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Effects of combined treatments with an oncogenic chemical and virus on cultured cells Hammerberg, Ole 1972

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THE EFFECTS OF COMBINED TREATMENTS WITH AN ONCOGENIC CHEMICAL AND VIRUS ON CULTURED CELLS by OLE HAMMERBERG B.Sc, University of British Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1972 In p resen t i ng t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree tha t the L i b r a r y sha l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy. I f u r t h e r agree t h a t permiss ion f o r ex tens ive copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed w i t h o u t my w r i t t e n pe rmiss ion . Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ABSTRACT Metaphase chromosome aberrations were studied in cultured embryonic Syrian hamster cells after exposure to one of two potent mutagenic chemi-cals in combination with Adenovirus 12. The cultured cells were grown in Minimal Essential Medium supplemented with fetal calf serum. A l l cells were retained in Arginine Deficient Medium during treatments. Exposure to 4-nitroquinoline-l-oxide (4NQ0) was followed at various intervals by Adenovirus 12 infection. Various concentrations of N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) were either followed or preceded at different time intervals by Adenovirus 12 infection. In a l l instances the rates of metaphase plates with chromosome abnormalities after combination treat-ments were approximately equal to the sums of the rates of abnormal plates induced by similar concentrations of each agent independently. A small proportion of the metaphase cells with chromosome abnormalities characteristic of both agents was observed, however, after combined treatments. The relationships between the rates and types of chromosome abnormalities induced by combination treatments and the rates of c e l l transformation as observed elsewhere but induced by similar treatments, are discussed. The rates of metaphase chromosome abnormalities induced by MNNG in vi r a l l y transformed embryonic Syrian hamster cells were also investigated. It was found that these rates were simply equal to the sums of the rates of chromosome abnormalities induced by MNNG in the nontransformed control cultures plus the spontaneous rates of chromosome abnormalities in the transformed hamster cells. The significance of these findings are also discussed. i i i TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES v i i i ACKNOWLEDGMENT ix INTRODUCTION 1 EXPERIMENTAL 7 A. MATERIALS AND METHODS 7 1. Types of cultured c e l l l i n e s used 7 2. O r i g i n and preparation of Adenovirus 12 7 3. Chemical mutagens used 7 4. Tissue culture media 7 5. Tissue cultures 8 6. Preparation of embryonic tis s u e cultures 8 7. Treatment of c e l l s with virus 9 8. Exposure of c e l l s to chemicals 9 9. Sampling 10 10. Cytologic preparations 10 11. ..Metaphase chromosome analysis 11 12. S t a t i s t i c s 13 B. RESULTS 1. Types of chromosome aberrations investigated 2. The e f f e c t s on embryonic Syrian hamster c e l l s of 4NQ0 treatments followed at various i n t e r v a l s by Ad 12 i n f e c t i o n 3. The e f f e c t s on embryonic Syrian hamster c e l l s of d i f f e r e n t concentrations of MNNG treatments followed at various i n t e r v a l s by Ad 12 i n f e c t i o n 4. The ef f e c t s on embryonic Syrian hamster c e l l s of Ad 12 i n f e c t i o n followed at various i n t e r v a l s by exposure to d i f f e r e n t concentrations of MNNG 5. The eff e c t s of MNNG treatments on transformed Syrian hamster c e l l s DISCUSSION REFERENCES V LIST OF TABLES Page Table 1 Types of chromosome exchanges induced by 2 hour treatment with 10"MNNG i n embryonic Syrian hamster c e l l s 16 Table 2a Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to 4NQ0 and Ad 12: vir u s t r e a t -ment at d i f f e r e n t i n t e r v a l s following chemical treatment 20 Table 2b Types of chromosome aberrations induced by the combined action of 4NQ0 and Ad 12 in primary embryonic Syrian hamster c e l l s : v irus treatment at d i f f e r e n t i n t e r v a l s following chemical treatment 21 Table 3 M i t o t i c rates i n embryonic Syrian hamster c e l l s at 27-32 hours a f t e r separate and combined treatments with 4NQ0 and Ad 12 22 Table 4a Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to MNNG and Ad 12: virus treatment at d i f f e r e n t i n t e r v a l s a f t e r chemical treatment 27 Table 4b Types of chromosome aberrations induced by the com-bined a c t i o n of MNNG and Ad 12 in primary embryonic Syrian hamster c e l l s : v i r u s treatment at d i f f e r e n t i n t e r v a l s following chemical treatments 28 Table 5a Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to MNNG and Ad 12: vir u s treatment 1 hour following treatment with concentra-tions of the chemical 29 v i Page Table 5b Types of chromosome aberrations induced by the com-bined a c t i o n of MNNG and Ad 12 i n primary embryonic Syrian hamster c e l l s : v i r u s treatment 1 hour follow-ing treatment with d i f f e r e n t concentrations of the chemical 30 Table 6 M i t o t i c rates i n embryonic Syrian hamster c e l l s at 27-32 hours a f t e r separate and combined treatments with MNNG and Ad 12 31 Table 7a Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to Ad 12 and MNNG: vir u s t r e a t -ment at d i f f e r e n t i n t e r v a l s preceding chemical t r e a t -ment 34 Table 7b Types of chromosome aberrations induced by the com-bined a c t i o n of Ad 12 and MNNG i n primary embryonic Syrian hamster c e l l s : v i r u s treatment at d i f f e r e n t i n t e r v a l s preceding chemical treatment Table 8a Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to Ad 12 and MNNG: vir u s t r e a t -ment 1 hour before treatment with d i f f e r e n t concentra-tions of the chemical 37 Table 8b Types of chromosome aberrations induced by the com-bined a c t i o n of Ad 12 and MNNG i n primary embryonic Syrian hamster c e l l s : v i r u s treatment 1 hour before treatment with d i f f e r e n t concentrations of the chemical 38 35 v i i Page Table 9 M i t o t i c rates i n embryonic Syrian hamster c e l l s at 27-32 hours a f t e r separate and combined treatments with Ad 12 and MNNG 40 Table 10a Chromosome aberrations i n transformed Syrian hamster c e l l s exposed to MNNG 44 Table 10b Types of chromosome aberrations induced by MNNG i n transformed Syrian hamster c e l l s 45 Table 11 M i t o t i c rates i n continuous and transformed hamster l i n e s 5 - 1 0 hours a f t e r treatments with MNNG 4 5 v i i i Page Figure 1 Classes of chromosome exchanges. 12 Figure 2 Types of chromosome aberrations investigated. 14 Figure 3 Percent c e l l s u r v i v a l of HEp-2 c e l l s a f t e r treatments with various concentrations of MNNG and 4NQ0. 18 Figure 4 Experimental design. V a r i a t i o n of inte r v a l s between 4NQ0 and Ad 12 treatments on embryonic Syrian hamster c e l l s . 19 Figure 5 Experimental design. V a r i a t i o n of inte r v a l s between d i f f e r e n t concentrations of MNNG and Ad 12 treatments on embryonic Syrian hamster c e l l s . 25 Figure 6 Frequency of metaphase plates with chromosome aberrations. 26 Figure 7 Experimental design. V a r i a t i o n of int e r v a l s between Ad 12 and d i f f e r e n t concentrations of MNNG treatment on embryonic Syrian Hamster c e l l s . 32 Figure 8 Frequency of metaphase plates with chromosome aberrations. 