is param eter.
As mentioned above in con trol hepatocytes g lu ta th io n e was dep leted during the is o la tio n procedure but resynthesis began r a p id ly fo llo w in g incubation o f hepatocytes in complete medium.
The a d d itio n o f 10 and
20mM paracetamol in h ib ite d the r a te o f resynthesis by 77% and 67% re s p e c tiv e ly but only a t a con centration o f 40mM paracetamol was d e p le tio n o f the le v e ls o f g lu ta th io n e to below the i n i t i a l co n tro l le v e ls in r a t hepatocytes observed.
Evidence from p re lim in a ry
experiments w ith paracetamol suggested th a t in h ib it io n o f the r a te o f resynthesis o f g lu ta th io n e occurred w ith in 5 minutes o f in c u b a tio n .
I t would th e re fo re appear th a t p ro te in synthesis and RNA synthesis are both in h ib ite d by paracetamol w ith the e f f e c t o f paracetamol on these two parameters being more marked than i t s e ffe c ts on c e llu la r g lu ta th io n e le v e ls .
There also appears to be no d ir e c t c o r r e la tio n
between c e llu la r g lu ta th io n e le v e ls and the e f f e c t o f paracetamol on the in h ib it io n o f p ro te in syn th esis.
This is supported by the f a c t
th a t i f hepatocytes are preincubated f o r one hour p r io r to the a d d itio n o f paracetam ol9 which allows the resynthesis o f g lu ta th io n e to p h ysio lo g ical le v e ls o r above, th ere was no marked d iffe re n c e in the dose dependent in h ib it io n o f p ro te in synthesis in r a t hepatocytes.
I t is o f in te r e s t to note here th a t i t has been suggested th a t g lu ta th io n e d e p le tio n per se may lead to l i p i d p ero x id a tio n (Anundi e t al_, 1979).
This hypothesis is fu r th e r supported by the r e s u lts o f
Younes and Siegers (1 9 8 1 ).
G lu ta th io n e , to g eth er w ith g lu ta th io n e
peroxidase, provides a defence mechanism w ith in the c e ll by removing hydrogen peroxide and hydroperoxides formed during l i p i d p e ro x id a tio n as w ell as e le c t r o p h ilic in term ed iates which may c o v a le n tly bind- to macromolecules o r i n i t i a t e l i p i d p e ro x id a tio n .
G lu tath io n e d e p le tio n
in is o la te d .h e p a to c y te s may lead to l i p i d p ero x id a tio n re s u ltin g in c e ll death p r io r to the a lk y la tio n o f macromolecules expressing i t s e l f as an im portant c e ll d e s tru c tiv e mechanism.
In the experim ents
c a rrie d out by Anundi e t £]_ (1979) paracetamol served as a p ro te c tiv e agent ag ain st l i p i d p e ro x id a tiv e a tta c k which may be due t o . i t s a f f i n i t y fo r the microsomal monooxygenase system and cytochrome P-450 hence reducing l i p i d peroxide form ation v ia the m etabolic a c tiv a tio n o f o th er su b s tra te s .
The in vivo s ig n ific a n c e o f the p ro te c tiv e a c tio n
o f paracetamol may only be s lig h t since i t also depletes g lu ta th io n e and is hep ato to xic,alth o u g h com petition f o r cytochrome P-450 may lead to reductions in a c tiv e m etab o lite production from both s u b s tra te s .
More d e ta ile d in v e s tig a tio n s in to p ro te in metabolism in d ic a te d th a t paracetamol also caused in h ib it io n o f both p ro te in degradation and p ro te in s e c re tio n .
Furthermore the in h ib it io n o f p ro te in synthesis
and p ro te in sec retio n was not s p e c ific to one major class o f p ro te in s , e .g . i n t r a c e ll u l a r or e x p o rt, but ra th e r was a general in h ib it io n . re s u lts from two experiments suggested th a t although synthesis was
The
in h ib ite d a t a concentration o f 40mM paracetamol the uptake o f amino acids in to the i n t r a c e ll u l a r amino acid pool was n o t.
This l a t t e r
fin d in g must however be tre a te d w ith some caution because subsequent in v e s tig a tio n o f the method used f o r these two experiments in d ic a te d th a t the hepatocytes may have been contaminated w ith la b e lle d amino acid since the q u a n tity o f in t r a c e ll u l a r la b e lle d amino acid counted f o r both the tre a te d and co n tro ls was unexpectedly high (> 3 0 0 0 DPM/ mg c e llu la r p ro te in ) and any d iffe re n c e s would not have been seen.
Paracetamol was also found to cause a dose- and tim e-dependent decrease in the 0 -d e e th y la tio n o f 7-ethoxycoum arin.
When the e f f e c t
o f d if f e r e n t concentrations o f paracetamol on the production o f the in d iv id u a l and conjugated m etabolites was in v e s tig a te d i t was found to have a more marked e f f e c t on sulphate conjugation than on glucuronic .a cid co n ju g atio n .
At 20mM paracetamol both conjugation re a c tio n s
were in h ib ite d m arkedly.
At 40mM paracetamol th ere was minimal
conjugation o f the m etabolites o f 7-ethoxycoumarin whereas the le v e l o f fre e 7-hydroxycoumarin was in c re a s in g .
Paracetamol (40mM) caused a ra p id decrease in c e l l u la r ATP le v e ls w ith in two minutes o f exposure.
I t also in h ib ite d endogenous
re s p ira tio n in a dose dependent manner. had l i t t l e
In c o n tra s t paracetamol
o r no e f f e c t on c y c lic AMP le v e ls .
'
F in a lly incubation o f is o la te d r a t hepatocytes w ith paracetamol a t 20 and 40mM, spiked w ith
[ ^ c ] -p aracetam o l, produced dose-
dependent covalent binding o f paracetamol (3 1 .8 and 65.4nmole paracetam ol/ mg p ro te in re s p e c tiv e ly ) o r i t s m etabolites to c e l l u la r macromolecules w ith in 5 minutes o f in cu b a tio n .
The r a te o f covalent binding tended
to plateau a f t e r the i n i t i a l ra p id phase.
Paracetamol has been shown to cause acute v a ria tio n s in a number o f c h a ra c te ris tic s o f r a t hepatocytes which are not a t tr ib u t a b le to leakage o f cofactors from the c e l l .
One o f the e a r l ie s t responses o f
the c e lls to exposure w ith high le v e ls o f paracetamol appears to be a marked f a l l
in ATP.
Marked reductions in endogenous r e s p ir a tio n
and p ro te in synthesis also occur q u ite r a p id ly .
These changes occur
p r io r to a marked d e p le tio n o f g lu ta th io n e and are probably r e v e rs ib le
i f exposure tim e is r e l a t i v e l y s h o rt.
Exposure f o r longer periods
o f tim e in prim ary c u ltu re however leads to ir r e v e r s ib le plasma membrane damage and subsequent c e ll death.
A number o f recent studies have been c a rrie d out using in v it r o systems to in v e s tig a te the metabolism and t o x ic it y o f paracetam ol. Is o la te d hepatocytes have been used by a number o f workers (Moldeus, 1978;
Moldeus e t al_, 1980;
Jo llo w , 1980;
McLean, 1978) where
hepatocytes from r a t , mouse o r hamster have been in v e s tig a te d .
