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MEASUREMENT OF OXIDATIVE STRESS-INDUCIBLE GENE EXPRESSION IN THE RAT ALVEOLAR MACROPHAGE CELL LINE NR8383 AS A TOXICOLOGICAL MODEL

by

GEOFFREY W. PATTON

A d is s e rta tio n submitted in p a rtia l fu lfillm e n t o f the requirements fo r the degree o f Doctor o f Philosophy Department o f Pathology and Laboratory Medicine College o f Medicine U n ive rsity o f South F lo rid a

A p ril 1997

Major Professor: Sue A. Shelley, Ph.D.


UMI Number: 9724018

UMI Microform 9724018 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI

300 North Zeeb Road Ann Arbor, MI 48103


Graduate School U n ive rsity o f South Florida Tampa, F lo rida

CERTIFICATE OF APPROVAL Ph.D. D isserta tion This is to c e r tify th a t the Ph.D. D isserta tion of

GEOFFREY W. PATTON

with a major in Pathology and Laboratory Medicine has been approved by the Examining Committee on March 24, 1997 as s a tis fa c to ry fo r the d isse rta tio n requirement fo r the Doctor o f Philosophy degree

Examining Committei Major Professor: Sue A. Shell Shelley,' Ph.D. Member: G loria C 2 .5 -fo ld increase in expression and s e n s itiz a tio n to 500 /*M H202 re fle cte d in an apparent 8 -fo ld induction, s e ve ra l-fo ld higher than H202 alone and s ig n ific a n t at p < 0.005 (see Discussion).

61


combined treatment o f 4500 /zM MS with 500 jzM H202.

RT-PCR o f control

samples evaluated by scanning densitometry at a d ilu tio n equivalent to .008 ng//zl (1:15,764 d ilu tio n ) o f RNA had a mean absorbance f o r (3-ACTIN o f 179 (n = 6) which was s t a t is t ic a lly in d istin g u ish a b le from samples treated only with 500 /zM H202 (mean = 177, n = 6 ).

When these two data

sets were combined and tested fo r sig nifican ce against (3-ACTIN expression level in samples treated with both 4500 /zM MS and 500 y.W H202 (mean = 8 6 , n = 5), the re s u lt was p o sitive at p < 0.01.

Thus, i t

appears th a t the combined treatment a c tiv e ly reduces (3-ACTIN mRNA le v e ls .

Since our re s u lts are normalized to the (3-ACTIN le ve ls between

co n tro ls and treated samples, the reduction in (3-ACTIN may account fo r the apparently greater GADD45 and GADD153 inductions in c e lls treated w ith both 4500 /zM H202 and MS.

This being the case, the GADD45 and

GADD153 induction le ve ls seen w ith the combined treatments may be the

maximum a tta in a ble fo r treatments o f H202 and 4500 /zM MS in th is c e ll 1i ne.

GADD34.

In te re s tin g ly , the apparent in h ib itio n o f GADD34 in

response to H202 was fu rth e r re fle cte d in the combined exposure treatments using H202 and MS in which there was a dose-response in h ib itio n to increasing concentrations o f H202 against the backdrop o f MS exposure (Figure 14).

As w ith the 7500 jzM MS treatment, the

combination o f 500 /zM H202 and 4500 /zM MS depressed GADD34 expression below the control le v e l.

62


G A D D 34 P e r c e n t In d u c tio n

CONTROL

4500

5 /4 5 0 0

5 0 /4 5 0 0

5 0 0 /4 5 0 0

M i c r o m o l a r C o n c e n t r a t io n o f H 2 0 2 / M e r c a p t o s u e c i n a t e

FIGURE 14.

The GADD34 response to combined treatments o f hydrogen

peroxide and mercaptosuccinate is negative, declining to approximately 50% o f the le ve ls at e ith e r sham-treated or treated only w ith MS.

63


HSP70.

HSP70, on the other hand, was responsive to 4500 jtM MS and

5 /zM H202/4500 /zM MS and was strongly induced at 50 /zM H202/4500 jzM MS and 500 jzM H202/4500 /zM MS (Figure 15).

The 50 n M H202/4500 *zM MS HSP70

induction was s t a t is t ic a lly s ig n ific a n t at p < 0.05 and the other combination treatment were s ig n ific a n t at p < 0.01.

HO-1.

Figure 16 shows th a t HO-1 was approximately equally induced

a t 4- to 6 -fo ld fo r the 4500 /zM MS, 5 /zM H202/4 500 jzM MS and 50 /zM H202/ 4500 n M MS treatments in contrast to the 9 -fo ld induction seen w ith 500 /zM H202 alone and the 12-fold induction seen w ith 500 /zM H202/4500 /zM MS.

64


8000

H S P 70 P e r c e n t In d u c tio n

In 6000

4000

2000 ln=2

[n=3|

CONTROL

4500

5 /4 5 0 0

M ic r o m o la r C o n c e n tr a tio n

FIGURE 15.