36 Figure 9 Experimental design. Different transformed Syrian hamster c e l l l i n e s treated with MNNG. 41 ix ACKNOWLEDGMENT I wish to thank Dr. H.F. S t i c h for h i s guidance throughout the preparation of th i s thesis project. I also wish to express my gratitude to Mrs. H.F. S t i c h for her technical assistance and to Dr. R.L. Noble for supplying research accommodations. - 1 -INTRODUCTION The f i r s t work on environmental carcinogenesis dates back to 1775 when Pott demonstrated that prolonged exposure to soot was the cause of cancer of the scrotum i n chimney sweeps. There now exist ample evidence that e x t r i n s i c factors are the d i r e c t or i n d i r e c t causes of many cancers (Freeman et a l . , 1971). While one area of environmental carcinogenesis i s concerned with the i d e n t i f i c a t i o n of such e x t r i n s i c agents, another area involves i n v e s t i g a t i n g the mechanisms by which agents produce malignant neoplasms. The mutagenic capacity of environmental carcinogenic agents has been of much interest as a possible cause of neoplastic transformation. There e x i s t three major groups of carcinogenic agents: chemical, v i r a l and ph y s i c a l . Although i t has been known for many years that chemical and physical car.cinogens are often highly mutagenic i n lower animals, i t was only recently confirmed that such agents are also highly mutagenic i n somatic mammalian c e l l s (Chu, 1968 and Kao et a l . , 1968). In the past decade much att e n t i o n has also been paid to the mutagenic capacity of oncogenic viruses (Stich & Yohn, 1967). Several investigators have shown that the oncogenic DNA viruses a c t u a l l y change the genetic information contained within the c e l l s they transform by covalently l i n k i n g some of t h e i r own DNA molecules with those of the host c e l l s (Doerfler., 1968 and Sambrook et a l . , 1968). The exact mechanism of t h i s i s s t i l l not known. Although the transforming mechanisms of the oncogenic RNA viruses are not as well understood, the recent discovery of a RNA dependent DNA polymerase (Temin & Mizutani, 1970 and Baltimore, 1970) indicates that RNA v i r u s transformation l i k e l y involves a genetic process. Such findings have given support to the "somatic c e l l mutation theory - 2 -of cancer". This theory i s not recent. It was f i r s t s e r i o u s l y considered by Bauer i n 1928, who postulated that cancer could be the product of a spontaneous or induced gene change i n a somatic c e l l . Bauer's proposal was supported by many other early investigators including Ludford (1930) and Lockhart-Mummery (1934). Berenblum (1944) and Berenblum & Shubik (1947, 1949) used the somatic mutation theory to explain the r e s u l t s from t h e i r croton o i l experiments. They discovered that a non-carcinogenic substance, such as the skin i r r i t a n t croton o i l , could e l i c i t a highly neoplastic response on the skin of mice which had previously been treated for an inadequate period with a single dose of a carcinogen such as dibenzarthracene. Treatment with ei t h e r of the chemicals alone, or the reverse order, f a i l e d to induce tumours to any s i g n i f i c a n t degree. They concluded that carcinogenesis is a genetic process which could be divided into two stages, which they termed the " i n i t i a t i n g stage" and the "promoting stage". The croton o i l experiments were the beginning of a new series of hypotheses and experimentations which have continued to present day. An abundant number of d i f f e r e n t combinations of neoplastic agents have been investigated both _in vivo and _in v i t r o . Such studies have been referred to under the t i t l e of "cocarcinogenesis". Salaman and Roe (1964) and Berenblum (1969) have since reviewed t h i s f i e l d . They pointed out that i t is important to recognize that cocarcinogenesis includes a wide range of phenomena i n which two or more agents may act together to enhance or reduce the growth of tumours. A cocarcinogenic agent i s any agent which a f f e c t s the tumour growth when combined either before, simultaneously or a f t e r another agent. Viruses are of p a r t i c u l a r i n t e r e s t as cocarcinogenic agents as they - 3 -are both abundant and universal i n the environment. Duran-Reynals (1963) and Salaman and Roe (1964) have reviewed the p o s s i b i l i t y of viruses being important cocarcinogens. Viruses have been discovered to act as c o c a r c i -nogens when combined with either chemical, physical or other v i r a l agents to produce a change i n neoplastic growth. Rous and Kidd (1936) were the f i r s t to s u c c e s s f u l l y demonstrate a v i r u s as a cocarcinogenic agent when they enhanced tumour growth by innoculating papilloma v i r u s into rabbits whose ears had previously been painted with tar. Many other experiments have been done since i n which various d i f f e r e n t viruses have been shown to have cocarcinogenic capacity. In vivo studies include combinations of the following viruses with various other neoplastic agents: Shope papilloma (Rous & Kidd, 1936 and Rous & Friedewald, 1944), Shope fibroma (Ahlstrom & Andrews, 1938), Rous sarcoma (Carr, 1942), polyoma (Rawson set a l . , 1961), v a c c i n i a (Duran-Reynals, 1957), and influenza (Imagawa, 1957). In a l l of these studies the combined treatment gave more rapid and also more frequent tumour production than the si n g l e agents. Although the tumours usually possess the c h a r a c t e r i s t i c s of those produced by the virus, they are often l o c a l i z e d where the chemical i s rendered most active. Salaman and Roe (1964) described the production of tumours induced during v i r a l cocarcinogenesis..."the whole course of oncogenesis may resemble that induced by a more v i r u l e n t v i r u s i n a s i m i l a r host, or by the same v i r u s i n a more susceptible host not treated with the chemical". V i r a l cocarcinogenesis i s now being studied more commonly i n the i n  v i t r o c e l l system. I f i t i s recognized that in v i t r o conditions are far d i f f e r e n t from those _in vivo and should be used as a complementary rather than as an a l t e r n a t i v e t o o l , the ti s s u e culture o f f e r s important advan-tages. I t i s both more f l e x i b l e , far less expensive and gives r e s u l t s - 4 -much faster. Whereas transformation experiments i n ti s s u e culture can be analyzed within days, tumour growth takes months i n test animals. V i r a l cocarcingoenic studies _in vivo include combination e f f e c t s of viruses and several agents. Viruses which have been combined with r a d i a t i o n include polyoma (Stoker, 1963) and SV 40 (Pollock & Todaro, 19 68; Kouri & Coggin, 1968 and Coggin et a l . , 1970); with chemicals, Rauschner leukaemia (Price et a l . , 1971), polyoma (Rawson et a l . , 1961), and SA 7 ( S t i c h & Casto, 1971); and with other viruses, Ad 12 (Butel et a l . , 1971) and murine sarcoma (Chirigos et a l . , 1968; Turner & Chirigos, 1969 and Turner et a l . , 1970). In a l l of these studies the combination enhanced the transforming frequency, while the transformed clones re-tained the v i r a l c h a r a c t e r i s t i c s . Although i t has been known for a long time that many oncogens are also potent inducers of metaphase chromosome aberrations (Stich & Yohn, 1970), any r e l a t i o n s h i p between cocarcinogenesis and associated chromo-some aberrations has never been studied. Such an i n v e s t i g a t i o n i s of int e r e s t since i t has been proposed that v i r a l cocarcinogenesis i s a genetic process (Pollock & Todaro, 1968 and Coggin, 1969). Assuming that there exists a r e l a t i o n s h i p between the a b i l i t y of agents to induce transformation and chromosome aberrations within the same system, i t may be expected that treatments with two agents which induce a synergestic increase i n the rate of transformation, would have one or more of the following e f f e c t s on the chromosome complement: 1. The proportions of c e l l s with chromosome abnormalities should be much higher than the sums of the proportions of c e l l s with chromosome aberrations induced by each agent independently. 2. The proportion of metaphase c e l l s with a c e r t a i n type of - 5 -chromosome aberrations should increase. Recently S t i c h and Casto (1971) discovered that t r e a t i n g primary embryonic Syrian hamster c e l l s _in v i t r o with the oncogen 4-nitroquinoline-1-oxide (4NQ0) followed by in f e c t ion with the simian Adenovirus SA 7 produced a 23 f o l d enhancement i n the rate of transformation over that induced by treatment with the chemical alone. The human adenovirus Ad 12 has very s i m i l a r properties to those of the simian adenovirus except for host s p e c i f i c i t y . In preliminary experiments Ad 12 has been found to induce a high rate of fragmentation, p u l v e r i z a t i o n and chromatid breaks but a low rate of exchange configurations i n hamster c e l l s . 