Chinese hamster V79 fibroblasts, frequently used as target cells in short-term tests for mutagenicity, do not possess measurable monooxygenase activity; in particular, enzymatic oxidation of testosterone (T) cannot be demonstrated. If these V79 cells, however, had been transfected with the cDNA-encoding rat liver cytochrome P-450IIB1 under control of the SV40 early promoter, they stably expressed monooxygenase activity. These so-called SD1 cells then oxidatively metabolized T at a rate of 27 pmol/mg protein/min, converting it to 16 alpha- and 16 beta-hydroxy-T as well as 4-androsten-3,17-dione as sole metabolites in a ratio of 1.1:1.0:1.6. The regio- and stereoselective conversion of T by SD1 cells, as well as the quantitative distribution of the metabolites, corresponds well with the results reported for pure cytochrome P-450IIB1 in a reconstituted system.

Cytochrome P-450 dependent hydroxylation of testosterone was measured in 7-day-old cultures of primary rat liver parenchymal cells. Determinations were carried out in monocultures of parenchymal cells and co-cultures of parenchymal cells with rat liver nonparenchymal epithelial cells, or mouse embryo fibroblasts. In the monoculture system, testosterone metabolism was drastically reduced and hardly measurable after 7 days in culture. In the co-culture systems, individual P-450 isoenzymes were stabilized on different levels. P-450s p and presumably c were well preserved, P-450 a was reduced but clearly measurable, P-450 h was totally lost whereas P-450s b and e were not measurable after 7 days (the activities of these isoenzymes however were already low in freshly isolated parenchymal cells). The results were independent of the cell line used for co-cultivation and of the method of parenchymal cell isolation, that is whether collagenase or EDTA was used as the agent for dissociating the cells from the liver. The results showed that the co-cultivation of liver parenchymal cells with other nonparenchymal cells significantly improved the differentiated status of the former. In this cell culture system however, not every parameter was equally well stabilized.

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The ability of isolated rat liver endothelial and Kupffer cells to activate benzo(a)pyrene (BP), trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (DDBP), trans-1,2-dihydroxy-1,2-dihydrochrysene (DDCH), and aflatoxin B, (AFB%) to mutagenic metabolites was assessed by means of a cell-mediated bacterial mutagenicity assay and compared with the ability of parenchymal cells to activate these compounds. Endothelial and Kupffer cells from untreated rats were able to activate AFB, and DDBP; DDBP was activated even in the absence of an NADPH-generating system. Pretreating the animals with Aroclor 1254 strongly enhanced the mutagenicity of the dihydrodiol, whereas the mutagenicity of AFB, showed a slight increase. BP and DDCH were only activated by endothelial and Kupffer cells isolated from Aroclor 1254-pretreated rats. Parenchymal cells from untreated animals activated all four carcinogens tested; Aroclor 1254 enhanced the parenchymal cell-mediated mutagenicity of BP and DDCH but did not affect that of DDBP and clearly reduced that of AFB,. The reduced mutagenicity of AFB, correlates with the decrease in the amount of 2a-hydroxytestosterone formed when testosterone was incubated with parenchymal cell microsomes from Aroclor 1254-pretreated rats (compared with microsomes from untreated animals): the formation of 2a-hydroxytestosterone is specifically catalyzed by cytochrome P-450h, a hemoprotein thought to be involved in the activation of AFB,. These results show that not only rat liver parenchymal cells, but also endothelial and Kupffer cells, activate several carcinogens to mutagenic metabolites.

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