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Micrococcus luteus UV-endonuclease-sensitive sites and sister-chromatid exchanges in Chinese hamster ovary cells
353
Mutation Research, 64 (1979) 353--356 © Elsevier/North-Holland Biomedical Press
Short Communication MICROCOCCUS LUTEUS UV-ENDONUCLEASE-SENSITIVE SITES AND SISTER-CHROMATID EXCHANGES IN CHINESE HAMSTER OVARY CELLS
R.J. REYNOLDS 1,., A.T. NATARAJAN 2,3 and P.H.M. LOHMAN 1
1 Medical Biological Laboratory TNO, Rijswijk Z.H., 2 Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden and 3 J.A. Cohen Institute of Radiation Protection and Radiopathology, Leiden (The Netherlands) (Received 2 March 1979) (Accepted 23 May 1979)
The reciprocal exchange of DNA between chromatids or sister chromatids (SCE) is readily induced in mammalian cells by many mutagenic and/or carcinogenic agents including alkylating chemicals and ionizing and ultraviolet radiations [6,12]. Due to the relative ease with which SCEs can be visualized and quantified in metaphase chromosomes by differential staining techniques and due to the sensitivity with which SCE can be induced at subtoxic concentrations of various mutagenic agents, SCE induction has become of interest as a possible indicator of mutagenic and/or carcinogenic potential. The mechanism of SCE induction as well as the primary lesions which lead to SCEs, however, remain to be elucidated. Far-UV radiation is an extensively studied agent with various biological consequences including the induction of SCEs. Cyclobutyl pyrimidine dimers, the predominant DNA photoproducts induced by far-UV radiation in vivo [10], have been implicated in a variety of UV-associated, biological processes that include cell killing [9], induction of chromosome aberrations [1], and tumor induction [3] in higher eukaryotes. The role of pyrimidine dimers in the induction of SCEs by UV radiation, if any, is not clear [4,11]. In the present study SCEs have been induced in Chinese hamster ovary (CHO) cells by two UV sources emitting radiation in different spectral regions. Frequencies of SCEs have been compared after exposure times that induce similar numbers of pyrimidine dimers as determined by an enzymatic assay that makes use of dimer-specific endonuclease activities from Micrococcus luteus (enzyme specific sites = ESS) as described previously [5,8]. Our results are compatible with the interpretation that SCEs arise from the presence of pyrim-
* Present address: Department of Pathology, Stanford University, Medical Center, Stanford, CA 94305 (U.S.A.)
354 idine dimers or some other lesions induced in equal numbers relative to dimers by both radiation sources. Materials and methods Chinese hamster ovary cells were grown at 37°C as monolayer cultures in Ham's F-10 medium (Flow) containing 15% fetal calf serum (Flow) and antibiotics. For ESS determinations this medium was supplemented with [Me-3H] thymidine ([3H]TdR; 0.25 pCi/ml) to prelabel cellular DNA. Far-UV radiation was obtained from a low-pressure-mercury lamp (Phillips TUV) at an incident dose rate of 0.39 J • m -2 • sec -1. Broad-spectrum, near-UV radiation was obtained from two "sunlight" fluorescent lamps (Westinghouse) and filtered through the lids of 60-mm plastic petri dishes (Greiner) to eliminate wavelengths below 290 nm (approx. dose rate: 2.5 J - m -2 • sec-1). Cells were irradiated simultaneously for both ESS and SCE determinations and were washed free of medium prior to irradiation as described previously [8]. For the determination of SCEs cells were incubated with fresh medium containing 5 pM 5-bromodeoxyuridine at 37°C in the dark and Colcemid (0.0001% final concentration; CIBA) was added 2 h prior to fixation at 24 or 28 h after irradiation. The cells were processed as air-dried preparations and were differentially stained with Hoechst 33258 and Giemsa stains [7]. All preparations were coded prior to SCE scoring. 25 cells were scored for each point and 3 independent experiments were performed. UV-endonuclease-sensitive sites were determined by a procedure modified from t h a t of Paterson et al. [5]. UV endonuclease was prepared from M i c r o c o c c u s l u t e u s according to the procedure of Carrier and Setlow and a preparation corresponding to their fraction II was used [2]. Results and discussion In Fig. 1 frequencies of SCEs in CHO cells irradiated with different doses of either far- or near-UV radiation are plotted as a function of the number of ESSs induced per 106 dalton of DNA. Evidence that ESSs are quantitative indicators of pyrimidine dimers in DNA has been presented previously [5,8]. Each point represents the average of 2 or 3 independent determinations. Separate culture dishes of CHO cells -- one unlabeled and one prelabeled with [3H]TdR -- were irradiated simultaneously so t h a t SCE and ESS determinations could be made for each exposure. Since far-UV radiation induces dimers with much greater efficiency t h a n does near-UV radiation, exposures were adjusted to yield comparable ESS frequencies. Both radiation sources have been shown previously to induce ESSs as a linear function of exposure time [8]. The data in Fig. 1 clearly indicate linear relationships between UV-induced SCEs and ESS frequency with both radiation sources. Furthermore the ratios of UV-induced SCEs to ESSs are indistinguishable over the dose ranges examined. Thus under our experimental conditions, the ability to induce SCEs appears to be related to the ability to induce ESSs. The role of pyrimidine dimers in SCE induction has been the subject of two other investigations. Wolff [11] found no reduction in the frequencies of SCEs
355 i
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,
I
i
,
I
I
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I
I
!