4NQ0 and N-methyl-N 1-nitro-N-nitrosoguanidine (MNNG) two powerful chemical oncogens and mutagens (Nakahura et a l . , 1957; Endo et a l . , 1961; Adelberg et a l . , 1965 and Sugimura et a l . , 1966), induce a high rate of exchanges but a low rate of chromatid breaks, p u l v e r i z a t i o n and fragmentations i n cultured hamster c e l l s at G-^  of the c e l l cycle (Kelly & Legator, 1970). Hence the v i r u s and the chemicals induce d i f f e r e n t kinds of chromosome abnormalities and th e i r independent a c t i v i t i e s within a metaphase plate can be i d e n t i f i e d . Since they are s i m i l a r to the agents whose cocarcino-genic properties were investigated by S t i c h and Casto, they provide an ideal system for studying the combined e f f e c t s of chemicals and viruses on chromosomes. In the present study several experiments were conducted to i n v e s t i -gate the combination e f f e c t s of the chemicals and Ad 12 on the metaphase chromosome complement: 1. Embryonic Syrian hamster c e l l s were treated with d i f f e r e n t concentrations of the chemical carcinogens followed at d i f f e r e n t i n t e r v a l s by i n f e c t i o n with Ad 12. - 6 -2. The procedure was reversed and the embryonic Syrian hamster c e l l s were infected by Ad 12 followed at various i n t e r v a l s by treatment with d i f f e r e n t concentrations of MNNG. In c e l l s transformed by viruses the genetic material of the virus, or at le a s t part of i t , i s incorporated into the DNA of the host c e l l (Westphal & Dulbecco, 1968). Since viruses are prone to destruction by the mutagen i f the l a t t e r i s applied a f t e r the v i r u s to the c e l l culture, i t i s more p r a c t i c a l to treat v i r u s transformed c e l l s with the chemical mutagen. I t i s also of in t e r e s t to determine i f such transformed c e l l cultures which contain c e l l s with metaphase chromosome aberrations, are more susceptible to induction of chromosome aberrations by a mutagenic chemical. C e l l l i n e s transformed by various oncogenic DNA viruses were therefore treated with MNNG and the r e s u l t s were compared to those of a continuous non-transformed hamster c e l l l i n e having undergone s i m i l a r treatments. - 7 -EXPERIMENTAL A. MATERIALS AND METHODS 1. The following types of cultured c e l l l i n e s were:used: (a) Primary or secondary cultures of embryonic Syrian hamster c e l l s . (b) Baby Hamster Kidney 21 c e l l s (BHK-21). (c) Rous sarcoma transformed Syrian hamster c e l l s . (d) Polyoma transformed Syrian hamster c e l l s . (e) SV 40 transformed Syrian hamster c e l l s . 2. O r i g i n and preparation of Adenovirus 12: The human Adenovirus 12 ( s t r a i n Huie) was o r i g i n a l l y obtained from Dr. R. Huebner of the National Cancer I n s t i t u t e , Bethesda, Md. The absence of adeno-associated v i r u s (AAV) was confirmed by ele c t r o n micro-scopic examination. The vir u s was grown i n KB suspension cultures and t i t r a t e d on primary human embryonic kidney c e l l s i n Medium 199, supple-mented with 2% f e t a l c a l f serum. 3. The following chemical mutagens were used: 4-nitroquinoline-l-oxide (4NQ0) was obtained from D a i i c h i Pure Chemicals Co. Ltd., Tokyo. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was obtained from A l d r i c h Chemical Co. Inc., Milwaukee, Wis. 4. Tissue culture media: A l l c e l l s were grown and maintained at 37°C i n Eagle's Minimum E s s e n t i a l Medium (MEM), buffered with Hank's s a l t s . The medium was supplemented with 107o f e t a l c a l f serum and the following a n t i b i o t i c s : - 8 -p e n i c i l l i n (100 units/ml.), streptomycin (100 mg/ml.), kanamycin (1%), and fungizone (1%). MEM d e f i c i e n t i n arginine was l a b e l l e d arginine d e f i c i e n t medium (ADM), I t was used to stop c e l l growth i n G^ of the c e l l cycle. A l l primary embryonic Syrian hamster c e l l s were maintained for two days i n ADM p r i o r to v i r u s and chemical treatment which res u l t e d i n exposure to the vi r u s and the chemical i n G^, a time of no DNA synthesis. (Freed & Schatz, 1969) The ADM also induced a p a r t i a l synchrony of the c e l l c y c l e which ensured a high l e v e l of mitosis at c e r t a i n i n t e r v a l s a f t e r the medium was changed to MEM. 5. Tissue cultures: A l l the c e l l l i n e s grew as a monolayer on glass surface. The stock cultures were grown i n 32 oz. p r e s c r i p t i o n glass b o t t l e s . Experimentation was performed i n Leighton tubes i n which the c e l l s grew on glass cover-s l i p s . The cov e r s l i p s were prepared by b o i l i n g i n 3 parts sulphuric a c i d to 1 part n i t r i c a c i d for two hours. The primary embryonic Syrian hamster c e l l s were seeded into the Leighton tubes at a c e l l density of 2xl0~* 4 c e l l s per tube, whereas the continuous l i n e s were seeded at 5x10 c e l l s per tube. This was necessary because of differences i n seeding e f f i c i e n c y . Treatment was started before contact i n h i b i t i o n to allow for better meta-phase chromosome preparations. C e l l s which were also maintained i n ADM were rinsed twice and transfered to clean Leighton tubes. The culture medium was often changed to maintain a pH close to 7.4. 6. Preparation of embryonic tis s u e cultures; Pregnant Syrian hamsters were k i l l e d and the e n t i r e u t e r i contain-ing 15-18 day embryos were removed. The embryos were separated from the membranes under s t e r i l e conditions. The heads and guts were removed and - 9 -the remainder was grated with a pair of blunt scissors. The cells were separated in sterile Bellco trypsinization bottles containing 0.257» trypsin in calcium low medium (15 mg/liter), by magnetic sti r r i n g for approximately \\ hours. After the trypsinization, 5-10% fetal calf serum was added to inactivate the action of the trypsin. Large pieces of tissue were removed from the suspension by f i l t e r i n g through sterile cheesecloth. The filtered suspension was centrifugedat 1000 r.p.m. for 5 min., the supernatant was discarded, and MEM with 107» F.C.S. and the antibiotics described was added. The pellet was resuspended thoroughly using vigor-ous pipetting. Finally the c e l l concentration was estimated using a haemocytometer and the desired number of cells were seeded in 32 oz. pre-scription bottles. A l l the continuous lines were obtained as monolayers from Flow Laboratories, Berkley, Calif. 7. Treatment of cells with virus: A multiplicity of lOx was used to induce the desired number of chro-mosome aberrations. The number of cells per coverslip was calculated from the seeding efficiency and the growth rate. The virus, suspended in Hank's buffered salt solution, was kept on ice whenever i t was removed from the freezer. It was diluted so a volume of approximately 0.2 ml. per Leighton tube was always added. During the absorption period, of either 3 or 4 hours at 37°C, the Leighton tubes were shaken repeatedly to ensure an even spread of the virus across the entire coverslip. The Leighton tubes were rinsed twice with ADM containing no serum before virus infection. 8. Exposure of cells to chemicals: 4NQ0 and MNNG were dissolved immediately before treatment. The - 10 -o chemicals were dissolved d i r e c t l y to give a 10- M s o l u t i o n i n 10 ml. ethanol. Ethanol was non-toxic at low concentrations (less than 1%) and a better solvent than water. The appropriate concentrations were made by s e r i a l d i l u t i o n s i n ADM or MEM. The c e l l s were exposed to 4NQ0 for \\ hours and MNNG for 2 hours. After mutagen treatment, a l l c e l l s were rinsed twice i n MEM without serum. 9. Sampling: The c e l l growth was arrested at the metaphase stage of mitosis by tre a t i n g with low concentrations of c o l c h i c i n e (Inoue, 1952). 0.1 ml. of 0.017o c o l c h i c i n e was added for 5 hours to each Leighton tube containing 1 ml. MEM. Colchicine is decomposable i n solu t i o n and should be prepared not more than 24 hours before treatment. Since ADM treatment p a r t i a l l y synchronizes the c e l l cycle, c o l c h i c i n e was added to d i f f e r e n t Leighton tubes of the same treatment set at various in t e r v a l s a f t e r ADM was changed to MEM. This was necessary to ensure that at l e a s t one set of preparations had a high rate of mitosis (see Figs. 4, 5 & 7). In the case of both virus and mutagen treatment, "time 0" always started with MEM addition. Since ADM was not used on the continuous l i n e s , "time 0" always started just a f t e r mutagen treatment i n experiments involving continuous culture l i n e s . 