249
Mutation Research, 48 (1977) 249--254
© Elsevier/North-Holland Biomedical Press
MUTAGENICITY OF POLYCYCLIC HYDROCARBONS. II. MONITORING GENETICAL HAZARDS OF CHRYSENE IN VITRO AND VIVO
A. BASLER, B. HERBOLD, S. PETER and G. R{~HRBORN Institut fur Humangenetik und Anthropologie der Universit~t Diisseldorf (GFR) (Received September 29th, 1976) (Revision received November 11th, 1976) (Accepted November 14th, 1976)
Summary Mutagenicity tests were performed with chrysene in the Salmonella/microsome test, NMRI-mice oocytes, bone-marrow cells and spermatogonia of Chinese hamsters. Only in mice oocytes was a weak but significant increase of structural chromosome aberrations observed. Correlations were found between weak carcinogenic and observed weak mutagenic activities of chrysene in vitro and in vivo.
Introduction We started correlation studies to evaluate the mutagenic activity of polycyclic hydrocarbons in various test systems. The present paper will discuss results obtained with chrysene in the Salmonella/microsome test, the metaphase II oocyte test in mice and two further cytogenetic tests in Chinese hamsters (Cricetulus griseus). To increase possible repair-dependent mutagenicity we also used caffeine in the microsome-mediated assay. Material and methods S a l m o n e l l a / m i c r o s o m e test C o m p o u n d s and solvents
Chrysene (purity 98%), purchased from EGA-Chemie, was dissolved in dimethylsulphoxide (DMSO). Doses used were 100, 10 and 1 pg per plate. Caffeine (Merck) was suspended in distilled water at a final concentration of 200 pg per plate.
250
Bacteria As indicator organisms, Salmonella typhimurium TA 1535 and TA 98 were used. They were selected by Ames et al. [2] and McCann et al. [9] and seemed to be most suitable for the microsome-mediated assay. With these strains, basepair substitutions as well as frameshift mutations can be detected.
Procedure Liver microsomes were obtained from 12-week-old male rats (Wistar II). The animals were pre-treated with sodium phenobarbital, 0.1% in drinking water, for 8 days. Water was changed daily. After this time the rats were killed by cervical dislocation and prepared as described by Ames et al. [2]. Centrifugation was done at 3100 rpm. For testing, 0.5 ml of S-9 Mix, 0.1 ml test solution and 0.1 ml bacterial suspension of an overnight culture were added to 2 ml soft agar (45°C) and plated on nutrient medium. The plates were examined after 48 h.of incubation at 37 ° C. In controls and caffeine experiments we used 6 plates, and in all chrysene examinations 4 plates, per dose. The titre was determined by bacterial dilutions of 10 -5 and 4 plates per concentration. Statistical evaluations were carried out by the Wilcoxon rank test. Significant mutagenicity was considered at P < 0.05.
Cytogenetic methods Application of chrysene Chrysene was suspended either in gum arabic and given per stomach tube or in foetal calf serum a n d injected i.p. The total a m o u n t of liquid was 0.5 ml.
Preparation of metaphase H oocytes We used 67 mice, 8--12 weeks old, of the NMRI strain in the experiment and 45 as control animals. To synchronize the oestrus the females were pre-treated with 1.5 IU pregnant mare's serum, and 48 h later with 2 IU human c h o r i o n i c gonadotropin. 1 h after the application of HCG, chrysene (450 mg/kg) was given per stomach tube. Metaphase II oocytes were prepared according to RShrborn and Hansmann [10] 16 h after application of HCG.
Preparation of spermatogonial chromosomes We used 8--12-week-old male Chinese hamsters. Chrysene was suspended in gum arabic and given per stomach tube. The hamsters were given twice 450 mg/ kg within a 24-h interval. The chromosomes were prepared either 6 h (Expt. a) or 24 h (Expt..b) after the last application. Chromosomes were prepared by the method of Hoo and Bowles [7]. 100 metaphases were analysed from each hamster.
Preparation of bone-marrow chromosomes In all experimental series we used the same number (2 of each sex) of 8--12week-old Chinese hamsters. One group of animals was treated twice with chrysene (450 mg/kg), suspended in gum arabic, within a 24-h interval. The chromosomes were prepared either 6 h (Expt. a) or 24 h (Expt. b) after the last
251 application. Another group was given five times 450 mg/kg within a val. Chrysene was suspended either in gum arabic (Expt. c) or in serum (Expt. d). The bone-marrow chromosomes were prepared 18 last application, b y the method of Schmid and Staiger [11]. From ster, 200 metaphases were analysed.
