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 GRANTS MT 217 B, MT 227 B AND MT 2715,

C O N T E N T S

P r e f a c e

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 c o n t r i b u t i o n

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 C0 2 - und Neodym-Laser 5

P.R. Zellner und C. Lazarides

Die Anwendung des C0 2 Lasers bei der Behandlung des Brand­verletzten 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 not die Iris des albinotischen und pigmentierten Kaninchens r e c e i v e d

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 Szinti- not graphie r e c e i v e d

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-Farbstoff- n o t Komplexen mit Farbstofflasern r e c e i v

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

J. Kühl

Tunable UV Laser o c

R.Beck

Development of a Holmium Laser for Use in Experimenta! Surgery 36

W. Rother n o t

Anforderungen an ein medizinisches Nd:Yag-Laser System recei ved

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-, C02-Laser) auf die Ohrmuschel des Kaninchens: vergleichende morphologische Untersuchungen

C. Frantzen und H.W. Schlösser

Untersuchungen über die Anwendbarkeit eines C02-Laser­gerätes als mikrochirurgisches Instrument bei Eingriffen am weiblichen Genitale (tierexperimentelle Studie am Uterushorn der Ratte)

A u t h o r s I n d e x

LASER-UV-MICROIRRADIATION ( X = 257 nm) OF CHINESE HAMSTER CELLS: EVIDENCE

OF UV-INDUCED CHROMOSOME ABERRATIONS WHICH DO NOT ORIGINATE AT THE SITES

OF PHOTOLESIONS IN THE CHROMATIN

C. Zorn, C. Cremer, T. Cremer and J. Zimmer I n s t i t u t für Humangenetik und Anthropologie der Universität Freiburg i.Br.

1. Introduction

Using a laser-uv-microbeam ( X = 257 nm) we have investigated the hypo-t h e s i s that photolesions i n the chromatin are the s i t e s at which most uv-induced chromosome aberrations originate (1,2), The hypothesis follows from two assumptions:

( i ) Photolesions produced i n uv-irradiated chromatin are decisive f o r the production of most uv-induced chromosome aberrations. ( i i ) A l l chromosome aberrations 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 photolesions, ( i i ) includes the p o s s i b i l i t y that an aberration, such as a chromatid break, occurs very near, but not exactly at the s i t e of a chromatin l e -sion (3) as well as the p o s s i b i l i t y that p r i m a r i l y undamaged s i s t e r chro-matids may be involved i n the f i n a l aberration by recombinational r e p a i r processes (2). The mechanism of aberration production by photolesions i s considered to include incomplete or f a u l t y repair processes at the s i t e s of these lesions (2). The percentage of lesions which f i n a l l y lead to a chromosome aberration may be very small. It i s important to note, that assumption ( i i ) does not necessarily follow from assumption ( i ) (see d i s -cussion).

The hypothesis has several consequences which can be tested by the use of a uv-microbeam. Most uv-induced chromosome aberrations should o r i g i n a t e at the s i t e s where uv-photons are absorbed i n the Chromatin. Photolesions i n the chromatin should not have any important influence on the y i e l d of aberrations produced i n other parts of chromatin not containing photole­sions« Chromatin lesions created by mutagens photochemically produced i n the karyoplasm or cytoplasm (1) should contribute very l i t t l e to the y i e l d of aberrations* In such a case the s i t e where i t creates a chromatin l e -sion and f i n a l l y a chromosome aberration may be s i g n i f i c a n t l y apart from each other. A non random d i s t r i b u t i o n of chromosome aberrations i s compatible with the hypothesis, since the p r o b a b i l i t y that a chromatin l e s i o n w i l l cause an aberration may be d i f f e r e n t i n d i f f e r e n t chromosome segments.

Griggs and Bender (4) have obtained evidence i n Xenopus c e l l s that a cer-t a i n c l a s s of DNA-photolesions, namely the pyrimidine cyclobutane dimer i s responsible for most uv-induced chromosome aberrations. Bender et a l . (2) have provided a model which suggests that the s i t e of dimer formation i s the s t a r t i n g point for the formation of uv-induced aberrations. With res -pect to t h i s model the above predictions can be formulated i n a more re-s t r i c t e d form, sub s t i t u t i n g pyrimidine cyclobutane dimer for photolesion.

