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DNA repair test supplemented with β-glucosidase
Cancer Letters, 12 (1981) 329-333 o Elsevier/North-Holland Scientific Publishers Ltd.
329
GENOTOXICITY OF CYCASIN IN THE HEPATOCYTE PRIMARY CULTURE/DNA REPAIR TEST SUPPLEMENTED WITH @GLUCOSIDASE
GARY M WILLIAMSa,*,
MICHAEL F. LASPIAa, HIDEKI MORIa and IWAO HIRONOb
aDivision of Experimental Pathology, Naylor Dana Institute for Disease Prevention, American Health Foundation, Dana Road, Valhalla, New York 10595 (U.S.A.) and bDepartment of Carcinogenesis and Cancer Susceptibility, Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-l Minato-Ku, Tokyo 108 (Japan) (Received 7 January 1981) (Accepted 26 January 1981)
SUMMARY
The genotoxicity of cycasin was examined in the standard hepatocyte primary culture (HPC)/DNA repair test and in the test supplemented with /3-glucosidase. Generally, no DNA repair was elicited by cycasin in the standard test except for one assay which showed a strong response. With the addition of /3-glucosidase to the test medium, cycasin elicited DNA repair with clear dependence on both dose and amount of /3-glucosidase. These results indicate that supplementation of the HPC/DNA repair test with the appropriate glucosidase should be useful in detecting potentially genotoxic glucosides and suggests that supplementation with other specific enzymes could compensate for extrahepatic biotransformation processes required prior to final activation by hepatocytes.
INTRODUCTION
The hepatocyte primary culture (HPC)/DNA repair test is a short term test for chemical carcinogens, which detects DNA damage by the production of autoradiographic DNA repair in freshly isolated, non-replicating hepatocytes [ 14-161. Since liver has the ability to metabolize all carcinogens known to require metabolic activation, except those that need a prior step of bacterial metabolism [ 131, the test, in principle, can detect all carcinogens activated to DNA damaging moieties except those dependent upon bacterial metabolism. Intestinal microflora are active in the metabolism of a variety of xeno*To whom reprint requests should be addressed.
330
biotics [lo], including chemical carcinogens [ 11,121. Indeed, bacterial metabolism appears to be essential to the carcinogenicity of cycasin in adult animals. Cycasin is the naturally occurring @-D-glucoside of the proximate carcinogen methylazoxymethanol (MAM). It has induced tumors in the liver, kidney and large intestine when fed to conventional animals [ 41, but did not induce tumors in germ-free animals [3]. The aglycone of cycasin, MAM, produced tumors in the liver, kidney and colon when injected i.p. in both conventional and germ-free animals [ 561. It is thought that cycasin is hydrolyzed to the carcinogenic aglycone by p-D-glucosidase produced by the intestinal microflora. Recently, cycasin, which was previously reported to be negative [9] in the Ames Salmonella/microsome test [ 11, was demonstrated by Matsushima et al. [ 71 to be mutagenic in a modified Ames test [ 201, in which p-glucosidase was added to the pre-incubation mixture. However, the response varied greatly among the 6 strains tested with only 1 strain giving strongly positive results. In the present study, we examined the genotoxicity of cycasin in the HPC/DNA repair test. Cycasin was generally negative in the standard HPC/ DNA repair test, but the addition of /3-glucosidase together with cycasin in the test medium consistently resulted in a strong positive response. Thus, the addition of specific bacterial enzymes to the .HPC/DNA repair test extends its capability to the detection of carcinogens normally thought of as requiring metabolism in vivo through biotransformation by intestinal flora. MATERIALS AND METHODS
Chemicals Purified cycasin was kindly provided by Dr. A. Kobayashi, Laboratory of Biochemistry and Nutritional Chemistry, Faculty of Agriculture, Kagoshima University, Japan. /3-Glucosidase (EC 3.2.1.21) from almonds (6.6 units/mg) was obtained from Sigma Chemical Company, St. Louis, MO. HPCIDNA repair test As described in detail [ 191, hepatocytes were isolated from the livers of adult Fischer strain rats (150-300 g) by procedures that yielded 200 X lo6 cells/100 g body wt that were greater than 85% viable. The isolated hepatocytes were allowed 2 h to attach on plastic coverslips in Williams’ medium E (WME) plus 10% calf serum after which the cultures were washed and exposed to the test compound and [3H]TdR (10 pCi/ml; 40-60 Ci/mmol) in WME for 20 h according to the standard protocol for this test [17,18]. Stock solutions of cycasin and p-glucosidase were prepared in DMSO and WME, respectively. A range of logarithmic doses was tested . which was narrowed to half log doses in successive experiments.