The
re s u lts o f Moldeus (1978) and McLean (1978) support our fin d in g o f the r e l a t i v e l y low s u s c e p t ib ilit y o f is o la te d r a t hepatocytes to the hep ato toxic e ffe c ts o f paracetam ol.
Moldeus (1978) id e n t if ie d
the form ation o f glucuronic a c id , su lp h a te , g lu ta th io n e and cystein e conjugates o f paracetamol by both the r a t and mouse.
His re s u lts
suggested th a t sulphation had a higher a f f i n i t y fo r paracetamol than glu cu ro n id atio n whereas glu cu ro n id atio n had a higher c a p a c ity .
The
le v e l o f sulphate conjugate formed by mouse hepatocytes was lower than th a t seen in the r a t whereas the form ation o f the g lu ta th io n e conjugate was f a s te r in the mouse.
His re s u lts also suggested th a t
the-mouse was more suscep tib le to the hepatotoxic e ffe c ts o f paracetam ol.
In r e la tio n to varying s u s c e p t ib ilit y o f d if f e r e n t animal species some re s u lts were obtained here f o r the hamster, a species which in vivo is more su s cep tib le to the to x ic e f f e c t o f paracetam ol.
P ro te in
synthesis was in h ib ite d in a dose-dependent manner by paracetam ol. The p r o f ile o f th is in h ib it io n was however s lig h t ly d if f e r e n t to th a t observed in r a t hepatocytes.
The e f f e c t o f lower concentrations
was more marked w ith the ID^q being about 5mM paracetamol (9mM in r a t ) and ImM causing about a
35% in h ib it io n .
Furtherm ore, an
in te re s tin g phenomenon was observed w ith the e ffe c ts o f paracetamol on the in t e g r it y o f the plasma membrane o f hamster hepatocytes w ith low concentrations being c o n s is te n tly more to x ic than h ig h er con cen tratio n s.
This phenomenon may be p a r t i a l l y exp lained by the
re s u lts o f Jollow (1980) who found th a t increasin g the co n centration o f paracetamol led to a g re a te r production o f glucuronic acid m etabolites than g lu ta th io n e conjugates.
This may be regarded as
a p ro te c tiv e mechanism since the a f f i n i t y o f paracetamol in the hamster is g re a te r fo r the g lu ta th io n e conjugation pathway and th e re fo re a t low concentrations more a c tiv e m etab o lite is produced.
The re s u lts reported have demonstrated th a t r a t hepatocytes can a c tiv e ly m etabolise exogenous s u b s tra te s .
Furthermore paracetamol
caused dose dependent reductions in g lu ta th io n e le v e ls and is c o v a le n tly bound to p ro te in .
I t may th e re fo re be assumed th a t
paracetamol was m etabolised by the system and a t high concentrations an a c tiv e m e ta b o lite was produced which formed a g lu ta th io n e conjugate.
As alread y discussed paracetamol caused dose dependent reductions in the a c t i v it y o f 7-ethoxycoumarin 0-d eeth ylase and a t low concentrations i t had a more marked e f f e c t on the sulphate conjugation o f 7-hydroxycoumarin than on glucuronic acid co n ju g atio n .
This
could be due to the f a c t th a t th ere was com petition f o r a v a ila b le a c tiv e sulphate or th a t paracetamol has a higher a f f i n i t y f o r the sulphotransferase than 7-hydroxycoumarin.
To p a r tic ip a te in sulphate
conjugation c e llu la r sulphate must f i r s t be a c tiv a te d to 3-phosphoadenosine 5'-phosphosulphate (PAPS) which req u ires two molecules o f ATP.
Sulphate conjugation is th e re fo re r e l a t i v e l y expensive in ATP
and since paracetamol has a h igher a f f i n i t y f o r sulphation than glucu ro n id atio n (Moldeus, 1978) exposure o f hepatocytes to high le v e ls o f paracetamol may r e s u lt in a d e p le tio n o f c e l l u la r ATP and su lp h ate.
The re s u lts reported here would tend to support t h is .
The form ation o f glucuronic acid and g lu ta th io n e conjugates also u t i l i s e ATP as an energy source, e ith e r d ir e c t ly or in d ir e c t l y .
As
supplies o f ATP become lim itin g the systems w ith the g re a te r a f f i n i t i e s fo r ATP w i l l be spared a t the expense o f those systems w ith poorer a ffin itie s .
The enzymes c a ta ly s in g the synthesis o f g lu ta th io n e have
a higher a f f i n i t y fo r ATP than those req u ired f o r the form ation o f a c tiv e sulphate (PAPS) (Jones, 1981).
A th e ra p e u tic dose o f 0 .6 - 1 g o f paracetamol in man leads to a con centration o f l-2mM paracetamol in the p o rta l plasma.
The
concentrations used in the experiments w ith is o la te d hepatocytes o f up to 40mM paracetamol are th e re fo re very high.
Exposure o f
hepatocytes to paracetamol might be expected to lead to p r i o r it y u t il is a t io n o f a v a ila b le ATP to enable m etabolic clearance o f paracetamol v ia more w ater soluble m etabolites which can be excreted from the c e l l .
At high concentrations the sulphate and glucuronic
acid pathways become satu rated (Moldeus, 1978) hence the metabolism o f paracetamol v ia the g lu ta th io n e conjugation pathway mediated by the microsomal monooxygenase system w i l l become more a c tiv e leading to a d e p le tio n o f c e llu la r g lu ta th io n e .
The f i r s t stage o f p ro te in synthesis also requires ATP to e s t e r if y in d iv id u a l amino acids to t h e ir corresponding tRNAs: Amino acid + tRNA + ATP
» ami noacyl-tRNA + AMP + PP^
A consequence o f d e p letio n o f c e llu la r ATP may w ell be an in h ib it io n o f p ro te in synthesis a t the a c tiv a tio n stage o f the tra n s la tio n process.
A
f u r th e r consequence o f th is may be a feedback in h ib it io n o f RNA synthesis since less would be required fo r p ro te in syn thesis.
Paracetamol w i l l cause gross t o x i c it y to hepatocytes i f the con centration is high enough and the exposure fo r long.enough.
P r io r
to the development o f serious c e ll damage paracetamol causes reductions in p ro te in syn th e s is , degradation and s e c re tio n , g lu ta th io n e , ATP, and basal r e s p ir a tio n .
The e ffe c ts o f a l l xen obiotics however are not
s im ila r as has been seen w ith re s u lts f o r phenobarbitone and s a fro le reported here.
For example Gluud £ t a]_ (1979) have shown th a t xanthine
and some o f i t s d e riv a tiv e s in h ib ite d the synthesis o f p ro tein s in is o la te d r a t hepatocytes a t the tra n s la tio n a l le v e l in is o la te d r a t hepatocytes w ith no e ffe c ts on c e l l u la r c y c lic AMP, p ro te in s e c re tio n , amino acid tra n s p o rt o r degradation o f p ro te in s . Furthermore M attei e t al_ (1979) have shown th a t d ie t h y l- and dim eth yln itrosam ine i n h ib it p ro te in synthesis in is o la te d r a t hepatocytes w ith no e f f e c t on the energy statu s o f the c e lls and th a t th is is a r e v e rs ib le process.
They suggest th a t the cap acity f o r these nitrosam ine
d e riv a tiv e s to in h ib it p ro te in synthesis in d ic ates th a t the hepatocytes
are capable o f m etabolising them to t h e i r a c tiv e m etabolites medi ated by cytochrome P -450.
