261
Mutation Research, 46 (1977) 261--264 © Elsevier/North-Holland Biomedical Press
ACTIVATION OF A P R O C A R C I N O G E N TO A MUTAGEN BY CELL-FREE EXTRACTS OF ANAEROBIC BACTERIA
ELENA C. McCOY, WILLIAM T. SPECK and HERBERT S. ROSENKRANZ Department of Microbiology, New York Medical College, Valhalla, N.Y. 10595 (U.S.A.)
(Received January 14th, 1977) (Revision received April 4th, 1977) (Accepted April 20th, 1977)
Summary The Salmonella mutagenicity assay can be coupled to cell-free preparations derived from anaerobic bacteria ( Clostridium perfringens and Bacteroides fragilis) to activate a procarcinogen to a mutagen. This activity is destroyed by heating and by digestion with pronase and it is sensitive to oxygen. These findings indicate that the Salmonella mutagenicity assay can be adapted to the study of the role of anaerobes in the activation of carcinogens.
Introduction The mutagenicity assay developed by Ames and his associates [2] is finding widespread acceptance as a prescreening procedure for detecting mutagens and potential carcinogens [4,5,11,12,14,20]. One of the chief advantages of this and other microbial assays is their flexibility and adaptibility to various practical and experimental protocols. Perhaps one of the most important examples of the versatility of these assay procedures was achieved when it was recognized that they are compatible with microsomal assay mixtures [1,10,21]. The addition of such cellular extracts to agar plates greatly extended the scope of these methods. More recently the use of livers from rats induced by Araclor [2], a polychlorinated biphenyl, led to a further increase in the sensitivity of the response. The use of such inducers also has a great physiological relevance to environmental carcinogenesis, as our human population is constantly challenged (and induced) by agents in our environment [3]. Another situation which exemplifies the versatility of microbial assay procedures derives from the observation that some mutagens displayed increased genetic activity when the initial period of incubation was carried o u t under anaerobic conditions. This effect which is frequently seen with nitro-containing substances probably is due to oxygen-labile character of the biologically
262 active intermediate (presumably the corresponding hydroxamate) [23]. With some test agents, mutagenicity can only be demonstrated when the incubation was carried out under anaerobic conditions (e.g. azathioprine, (6-[(1-methyl4 nitroimidazol-5-yl) thio]purine)) [23]. The present report deals with the demonstration that the Salmonella mutagenicity assay can be coupled to extracts from anaerobic bacteria to activate procarcinogens. This demonstration is timely as recent studies have indicated a relationship between diet and the incidence of certain cancers [6,24]. These correlations may reflect the fact that certain diets affect the colonization of the gastrointestinal tract with an anaerobic flora which may be active in metabolizing normal nutrients or their metabolites to carcinogenic substances [7,13, 15,16,19]. This possibility is reinforced by the finding that germ-free animals are refractory to the carcinogenic action of certain chemicals [8,17,18]. Although the liver is indeed a very important metabolic organ, it is not always realized that the gastrointestinal tract behaves similarly. Filled with bacteria, primarily anaerobes, which o u t n u m b e r the aerobes by a factor of one thousand (1011 anaerobes/g), and which are u n d o u b t e d l y active in metabolizing (and activating) procarcinogens. However, in the usual bioassay procedures we take into consideration only the role of the liver in metabolizing procarcinogens and ignore the role of the gastrointestinal tract and its anaerobic bacterial flora. In view of this deficiency, studies were initiated to determine whether anaerobes could be coupled to the Salmonella mutagenicity assay in a manner similar to that used for liver microsomes. Materials and methods
Bacterial strains Clinical isolates of Clostridium perfringens and Bacteroides [ragilis were obtained from the Columbia Presbyterian Medical Center, New York, N.Y. and Salmonella typhimurium TA1538 [2] from Dr. Bruce N. Ames (Department of Biochemistry, University of California, Berkeley, Calif.). Media and chemicals Strains of Cl. perfringens and B. fragilis were grown anaerobically (Gas-Pack, BBL, Becton, Dickinson and Co., Cockeyville, Md.) in Columbia broth (BBL, Becton, Dickinson and Co., Cockeyville, Md., 35 g/l). 2-Aminofluorene was purchased from Starks Assoc. Inc., Buffalo, N.Y. and pronase from Cal Biochem, San Diego, Calif. All other materials were of the highest purity commercially available. Ex tract preparation Cl. perfringens and B. fragilis cells from overnight cultures were harvested by centrifugation at 4°C and washed with 0.1 M phosphate buffer, pH 7.4. The washed cells were resuspended in cold buffer and treated with the Sonic Dismembrator (Artek Systems Corp., Farmingdale, N.Y.) for three 1-min periods. The resulting suspension was centrifuged at 17,370 X g for 15 min and the supernatant fluids were collected by aspiration, filtered through Millex (Millipore, Bedford, Mass.) membrane filters and either used directly or stored at
263 --20C. For preparation of heated extracts, samples were placed in a water bath (80°C) for 10 min and centrifuged at 17,370 × g for 10 min prior to filtration.