5 0 /4 5 0 0

5 0 0 /4 5 0 0

o f H 2 0 2 /M e r c a p to s u c c in a te

HSP70 is induced equally about 4 .5 -fo ld by 4500 y.W

mercaptosuccinate alone and MS followed by 5 /nM hydrogen peroxide but is stro n g ly induced at higher H202 le v e ls .

The 6 0 -fo ld induction at 500 /zM

H202/4500 /iM MS is s t a t is t ic a lly s ig n ific a n t at p < 0.05.

Note th a t the

Y-axis scale is changed compared to Figure 14.

65


H O —1 P e r c e n t I n d u c t i o n

1500

r~

1200

-

CONTROL

4500

5 /4 5 0 0

5 0 /4 5 0 0

5 0 0 /4 5 0 0

M ic r o m o la r C o n c e n t r a t io n o f H 2 0 2 /M e r c a p t o s u c c in a t e

FIGURE 16.

HO-1 is also induced by mercaptosuccinate and appears to be

also s tro n g ly induced by the maximal combined treatments.

Note th a t the

Y-axis scale is changed compared to Figure 15.

66


DISCUSSION

The purpose o f th is in ve stig a tio n was to seek a mechanistic model fo r examining the b io lo g ic a l e ffe c ts o f a ir p o llu tio n .

Chemicals

s u ffic ie n tly to x ic to a rre st the c e ll cycle often do so by damaging DNA in ways that are im portant causative fa cto rs in the carcinogenic process and which abrogate normal p rote ctive functions in c e lls .

Hydrogen

peroxide, which fre e ly permeates the nuclear as well as other c e llu la r membranes, does not react extensively w ith DNA but, under su ita b le conditions, forms hydroxyl radicals via the Fenton reaction involving tra n s itio n metal ions and causes DNA damage in the forms o f oxidized nucleotides and s in g le strand breaks (Mello Filho and Meneghini, 1984). GADD gene expression in response to such DNA damage in other c e ll

systems is re a d ily observable in 4 hours (Fornace e t a l . , 1988).

The

genotoxic stress o f DNA damage can occur during ph ysiolo gic processes (e .g ., inflammation, reperfusion in ju r y , chemotherapy) or as a re s u lt o f environmental p o llu ta n t exposure and can e l i c i t a complex gene expression response in mammalian c e lls , which may re s u lt in growth in h ib itio n , enhanced protection or tolerance, and a lte re d mutagenicity (Fornace, 1992).

Our in te re s t is in the a b ilit y to detect early

response to DNA damage as a biomarker fo r environmental oxid ative stre ss.

67

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GADD gene expression and induction have been reported in a ll normal mammalian c e ll types examined; however, th is is the f i r s t d e s c rip tio n o f the baseline and induction le ve ls fo r alveolar macrophages.

The present study has shown the expression p r o file s o f

fiv e o xid a tive stress-responsive genes in normal NR8383 ra t a lv e o la r macrophages and NR8383 tre a te d with H202 w ith and without GPx in h ib itio n by mercaptosuccinate.

The re s u lts raise the p o s s ib ility th a t these

genes play important ro le s in the pathogenesis o f pulmonary diseases in v o lv in g the in h a la tio n o f p a rtic le s capable o f inducing o xid a tive stre s s .

Alveolar macrophages are p a rtic u la r ly endowed w ith fu n ctio n al

a b ilit ie s th a t increase the r is k of exposure to oxidative stress through phagocytosis o f radical-m ediating chemicals such as iron and s ilic a from inhaled p a rtic le s .

As phagocytes, they also generate elevated

endogenous reactive o x id a tiv e species through the re sp ira to ry burst associated w ith phagocytosis.

Although human alveolar macrophages are

more s e n s itiv e to noxious substances from airborne p a rticu la te s than ra t a lv e o la r macrophages when phagocytotic a b ilit y and ce ll v ia b ilit y are quantitated (Hadnagy et a l . , 1993), the a v a ila b ilit y o f the NR8383 ra t a lv e o la r macrophage c e ll lin e provides considerable economic and lo g is t ic e ffic a c y . Evaluation o f the responses by each o f the genes in dica tes complex association w ith loss o f c e ll p r o life r a tiv e capacity, supporting the view th a t there is a response to the to x in s .

In cases such as the MS

treatm ents, the response is bell-shaped ( i . e . , quadratic) in th a t induction declines at the highest (7500 fiH) treatment.

Likewise, gene

expression le ve ls c o n s is te n tly increase across the spectrum o f combined 68


treatments, except fo r 6ADD34.

The apparent adaptive response to doses

s u ffic ie n tly high to cause growth r e s tr ic tio n would seem to be exceeded at some o f the high treatments concentrations/com binations.

Gene

expression elevated 2- to 4 -fo ld at e q u ito xic doses o f H202 (500 ^M) and MS (4500 n M) occurred fo r GADD45, GADD153, HO-1, and HSP70, w ith the la tte r two responding stronger to the H202.

This e ffe c t was am plified

at the highest combined treatment, to some degree possibly a ttrib u ta b le to loss o f (3-ACTIN.