The N -hyd ro xylation o f paracetamol is also mediated by cytochrome P-450 w ith a requirem ent fo r NADPH and oxygen.
High concentrations o f
paracetamol w i l l lead to s a tu ra tio n o f the sulphate and glucuronic acid conjugation pathways r e s u ltin g in more being m etabolised by cytochrome P-450 dependent pathways to a c tiv e m e ta b o lite s .
Paracetamol
a t a concentration o f lOmM,which was shown to have n e g lig ib le e ffe c ts on the plasma membrane o f is o la te d hepatocytes but in h ib ite d p ro te in synthesis by about 50% was in v e s tig a te d cytoch em ically in c u ltu re s . I t was found to cause s lig h t reductions in G6PDH a c t i v i t i e s w ith in 6 hours) w ith more marked e ffe c ts on SDH a c t i v i t i e s by about 60% w ith in 6 hou rs).
The f a l l
(30% (reduced
in SDH a c t i v it y is in d ic a tiv e
o f m itochondrial e ffe c ts o f paracetamol which would cause a red u c tio n in the production o f ATP and a reduced oxygen consumption by the hepatocyte.
The e f f e c t o f lOmM paracetamol on
diaphorase
however was a marked increase in a c t i v it y to about 200% o f the control value w ith in 6 hours.
This r e s u lt may suggest th a t the response o f the c e ll to a p o t e n t ia lly to x ic chemical is to a c tiv a te the microsomal monooxygenase system to remove the in s u lt more ra p id ly v ia i t s matabolism.
This hypothesis
is fu r th e r supported by the evidence obtained which in d ic a te d th a t low le v e ls o f paracetamol enhanced the r a te o f p ro te in synthesis which could .provide the c e ll w ith an increased source o f enzymes and haemoproteins necessary to accommodate the increased a c t i v i t y o f the microcomal monooxygenase system or to r e p a ir damaged macromolecules. This re d ire c tio n o f the c e lls a c t i v i t y could r e s u lt in a compensatory slowing o f o th er anabolic and c a ta b o lic c e llu la r processes.
The
reduction in ATP s u p p lie s , supported by the reduced m itochondrial a c t i v i t y o f the c e l l , w i l l in i t s e l f lead to a slowing o f p ro te in metabolism w ith reductions in p ro te in degradation as w e ll as p ro te in syn thesis.
The a c tiv e m etabolites produced by the microsomal monooxygenase system w il l be in a c tiv a te d by conjugation w ith g lu ta th io n e u n til the c e l l u la r
g lu ta th io n e is reduced to a le v e l where i t cannot provide th is p ro te c tiv e a c tio n .
This w il l
r e s u lt in covalent binding o f a c tiv e
m e ta b o lite to c e llu la r macromolecules which may include p ro te in s , enzymes, RNA, DNA, l i p i d s , and low m olecular w eight substances lik e ATP and UDPG ( G i l l e t t e , 1981).
Covalent binding per se probably does
not lead to c e ll death since the c e ll is u s u a lly able to r e p a ir the damage and degrade the c o v a le n tly bound products.
A consequence o f
chronic exposure to high le v e ls w i l l however lead to damage from which the c e ll cannot recover w ith r e s u lta n t c e ll death.
There may be o th e r mechanisms d ir e c t ly o r in d ir e c t ly involved in the o v e ra ll to x ic response o f the c e ll to paracetamol in clu d in g e ffe c ts on Ca
4H"
f lu x and l i p i d p e ro x id a tio n .
I t is c le a r however th a t once the
le v e l o f chemical in s u lt to the c e ll increases beyond a c r i t i c a l le v e l (as y e t unknown) the compensatory mechanisms o f the c e ll'w h ic h can p ro te c t i t from t o x ic it y may f a i l
to op e ra te.
Furthermore the
to x ic ac tio n o f paracetamol on the l i v e r c e ll is not a s in g le mechanism but a composite o f a number o f p ath o lo g ical changes which are s t i l l
5 .3 .2
not f u l l y understood.
A n ilin e analogues
A v a r ie ty o f s tru c tu ra l analogues o f a n ilin e r e la te d to paracetam ol, e .g . a n ilin e , phenacetin and a c e ta n ilid e were also in v e s tig a te d fo r t h e i r e ffe c ts on p ro te in synthesis in suspensions o f is o la te d r a t hepatocytes.
They each caused a dose-dependent in h ib it io n although
the concentrations which caused a 50% in h ib it io n ( ID^ q ) v a rie d (Gwynn e t a]_, 1979).
Chemical
ID5Q(mM)
Paracetamol
9
Phenacetin
3
A c e ta n ilid e A n ilin e
.
6 .5 13.5
The metabolism o f these fo u r compounds in vivo is in te r r e la t e d (see Fig. 5 ) .
Phenacetin, a c e ta n ilid e and paracetamol a l l have an a lg e sic
and a n tip y r e tic p ro p e rtie s although the a c t i v it i e s o f phenacetin and a c e ta n ilid e are probably v ia t h e i r m etabolic conversion to paracetam ol. The ta r g e t organ f o r paracetamol t o x ic it y in vivo is the l i v e r whereas fo r phenacetin i t is p r im a rily the kidney o r blood causing renal n ec ro sis , or methaemaglobimaemia also caused by a c e ta n ilid e .
A ll fo u r
analogues, however, are hep ato toxic under the r ig h t experim ental c o n d itio n s .
In v e s tig a tio n s by Nelson et_ al_ (1981) on the metabolism
o f phenacetin in d ic a te d th a t phenacetin can be m etabolised to both re a c tiv e a r y la tin g and a lk y la tin g agents by hamsters.
Furthermore the
m u ltip le pathways leading to re a c tiv e m e ta b o lite form ation from phenacetin are d if f e r e n t to those fo r paracetam ol.
The o u tlin e in Figure 5 suggests th a t paracetamol would have the g re a te s t c a p acity f o r a c tiv e m etab o lite production in the l i v e r since f o r the o th e r th ree analogues more m etabolic steps are re q u ire d before a c tiv e m e tab o lite production becomes a major f a c t o r .
A c tive
m etab o lite form ation from phenacetin in vivo is probably v ia the kidney ra th e r than by the l i v e r which may also be the case f o r a c e ta n ilid e and a n ilin e .
I t would th e re fo re appear th a t th ere is no d ir e c t
c o rr e la tio n between a c tiv e m etab o lite form ation and the e f f e c t o f compounds on l i v e r p ro te in synthesis since phenacetin and a c e ta n ilid e are s lig h t ly more e f fe c tiv e in h ib ito r s than paracetam ol.
The
in h ib it io n o f p ro te in synthesis may be re la te d to o th e r e ffe c ts o f the compounds on the m itochondria o r in d ir e c t e ffe c ts on c e l l u la r ATP le v e ls .
5 .3 .3
S a fro le
S a fro le (4 -a lly l-l,2 -m e th y le n e d io x y b e n z e n e ) is a n a tu r a lly o ccurring substance which has been used as an a r t i f i c i a l fla v o u rin g agent in foods.