3o - 2

It i s s t i l l a matter of controversy, however, whether t h i s dimer should be considered the only important class of photolesions decisive for prac-t i c a l l y a l l uv-induced chromosome aberrations i n a l l eukaryote c e l l s (5, 6 , 7 ) .

According to the general formulation of the hypothesis any photolesion i n -troduced either i n the DNA or in the protein moiety of the chromatin may be considered to contribute to the y i e l d of uv-induced chromosome aberra­tions.

2. Experimental methods and r a t i o n a l e

Chinese hamster c e l l s were microirradiated during interphase ei t h e r i n the nucleus or in the cytoplasm and chromosome spreads were obtained i n s i t u a f t e r d i f f e r e n t incubation 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 material and culture conditions have been described elsewhere (8,9,10,11). UV-microirra-d i a t i o n alone did not induce s u f f i c i e n t numbers of chromosome aberrations at a convenient uv-dose (Fig. l d ) . At higher doses the mitotic index de-creased strongly so that the microbeam procedure became unsuitable. The y i e l d of aberrations i n c e l l s microirradiated i n the nucleus, however, was greatly increased by addition of Coffeine (0.5 - 2 mM) to the culture me­dium during the incubation period ( F i g . l e ) . Coffeine i s known to poten-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 aberrations i n rodent c e l l s , when i t i s present during the sub-sequent S-phase a f t e r i r r a d i a t i o n (13). The number of aberrations i n con-t r o l c e l l s and i n c e l l s microirradiated in the cytoplasm was small and not s i g n i f i c a n t l y increased by Coffeine at the indicated concentrations ( F i g . la-c,6a). The hypothesis predictsthat those interphase chromosomes which l i e i n the microirradiated part of the nucleus and receive uv-induced pho­tolesions, should show aberrations at metaphase, depending on the number of photolesions and the stage of the c e l l cycle at i r r a d i a t i o n . Those chro­mosomes or chromosome segments which are situated i n the unirradiated part of a nucleus during m i c r o i r r a d i a t i o n and bear few or no photolesions should show very few or no aberrations. An increase of the size of the i r r a d i a t e d nuclear area maintaining 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 interphase 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 aberrations i n metaphase spreads, r e f l e c t i n g the d i s t r i b u -tion of photolesions.

3. Results

3.1. D i s t r i b u t i o n of chromatin photolesions within microirradiated n u c l e i .

For the Interpretation of the r e s u l t s of a chromosome analysis of micro­i r r a d i a t e d c e l l s i t i s important to know the d i s t r i b u t i o n of chromatin pho­tolesions within the nucleus obtained by uv-microirradiation. A method to measure th 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 evaluation of the d i s t r i b u t i o n was obtained i n d i r e c t l y by t h e o r e t i c a l considerations 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 available indicate that most of the t o t a l uv-dose absorbed by a c e l l i s absorbed within the microirradiated area. Consequently most of the photolesions have to be pro­duced there, while the number of photiesions produced outside t h i s area by d i f f r a c t i o n and dispersion of the uv-microbeam i s small.

3o - 3

a b c d • 1 • i.ni

A B C D E A B C D E A B C D E A B C D E A B C D E

F i g . 1; Chromosome damage a f t e r laser-uv-microirradiation i n the nucleus and the cytoplasm and posttreatment with/without Coffeine.

C e l l s of a V-79-subline of the Chinese hamster (10) were m i c r o i r r a ­diated ( X = 257.3 nm) at one s i t e and thereafter incubated i n the presence/absence of Coffeine. Colchicine was added 3 hours be-fore i n s i t u chromosome preparation which was performed 13 hours a f t e r i r r a d i a t i o n . Scorable mitotic figures obtained were c l a s s i -f i e d according to the following c r i t e r i a :

A: No chromosome damage; B: only Single defects (1-2 breaks,gaps); C: some chromosomes severely damaged, the majority being i n t a c t ; D: most chromosomesdamaged, at le a s t one chromosome remaining i n ­t a c t ; E: a l l chromosomes damaged up to complete d i s i n t e g r a t i o n .

Abscissa: type of damaged mitotic f i g u r e . Ordinate; proportion of each t y p e ( % ) .