A tte stin g against th is view, however, is the

continued d ire c tio n w ith loss o f GADD34 at the high combined treatm ents. Thus, through a realm o f gene inductions, NR8383 respond to the oxidative challenge o f H202 or MS. Because i t is d i f f i c u l t to separate the genotoxic e ffe cts o f treatment agents from the grow th-arresting e ffe c ts , i t is important to determine the influence o f such agents on the growth and v ia b ilit y o f c e lls (Crawford e t a /., 1996).

I t was fo r th is reason th a t H202 and MS

treatment le v e ls were selected which yielded comparable in h ib itio n o f c e ll growth in h ib itio n at which to compare GADD and heat shock gene response.

Thus, d iffe r e n tia l gene expression can properly be a ttrib u te d

to the respective treatments and not differences in cytopathic e ffe c ts . In the current work, GADD45 expression increased 5 -fo ld when c e lls were treated w ith 500 /xM H202 and GADD153 expression increased 2 .5 -fo ld , a level 50% th a t o f GADD45.

Heat shock genes HSP70 and H0-1 rose

comparably, w hile GADD34 a ctu a lly decreased.

Since glutathione is a

p rin cip al biomolecule by which c e lls p rotect themselves against oxidative damage such as that produced by H202, i t is reasonable to expect th a t in h ib itio n o f GPx would produce s im ila r GADD gene expression 69


changes as those seen w ith exogenous H202.

When GPx was in h ib ite d w ith

1500-4500 /zM MS, GADD45 expression was induced approximately 3 -fo ld and GADD153 approximately 2- to 2 .5 -fo ld .

These re su lts support a ro le fo r

GPx in p ro te ctin g c e lls from genotoxic damage.

In ad dition , HSP70 and

HO-1 gene expression were also increased in response to MS.

Combined

exposures o f H202 w ith MS resulted in even greater induction f o r a ll but GADD34, demonstrating modulation o f the a n tio xida nt pathway p ro te ctin g

DNA from o xid a tive damage that can occur at sub-lethal to x ic exposures. Indeed, elevated le ve ls o f GPx in sta b ly-tra n sfe cte d c e lls have been shown to p rote ct against UVB-induced DNA strand breaks mediated in part by H202 (Ghosh e t a l . , 1993). In the absence o f oxidant s tim u li, the basal level o f GADD45 was higher than GADD153 when normalized to 0-ACTIN (Table 1 Control Means) and HO-1 > GADD34 > HSP70 (Table 2).

When normalized to &-ACTIN, GADD45

expression increased to a greater degree than GADD153, and GADD34 decreased w hile HSP70 and H0-1 both increased to comparable degrees. However, the known promoter sequences do not support a common re la tio n s h ip fo r these patterns o f induction (Table 3).

T ra d itio n a lly ,

i t has been popular to examine gene response fo r common s tim u li as a

70



Table 3. Regulatory elements reported fo r the o x id a tiv e stress-response genes in th is study.

IL -6E /

CCAATT StRE

TRE HpoRE

TPA

HprRE

USE p53 NFkB C/EBP-6 AP-1 AP-2 ARE (HIF-1) HSE MTE CdRE CAAT SP1 TATA PRE (STAT)

GADD34 (none reported)

++

GADD45 2

GADDI5 3

+

(-)

HSP70-1 HO-1

+

+

+x3 +

+x3 +x5

+

p o s itiv e inducer or enhancer sequence

++

strong p o s itiv e induction

x#

m u ltip le site s

0

known not to be present

(-) p53 in h ib its a C/EBP-0 by competition

+

+

+x2

+

+x2 +

+

+X2 0

+ +

p re d ic to r o f common pathways o f induction but i t is ra p id ly becoming c le a r th a t there are numerous complex routes, often with various peptide subunits competing fo r stim ula tory or in h ib ito ry p o sitio n s.

Thus, at

th is time i t seems prudent to await f u lle r d e scrip tio n o f regulatory sequence fu n c tio n a litie s and to focus on patterns o f induction to classes o f toxins as a means to understand mechanisms and possible remedies. The unique reaction o f GADD34 to H202 is contrasted w ith the m ild induction to low MS concentrations and suggests th a t there may e ith e r be differences between H202 and MS in the s p e c ific means o f to x ic it y or there may be m u ltip le sig n a lin g elements involved, or both. conundrum remains to be elucidated.

This

S t i l l , the purported ro le o f GADD34

in regulation o f p rote in synthesis (Chow and Roizman, 1994; Yanez et a7., 1995) may be re fle c te d in the patterns o f induction seen w ith combined treatments w ith H202 and MS.

The apparent reduction in GADD34

mRNA at 500 /xM H202 a fte r 4500 /xM MS conceivably could represent a threshold level at which p rote in synthesis re g u la tio n is reversed and new synthesis may no longer be permitted.

These suggestions are

speculative, at best, at th is time pending confirm ation o f the GADD34 PCR product sequence and b e tte r information on the ro le of the Gadd34 p ro te in .

However, the c le a r differences in induction patterns seen w ith

th is gene compared to the other GADD genes endorses the view th a t genomic response to stress necessarily must include down-regulation o f some genes while discordant genes are up-regulated. GADD153 is normally low in expression and ra p id ly increases in

response to oxidative stress (Guyton et a l . , 1996).