I t is hepatotoxic (Crampton e t a l , 1977) and has also been
CH
2
CHCH
2
F ig u r e 5:
The m e t a b o lic r e l a t i o n s h i p o f a n i l i n e and some analo gues
d e a c e ty la tio n ■>
Phenacetin
A c e ta n ilid e
p-Phenetedine
A n ilin e
N-Hydroxy Phenacetin .
Paracetamol
a c tive, m etabolites
p-Aminophenol
a c tiv e m e ta b o lite s
\]/
4.GHS covalent binding
N/
c e ll death
co valen t binding ?
V i/
c e ll damage
found to be a weak hepatocarcinogen in ra ts and mice (Long £ t aT_, 1963). These e ffe c ts are considered to be due to the form ation o f a c tiv e m e ta b o lite s .
One o f the m etabolic fa te s o f s a fr o le is conversion in
vivo to i t s 1-hydroxy analogue and e x c re tio n in the u rin e as the O-glucuronide (B orchert e t
al_9 1973).
The l i k e l y a c tiv a tio n pathway
is v ia the form ation o f 1-hydroxy s a f r o le , mediated by cytochrome P -450, which is then e s t e r if ie d to form an e le c t r o p h ilic r e a c ta n t.
A c h a r a c te r is tic p roperty o f s a fro le is th a t i t is capable o f form ing m etabolic products which complex w ith cytochrome P-450.
These t ig h t
complexes (product-adduct complexes) which are id e n t if ia b le s p e c tro p h o to m e tric a lly , e f f e c t iv e ly decrease the c e l l u la r content o f fu n c tio n a l cytochrome P -450, thereby in h ib it in g the metabolism o f a number o f exogenous (and endogenous) sub strates (Elcombe e t
a\_9 1979;
W errin g lo er and Estabrook, 1979).
Because
i t is l i k e l y th a t the h e p a to to x ic ity o f s a f r o le ,in common w ith
paracetam ol, is probably mediated v ia m etabolic a c tiv a tio n i t was considered ap p ro p riate to use s a fro le as a model f o r the in v e s tig a tio n o f the e a r ly to x ic changes which can occur in is o la te d hepatocytes.
Q u a n tita tiv e ly s a fro le was more hep ato toxic to is o la te d hepatocytes than paracetam ol.
At 0.1 and l.OmM th e re were n e g lig ib le e ffe c ts on
trypan blue exclusion and LDH leakage w ith in two and a h a lf hours o f incubation but a t 5 and lOmM th ere was evidence w ith in 10 minutes o f a ra p id ly induced plasma membrane damage. RNA synthesis were also in h ib ite d .
P ro tein .synthesis and
Since marked in h ib it io n did
not occur u n t il the con centration o f s a fr o le was g re a te r than ImM the observed reductions in p ro te in and RNA synthesis may be d ir e c t ly r e la te d to a reduction in c e ll v i a b i l i t y .
Experiments were not c a r r ie d
out to determine whether the e ffe c ts o f s a fro le on p ro te in synthesis in is o la te d hepatocytes were r e v e rs ib le .
At a concentration o f ImM s a fr o le in h ib ite d the resynthesis o f g lu ta th io n e . lOmM caused a marked d e p le tio n o f g lu ta th io n e ,a s w e ll as causing a marked decrease in ATP le v e ls (to 17% o f co n tro l valu es) w ith in two minutes o f exposure.
The ra te o f endogenous r e s p ir a tio n
was not a ffe c te d however by ImM s a fr o le .
S a fro le was not in v e s tig a te d
a t higher concentrations f o r i t s e ffe c ts on re s p ira tio n since under such conditions i t was seen to cause rap id plasma membrane damage. U n like the s itu a tio n w ith s a fr o le the con centration o f paracetamol which'caused a 50% in h ib it io n o f p ro te in synthesis (^lOm M ) was found to cause decreases in endogenous re s p ir a tio n as w e ll.
This may in d ic a te
th a t th ere is not an o b lig a to ry c o rr e la tio n between endogenous re s p ira tio n and p ro te in synthesis in terms o f the to x ic response by the l i v e r c e l l .
C y c lic AMP le v e ls were also reduced by lOmM s a fr o le
however th is e f f e c t may sim ply r e f le c t the plasma membrane damage caused by s a fr o le .
The metabolism o f 7-ethoxycoumarin was in h ib ite d in a dose-dependent manner.
The p a tte rn o f in h ib it io n woujd suggest th a t once produ ct-
adduct form ation w ith cytochrome P-450 was completed the le v e l o f in h ib it io n remained a t a steady s t a t e .
At a lOmM co n centration o f
s a fro le the metabolism o f 7-ethoxycoumarin was com pletely in h ib ite d which could be due to e ith e r product-adduct form ation w ith a l l a v a ila b le cytochrome P-450 o r c o fa c to r leakage due to plasma membrane damage. ■
This p a tte rn o f in h ib it io n was d if f e r e n t to th a t seen fo r paracetamol which does not form product-adduct complexes w ith cytochrome P-450. The in h ib it io n o f 7-ethoxycoumarin by paracetamol was .both dose- and tim e-dependent.
The major w ater so lu b le m etab o lite o f s a fr o le in vivo
is the glucuronide th e re fo re the metabolism o f s a fr o le has a low requirem ent f o r a c tiv e sulphate (PAPS).
The marked d e p le tio n o f ATP
by s a fro le (lOmM) is u n lik e ly to be due to the increased form ation Of PAPS but ra th e r to plasma membrane damage.
The attem pt to in v e s tig a te the e ffe c ts o f s a fro le (ImM) on p ro te in synthesis in c u ltu re d hepatocytes was la r g e ly unsuccessful.
S a fro le
caused the ra p id detachment o f hepatocytes from the c u ltu re surface although i t d id *n o t appear to im p air the v i a b i l i t y o f these hepatocytes over one hour o f in c u b a tio n .
The t o x i c it y o f s a fro le to is o la te d
hepatocytes may be re la te d to more s p e c ific e ffe c ts on the plasma
membrane r e s u ltin g in rap id changes in it s a f f i n i t y o f s a fro le fo r p la s t ic .
in t e g r it y or to the
Ir r e v e r s ib le plasma membrane
damage occurred ra p id ly a t concentrations g re a te r than ImM s a fr o le . The reductions in g lu ta th io n e , an in h ib ite d r a te o f synthesis seen a t ImM and marked d e p le tio n a t lOmM, may be re la te d to a c tiv e m e tab o lite production.
S a fro le is also an inducer o f the microsomal
monooxygenase enzyme system.
Cytochrome P-450 mediates the production
o f an a c tiv e m e ta b o lite o f s a fr o le , probably an epoxide and the reductions in g lu ta th io n e may be re la te d to the fu r th e r metabolism o f th is epoxide v ia co n ju g atio n .
D ih yd ro safro le also depletes
g lu ta th io n e in hepatocytes (personal communication: Benford, 19 8 1 ). This a c tiv e m e ta b o lite is the undoubted cause o f the t o x ic it y o f s a fro le i_n v iv o .
5 .3 .4
Phenobarbitone
Phenobarbitone is a member o f the b a rb itu ra te class o f drugs which cause CNS depression in vivo and hence sed atio n .
I t has an a d d itio n a l
im portant property in th a t i t induces the a c t i v it y o f the microsomal monooxygenase system by increasing the amounts o f cytochrome P-450 and a number o f o th e r microsomal enzymes.