24-h interfoetal calf h after the each ham-
Statistics Statistics were performed with the ×2 test. Significance was considered if P ~< 0.02. Results As shown in Table I and II, no increase of mutant colonies in Salmonella typhimurium TA 1535 and TA 98 was produced by caffeine alone. Chrysene alone was not mutagenic in either tester strain even at the highest dose. With respect to possible synergistic effects between chrysene and caffeine, no increase of mutation was detected in Salmonella typhimurium TA 1535 (Table I). Only in experiments with Salmonella typhimurium TA 98 did we observe a significant mutagenic activity of chrysene together with caffeine at dose levels of 10 and 200 pg respectively per plate (Table II). This difference is significant compared with the control experiment but n o t in comparison with chrysene alone. In all tests there was a slight toxic effect of chrysene at 100 pg per plate.
Bone marrow cells The result of the 4 bone-marrow test series are summarized in Table III. Neither an oral nor an intraperitoneal application of chrysene yielded a significant increase of chromosomal aberrations. Nor did chromosome preparations made at different intervals after the last application influence the aberration rate. Spermatogonia In spermatogonia of Chinese hamsters a low but not significant increase in chromosomal aberrations, excluding gaps, was found. TABLE I RESULTS OBSERVED WITH SALMONELLA
TYPHIMURIUM TA 1535
Liver e n z y m e s i n d u c e d b y s o d i u m phenobarbital. Substance
Dose per plate (mg)
Mutants per plate
Titre
Mutation frequency
Mutation f r e q u e n c y ( E x p t . ) Mutation f r e q u e n c y ( C o n t r . )
Caffeine
0.000 0.300
19.25 20.17
2.39 × 108 2.48 X 108
8.07 X 10 - 7 8 . 1 2 X 10 - 7
1.00 1.01
Chrysene
0.000 0.001 0.010 0.100
15.75 22.25 16.50 22.00
2.17 2.12 2.15 1.20
7.27 1.05 7.68 1.83
10 -7 10 -7 10 -7 10 -6
1.00 1.44 1.06 2.52
Chrysene + Caffeine
0 . 0 0 1 + 0.2 0.010+0.2 0 . 1 0 0 + 0.2
23.00 26.75 18.00
2.43 X 108 2.32 X 1 0 8 1 . 2 0 X 108
9.47 X 10 - 7 1 . 1 5 X 1 0 -6 1.50 X 10 -6
1.30 1.58 2.06
X X X X
108 108 108 108
X X X X
252
T A B L E II RESULTS WITH SALMONELLA
T Y P H I M U R I U M T A 98
Liver enzymes induced by sodium phenobarbital. Substance
Dose p e r plate (mg)
Mutants per plate
Titre
Mutation frequency
Mutation frequency (Expt.) Mutation frequency (Contr.)
Caffeine
0.000 0.200
30.25 36.00
1.60 X 108 1.56 X 108 ×
1.89 × 10 - 6 2.31 X 10 -5
1.00 1.22
Chrysene
0.000 0.001 0.010 0.100
35.50 32.75 33.50 27.75
1.69 1.67 1.73 9.00
2.10 1.96 1.93 3.08
X 10 -6 × 10 -6 )< 10 -6 )< 10 - 6
1.00 0.93 0.92 1.47
Chrysene+ caffeine
0.001 + 0.2 0.010 + 0.2 0.100+0.2
31.75 43.00 36.25
1.55 X 108 1.40 X 108 9.25 X 1 0 7
2.05 X 10 -6 3.07 X 10 -6 3.92 X 1 0 -6
0.98 1.46 1.87
TABLE
X 108 × 108 X 108 X 107
III
CHROMOSOMAL
ABERRATIONS
OF CHINESE
HAMSTER
BONE-MARROW
CELLS
Expts. a and b. T w o applications of chrysene, at 4 5 0 mg/kg, suspended in g u m arabic and given per stomach tube within a 24-h interval. Expt. a. C h r o m o s o m e preparations 6 h after the last application. Expt. b. C h r o m o s o m e preparations 24 h after the last application. Expts. c and d. Five applications of chrysene at 4 5 0 mg/kg. C h r o m o s o m e preparations 16 h after the last application. Expt. c. Suspended in g u m arabic and given per stomach tube. Expt. d. Suspended in foetal calf serum and injected intraperitoneally. Series
Control Expt. a Expt. b ExpL c Expt. d
No. o f animals
4 4 4 4 4
No. o f m e t a - A b e r r a n t phases incl. gaps analysed No. %
Aberrant excl. gaps %
Breaks a n d fragments
Deletions
No.
