cDNA-expressed human cytochrome P450 isozymes. Inactivation by porphyrinogenic xenobiotics

Abstract

A number of xenobiotics are known to exert their porphyrinogenic effects in rodents and chick embryos through mechanism-based inactivation of certain cytochrome P450 (P450) isozymes. To facilitate the extrapolation of results from test animals to humans, we have assessed the ability of three prototype porphyrinogenic compounds-namely, 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (DDEP), 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), and allylisopropylacetamide (AIA)-to cause mechanism-based inactivation of cDNA-expressed human P450s 1A1, 1A2, 2C9-Arg144 (2C9), 2D6-Val374 (2D6), and 3A4 in microsomes from human lymphoblastoid cell lines (Gentest Corp., Woburn, MA). The following catalytic markers of human P450 isozymes were used: ethoxyresorufin O-deethylase (P450s 1A1 and 1A2), diclofenac 4-hydroxylation (P4502C9), dextromethorphan O-demethylase (P4502D6), and testosterone 6 beta-hydroxylation (P4503A4). We found that DDEP and TTMS caused mechanism-based inactivation of cDNA-expressed human P450s 1A1, 1A2, and 3A4, whereas only DDEP was able to cause mechanism-based inactivation of cDNA-expressed human P4502C9; neither xenobiotic caused mechanism-based inactivation of cDNA-expressed human P4502D6. A comparison of the human P450 isozyme data with results previously obtained in rat and chick embryo liver showed a close correspondence between the results obtained with P450s 1A and 3A, but not the P4502C subfamily. Because several rat isozymes (P450s 2A1, 2B1, 2C6, 2C11, and 3A1) undergo inactivation by AIA, it was noteworthy that AIA did not inactivate any of the cDNA-expressed human P450 isozymes. Because mechanism-based inactivation of P450 isozymes is related to the porphyrinogenicity of xenobiotics, our results demonstrate the importance of supplementing studies of mechanism-based inactivation of P450 isozymes in animal models with similar studies on cDNA-expressed human P450 isozymes.