Blutstillung mit dem Infrarot-Kontakt-Koagulator am Nieren- ... Light Absorption in Blood. 14 .... at the sites where uv-photons are absorbed in the Chromatin.
GESELLSCHAFT FÜR STRAHLEN- UND UMWELTFORSCHUNG MBH BEREICH PROJEKTTRÄGERSCHAFTEN PROJEKT LASER IN MEDIZIN UND BIOLOGIE INGOLSTÄDTER LANDSTRASSE 1
D-8042
NEUHERBERG
PROCEEDINGS OF THE SYMPOSIUM LASERS IN MEDICINE AND BIOLOGY NEUHERBERG, JUNE 22-25, 1977 GSF - BERICHT BPT 5
THE
SYMPOSIUM ON LASERS IN MEDICINE AND BIOLOGY IS SUPPORTED
BY THE FEDERAL MINISTRY OF RESEARCH AND TECHNOLOGY (BMFT) AS ARE THE RESEARCH EFFORTS REPRESENTED BY THE CONTRIBUTIONS
2-11, 13-17, 19, 23, 26-28, 33-40 UNDER MT 227 B AND MT 2715,
GRANTS
MT 217 B,
CONTENTS
Preface n u m b e r of
S e c t i o n , a u t h o r s , t i t le
contr ibution
M.L. Wolbarsht, Duke University, N.C. Trends in Biomedical Laser Application - Present and Future
1
LASERS IN SURGERYI Chairman: R. Verschueren
P. Kiefhaber, G. Nath, K. Moritz, W. Gorisch and A. Kreitmair Endoscopical Control of Massive Gastrointestinal Hemorrhage With a High Power Neodymium-Yag Laser
2
G. Staehler, A. Hofstetter und W. Siepe Endoskopische Laserbestrahlung von Harnblasentumoren
3
F. Leheta, J. Wilske und R.C. McCord Erste klinische Erfahrungen mit dem Nd-Yag-Laser in der Neurochirurgie
4
O.J. Beck, J. Wilske und J. Schönberger Gewebsveränderungen nach Laseranwendung am Kaninchen* hirn. Ergebnisse mit C 0 - und Neodym-Laser 2
5
P.R. Zellner und C. Lazarides Die Anwendung des C 0 Lasers bei der Behandlung des Brandverletzten 2
6
D.H. Sliney, US Army Environmental Hygiene Agency, Aberdeen Proving Ground Md. Developments in Laser Safety
7
LASERS IN SURGERY II Chairman: R. Verschueren
W. Gorisch, K.P. Boergen, R.C. McCord and W. Weinberg Temperature Measurement of Laser Irradiated Mesenterial Blood Vessels of the Rabbit
8
R.C. McCord, W. Weinberg, W. Gorisch, F. Leheta and J.L. Schönberger Thermal Effects in Laser Irradiated Biological Tissue
9
E. Keiditsch, E. Langer, G. Staehler, A. Hofstetter und H. Ebner Histologische Veränderungen an der Kaninchenharnblase nach Laserbestrahlung
1 0
A. Hofstetter, G. Staehler und H.E. Meilin Blutstillung mit dem Infrarot-Kontakt-Koagulator am Nierenparenchym
n
LASERS IN OPHTALMOLOGY Chairman: F. Fankhauser E. v.d. Zypen, H. Bebie und F. Fankhauser Die morphologischen Wirkungen von Hochleistungslasern auf die Iris des albinotischen und pigmentierten Kaninchens
not received
F. Dannheim and B. Rassow Lesions of the Anterior Segment of the Eye by Lasers of Different Wavelength
13
H. Welsch, R. Birngruber, K.-P. Boergen, V..P. Gabel and F. Hillenkamp The Influence of Scattering on the Wavelength dependent Light Absorption in Blood
14
K.-P. Boergen, R. Birngruber, V.-P. Gabel and F. Millenkamp Experimental Studies on Controlled Closure of Small Vessels by Laser Irradiation R. Birngruber, V.-P. Gabel, und F. Hillenkamp Time Dependence of the Retina-Reaction during and after Lasercoagulation with Various Coagulation Parameters W. Weinberg, V.-P. Gabel, R. Birngruber und R.C. McCord Zur Messung der Korrelation von Temperaturverteilung und Lichtreflexion am Augenhintergrund bei Laser-Koagulation A. Lewis, Cornell University, Ithaca, N.Y. Primary Photophysical and Photochemical Processes in Visual Excitation
LASERS IN DIAGNOSTICS Chairman: R. Kaufmann M. Achatz and G. Seger Development of a Process for Rapid Serum Electrophoresis on the Basis of Light-Beating Spectroscopy R. Steiner, R. Hartmann, N. Hof mann and R. Kaufmann Motility Measurements on Human Spermatozoa by Laser Light Beating Technique C.E. Riva, G.T. Feke and B. Eberli Blood Velocity in Human Retinal Vessels H. Weiss, E. Klotz, R. Linde and U. Tiemens Flashing Tomosynthesis
E.R. Reinhardt, G. Laub, W.H. Bloss und K. Müller-Schauenburg Anwendung von Zonenplatten zur Abbildung in der Szintigraphie
not received
H. Albrecht, G. Müller and M. Schaldach Raman Gas Spectroscopy for Biomedical Applications
25
LASERS IN DENTISTRY Chairman: W. Ketterl
R. Beck, H. Heide, H.J. Kinkel and R. König Investigations into the Fusion of Dental Filling Cement with the Dental Enamel by Means of Lasers
26
P. Lenz, H. Gilde, U.-J. Pyttel und M. Harter Versiegelungsversuche an Zahnhartsubstanzen mit Laserstrahlen
27
J. Vahl, H. van Benthem und K.-J. Reinhardt Laserexperimente in der Zahnmedizin
28
LASERS IN CELLULAR
RESEARCH
Chairman: M. Berns
M.W. Berns, R.K. Strahs, S.P. Peterson, K.G. Gilmer-Waymire and S. Brenner Laser Microirradiation of Cells
29
C. Zorn, C. Cremer, T. Ctemer and J. Zimmer Laser UV-Microirradiation of Mammalian cells: A Test of Models for the Mechanism of UV-lnduced Chromosome Aberrations
30
A. Anders Spektroskopische Untersuchungen an Nukleinsäure-FarbstoffKomplexen mit Farbstofflasern
not receiv
R. Nitsche, F. Hillenkamp, R. Kaufmann, E. Unsöld, H. Vogt and R. Wechsung The LAMMA Instrument: A new Laser Microprobe Mass Analyzer for Biomedical Purposes
32