Oxidative stress

72


strongly induces the expression o f C-FOS and C-JUN and AP-1 binding of the heterodim eric p a ir to DNA is m ild ly stim ulated by H202 (Devray e t a l . , 1991).

Both HO-1 and GADD153 are known to be regulated by AP-1

and co-regulation o f other genes by these may occur through carbon monoxide released by the action o f heme oxygenase or Gaddl53 protein competition w ith C/EBP-/?.

A n t i o x i d a t i v e P r ot e c t i on

In the cu rre n t work, we have shown th a t NR8383 a lv e o la r macrophages have basal stress gene expression tha t responds w ith in 4 hours a fte r o x id a tiv e challenge by H202 or MS.

By co n tra st, GADD34 is

weakly induced, i f at a l l, and decreases w ith the combined o xidative stress o f H202 w ith MS pretreatment, contrary to it s previously described ro le .

The c e llu la r response to H202-induced o xid a tiv e stress

is not re s tric te d to GADD genes.

In h ib itio n o f DNA synthesis by

g lutathione depletion o f T lymphocytes increased HSP70 (heat shock protein 70) and GADD153 mRNA levels (Walsh e t a l . , 1995).

Further, i t

previously has been shown that overexpression o f CuZn-superoxide dismutase produces an oxidative stress h y p e rs e n s itiv ity which can be blocked by overexpression o f GPx (Amstad et a l . , 1994) thus underscoring the fa c t th a t small imbalances in physiological a c tiv itie s o f antioxidants can a lte r oxidant-induced DNA damage and patterns o f ce ll death.

As mentioned previously, the GADD genes f i r s t cloned (A34, A45,

and A153; la te r named GADD34, GADD45, and GADD153) do not respond to heat shock (Fornace et a l ., 1989).

Yet, we have shown th a t the heat 73


shock genes HSP70-1 and HO-1 are also stro n g ly induced by endogenous or exogenous H202-stim ulated oxidative stress.

These gene products lik e ly

serve a n tio x id a tio n p ro te ctive functions and may co-regulate other genes through disparate mechanisms cu rre n tly being elucidated by many la b o ra to rie s . In b a cte ria such as E. c o l i and Salmonella typhimurium, s p e c ific o xidative stress prote ctio n genes are up-regulated by the potent SOS response, superoxide ( SoxR regulon) or by H202 ( OxyR regulon) mediated by "sensing" p ro te in s.

Nothing sim ila r has ye t been found fo r

eukaryotes but the a c tiv a tio n o f AP-1 subunits c - f o s and c-j un , NF-jc-B, e r g l , B c l - 2 , p53 and possibly other tra n s c rip tio n fa c to rs , promoters,

and regulators o f gene expression lend support to the consideration th a t H202 and other ROS modulate signal transduction through redox a lte ra tio n s o f s ig n a llin g proteins or through the a b ilit y to a ffe c t c e llu la r GSH (Burdon, 1995).

Further evidence o f the enigmatic nature

o f signal transduction in alveolar macrophages is re fle c te d in the report th a t a d iffu s ib le spore product o f A s p e r g i l l u s fumigatus s p e c ific a lly in h ib ite d the a ctiva tio n o f NF-kappa B and AP-1, both required fo r the coordinate upregulation o f tra n s c rip tio n of the proinflammatory cytokines TNF alpha, IL-1 beta, and IL-6 (Nicholson et a l . , 1996).

The relevance o f the present model system to the s itu a tio n in vivo remains to be in ve stig a te d.

C e rtainly, the 500 pH hydrogen peroxide

concentration used to in h ib it p ro life ra tio n is lik e ly higher than would be encountered in viv o, even at sites o f inflammation.

Cells produce

hydrogen peroxide during c e llu la r re sp ira tio n and phagocytes generate 74


additional hydrogen peroxide in phagosomes as part o f the re s p ira to ry bu rst.

Low le ve ls o f H202 promote growth response in many rodent and

human c e ll types; s tim u la tin g the expression o f immediate e a rly response genes c-fos , c -j u n and e r g l depending on the c e ll type, species and redox state o f the c e lls (Burdon, 1995).

The in tr a c e llu la r steady-state

level o f H202 has been stated as 10‘8 to 10'9 M (Mello F ilho , 1984; Boveris and Chance, 1973) w ith up to 0.5 nmol/h/104 c e lls being released by diverse types o f tumor c e lls (Burdon, 1995).

Isolated ra t peritoneal

macrophages exposed fo r 1 hr to surgical glove starch p a rtic le s released as much as 2878 nM/106 c e lls H202 (Renz et a l ., 1993).

NR8383 a lve o la r

macrophages produce 5-20 nmol/106 c e lls 4 hr fo llow in g in it ia t io n o f incubation w ith P. c a r i n i i and 30-40 nmol/106 c e lls i f stim ulated with zymosan (Hidalgo at a l ., 1992). However, these are iso lated measurements o f the dynamic processes o f production versus n e u tra liz a tio n .