The t o t a l number of mit o t i c figures 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 of the cytoplasm (1/8 sec), 1 mM Coffeine (87) d) m i c r o i r r a d i a t i o n of the nucleus (1/8 sec), no Coffeine (29) e) m i c r o i r r a d i a t i o n of the nucleus (1/8 sec), 1 mM Coffeine (47).

The size of the fluorescent area induced by the microbeam i n the object p l a ­ne and the si z e of v i s i b l e l e sions produced i n unstained nuclei of l i v i n g c e l l s , as well as i n stained c e l l specimens i s very s i m i l a r (8,12). The s i ­ze of the le s i o n detectable i n the l i g h t microssope produced with the U l t r a -f l u a r 32x/Ph, used i n a l l experiments described i n the present 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 further indicated by electron microscopic Obser­vation (14) and by the fi n d i n g that unscheduled DNA synthesis can be indu­ced s e l e c t i v e l y at the i r r a d i a t i o n s i t e ( F i g . 2) (15).

Autoradiographs of microirradiated nuclei which were not i n S-phase during the subsequent incubation period with ^HTdR show that the number of s i l v e r grains/|im2 over the microirradiated s i t e c l e a r l y increases with dose, but f a l l s to the small l e v e l of control nuclei within a distance of 6 u.m from the center of the microirradiated area (data not shown). The area covered densely with s i l v e r grains was found to be approx. 3.6 + 1.0 |im i n diame­ter. This area i s somewhat larger than the si z e of the fluorescent spot suggesting that a s i g n i f i c a n t number of DNA-lesions giving r i s e to DNA

3o - 4

repair synthesis i s produced within the whole cone of uv-rays within the nucleus, A large part of the chromatin of microirradiated nuclei as shown i n F i g . 2 did not perform any uv-induced unscheduled DNA synthesis. It has been shown that the amount of unscheduled DNA synthesis 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 incident photons and not on the area of i r r a d i a t i o n (15).

These data suggest that a considerable proportion of uv-induced DNA lesions produced outside the microirradiated area and giving rise to DNA repair syn­thesis, should be detected by the autoradiographic method. Our r e s u l t s i n -dicate, therefore, that the large majority of such le s i o n s i s produced within a radius of less than 6 u.m from the center 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 laser-uv-microirradiation of the nucleus and subsequent incubation with 3|-|_Thymidine for 1 hour. Unscheduled DNA synthesis at the microirradiated s i t e i s indicated by s i l v e r grains(arrows).

Figs.3-5: M i t o t i c figures obtained a f t e r laser-uv-microirradiation of nuclei of V-79 c e l l s and posttreatment with Coffeine. (For ex-perimental d e t a i l see F i g . 1) F i g . 3: chromosome damage i n a li m i t e d area (arrows ) (type C), Figs. 4,5: generalized chromosome damage (type E). Fig. 5 shows an example of complete chromosomal d i s i n t e g r a t i o n

Bars indicate 10 p.m.

3o - 5

3.2. Chromosome aberrations obtained i n metaphase spreads from c e l l s micro­

i r r a d i a t e d i n the nucleus.

Five types A - E of mitotic figures obtained from c e l l s microirradiated in the nucleus and thereafter incubated i n the presence of Coffeine were ten-t a t i v e l y distinguished (see legend of F i g . 1). After i r r a d i a t i o n of one small part of the nucleus a cert a i n number of mitotic figures showed a small number of damaged chromosomes (type C, Figs. le, 6). In the large majority 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 less concentrated i n one small area (Fig. 3). This type of damage has not been observed a f t e r uv-i 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 hypothesis. Karyotypes obtained from metaphase spreads showing t h i s type of damage re-vealed that 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 far are consistent with the hypothesis that i n h i b i t i o n of post r e p l i c a t i o n repair at the s i t e s of photolesions caused aberrations to appear i n microirradiated chromosomes (16,11).