DEVELOPMENT OF LASERS AND OPTICAL SYSTEMS FOR MEDICAL APPLICATIONS Chairman: M. Maler G. Nath, A. Kreitmair, P. Kiefhaber und K. Moritz Transmissionssysteme für Laser- und intensive Lichtstrahlung
33
H. Wurster Endoskop zur Lasertherapie
3
4
o
c
J. Kühl Tunable UV Laser R.Beck Development of a Holmium Laser for Use in Experimenta! Surgery
36
W. Rother Anforderungen an ein medizinisches Nd:Yag-Laser System
not
recei v e d
LASERS IN SURGERY
III
Chairman: F. Hillenkamp
G. Königsmann, E. Karbe and R. Beck Application of the CO Laser and the Ho Laser as a Surgical Instrument compared with other IR Lasers and Conventional Instruments J. Buchholz, B. Grotelüschen and H. Welling Liver Surgery with Nd:YAG Laser Radiation H. -H. Horch, R.C. McCord, E. Schäffer und L. Ruprecht Zur Frage der Laser-Osteotomie A. Karduck, H.-G. Richter, A. Wandhöfer, G. Kauffmann und G. v. Bally Wirkung von Laserstrahlung verschiedener Wellenlängen (Nd:YAG-, C0 -Laser) auf die Ohrmuschel des Kaninchens: vergleichende morphologische Untersuchungen 2
C. Frantzen und H.W. Schlösser Untersuchungen über die Anwendbarkeit eines C0 -Lasergerätes als mikrochirurgisches Instrument bei Eingriffen am weiblichen Genitale (tierexperimentelle Studie am Uterushorn der Ratte) 2
Authors
Index
LASER-UV-MICROIRRADIATION ( X OF
= 257
nm)
OF CHINESE HAMSTER CELLS: EVIDENCE
UV-INDUCED CHROMOSOME ABERRATIONS WHICH DO OF PHOTOLESIONS IN THE
NOT
ORIGINATE AT THE
CHROMATIN
C. Zorn, C. Cremer, T. Cremer and J . Zimmer I n s t i t u t für Humangenetik und A n t h r o p o l o g i e der Universität 1.
SITES
Freiburg i.Br.
Introduction
U s i n g a laser-uv-microbeam ( X = 257 nm) we have i n v e s t i g a t e d the hypot h e s i s t h a t p h o t o l e s i o n s i n the chromatin are the s i t e s at which most uvi n d u c e d chromosome a b e r r a t i o n s o r i g i n a t e (1,2), The h y p o t h e s i s f o l l o w s from two assumptions: ( i ) P h o t o l e s i o n s produced i n u v - i r r a d i a t e d chromatin are d e c i s i v e f o r the p r o d u c t i o n of most uv-induced chromosome a b e r r a t i o n s . ( i i ) A l l chromosome a b e r r a t i o n s which depend on the formation of photol e s i o n s i n the chromatin o r i g i n a t e at the s i t e s of the p h o t o l e s i o n s , ( i i ) i n c l u d e s the p o s s i b i l i t y t h a t an a b e r r a t i o n , such as a chromatid break, occurs very near, but not e x a c t l y at the s i t e of a chromatin l e s i o n (3) as w e l l as the p o s s i b i l i t y t h a t p r i m a r i l y undamaged s i s t e r c h r o m a t i d s may be i n v o l v e d i n the f i n a l a b e r r a t i o n by r e c o m b i n a t i o n a l r e p a i r p r o c e s s e s ( 2 ) . The mechanism of a b e r r a t i o n p r o d u c t i o n by p h o t o l e s i o n s i s c o n s i d e r e d t o i n c l u d e i n c o m p l e t e or f a u l t y r e p a i r processes a t the s i t e s of t h e s e l e s i o n s ( 2 ) . The percentage of l e s i o n s which f i n a l l y l e a d t o a chromosome a b e r r a t i o n may be very s m a l l . I t i s important to note, t h a t assumption ( i i ) does not n e c e s s a r i l y f o l l o w from assumption ( i ) (see d i s cussion). The h y p o t h e s i s has s e v e r a l consequences which can be t e s t e d by the use of a uv-microbeam. Most uv-induced chromosome a b e r r a t i o n s should o r i g i n a t e a t the s i t e s where uv-photons are absorbed i n the Chromatin. P h o t o l e s i o n s i n the chromatin should not have any important i n f l u e n c e on the y i e l d of a b e r r a t i o n s produced i n o t h e r p a r t s of chromatin not c o n t a i n i n g p h o t o l e sions« Chromatin l e s i o n s c r e a t e d by mutagens p h o t o c h e m i c a l l y produced i n the karyoplasm or cytoplasm (1) should c o n t r i b u t e very l i t t l e t o the y i e l d of a b e r r a t i o n s * In such a case the s i t e where i t c r e a t e s a chromatin l e s i o n and f i n a l l y a chromosome a b e r r a t i o n may be s i g n i f i c a n t l y a p a r t from each o t h e r . A non random d i s t r i b u t i o n of chromosome a b e r r a t i o n s i s compatible with the h y p o t h e s i s , s i n c e the p r o b a b i l i t y t h a t a chromatin l e s i o n w i l l cause an a b e r r a t i o n may be d i f f e r e n t i n d i f f e r e n t chromosome segments. G r i g g s and Bender (4) have o b t a i n e d evidence i n Xenopus c e l l s t h a t a c e r t a i n c l a s s of DNA-photolesions, namely the p y r i m i d i n e cyclobutane dimer i s r e s p o n s i b l e f o r most uv-induced chromosome a b e r r a t i o n s . Bender e t a l . (2) have p r o v i d e d a model which suggests t h a t the s i t e of dimer formation i s the s t a r t i n g p o i n t f o r the formation of uv-induced a b e r r a t i o n s . With r e s p e c t t o t h i s model the above p r e d i c t i o n s can be formulated i n a more r e s t r i c t e d form, s u b s t i t u t i n g p y r i m i d i n e cyclobutane dimer f o r p h o t o l e s i o n .