In vivo t h is re s u lts in
l i v e r enlargem ent, and both in vivo and in v it r o in the increased ra te o f microsomal monooxygenase mediated metabolism o f many x e n o b io tic s .
This property o f phenobarbitone has led to i t s extensive use as a tool both fo r in vivo and in v it r o experiments on drug metabolism and t o x i c it y . For example phenobarbitone p re tre a te d preparations are commonly used to assess the c o n trib u tio n o f a c tiv e m etab o lites to c e llu la r t o x i c i t y .
It
was th e re fo re ap p ro p riate to determine what e ffe c ts phenobarbitone has on the v i a b i l i t y c h a ra c te ris tic s o f is o la te d r a t hepatocytes as i t may in flu e n c e the t o x ic it y o f xenobiotics in o th er ways than simply enhancing the ra te o f o x id a tiv e metabolism.
In experiments on trypan blue exclusion and LDH release th ere was no evidence o f marked t o x ic it y a f t e r one hours incubation w ith the concentrations o f up to 20mM th a t were in v e s tig a te d .
A fte r th ree
hours, however, 20mM caused a reduction o f up to 70% in c e ll v i a b i l i t y
although the e ffe c ts o f 1 and lOmM phenobarbitone were in d is tin g u is h a b le from the c o n tro ls .
S im ila r e ffe c ts were seen w ith LDH re le a s e .
I t was in te re s tin g to fin d th a t phenobarbitone caused a dose dependent in h ib it io n o f t o ta l p ro te in synthesis and t o ta l RNA s y n th e s is , s im ila r to th a t found f o r paracetam ol, although the concentration o f pheno barbitone which caused a 50% in h ib it io n was lo w er, because i t is w ell e stab lish ed th a t both in vivo and in v it r o phenobarbitone'causes an increase in l i v e r microsomal p ro te in .
This r e s u lt is however supported
by the re s u lts o f Tuma e t al_ (1978) who also demonstrated th a t sec retio n o f glycop ro teins by r a t l i v e r s lic e s was im paired by phenobarbitone.
They suggested th a t the in h ib it io n o f sec reto ry
p ro tein s may be an a d d itio n a l fa c to r c o n trib u tin g to l i v e r enlargement as a r e s u lt o f treatm ent w ith phenobarbitone.
Although phenobarbitone was found to be more to x ic to c u ltu re s a f t e r chronic exposure f o r a few days,a con centration o f l-2mM allowed continued c e ll attachm ent.
A concentration o f ImM phenobarbitone
was found to cause marked increases in the a c t i v i t y o f the microsomal monooxygenase system demonstrated by the increased a c t i v i t y o f 7 ethoxycoumarin 0-d e e th y la s e .
Furthermore 2mM phenobarbitone produced
marked increases in the a c t i v it y o f NADPI^ diaphorase, G6PDH and SDH a c t i v it i e s which were demonstrated cyto ch em ically.
Phenobarbitone a t a concentration o f 4mM caused about a 50% in h ib it io n o f p ro te in synthesis and in h ib ite d the resynthesis o f g lu ta th io n e w ith in the hepatocyte.
At a high con centration (20mM) which caused
about a 90% in h ib it io n o f p ro te in synthesis the e f f e c t on g lu ta th io n e was s im ila r to th a t o f 4mM phenobarbitone.
The le v e ls o f g lu ta th io n e
were not depleted below the o r ig in a l control le v e ls by phenobarbitone. D ire c t d o s e -re la te d d e p le tio n o f g lu ta th io n e by phenobarbitone th e re fo re did not occur instead an in h ib it io n o f the resynthesis which is maximal a t concentrations o f phenobarbitone g re a te r than 4mM. This may be re la te d to the e ffe c ts o f phenobarbitone on the e le c tro n tra n s p o rt chain where i t probably acts as an uncoupler causing a reduced production o f ATP and reduced oxygen consumption, supported by the re s u lts obtained here.
The la rg e reduction in the le v e l o f
ATP may be responsible f o r the in h ib it io n o f the resynthesis o f g lu ta th io n e .
Phenobarbitone th e re fo re causes inductio n and possibly a c tiv a tio n o f c e rta in haemoproteins and enzymes in is o la te d hepatocytes.
I t may
be th a t a f t e r acute exposure the c e ll switches to support the enhanced synthesis o f these pro tein s due to the s p e c ific a f f i n i t i e s o f phenobarbitone fo r the microsomal monooxygenase system.
As a
r e s u lt the synthesis o f o th er p ro tein s and RNAs not req u ired may be in h ib ite d r e s u ltin g in an o v e ra ll in h ib it io n o f t o ta l p ro te in and RNA syn th esis.
Experiments w ith phenobarbitone in c u ltu re s
suggested th a t the marked e f f e c t on p ro te in synthesis over one hour in suspensions o f hepatocytes was not m aintained on longer term exposure.
The re s u lts may even be in te rp re te d to suggest th a t
exposure to 5mM phenobarbitone fo r e ig h t hours caused an increase in p rotein' synthesis between 24 and 48 hours o f c u ltu re although too much w eight should not be placed on the re s u lts o f th is experim ent since the re s u lts were very v a r ia b le .
A fu r th e r im p lic a tio n from the re s u lts
w ith phenobarbitone is th a t p ro te in synthesis in h ib it io n is not d ir e c t ly re la te d to d e p le tio n o f g lu ta th io n e le v e ls .
5 .3 .5
Carbon te tr a c h lo rid e
Carbon te tr a c h lo rid e is one o f the c la s s ic a l model hepatotoxins and much work has been dedicated to e lu c id a tin g the mechanism o f i t s h e p a to to x ic ity .
Carbon te tr a c h lo rid e is m etabolised by the microsomal
monooxygenase system to a fre e ra d ic a l CC1^*•
I t was thought th a t
th is a c tiv e species was the cause o f l i p i d p e ro x id a tiv e a tta c k on c e llu la r membranes r e s u ltin g in c e ll death.
S la te r e t a t (1980)
have demonstrated, w ith the use o f pulse r a d io ly s is , th a t CCl^* is produced by the l i v e r c e ll and although r e l a t i v e l y a c tiv e and capable o f c o v a le n tly binding to c e llu la r macromolecules i t is not the p e ro x id a ti species.
CClg
is fu r th e r m etabolised by an oxygen dependent re a c tio n
to produce a h ig h ly re a c tiv e pero^yradical (CCl^O^’ ) .
This l a t t e r
species is more l i k e l y to be responsible fo r c e ll death due to l i p i d p eroxidation and p ro te in damage.
The high r e a c t iv it y o f th is species
w i l l lead to damage occurring a t i t s s it e o f form ation in the endoplasmic
re tic u lu m .
The less re a c tiv e CC13 * and secondary products can
d iffu s e from the s it e o f a c tiv a tio n and cause c e ll damage a t other s ite s .
Kieczka and Kappus (1980) provide fu r th e r support f o r the oxygen dependence o f the form ation o f l i p i d peroxides w ith the suggestion th a t carbon te tr a c h lo rid e competes fo r the oxygen binding s ite s on cytochrome P-450.
Low le v e ls o f oxygen w il l allo w g re a te r access
o f carbon te tr a c h lo rid e f o r metabolism to a c tiv e m etab o lites y e t a low oxygen con centration w i l l i n h ib it the form ation o f a p e ro x id a tiv e species.