10. Cytologic preparations: After metaphase arrest, the c e l l cultures were placed i n a hypotonic l7o sodium c i t r a t e s o l u t i o n for 14 minutes. F i x a t i o n was c a r r i e d out for 10 minutes i n f i x a t i v e c o n s i s t i n g of 1 part a c e t i c a c i d to 3 parts ethanol. The c e l l s were stained with 2% orcein i n 50% acetic acid for 5 minutes. The s t a i n was refluxed for 6 - 11 -hours i n the a c e t i c acid and double f i l t e r e d twice before using. The stained preparations were washed i n alcohol, butanol and x y l o l before being mounted, with the c e l l s facing down, with Permount on microscopic s i ides. 11. Metaphase chromosome analysis: A l l the s l i d e preparations were i n i t i a l l y screened i n order to determine which of the c o l c h i c i n e treatments produced the highest rate of mitosis. Only the preparations from a si n g l e c o l c h i c i n e treatment were compared as the rates of chromosome damage i n the same experiment would be al t e r e d i f sampled even a few hours apart. A minimum of 100 metaphase plates per s l i d e were investigated. The chromosomes of a c e l l were considered fragmented i f more than ten isochromatid breaks were observed (Fig. 2c). Metaphase plates with fragmented or pulverized chromosomes were included under "Metaphase plates with chromatid breaks". Chromosome breaks which were smaller than the width of the chromatid strand were considered gaps and were not counted as breaks ( F i g . 2b). A l l translocations, r i n g chromosomes and other chromosome config-urations r e s u l t i n g from exchanges were included under "Metaphase-plates with chromatid exchanges". The types of chromatid exchanges induced by MNNG were investigated using the procedure developed by M.M. Cohen (1969). The chromatid ex-changes were c l a s s i f i e d as f i v e types. Classes 1, 2 and 3 are homologous chromosome exchanges (Fig. 1). Class 4 i s heterogenous and class 5 consists of multichromosome configurations. A l l other chromosome abnormalities such as c o i l i n g d e f i c i e n c i e s , C-mitosis and other rare aberrations were not considered. P o l y p l o i d - 12 C L A S S 4 Figure 1: Classes of chromosome exchanges as proposed by M.M. Cohen. Classes 1, 2 and 3 consist of homologous exchanges while class 4 i s a heterogenous exchange. - 13 -c e l l s which were more common i n the continuous c e l l cultures were ignored. 12. S t a t i s t i c s : The standard deviations of the l i m i t s were calculated i n two samples which were chosen due to t h e i r r e l a t i v e l y high and low rates of metaphase chromosome aberrations. This exercise was performed s o l e l y for the purpose of demonstrating the error i n counting. It was assumed that the error i n counting was approximately s i m i l a r throughout the present inves-t i g a t i o n . The two samples were analyzed four times each. In each analysis not less than 100 metaphase plates were investigated. The sin-arc trans-formation was used to transform the percentage data into a t - d i s t r i b u t i o n . The l i m i t s of the standard deviation was derived as follows: Standard deviation of the l i m i t s = + T-CJ^where Q = standard deviation of the mean at a p r o b a b i l i t y of 0.05. The two samples were found to have sample means of 66.3% and 13.87,,. The standard deviations of the high and low sample means were O = 1.01 and 0(j= 1.26, and the deviation of the l i m i t s were + 3.2 and + 4.0 r e s p e c t i v e l y at a p r o b a b i l i t y of p = 0.05. The best estimates of the two population means would therefore 1 be expressed as 66.3 + 3.2% and 13.8 + 4.0%. B. RESULTS 1. Types of chromosome aberrations investigated: The d i f f e r e n t types of chromosome aberrations which were observed are i l l u s t r a t e d i n Figs. 2a-2g. Normal embryonic Syrian hamster c e l l s (a) Normal chromosome complement of an embryonic Syrian hamster c e l l . (b) Metaphase plate i l l u s t r a t i n g a chromatid gap (I) and a chromatid break ( I I ) . (c) Metaphase plate containing chromosome fragments r e s u l t i n g from isochromatid breaks. (d) Metaphase plate containing an exchange configuration between two chromosomes. (e) Metaphase plate containing an exchange configuration between several chromosomes. (f) Metaphase plate containing pulverized chromosomes. (g) Metaphase plate with both chromatid breaks (I) and exchanges (II) . - 15 -contained a t o t a l of 44 chromosomes (Fig. 2a). The c r i t e r i o n for d i s t i n g u i s h i n g gaps from chromatid breaks (Fig. 2b) has been described i n "Materials and Methods". Chromosome fragments r e s u l t i n g from i s o -chromatid breaks are i l l u s t r a t e d i n F i g . 2c. Isochromatid breaks were recorded as two chromatid breaks. Chromatid exchanges involved two chromosomes or multichromosome configurations (Fig. 2d and 2e). Meta-phase c e l l s with pulverized chromosomes (Fig. 2f) were recorded as having two chromatid breaks per pulverized chromosome. Metaphase c e l l s with both chromatid exchanges and breaks (Fig. 2g) were encountered a f t e r treatments with both chemical and v i r u s . The types of chromatid exchanges induced by a 2 hour treatment of 10~^M MNNG in embryonic Syrian hamster c e l l s were investigated (Table 1). Of the t o t a l number of exchange configurations, 257, involved more than two chromosomes, 587, involved non-homologous chromosome pairs and 167, involved homologous chromosome pa i r s . At no time did exposure to either chemical induce any pulverized chromosomes i n any of the c e l l l i n e s , 2. The ef f e c t s on embryonic Syrian hamster c e l l s of 4NQ0 treatments followed at various i n t e r v a l s by Ad 12 i n f e c t i o n : Cloning experiments (see "Materials and Methods") were employed to estimate the toxic e f f e c t s of 4NQ0 on HEp-2 c e l l s . 95% of the c e l l s survived a f t e r a 90 minute treatment with 5x10 ^ M 4NQ0 whereas only 18% -6 survived a f t e r a 90 minute treatment with 2x10 M 4NQ0 (Fig. 3). Approx-imately 50% c e l l s u r v i v a l occurred a f t e r exposure to 8x10"^ 4NQ0 for 90 minutes. This same treatment was chosen for the present study. After t e s t i n g several v i r u s m u l t i p l i c i t i e s at an absorption period of 4 hours, - 16 -Table 1. Types of chromosome exchanges induced by 2 hour treatment with 10"-> M MNNG in embryonic Syrian hamster c e l l s . Types of Chromosome Exchanges1 Class Number Observed Percent of Total Number of Exchanges Homologous' 13 ) ) 10 ) 32 ) 9 ) 16 Non-homologous 117 59 Multichromosome configurations 26 25 See Figure 1. 2 In some cases an ambiguity of i d e n t i f y i n g the homologous chromosomes could not be resolved. - 17 -a m u l t i p l i c i t y of lOx was found to induce 30-37% metaphase plates with chromosome aberrations at 27-32 hours a f t e r v i r u s absorption. A l l preparations were retained i n ADM from 48 hours p r i o r u n t i l the end of treatments (Fig. 4). A l l the preparations exposed to both the chemical and the vir u s were treated at the same time with the chemical and at d i f f e r e n t time in t e r v a l s afterwards with the v i r u s . The c e l l s were exposed to c o l c h i c i n e from 22-27 and from 27-32 hours a f t e r v i r u s absorption. The preparations from the l a t t e r c o l c h i c i n e treatment were analyzed for chromosome aberrations (Tables 2a and 2b). Table 2a shows the percents of metaphase c e l l s with chromosome aberrations a f t e r separate and combined chemical and v i r a l treatments. Exposure to both agents produced approximately the sums of the metaphase plates with chromosome aberrations induced by each agent independently. As the time i n t e r v a l s between chemical treatment and v i r a l i n f e c t i o n increased, the rate'of metaphase plates with chromosome aberrations induced by both agents, decreased from 55%, to 38%. From 97%, to 100% of the abnormal metaphase plates contained chromatid breaks a f t e r Adenovirus 12 i n f e c t i o n (Table 2b). Treatment with 4NQ0 induced exchanges i n 80% to 95% of the metaphase plates with chromosome aberrations. The combined treatments i n -duced a higher rate of chromatid breaks than exchanges i n the abnormal metaphase c e l l s . Metaphase plates with both chromatid breaks and exchanges were observed only a f t e r treatments with both the chemical and the v i r u s . The percent of c e l l s i n the metaphase stage of c e l l mitosis i s commonly r e f e r r e d to as the mitotic rate. In Table 3 i t i s shown that by comparing the mitot i c rates of the treated preparations to those of the controls, the 90 minute 8xl0~^M 4NQ0 treatment decreased the mitotic rate nearly f i v e times whereas the Adenovirus 12 treatment increased the mitotic - 18 -Concent ra t ion of Mutagens (M) Figure 3: Percent c e l l s u r v i v a l of HEp-2 c e l l s a f t e r treatment with various concentrations of 4NQ0 and MNNG (for 90 minutes and 120 minutes r e s p e c t i v e l y ) . 