3o -
2
It i s s t i l l a matter of c o n t r o v e r s y , however, whether t h i s dimer s h o u l d be c o n s i d e r e d the only important c l a s s of p h o t o l e s i o n s d e c i s i v e f o r p r a c t i c a l l y a l l uv-induced chromosome a b e r r a t i o n s i n a l l eukaryote c e l l s (5, 6,7). A c c o r d i n g to the g e n e r a l f o r m u l a t i o n of the h y p o t h e s i s any p h o t o l e s i o n i n troduced e i t h e r i n the DNA or i n the p r o t e i n moiety of the chromatin may be c o n s i d e r e d t o c o n t r i b u t e to the y i e l d of uv-induced chromosome a b e r r a tions. 2. Experimental
methods and
rationale
Chinese hamster c e l l s were m i c r o i r r a d i a t e d d u r i n g i n t e r p h a s e e i t h e r i n the nucleus or i n the cytoplasm and chromosome spreads were o b t a i n e d i n s i t u a f t e r d i f f e r e n t i n c u b a t i o n times. The laser-uv-microbeam, the m i c r o i r r a d i a t i o n procedure, c e l l m a t e r i a l and c u l t u r e c o n d i t i o n s have been d e s c r i b e d elsewhere (8,9,10,11). U V - m i c r o i r r a d i a t i o n alone d i d not induce s u f f i c i e n t numbers of chromosome a b e r r a t i o n s at a convenient uv-dose ( F i g . l d ) . At h i g h e r doses the m i t o t i c index decreased s t r o n g l y so t h a t the microbeam procedure became u n s u i t a b l e . The y i e l d of a b e r r a t i o n s i n c e l l s m i c r o i r r a d i a t e d i n the nucleus, however, was g r e a t l y i n c r e a s e d by a d d i t i o n of C o f f e i n e (0.5 - 2 mM) t o the c u l t u r e medium d u r i n g the i n c u b a t i o n p e r i o d ( F i g . l e ) . C o f f e i n e i s known t o p o t e n t i a t e s y n e r g i s t i c a l l y the e f f e c t of u v - i r r a d i a t i o n on the formation of chromosome a b e r r a t i o n s i n rodent c e l l s , when i t i s present d u r i n g the subsequent S-phase a f t e r i r r a d i a t i o n (13). The number of a b e r r a t i o n s i n cont r o l c e l l s and i n c e l l s m i c r o i r r a d i a t e d i n the cytoplasm was s m a l l and not s i g n i f i c a n t l y i n c r e a s e d by C o f f e i n e at the i n d i c a t e d c o n c e n t r a t i o n s ( F i g . l a - c , 6 a ) . The h y p o t h e s i s p r e d i c t s t h a t those i n t e r p h a s e chromosomes which l i e i n the m i c r o i r r a d i a t e d p a r t of the nucleus and r e c e i v e uv-induced phot o l e s i o n s , should show a b e r r a t i o n s at metaphase, depending on the number of p h o t o l e s i o n s and the stage of the c e l l c y c l e at i r r a d i a t i o n . Those c h r o mosomes or chromosome segments which are s i t u a t e d i n the u n i r r a d i a t e d p a r t of a nucleus d u r i n g m i c r o i r r a d i a t i o n and bear few or no p h o t o l e s i o n s should show very few or no a b e r r a t i o n s . An i n c r e a s e of the s i z e of the i r r a d i a t e d n u c l e a r area m a i n t a i n i n g a constant t o t a l i r r a d i a t i o n energy, with any d i s t r i b u t i o n of i n t e r p h a s e chromosomes, should r e s u l t i n very d i f f e r e n t d i s t r i b u t i o n s of a b e r r a t i o n s i n metaphase spreads, r e f l e c t i n g the d i s t r i b u t i o n of p h o t o l e s i o n s .
3.
Results
3.1.
D i s t r i b u t i o n of chromatin p h o t o l e s i o n s w i t h i n m i c r o i r r a d i a t e d n u c l e i .