331
At the end of incubation, the cultures were washed, fixed in acetic acid, ethanol, mounted on slides, dipped in NTB-2 photographic emulsion and stored for 7 days at 4°C. Autoradiographs were then developed and stained in hematoxylin. Autoradiographic grains were counted on an Artek 880 counter with microscopic attachment. The highest cytoplasmic background count for each cell was subtracted from the nuclear count to obtain the net nuclear grains due to DNA repair synthesis. Counts on 12 cells were made or until significant counts were obtained to give a coefficient of variation of 0.05. The data are expressed as the mean + standard deviation of the average net nuclear grain counts from triplicate coverslips. A result is considered positive when the mean + standard deviation of average net nuclear grain counts exceed 5 grains/nucleus in replicate experiments [ 151. RESULTS
Cycasin was negative in 3 assays using the standard HPC/DNA repair test. In 1 assay, however, a strong DNA repair response (i.e. 31.7 + 6.6 grains/ nucleus) occurred, which could result from a preparation with the capability to metabolize cycasin, either through endogenous activity or by contamination with bacteria. These results, in general, therefore correspond with the limited capability of adult liver for activating cycasin [ 81. To further evaluate the genotoxicity of cycasin, it was tested in the HPC/DNA repair test supplemented with P-glucosidase. Cycasin, with the TABLE 1 GENOTOXICITY OF CYCASIN IN THE HPC/DNA REPAIR SUPPLEMENTED WITH D-GLUCOSIDASE Cycasin (M)
Grains/nucleu.? _
0 5 x 10-3 10-3 5 x lo-’ 1o-4 10-s 0.1% DMSO Control
-
p-Glucosidase (units/ml)
0.4 0.1 0.5 0.5 0.2
0.75 f f f * f 0
0.3 0.1 0.6 0.6 0.3
52.1 17.8 0.4 0.1 0.7
1.5 f f * f f
7.5 6.6 0.1 0.1 0.4
44.7 37.0 1.2 0.2 0.1
f f * f *
2.3 5.6 1.0 0.4 0.1
3.0
7.5
15.0
Toxicb 60.5 f 12.7 12.1 f 1.4 6.4 f 1.1 0.1 f 0.1
Toxicb 85.5 * 73.8 30.4 f 6.9 11.4 f 6.0 0.1 * 0.1
Toxicb 30.5 * 18.7 31.3 * 4.1 19.7 f 6.4 0.9 f 0.9
II Mean * S.D. of triplicate coverslips exposed to cycasin and fl-glucosidase plus [ 3H]TdR for 20 h. b No cells remaining attached to coverslip.
332
addition p-glucosidase to the test medium elicited a strong positive response (Table 1) which was confirmed in repeat experiments. Although p-glucosidase has maximum activity at pH 5.0 [ 21, it was not necessary to alter the pH of the test medium from 7.35 to obtain a strong response. The amount of DNA repair was dose-dependent (Table 1). Moreover, the amount of DNA repair induced at a given dose was proportionate to the concentration of /3-glucosidase in the test medium reaching a maximum with 7.5 units/ml p-glucosidase at, 10m3 M cycasin. DISCUSSION
The present results have demonstrated that while cycasin is generally negative in the HPC/DNA repair test, addition of /3-glucosidaseto the culture medium results in a genotoxic effect which is related to both the dose of cycasin and the concentration of @glucosidase. These results suggest that supplementation of the culture medium with the appropriate glucosidase should be useful in detecting potentially genotoxic glucosides. The single positive response induced by cycasin in the standard HPC/ DNA repair test is of some interest. Animal studies have indicated that the intestinal microflora are essential in the activation in vivo of cycasin [ 3,4], but Beck and Toppel [2] found that rat liver does possess intracellular p-glucosidase which is bound to lysomal membranes. Therefore, it is possible that under some conditions rat liver can endogenously metabolize cycasin to the genotoxic proximate carcinogen MAM. With the addition of p-glucosidase to the standard HPC/DNA repair test to simulate glucoside hydrolysis of cycasin by intestinal flora, the HPC/ DNA repair test proved to be a sensitive indicator of the carcinogenicity of cycasin in vivo. This finding in support of that of Matsushima et al. [ 71 clearly indicates that media supplementation with specific enzymes, including those of bacterial origin, has an important role in the development of systems in vitro for detecting the broadest possible spectrum of agents requiring biotransformation for their mutagenic/carcinogenic activity. ACKNOWLEDGEMENTS
We thank Mrs. Linda Stempel for preparing the manuscript. This work was supported by contract NOl-CP-55705 from the National Cancer Institute. REFERENCES
1 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens
and mutagens with the SaZmoneZla/mammalian microsome mutagenicity test. Mutat. Res., 31, 347-364. 2 Beck, C. and Tappel, A.L. (1968) Rat-liver lysosomal Pglucosidase: A membrane enzyme. Biochim. Biophys. Acta, 151,159-164.
