Effect of inter-individual variability in human liver cytochrome P450 isozymes on cyclophosphamide-induced micronucleus formation

Abstract

We investigated the relationship between metabolic activities of cytochrome P450 (CYP) isozymes present in microsomal fractions derived from the livers of 78 donors and micronucleus induction by cyclophosphamide (CPA). Consequently, a wide inter-individual variation in CYP activities was observed among the 78 donors. The CYP activities were partially correlated with the metabolic phenotypes predicted for the donors based on their single nucleotide polymorphisms. In addition, CPA induced micronucleus formation was seen for 47 out of 52 donors whose samples were tested with CPA doses ranging from 18.8 to 100 μg/mL. The CPA dose at which micronucleated cells were observed varied among the donors. Furthermore, a close correlation was identified between the catalytic activities of the CYP2B6, CYP2C9, CYP2C19, and CYP3A4 isozymes and micronucleus induction by CPA. To elucidate the mechanism underlying CPA-induced micronucleus formation in vitro tests were conducted on expression systems of CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Additionally, the metabolites of CPA generated by the expression systems were quantified by a liquid chromatography tandem mass spectrometer. Interestingly, several metabolites including the 4-hydroxyl form of CPA (4-OH-CPA) and phosphamide mustard were detected in the CYP2B6, CYP2C19, and CYP3A4 expression systems, but not in the CYP2C9 and CYP2D6 system. The presence of these metabolites was correlated with micronucleus induction by CPA. The absence of CPA metabolites in the CYP2C9 expression system might be associated with the lower 4-hydroxylase activity of this system.The present results suggest that inter-individual variability in the metabolic capacity of each donor was associated with potential micronucleus induction due to CPA. Additionally, CPA metabolites like 4-OH-CPA and phosphamide mustard produced by human CYP2B6, CYP2C9, CYP2C19, and CYP3A4 are suggested to be major determinants of micronucleus induction by CPA.