800 800 800 800 800
4 6 4 6 6
0.50 0.75 0.50 0.75 0.75
4 5 3 5 5
0 1 1 1 1
11 18 20 16 19
1.38 2.25 2.50 2.00 2.38
No. of m e t a p h a s e s w i t h
TABLE IV CHROMOSOMAL ABERRATIONS OF CHINESE HAMSTER SPERMATOGONIA E x p t s . a a n d b. T w o a p p l i c a t i o n s o f c h r y s e n e ( 4 5 0 m g / k g ) ; s u s p e n d e d in g u m a r a b i c a n d given p e r s t o m a c h t u b e w i t h i n a 24-h interval. E x p t . a. C h r o m o s o m e p r e p a r a t i o n s 6 h a f t e r the last a p p l i c a t i o n . E x p t . b. C h r o m o s o m e p r e p a r a t i o n s 24 h a f t e r the last a p p l i c a t i o n . Series
Control Expt. a Expt. b
No. of animals
3 3 3
No. of m e t a phases analysed
300 300 300
Aberrant incl. gaps
Aberrant excl. gaps
No. o f m e t a p h a s e s w i t h
%
No.
%
Breaks a n d fragments
Deletions
No. 22 18 15
7.33 6.00 5.00
4 3 7
1.33 2.66 2.33
3. 8 7
1 0 0
253 TABLE V F R E Q U E N C I E S AND TYPES OF A B E R R A T I O N OBSERVED IN M E T A P H A S E II OOCYTES Series
Control Experiment
No. of animals
No. o f metaphases
45 67
122 108
% Structural aberrant MII
0.82 5.56
No. of M I I w i t h
Satellite associations
Breaks a n d fragments
1 2
0 4
Oocytes of NMRI mice Mutagenicity tests in mice oocytes have been shown in earlier investigations [4,10] to be a test system with high sensitivity. Before ovulation, female mice were treated with the test compound. The chromosomes were analysed shortly after ovulation at the time of metaphase II. By this method it is possible to recognize all types of induced structural and numerical chromosomal aberrations. All the possible cytogenetic effects, induced by chrysene, should have been detectable by this test system. As seen in Table V, in this test system a single dose of chrysene (450 mg/kg) led to a weak (P < 0.04) increase of structural aberrations from 0.82% in the control up to 5.56%. Discussion Using the Salmonella/microsome test, we found no mutagenic activity of chrysene. But it had been reported by McCann et al. [9] that this agent is a strong and powerful mutagen in the Salmonella/microsome test. They used ratliver enzymes activated by Aroclor 1254 and Salmonella typhimurium TA 100 and TA 98. For both strains they found an increase of mutants per plate up to 1670 using 0.01 mg Aroclor. We suppose that our negative result is due to an insufficient induction of liver enzymes by sodium phenobarbital in comparison with Aroclor 1254 used by McCann et al. [9]. This effect may be explained by different activation of cytochromes with the two compounds. Conney [5] reported that phenobarbital activates c y t o c h r o m e P 450, whereas Aroclor 1254 activates P 450 as well as P 448 [1]. Therefore it seems, according to Guttenplan et al. [6], that active cytochrome P-448 is necessary to demonstrate the mutagenic activity of polycyclic hydrocarbons in the Salmonella/microsome test. These results cannot be extrapolated to man because the dose-effects relationship in the Salmonella/microsome test cannot be correlated with doses in mammals and man in vivo. Therefore experiments were performed in mitotic and meiotic cells in mammals in vivo. The sensitivity of these cytogenetic test systems and animal strains to polycyclic hydrocarbons was demonstrated in earlier investigations with 3,4-benzopyrene (B(a)P) [4]. A single dose of B(a)P at 450 mg/kg led to 5.33% structural aberrant m II oocytes including gaps. Two doses of B(a)P at 450 mg/kg given to Chinese hamsters led to 1.5% chromosomal aberrations, excluding gaps, in bone-marrow cells and 3.5% including gaps. As described above, in bone-marrow cells and spermatogonia of Chinese hamsters no mutagenic effect