30
Q. t~ W (D
10
i
000
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004 008 0.12 016 Endonudease-sensitive sites per 106 daltons
Fig. 1. T h e r e l a t i o n s h i p b e t w e e n t h e o c c u r r e n c e o f . U V - e n d o n u c l e a s e - s e n s i t i v e sites ( E S S ) and t h e f r e q u e n cies o f s i s t e r - c h r o m a t i d e x c h a n g e s (SCEs) in C H O cells i r r a d i a t e d w i t h far- ( e ) o r w i t h n e a r - (~) U V radiation. D a t a p o i n t s are a v e r a g e s o f 2 o r 3 i n d e p e n d e n t d e t e r m i n a t i o n s . Bars d e n o t e s t a n d a r d d e v i a t i o n s f o r points representing the average of 3 determinations.
upon photoreactivating treatment of embryonic chick fibroblasts despite a reduction in DNA dimer content. He therefore concluded t h a t some minor p h o t o p r o d u c t and n o t pyrimidine dimers were responsible for the induction of SCEs. On the other hand, Kato [4] demonstrated that the frequencies o f UVinduced SCEs in rat kangaroo cells are reduced on photoreactivation with visible light, which suggests that pyrimidine dimers are involved in the develo p m e n t o f SCEs. In our studies a comparable effect was obtained, viz. a considerable reduction by photoreactivation in the frequencies of b o t h SCEs and ESSs induced in embryonic chick cells by doses of 254 nm UV between I and 5 J • m -2 (Natarajan and Van Zeeland, unpublished results). These data are consistent with the relationship presented here between the number of dimers and the number of SCEs; they strongly s u g g e s t - but do n o t prove -- that dimers are related to SCE induction. A substantial number of dimers has to be induced in the DNA of CHO cells, however, before one SCE will occur; when the DNA c o n t e n t of a CHO cell is 5" 10 -6 pg (the average value for rodent cells, see [13]), which would correspond with 3 • 1012 daltons, about 20 000 pyrimidine dimers have to be introduced for each additional SCE (calculation based on the line in Fig. 1). The results presented do not disagree with the hypothesis t h a t the induction of SCEs just as other biological effects, such as cell survival and chromosome aberrations [ 1 , 9 ] , is related to the induction of pyrimidine dimers in UV irradiated rodent cells. Acknowledgement This investigation was financially supported by the Euratom grants 200-76BIO N (Rijswijk) and 052-64-I-BIAN (Leiden).