Chemical and mutagenicity assays Proteins were determined by the method of L o w r y et al. [9] using a standard of bovine serum albumin. Mutagenicity testing was performed using the m e t h o d described b y Ames et al. [2]. The mutagenicity plates were incubated in Gas-Pack jars (BBL, Becton, Dickinson and Co., Cockeysville, Md.) for 18 h at 37°C and this was followed by 48 h of aerobic incubation. All these incubations were carried o u t in the dark [22]. Results and discussion Using the procedures described above, it could be demonstrated (Table 1) that cell-free extracts prepared from the predominant gastrointestinal flora (Cl. perfringens and B. fragilis) were both active in metabolizing a procarcinogen (2-aminofluorene) to a substance mutagenic for Salmonella typhimurium. No such activity was demonstrable when extracts from the anaerobic bacteria were omitted, heated to 80°C or when pronase (0.1 mg/plate) was incorporated into the plate assay. Incubation in the presence of extracts b u t under aerobic conditions resulted in minimal activity (Table 1). Stored extracts assayed 1--4 weeks after preparation exhibited reduced activity. However, it should be noted that these samples were not stored in an anaerobic environment. TABLE I ACTIVATION TERIA
OF
2-AMINOFLUORENE
BY C E L L - F R E E
EXTRACTS
FROM
ANAEROBIC
Amount of extract (rag p r o t e i n p e r plate)
Condition of incubation
Clostridium perfringens Clostridium perfringens
1.0 2.0
Anaerobic Anaerobic
5
288 505
H e a t e d Cl. p e r f r i n g e n s H e a t e d Cl. p e r f r i n g e n s
1.0 2.0
Anaerobic Anaerobic
12
21 16
B a c t e r o i d e s fragilis B a c t e r o i d e s fragilis
1.0 2.0
Anaerobic Anaerobic
5
345 549
H e a t e d B. fragilis H e a t e d B. fragilis
1.0 2.0
Anaerobic Anaerobic
9
14 17
0 0
Anaerobic Aerobic
10 4
18 34
B. fragilis B. fragilis
0.5 0.5
Anaerobic Aerobic
4 9
533 68
CI. p e r f r i n g e n s Cl. p e r f r i n g e n s
0.8 0.8
Anaerobic Aerobic
5 8
424 56
Source of extract
None None
a 25 ~g 2 - A m i n o f l u o r e n e .