254
was detected after application of chrysene. Only the test system with the highest sensitivity, metaphase II oocytes, showed a weak increase of structural aberrations. For years, carcinogenesis and mutagenesis have been correlated. Mutated somatic cells are suspected to be the first step in the development of cancer. When cancerogenic and mutagenic activities of B(a)P and chrysene are compared, these correlations are seen too. B(a)P, a polycyclic hydrocarbon with high mutagenic activity, shows a carcinogenicity index of > 70 [3,8]. The index of chrysene on the contrary is < 5 [3]. Acknowledgements For technical assistance we are grateful to Mrs. Sezer. Our investigation was supported by the Bundesministerium fiir Forschung und Technologie. References 1 Alvares, D.R., D.R. Bickers and A. Kappas, Polychlorinated biphenyls: a n e w type of induces of cytoc h r o m e P-448 in the liver,Proc. Natl. Acad. Sci. U S A , 70 (1973) 1321--1325 2 Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. U S A , 70 (1973) 2281--2285. 3 Arcos, J.C. and M.F. Argus, Molecular geometry and carcinogenic activity of aromatic compounds. N e w p e r s p e c t i v e s , A d v . C a n d e r R e s . , 11 ( 1 9 6 8 ) 3 0 5 - - 4 7 1 . 4 Basler, A. a n d G. R o h r b o r n , C h r o m o s o m e a b e r r a t i o n s in o o c y t e s o f N M R I - m i e e a n d b o n e m a r r o w cells o f C h i n e s e h a m s t e r s i n d u c e d w i t h 3 , 4 - b e n z o p y r e n e , M u t a t i o n R e s . , 3 8 ( 1 9 7 6 ) 3 2 7 - - 3 3 2 . 5 C o n n e y , A . H . , P h a r m a c o l o g i c a i m p l i c a t i o n s o f m i c r o s o m a l e n z y m e i n d u c t i o n , P h a r m a c o l . R e v . , 19 (1967) 317--366. 6 G u t t e n p l a n , J . B . , F. H u t t e r e r a n d A . J . G a r r o , E f f e c t s o f c y t o c h r o m e P - 4 4 8 a n d P - 4 5 0 i n d u c e r s o n microsomal dimethylnitrosamine demethylase activity and the capacity of isolated microsomes to a c t i v a t e d i m e t h y l n i t r o s a m i n e t o a m u t a g e n , M u t a t i o n Res., 3 5 ( 1 9 7 6 ) 4 1 5 - - - 4 2 2 . 7 HOD, S.S. a n d C . A . B o w l e s , A n a i r - d r y i n g m e t h o d f o r p r e p a r i n g m e t a p h a s e c h r o m o s o m e s f r o m t h e s p e r m a t o g o n i a l cells o f r a t s a n d m i c e , M u t a t i o n R e s . , 13 ( 1 9 7 1 ) 8 5 - - 8 8 . 8 Iball, J., T h e relative p o t e n c y o f c a r c i n o g e n i c c o m p o u n d s , A m e r . J. C a n c e r , 3 5 ( 1 9 3 9 ) 1 8 8 - - 1 9 0 . 9 M c C a n n , J., E. C h o i , E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e S a l m o n e U a / m i c r o s o m e t e s t : a s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. U S A , 7 2 ( 1 9 7 5 ) 5 1 3 5 - - 5 1 3 9 . 10 R o h r b o r n , G. a n d I. H a n s m a n n , I n d u c e d c h r o m o s o m e a b e r r a t i o n s in u n f e r t i l i z e d o o c y t e s o f m i c e , H u m a n g e n e t i k , 13 ( 1 9 7 1 ) 1 8 4 - - 1 9 8 . 11 S c h m i d , W. a n d G . R . S t a i g e r , C h r o m o s o m e s t u d i e s o n b o n e m a r r o w f r o m c h i n e s e h a m s t e r s t r e a t e d with benzodiazepine tranquillizers and cyclophosphamide, Mutation Res., 7 (1969) 99--108.