Concentrations o f H202 less than

300 /xM had no e ffe c t on fib ro b la s t trypan blue exclusion but DNA synthesis is a ll but o b lite ra te d ir r e v e r s ib ly at >200 Ames, 1994).

(Chen and

Indeed, since alve o la r macrophages are known to contain

large amounts o f a n ti oxidants, McDonald et a l . , (1991) determined th a t as few as 2.5 x 105 is o la te d ra b b it alve o la r macrophages could scavenge 200 mM H202 in 30 min.

Thus, w hile serum and other medium components,

c e ll types, species, gender and other fa cto rs can d ir e c tly a ffe c t experimental re s u lts , i t remains la rg e ly unclear to what degree in v i t r o exposures using exogenous H202 can model the molecular mechanisms underlying gene expression in vivo (Burdon, 1995).

S t i l l , oxid ative

75


damage may a ffe c t c r it i c a l c e llu la r metabolic pathways through DNA nicks and irre v e rs ib le p ro te in degradation observable as changes in stre ssresponsive mRNA le ve ls in advance o f other hallmarks o f o xid a tiv e damage such as lip id peroxidation and necrosis. The current re s u lts are consistent w ith reported inductions in other model systems such as the above-mentioned 300 n M H202 treatments o f fib ro b la s ts which had no e ffe c t on c e ll v ia b ilit y but e s s e n tia lly o b lite ra te d DNA synthesis (Chen and Ames, 1994) and w ith the known increase in both p ro te in level and tra n s c rip tio n a l a c t iv it y o f p53 during oxidative stre ss, given th a t Gadd45 is a downstream e ffe c to r fo r p53 (Zhan et a l . , 1995).

Gadd45 and p21Waf1/c,p1 act as downstream

e ffe c to rs o f both p53-dependent and p53-independent DNA damage-response pathways to cause growth a rre st pending DNA re p a ir (Zhan et a l ., 1995; Kearsey et a l . , 1995).

Gadd45 and p2 i w“n/c,p1 may compete (Chen e t a l . ,

1995; Kearsey et a l . , 1995) fo r binding to PCNA ( p ro life ra tin g c e ll nuclear antigen) which binds c y c lin D1 (Smith et a l ., 1994).

The Gadd45

homologue MyD118 and p ossibly other proteins also function in s im ila r but d is tin c t pathways c o n tro llin g suppression o f c e llu la r growth and apoptosis (Vairapandi e t a l . , 1996).

Taken together, these fa c ts

support the idea th a t the in te ra c tio n s help coordinate the c e ll cycle and DNA re p a ir (Hall et a l ., 1995).

Also, i t is lik e ly th a t GADD45 and

GADD153 s y n e rg is tic a lly c o n trib u te to growth in h ib itio n as

overexpression demonstrates an ad d itive e ffe c t (Zhan et a l . , 1994). The c e ll lin e , NR8383, chosen as the basic te s t system fo r th is model, has been well characterized by others and recently was u tiliz e d to study changes in inflammatory cytokine gene expression d ifferen ce s 76


between in fe c tio n by two s tra in s o f reovirus (Farone et a l . , 1996).

The

purpose o f th e ir study was to compare the induction o f proinflammatory cytokine mRNA and p ro te in expression in vivo and in v i t r o .

I t was found

that the reovirus type 3 Dearing, which causes a prominent neutrophil in flu x in a ra t model o f v ir a l pneumonia, produced greater mRNA and protein le v e ls o f macrophage inflammatory protein 2 (MIP-2) and tumor necrosis fa c to r alpha than the le s s -in fe c tio u s type 1 Lang reovirus in both animals and NR8383 c e lls .

UV-inactivated v iru s was non-stim ulatory

and b a cte ria l 1ipopolysaccharide was syn e rg istic fo r MIP-2 and KC.

In

sum, NR8383 respond to these two v ir a l stra ins in a pattern s im ila r to primary a lve o la r macrophages; demonstrating th a t the normal in tr a c e llu la r pathways th a t detect and respond to in fe c tio u s challenge function ty p ic a lly .

Since the exact nature o f the im m ortalization o f

such c e lls is not yet known, the fa c t that NR8383 c e lls s t i l l demonstrate c r it ic a l macrophage functions supports the v a lid it y o f the selection o f th is c e ll lin e fo r modelling gene expression responses.

Patterns o f Gene Expression

DNA s u p e r-s h ift assays (Kurata, 1996) im plicated the o xid a tive a ctiv a tio n o f NF-kappa B over the TPA-Responsive Element (TRE) m o tif as being responsible fo r the HO-1 induction in Ml c e lls , suggesting th a t the same mechanism may be acting in the current induction o f HO-1 in NR8383 c e lls .