BAC-
Mutants per plate No 2-aminofluorene
+ 2-aminofluorene a
264
The present findings which indicate that the Salmonella mutagenicity assay can be adapted to the study of the role of anaerobic flora in the activation of chemical carcinogens will undoubtedly stimulate a number of such investigations. Acknowledgement This investigation was supported by the Environmental Protection Agency (68-01-3246) the National Institute of Environmental Sciences (NO1-ES-2124) and the National Cancer Institute (NO-1-CP-65855). References 1 A m e s , B.N., E. D u r s t o n , E. Y a m a s a k i a n d F.D. L e e , C a r c i n o g e n s as m u t a g e n s : a s i m p l e test s y s t e m c o m b i n i n g liver h o m o g e n a t e s for a c t i v a t i o n and b a c t e r i a for d e t e c t i o n , P r o c . Natl. A c a d . Sci. ( U . S . A . ) , 70 ( 1 9 7 3 ) 2 2 8 1 - - 2 2 8 5 . 2 A m e s , B.N., J. M c C a n n and E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h the S a l m o n e l l a ] m a m m a l i a n m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 C o n n e y , A . H . a n d J.J. Burns, M e t a b o l i c i n t e r a c t i o n a m o n g e n v i r o n m e n t a l c h e m i c a l s a n d drugs., Science, 178 (1972) 576--586. 4 d e S e r r e s , F.J., T h e utility of s h o r t - t e r m tests f o r m u t a g e n i c i t y , M u t a t i o n Res., 38 ( 1 9 7 6 ) 1 - - 2 . 5 d e S e r r e s , F..J., P r o s p e c t s for a r e v o l u t i o n in the m e t h o d s of t o x i c o l o g i c a l e v a l u a t i o n , M u t a t i o n Res., 38 ( 1 9 7 6 ) 1 6 5 - - 1 7 6 . 6 Hill, M.J., B a c t e r i a a n d the e t i o l o g y of c o l o n c a n c e r , C a n c e r , 34 ( 1 9 7 4 ) 8 1 5 - - 8 1 8 . 7 Hill, M.J., M e t a b o l i c e p i d e m i o l o g y of d i e t a r y f a c t o r s in large b o w e l c a n c e r , C a n c e r Res., 35 ( 1 9 7 5 ) 3398--3402. 8 L a q u e u e r , G . L . , C o n t r i b u t i o n s of i n t e s t i n a l m i c r o f l o r a to c a r c i n o g e n e s i s , in W.J. B u r d e t t e ( e d . ) , Carc i n o m a o f t h e C o l o n a n d A n t e c e d e n t E p i t h e l i u m , C.C. T h o m a s , Springfield, Ill., 1 9 7 0 , pp. 3 0 5 - - 3 1 5 . 9 L o w r y , O.H., N.J. R o s e b r o u g h , A.L. F a r r and R.J. R a n d a l l , P r o t e i n m e a s u r e m e n t s with the Folin r e a g e n t , J. Biol. C h e m . , 193 ( 1 9 5 1 ) 2 6 5 - - 2 7 5 . 10 Malling, H . V . , D i m e t h y l n i t r o s a m i n e : f o r m a t i o n of m u t a g e n i c c o m p o u n d s by i n t e r a c t i o n w i t h m o u s e liver m i c r o s o m e s , M u t a t i o n Res., 13 ( 1 9 7 1 ) 4 2 5 - - 4 2 9 . 11 M c C a n n , J. a n d B.N. A m e s , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in the S a l m o n e l l a / m i c r o s o m e test: assay of 3 0 0 c h e m i c a l s : discussion, Proc. Natl. A c a d . Sci. U.S., 73 ( 1 9 7 6 ) 9 5 0 - - 9 5 4 . 