Mutation Research, 48 (1977) 249--254
© Elsevier/North-Holland Biomedical Press
MUTAGENICITY OF POLYCYCLIC HYDROCARBONS. II. MONITORING GENETICAL HAZARDS OF CHRYSENE IN VITRO AND VIVO
A. BASLER, B. HERBOLD, S. PETER and G. R{~HRBORN Institut fur Humangenetik und Anthropologie der Universit~t Diisseldorf (GFR) (Received September 29th, 1976) (Revision received November 11th, 1976) (Accepted November 14th, 1976)
Summary Mutagenicity tests were performed with chrysene in the Salmonella/microsome test, NMRI-mice oocytes, bone-marrow cells and spermatogonia of Chinese hamsters. Only in mice oocytes was a weak but significant increase of structural chromosome aberrations observed. Correlations were found between weak carcinogenic and observed weak mutagenic activities of chrysene in vitro and in vivo.
Introduction We started correlation studies to evaluate the mutagenic activity of polycyclic hydrocarbons in various test systems. The present paper will discuss results obtained with chrysene in the Salmonella/microsome test, the metaphase II oocyte test in mice and two further cytogenetic tests in Chinese hamsters (Cricetulus griseus). To increase possible repair-dependent mutagenicity we also used caffeine in the microsome-mediated assay. Material and methods S a l m o n e l l a / m i c r o s o m e test C o m p o u n d s and solvents
Chrysene (purity 98%), purchased from EGA-Chemie, was dissolved in dimethylsulphoxide (DMSO). Doses used were 100, 10 and 1 pg per plate. Caffeine (Merck) was suspended in distilled water at a final concentration of 200 pg per plate.
250
Bacteria As indicator organisms, Salmonella typhimurium TA 1535 and TA 98 were used. They were selected by Ames et al. [2] and McCann et al. [9] and seemed to be most suitable for the microsome-mediated assay. With these strains, basepair substitutions as well as frameshift mutations can be detected.
Procedure Liver microsomes were obtained from 12-week-old male rats (Wistar II). The animals were pre-treated with sodium phenobarbital, 0.1% in drinking water, for 8 days. Water was changed daily. After this time the rats were killed by cervical dislocation and prepared as described by Ames et al. [2]. Centrifugation was done at 3100 rpm. For testing, 0.5 ml of S-9 Mix, 0.1 ml test solution and 0.1 ml bacterial suspension of an overnight culture were added to 2 ml soft agar (45°C) and plated on nutrient medium. The plates were examined after 48 h.of incubation at 37 ° C. In controls and caffeine experiments we used 6 plates, and in all chrysene examinations 4 plates, per dose. The titre was determined by bacterial dilutions of 10 -5 and 4 plates per concentration. Statistical evaluations were carried out by the Wilcoxon rank test. Significant mutagenicity was considered at P < 0.05.
Cytogenetic methods Application of chrysene Chrysene was suspended either in gum arabic and given per stomach tube or in foetal calf serum a n d injected i.p. The total a m o u n t of liquid was 0.5 ml.
Preparation of metaphase H oocytes We used 67 mice, 8--12 weeks old, of the NMRI strain in the experiment and 45 as control animals. To synchronize the oestrus the females were pre-treated with 1.5 IU pregnant mare's serum, and 48 h later with 2 IU human c h o r i o n i c gonadotropin. 1 h after the application of HCG, chrysene (450 mg/kg) was given per stomach tube. Metaphase II oocytes were prepared according to RShrborn and Hansmann [10] 16 h after application of HCG.
Preparation of spermatogonial chromosomes We used 8--12-week-old male Chinese hamsters. Chrysene was suspended in gum arabic and given per stomach tube. The hamsters were given twice 450 mg/ kg within a 24-h interval. The chromosomes were prepared either 6 h (Expt. a) or 24 h (Expt..b) after the last application. Chromosomes were prepared by the method of Hoo and Bowles [7]. 100 metaphases were analysed from each hamster.
Preparation of bone-marrow chromosomes In all experimental series we used the same number (2 of each sex) of 8--12week-old Chinese hamsters. One group of animals was treated twice with chrysene (450 mg/kg), suspended in gum arabic, within a 24-h interval. The chromosomes were prepared either 6 h (Expt. a) or 24 h (Expt. b) after the last
251 application. Another group was given five times 450 mg/kg within a val. Chrysene was suspended either in gum arabic (Expt. c) or in serum (Expt. d). The bone-marrow chromosomes were prepared 18 last application, b y the method of Schmid and Staiger [11]. From ster, 200 metaphases were analysed.