Other mitotic figures were unexpectedly found where most (type D) or even a l l chromosomes (type E) were damaged (Figs. 4,5). While some aberrations were expected to appear i n chromatin outside the microirradiated area be-cause of the small number of photolesions which might be introduced i n t h i s chromatin, there i s no apparent reason why the microirradiated chromosomes or 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 microirradiated 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 that the proportion of type D and type E increase with dose. The Coffeine concentration and the stage of the c e l l cycle are other im­portant parameters i n determining whether in t a c t (type A) or severely dama­ged mitotic figures (types C - E) are obtained (data not shown). The number of photolesions produced i n any interphase chromosome i s not a s u f f i c i e n t c r i t e r i o n to predict the p r o b a b i l i t y of the number of aberra­tions i n mitosisoriginating at t h i s chromosome. This conclusion i s suppor-ted by the following comparison. At the lowest dose (1/125 sec) the concen­t r a t i o n of photolesions i n the microirradiated part of the nucleus i s s t i l l rather high, the energy density within the focus being i n the order of 750 ergs/mm^. Unscheduled DNA synthesis at the mi c r o i r r a d i a t i o n s i t e of such nuclei could be detected, but there was no s i g n i f i c a n t y i e l d of uv-in­duced chromosome aberrations (Fig. 6b). At higher doses, however, chromoso­me d i s i n t e g r a t i o n was obtained even i n unirradiated parts of m i c r o i r r a d i a ­ted n u c l e i , although the data described i n 3.1 suggest a much lower con­centration of photolesions i n these unirradiated parts at any dose, as com-pared with the concentration of photolesions in chromatin microirradiated 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 small part of the nucleus were compa-red with those of a larger part ( F i g . 6b-e). For any investigated uv-dose the percentages of types A - E were found s i m i l a r i n both types of experi-ments.

In further experiments m i c r o i r r a d i a t i o n of a small part of the nucleus was compared with m i c r o i r r a d i a t i o n of whole nuclei at the same dose (1/15 sec) (unpublished data). Under the experimental conditions of these l a t t e r ex­periments the percentages for type C and D were very low. The percentages

2c - 6

for type E were found to be 40 % and 37 %. These data indicate that demo-l i t i o n of a l l chromosomes may be achieved in 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 photolesions.

a b i

c d e

Ii; l l Ü . i . i : l!h:,:|i J_j L l i ± 1 " " - 1 1

A B C D E A B C D E A B C D E A B C D E A B C D E

Fig. 6: Chromosome dar.iage af t e r laser-uv-microirradiation of nuclei with d i f f e r e n t doses and posttieatment with 2 mM Coffeine. For ex 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 of mitotic figures see F i g . 1. Abscissa: type of damaged mitotic f i g u r e . Ordinate: proportion of each t y p e ( % )

I : data obtained with the uv-focus placed inside the c e l l nucleus (as i n experiments of F i g . 1);

i : data obtained with the uv-focus placed above the c e l l , r e-J s u l t i n g in 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 gives the i r r a d i a t i o n time, the second and t h i r d number give the number of analyzed mitotic figures using the i r r a ­d i a t i o n modes with the focus inside ( | ) and above the c e l l ( ! ), respectively. a) no i r r a d i a t i o n , 142 mitotic figures, b) 1/125 sec, 3/18, c) 1/60 sec, 28/19, d) 1/30 sec, 52/65, e) 1/15 sec, 30/60.

4. Discussion

M i t o t i c figures showing d i s i n t e g r a t i o n of a l l chromosomes induced by micro­i r r a d i a t i o n of a small part of the nucleus do not support the hypothesis I that photolesions i n the chromatin are the only important s i t e s at which uv-induced chromosome aberrations can o r i g i n a t e . To f i t our r e s u l t s with the hypothesis, we have to assume that the probabi-l i t y of a Single photolesion to create an aberration 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 other photolesions i n the nucleus. If the t o t a l number of photolesions i s high enough, then very few photole­sions i n a chromosome not detectable by the autoradiographic method d e s c r i -bed i n 3.1., could be s u f f i c i e n t to tr i g g e r i t s complete demolition. In cases where a l l chromosomes are completely disintegrated (Fig. 5), the much higher concentration of photolesions i n microirradiated chromosomes cannot be distinguished by c y t o l o g i c a l Observation. This Interpretation, however, does not s a t i s f a c t o r i l y explain why microirradiated chromosomes do not show a more severe d i s i n t e g r a t i o n than unirradiated ones i n many mitotic f i g u ­res of type E, where chromosome demolition i s not complete (Fig. 4).