12 M c C a n n , J., E. Choi, E. Y a m a s a k i and Bruce N. A m e s , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in the S a l m o n e l l a / m i c r o s o m e test: assay of 3 0 0 c h e m i c a l s , Proc. Natl. A c a d . Sci. U.S., 72 ( 1 9 7 5 ) 5 1 3 5 - 5139. 13 M o o r e , W.E.C. a n d L . V . H o l d c m a n , Discussion of c u r r e n t b a c t e r i o l o g i c a l inw~stigations of the r e l a t i o n ship b e t w e e n intestinal flora, diet and c o l o n c a n c e r , C a n c e r Res., 35 ( 1 9 7 5 ) 3 4 1 8 - - 3 4 2 0 . 14 P u r c h a s e , I . F . H . , F. L o n g s t a f f , J. A s h b y , J . A . Styles, D. A n d e r s o n , D.A. L e f e v r e and F.R. W e s t w o o d , E v a l u a t i o n of six s h o r t t e r m tests f o r d e t e c t i n g organic c h e m i c a l c a r c i n o g e n s a n d r e c o m m e n d a t i o n s for t h e i r use, N a t u r e ( L o n d . ) , 2 6 4 ( 1 9 7 6 ) 6 2 4 - - 6 2 7 . 15 R e d d y , B.S., A. M a s t r o m a r i n o a n d E.L. W y n d e r , F u r t h e r leads on m e t a b o l i c e p i d e m i o l o g y of large b o w e l c a n c e r , C a n c e r Res., 3 5 ( 1 9 7 5 ) 3 4 0 3 - - 3 4 0 6 . 16 R e d d y , B.S., T. N a r i s a w a , R. M a r o n p o t , J . H . W e i s b u r g e r a n d E.L. W y n d e r , A n i m a l m o d e l s for t h e s t u d y of d i e t a r y f a c t o r s and c a n c e r of the large b o w e l , C a n c e r Res., 35 ( 1 9 7 5 ) 3 4 2 1 - - 3 4 2 6 . 17 R e d d y , B.S., T. Narisawa, P. Wright, D. V u k u s i c h , J . H . W e i s b u r g e r a n d E.L. W y n d e r , Colon c a r c i n o genesis w i t h a z o x y m e t h a n e and d i m e t h y l h y d r a z i n e in g e r m - f r e e rats, C a n c e r Res., 35 ( 1 9 7 5 ) 2 8 7 - 290. 18 R e d d y , B.S., J . H . Weisburger, T. N a r i s a w a and E.L. W y n d e r , C o l o n c a r c i n o g e n e s i s in g e r m - f r e e rats w i t h 1 , 2 - d i m e t h y l h y d r a z i n e a n d N-methyl-Nr-nitro-N-nitrosoguanidine, C a n c e r Res., 34 ( 1 9 7 4 ) 2 3 6 8 - 2372. 19 Redd~y, B.S., J . H . Weisburgcr and E.L. W y n d e r , E f f e c t of high risk and low risk diets for c o l o n carc i n o g e n e s i s o n fecal m i c r o f l o r a a n d steroids in m a n , J. N u t r . , 1 0 5 ( 1 9 7 5 ) 8 7 6 - - 8 8 4 . 20 R o s e n k r a n z , H.S., A s p e c t s of m i c r o b i o l o g y in c a n c e r r e s e a r c h , A n n . Rev. Microbiol., 27 ( 1 9 7 3 ) 3 8 3 - 401. 21 Slater, E.E., M.D. A n d e r s o n a n d H.S. R o s e n k r a n z , R a p i d d e t e c t i o n of m u t a g e n s and c a r c i n o g e n s , Cancer Res., 31 ( 1 9 7 1 ) 9 7 0 - - 9 7 3 . 22 S p e c k , W.T. a n d H.S. R o s e n k r a n z , Base s u b s t i t u t i o n m u t a t i o n s i n d u c e d in S a l m o n e l l a strains by visible light ( 4 5 0 r a m ) , P h o t o c h e m P h o t o b i o l . , 21 ( 1 9 7 5 ) 3 6 9 - - 3 7 1 . 23 S p e c k , W.T. a n d H.S. R o s e n k r a n z , M u t a g e n i c i t y of a z a t h i o p r i n e , C a n c e r Res., 36 ( 1 9 7 6 ) 1 0 8 - - 1 0 9 . 24 W y n d e r , E.L., N u t r i t i o n a n d C a n c e r , Fed. P r o c . , 35 ( 1 9 7 6 ) 1 3 0 9 - - 1 3 1 5 .
