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Demonstration of the clastogenic and SCE-inducting capacity of indirect-acting mutagens in mammalian cell lines with specific metabolizing competence
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up to 0.85 mM or in the presence of N-OH-2-acetylaminofluorene or benzidine plus $9 fraction (under conditions for the positive hepatocyte UDS). Colon cells in suspension, exposed to M N N G (up to 3 mM) for 2 h also showed tittle evidence of UDS but did exhibit some S-phase cells which were not observed in culture. At very high M N N G levels (3 mM) only low amounts of UDS were seen in non-repricating rat colon cells obtained by partial pronase digestion [2]. Under conditions in which hepatocytes show extensive D N A repair, colon mucosal cells exhibit little or unmeasurable UDS even at much higher dose levels of M N N G and suggest a relatively low capacity to repair. The results are in accord with minimal D N A repair in intestinal cells in comparison to hepatocytes of fish [3]. [1] Furihata, C., and T. Matsushima (1986) CRC Crit. Rev. Toxicol., 17, 245. [2] Mori, H. et al. (1984) J. Toxicol. Sci., 9, 23. [3] Walton, D.G. et al. (1984) Cancer Res., 44, 1121.
71 Salassidis, K., U. Kulka, E. Schmid, D. Paul a and M. Bauchinger, GSF Inst. fiir Strahlenbiologie, D-8042 Neuherberg and 1 Fraunhofer Inst. fiar Aerosolforschung, D-3000 Hannover (F.R.G.)
mosome preparations were made with standard procedures and stained with FPG. In rat hepatocytes the incidence of CA was significantly increased, compared to untreated controls, by AFB 1 (10-fold), B(a)P (3-fold) and DMBA (2.5-fold). In mouse hepatocytes a significant clastogenic effect was observed for B(a)P (20-fold) and DMBA (3fold). A doubling of SCE frequencies, accepted as a positive response, was observed in the rat cell line for AFB a and CP and in the mouse line for AFB t only. In experiments with V79 Chinese hamster cells lacking P-450 activity biotransformation of the test substances indicated by the induction of CA and SCE could be only achieved in the presence of a metabolically active $9 mix system. The results suggest the presence of liverspecific cytochrome P-450-dependent monooxygenases in both cell lines which provide metaboric activation of indirect-acting mutagens. The variable response of the cell lines to the treatment with the 4 structurally different promutagens may be an indication of the expression of different forms of cytochrome P-450 and of variable competing activating and inactivating mechanisms.
72 Glatt, H.R., S. Dogra, C. Janssens, J. Doehmer and F. Oesch, Institute of Toxicology, University of Mainz, D-6500 Mainz (F.R.G.)
Demonstration of the clastogenic and SCE-inducing capacity of indirect-acting mutagens in mammalian cell lines with specific metabolizing competence
Transfection and stable expression of genes encoding xenobiotic-metabolizing enzymes in V79 cells
Cell tines of polyoma-transformed fetal rat hepatocytes and fetal mouse hepatocytes were used as a test system for the induction of chromosome aberrations (CA) and sister-chromatid exchanges (SCE) by indirect-acting carcinogens. As test substances requiring metabolic activation by different forms of cytochrome P-450-dependent monooxygenases to express cytogenetac effects, aflatoxin B t (AFBt), cyclophosphamide (CP), benzo[a]pyrene (B(a)P) and 7,12-dimethylbenz[a]anthracene (DMBA) were used. Exponentially growing cells were treated for 25-29 h with the test substances and Colcemid for the final 3 h. Chro-
V79 Chinese hamster cells are widely used for mutagenicity testing, but have the serious limitation that they do not express cytochromes P-450, which are required for the activation of many promutagens. Full-length c D N A clones encoding the cytochromes P-450IA1, P-450IA2 and P450IIB1 under the control of the SV40 early promoter were constructed and co-introduced with the selection marker neomycin phosphotransferase into V79 cells. Selected clonal cell lines were examined for the presence of the corresponding cytochrome P-450 cDNA, RNA and protein, using Southern, Northern and Western blotting tech-
up to 0.85 mM or in the presence of N-OH-2-acetylaminofluorene or benzidine plus $9 fraction (under conditions for the positive hepatocyte UDS). Colon cells in suspension, exposed to M N N G (up to 3 mM) for 2 h also showed tittle evidence of UDS but did exhibit some S-phase cells which were not observed in culture. At very high M N N G levels (3 mM) only low amounts of UDS were seen in non-repricating rat colon cells obtained by partial pronase digestion [2]. Under conditions in which hepatocytes show extensive D N A repair, colon mucosal cells exhibit little or unmeasurable UDS even at much higher dose levels of M N N G and suggest a relatively low capacity to repair. The results are in accord with minimal D N A repair in intestinal cells in comparison to hepatocytes of fish [3]. [1] Furihata, C., and T. Matsushima (1986) CRC Crit. Rev. Toxicol., 17, 245. [2] Mori, H. et al. (1984) J. Toxicol. Sci., 9, 23. [3] Walton, D.G. et al. (1984) Cancer Res., 44, 1121.