356
References 1 B e n d e r , M . A . , H . G . Griggs a n d P.L. W a l k e r , M e c h a n i s m s o f c h r o m o s o m a l a b e r r a t i o n p r o d u c t i o n , I. A b e r r a t i o n i n d u c t i o n b y u l t r a v i o l e t light, M u t a t i o n R e s . , 2 0 ( 1 9 7 3 ) 3 8 7 - - 4 0 2 . 2 Carrier, W.L., and R.B. Setlow, Endonuclease from Micrococcus luteus which has activity toward ultraviolet-irradiated deoxyribonucleic acid: purification and properties, J. Bacteriol., 102 (1970) 178--186. 3 H a r t , R . W . , R . B . S e t l o w a n d A . D . W o o d h e a d , E v i d e n c e t h a t p y r i m i d i n e d i m e r s in D N A c a n give rise t o t u m o r s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 4 ( 1 9 7 7 ) 5 5 7 4 - - 5 5 7 8 . 4 K a t o , H., P h o t o r e a c t i v a t i o n o f sister c h r o m a t i d e x c h a n g e s i n d u c e d b y u l t r a v i o l e t i r r a d i a t i o n , N a t u r e (London), 249 (1974) 552--553. 5 P a t e r s o n , M . C . , P.H.M. L o h m a n a n d M.L. S l u y t e r , Use o f a n U V e n d o n u c l e a s e f r o m M i c r o c o c c u s l u t e u s t o m o n i t o r t h e p r o g r e s s o f D N A r e p a i r in U V - i r r a d i a t e d h u m a n cells, M u t a t i o n R e s . , 1 9 ( 1 9 7 3 ) 245--256. 6 P e r r y , P., a n d H . J . E v a n s , C y t o l o g i c a l d e t e c t i o n o f m u t a g e n / c a r c i n o g e n e x p o s u r e b y sister c h r o m a t i d exchange, Nature (London), 258 (1975) 121--125. 7 P e r r y , P., a n d S. W o l f f , N e w G i e m s a m e t h o d f o r t h e d i f f e r e n t i a l s t a i n i n g o f sister c h r o m a t i d s , N a t u r e (London), 251 (1974) 156--158. 8 R e y n o l d s , R . J . , a n d P . H . M . L o h m a n , M i c r o c o c c u s l u t e u s U V - e n d o n u c l e a s e - s e n s i t i v e sites in far- a n d n e a r * U V - i r r a d i a t e d C h i n e s e h a m s t e r o v a r y cells, in: P.C. H a n a w a l t , E.C. F r i e d b e r g a n d C.F. F o x ( E d s . ) , I C N - - U C L A S y m p o s i a o n M o l e c u l a r a n d C e l l u l a r B i o l o g y , D N A R e p a i r M e c h a n i s m s , V o l . 9, A c a d e m i c Press, N e w Y o r k , 1 9 7 8 , p p . 2 7 - - 3 0 . 9 R o t h m a n , R . H . , a n d R . B . S e t l o w , A n a c t i o n s p e c t r u m f o r cell killing a n d p y r i m i d i n e d i m e r f o r m a t i o n in C h i n e s e h a m s t e r V-79 cells, P h o t o c h e m . P h o t o b i o l . , 2 9 ( 1 9 7 8 ) 5 7 - - 6 1 . 1 0 S e t l o w , R . B . , P h o t o p r o d u c t s in D N A i r r a d i a t e d in vivo, P h o t o c h e m . P h o t o b i o l . , 7 ( 1 9 6 8 ) 6 4 3 - - 6 4 9 . 11 W o l f f , S., R e l a t i o n b e t w e e n D N A r e p a i r , c h r o m o s o m e a b e r r a t i o n s a n d sister c h r o m a t i d e x c h a n g e s , in: P.C. H a n a w a l t , E.C. F r i e d b e r g a n d C.F. F o x ( E d s . ) , I C N - - U C L A S y m p o s i a o n M o l e c u l a r a n d C e l l u l a r B i o l o g y , D N A R e p a i r M e c h a n i s m s , Vol. 9, A c a d e m i c Press, N e w Y o r k , 1 9 7 8 , p p . 7 5 1 - - 7 6 0 . 12 W o l f f , S., a n d S. T a k e h i s j a , I n d u c t i o n o f sister c h r o m a t i d e x c h a n g e s in m a m m a l i a n cells b y l o w c o n c e n t r a t i o n s o f m u t a g e n i c c a r c i n o g e n s t h a t r e q u i r e m e t a b o l i c a c t i v a t i o n as well as t h o s e t h a t d o n o t , in: D. S c o t t , B.A. B r i d g e s a n d F . H . S o b e l s ( E d s . ) , P r o g r e s s in G e n e t i c T o x i c o l o g y , E l s e v i e r / N o r t h - H o l land, Amsterdam, 1977, pp. 193--200. 13 H a n d b o o k o f B i o c h e m i s t r y , S e l e c t e d D a t a f o r M o l e c u l a r B i o l o g y , 2 n d E d n . , C h e m i c a l R u b b e r C o m p . , 1970, H-112.