The few re s u lts reported here fo r carbon te tr a c h lo rid e in d ic a te th a t i t is markedly to x ic to the plasma membrane o f is o la te d r a t hepatocytes and causes reductions in oxygen consumption and p ro te in syn th e sis . In d ic a tio n s were also seen th a t low le v e ls produced increases in NADPH^ diaphorase a c t i v it y suggesting the i n i t i a t i o n o f s e l f p ro te c tiv e mechanisms w ith in the l i v e r c e l l .
There was also a suggestion from
cytochemical in v e s tig a tio n s , although not q u a n tifie d , th a t cyclohexim ide. had a m ild p ro te c tiv e actio n a g a in s t the t o x i c it y . o f carbon te tr a c h lo r id e in c u ltu re s .
The p ro te c tiv e e f f e c t o f cycloheximide is supported by the re s u lts o f Castro e t al_ (1 9 7 7 ), Lindstrom and Anders (1977) and Popp e t al_ (1 9 7 8 ). The concept th a t p ro te in synthesis plays an a c tiv e r o le in the production o f c e l l u la r damage produced by carbon te tr a c h lo r id e is probably however too s im p lis t ic .
Carbon te tr a c h lo rid e induced l i v e r
in ju r y is probably a composite o f more than one p a th o lo g ical change.
Endogenous p ro te c tio n ag ain st l i p i d p e ro x id a tio n may w ell be mediated by g lu ta th io n e v ia g lu ta th io n e peroxidase.
Lowering o f g lu ta th io n e
le v e ls by d ie th y l maleate has been associated w ith enhanced malondialdehyde form ation by carbon te tr a c h lo rid e (Lindstrom e t al_, 19 7 8 ). D ieth yl maleate alone however produces l i p i d peroxidation- and g lu ta th io n e d e p letio n (Stacey and P r ie s t ly , 1978) whereas only high doses o f carbon te tr a c h lo rid e ( 2 0 ^ 1 /m l) produced g lu ta th io n e d e p le tio n and c e ll damage w ith o u t concomitant l i p i d p ero x id a tio n .
P o li e t al (1979) have re c e n tly confirmed th a t carbon te tr a c h lo r id e stim u lates l i p i d pero xid atio n in is o la te d hepatocytes.
I t also
in h ib it s both p ro te in synthesis and p ro te in and lip o p ro te in s e c retio n and stim u lates f a t accumulation w ith in l i v e r c e l l s , a l l s im ila r fin d in g s to those seen a f t e r in vivo treatm en t w ith carbon t e tr a c h lo r id e .
I t was also confirmed th a t the increase in l i p i d
p ero xid atio n is mediated by i t s microsomal monooxygenase metabolism.
5 .3 .6
A summary o f some postulated mechanisms o f t o x i c it y
Is o la te d hepatocytes in suspension o r prim ary monolayer c u ltu re have been used here to in v e s tig a te the e ffe c ts o f paracetam ol, phenobarbitone and s a fro le on the l i v e r .
Chem ically induced changes
in a number o f parameters have been id e n t if ie d which may or may not be re la te d to t o x i c it y .
Examination o f the changes caused in d ic a te s th a t th ere is one sequence o f events which is common to a l l th ree compounds. by a rap id f a l l
This is c h a ra c te ris e d
in ATP le v e ls follow ed by in h ib it io n o f p ro te in synthesis
and RNA syn thesis.
Furthermore ir r e v e r s ib le plasma membrane damage was
observed in is o la te d hepatocytes a f t e r exposure to a l l th ree compounds: 4ATP
^ p r o t e in s y n th e s is
^RNA synthesis — »; c e ll death
S a fro le was found to have the most marked to x ic e ffe c ts in hepatocytes w ith ir r e v e r s ib le plasma membrane damage
occurring a f t e r a sh o rt
exposure time to r e l a t i v e l y low co n cen tratio n s .
Phenobarbitone was
to x ic to hepatocytes in suspension and c u ltu re although ir r e v e r s ib le plasma membrane damage occurred l a t e r than w ith s a fr o le .
The e ffe c ts
o f paracetamol in suspensions o f r a t hepatocytes were in c o n s is te n t. Marked t o x ic it y was seen, however, in c u ltu re d hepatocytes a f t e r prolonged exposure to high co n cen tratio n s. Scale o f t o x ic it y : S a fro le
>
Phenobarbitone
>
Paracetamol
Paracetamol caused marked reductions in ATP and p ro te in synthesis a t a concentration o f 40mM.
The in h ib it io n o f p ro te in synthesis was found
to be r e v e rs ib le , however, in c u ltu res which were exposed to paracetamol
i
f o r sh o rt periods o f tim e.
I t is th e re fo re probable th a t the reductions
in ATP are also r e v e rs ib le .
Furthermore i t is u n lik e ly th a t reductions
in p ro te in synthesis and RNA s yn th e sis , or ATP are d ir e c t causes o f ir r e v e r s ib le plasma membrane damage:
c e ll death
p ro te in synthesi~ ^ c e ll death nI rNA
synthesis
The microsomal monooxygenase (MMO) enzyme system is involved in the metabolism o f paracetam ol, s a fro le and phenobarbitone.
The m ajor
m etabolic pathways fo r paracetamol are v ia metabolism to the sulphate and glucuronic acid conjugates.
At high co n cen tratio n s , however,
these pathways become satu rated and metabolism v ia the microsomal monooxygenase system becomes a major pathway producing more to x ic ra th e r than less to x ic m etabolites in the form o f a c tiv e a r y la tin g or a lk y la tin g species.
S a fro le forms a product-adduct complex w ith
cytochrome' P-450 hence the MMO enzyme system plays a m ajor ro le in the metabolism o f s a fro le probably v ia form ation o f an epoxide.
One minor
m etab o lite o f phenobarbitone may be a d ih ydrodiol which would re q u ire i n i t i a l metabolism to an epoxide d e r iv a tiv e .
A ctive m e ta b o lite form ation
may also in vo lve the production o f r e a c tiv e oxygen species.
R eactive
e le c tr o p h ile s , in a c tiv a te d by conjugation w ith g lu ta th io n e catalyse d by g lu ta th io n e tra n s fe ra s e o r peroxidase, w i l l dep lete i n t r a c e ll u l a r g lu ta th io n e : RH NADPH cytochrome P-450 NADH ROH l i p i d p e ro x id a tio n and c e ll death
GSH \
peroxidase GSSG
GSH-
>
RSG
GSSG = o xid ised g lu ta th io n e
Paracetamol and s a fro le both caused dose-dependent d e p le tio n g lu ta th io n e .
of
Phenobarbitone, however, reduced the ra te o f resynthesis
o f g lu ta th io n e but did not a c tu a lly dep lete i t in a dose-dependent manner.
I t has been postu lated th a t d e p le tio n o f g lu ta th io n e alone may
lead to c e ll death v ia l i p i d p e ro x id a tio n .
Prolonged treatm en t w ith
paracetamol may th e re fo re r e s u lt in c e ll death due to l i p i d p e ro x id a tio n . ,/
A ctive a r y la tin g or a lk y la tin g m etabolites may c o v a le n tly bind to p ro te in o r o th e r c e l l u la r m a te r ia l.
High concentrations o f paracetamol
were shown here to cause ra p id dose-dependent binding o f d ru g -re la te d m a te ria l to p ro te in s .