For the I n t e r p r e t a t i o n of the r e s u l t s of a chromosome a n a l y s i s of m i c r o i r r a d i a t e d c e l l s i t i s important t o know the d i s t r i b u t i o n of chromatin phot o l e s i o n s w i t h i n the nucleus o b t a i n e d by u v - m i c r o i r r a d i a t i o n . A method to measure t h i s d i s t r i b u t i o n d i r e c t l y was not a v a i l a b l e , but an e v a l u a t i o n of the d i s t r i b u t i o n was o b t a i n e d i n d i r e c t l y by t h e o r e t i c a l c o n s i d e r a t i o n s and the r e s u l t s of d i f f e r e n t experiments (8,12). A l l data a v a i l a b l e i n d i c a t e t h a t most of the t o t a l uv-dose absorbed by a c e l l i s absorbed w i t h i n the m i c r o i r r a d i a t e d a r e a . Consequently most of the p h o t o l e s i o n s have t o be p r o duced t h e r e , while the number of p h o t i e s i o n s produced o u t s i d e t h i s area by d i f f r a c t i o n and d i s p e r s i o n of the uv-microbeam i s s m a l l .
3o - 3
b
a
1
ABCDE
Fig.
1;
•
ABCDE
c
ABCDE
d
ABCDE
• i.ni ABCDE
Chromosome damage a f t e r l a s e r - u v - m i c r o i r r a d i a t i o n i n t h e nucleus and the cytoplasm and posttreatment with/without C o f f e i n e . C e l l s of a V - 7 9 - s u b l i n e of the Chinese hamster (10) were m i c r o i r r a d i a t e d ( X = 257.3 nm) a t one s i t e and t h e r e a f t e r i n c u b a t e d i n the presence/absence o f C o f f e i n e . C o l c h i c i n e was added 3 hours bef o r e i n s i t u chromosome p r e p a r a t i o n which was performed 13 hours a f t e r i r r a d i a t i o n . S c o r a b l e m i t o t i c f i g u r e s o b t a i n e d were c l a s s i f i e d according t o the f o l l o w i n g c r i t e r i a : A: No chromosome damage; B: o n l y Single d e f e c t s (1-2 b r e a k s , g a p s ) ; C: some chromosomes s e v e r e l y damaged, t h e m a j o r i t y being i n t a c t ; D: most chromosomesdamaged, a t l e a s t one chromosome remaining i n t a c t ; E: a l l chromosomes damaged up t o complete d i s i n t e g r a t i o n . A b s c i s s a : type of damaged m i t o t i c f i g u r e . O r d i n a t e ; p r o p o r t i o n o f each t y p e ( % ) . The t o t a l number o f m i t o t i c f i g u r e s c l a s s i f i e d as type A - E i s given i n parentheses. a) No i r r a d i a t i o n , no Coffeine (114) b) no i r r a d i a t i o n , 1 mM Coffeine (82) c ) m i c r o i r r a d i a t i o n o f t h e cytoplasm (1/8 s e c ) , 1 mM Coffeine (87) d) m i c r o i r r a d i a t i o n o f t h e n u c l e u s (1/8 s e c ) , no Coffeine (29) e) m i c r o i r r a d i a t i o n o f t h e n u c l e u s (1/8 s e c ) , 1 mM Coffeine ( 4 7 ) .
The s i z e o f t h e f l u o r e s c e n t a r e a induced by t h e microbeam i n t h e o b j e c t p l a ne and the s i z e o f v i s i b l e l e s i o n s produced i n u n s t a i n e d n u c l e i of l i v i n g c e l l s , as w e l l as i n s t a i n e d c e l l specimens i s v e r y s i m i l a r (8,12). The s i ze of the l e s i o n d e t e c t a b l e i n t h e l i g h t microssope produced with t h e U l t r a f l u a r 32x/Ph, used i n a l l experiments described i n t h e p r e s e n t paper, i s approx. 1 |im i n diameter. A l o c a l i z e d type of chromatin damage obtained by the microbeam procedure i s f u r t h e r i n d i c a t e d by e l e c t r o n m i c r o s c o p i c Observ a t i o n (14) and by t h e f i n d i n g t h a t unscheduled DNA s y n t h e s i s can be i n d u ced s e l e c t i v e l y a t t h e i r r a d i a t i o n s i t e ( F i g . 2) (15). Autoradiographs o f m i c r o i r r a d i a t e d n u c l e i which were not i n S-phase d u r i n g the subsequent i n c u b a t i o n p e r i o d with ^HTdR show t h a t the number o f s i l v e r grains/|im2 over the m i c r o i r r a d i a t e d s i t e c l e a r l y i n c r e a s e s with dose, but f a l l s t o t h e s m a l l l e v e l o f c o n t r o l n u c l e i w i t h i n a d i s t a n c e of 6 u.m from the c e n t e r of the m i c r o i r r a d i a t e d a r e a (data not shown). The a r e a covered densely with s i l v e r g r a i n s was found t o be approx. 3.6 + 1.0 |im i n diamet e r . T h i s a r e a i s somewhat l a r g e r than t h e s i z e o f t h e f l u o r e s c e n t spot s u g g e s t i n g t h a t a s i g n i f i c a n t number of DNA-lesions g i v i n g r i s e t o DNA
3o -
4
r e p a i r s y n t h e s i s i s produced w i t h i n the whole cone of uv-rays w i t h i n the nucleus, A l a r g e p a r t of the chromatin of m i c r o i r r a d i a t e d n u c l e i as shown i n F i g . 2 d i d not perform any uv-induced unscheduled DNA s y n t h e s i s . I t has been shown that the amount o f unscheduled DNA s y n t h e s i s induced by mic r o i r r a d i a t i o n of the nucleus depends only on the t o t a l number of i n c i d e n t photons and not on the a r e a of i r r a d i a t i o n ( 1 5 ) . These data suggest t h a t a c o n s i d e r a b l e p r o p o r t i o n of uv-induced DNA l e s i o n s produced o u t s i d e the m i c r o i r r a d i a t e d a r e a and giving rise t o DNA r e p a i r synt h e s i s , should be d e t e c t e d by the a u t o r a d i o g r a p h i c method. Our r e s u l t s i n d i c a t e , t h e r e f o r e , that the l a r g e m a j o r i t y of such l e s i o n s i s produced w i t h i n a r a d i u s of l e s s than 6 u.m from the c e n t e r of the i r r a d i a t i o n s i t e .
