The use of transgenic cell lines in mutagenicity testing

The availability of cell lines stably expressing cytochrome P450s, has provided the possibility to study the metabolism of compounds by an individual cytochrome P450 as well as cytochrome P450-mediated (pro)mutagen activation. This may lead to a better understanding of a compound's mutagenicity in the target species, and to improved preselection of compounds that still have to be tested in laboratory animals, ideally resulting in a reduction of animal experimentation. NIH/3T3 cell lines expressing cytochrome P450 cDNAs were obtained by using retroviral infection. In addition, a shuttle vector was constructed, carrying the bacterial beta-galactosidase (lacZ) gene as a reporter gene for mutations. This construct was introduced into the cytochrome P450 expressing cell lines, and after exposure of the cell lines to (pro)mutagens, mutagenicity was assessed by determining the functionality of the lacZ gene by complementation in lacZ-deficient bacteria. The mutation frequency of ethylmethanesulphonate increased in all the cell lines tested and was not different from the parental NIH/3T3 cells, demonstrating that expression of human CYP enzymes had no influence on the sensitivity of the cells towards mutagens. The mutation frequency of the naturally occurring mycotoxin, ochratoxin A, was increased only in cell lines expressing human CYP1A1, CYP1A2, CYP2C10 and CYP3A4, but not in vector-transfected NIH-3T3 cells nor in CYP2D6- and CYP2E1-expressing cells. The system described is sensitive and versatile and could be a valuable tool in the assessment of the relevance of human metabolism and the bioactivation of mutagenic compounds.