The ro le o f increased AP-1 binding a c tiv ity in HO-1

induction fo llo w in g hyperoxia as a model o f oxidant-induced lung in ju ry in ra ts fu rth e r supports th is contention in th a t elevated HO-1 gene 77


expression was seen in vivo in bronchoalveolar epithelium , in t e r s t it ia l and inflammatory c e lls and in v i t r o in cultured e p ith e lia l c e lls , fib ro b la s ts , macrophages, and smooth muscle c e lls (Lee et a l . , 1996). However, recent work has suggested that several classes o f inducers may be involved in th a t an extended AP-1 binding s ite resembling the maf oncogene and the tra n s c rip tio n fa c to r NF-E2 along w ith the AP-1 s ite (Inamdar et a l . , 1996) and antioxidant-responsive elements (ARE) (Prestera, 1995) can regulate in d u c ib ility in some models.

I n t e r r e l a t i o n s Between Stress-Response Genes

NR8383 c e lls in c u ltu re appear to p ro life ra te norm ally and respond predictably to treatment w ith chemicals known to cause o x id a tiv e -s tre s s induced growth in h ib itio n .

This work has shown th a t the to x ic e ffe c ts

o f H202, presumably through hydroxyl radical damage observed in the reduced p ro life r a tio n o f NR8383 alveolar macrophages, are associated w ith induction o f GADD45 and GADD153.

The overall response p ro te ctive

o f the genome lie s in delaying c e ll cycle progression u n til DNA re p a ir can be accomplished (Gately et a l . , 1994; Luethy and Holbrook, 1992). The demonstrated o xid a tive induction o f tra n s c rip ts fo r GADD153 in addition to GADD45 and the heat shock genes HSP70 and HO-1 supports the view that H202 in th is model is stim ulating a generalized c e llu la r response to DNA damage.

GADD153 induction is known to be mediated in

part by an AP-1 binding s ite in the promoter region (Guyton et a l . , 1996), a widespread genomic regulatory domain fo r inducible genes. M ichiels and Remade (1988) showed th a t chemical in h ib itio n o f GPx 78


in h ib ite d v i a b i l i t y o f cultured human WI-38 fib ro b la s ts such th a t 21-30% in h ib itio n o f GPx by 1 micromolar MS re s u lts in 50% reduction in c e ll v i a b ilit y and a t 50 /xM, the c e lls die w ith in 7 days when plated at 100 cells/cm 2 w ith 10% FBS.

With the NR8383 r a t a lv e o la r macrophages in our

hands, 4500 /xM MS was required to achieve 50% reduction in p r o life r a tio n , the d ifference possibly a ttrib u ta b le to the la rg e r number o f c e lls (4 x 105/m l) in our system.

An a lte rn a tiv e explanation could

stem from d iffe re n ce s in antioxidant le ve ls in phagocytes compared with fib ro b la s ts , as previously mentioned. The apparent a d d itive co n trib u tio n to GADD gene response by the combination o f MS w ith H202 treatments in the current work suggests surplus capacity fo r c e llu la r reaction to more severe DNA damage than seen w ith e ith e r MS or H202 alone.

However, the decreased amount of 0-

ACTIN at severe treatment levels lik e ly accounts fo r most o f the

a d d itiv e e ffe c ts .

There are several evident hypotheses fo r how (3-ACTIN

gene expression could be affected by severe treatments in th is system. In studies o f (3-ACTIN gene tra n s c rip tio n in iso la te d r a t hepatocytes, i t has been seen th a t glutamine-induced c e ll sw elling stim ulated (.3-ACTIN tra n s c rip tio n (Husson et a l . , 1996).

These authors found th a t

hypoosmolarity increased expression w hile hyperosm olarity decreased /3ACTIN mRNA, suggesting th a t i f the o xidative stress treatm ents in our

system were d is ru p tin g the c e lls ’ perm eability which would lead to sw e llin g , an opposite e ffe c t would occur. could respond d iffe r e n tly than hepatocytes.

A lte rn a tiv e ly , macrophages In ra t a lv e o la r macrophages

exposed to A s p e r g i l l u s extracts, (3-ACTIN is unaffected despite sp e cific in h ib itio n o f NF-kappa B and AP-1 tra n s c rip tio n fa c to rs (Nicholson et 79


a l . , 1996).

Thus, i t would appear th a t only where o xid a tive stress is

s u ffic ie n t to cause widespread damage does f3-ACTIN gene expression decline w hile other induced gene levels remain high. There remain many unresolved questions before phagocytes can re a d ily be used to study p a rtic u la te t o x ic it y .

I t is important to know

i f metals or other p a rtic le constituents d ir e c tly a ffe c t post-treatm ent aspects o f the assay such as amounts o f RNA is o la te d , reverse tra n scrip ta se a c t iv it y or PCR e ffic ie n c y , to name a few.

For example,

in one study the usual te s t o f gene in d u c ib ility re ly in g on a m eta lloth io n e in promoter was unusable due to s tim u la tio n o f the gene by the metal ion in the m etallothionein. using the appropriate co n tro ls. begs fu rth e r study.

These points can be addressed

Also, the pathobiology o f macrophages

Gaucher’ s Disease (gene: GBA) is a metabolic

disease characterized by glucocerebroside-loaded phagocytes; saturated phagocytes might be analogous to p a rtic le -la d e n a lve o la r macrophages in terms o f abrogating fu nction.