\
\
F i g . 2:
Autoradiograph of two f i b r o b l a s t o i d Chinese hamster c e l l s d e r i ved from lung t i s s u e (11) a f t e r l a s e r - u v - m i c r o i r r a d i a t i o n of the nucleus and subsequent i n c u b a t i o n with 3|-|_Thymidine f o r 1 hour. Unscheduled DNA s y n t h e s i s a t the m i c r o i r r a d i a t e d s i t e i s i n d i c a t e d by s i l v e r g r a i n s ( a r r o w s ) .
Figs.3-5:
M i t o t i c f i g u r e s o b t a i n e d a f t e r l a s e r - u v - m i c r o i r r a d i a t i o n of n u c l e i of V-79 c e l l s and posttreatment with C o f f e i n e . ( F o r exp e r i m e n t a l d e t a i l see F i g . 1) F i g . 3: chromosome damage i n a l i m i t e d a r e a (arrows ) (type C), F i g s . 4,5: g e n e r a l i z e d chromosome damage (type E ) . F i g . 5 shows an example of complete chromosomal d i s i n t e g r a t i o n Bars i n d i c a t e 10 p.m.
3o 3.2.
5
Chromosome a b e r r a t i o n s o b t a i n e d i n metaphase spreads from c e l l s microi r r a d i a t e d i n the n u c l e u s .
F i v e types A - E of m i t o t i c f i g u r e s o b t a i n e d from c e l l s m i c r o i r r a d i a t e d i n the nucleus and t h e r e a f t e r i n c u b a t e d i n the presence of C o f f e i n e were t e n t a t i v e l y d i s t i n g u i s h e d (see legend of F i g . 1). A f t e r i r r a d i a t i o n of one s m a l l p a r t of the nucleus a c e r t a i n number of m i t o t i c f i g u r e s showed a s m a l l number of damaged chromosomes (type C, F i g s . l e , 6 ) . In the l a r g e m a j o r i t y of these cases the damaged chromosomes were c l e a r l y not randomly d i s t r i b u ted over the whole metaphase spread, but more or l e s s c o n c e n t r a t e d i n one s m a l l area ( F i g . 3 ) . T h i s type of damage has not been observed a f t e r uvi r r a d i a t i o n of whole c e l l s (11) and i s i n agreement with the h y p o t h e s i s . Karyotypes o b t a i n e d from metaphase spreads showing t h i s type of damage r e v e a l e d t h a t d i f f e r e n t chromosomes were damaged i n d i f f e r e n t c e l l s (11). The r e s u l t s so f a r are c o n s i s t e n t with the h y p o t h e s i s t h a t i n h i b i t i o n of post r e p l i c a t i o n r e p a i r at the s i t e s of p h o t o l e s i o n s caused a b e r r a t i o n s t o appear i n m i c r o i r r a d i a t e d chromosomes (16,11). Other m i t o t i c f i g u r e s were unexpectedly found where most (type D) or even a l l chromosomes (type E) were damaged ( F i g s . 4,5). While some a b e r r a t i o n s were expected to appear i n chromatin o u t s i d e the m i c r o i r r a d i a t e d area because of the s m a l l number of p h o t o l e s i o n s which might be i n t r o d u c e d i n t h i s chromatin, t h e r e i s no apparent reason why the m i c r o i r r a d i a t e d chromosomes o r chromosome segments themselves should not show a much more pronounced e f f e c t . Using t h i s c r i t e r i o n m i c r o i r r a d i a t e d chromosomes can be t e n t a t i v e l y i d e n t i f i e d i n some but not i n a l l cases c l a s s i f i e d as type D and E. F i g . 6 shows t h a t the p r o p o r t i o n of type D and type E i n c r e a s e with dose. The C o f f e i n e c o n c e n t r a t i o n and the stage of the c e l l c y c l e are o t h e r imp o r t a n t parameters i n d e t e r m i n i n g whether i n t a c t (type A) or s e v e r e l y damaged m i t o t i c f i g u r e s (types C - E) are o b t a i n e d (data not shown). The number of p h o t o l e s i o n s produced i n any i n t e r p h a s e chromosome i s not a s u f f i c i e n t c r i t e r i o n t o p r e d i c t the p r o b a b i l i t y of the number of a b e r r a t i o n s i n m i t o s i s o r i g i n a t i n g at t h i s chromosome. T h i s c o n c l u s i o n i s supported by the f o l l o w i n g comparison. At the lowest dose (1/125 sec) the concent r a t i o n of p h o t o l e s i o n s i n the m i c r o i r r a d i a t e d p a r t of the nucleus i s s t i l l r a t h e r high, the energy d e n s i t y w i t h i n the focus b e i n g i n the o r d e r of 750 ergs/mm^. Unscheduled DNA s y n t h e s i s at the m i c r o i r r a d i a t i o n s i t e of such n u c l e i c o u l d be d e t e c t e d , but t h e r e was no s i g n i f i c a n t y i e l d of u v - i n duced chromosome a b e r r a t i o n s ( F i g . 