4NQ0 6 — ; MNNG — • 4 N Q O A d 12 co lch ic ine co lch ic ine _0 2 4 6 8 10 12 14 16 18 2 0 22 2 4 2 6 2 8 3 0 32 A D M 4 N Q Q 4UQO A d 12 c o l c h i c i n e co l ch i c ine 0 2 4 6 A D M 15 2 4 6 8 10 V2 18 2 0 2 2 2 4 2 6 2 8 3 0 32 A d 12 co lch ic ine co lch ic ine 0 2 4 6 8 10 12 A D M r inse 2 x M E M a d d e d 0 2 4 6 18 20 2 2 2 4 2 6 2 8 3 0 3 2 f i x a t i o n f i x a t i o n s t a i n i n g s ta in ing Figure 4: Experimental design. V a r i a t i o n of int e r v a l s between 4NQ0 and Ad 12 treat ments of embryonic Syrian hamster c e l l s . - 20 -Table 2a. Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to 4-nitroquinoline-l-oxide and Adenovirus 12: vi r u s treatment at d i f f e r e n t i n t e r v a l s following chemical treatment. Interval Between Total Number of Metaphase Plates 4NQ0 and Ad 12 Metaphase Plates with Chromosome Treatment Treatment Investigated Aberrations "L Control 150 2 Ad 12 100 37 1 hour 8xl0" 7M 4NQ0 100 20 8xl0" 7M 4NQ0 and Ad 12 100 55 Ad 12 100 32 6 hours 8x10"7M 4NQ0 100 20 8xl0 _ 7M 4NQ0 and Ad 12 100 49 Ad 12 100 30 12 hours 8xl0" 7M 4NQ0 100 11 8xl0~ 7M 4NQ0 and Ad 12 100 38 Table 2b. Types of chromosome aberrations induced by the combined a c t i o n of 4 - n i t r o q u i n o l i n e - l -oxide and Adenovirus 12 i n primary embryonic Syrian hamster c e l l s : v i r u s treatment at di f f e r e n t intervals following chemical treatment. Metaphase Plates Metaphase Plates Metaphase Plates Interval Between with Chromatid with Chromatid with both Chroma-4NQ0 and Ad 12 Exchanges Breaks t i d Breaks and Treatment Treatment (%} (%} Exchanges (%) Control Ad 12 0 100 0 1 hour 8 x l 0 - 7 M 4NQ0 95 5 0 8X10"7M 4NQ0 and Ad 12 16 89 5 Ad 12 3 97 0 6 hours 8x10-7M 4NQ0 90 10 0 8 x l 0 - 7 M 4NQ0 and Ad 12 10 100 10 Ad 12 0 100 0 12 hours 8xl0" 7M 4NQ0 80 20 0 8x10 - 7M 4NQO and Ad 12 16 84 0 22 Table 3. M i t o t i c rates i n embryonic Syrian hamster c e l l s at 27-32 hours a f t e r separate and combined treatments with 4-nitorquinoline--1-oxide and Adenovirus 12. Treatments M i t o t i c Rate (%) Control 2.6 Ad 12 (lOx) 3.3 8xlO" 7M 4NQ0 0.6 8xl0" 7M 4NQ0 and Ad 12 1 1.1 Chemical treatment was followed 1 hour l a t e r by vi r u s i n f e c t i o n . - 23 -rate. The combined expsoure to the chemical followed one hour l a t e r by vir u s i n f e c t i o n resulted i n a decrease i n the mito t i c rate when compared to that of the control. 3. The ef f e c t s on embryonic Syrian hamster c e l l s of d i f f e r e n t concentra-tions of MNNG treatments followed at various i n t e r v a l s by Ad 12 inf e c t i o n : The design of this experiment was si m i l a r to that described for the experiment i n v e s t i g a t i n g the e f f e c t s of 4NQ0 and Ad 12 on metaphase chrom-osomes (see Results Section 2). Exposure of embryonic Syrian hamster c e l l s to MNNG was followed at various i n t e r v a l s by Ad 12 i n f e c t i o n (Fig. 5). In t h i s experiment the c e l l s were exposed to MNNG for 2 hours, however, rather than 90 minutes as was the case i n the experiment with 4NQ0. The v i r a l m u l t i p l i c i t y was lOx and the absorption period lasted 4 hours. The c e l l s were arrested i n metaphase by c o l c h i c i n e treatments from 22-27 hours a f t e r v i r u s i n f e c t i o n . The preparations from the l a t t e r c o l c h i c i n e period were analyzed. In addit i o n to the v a r i a t i o n i n the time i n t e r v a l s between the chemical and the v i r a l treatments, the effects: of varying concentra-tions of MNNG were also investigated. Three d i f f e r e n t concentrations of MNNG were used: 2.5xlO~^M, 5xl0~^M and 10"^M. These chemical concentra-tions were previously found to induce approximately 75%, 65% and 55% c e l l s u r v i v a l . r e s p e c t i v e l y as estimated from cloning experiments of HEp-2 c e l l s (see Materials and Methods) i l l u s t r a t e d i n F i g . 2. 4NQ0 treatments were generally more toxic than s i m i l a r MNNG treatments i n the HEp-2 c e l l s . The percents of metaphase plates with chromosome aberrations induced by the combined e f f e c t s of MNNG and Ad 12 were less than the sums of the percents of metaphase plates with chromosome damage induced by the chemical - 24 -and the v i r u s independently (see Tables 4a and 5a). The frequency of metaphase plates with chromosome aberrations induced by both agents, were d i r e c t l y proportional to the increasing concentrations of the chemical i f the v i r a l m u l t i p l i c i t y remained constant (Fig. 6). The types of chromosome damage induced by the various treatments are i l l u s t r a t e d i n Tables 4b and 5b. From 89-100% of the abnormal meta-phase plates;.of the preparations treated with MNNG contained chromatid exchanges. The Ad 12 induced chromatid breaks i n 96-1007., of the abnormal metaphase plates. Except a f t e r treatment with 1 0 " % MNNG i n just one preparation, only preparations exposed to both the chemical and the v i r u s contained metaphase plates with both chromatid breaks and exchanges. Aft e r treatment with both agents the frequency of abnormal metaphase plates with both chromatid breaks and exchanges increased when the con-centrations of MNNG were increased (Table 5b), whereas i t decreased as the time i n t e r v a l s between the chemical and v i r a l treatments were increased (Table 4b). The mit o t i c rates of the metaphase c e l l s were estimated at 27-32 hours a f t e r separate and combined treatments with MNNG and Ad 12 (Table 6). The preparations treated with either the chemical or both the chemical and Ad 12, had metaphase mitotic rates of approximately h a l f that of the con t r o l . 4. The ef f e c t s on embryonic Syrian hamster c e l l s of Ad 12 i n f e c t i o n followed at various i n t e r v a l s by exposure to d i f f e r e n t concentrations of MNNG: The design of th i s experiment i s i l l u s t r a t e d i n F i g . 7. The c e l l s were infected by Ad 12 at a vi r u s m u l t i p l i c i t y of lOx for an absorption M N N G A d 12 co lch ic ine co lch ic ine J J 2 4 6 8 10 12 14 16 18 2 0 22 2 4 2 6 2 8 3 0 32 A d 12 < co l ch i c ine co lch ic ine A D M M N N Q 1 6 i M N N G A D M ^6 2 4 6 8 10 V2 18 2 0 22 24 2 6 2 8 3 0 32 A d 12 co lch ic ine co lch ic ine 0 2 8 10 12 0 2 A D M r inse 2 x M E M a d d e d 18 20 22 24 2 6 2 8 3 0 3 2 f i x a t i o n f i x a t i o n s t a i n i n g s ta in ing Figure 5: Experimental design. V a r i a t i o n of int e r v a l s between d i f f e r e n t concentra-tions of MNNG and Ad 12 treatments on embryonic Syrian hamster c e l l s . - 26 -10CL M N N G concentrat ions (moles) Figure 6: Frequency of metaphase plates with chromosome abnormalities. MNNG — • — ; MNNG followed by Ad 12 i n f e c t i o n — A . — - 27 -Table 4a. Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to N-methyl-N'-nitro-N-nitrosoguanidine and Adenovirus 12: virus treatment at d i f f e r e n t i n t e r v a l s a f t e r chemical treatment. Interval Between MNNG and Ad 12 Treatment Treatment Total Number of Metaphase Plates Investigated Metaphase Plates with Chromosome Aberrations % Control 150 1 hour Ad 12 10"5M MNNG 10"5M MNNG and Ad 12 100 100 100 44 32 66 6 hours Ad 12 lQ-^M MNNG 10"5M MNNG and Ad 12 100 100 100 32 28 60 12 hours Ad 12 10"5M MNNG 10"JM MNNG and Ad 12 100 100 100 27 29 35 Table 4b. Types of chromosome aberrations induced by the combined a c t i o n of N-methyl-N 1-nitro-N-nitrosoguanidine and Adenovirus 12 in primary embryonic Syrian hamster c e l l s : v i r u s treatment at d i f f e r e n t intervals following chemical treatment. Interval Between 4NQ0 and Ad 12 Treatment Treatment Metaphase Plates with Chromatid Exchanges q>  Metaphase Plates with Chromatid Breaks Metaphase Plates with both Chroma-t i d Breaks and Exchanges (%) Control 1 hour Ad 12 10-5M MNNG 10-5M MNNG and Ad 12 0 100 60 100 6 70 0 6 30 6 hours Ad 12 10-5M MNNG 10 - 5M MNNG and Ad 12 3 93 47 97 7 70 0 0 17 12 hours Ad 12 10-5M MNNG 10-5M MNNG and Ad 12 4 93 51 96 7 63 0 0 14 - 29 -Table 5a. Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to N-methyl-N'-nitro-N-nitrosoguanidine and Adenovirus 12: virus treatment 1 hour following treatment with d i f f e r e n t concentrations of the chemical. Treatment Total Number of Metaphase Plates Investigated Metaphase Plates with Chromosome Aberrat ions /o Control 150 Ad 12 100 44 2.5x10 M MNNG 2.5xlO" 6M MNNG 100 100 46 5xlO _ 6M MNNG 5xlO" 6M MNNG and Ad 12 100 100 20 55 10"JM MNNG 10~5M MNNG and Ad 12 100 100 32 66 Table 5b. Types of chromosome aberrations induced by the combined a c t i o n of N-methyl-N'-nitor-N-nitrosoguanidine and Adenovirus 12 in primary embryonic Syrian hamster c e l l s : v i r u s treatment 1 hour following treatment with d i f f e r e n t concentrations of the chemical. Treatment Metaphase Plates with Chromatid Exchanges a) Metaphase Plates with Chromatid Breaks (%) Metaphase Plates with both Chromatid Breaks and Exchanges (7o) Control Ad 12 0 100 2.5xl0"°M MNNG 2.5xl0"°M MNNG and Ad 12 89 17 11 87 0 4 5xl0~ 6M MNNG 5x10 M MNNG and Ad 12 100 31 0 84 0 15 10"JM MNNG 10 M MNNG and Ad 12 100 61 6 68 29 - 31 -Table 6. M i t o t i c rates i n embryonic Syrian hamster c e l l s at 27-32 hours a f t e r separate and combined treatments with N-methyl-N'-nitro-N-nitrosoguanidine and Adenovirus 12. Treatments M i t o t i c Rate (%,) Control 4.6 Ad 12 4.0 10"5M MNNG 1.4 10"5M MNNG and Ad 12 1 1.7 i Chemical treatment was followed 1 hour l a t e r by vir u s i n f e c t i o n . In terval between A d 12 and M N N G t r e a t m e n t 16hrs. 12hrs. 7hrs. 4hrs. 1 hr. 20 A d 12 18 16 A d 12 14 Ad 12 . A d 12 12 Ad 12 M N N G 8 A D M r i n s e 2x M E M a d d e d c o l c h i c i n e c o l c h i c i n e 22 2 4 2 6 28 3 0 3 2 f i x a t i o n s t a i n i n g f i x a t i o n s t a i n i n g I Figure 7: Experimental design. V a r i a t i o n of intervals between Ad 12 and d i f f e r e n t centrations of MNNG treatment on embryonic Syrian hamster c e l l s . - 33 -period of 3 hours. Virus i n f e c t i o n occurred at the following i n t e r v a l s before exposure to MNNG: 1 hour, 4 hours, 7 hours, 12 hours and 16 hours. A l l the preparations were simultaneously exposed for 2 hours to MNNG. 6 "6 The d i f f e r e n t MNNG concentrations used were 2.5x10" M, 5x10 M and 10"^M. A l l the preparations were retained i n ADM for 48 hours u n t i l the end of the treatments. The c e l l s were exposed to c o l c h i c i n e from 22-27 hours and from 27-32 hours a f t e r the MNNG treatments. Metaphase chromo-somes from the l a t t e r c o l c h i c i n e period were analyzed. Virus i n f e c t i o n alone induced chromosome damage i n 21-327, of the metaphase plates (Table 7a). The differences i n time i n t e r v a l s between treatments, and hence from v i r u s i n f e c t i o n s to sampling, did not have any s i g n i f i c a n t e f f e c t s on the rates of metaphase plates with chromosome aberrations induced e i t h e r by the v i r u s or both agents combined. The rates of metaphase c e l l s with chromosome aberrations induced by both agents were higher than those induced by the v i r u s or chemical independently but lower than the sum of the damages induced by these controls. The types of chromosome damage encountered i n t h i s experiment are recorded i n Table 7b. The chemical induced exchanges i n 977, of the abnormal metaphase plates and the virus induced breaks i n 97-1007, of the abnormal metaphase plates. Various treatments with both the v i r u s and the chemical induced abnormal metaphase plates with 50-747, chromosome exchanges and 44-657, chromatid breaks. Metaphase plates with both chromatid breaks and exchanges were observed to constitute 7-217, of the abnormal metaphase plates but only a f t e r treatments with both agents. The rates of metaphase plates with chromosome aberrations in c e l l s infected by Ad 12 followed 1 hour l a t e r with exposure to various concen-tr a t i o n s of MNNG are recorded i n Table 8a. The preparations exposed to - 34 -Table 7a. Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to Adenovirus 12 and N-methyl-N 1-nitro-N-nitrosoguanidine: v i r u s treatment at d i f f e r e n t i n t e r v a l s preceding chemical treatment. Interval Between Ad 12 and MNNG Treatment Treatment Total Number of Metaphase Plates Metaphase Plates with Chromosome Investigated Aberrations % Control 150 5xlO _ 6M MNNG 100 30 1 hour Ad 12' Ad 12 and 5x10 UM MNNG -6, 100 100 21 38 4 hours Ad 12 Ad 12 and 5x10 _ 6M MNNG 100 100 31 46 7 hours Ad 12 Ad 12 and 5xl0"°M MNNG 100 100 30 43 12 hours Ad 12 Ad 12 and 5xlO"°M MNNG 100 100 32 37 16 hours Ad 12 6, Ad 12 and 5x10 UM MNNG 100 100 28 46 Table 7b. Types of chromosome aberrations induced by the combined a c t i o n of Adenovirus 12 and N-methyl-N'-nitro-N-nitrosoguanidine in primary embryonic Syrian hamster c e l l s : v i r u s treatment at d i f f e r e n t intervals preceding chemical treatment. Interval Between Ad 12 and MNNG Treatment Treatment Metaphase Plates with Chromatid Exchanges a) Metaphase Plates with Chromatid Breaks (%) Metaphase Plates with both Chroma-t i d Breaks and Exchanges (%) Control 5x10 UM MNNG 97 0 1 hour Ad 12 Ad 12 and 5x10"6M MNNG 0 68 100 53 0 21 4 hours Ad 12 Ad 12 and 5xl0" 6M MNNG 3 74 97 43 0 17 7. hours Ad 12 Ad 12 and 5xl0" 6M MNNG 0 65 100 44 0 12 hours Ad 12 Ad 12 and 5xlO" bM MNNG 3 54 97 65 0 19 16 hours Ad 12 -6, Ad 12 and 5x10 UM MNNG 0 50 100 57 0 7 - 36 -Figure 8: Frequency of metaphase plates with chromosome abnormalities. MNNG — • — ; Ad 12 i n f e c t i o n followed by MNNG treatment — A — - 37 -Table 8a. Chromosome aberrations i n primary embryonic Syrian hamster c e l l s exposed to Adenovirus 12 and N-methyl-N'-nitro-N-nitrosoguanidine: v i r u s treatment 1 hour before treatment with d i f f e r e n t concentrations of the chemical. Total Number of Metaphase Plates Metaphase Plates with Chromosome Treatment Investigated Aberrations 7„ Control 150 2 Ad 12 100 21 2.5xl0 _ 6M MNNG 100 10 Ad 12 and 2. 5x10^'6M MNNG 100 22 5xl0" 6M MNNG 100 30 Ad 12 and 5xlO" 6M MNNG 100 38 10 "5M MNNG 100 44 Ad 12 and 10"5M MNNG 100 49 Table 8b. Types of chromosome aberrations induced by the combined a c t i o n of Adenovirus 12 and N-methyl-N'-nitro-N-nitrosoguanidine i n primary embryonic Syrian hamster c e l l s : v i r u s treatment 1 hour before treatment with d i f f e r e n t concentrations of the chemical. Treatment Metaphase Plates with Chromatid Exchanges (%) Metaphase Plates with Chromatid Breaks Metaphase Plates with both Chromatid Breaks and Exchanges (70) Control Ad 12 0 100 2.5xl0" 6M MNNG Ad 12 and 2.5xlO"°M MNNG 100 36 0 73 0 9 5x10 M MNNG Ad 12 and 5xl0 _ 6M MNNG 97 67 3 54 0 21 10 M^ MNNG Ad 12 and 10"JM MNNG 100 94 5 39 5 33 - 39 -the chemical alone and those exposed to both agents, a l l showed an increase i n the number of metaphase plates with aberrations as the concentrations of MNNG increased (see F i g . 8). In Table 8b the v i r u s and the chemical are shown to have r e s p e c t i v e l y induced nearly e x c l u s i v e l y chromatid breaks and chromatid exchanges. The preparations exposed to the combined treat-ments showed an increase i n the percents of abnormal metaphase plates with chromatid exchanges as the concentrations of MNNG were increased, while the percent of metaphase plates with chromatid breaks decreased. An increase i n the percents of abnormal metaphase plates with both chromatid exchanges and breaks, a f t e r exposure to both agents, also increased as the concentrations of MNNG were increased. Only a 2 hour treatment with 1 0 " % MNNG of the preparations exposed as a singl e agent, could induce abnormal metaphase plates with both exchanges and breaks. The mit o t i c rates at 27-32 hours a f t e r separate or combined treat-ments were investigated (Table 9). The preparations infected by Ad 12 for 3 hours contained mit o t i c rates nearly three times higher than that of the c o n t r o l . Exposure to the chemical caused a decrease i n the mitotic rate. Virus i n f e c t i o n preceding exposure to MNNG gave r i s e to mitotic rates higher than that of the control. 5. The ef f e c t s of MNNG treatments on transformed Syrian hamster c e l l s : Transformed Syrian hamster c e l l l i n e s were treated with 10 "*M MNNG for 2 hours. Because of the i n a b i l i t y of ADM to block the SV 40 trans-formed c e l l s i n G-^ , none of the c e l l l i n e s i n t h i s experiment were re-tained i n ADM before chemical treatment. BHK-21, a non-malignant contin-uous Syrian hamster c e l l l i n e , was used as a control. The design of t h i s experiment i s i l l u s t r a t e d i n F i g . 9. The MEM was changed 6-'hours before - 40 -Table 9. M i t o t i c rates i n embryonic Syrian hamster c e l l s at 27-32 hours a f t e r separate and combined treatments with Adenovirus 12 and N-methyl-N'-nitro-N-nitrosoguanidine. Treatments M i t o t i c Rate (%,) Control 2.7 Ad 12 6.8' 10"5M MNNG 1.2 Ad 12 1and 10"5M MNNG 3.6; 1 Virus i n f e c t i o n followed 1 hour l a t e r with chemical treatment. MNNG c o I c h i c i n e hr.