Mutation Research, 64 (1979) 353--356 © Elsevier/North-Holland Biomedical Press
Short Communication MICROCOCCUS LUTEUS UV-ENDONUCLEASE-SENSITIVE SITES AND SISTER-CHROMATID EXCHANGES IN CHINESE HAMSTER OVARY CELLS
R.J. REYNOLDS 1,., A.T. NATARAJAN 2,3 and P.H.M. LOHMAN 1
1 Medical Biological Laboratory TNO, Rijswijk Z.H., 2 Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden and 3 J.A. Cohen Institute of Radiation Protection and Radiopathology, Leiden (The Netherlands) (Received 2 March 1979) (Accepted 23 May 1979)
The reciprocal exchange of DNA between chromatids or sister chromatids (SCE) is readily induced in mammalian cells by many mutagenic and/or carcinogenic agents including alkylating chemicals and ionizing and ultraviolet radiations [6,12]. Due to the relative ease with which SCEs can be visualized and quantified in metaphase chromosomes by differential staining techniques and due to the sensitivity with which SCE can be induced at subtoxic concentrations of various mutagenic agents, SCE induction has become of interest as a possible indicator of mutagenic and/or carcinogenic potential. The mechanism of SCE induction as well as the primary lesions which lead to SCEs, however, remain to be elucidated. Far-UV radiation is an extensively studied agent with various biological consequences including the induction of SCEs. Cyclobutyl pyrimidine dimers, the predominant DNA photoproducts induced by far-UV radiation in vivo [10], have been implicated in a variety of UV-associated, biological processes that include cell killing [9], induction of chromosome aberrations [1], and tumor induction [3] in higher eukaryotes. The role of pyrimidine dimers in the induction of SCEs by UV radiation, if any, is not clear [4,11]. In the present study SCEs have been induced in Chinese hamster ovary (CHO) cells by two UV sources emitting radiation in different spectral regions. Frequencies of SCEs have been compared after exposure times that induce similar numbers of pyrimidine dimers as determined by an enzymatic assay that makes use of dimer-specific endonuclease activities from Micrococcus luteus (enzyme specific sites = ESS) as described previously [5,8]. Our results are compatible with the interpretation that SCEs arise from the presence of pyrim-
* Present address: Department of Pathology, Stanford University, Medical Center, Stanford, CA 94305 (U.S.A.)
354 idine dimers or some other lesions induced in equal numbers relative to dimers by both radiation sources. Materials and methods Chinese hamster ovary cells were grown at 37°C as monolayer cultures in Ham's F-10 medium (Flow) containing 15% fetal calf serum (Flow) and antibiotics. For ESS determinations this medium was supplemented with [Me-3H] thymidine ([3H]TdR; 0.25 pCi/ml) to prelabel cellular DNA. Far-UV radiation was obtained from a low-pressure-mercury lamp (Phillips TUV) at an incident dose rate of 0.39 J • m -2 • sec -1. Broad-spectrum, near-UV radiation was obtained from two "sunlight" fluorescent lamps (Westinghouse) and filtered through the lids of 60-mm plastic petri dishes (Greiner) to eliminate wavelengths below 290 nm (approx. dose rate: 2.5 J - m -2 • sec-1). Cells were irradiated simultaneously for both ESS and SCE determinations and were washed free of medium prior to irradiation as described previously [8]. For the determination of SCEs cells were incubated with fresh medium containing 5 pM 5-bromodeoxyuridine at 37°C in the dark and Colcemid (0.0001% final concentration; CIBA) was added 2 h prior to fixation at 24 or 28 h after irradiation. The cells were processed as air-dried preparations and were differentially stained with Hoechst 33258 and Giemsa stains [7]. All preparations were coded prior to SCE scoring. 25 cells were scored for each point and 3 independent experiments were performed. UV-endonuclease-sensitive sites were determined by a procedure modified from t h a t of Paterson et al. [5]. UV endonuclease was prepared from M i c r o c o c c u s l u t e u s according to the procedure of Carrier and Setlow and a preparation corresponding to their fraction II was used [2]. Results and discussion In Fig. 1 frequencies of SCEs in CHO cells irradiated with different doses of either far- or near-UV radiation are plotted as a function of the number of ESSs induced per 106 dalton of DNA. Evidence that ESSs are quantitative indicators of pyrimidine dimers in DNA has been presented previously [5,8]. Each point represents the average of 2 or 3 independent determinations. Separate culture dishes of CHO cells -- one unlabeled and one prelabeled with [3H]TdR -- were irradiated simultaneously so t h a t SCE and ESS determinations could be made for each exposure. Since far-UV radiation induces dimers with much greater efficiency t h a n does near-UV radiation, exposures were adjusted to yield comparable ESS frequencies. Both radiation sources have been shown previously to induce ESSs as a linear function of exposure time [8]. The data in Fig. 1 clearly indicate linear relationships between UV-induced SCEs and ESS frequency with both radiation sources. Furthermore the ratios of UV-induced SCEs to ESSs are indistinguishable over the dose ranges examined. Thus under our experimental conditions, the ability to induce SCEs appears to be related to the ability to induce ESSs. The role of pyrimidine dimers in SCE induction has been the subject of two other investigations. Wolff [11] found no reduction in the frequencies of SCEs
355 i
]
,
I
i
,
I
I
]
I
I
!