6b). At h i g h e r doses, however, chromosome d i s i n t e g r a t i o n was o b t a i n e d even i n u n i r r a d i a t e d p a r t s of m i c r o i r r a d i a ted n u c l e i , although the data d e s c r i b e d i n 3.1 suggest a much lower conc e n t r a t i o n of p h o t o l e s i o n s i n these u n i r r a d i a t e d p a r t s at any dose, as compared with the c o n c e n t r a t i o n of p h o t o l e s i o n s i n chromatin m i c r o i r r a d i a t e d with the lowest dose. The e f f e c t s of m i c r o i r r a d i a t i o n of a s m a l l p a r t of the nucleus were compared with those of a l a r g e r p a r t ( F i g . 6b-e). For any i n v e s t i g a t e d uv-dose the percentages of types A - E were found s i m i l a r i n both types of e x p e r i ments. In f u r t h e r experiments m i c r o i r r a d i a t i o n of a s m a l l p a r t of the nucleus was compared with m i c r o i r r a d i a t i o n of whole n u c l e i at the same dose (1/15 sec) (unpublished d a t a ) . Under the e x p e r i m e n t a l c o n d i t i o n s of these l a t t e r experiments the percentages f o r type C and D were very low. The percentages
2c
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for type E were found to be 40 % and 37 %. These data i n d i c a t e that demol i t i o n of a l l chromosomes may be achieved i n s e n s i t i v e c e l l s with any d i s t r i b u t i o n of a given number of p h o t o l e s i o n s .
a
J_j
A B C D E
Fig.
6:
Ii;
Lli±
1
A B C D E
" "
-
A B C D E
e
d
c
b
i
1
llÜ.i.i: A B C D E
1
l!h:,:|i A B C D E
Chromosome dar.iage a f t e r l a s e r - u v - m i c r o i r r a d i a t i o n of n u c l e i with d i f f e r e n t doses and posttieatment with 2 mM C o f f e i n e . For e x p e r i mental d e t a i l s and C l a s s i f i c a t i o n o f m i t o t i c f i g u r e s see F i g . 1. A b s c i s s a : type of damaged m i t o t i c f i g u r e . O r d i n a t e : p r o p o r t i o n of each t y p e ( % )
I
: data o b t a i n e d with the uv-focus p l a c e d i n s i d e the c e l l nucleus (as i n experiments o f F i g . 1);
i : data o b t a i n e d with the uv-focus p l a c e d above the c e l l , r e J s u l t i n g i n an i r r a d i a t i o n f i e l d of approx. 4 \jm diameter. The f i r s t number g i v e s number g i v e the number d i a t i o n modes with the respectively. a) no i r r a d i a t i o n , 142 c) 1/60 sec, 28/19, d)
the i r r a d i a t i o n time, the second and t h i r d of analyzed m i t o t i c f i g u r e s u s i n g the i r r a focus i n s i d e ( | ) and above the c e l l ( ! ), m i t o t i c f i g u r e s , b) 1/125 sec, 3/18, 1/30 sec, 52/65, e) 1/15 s e c , 30/60.
4. D i s c u s s i o n M i t o t i c f i g u r e s showing d i s i n t e g r a t i o n of a l l chromosomes induced by m i c r o i r r a d i a t i o n of a s m a l l p a r t of the nucleus do not support the h y p o t h e s i s I t h a t p h o t o l e s i o n s i n the chromatin a r e the o n l y important s i t e s a t which uv-induced chromosome a b e r r a t i o n s can o r i g i n a t e . To f i t our r e s u l t s with the h y p o t h e s i s , we have t o assume that the p r o b a b i l i t y of a S i n g l e p h o t o l e s i o n t o c r e a t e an a b e r r a t i o n depends on the t o t a l number, but not on the d i s t r i b u t i o n of o t h e r p h o t o l e s i o n s i n the n u c l e u s . If the t o t a l number of p h o t o l e s i o n s i s high enough, then very few p h o t o l e s i o n s i n a chromosome not d e t e c t a b l e by the a u t o r a d i o g r a p h i c method d e s c r i bed i n 3.1., c o u l d be s u f f i c i e n t t o t r i g g e r i t s complete d e m o l i t i o n . In cases where a l l chromosomes a r e completely d i s i n t e g r a t e d ( F i g . 5 ) , the much h i g h e r c o n c e n t r a t i o n of p h o t o l e s i o n s i n m i c r o i r r a d i a t e d chromosomes cannot be d i s t i n g u i s h e d by c y t o l o g i c a l O b s e r v a t i o n . T h i s I n t e r p r e t a t i o n , however, does not s a t i s f a c t o r i l y e x p l a i n why m i c r o i r r a d i a t e d chromosomes do not show a more severe d i s i n t e g r a t i o n than u n i r r a d i a t e d ones i n many m i t o t i c f i g u res of type E, where chromosome d e m o l i t i o n i s not complete ( F i g . 4 ) .