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To i n t e r p r e t e our r e s u l t s we now consider 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 aberrations can occur at chromatin s i t e s which do not contain photolesions. Both the s i t e s of photolesions and these other s i t e s contribute to the t o t a l y i e l d of uv-induced chromosome aberrations. The proportion of aberrations obtained from each source i n any c e l l may depend on some unknown properties of the c e l l and on the experimental con-d i t i o n s . At least one of the two assumptions leading to hypothesis I i s not compatible with hypothesis I I . Two new assumptions (a) and (b) may be formulated as a l t e r n a t i v e s .

(a) Besides photolesions i n the chromatin other photochemical reactions are also important for the formation of uv-induced chromosome aberrations. In t h i s case assumption ( i i ) may s t i l l be v a l i d . There are several p o s s i -b i l i t i e s . 1. Mutagens may be produced i n the microirradiated part of the karyoplasm and create chromatin lesions anywhere in the nucleus (1). 2. UV-damage of the nuclear envelope may lead to uncontrolled fluxes of important substances i n both d i r e c t i o n s at the microirradiated s i t e . Such an e f f e c t , as well as an uv-dependent formation of toxic substances may lead to severe changes i n the i n t e r n a l milieu of the whole nucleus which may become more s e n s i t i v e to the action of c a f f e i n e . It i s known that Coffeine at high con-centrations ( ^ 10 mM) induces chromosome d i s i n t e g r a t i o n i n unirradiated c e l l s (17 and our unpublished data).

A l t e r n a t i v e l y , assumption ( i ) may be supposed to be v a l i d . Then assumption ( i i ) has to be abandoned and assumption (b) may be formulated: A s i g n i f i ­cant number of chromosome aberrations which depend on the formation of photolesions i n the chromatin do not o r i g i n a t e at the s i t e s of the photole­sions. Again several p o s s i b i l i t i e s may be considered.

1. The genetic Information at the irradiated part of a nucleus including genes important for the morphological i n t e g r i t y of a l l mitotic chromosomes may be blocked by t r a n s c r i p t i o n terminating photolesions i n a dose depen-dent way (18). A codogenic Strand of such genes free from t r a n s c r i p t i o n terminating lesions may be necessary sh o r t l y a f t e r semiconservative DNA r e p l i c a t i o n of these genes and obtained by DNA repair processes, 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 correct time by In­h 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 required d i s t r i b u t e d throughout the nucleus to explain the frequent occurrence of type E at higher doses (Figs. le, 6e).

2. Increased DNA degradation a f t e r u v - i r r a d i a t i o n has been reported for mammalian c e l l Systems and several mutants i n bacteria (19,20). These f i n -dings may j u s t i f y a speculation whether generalized chromosomal demolition could be due to the uncontrolled action of an uv-induced nuclease. Recent-l y , i n h i b i t i o n of poly(ADPR) synthase by caffeine has been reported (21). A possible r o l e of such enzyme(s) in the control of nucleases i s exempli-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 -ation (22). 3. Finally, the possible rol e of competition between s i t e s of post r e p l i ­cation r e p a i r and DNA r e p l i c a t i o n points for some limiting enzyme(s) may be considered. Both s i t e s provide d i s c o n t i n u i t i e s i n newly synthesized Strands of DNA (23,25) and are known to be affected by caffeine. The mole-cular mechanism may involve binding of caffeine to Single stranded DNA

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regions (24) which occur at both s i t e s . The function of l i a i t i n g enzyme(s) binding to these s i t e s and involved both in an accurate Performance of se-miconservative DNA synthesis and i n post r e p l i c a t i o n repair may be impaired in the presence of c a f f e i n e . I n h i b i t i o n by dire c t binding of caffeine to such l i m i t i n g enzyme(s) may also be considered (17). At a low concentration of caffeine the function of the enzyme(s) may s t i l l s u f f i c e to avoid chromosomal aberrations occuring in unirradiated c e l l s . A c e r t a i n number of DNA photolesions, however, with any d i s t r i b u t i o n of the lesions i n the nucleus, may provide so many addi-t i o n a l s i t e s of binding for l i m i t i n g enzyme(s) that faulty DNA synthesis occurs at DNA r e p l i c a t i o n points and post r e p l i c a t i o n repair becomes i n -complete or f a u l t y at other s i t e s of chromatin lesions which occur spon-taneously during the c e l l cycle (23).