Mutation Research, 46 (1977) 261--264 © Elsevier/North-Holland Biomedical Press
ACTIVATION OF A P R O C A R C I N O G E N TO A MUTAGEN BY CELL-FREE EXTRACTS OF ANAEROBIC BACTERIA
ELENA C. McCOY, WILLIAM T. SPECK and HERBERT S. ROSENKRANZ Department of Microbiology, New York Medical College, Valhalla, N.Y. 10595 (U.S.A.)
(Received January 14th, 1977) (Revision received April 4th, 1977) (Accepted April 20th, 1977)
Summary The Salmonella mutagenicity assay can be coupled to cell-free preparations derived from anaerobic bacteria ( Clostridium perfringens and Bacteroides fragilis) to activate a procarcinogen to a mutagen. This activity is destroyed by heating and by digestion with pronase and it is sensitive to oxygen. These findings indicate that the Salmonella mutagenicity assay can be adapted to the study of the role of anaerobes in the activation of carcinogens.
Introduction The mutagenicity assay developed by Ames and his associates [2] is finding widespread acceptance as a prescreening procedure for detecting mutagens and potential carcinogens [4,5,11,12,14,20]. One of the chief advantages of this and other microbial assays is their flexibility and adaptibility to various practical and experimental protocols. Perhaps one of the most important examples of the versatility of these assay procedures was achieved when it was recognized that they are compatible with microsomal assay mixtures [1,10,21]. The addition of such cellular extracts to agar plates greatly extended the scope of these methods. More recently the use of livers from rats induced by Araclor [2], a polychlorinated biphenyl, led to a further increase in the sensitivity of the response. The use of such inducers also has a great physiological relevance to environmental carcinogenesis, as our human population is constantly challenged (and induced) by agents in our environment [3]. Another situation which exemplifies the versatility of microbial assay procedures derives from the observation that some mutagens displayed increased genetic activity when the initial period of incubation was carried o u t under anaerobic conditions. This effect which is frequently seen with nitro-containing substances probably is due to oxygen-labile character of the biologically
262 active intermediate (presumably the corresponding hydroxamate) [23]. With some test agents, mutagenicity can only be demonstrated when the incubation was carried out under anaerobic conditions (e.g. azathioprine, (6-[(1-methyl4 nitroimidazol-5-yl) thio]purine)) [23]. The present report deals with the demonstration that the Salmonella mutagenicity assay can be coupled to extracts from anaerobic bacteria to activate procarcinogens. This demonstration is timely as recent studies have indicated a relationship between diet and the incidence of certain cancers [6,24]. These correlations may reflect the fact that certain diets affect the colonization of the gastrointestinal tract with an anaerobic flora which may be active in metabolizing normal nutrients or their metabolites to carcinogenic substances [7,13, 15,16,19]. This possibility is reinforced by the finding that germ-free animals are refractory to the carcinogenic action of certain chemicals [8,17,18]. Although the liver is indeed a very important metabolic organ, it is not always realized that the gastrointestinal tract behaves similarly. Filled with bacteria, primarily anaerobes, which o u t n u m b e r the aerobes by a factor of one thousand (1011 anaerobes/g), and which are u n d o u b t e d l y active in metabolizing (and activating) procarcinogens. However, in the usual bioassay procedures we take into consideration only the role of the liver in metabolizing procarcinogens and ignore the role of the gastrointestinal tract and its anaerobic bacterial flora. In view of this deficiency, studies were initiated to determine whether anaerobes could be coupled to the Salmonella mutagenicity assay in a manner similar to that used for liver microsomes. Materials and methods
Bacterial strains Clinical isolates of Clostridium perfringens and Bacteroides [ragilis were obtained from the Columbia Presbyterian Medical Center, New York, N.Y. and Salmonella typhimurium TA1538 [2] from Dr. Bruce N. Ames (Department of Biochemistry, University of California, Berkeley, Calif.). Media and chemicals Strains of Cl. perfringens and B. fragilis were grown anaerobically (Gas-Pack, BBL, Becton, Dickinson and Co., Cockeyville, Md.) in Columbia broth (BBL, Becton, Dickinson and Co., Cockeyville, Md., 35 g/l). 2-Aminofluorene was purchased from Starks Assoc. Inc., Buffalo, N.Y. and pronase from Cal Biochem, San Diego, Calif. All other materials were of the highest purity commercially available. Ex tract preparation Cl. perfringens and B. fragilis cells from overnight cultures were harvested by centrifugation at 4°C and washed with 0.1 M phosphate buffer, pH 7.4. The washed cells were resuspended in cold buffer and treated with the Sonic Dismembrator (Artek Systems Corp., Farmingdale, N.Y.) for three 1-min periods. The resulting suspension was centrifuged at 17,370 X g for 15 min and the supernatant fluids were collected by aspiration, filtered through Millex (Millipore, Bedford, Mass.) membrane filters and either used directly or stored at
263 --20C. For preparation of heated extracts, samples were placed in a water bath (80°C) for 10 min and centrifuged at 17,370 × g for 10 min prior to filtration.