30
Q. t~ W (D
10
i
000
I
004 008 0.12 016 Endonudease-sensitive sites per 106 daltons
Fig. 1. T h e r e l a t i o n s h i p b e t w e e n t h e o c c u r r e n c e o f . U V - e n d o n u c l e a s e - s e n s i t i v e sites ( E S S ) and t h e f r e q u e n cies o f s i s t e r - c h r o m a t i d e x c h a n g e s (SCEs) in C H O cells i r r a d i a t e d w i t h far- ( e ) o r w i t h n e a r - (~) U V radiation. D a t a p o i n t s are a v e r a g e s o f 2 o r 3 i n d e p e n d e n t d e t e r m i n a t i o n s . Bars d e n o t e s t a n d a r d d e v i a t i o n s f o r points representing the average of 3 determinations.
upon photoreactivating treatment of embryonic chick fibroblasts despite a reduction in DNA dimer content. He therefore concluded t h a t some minor p h o t o p r o d u c t and n o t pyrimidine dimers were responsible for the induction of SCEs. On the other hand, Kato [4] demonstrated that the frequencies o f UVinduced SCEs in rat kangaroo cells are reduced on photoreactivation with visible light, which suggests that pyrimidine dimers are involved in the develo p m e n t o f SCEs. In our studies a comparable effect was obtained, viz. a considerable reduction by photoreactivation in the frequencies of b o t h SCEs and ESSs induced in embryonic chick cells by doses of 254 nm UV between I and 5 J • m -2 (Natarajan and Van Zeeland, unpublished results). These data are consistent with the relationship presented here between the number of dimers and the number of SCEs; they strongly s u g g e s t - but do n o t prove -- that dimers are related to SCE induction. A substantial number of dimers has to be induced in the DNA of CHO cells, however, before one SCE will occur; when the DNA c o n t e n t of a CHO cell is 5" 10 -6 pg (the average value for rodent cells, see [13]), which would correspond with 3 • 1012 daltons, about 20 000 pyrimidine dimers have to be introduced for each additional SCE (calculation based on the line in Fig. 1). The results presented do not disagree with the hypothesis t h a t the induction of SCEs just as other biological effects, such as cell survival and chromosome aberrations [ 1 , 9 ] , is related to the induction of pyrimidine dimers in UV irradiated rodent cells. Acknowledgement This investigation was financially supported by the Euratom grants 200-76BIO N (Rijswijk) and 052-64-I-BIAN (Leiden).