Furthermore covalent binding o f s a fr o le and is o s a fro le
is known to occur a t le v e ls below those which cause t o x i c it y .
Thus i t
is u n lik e ly th a t covalent binding alone causes c e ll death.
Oxygen consumption was reduced by both paracetamol and phenobarbitone, but not by s a f r o le , a t the concentrations in v e s tig a te d here. was also found to i n h ib it succinate dehydrogenase a c t i v i t y .
Paracetamol The
mitochondrion may, th e r e fo r e , be su s cep tib le to the d ir e c t e ffe c ts o f paracetamol and phenobarbitone but is u n lik e ly to be a d ir e c t ta r g e t f o r the to x ic e ffe c ts o f s a fr o le .
The p ro p e rtie s o f these th ree compounds d i f f e r f u r th e r in th a t phenobarbitone and s a fr o le are both inducers o f the microsomal monooxygenase enzyme system.
Phenobarbitone induces cytochrome P-450 whereas s a fr o le induces
a novel form o f cytochrome P-450.
Phenobarbitone's r o le as an inducing
agent was supported here by i t s e ffe c ts on 7-ethoxycoumarin 0-d eeth ylase and NADPH2 diaphorase a c t i v it i e s in c u ltu re d hepatocytes.
Phenobarbitone
also caused increases in the a c t i v it y o f glucose-6-phosphate dehydrogenase and succinate dehydrogenase.
Paracetamol caused increases in the a c t i v i t y
o f NADPH2 diaphorase but decreases in glucose-6-phosphate dehydrogenase and succinate dehydrogenase.
Substrates w ith an a f f i n i t y f o r cytochrome
P-450 may cause a c tiv a tio n o f the MMO enzyme system which could e x p la in the e f f e c t o f paracetamol on NADPHg diaphorase a c t i v i t y .
The e ffe c ts
o f s a fro le on these parameters were not in v e s tig a te d in c u ltu re d hepatocytes since s a fr o le caused rap id detachment o f c e lls from the c u ltu r e s u rfa c e .
One would a n t ic ip a te , however, th a t concentrations o f s a f r o le ,
which did not cause rapid t o x i c it y , would increase the a c t i v it y o f 7-ethoxycoumarin 0-deethylase and NADPl^ diaphorase in hepatocytes.
The re s u lts obtained here have not id e n t if ie d the d ir e c t cause o f c e ll death although they would tend to suggest th a t i t is not due to one fa c to r alone but the combination o f a number o f fa c to r s .
In other,
words chem ically induced t o x ic it y is re la te d to the le v e l o f in s u lt and the du ratio n o f exposure and a reduced cap acity o f the hepatocyte to r e p a ir o r reverse chem ically induced to x ic changes.
A number o f common mechanisms have been id e n t if ie d which may be r e la te d to the c e ll death caused by paracetam ol, s a fro le and phenobarbitone:
T co valen t binding • T l i p i d p ero x id a tio n ?
^ 4 > p ro tein synthesis - iRNA synthesis
------------------ ^ ? ----------------> nec ( l a b i l i t y to re p a ir damage) ce
iGSH iATP
The rap id decrease in ATP le v e ls was the e a r l ie s t change id e n t if ie d in the studies c a rrie d out here.
I t is not known, however, whether th is
was a d ir e c t e f f e c t o f the chemicals in v e s tig a te d or whether i t was secondary to an e a r l i e r chem ically induced change.
Furthermore th e re
might be o th er changes occurring which are c a u s a lly r e la te d to c e ll death which have not been id e n t if ie d .
More d e ta ile d in v e s tig a tio n s in to the e ffe c ts on ATP may y i e l d f u r th e r in fo rm atio n in to mechanisms o f t o x i c it y .
Other areas which might be
looked a t in hepatocytes are the r e la tio n s h ip between g lu ta th io n e le v e ls and the onset o f l i p i d pero xid atio n and the e f f e c t o f chemicals on calcium f lu x o r the tra n s p o rt o f o th e r e s s e n tia l e le c t r o ly te s .
C H A P T E R
6
C O N C L U S I O N S
CONCLUSIONS 6-1
the value of is o la te d hepatocytes for stu d ying the t o x ic
EFFECTS OF CHEMICALS Is o la te d hepatocytes have a number o f advantages over the use o f whole animals fo r in v e s tig a tin g the to x ic e ffe c ts o f chemicals which may be summarised as fo llo w s : 1.
Less animals required
2.
Sm aller tis s u e samples th e re fo re a sm alle r requirem ent fo r chemicals
3.
A la rg e number o f rep ro d u cib le samples from one animal
4.
The tis s u e is removed from the in flu en c es o f hormonal c o n tr o l, and the environment
5.
The n u trie n ts may be c a r e fu lly defined in the media used f o r incubations
6.
The " d ie t" is e a s ily manipulated v ia media v a r ia tio n
7.
The o v e ra ll cost o f experiments is le s s .
These advantages are supplemented by the technique used here in th a t comparison between d if f e r e n t species in clu d in g humans is fa c ilita te d .
Furthermore the h is to r ic a l data base f o r these
p re p a ra tio n s , g iv in g support to the s i m il a r it i e s o f is o la te d hepatocytes to l i v e r tis s u e in v iv o , is in c re a s in g .
There are however a number o f
disadvantages to using is o la te d hepatocytes some o f which have been h ig h lig h te d by the re s u lts reported here.
The l i v e r is a complex tis s u e made up o f a number o f d if f e r e n t c e ll types and areas.
The to x ic e ffe c ts o f chemicals are o fte n r e s t r ic te d
to one area o f the l i v e r , e .g . paracetamol causes c e n tr ilo b u la r n ecro sis.
Chemicals whose t o x ic it y is caused by h ig h ly r e a c tiv e
m etabolites produced by the microsomal monooxygenase system are more l i k e l y to produce c e n tr ilo b u la r necrosis since cytochrome P-450 is more p reve lan t in the c e n tr ilo b u la r hepatocytes (Gooding e t al.» 1978). Furthermore Smith e t al_ (1979) demonstrated an uneven d is t r ib u t io n o f g lu ta th io n e w ith in the l i v e r lobule by cytochemical means fin d in g th a t the c e n tr ilo b u la r c e lls contained less reduced g lu ta th io n e than o th e r regions in the l i v e r lo b u le which may fu r th e r support the s u s c e p t ib ilit y
o f these c e lls to the to x ic e ffe c ts o f m e ta b o lic a lly a c tiv a te d chem icals.
A suspension o f hepatocytes contains a homogeneous m ixture o f l i v e r c e ll types from the d if f e r e n t regions o f the l i v e r lo b u le .
I t is
th e re fo re im possible to id e n t if y h is t o lo g ic a lly the s it e w ith in the l i v e r a t which damage is o cc u rrin g .
I t is also possible th a t
hepatocytes may lose t h e i r in d iv id u a l s p e c ific fu n ctio n s in is o la t io n . Furthermore i f the e ffe c ts o f a chemical are s lig h t in the acute phase, or even the chronic phase, these w i l l be q u a n tit a tiv e ly d ilu te d out by the presence o f la rg e numbers o f unaffected c e l l s . A to x ic e f f e c t may th e re fo re need to be q u ite marked before i t is detected in a c e ll suspension.
I f the chemical is a ffe c tin g a l l the
c e lls to a s im ila r e x te n t then i t is possible th a t the to x ic e f f e c t in v it r o d if f e r s from th a t in v iv o .
