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Development of Human Cytochrome P450-expressing Cell Lines: Application in Mutagenicity Testing of Ochratoxin A

Research Institute of Toxicology [E. M. d. G., I. G. A. M. H., W. S., G. J. H.], Department of Veterinary Pharmacy, Pharmacology, and Toxicology [M. J. B., J. F. G.], Utrecht University, Yalelaan 2, P.O. Box 80.176, NL-3508 TD Utrecht, the Netherlands

With the aim to assess the involvement of distinct forms of cytochrome P450 enzymes in the activation of procarcinogens, we have developed by means of retroviral infection a series of NIH/3T3 cell lines stably expressing human CYP1A1, CYP1A2, CYP2C10, CYP2D6, and CYP2E1 cDNA. The levels of cytochrome P450 enzyme activities were determined using specific substrates. An increase in specific catalytic activity could be observed in all cell lines compared to background activity in vector-infected cells. Furthermore, we developed a test system in which we are able to combine P450-expressing cells with a shuttle vector containing the lacZ' gene, which serves as a reporter gene for mutations.

Using this system, we investigated the cytotoxicity and mutagenicity of the mycotoxin ochratoxin A. Natural occurrence of ochratoxin A in food commodities has been linked to an increased incidence of urinary tract tumors in certain geographic regions. Although biotransformation seems to play a crucial role in ochratoxin A toxicity, the possible contribution of metabolites to genotoxicity and carcinogenicity remained unelucidated.

We have demonstrated that the mutation frequency of ochratoxin A was increased dependent upon concentration in NIH/3T3 cell lines, stably expressing human CYP1A1, CYP1A2, CYP2C10, and CYP3A4. In contrast, neither in vector-infected NIH/3T3 cells nor in CYP2D6- and CYP2E1-expressing cells was an increase of mutation frequency observed.

¹ To whom requests for reprints should be addressed. Phone: 31-30-2535400; Fax: 31-30-2535077.

Received 8/ 1/95. Accepted 11/ 8/95.

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