-2 rinse 2x 10 t fixation staining i Figure 9: Experimental design. Different transformed Syrian hamster c e l l l i n e s treated with MNNG. (These c e l l l i n e s were not maintained i n ADM). - 42 -chemical treatment. Colchicine treatments were from 5-10 hours, 15-20 hours and 25-30 hours a f t e r the chemical treatments. The samples of the f i r s t of these c o l c h i c i n e periods were analyzed. The experiment was repeated a second time. The former experiment was l a b e l l e d "Experiment A" and the l a t t e r was labelled"Experiment B". The r e s u l t s of both experiments were recorded independently. The percents of metaphase plates with chromosome damage are i l l u s -trated i n Table 10a. Spontaneous rates of chromosome damage i n the trans-formed Syrian hamster c e l l l i n e s were observed i n both experiments. The polyoma transformed c e l l s contained the lowest rates of spontaneous meta-phase chromosome aberrations of the d i f f e r e n t transformed c e l l l i n e s , whereas the SV 40 transformed c e l l s contained the highest rates. The BHK-21 c e l l s contained only 2°L metaphase plates with chromosome aberra-tions i n Experiment A while no metaphase plates with chromosome aberra-tions were observed i n Experiment B. Two hour treatments with 10"^M MNNG induced metaphase chromosome aberrations i n a l l the c e l l l i n e s . It was observed i n both experiments that a f t e r subtracting the spontaneous rates of metaphase plates with chromosome aberrations, both the treated trans-formed c e l l l i n e s and the treated BHK-21 c e l l l i n e contained approximately equal rates of MNNG induced chromosome aberrations. Table 10b i l l u s t r a t e d the types of metaphase chromosome damage observed i n the transformed Syrian hamster c e l l s . In a l l the treated c e l l l i n e s there existed a predominance of metaphase plates with chromatid exchanges. The spontaneous rates of metaphase plates with chromosome aberrations i n the polyoma transformed and the BHK-21 c e l l s , were too low to record any meaningful estimates of t h e i r types of chromosome aberrations. It was observed i n both experiments that the Rous sarcoma transformed and - 43 -and the SV 40 transformed c e l l l i n e s contained metaphase plates with chromosome aberrations having both chromosome breaks and exchanges. The rates of mitosis of the preparations of the various c e l l l i n e s are recorded i n Table 11. Treatments with MNNG reduced the rates of mitosis greatly i n a l l the c e l l l i n e s investigated except for the SV 40 transformed Syrian hamster c e l l s . Table 10a. Chromosome aberrations i n transformed Syrian hamster c e l l s exposed to N-methyl-N'-nitro-N-nitrosoguanidine. BHK-21 BHK-21 exposed to 10"5M MNNG for 2 hours Rous sarcoma transformed Rous sarcoma transformed exposed to 10"5M MNNG for 2 hours Polyoma transformed Total Number of Metaphase Plates Investigated 200 200 200 250 Experiment A Metaphase Plates With Chromosome Aberrat. (7.) 2 35 9 40 Experiment B Metaphase Plates With Chromosome Aberrat. (%) 0 28 13 44 200 Polyoma transformed exposed to 10"5M MNNG 200 29 33 for 2 hours SV 40 transformed 200 14 12 SV 40 transformed exposed to 10 _ 5M MNNG 200 55 46 for 2 hours Table 10b. Types of chromosome aberrations induced by N-methyl-N'-nitro-N-nitrosoguanidine i n transformed Syrian hamster c e l l s . Metaphase Plates With Chromatid Exchanges (7=) (Exp. A) (Exp. B) Metaphase Plates With Chromatid Breaks (Exp. A) (%) (Exp. B) Metaphase Plates With Both Chromatid Breaks and Exchanges (%) (Exp. A) (Exp. B) BHK-21 BHK-21 exposed to 10~5M MNNG for 2 hours 91 96 14 Rous sarcoma transformed 55 Rous sarcoma transformed exposed to 10 _ 5M MNNG 73 for 2 hours 70 82 45 37 30 24 0 10 Polyoma transformed Polyoma transformed exposed to 10 _ 5M MNNG for 2 hours 93 96 SV 40 transformed SV 40 transformed exposed to 10"5M MNNG for 2 hours 71 87 43 93 43 29 57 13 14 16 - 46 " Table 11. M i t o t i c rates i n continuous and transformed hamster l i n e s 5-10 hours a f t e r treatments with N-methyl-N'-nitro-nitrosoguanidine. Treatments and C e l l Lines M i t o t i c Rate (%) BHK-21 13.0 BHK-21 exposed to 10"5M MNNG for 2 hours 8.1 Rous sarcoma transformed 8.8 Rous sarcoma transformed expos ed to 10 _ 5M MNNG 2.8 for 2 hours Polyoma transformed 8.1 Polyoma transformed exposed to 1 0 " % MNNG 2.1 for 2 hours SV 40 transformed 11.1 SV 40 transformed exposed to 10"5M MNNG 11.0 for 2 hours - 47 -DISCUSSION In regard to the classes of chromosome exchanged induced by MNNG, i t can be argued that class 1 i s not a true chromosome exchange involving breakage but only a chromosome association. However, Cohen (1969) has already shown by c y t o l o g i c a l means that class 1 may be the r e s u l t of a true exchange event. Many chromosomes of s i m i l a r sizes and respective chromatid lengths are present i n the Syrian hamster chromosome complement (Lehman et a l . , 1963). In several cases i t was therefore ambiguous as to which classes the exchange configurations belonged. In order to suggest whether chromo-some exchanges occur randomly or whether they p r e f e r e n t i a l l y occur amongst homologous chromosomes, the i n d i v i d u a l chromosomes involved i n the exchange configurations must necessarily be i d e n t i f i e d . Since t h i s was not accom-plished i t is not possible to conclude from t h i s study whether or not MNNG induces exchange configurations from a random assortment of chromosomes. Although i t was o r i g i n a l l y intended to use 4NQ0 i n a l l these experi-ments, preliminary observations indicated that t h i s chemical was extremely tox i c . 4NQ0 treatment was also found to greatly reduce the mit o t i c rate at a s p e c i f i c i n t e r v a l a f t e r treatment. Although the MNNG treatments also reduced the mit o t i c rate, the e f f e c t s were not as d r a s t i c . The re-duction of the mitotic rate a f t e r MNNG treatment i s due to the delay i n growth of the c e l l s i n G-^ , S, and probably also G2 of the c e l l cycle (Barranco & Humphrey, 1971 and Ke l l y & Legator, 1970). The same i s expected to be true of 4NQ0. This delay i n growth r e s u l t s i n a reduced m i t o t i c rate at a s p e c i f i c time i n t e r v a l a f t e r treatment as compared to the controls since the c e l l s i n both the controls and treatments were i n i t i a l l y arrested i n Gl while being maintained i n ADM. - 48 -A l l the experiments involving treatments of embryonic Syrian hamster c e l l s with both Ad 12 and MNNG or Ad 12 and 4NQ0, indicated that the v i r u s and the chemicals induced metaphase chromosome aberrations independently of each other. The t o t a l rates of metaphase chromosome aberrations a f t e r combined treatments with both the virus and either chemical were s t r i c t l y a d d i t i v e of the independent e f f e c t s of the v i r u s and the chemicals alone. The add i t i v e e f f e c t s were also independent on the order of treatments, the concentrations of the chemicals, and the i n t e r v a l s between treatments by the two agents. In no instance was there any i n d i c a t i o n of a syner-gestic e f f e c t on the rate of metaphase chromosome aberrations a f t e r com-bined treatments with either chemical and the v i r u s as had previously been observed i n regard to the rate of transformation _in v i t r o - ( S t i c h & Casto, 1971 and S t i c h et a l . , 1971). However, a f t e r treatments with either chemical and Ad 12 a propor-t i o n of the abnormal metaphase c e l l s contained both chromatid exchanges and chromatid breaks. Since i t was observed that the chemicals induced nearly e x c l u s i v e l y chromatid exchanges and the v i r u s induced nearly e x c l u s i v e l y chromatid breaks, i t can be assumed that both agents induced chromosome aberrations i n those metaphase plates containing about s i m i l a r proportions of both chromatid breaks and exchanges. In addition, the proportions of metaphase plates with both types of chromosome aberrations were approximately the values expected from the separate and random e f f e c t s of the two agents concerned ( i . e . the product of the f r a c t i o n of metaphase plates with chromatid breaks and the f r a c t i o n of metaphase plates with chromatid exchanges). A decrease i n the e f f e c t of either one or both agents was hence accompanied by a decrease i n the proportion of metaphase plates containing both v i r a l l y and chemically induced metaphase chromosome - '49 -aberrations. The proportion of c e l l s i n which both agents were ac t i v e is the product of the f r a c t i o n s of c e l l s a f f e c t e d by each agent independently. I f the f r a c t i o n of c e l l s affected by each agent i s then doubled, the proportion of c e l l s i n which both agents are a c t i v e i s increased fourfold. Since the c e l l s i n which both agents were, ac t i v e are expected to be more susceptible to transformation, an increase i n the concentration