3o
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7
To i n t e r p r e t e our r e s u l t s we now c o n s i d e r a second h y p o t h e s i s ( l l ) : A s i g n i f i c a n t number of a b e r r a t i o n s can occur at chromatin s i t e s which do not c o n t a i n p h o t o l e s i o n s . Both the s i t e s of p h o t o l e s i o n s and these o t h e r s i t e s c o n t r i b u t e to the t o t a l y i e l d of uv-induced chromosome a b e r r a t i o n s . The p r o p o r t i o n of a b e r r a t i o n s o b t a i n e d from each source i n any c e l l may depend on some unknown p r o p e r t i e s of the c e l l and on the e x p e r i m e n t a l cond i t i o n s . At l e a s t one of the two assumptions l e a d i n g t o h y p o t h e s i s I i s not c o m p a t i b l e with h y p o t h e s i s I I . Two new assumptions (a) and (b) may be f o r m u l a t e d as a l t e r n a t i v e s . (a) B e s i d e s p h o t o l e s i o n s i n the chromatin o t h e r photochemical r e a c t i o n s are a l s o important f o r the f o r m a t i o n of uv-induced chromosome a b e r r a t i o n s . In t h i s case assumption ( i i ) may s t i l l be v a l i d . There are s e v e r a l p o s s i b i l i t i e s . 1. Mutagens may be produced i n the m i c r o i r r a d i a t e d p a r t of the karyoplasm and c r e a t e chromatin l e s i o n s anywhere i n the nucleus ( 1 ) . 2. UVdamage of the n u c l e a r envelope may l e a d t o u n c o n t r o l l e d f l u x e s of important substances i n both d i r e c t i o n s at the m i c r o i r r a d i a t e d s i t e . Such an e f f e c t , as w e l l as an uv-dependent f o r m a t i o n of t o x i c substances may l e a d t o severe changes i n the i n t e r n a l m i l i e u of the whole nucleus which may become more s e n s i t i v e t o the a c t i o n of c a f f e i n e . I t i s known t h a t C o f f e i n e at high conc e n t r a t i o n s ( ^ 10 mM) i n d u c e s chromosome d i s i n t e g r a t i o n i n u n i r r a d i a t e d c e l l s (17 and our unpublished d a t a ) . A l t e r n a t i v e l y , assumption ( i ) may be supposed t o be v a l i d . Then assumption ( i i ) has t o be abandoned and assumption (b) may be formulated: A s i g n i f i cant number of chromosome a b e r r a t i o n s which depend on the formation of p h o t o l e s i o n s i n the chromatin do not o r i g i n a t e at the s i t e s of the p h o t o l e s i o n s . A g a i n s e v e r a l p o s s i b i l i t i e s may be c o n s i d e r e d . 1. The g e n e t i c I n f o r m a t i o n at the irradiated p a r t of a nucleus including genes important f o r the m o r p h o l o g i c a l i n t e g r i t y of a l l m i t o t i c chromosomes may be b l o c k e d by t r a n s c r i p t i o n t e r m i n a t i n g p h o t o l e s i o n s i n a dose dependent way ( 1 8 ) . A codogenic Strand of such genes f r e e from t r a n s c r i p t i o n t e r m i n a t i n g l e s i o n s may be necessary s h o r t l y a f t e r s e m i c o n s e r v a t i v e DNA r e p l i c a t i o n of these genes and o b t a i n e d by DNA r e p a i r p r o c e s s e s , but c a f f e ine might prevent such genes from t r a n s c r i p t i o n at the c o r r e c t time by I n h i b i t i o n of post r e p l i c a t i o n r e p a i r . Many such genes would be r e q u i r e d d i s t r i b u t e d throughout the nucleus t o e x p l a i n the frequent o c c u r r e n c e of type E at h i g h e r doses ( F i g s . l e , 6 e ) . 2. I n c r e a s e d DNA d e g r a d a t i o n a f t e r u v - i r r a d i a t i o n has been r e p o r t e d f o r mammalian c e l l Systems and s e v e r a l mutants i n b a c t e r i a (19,20). These f i n dings may j u s t i f y a s p e c u l a t i o n whether g e n e r a l i z e d chromosomal d e m o l i t i o n c o u l d be due t o the u n c o n t r o l l e d a c t i o n of an uv-induced n u c l e a s e . Recentl y , i n h i b i t i o n of poly(ADPR) synthase by c a f f e i n e has been r e p o r t e d (21). A p o s s i b l e r o l e of such enzyme(s) i n the c o n t r o l of nucleases i s e x e m p l i f i e d by the i n h i b i t i o n of a C a Mg++ dependent endonuclease by ADP r i b o s y l a t i o n (22). 3. F i n a l l y , the possible r o l e of c o m p e t i t i o n between s i t e s of post r e p l i c a t i o n r e p a i r and DNA r e p l i c a t i o n p o i n t s f o r some l i m i t i n g enzyme(s) may be c o n s i d e r e d . Both s i t e s p r o v i d e d i s c o n t i n u i t i e s i n newly s y n t h e s i z e d Strands of DNA (23,25) and are known to be a f f e c t e d by c a f f e i n e . The molec u l a r mechanism may i n v o l v e b i n d i n g of c a f f e i n e t o Single stranded DNA + +
3o -
8