The is o la tio n procedure may
also introduce a fu r th e r problem o f r e la t iv e re s istan ce to damage by adaptation o f hepatocytes to t h e i r new environment.
Some o f these problems can be overcome by using c u ltu re d hepatocytes since a h is to lo g ic a l exam ination can be made and cytochem istry can be used to id e n t if y changes in p a r t ic u la r c e ll types.
I t is apparent from the re s u lts reported here th a t th e re is a marked v a r i a b i l i t y in the preparations o f hepatocyte suspensions and c u ltu r e s . This v a r i a b i l i t y is less o f a problem in terms o f the q u a n tita tio n o f fu n c tio n a l parameters l ik e c o fa c to r le v e ls and m etabolic c a p a c ity than in the response to to x ic e ffe c ts o f chem icals.
To c l a r i f y
th is p o in t i t was apparent from these re s u lts th a t the hepatocytes derived from some animals were f a r more suscep tib le to the to x ic e ffe c ts o f paracetamol than o th e rs .
We are th e re fo re unable to
remove the in d iv id u a l v a r ia tio n between animals when using hepatocytes in v it r o since they not only fu n c tio n as they would in vivo but also m aintain the in d iv id u a l c h a ra c te r is tic s o f the animal from which they were d e riv e d .
This problem may be p a r t i a l l y
overcome by m aintaining more rigorous standards f o r animal husbandry and animal s e le c tio n .
The f a c t th a t hepatocytes in an is o la te d s itu a tio n are removed from the in vivo hormonal co n tro ls and a blood supply is probably a major advantage i f one is try in g to c h a ra c te ris e a w ell defined m etabolic pathway o r p h y sio lo g ical fu n ctio n o f the l i v e r .
I t is
c le a r ly apparent however th a t mechanisms o f t o x ic it y are not e a s ily defined and co n sis t o f many v a ria b le s .
Furthermore in vivo the
pharm acokinetic d is tr ib u tio n o f a drug and i t s m etab o lites can play a major ro le in i t s t o x i c i t y , and the body has o v e ra ll defense mechanisms which although undefined c le a r ly a id in reve rs in g t o x i c it y .
The in vivo blood supply provides a continued source o f n u trie n ts and oxygen to the l i v e r c e l l , and serves to remove r a p id ly components produced by the l i v e r in clu d in g p ro tein s and drug m e ta b o lite s .
The
b ile and lymph also provide fu r th e r pools in to which the l i v e r c e ll may excrete or s e c re te .
The l i v e r is th e re fo re under continuous f lu x .
The suspension o f hepatocytes and c u ltu re s used here s u ffe r from the disadvantage o f not being under continuous f lu x thereby su b jec tin g the l i v e r c e ll to high concentrations o f p o t e n t ia lly to x ic chemical f o r abnormally, prolonged periods o f tim e.
Furthermore the incubation
media may concentrate unwanted to x ic by-products produced by the l i v e r which may enhance the to x ic response o f the c e ll and in flu e n c e the m echanistic pathway by which t o x i c it y occurs.
Although the disadvantages.discussed here may seem numerous- and insurmountable the use o f is o la te d hepatocytes has a wide p o t e n t ia l. The research reported here serves to i l l u s t r a t e th a t the system in use needs to be w ell defined before s u b jec tin g i t to to x ic chem icals. Once the control hepatocyte in v it r o is more f u l l y understood in te r p r e ta tio n o f to x ic changes should prove to be more e a s ily accomplished.
The use o f hepatocytes as a c y to to x ic screen is now
being in v e s tig a te d .
Tyson e t al_ (1980) compared the e ffe c ts o f 23
chemicals on GOT release fin d in g a good c o rre la tio n between the e ffe c ts seen in v i t r o , in hepatocytes prepared by the method used h e re , and those reported in v iv o .
This type o f screening f o r chemicals is n o t,
however, very in fo rm a tiv e and can give m isleading r e s u lts .
I t is
hoped th a t i t w il l be some tim e, i f a t a l l , before th is type o f ro u tin e
te s tin g is adopted fo r screening the t o x ic it y o f chem icals.
Continued
in v e s tig a tio n s in hepatocytes however may lead to the p ro v is io n o f a more s u ita b le model than the whole animal fo r e lu c id a tin g the mechanisms by which chemicals are to xic,w h ich is f a r more valu ab le than ju s t knowing whether or not they are to x ic .
Future in v e s tig a tio n s in to mechanisms o f t o x ic it y w i l l probably be more productive i f g re a te r a tte n tio n is given to the use o f is o la te d hepatocytes in prim ary monolayer c u ltu re ra th e r than in suspension.
Cultured
hepatocytes allo w g re a te r f l e x i b i l i t y w ith the a p p lic a b ilit y o f a w ider range o f experim ental techniques.
To achieve th is end the q u a lity o f c u ltu re s must f i r s t be improved to enable r e p r o d u c ib ility o f preparations w ith s im ila r p ro p e rtie s , la r g e r numbers o f c e lls attached and a more p re d ic ta b le l i f e
span.
F u rth er
in v e s tig a tio n s in to the id e n t it y and a c t i v it y o f the MMO enzymes present in c u ltu re w ith the aim o f o b tain in g a s im ila r p r o f ile to th a t present in vivo would provide a more r e lia b le system to work w ith .
F u rth er improvements may be made by .using animals o f b e tte r q u a lity or even germ fr e e anim als.
Consideration o f d ie t and environm ental
conditions p r io r to s a c r if ic e may also help to produce p rep aratio n s o f a b e tte r q u a lity .
There is a need to re d e fin e the c o n stitu en ts o f the media used during the is o la tio n procedure and f o r incubation o f the c u ltu re s .
C areful
m anipulation o f the c o n stitu en ts w ith a con sid eration o f o th e r supplements may d efin e more s u ita b le media.
This is e s p e c ia lly re le v e n t to the le v e ls
o f g lu ta th io n e and cytochrome P-450 w ith in the hepatocytes.
The a v a i l a b i l i t y o f a w ell defined c u ltu re system w i l l provide a more r e lia b le in v it r o model fo r the in v e s tig a tio n o f chem ically induced t o x i c it y .
The w ider range o f techniques th a t may be used to a id these
in v e s tig a tio n s include phase co n tra s t e le c tro n microscopy and the a p p lic a tio n o f q u a n tita tiv e cytochemical analyses, in a d d itio n to the enzyme marker approach used here.
The use o f hepatocytes is o la te d from
g e n e tic a lly d if f e r e n t s tra in s o f ra ts or the g re a te r use o f s e le c tiv e
m etabolic in h ib ito r s may provide fu r th e r in fo rm a tio n .
More d e ta ile d tim e
sequencing o f chem ically induced changes may enable more p o s itiv e conclusions to be made on the mechanisms o f t o x i c it y .
The re s u lts from w ell designed and c a r e fu lly executed experiments may e v e n tu a lly lead to the e lu c id a tio n o f the mechanisms leading to chem ically induced c e ll death.
Furthermore the v a lid a tio n o f is o la te d
hepatocytes as a tool f o r in v e s tig a tin g the to x ic e ffe c ts o f chemicals may enable t h e i r use f o r p re d ic tin g more a c c u ra te ly the e ffe c ts o f chemicals in man.
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