333 3 Laqueur, G.L. (1964) Carcinogenic effects of cycad meal and cycasin, methylazoxymethanol glycoside, in rats and effects of cycasin in germfree rats. Fed. Proc., 23, 1386-1388. 4 Laqueur, G.L. (1965) The induction of intestinal neoplasms in rats with the glycoside cycasin and its aglycone. Virchows Arch. A. 340,151-163. 5 Laqueur, G.L. and Matsumoto, H. (1966) Neoplasms in female Fischer rats following intraperitoneal injection of methylazoxymethanol. J. Natl. Cancer Inst., 37, 217232. 6 Laqueur, G.L., McDanile, E.G. and Matsumoto, H. (1967) Tumor induction in germfree rats with methylazoxymethanol (MAM) and synthetic MAM acetate. J. Natl. Cancer Inst., 39,355-371. 7 Matsushima, T., Matsumoto, H., Shirai, A., Sawamura, M. and Sugimura, T. (1979) Mutagenicity of the naturally occurring carcinogen cycasin and synthetic methylaxoxymethanol conjugates in Salmonella typhimurium. Cancer Res., 39, 3780-3782. 8 Matsumoto, H., Nagata, Y., Nishimura, E.T., Bristol, R. and Haber, M. (1972) p-Glucosidase modulation in preweanling rats and its association with tumor induction by cycasin. J. Natl. Cancer Inst., 49, 423-434. 9 McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. ROC. Natl. Acad. Sci. U.S.A., 72, 5135-5139. 10 Scheline, R.R. (1973) Metabolism of foreign compounds by gastrointestinal microorganisms. Pharmacol. Rev., 26,451-523. 11 Weisburger, J.H., Grantham, P.H., Horton, R.E. and Weisburger, E.K. (1970) Metabolism of the carcinogen N-hydroxy-N-2-fluorenyl-a&amide in germ-free rats. Biochem. Pharmacol., 19,151-162. 12 Wheeler, L.A., Soderberg, F.B. and Goldman, P. (1975) The reduction of N-hydroxy4acetylaminobiphenyl by the intestinal microflora of the rat. Cancer Res., 35,29622968. 13 Weisburger, J.H. and Williams, GM. (1981) Metabolism of chemical carcinogens. In: Cancer, a comprehensive treatise, 2nd edn. Vol. I. Editor: F.F. Becker. Plenum Press, New York (in press). 14 Williams, G.M. (1976) Carcinogen-induced DNA repair in primary rat liver cell cultures: a possible screen for chemical carcinogens. Cancer Letters, 1, 231-236. 15 Williams, G.M. (1977) Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res., 37, 1845-1851. 16 Williams, G.M. Further improvements in the hepatocyte primary culture DNA repair test for carcinogens: detection of carcinogenic biphenyl derivatives. Cancer Letters, 4,69-75. 17 Williams, G.M. (1980) The detection of chemical mutagens/carcinogens by DNA repair and mutagenesis in liver cultures. In: Chemical Mutagens, Vol. VI, pp. 6179. Editors: F.J. DeSerres and A. Hollaender. Plenum Press, New York. 18 Williams, GM. (1980) Liver culture indicators for the detection of chemical carcinogens. In: Short Term Tests for Chemical Carcinogens, pp. 581-609. Editors: R.H.C. San and H.F. Stich. Springer-Verlag, NY. 19 Williams, GM., Bermudez, E. and Scaramuzzino, D. (1977) Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro, 13,809-817. 20 Yahagi, T., Nagao, M., Seino, Y., Matsushima, T., Sugimura, T. and Okada, M. (1977) Mutagenicity of N-nitrosamine on Salmonella. Mutat. Res., 48, 121-130.