r e g i o n s (24) which occur at both s i t e s . The f u n c t i o n of l i a i t i n g enzyme(s) b i n d i n g to these s i t e s and i n v o l v e d both i n an a c c u r a t e Performance of sem i c o n s e r v a t i v e DNA s y n t h e s i s and i n post r e p l i c a t i o n r e p a i r may be impaired i n the presence of c a f f e i n e . I n h i b i t i o n by d i r e c t b i n d i n g of c a f f e i n e to such l i m i t i n g enzyme(s) may a l s o be c o n s i d e r e d (17). At a low c o n c e n t r a t i o n of c a f f e i n e the f u n c t i o n of the enzyme(s) may s t i l l s u f f i c e to a v o i d chromosomal a b e r r a t i o n s o c c u r i n g i n u n i r r a d i a t e d c e l l s . A c e r t a i n number of DNA p h o t o l e s i o n s , however, with any d i s t r i b u t i o n of the l e s i o n s i n the nucleus, may p r o v i d e so many a d d i t i o n a l s i t e s of b i n d i n g f o r l i m i t i n g enzyme(s) t h a t f a u l t y DNA s y n t h e s i s occurs at DNA r e p l i c a t i o n p o i n t s and post r e p l i c a t i o n r e p a i r becomes i n complete or f a u l t y at other s i t e s of chromatin l e s i o n s which occur spontaneously d u r i n g the c e l l c y c l e (23). D i f f e r e n t s i t e s may p r o v i d e d i f f e r e n t p r o b a b i l i t i e s of causing a chromosome a b e r r a t i o n , but the p r o b a b i l i t y f o r any s i t e depends on the number of a l l other s i t e s , present i n the nucleus at the same time. A c c o r d i n g to t h i s model uv-induced chromosome a b e r r a t i o n s o r i g i n a t e at the s i t e s provided by t h r e e d i f f e r e n t s o u r c e s : p h o t o l e s i o n s i n the chromatin, other chromatin l e s i o n s and DNA r e p l i c a t i o n p o i n t s . The c o n t r i b u t i o n of each source to the t o t a l y i e l d of uv-induced a b e r r a t i o n s may vary w i t h the experimental c o n d i t i o n s . Our model does not exclude the p o s s i b i l i t y that i n the absence of c a f f e i n e most a b e r r a t i o n s would occur at the s i t e s of p h o t o l e s i o n s ( 2 ) . I t g i v e s an e x p l a n a t i o n why u v - l i g h t produces chromosome a b e r r a t i o n s i n a S-phase dependent mode (2,13). I t would a l s o e x p l a i n the f i n d i n g that the change from c e l l s c o n t a i n i n g no or few a b e r r a t i o n s t o c e l l s with m u l t i p l e a b e r r a t i o n s i s r a t h e r abrupt i n experiments where uv i s combined with c a f f e i n e (16). The model i s compatible with the f i n d i n g of G r i g g s and Bender (4) that phot o r e a c t i v a t i o n can prevent most uv-induced chromosome a b e r r a t i o n s . On the o t h e r hand the above c o n s i d e r a t i o n s i n d i c a t e t h a t p h o t o r e a c t i v a t i o n i s not s u f f i c i e n t evidence f o r the model of Bender et a l . ( 2 ) . T h i s i n v e s t i g a t i o n was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 46). P a r t s of t h i s i n v e s t i g a t i o n w i l l be presented i n the d o c t o r a l d i s s e r t a t i o n of C. Zorn to be submitted to the f a c u l t y of B i o l o g y , U n i v e r s i t y of F r e i b u r g i . B r . References 1) C.Auerbach, Mutation Research, Problems, R e s u l t s and pp. 159-255. Chapman and H a l l , London (1976). 2) M.A.Bender, H.G.Griggs and
Perspectives,
P.L.Walker, Mutat.Res. 20,
387-402 (1973).
3) R.B.Painter, i n : M o l e c u l a r Mechanisms f o r Repair of DNA, Part B (P.C. Hanawalt and R.B.Setlow, Eds.), pp. 595-600. Plenum Press, New York and London (1975). 4) H.G.Griggs and
M.A.Bender, Science J79,
5) S.Wolff, Photophysiology
7,
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9) C.Zorn, C.Cremer and T.Cremer, i n : Proceedings of the T e c h n i c a l program E l e c t r o - O p t i c s I n t e r n a t i o n a l '74 Conference (M.S.Kiver, Ed.), pp. 203-2 K i v e r Communications, Surrey (1974). 10) C.Cremer, T.Cremer, C.Zorn, and L . S c h o e l l e r , Radiat.Res. 66, (1976).
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11) C.Zorn, T.Cremer, C.Cremer, and J.Zimmer, Hum.Genet. 35, 83-89 (1976). 12) C.Cremer, D i s s e r t a t i o n , U n i v e r s i t ä t F r e i b u r g i . B r . (1976). 13) B.A.Kihlman, S . S t u r e l i d , B.Hartley-Asp, and K . N i l s s o n , Mutat.Res. 26, 105-122 (1974). 14) G.Moreno and F.Vinzens, Exp. C e l l Res. 56, 75-83 (1969). 15) G.Moreno and C . S a l e t , Radiat.Res. 58, 52-59 (1974). 16) K . N i l s s o n and A.R.Lehmann, Mutat.Res. 30, 255-266 (1975). 17) B.A.Kihlman, Mutat.Res. 26, 53-71 18) W.Sauerbier,
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19) J . R . W i l l i a m s , Adv.
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20) L.J.Gudas, J . M o l . B i o l . J04, 567-587 (1976). 21) M.I.Davies, H . H a l l d o r s s o n , S . S h a l l , and C.J.Skidmore, A b s t r a c t book of the 8th Meeting of the European Study Group f o r C e l l Proliferation, Montreux, October 12-15 (1976). 22) H . H i l z and P.Stone, Rev.Physiol.Biochemie.Pharmacol. 23) J.W.Drake and R.H.Baltz,
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