Chemical and mutagenicity assays Proteins were determined by the method of L o w r y et al. [9] using a standard of bovine serum albumin. Mutagenicity testing was performed using the m e t h o d described b y Ames et al. [2]. The mutagenicity plates were incubated in Gas-Pack jars (BBL, Becton, Dickinson and Co., Cockeysville, Md.) for 18 h at 37°C and this was followed by 48 h of aerobic incubation. All these incubations were carried o u t in the dark [22]. Results and discussion Using the procedures described above, it could be demonstrated (Table 1) that cell-free extracts prepared from the predominant gastrointestinal flora (Cl. perfringens and B. fragilis) were both active in metabolizing a procarcinogen (2-aminofluorene) to a substance mutagenic for Salmonella typhimurium. No such activity was demonstrable when extracts from the anaerobic bacteria were omitted, heated to 80°C or when pronase (0.1 mg/plate) was incorporated into the plate assay. Incubation in the presence of extracts b u t under aerobic conditions resulted in minimal activity (Table 1). Stored extracts assayed 1--4 weeks after preparation exhibited reduced activity. However, it should be noted that these samples were not stored in an anaerobic environment. TABLE I ACTIVATION TERIA
OF
2-AMINOFLUORENE
BY C E L L - F R E E
EXTRACTS
FROM
ANAEROBIC
Amount of extract (rag p r o t e i n p e r plate)
Condition of incubation
Clostridium perfringens Clostridium perfringens
1.0 2.0
Anaerobic Anaerobic
5
288 505
H e a t e d Cl. p e r f r i n g e n s H e a t e d Cl. p e r f r i n g e n s
1.0 2.0
Anaerobic Anaerobic
12
21 16
B a c t e r o i d e s fragilis B a c t e r o i d e s fragilis
1.0 2.0
Anaerobic Anaerobic
5
345 549
H e a t e d B. fragilis H e a t e d B. fragilis
1.0 2.0
Anaerobic Anaerobic
9
14 17
0 0
Anaerobic Aerobic
10 4
18 34
B. fragilis B. fragilis
0.5 0.5
Anaerobic Aerobic
4 9
533 68
CI. p e r f r i n g e n s Cl. p e r f r i n g e n s
0.8 0.8
Anaerobic Aerobic
5 8
424 56
Source of extract
None None
a 25 ~g 2 - A m i n o f l u o r e n e .