329
GENOTOXICITY OF CYCASIN IN THE HEPATOCYTE PRIMARY CULTURE/DNA REPAIR TEST SUPPLEMENTED WITH @GLUCOSIDASE
GARY M WILLIAMSa,*,
MICHAEL F. LASPIAa, HIDEKI MORIa and IWAO HIRONOb
aDivision of Experimental Pathology, Naylor Dana Institute for Disease Prevention, American Health Foundation, Dana Road, Valhalla, New York 10595 (U.S.A.) and bDepartment of Carcinogenesis and Cancer Susceptibility, Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-l Minato-Ku, Tokyo 108 (Japan) (Received 7 January 1981) (Accepted 26 January 1981)
SUMMARY
The genotoxicity of cycasin was examined in the standard hepatocyte primary culture (HPC)/DNA repair test and in the test supplemented with /3-glucosidase. Generally, no DNA repair was elicited by cycasin in the standard test except for one assay which showed a strong response. With the addition of /3-glucosidase to the test medium, cycasin elicited DNA repair with clear dependence on both dose and amount of /3-glucosidase. These results indicate that supplementation of the HPC/DNA repair test with the appropriate glucosidase should be useful in detecting potentially genotoxic glucosides and suggests that supplementation with other specific enzymes could compensate for extrahepatic biotransformation processes required prior to final activation by hepatocytes.
INTRODUCTION
The hepatocyte primary culture (HPC)/DNA repair test is a short term test for chemical carcinogens, which detects DNA damage by the production of autoradiographic DNA repair in freshly isolated, non-replicating hepatocytes [ 14-161. Since liver has the ability to metabolize all carcinogens known to require metabolic activation, except those that need a prior step of bacterial metabolism [ 131, the test, in principle, can detect all carcinogens activated to DNA damaging moieties except those dependent upon bacterial metabolism. Intestinal microflora are active in the metabolism of a variety of xeno*To whom reprint requests should be addressed.
330
biotics [lo], including chemical carcinogens [ 11,121. Indeed, bacterial metabolism appears to be essential to the carcinogenicity of cycasin in adult animals. Cycasin is the naturally occurring @-D-glucoside of the proximate carcinogen methylazoxymethanol (MAM). It has induced tumors in the liver, kidney and large intestine when fed to conventional animals [ 41, but did not induce tumors in germ-free animals [3]. The aglycone of cycasin, MAM, produced tumors in the liver, kidney and colon when injected i.p. in both conventional and germ-free animals [ 561. It is thought that cycasin is hydrolyzed to the carcinogenic aglycone by p-D-glucosidase produced by the intestinal microflora. Recently, cycasin, which was previously reported to be negative [9] in the Ames Salmonella/microsome test [ 11, was demonstrated by Matsushima et al. [ 71 to be mutagenic in a modified Ames test [ 201, in which p-glucosidase was added to the pre-incubation mixture. However, the response varied greatly among the 6 strains tested with only 1 strain giving strongly positive results. In the present study, we examined the genotoxicity of cycasin in the HPC/DNA repair test. Cycasin was generally negative in the standard HPC/ DNA repair test, but the addition of /3-glucosidase together with cycasin in the test medium consistently resulted in a strong positive response. Thus, the addition of specific bacterial enzymes to the .HPC/DNA repair test extends its capability to the detection of carcinogens normally thought of as requiring metabolism in vivo through biotransformation by intestinal flora. MATERIALS AND METHODS
Chemicals Purified cycasin was kindly provided by Dr. A. Kobayashi, Laboratory of Biochemistry and Nutritional Chemistry, Faculty of Agriculture, Kagoshima University, Japan. /3-Glucosidase (EC 3.2.1.21) from almonds (6.6 units/mg) was obtained from Sigma Chemical Company, St. Louis, MO. HPCIDNA repair test As described in detail [ 191, hepatocytes were isolated from the livers of adult Fischer strain rats (150-300 g) by procedures that yielded 200 X lo6 cells/100 g body wt that were greater than 85% viable. The isolated hepatocytes were allowed 2 h to attach on plastic coverslips in Williams’ medium E (WME) plus 10% calf serum after which the cultures were washed and exposed to the test compound and [3H]TdR (10 pCi/ml; 40-60 Ci/mmol) in WME for 20 h according to the standard protocol for this test [17,18]. Stock solutions of cycasin and p-glucosidase were prepared in DMSO and WME, respectively. A range of logarithmic doses was tested . which was narrowed to half log doses in successive experiments.