24-h interfoetal calf h after the each ham-
Statistics Statistics were performed with the ×2 test. Significance was considered if P ~< 0.02. Results As shown in Table I and II, no increase of mutant colonies in Salmonella typhimurium TA 1535 and TA 98 was produced by caffeine alone. Chrysene alone was not mutagenic in either tester strain even at the highest dose. With respect to possible synergistic effects between chrysene and caffeine, no increase of mutation was detected in Salmonella typhimurium TA 1535 (Table I). Only in experiments with Salmonella typhimurium TA 98 did we observe a significant mutagenic activity of chrysene together with caffeine at dose levels of 10 and 200 pg respectively per plate (Table II). This difference is significant compared with the control experiment but n o t in comparison with chrysene alone. In all tests there was a slight toxic effect of chrysene at 100 pg per plate.
Bone marrow cells The result of the 4 bone-marrow test series are summarized in Table III. Neither an oral nor an intraperitoneal application of chrysene yielded a significant increase of chromosomal aberrations. Nor did chromosome preparations made at different intervals after the last application influence the aberration rate. Spermatogonia In spermatogonia of Chinese hamsters a low but not significant increase in chromosomal aberrations, excluding gaps, was found. TABLE I RESULTS OBSERVED WITH SALMONELLA
TYPHIMURIUM TA 1535
Liver e n z y m e s i n d u c e d b y s o d i u m phenobarbital. Substance
Dose per plate (mg)
Mutants per plate
Titre
Mutation frequency
Mutation f r e q u e n c y ( E x p t . ) Mutation f r e q u e n c y ( C o n t r . )
Caffeine
0.000 0.300
19.25 20.17
2.39 × 108 2.48 X 108
8.07 X 10 - 7 8 . 1 2 X 10 - 7
1.00 1.01
Chrysene
0.000 0.001 0.010 0.100
15.75 22.25 16.50 22.00
2.17 2.12 2.15 1.20
7.27 1.05 7.68 1.83
10 -7 10 -7 10 -7 10 -6
1.00 1.44 1.06 2.52
Chrysene + Caffeine
0 . 0 0 1 + 0.2 0.010+0.2 0 . 1 0 0 + 0.2
23.00 26.75 18.00
2.43 X 108 2.32 X 1 0 8 1 . 2 0 X 108
9.47 X 10 - 7 1 . 1 5 X 1 0 -6 1.50 X 10 -6
1.30 1.58 2.06
X X X X
108 108 108 108
X X X X
252
T A B L E II RESULTS WITH SALMONELLA
T Y P H I M U R I U M T A 98
Liver enzymes induced by sodium phenobarbital. Substance
Dose p e r plate (mg)
Mutants per plate
Titre
Mutation frequency
Mutation frequency (Expt.) Mutation frequency (Contr.)
Caffeine
0.000 0.200
30.25 36.00
1.60 X 108 1.56 X 108 ×
1.89 × 10 - 6 2.31 X 10 -5
1.00 1.22
Chrysene
0.000 0.001 0.010 0.100
35.50 32.75 33.50 27.75
1.69 1.67 1.73 9.00
2.10 1.96 1.93 3.08
X 10 -6 × 10 -6 )< 10 -6 )< 10 - 6
1.00 0.93 0.92 1.47
Chrysene+ caffeine
0.001 + 0.2 0.010 + 0.2 0.100+0.2
31.75 43.00 36.25
1.55 X 108 1.40 X 108 9.25 X 1 0 7
2.05 X 10 -6 3.07 X 10 -6 3.92 X 1 0 -6
0.98 1.46 1.87
TABLE
X 108 × 108 X 108 X 107
III
CHROMOSOMAL
ABERRATIONS
OF CHINESE
HAMSTER
BONE-MARROW
CELLS
Expts. a and b. T w o applications of chrysene, at 4 5 0 mg/kg, suspended in g u m arabic and given per stomach tube within a 24-h interval. Expt. a. C h r o m o s o m e preparations 6 h after the last application. Expt. b. C h r o m o s o m e preparations 24 h after the last application. Expts. c and d. Five applications of chrysene at 4 5 0 mg/kg. C h r o m o s o m e preparations 16 h after the last application. Expt. c. Suspended in g u m arabic and given per stomach tube. Expt. d. Suspended in foetal calf serum and injected intraperitoneally. Series
Control Expt. a Expt. b ExpL c Expt. d
No. o f animals
4 4 4 4 4
No. o f m e t a - A b e r r a n t phases incl. gaps analysed No. %
Aberrant excl. gaps %
Breaks a n d fragments
Deletions
No.