BAC-
Mutants per plate No 2-aminofluorene
+ 2-aminofluorene a
264
The present findings which indicate that the Salmonella mutagenicity assay can be adapted to the study of the role of anaerobic flora in the activation of chemical carcinogens will undoubtedly stimulate a number of such investigations. Acknowledgement This investigation was supported by the Environmental Protection Agency (68-01-3246) the National Institute of Environmental Sciences (NO1-ES-2124) and the National Cancer Institute (NO-1-CP-65855). References 1 A m e s , B.N., E. D u r s t o n , E. Y a m a s a k i a n d F.D. L e e , C a r c i n o g e n s as m u t a g e n s : a s i m p l e test s y s t e m c o m b i n i n g liver h o m o g e n a t e s for a c t i v a t i o n and b a c t e r i a for d e t e c t i o n , P r o c . Natl. A c a d . Sci. ( U . S . A . ) , 70 ( 1 9 7 3 ) 2 2 8 1 - - 2 2 8 5 . 2 A m e s , B.N., J. M c C a n n and E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h the S a l m o n e l l a ] m a m m a l i a n m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 C o n n e y , A . H . a n d J.J. Burns, M e t a b o l i c i n t e r a c t i o n a m o n g e n v i r o n m e n t a l c h e m i c a l s a n d drugs., Science, 178 (1972) 576--586. 4 d e S e r r e s , F.J., T h e utility of s h o r t - t e r m tests f o r m u t a g e n i c i t y , M u t a t i o n Res., 38 ( 1 9 7 6 ) 1 - - 2 . 5 d e S e r r e s , F..J., P r o s p e c t s for a r e v o l u t i o n in the m e t h o d s of t o x i c o l o g i c a l e v a l u a t i o n , M u t a t i o n Res., 38 ( 1 9 7 6 ) 1 6 5 - - 1 7 6 . 6 Hill, M.J., B a c t e r i a a n d the e t i o l o g y of c o l o n c a n c e r , C a n c e r , 34 ( 1 9 7 4 ) 8 1 5 - - 8 1 8 . 7 Hill, M.J., M e t a b o l i c e p i d e m i o l o g y of d i e t a r y f a c t o r s in large b o w e l c a n c e r , C a n c e r Res., 35 ( 1 9 7 5 ) 3398--3402. 8 L a q u e u e r , G . L . , C o n t r i b u t i o n s of i n t e s t i n a l m i c r o f l o r a to c a r c i n o g e n e s i s , in W.J. B u r d e t t e ( e d . ) , Carc i n o m a o f t h e C o l o n a n d A n t e c e d e n t E p i t h e l i u m , C.C. T h o m a s , Springfield, Ill., 1 9 7 0 , pp. 3 0 5 - - 3 1 5 . 9 L o w r y , O.H., N.J. R o s e b r o u g h , A.L. F a r r and R.J. R a n d a l l , P r o t e i n m e a s u r e m e n t s with the Folin r e a g e n t , J. Biol. C h e m . , 193 ( 1 9 5 1 ) 2 6 5 - - 2 7 5 . 10 Malling, H . V . , D i m e t h y l n i t r o s a m i n e : f o r m a t i o n of m u t a g e n i c c o m p o u n d s by i n t e r a c t i o n w i t h m o u s e liver m i c r o s o m e s , M u t a t i o n Res., 13 ( 1 9 7 1 ) 4 2 5 - - 4 2 9 . 11 M c C a n n , J. a n d B.N. A m e s , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in the S a l m o n e l l a / m i c r o s o m e test: assay of 3 0 0 c h e m i c a l s : discussion, Proc. Natl. A c a d . Sci. U.S., 73 ( 1 9 7 6 ) 9 5 0 - - 9 5 4 . 