The use of primary rat hepatocytes to achieve metabolic activation of promutagens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase mutational assay.

A method is described in which primary rat hepatocytes have been cocultured with Chinese hamster ovary (CHO) cells to provide metabolic activation of promutagens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fischer-344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum-free medium. The survival and 6-thioguanine-resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B1 (AFB1) 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(A)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB1 was approximately 8 days. Primary cell-mediated mutagenesis may be useful in elucidating metabolic pathways important in the production and detoxification of genotoxic products in vivo.

The effect of 2,4-diaminotoluene and isomers of dinitrotoluene on unscheduled DNA synthesis in primary rat hepatocytes.

The important industrial chemicals 2,4-dinitrotoluene (DNT) and 2,4-diaminotoluene (DAT) are hepatocarcinogens in rats. Technical grade DNT contains approximately 76% 2,4-DNT, 19% 2,6-DNT, and lesser amounts of the other isomers. The ability of 2,4-DAT, technical grade 2,4-DNT, and the purified isomers 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 3,4-DNT and 3,5-DNT to damage the DNA of primary rat hepatocytes was examined. Male Fischer-344 rats were perfused in situ, single cell suspensions were obtained after liver dissociation, and cultures of these cells were treated in the presence of 3H-thymidine. Autoradiography was employed to visualize label incorporation following repair of DNA. At the nontoxic (as judged by cell morphology) doses of 1 x 10(-4) M and below, only 2,4-DAT induced a significant response (ie an average greater than 5 grains net/nucleus). The activity seen with 2,4-DAT suggests that damage to the DNA of the hepatocytes may play a role in its carcinogenic activity and is consistent with the proposal that the induction of DNA repair in primary hepatocytes is of value in predicting the activity of aromatic amino compounds. However, the carcinogenic activity of the dinitrotoluenes was not reflected as DNA repair in the isolated hepatocyte, indicating that additional factors involving the whole animal also play a role in the mechanism of action of DNT.