Does senescence or quiescence occur in

saturated phagocytes and are stress-response genes in d u cib le by toxins under such conditions?

More inform ation on the in d u c ib ilit y of c u ltu re

c e ll stress genes both "per passage" and at various growth phases would be h e lp fu l.

Also o f in te re s t would be changes in stress gene

responsiveness fo llo w in g v ir a l in fe c tio n o f macrophages.

Oxidative Stress-Resistant Cells

A number o f recent studies have examined c e ll lin e s and tumor c e lls fo r the acquired resistance to oxida tive stre ss.

Hamster d ip lo id

80


fib ro b la s ts (HA-1) and c e ll lin e s iso lated fo r resistance to e ith e r H202 (0C14) or 02 (02R95) were examined fo r GADD153, c -ju n , heme oxygenase-1, and HSP70 since the re s is ta n t c e ll lin e s were already known to have augmented a n tio xid a n t defenses in the forms o f g lu ta th io n e content and enhanced catalase, superoxide dismutase, and g lu ta th io n e peroxidase a c tiv itie s .

Northern analysis showed increased basal HO-1, c - j u n , and

GADD153 w ith no d ifference in HSP70 leading to the conclusion th a t the

elevated gene expression may play a ro le both in the H202 resistance phenotype as w ell as providing an ad d itio n a l mechanism fo r enhanced ce ll survival in clonogenic studies (Guyton et a l ., 1996). That induction o f stress response genes in normal c e lls may be b e n e ficia l has been suggested by protein measurement using human peripheral blood leukocytes exposed to H202 or the H202-generating system xanthine oxidase/hypoxanthine in which a 27 kDa p ro te in and a 32 kDa p rotein (p u ta tiv e ly HO-1) were expressed (Marini et a l ., 1996). Pre-treatment w ith mild hyperthermia or o xidative stress increased survival o f c e lls exposed to high doses o f oxidants w ith maintenance of p r o life r a tiv e capacity underscoring the p ro te ctive ro le o f o xidative stress-responsive genes in preventing and repa irin g damage. The ubiquitous protection provided by stress response genes lik e HO-1 has been fu rth e r demonstrated in renal BSC-1 c e lls where both H202 and hemin can induce HO-1, presumably through a common pathway in that hemin was not a d d itiv e to H202 c y to to x ic ity (da S ilva e t a l . , 1996).

As

w ith the hyperthermia and oxidative stress study mentioned above, long term (24 hours) exposure o f the BSC-1 c e lls to both hemin and H202 conveyed resistance to a subsequent acute oxidative stress.

Thus, i t is

81


becoming c le a r th a t the lung’ s adaptive and/or p rote ctive response to oxid a tive stress may re ly h ea vily on heat shock-type ( i. e . , HO-l and HSP70) and GADD gene in d u c ib ility (Choi and Alam , 1996).

I t remains to

be determined i f long-term stress gene expression confers possible b enefits o f protection from disease.

Biomarkers

Various d e fin itio n s have been applied to the concept o f biomarkers fo r environmental exposures (Borm, 1994).

At the core o f th is concept

is a mechanistic connection between dose and relevant e ffe cts (Shulte, 1996).

Using known o xid a tive stress-responsive RNA induction appears

valuable and se n sitive as a m echanistically-based, biom arker-oriented approach fo r environmental p a rtic u la te c y to to x ic ity .

While the nature

o f antioxidant protection against g en otoxicity remains complex, the approach o f quantifying the c e lls ’ own genomic response to DNA damage seems e ffica cio u s fo r studying o xid a tive to x ic ity from causes such as environmental p a rtic u la te s .

As ju s t one example, various mineral fib e rs

could be d iffe re n tia te d at e q u ito xic exposures fo r exponentially-growing human-hamster hybrid c e lls as determined by the induction o f HO p rotein (Suzuki and Hei, 1996). One challenge facing researchers seeking to understand the c e llu la r consequences o f a ir p o llu tio n is the inherent complexity o f p a rtic u la te s th a t may have both soluble and insoluble constituents (Crouch, 1990).

Soluble components have been found to be the to x ic

mediators o f residual o il f l y ash (ROFA) but n e u tra liz a tio n o f the acid 82


ROFA in s t ille d IT in to ra ts potentiated lung c e ll in ju ry as measured by p ro te in , lactose dehydrogenase, albumin, and c e llu la r content consistent w ith the formation o f secondary, apparently more to x ic p a rtic le s (Dreher et a l . , 1996).

RT-PCR to detect genes encoding inflammatory mediators

demonstrated increases in fib ro n e c tin , vascular c e ll adhesion molecule1, E -se le ctin , IL-ljS , IL-5, IL-6, and macrophage inflammatory prote in -2 (MIP-2), w ith no increase in TNF-a.

An in v i t r o system using phagocytes

provides an opportunity to more clo se ly mimic the in vivo s itu a tio n where c e lls may be subjected to soluble and in so lu ble m aterials.