12 M c C a n n , J., E. Choi, E. Y a m a s a k i and Bruce N. A m e s , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in the S a l m o n e l l a / m i c r o s o m e test: assay of 3 0 0 c h e m i c a l s , Proc. Natl. A c a d . Sci. U.S., 72 ( 1 9 7 5 ) 5 1 3 5 - 5139. 13 M o o r e , W.E.C. a n d L . V . H o l d c m a n , Discussion of c u r r e n t b a c t e r i o l o g i c a l inw~stigations of the r e l a t i o n ship b e t w e e n intestinal flora, diet and c o l o n c a n c e r , C a n c e r Res., 35 ( 1 9 7 5 ) 3 4 1 8 - - 3 4 2 0 . 14 P u r c h a s e , I . F . H . , F. L o n g s t a f f , J. A s h b y , J . A . Styles, D. A n d e r s o n , D.A. L e f e v r e and F.R. W e s t w o o d , E v a l u a t i o n of six s h o r t t e r m tests f o r d e t e c t i n g organic c h e m i c a l c a r c i n o g e n s a n d r e c o m m e n d a t i o n s for t h e i r use, N a t u r e ( L o n d . ) , 2 6 4 ( 1 9 7 6 ) 6 2 4 - - 6 2 7 . 15 R e d d y , B.S., A. M a s t r o m a r i n o a n d E.L. W y n d e r , F u r t h e r leads on m e t a b o l i c e p i d e m i o l o g y of large b o w e l c a n c e r , C a n c e r Res., 3 5 ( 1 9 7 5 ) 3 4 0 3 - - 3 4 0 6 . 16 R e d d y , B.S., T. N a r i s a w a , R. M a r o n p o t , J . H . W e i s b u r g e r a n d E.L. W y n d e r , A n i m a l m o d e l s for t h e s t u d y of d i e t a r y f a c t o r s and c a n c e r of the large b o w e l , C a n c e r Res., 35 ( 1 9 7 5 ) 3 4 2 1 - - 3 4 2 6 . 17 R e d d y , B.S., T. Narisawa, P. Wright, D. V u k u s i c h , J . H . W e i s b u r g e r a n d E.L. W y n d e r , Colon c a r c i n o genesis w i t h a z o x y m e t h a n e and d i m e t h y l h y d r a z i n e in g e r m - f r e e rats, C a n c e r Res., 35 ( 1 9 7 5 ) 2 8 7 - 290. 18 R e d d y , B.S., J . H . Weisburger, T. N a r i s a w a and E.L. W y n d e r , C o l o n c a r c i n o g e n e s i s in g e r m - f r e e rats w i t h 1 , 2 - d i m e t h y l h y d r a z i n e a n d N-methyl-Nr-nitro-N-nitrosoguanidine, C a n c e r Res., 34 ( 1 9 7 4 ) 2 3 6 8 - 2372. 19 Redd~y, B.S., J . H . Weisburgcr and E.L. W y n d e r , E f f e c t of high risk and low risk diets for c o l o n carc i n o g e n e s i s o n fecal m i c r o f l o r a a n d steroids in m a n , J. N u t r . , 1 0 5 ( 1 9 7 5 ) 8 7 6 - - 8 8 4 . 20 R o s e n k r a n z , H.S., A s p e c t s of m i c r o b i o l o g y in c a n c e r r e s e a r c h , A n n . Rev. Microbiol., 27 ( 1 9 7 3 ) 3 8 3 - 401. 21 Slater, E.E., M.D. A n d e r s o n a n d H.S. R o s e n k r a n z , R a p i d d e t e c t i o n of m u t a g e n s and c a r c i n o g e n s , Cancer Res., 31 ( 1 9 7 1 ) 9 7 0 - - 9 7 3 . 22 S p e c k , W.T. a n d H.S. R o s e n k r a n z , Base s u b s t i t u t i o n m u t a t i o n s i n d u c e d in S a l m o n e l l a strains by visible light ( 4 5 0 r a m ) , P h o t o c h e m P h o t o b i o l . , 21 ( 1 9 7 5 ) 3 6 9 - - 3 7 1 . 23 S p e c k , W.T. a n d H.S. R o s e n k r a n z , M u t a g e n i c i t y of a z a t h i o p r i n e , C a n c e r Res., 36 ( 1 9 7 6 ) 1 0 8 - - 1 0 9 . 24 W y n d e r , E.L., N u t r i t i o n a n d C a n c e r , Fed. P r o c . , 35 ( 1 9 7 6 ) 1 3 0 9 - - 1 3 1 5 .