331
At the end of incubation, the cultures were washed, fixed in acetic acid, ethanol, mounted on slides, dipped in NTB-2 photographic emulsion and stored for 7 days at 4°C. Autoradiographs were then developed and stained in hematoxylin. Autoradiographic grains were counted on an Artek 880 counter with microscopic attachment. The highest cytoplasmic background count for each cell was subtracted from the nuclear count to obtain the net nuclear grains due to DNA repair synthesis. Counts on 12 cells were made or until significant counts were obtained to give a coefficient of variation of 0.05. The data are expressed as the mean + standard deviation of the average net nuclear grain counts from triplicate coverslips. A result is considered positive when the mean + standard deviation of average net nuclear grain counts exceed 5 grains/nucleus in replicate experiments [ 151. RESULTS
Cycasin was negative in 3 assays using the standard HPC/DNA repair test. In 1 assay, however, a strong DNA repair response (i.e. 31.7 + 6.6 grains/ nucleus) occurred, which could result from a preparation with the capability to metabolize cycasin, either through endogenous activity or by contamination with bacteria. These results, in general, therefore correspond with the limited capability of adult liver for activating cycasin [ 81. To further evaluate the genotoxicity of cycasin, it was tested in the HPC/DNA repair test supplemented with P-glucosidase. Cycasin, with the TABLE 1 GENOTOXICITY OF CYCASIN IN THE HPC/DNA REPAIR SUPPLEMENTED WITH D-GLUCOSIDASE Cycasin (M)
Grains/nucleu.? _
0 5 x 10-3 10-3 5 x lo-’ 1o-4 10-s 0.1% DMSO Control
-
p-Glucosidase (units/ml)
0.4 0.1 0.5 0.5 0.2
0.75 f f f * f 0
0.3 0.1 0.6 0.6 0.3
52.1 17.8 0.4 0.1 0.7
1.5 f f * f f
7.5 6.6 0.1 0.1 0.4
44.7 37.0 1.2 0.2 0.1
f f * f *
2.3 5.6 1.0 0.4 0.1
3.0
7.5
15.0
Toxicb 60.5 f 12.7 12.1 f 1.4 6.4 f 1.1 0.1 f 0.1
Toxicb 85.5 * 73.8 30.4 f 6.9 11.4 f 6.0 0.1 * 0.1
Toxicb 30.5 * 18.7 31.3 * 4.1 19.7 f 6.4 0.9 f 0.9
II Mean * S.D. of triplicate coverslips exposed to cycasin and fl-glucosidase plus [ 3H]TdR for 20 h. b No cells remaining attached to coverslip.
332
addition p-glucosidase to the test medium elicited a strong positive response (Table 1) which was confirmed in repeat experiments. Although p-glucosidase has maximum activity at pH 5.0 [ 21, it was not necessary to alter the pH of the test medium from 7.35 to obtain a strong response. The amount of DNA repair was dose-dependent (Table 1). Moreover, the amount of DNA repair induced at a given dose was proportionate to the concentration of /3-glucosidase in the test medium reaching a maximum with 7.5 units/ml p-glucosidase at, 10m3 M cycasin. DISCUSSION
The present results have demonstrated that while cycasin is generally negative in the HPC/DNA repair test, addition of /3-glucosidaseto the culture medium results in a genotoxic effect which is related to both the dose of cycasin and the concentration of @glucosidase. These results suggest that supplementation of the culture medium with the appropriate glucosidase should be useful in detecting potentially genotoxic glucosides. The single positive response induced by cycasin in the standard HPC/ DNA repair test is of some interest. Animal studies have indicated that the intestinal microflora are essential in the activation in vivo of cycasin [ 3,4], but Beck and Toppel [2] found that rat liver does possess intracellular p-glucosidase which is bound to lysomal membranes. Therefore, it is possible that under some conditions rat liver can endogenously metabolize cycasin to the genotoxic proximate carcinogen MAM. With the addition of p-glucosidase to the standard HPC/DNA repair test to simulate glucoside hydrolysis of cycasin by intestinal flora, the HPC/ DNA repair test proved to be a sensitive indicator of the carcinogenicity of cycasin in vivo. This finding in support of that of Matsushima et al. [ 71 clearly indicates that media supplementation with specific enzymes, including those of bacterial origin, has an important role in the development of systems in vitro for detecting the broadest possible spectrum of agents requiring biotransformation for their mutagenic/carcinogenic activity. ACKNOWLEDGEMENTS
We thank Mrs. Linda Stempel for preparing the manuscript. This work was supported by contract NOl-CP-55705 from the National Cancer Institute. REFERENCES
1 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens
and mutagens with the SaZmoneZla/mammalian microsome mutagenicity test. Mutat. Res., 31, 347-364. 2 Beck, C. and Tappel, A.L. (1968) Rat-liver lysosomal Pglucosidase: A membrane enzyme. Biochim. Biophys. Acta, 151,159-164.