This

has previously been suggested in the context o f human peripheral blood monocytes allowed to d iffe re n tia te in 10- to 14-day cu lture and then exposed to p a rtic le s fo r phagocytosis studies (Hadnagy et a l . , 1993). While e ffe c tiv e in terms o f good agreement w ith rodent in vivo in h a la tio n experiments, the missing component is inform ation on the mechanistic connection between reduced phagocytic capacity and s p e c ific to x in s .

RT-PCR provides a sensitive to o l to v is u a liz e early c e llu la r

response to genotoxic agents at concentrations below those th a t trig g e r apoptosis or produce necrosis.

These molecular methods provide a means

to move beyond Lethal Dose50-s ty le experimental approaches toward studies y ie ld in g mechanistic to x ic o lo g ic a l knowledge by determining the genomic response propagated by s p e c ific genes o f in te re s t.

Also, new

avenues to understanding species d iffe re nces w ith reduced animal use may be possible. Risk assessment o f a ir p o llu tio n e ffe c ts , e sp ecia lly those a ttrib u te d to inhalable p a rtic u la te s , remains a controversial issue in sp ite o f substantial and convincing epidem iological studies pending 83


determ ination o f the molecular mechanisms (Dockery e t a l . , 1993; Styer e t a l . , 1995; Samet et a l . , 1994).

Certain groups in the population are

designated, fo llo w in g the Environmental P rotection Agency guidelines, as a t- r is k populations (American Lung Association, 1994).

They include

asthmatics, fo r which there is strong evidence o f th e ir s e n s itiv ity to p a rtic u la te s (Dockery and Pope, 1994), others w ith p re -e xistin g re s p ira to ry disease including chronic o b stru ctive pulmonary disease, pre-adolescent ch ild re n (those less than 13 years old) and the e ld e rly (65 or more years o f age).

In a d d itio n , a ir p o llu tio n is not evenly

d is trib u te d across demographic groups, often w ith exposure and associated health ris k s fa llin g on populations th a t are poor and nonwhite (Sexton et a l . , 1993).

Further, a d e ficien cy o f many

to x ic o lo g ic a l studies is the focus on normal healthy organisms whereas to x ic e ffe c ts may be more pronounced and s ig n ific a n t fo r n u tr itio n a lly or disease-compromised in d ivid u a ls.

This view is supported by recent

evidence th a t selenium -deficient MDBK c e lls , a bovine renal e p ith e lia l c e ll lin e , were more susceptible to both hydrogen peroxide- and cumene hydroperoxide-induced apoptosis which could be o ffs e t by supplementation w ith selenium or catalase (Kayanoki et a l . , 1996).

The a pp lica tion o f

techniques inco rpo ra tin g the growing understanding o f biomolecular re la tio n s h ip s and pathways o ffe rs a uniquely inform ative model approach fo r evaluating the g enotoxicity o f environmental p a rtic u la te s while providing new in s ig h ts into the su b ce llu la r mechanisms o f to x ic ity .

84


An Inorganic Model o f Oxidative Stress

Patterns o f induction fo r eq uito xic treatments as evaluated by c e ll growth in h ib itio n were determined and support the contention th a t o xid a tive challenge is met with a se n sitive spectrum o f genomic responses in vo lvin g c e ll cycle in h ib ito rs , p rote in degradation pathways, and p ro te c tiv e mechanisms th a t together demarcate the boundaries between c e ll survival or death (Figure 17).

The approach taken has attempted to

d is tin g u is h between organic peroxidation e ffe c ts as characterized by te rt- b u ty l hydroperoxide w ith it s hallmark o f lip id peroxidation and inorganic peroxidation using exogenous and endogenous hydrogen peroxide. We are in c lin e d to view our work as an inorganic model of oxid ative stress but th is remains to va lid ated .

S im ila r studies are planned to

evaluate to x ic metals, GPx in h ib itio n by selenium depletion, and other means o f inducing oxidative stress at the elemental le v e l.

Also

important fo r e sta b lish in g the e ffic a c y o f th is model w ill be determ ination o f the e ffe c ts o f phagocytosis on stress gene in duction. I t may be postulated th a t phagocytosis o f in e r t p la s tic beads, fo r example, might not stim ulate GADD and heat shock gene expression, whereas phagocytosis o f s ilic a , laboratory-generated soot, or ambient environmental p a rtic le s might trig g e r a genotoxic response.

Further, i t

w ill be useful to compare macrophages isolated from healthy humans or other species and to compare macrophages from organs other than the lungs.

There would appear to be broad possible applications th a t would

b e n e fit from th is type o f comparative inform ation.

The future awaits.

85


Reactive

Oxygen

S p e c ie s

TARGET INTERACTION P r o t e i n . ONA. L i p i d s CELL Activation

of s i g n a l i n g

Induction

R ESPONSE molecules;

of o x i d a t i v e

stress - response

BIOLOGICAL

Aberrant

Adaptation proliferation

Maintenance

transcription

to

genes

ENDPOINTS

oxidant

of normal

stress Cytotoxicity/cell

death

function

A fter

FIGURE 17.

factors

J a n s s e n ,

e t a I. 1 9 9 3

Primary events in the c e llu la r outcomes of oxidative in ju ry .

86


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