of the chemical and an increase i n the i n f e c t i v i t y of the v i r u s should d r a s t i c -a l l y increase the synergestic e f f e c t on the rate of transformation. Such e f f e c t s have been observed i n v i r a l and chemical cocarcinogenesis experiments (Price et a l . , 1971). Since i t can be assumed from the cocarcinogenesis experiments of S t i c h & Casto (1971) that exposure to both a v i r u s and a chemical may induce a synergestic e f f e c t on the transformation rate i n cultured embryonic Syrian hamster c e l l s , the question a r i s e s as to whether a r e l a t i o n s h i p e x i s t s between induced metaphase chromosome aberrations and the rate of neoplastic transformation. The proportion of metaphase plates with chromosome aberrations a f t e r a population of cultured c e l l s has been exposed to an oncogenic agent, may be considered an index of the e f f e c t of the agent. There may therefore e x i s t a d i r e c t r e l a t i o n s h i p between the rate of metaphase c e l l s with chromosome aberrations and the rate of neoplastic transformation a f t e r treatment with a s i n g l e agent. The synergestic increase i n the rate of neoplastic transformation of cultured c e l l s a f t e r exposure to two agents may be due to a great increase i n the s u s c e p t i b i l i t y to transformation of those c e l l s i n which both agents induced t h e i r e f f e c t s . The number of such c e l l s may be d i r e c t l y r e l a t e d to the percent of metaphase plates with both v i r a l l y and chemically induced - 50 -chromosome aberrations. Hence, neoplastic transformation may be produced by two mechanisms i n experiments involving two carcinogenic agents: 1. A few transformed clones may have been formed by the a c t i o n of a s i n g l e carcinogenic agent. 2. Most of the transformed clones have been induced by the action of the two agents. The mechanism by which two agents may potentiate each other to en-hance the production of neoplastic transformation has been the subject of some speculation. Ashley (1969) and Knudson (1971) have recently c a l -culated by s t a t i s t i c a l means that several somatic c e l l mutagenic events were involved i n the production of the cancers they investigated. Berenblum (1969) has postulated for a long time that more than one stage i s involved i n the production of cancer. He believes that the f i r s t of the stages, the i n i t i a t i n g stage, i s the consequence of the mutagenic e f f e c t of the chemical which produces the f i r s t neoplastic c e l l d i v i s i o n . The second stage promotes t h i s f i r s t d i v i s i o n to produce neoplastic growth. In l i n e with these hypotheses are recent speculations on the mechanism involved i n the production of the synergestic increase i n the rate of transformation a f t e r treatment with a mutagenic agent (either a chemical or radiation) followed by v i r a l i n f e c t i o n . Several investigators (Coggin, 1969; Pollock & Todaro, 1968 and S t i c h et a l . , 1971) have suggested that c e l l s pretreated with mutagenic agents become more susceptible to trans-formation by viruses since the mutagenic agents may induce host c e l l DNA breakage which permits v i r a l DNA to be more e a s i l y covalently linked with the host c e l l DNA. V i r a l DNA incorporation into that of the host c e l l during transformation has been demonstrated by several investigators (Doerfler, 1968; Sambrook et a l . , 1968 and Westphal & Dulbecco, 1968). - 51 -Whether v i r a l DNA i s incorporated more e a s i l y during v i r a l cocarcinogenesis i s not known, however, but two observations support such a suggestion: A. A f t e r the chemically induced DNA damage i n the host c e l l has been repaired, the oncogenic v i r u s f a i l s to induce a synergestic increase i n the rate of transformation (Stich & Casto, 1971). B. The transformed clones appear morphologically l i k e those produced by the oncogenic v i r u s alone. (Salaman & Roe, 1964). Although a r e l a t i o n s h i p between the proportion of metaphase plates with chromosome aberrations c h a r a c t e r i s t i c of both agents and the syner-gestic increase i n the rate of transformation may e x i s t , i t i s impossible to r e a l i s t i c a l l y speculate on any r e l a t i o n s h i p between the mechanisms involved i n the production of metaphase chromosome aberrations and c e l l u l a r transformation. The structure of metaphase chromosomes i s s t i l l far from understood and the mechanisms by which metaphase chromosomes are produced remain equally obscure. Due to the observations that substances of t o t a l l y d i f f e r e n t natures may induce chromosome aberrations, i t i s believed that more than one mechanism may be involved (Dubinin & Soyter, 1969 and Heddle & Bodycote, 1970). The fact that many carcinogenic agents can induce metaphase chromosome damage i s probably only coincidental and not intimately associated with the process of carcinogenesis. It has been shown that treatment with an oncogenic chemical and a v i r u s r e s u l t i n g i n cocarcinogenesis i n vivo, i s i r r e v e r s i b l e . No increase i n the rate of transformation occurred i f the v i r u s i n f e c t i o n was followed by treatment with the chemical (Berenblum, 1947). Although the revers-i b i l i t y of such a procedure has not been tested _in v i t r o due to the i n -a c t i v a t i n g e f f e c t of the chemical on the v i r u s , the procedure i s expected to be i r r e v e r s i b l e i f the hypothesis involving-more e f f i c i e n t v i r a l DNA - 52- -incorporation is correct. In this study, however, the agents had s i m i l a r e f f e c t s on the metaphase chromosomes regardless of the order of trea t -ments. This may therefore further i l l u s t r a t e that the processes involved i n the formation of metaphase chromosome aberrations and neoplastic trans-formation are unrelated. Lately evidence has accumulated suggesting that c e l l s obtained from humans s u f f e r i n g from genetic syndromes are more e a s i l y transformed i n culture by oncogenic viruses and other oncogenic agents than controls (Todaro et a l . , 1966 and Aaronson & Todaro, 1968). Swift (1971) recently calculated that humans which are either heterozygous or homozygous car-r i e r s of a disease such as Fanconi's anaemia suffer from an increased rate of malignant neoplasma. He suggested that a l l such diseases may account for more than 157, of a l l cancerous i n d i v i d u a l s . Embryonic Syrian hamster c e l l s transformed by oncogenic viruses are e s s e n t i a l l y c e l l s a l t e r e d g e n e t i c a l l y since they contain v i r a l DNA. It was postulated that since g e n e t i c a l l y altered c e l l s have been demonstrated to be more susceptible to transformation, transformed c e l l l i n e s may also be more susceptible to chemically induced chromosome aberrations assuming a r e l a t i o n s h i p exists between the induction of chromosome aberrations and trans format ion. The transformed c e l l l i n e s used i n these experiments did not have any increased s u s c e p t i b i l i t y to chemically induced chromosome abnormal-i t i e s i f compared to the rate of chromosome aberrations induced i n the nontransformed continuous c e l l l i n e BHK-21. In repeated experiments, the differences i n the rates of metaphase chromosome aberrations induced by the chemical i n the various c e l l l i n e s was due to the differences i n the spontaneous chromosome abnormalities occurring i n the transformed Syrian - 53 -hamster l i n e s . Virus transformed c e l l l i n e s have been shown to contain both spontaneous chromosome abnormalities and unstable chromosome comple-ments (Cooper & Black, 1964 and Weinstein & Moorhead, 1966). MNNG induced a much higher incidence of chromatid breaks i n the experiments employing transformed c e l l l i n e s than in those where embryonic Syrian hamster c e l l s were used. This was because the embryonic Syrian hamster c e l l s were always treated i n arginine d e f i c i e n t medium which arrests c e l l s i n G^ (Freed & Schatz, 1969), whereas the transformed c e l l s were treated i n normal growth medium (MEM). C e l l s i n G\ i n which chromo-some aberrations are induced by MNNG, w i l l contain exchange configurations by metaphase. Chromosome aberrations induced during S phase and G2, however, w i l l r e s u l t i n chromatid breaks by metaphase (Stich et a l . , 1971). - 54 -REFERENCES Aaronson, S.A. and Todaro, G.J. 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