71 Salassidis, K., U. Kulka, E. Schmid, D. Paul a and M. Bauchinger, GSF Inst. fiir Strahlenbiologie, D-8042 Neuherberg and 1 Fraunhofer Inst. fiar Aerosolforschung, D-3000 Hannover (F.R.G.)
mosome preparations were made with standard procedures and stained with FPG. In rat hepatocytes the incidence of CA was significantly increased, compared to untreated controls, by AFB 1 (10-fold), B(a)P (3-fold) and DMBA (2.5-fold). In mouse hepatocytes a significant clastogenic effect was observed for B(a)P (20-fold) and DMBA (3fold). A doubling of SCE frequencies, accepted as a positive response, was observed in the rat cell line for AFB a and CP and in the mouse line for AFB t only. In experiments with V79 Chinese hamster cells lacking P-450 activity biotransformation of the test substances indicated by the induction of CA and SCE could be only achieved in the presence of a metabolically active $9 mix system. The results suggest the presence of liverspecific cytochrome P-450-dependent monooxygenases in both cell lines which provide metaboric activation of indirect-acting mutagens. The variable response of the cell lines to the treatment with the 4 structurally different promutagens may be an indication of the expression of different forms of cytochrome P-450 and of variable competing activating and inactivating mechanisms.
72 Glatt, H.R., S. Dogra, C. Janssens, J. Doehmer and F. Oesch, Institute of Toxicology, University of Mainz, D-6500 Mainz (F.R.G.)
Demonstration of the clastogenic and SCE-inducing capacity of indirect-acting mutagens in mammalian cell lines with specific metabolizing competence
Transfection and stable expression of genes encoding xenobiotic-metabolizing enzymes in V79 cells
Cell tines of polyoma-transformed fetal rat hepatocytes and fetal mouse hepatocytes were used as a test system for the induction of chromosome aberrations (CA) and sister-chromatid exchanges (SCE) by indirect-acting carcinogens. As test substances requiring metabolic activation by different forms of cytochrome P-450-dependent monooxygenases to express cytogenetac effects, aflatoxin B t (AFBt), cyclophosphamide (CP), benzo[a]pyrene (B(a)P) and 7,12-dimethylbenz[a]anthracene (DMBA) were used. Exponentially growing cells were treated for 25-29 h with the test substances and Colcemid for the final 3 h. Chro-
V79 Chinese hamster cells are widely used for mutagenicity testing, but have the serious limitation that they do not express cytochromes P-450, which are required for the activation of many promutagens. Full-length c D N A clones encoding the cytochromes P-450IA1, P-450IA2 and P450IIB1 under the control of the SV40 early promoter were constructed and co-introduced with the selection marker neomycin phosphotransferase into V79 cells. Selected clonal cell lines were examined for the presence of the corresponding cytochrome P-450 cDNA, RNA and protein, using Southern, Northern and Western blotting tech-