800 800 800 800 800
4 6 4 6 6
0.50 0.75 0.50 0.75 0.75
4 5 3 5 5
0 1 1 1 1
11 18 20 16 19
1.38 2.25 2.50 2.00 2.38
No. of m e t a p h a s e s w i t h
TABLE IV CHROMOSOMAL ABERRATIONS OF CHINESE HAMSTER SPERMATOGONIA E x p t s . a a n d b. T w o a p p l i c a t i o n s o f c h r y s e n e ( 4 5 0 m g / k g ) ; s u s p e n d e d in g u m a r a b i c a n d given p e r s t o m a c h t u b e w i t h i n a 24-h interval. E x p t . a. C h r o m o s o m e p r e p a r a t i o n s 6 h a f t e r the last a p p l i c a t i o n . E x p t . b. C h r o m o s o m e p r e p a r a t i o n s 24 h a f t e r the last a p p l i c a t i o n . Series
Control Expt. a Expt. b
No. of animals
3 3 3
No. of m e t a phases analysed
300 300 300
Aberrant incl. gaps
Aberrant excl. gaps
No. o f m e t a p h a s e s w i t h
%
No.
%
Breaks a n d fragments
Deletions
No. 22 18 15
7.33 6.00 5.00
4 3 7
1.33 2.66 2.33
3. 8 7
1 0 0
253 TABLE V F R E Q U E N C I E S AND TYPES OF A B E R R A T I O N OBSERVED IN M E T A P H A S E II OOCYTES Series
Control Experiment
No. of animals
No. o f metaphases
45 67
122 108
% Structural aberrant MII
0.82 5.56
No. of M I I w i t h
Satellite associations
Breaks a n d fragments
1 2
0 4
Oocytes of NMRI mice Mutagenicity tests in mice oocytes have been shown in earlier investigations [4,10] to be a test system with high sensitivity. Before ovulation, female mice were treated with the test compound. The chromosomes were analysed shortly after ovulation at the time of metaphase II. By this method it is possible to recognize all types of induced structural and numerical chromosomal aberrations. All the possible cytogenetic effects, induced by chrysene, should have been detectable by this test system. As seen in Table V, in this test system a single dose of chrysene (450 mg/kg) led to a weak (P < 0.04) increase of structural aberrations from 0.82% in the control up to 5.56%. Discussion Using the Salmonella/microsome test, we found no mutagenic activity of chrysene. But it had been reported by McCann et al. [9] that this agent is a strong and powerful mutagen in the Salmonella/microsome test. They used ratliver enzymes activated by Aroclor 1254 and Salmonella typhimurium TA 100 and TA 98. For both strains they found an increase of mutants per plate up to 1670 using 0.01 mg Aroclor. We suppose that our negative result is due to an insufficient induction of liver enzymes by sodium phenobarbital in comparison with Aroclor 1254 used by McCann et al. [9]. This effect may be explained by different activation of cytochromes with the two compounds. Conney [5] reported that phenobarbital activates c y t o c h r o m e P 450, whereas Aroclor 1254 activates P 450 as well as P 448 [1]. Therefore it seems, according to Guttenplan et al. [6], that active cytochrome P-448 is necessary to demonstrate the mutagenic activity of polycyclic hydrocarbons in the Salmonella/microsome test. These results cannot be extrapolated to man because the dose-effects relationship in the Salmonella/microsome test cannot be correlated with doses in mammals and man in vivo. Therefore experiments were performed in mitotic and meiotic cells in mammals in vivo. The sensitivity of these cytogenetic test systems and animal strains to polycyclic hydrocarbons was demonstrated in earlier investigations with 3,4-benzopyrene (B(a)P) [4]. A single dose of B(a)P at 450 mg/kg led to 5.33% structural aberrant m II oocytes including gaps. Two doses of B(a)P at 450 mg/kg given to Chinese hamsters led to 1.5% chromosomal aberrations, excluding gaps, in bone-marrow cells and 3.5% including gaps. As described above, in bone-marrow cells and spermatogonia of Chinese hamsters no mutagenic effect