333 3 Laqueur, G.L. (1964) Carcinogenic effects of cycad meal and cycasin, methylazoxymethanol glycoside, in rats and effects of cycasin in germfree rats. Fed. Proc., 23, 1386-1388. 4 Laqueur, G.L. (1965) The induction of intestinal neoplasms in rats with the glycoside cycasin and its aglycone. Virchows Arch. A. 340,151-163. 5 Laqueur, G.L. and Matsumoto, H. (1966) Neoplasms in female Fischer rats following intraperitoneal injection of methylazoxymethanol. J. Natl. Cancer Inst., 37, 217232. 6 Laqueur, G.L., McDanile, E.G. and Matsumoto, H. (1967) Tumor induction in germfree rats with methylazoxymethanol (MAM) and synthetic MAM acetate. J. Natl. Cancer Inst., 39,355-371. 7 Matsushima, T., Matsumoto, H., Shirai, A., Sawamura, M. and Sugimura, T. (1979) Mutagenicity of the naturally occurring carcinogen cycasin and synthetic methylaxoxymethanol conjugates in Salmonella typhimurium. Cancer Res., 39, 3780-3782. 8 Matsumoto, H., Nagata, Y., Nishimura, E.T., Bristol, R. and Haber, M. (1972) p-Glucosidase modulation in preweanling rats and its association with tumor induction by cycasin. J. Natl. Cancer Inst., 49, 423-434. 9 McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. ROC. Natl. Acad. Sci. U.S.A., 72, 5135-5139. 10 Scheline, R.R. (1973) Metabolism of foreign compounds by gastrointestinal microorganisms. Pharmacol. Rev., 26,451-523. 11 Weisburger, J.H., Grantham, P.H., Horton, R.E. and Weisburger, E.K. (1970) Metabolism of the carcinogen N-hydroxy-N-2-fluorenyl-a&amide in germ-free rats. Biochem. Pharmacol., 19,151-162. 12 Wheeler, L.A., Soderberg, F.B. and Goldman, P. (1975) The reduction of N-hydroxy4acetylaminobiphenyl by the intestinal microflora of the rat. Cancer Res., 35,29622968. 13 Weisburger, J.H. and Williams, GM. (1981) Metabolism of chemical carcinogens. In: Cancer, a comprehensive treatise, 2nd edn. Vol. I. Editor: F.F. Becker. Plenum Press, New York (in press). 14 Williams, G.M. (1976) Carcinogen-induced DNA repair in primary rat liver cell cultures: a possible screen for chemical carcinogens. Cancer Letters, 1, 231-236. 15 Williams, G.M. (1977) Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res., 37, 1845-1851. 16 Williams, G.M. Further improvements in the hepatocyte primary culture DNA repair test for carcinogens: detection of carcinogenic biphenyl derivatives. Cancer Letters, 4,69-75. 17 Williams, G.M. (1980) The detection of chemical mutagens/carcinogens by DNA repair and mutagenesis in liver cultures. In: Chemical Mutagens, Vol. VI, pp. 6179. Editors: F.J. DeSerres and A. Hollaender. Plenum Press, New York. 18 Williams, GM. (1980) Liver culture indicators for the detection of chemical carcinogens. In: Short Term Tests for Chemical Carcinogens, pp. 581-609. Editors: R.H.C. San and H.F. Stich. Springer-Verlag, NY. 19 Williams, GM., Bermudez, E. and Scaramuzzino, D. (1977) Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro, 13,809-817. 20 Yahagi, T., Nagao, M., Seino, Y., Matsushima, T., Sugimura, T. and Okada, M. (1977) Mutagenicity of N-nitrosamine on Salmonella. Mutat. Res., 48, 121-130.