356
References 1 B e n d e r , M . A . , H . G . Griggs a n d P.L. W a l k e r , M e c h a n i s m s o f c h r o m o s o m a l a b e r r a t i o n p r o d u c t i o n , I. A b e r r a t i o n i n d u c t i o n b y u l t r a v i o l e t light, M u t a t i o n R e s . , 2 0 ( 1 9 7 3 ) 3 8 7 - - 4 0 2 . 2 Carrier, W.L., and R.B. Setlow, Endonuclease from Micrococcus luteus which has activity toward ultraviolet-irradiated deoxyribonucleic acid: purification and properties, J. Bacteriol., 102 (1970) 178--186. 3 H a r t , R . W . , R . B . S e t l o w a n d A . D . W o o d h e a d , E v i d e n c e t h a t p y r i m i d i n e d i m e r s in D N A c a n give rise t o t u m o r s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 4 ( 1 9 7 7 ) 5 5 7 4 - - 5 5 7 8 . 4 K a t o , H., P h o t o r e a c t i v a t i o n o f sister c h r o m a t i d e x c h a n g e s i n d u c e d b y u l t r a v i o l e t i r r a d i a t i o n , N a t u r e (London), 249 (1974) 552--553. 5 P a t e r s o n , M . C . , P.H.M. L o h m a n a n d M.L. S l u y t e r , Use o f a n U V e n d o n u c l e a s e f r o m M i c r o c o c c u s l u t e u s t o m o n i t o r t h e p r o g r e s s o f D N A r e p a i r in U V - i r r a d i a t e d h u m a n cells, M u t a t i o n R e s . , 1 9 ( 1 9 7 3 ) 245--256. 6 P e r r y , P., a n d H . J . E v a n s , C y t o l o g i c a l d e t e c t i o n o f m u t a g e n / c a r c i n o g e n e x p o s u r e b y sister c h r o m a t i d exchange, Nature (London), 258 (1975) 121--125. 7 P e r r y , P., a n d S. W o l f f , N e w G i e m s a m e t h o d f o r t h e d i f f e r e n t i a l s t a i n i n g o f sister c h r o m a t i d s , N a t u r e (London), 251 (1974) 156--158. 8 R e y n o l d s , R . J . , a n d P . H . M . L o h m a n , M i c r o c o c c u s l u t e u s U V - e n d o n u c l e a s e - s e n s i t i v e sites in far- a n d n e a r * U V - i r r a d i a t e d C h i n e s e h a m s t e r o v a r y cells, in: P.C. H a n a w a l t , E.C. F r i e d b e r g a n d C.F. F o x ( E d s . ) , I C N - - U C L A S y m p o s i a o n M o l e c u l a r a n d C e l l u l a r B i o l o g y , D N A R e p a i r M e c h a n i s m s , V o l . 9, A c a d e m i c Press, N e w Y o r k , 1 9 7 8 , p p . 2 7 - - 3 0 . 9 R o t h m a n , R . H . , a n d R . B . S e t l o w , A n a c t i o n s p e c t r u m f o r cell killing a n d p y r i m i d i n e d i m e r f o r m a t i o n in C h i n e s e h a m s t e r V-79 cells, P h o t o c h e m . P h o t o b i o l . , 2 9 ( 1 9 7 8 ) 5 7 - - 6 1 . 1 0 S e t l o w , R . B . , P h o t o p r o d u c t s in D N A i r r a d i a t e d in vivo, P h o t o c h e m . P h o t o b i o l . , 7 ( 1 9 6 8 ) 6 4 3 - - 6 4 9 . 11 W o l f f , S., R e l a t i o n b e t w e e n D N A r e p a i r , c h r o m o s o m e a b e r r a t i o n s a n d sister c h r o m a t i d e x c h a n g e s , in: P.C. H a n a w a l t , E.C. F r i e d b e r g a n d C.F. F o x ( E d s . ) , I C N - - U C L A S y m p o s i a o n M o l e c u l a r a n d C e l l u l a r B i o l o g y , D N A R e p a i r M e c h a n i s m s , Vol. 9, A c a d e m i c Press, N e w Y o r k , 1 9 7 8 , p p . 7 5 1 - - 7 6 0 . 12 W o l f f , S., a n d S. T a k e h i s j a , I n d u c t i o n o f sister c h r o m a t i d e x c h a n g e s in m a m m a l i a n cells b y l o w c o n c e n t r a t i o n s o f m u t a g e n i c c a r c i n o g e n s t h a t r e q u i r e m e t a b o l i c a c t i v a t i o n as well as t h o s e t h a t d o n o t , in: D. S c o t t , B.A. B r i d g e s a n d F . H . S o b e l s ( E d s . ) , P r o g r e s s in G e n e t i c T o x i c o l o g y , E l s e v i e r / N o r t h - H o l land, Amsterdam, 1977, pp. 193--200. 13 H a n d b o o k o f B i o c h e m i s t r y , S e l e c t e d D a t a f o r M o l e c u l a r B i o l o g y , 2 n d E d n . , C h e m i c a l R u b b e r C o m p . , 1970, H-112.