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was detected after application of chrysene. Only the test system with the highest sensitivity, metaphase II oocytes, showed a weak increase of structural aberrations. For years, carcinogenesis and mutagenesis have been correlated. Mutated somatic cells are suspected to be the first step in the development of cancer. When cancerogenic and mutagenic activities of B(a)P and chrysene are compared, these correlations are seen too. B(a)P, a polycyclic hydrocarbon with high mutagenic activity, shows a carcinogenicity index of > 70 [3,8]. The index of chrysene on the contrary is < 5 [3]. Acknowledgements For technical assistance we are grateful to Mrs. Sezer. Our investigation was supported by the Bundesministerium fiir Forschung und Technologie. References 1 Alvares, D.R., D.R. Bickers and A. Kappas, Polychlorinated biphenyls: a n e w type of induces of cytoc h r o m e P-448 in the liver,Proc. Natl. Acad. Sci. U S A , 70 (1973) 1321--1325 2 Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. U S A , 70 (1973) 2281--2285. 3 Arcos, J.C. and M.F. Argus, Molecular geometry and carcinogenic activity of aromatic compounds. N e w p e r s p e c t i v e s , A d v . C a n d e r R e s . , 11 ( 1 9 6 8 ) 3 0 5 - - 4 7 1 . 4 Basler, A. a n d G. R o h r b o r n , C h r o m o s o m e a b e r r a t i o n s in o o c y t e s o f N M R I - m i e e a n d b o n e m a r r o w cells o f C h i n e s e h a m s t e r s i n d u c e d w i t h 3 , 4 - b e n z o p y r e n e , M u t a t i o n R e s . , 3 8 ( 1 9 7 6 ) 3 2 7 - - 3 3 2 . 5 C o n n e y , A . H . , P h a r m a c o l o g i c a i m p l i c a t i o n s o f m i c r o s o m a l e n z y m e i n d u c t i o n , P h a r m a c o l . R e v . , 19 (1967) 317--366. 6 G u t t e n p l a n , J . B . , F. H u t t e r e r a n d A . J . G a r r o , E f f e c t s o f c y t o c h r o m e P - 4 4 8 a n d P - 4 5 0 i n d u c e r s o n microsomal dimethylnitrosamine demethylase activity and the capacity of isolated microsomes to a c t i v a t e d i m e t h y l n i t r o s a m i n e t o a m u t a g e n , M u t a t i o n Res., 3 5 ( 1 9 7 6 ) 4 1 5 - - - 4 2 2 . 7 HOD, S.S. a n d C . A . B o w l e s , A n a i r - d r y i n g m e t h o d f o r p r e p a r i n g m e t a p h a s e c h r o m o s o m e s f r o m t h e s p e r m a t o g o n i a l cells o f r a t s a n d m i c e , M u t a t i o n R e s . , 13 ( 1 9 7 1 ) 8 5 - - 8 8 . 8 Iball, J., T h e relative p o t e n c y o f c a r c i n o g e n i c c o m p o u n d s , A m e r . J. C a n c e r , 3 5 ( 1 9 3 9 ) 1 8 8 - - 1 9 0 . 9 M c C a n n , J., E. C h o i , E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e S a l m o n e U a / m i c r o s o m e t e s t : a s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. U S A , 7 2 ( 1 9 7 5 ) 5 1 3 5 - - 5 1 3 9 . 10 R o h r b o r n , G. a n d I. H a n s m a n n , I n d u c e d c h r o m o s o m e a b e r r a t i o n s in u n f e r t i l i z e d o o c y t e s o f m i c e , H u m a n g e n e t i k , 13 ( 1 9 7 1 ) 1 8 4 - - 1 9 8 . 11 S c h m i d , W. a n d G . R . S t a i g e r , C h r o m o s o m e s t u d i e s o n b o n e m a r r o w f r o m c h i n e s e h a m s t e r s t r e a t e d with benzodiazepine tranquillizers and cyclophosphamide, Mutation Res., 7 (1969) 99--108.