Different s i t e s may provide 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 aberration, but the p r o b a b i l i t y for 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. According to this model uv-induced chromosome aberrations o r i g i n a t e at the s i t e s provided by three d i f f e r e n t sources:photolesions i n the chromatin, other chromatin lesions and DNA r e p l i c a t i o n points. The contribution of each source to the t o t a l y i e l d of uv-induced aberrations may vary with the experimental condi-tio n s . Our model does not exclude the p o s s i b i l i t y that in the absence of caffeine most aberrations would occur at the s i t e s of photolesions (2). It gives an explanation why uv-light produces chromosome aberrations in a S-phase dependent mode (2,13). It would also explain the finding that the change from c e l l s containing no or few aberrations to c e l l s with multiple aberrations i s rather abrupt i n experiments where uv i s combined with caffeine (16).

The model i s compatible with the finding of Griggs and Bender (4) that pho-t o r e a c t i v a t i o n can prevent most uv-induced chromosome aberrations. On the other hand the above considerations indicate that photoreactivation i s not s u f f i c i e n t evidence for the model of Bender et a l . (2).

This i n v e s t i g a t i o n was supported by grants from the Deutsche Forschungs­gemeinschaft (SFB 46). Parts of t h i s i n v e s t i g a t i o n w i l l be presented in the doctoral d i s s e r t a t i o n of C. Zorn to be submitted to the faculty of Biology, University of Freiburg i.Br.

References

1) C.Auerbach, Mutation Research, Problems, Results and Perspectives, pp. 159-255. Chapman and H a l l , London (1976).

2) M.A.Bender, H.G.Griggs and P.L.Walker, Mutat.Res. 20, 387-402 (1973).

3) R.B.Painter, i n : Molecular Mechanisms for 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, 86-88 (1973).

5) S.Wolff, Photophysiology 7, 189-205 (1972).

6) R.B.Painter, Photophysiology 5, 169-189 (1970).

7) J.Kiefer and I.Wienhard, i n : U l t r a v i o l e t t e Strahlen (J.Kiefer, Ed.), pp. 445-565. De Gruyter, B e r l i n - New York (1976).

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8) C.Cremer, C.Zorn and T.Cremer, Micr.Acta 75, 331-337 (1974).

9) C.Zorn, C.Cremer and T.Cremer, i n : Proceedings of the Technical program Electro-Optics International '74 Conference (M.S.Kiver, Ed.), pp. 203-2 Kiver Communications, Surrey (1974).

10) C.Cremer, T.Cremer, C.Zorn, and L.Schoeller, Radiat.Res. 66, 106-121 (1976).

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 , Universität Freiburg i .Br. (1976).

13) B.A.Kihlman, S.Sturelid, B.Hartley-Asp, and K.Nilsson, 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.Salet, Radiat.Res. 58, 52-59 (1974).

16) K.Nilsson and A.R.Lehmann, Mutat.Res. 30, 255-266 (1975).

17) B.A.Kihlman, Mutat.Res. 26, 53-71 (1974).

18) W.Sauerbier, Adv. i n Radiat.Biol. 6, 49-106 (1976).

19) J.R.Williams, Adv. i n Radiat.Biol. 6, 161-210 (1976).

20) L.J.Gudas, J.Mol.Biol. J04, 567-587 (1976).

21) M.I.Davies, H.Halldorsson, S.Shall, and C.J.Skidmore, Abstract book of the 8th Meeting of the European Study Group for C e l l P r o l i f e r a t i o n , Montreux, October 12-15 (1976).

22) H.Hilz and P.Stone, Rev.Physiol.Biochemie.Pharmacol. 76, 1-58 (1976).

23) J.W.Drake and R.H.Baltz, Ann.Rev.Biochem. 45, 11-37 (1976).

24) A.R.Lehmann, Biophys.J. J2, 1316-1325 (1972).

25) A.B.Blumenthal and E.J.Clark, Exp.Cell Res. J05, 15-26 (1977).