The Kinetics of Aflatoxin B1Oxidation by Human cDNA-Expressed and Human Liver Microsomal Cytochromes P450 1A2 and 3A4

Abstract

The combined presence of CYP1A2 and 3A4, both of which oxidize aflatoxin B1(AFB1) to the reactive aflatoxin B1-8,9-epoxide (AFBO) and to hydroxylated inactivation products aflatoxin M1(AFM1) and aflatoxin Q1(AFQ1), substantially complicates the kinetic analysis of AFB1oxidation in human liver microsomes. In the present study, we examine the reaction kinetics of AFB1oxidation in human liver microsomes (HLMs,N= 3) and in human CYP3A4 and CYP1A2 cDNA-expressed lymphoblastoid microsomes for the purpose of identifying the CYP isoform(s) responsible for AFB1oxidation at low substrate concentrations approaching those potentially encountered in the diet. AFBO formation by cDNA-expressed human CYP1A2 followed Michaelis–Menten kinetics (Km= 41 μM, Vmax= 2.63 nmol/min/nmol P450). Furthermore, the portion of AFBO formed in HLMs which was eliminated by furafylline, a specific mechanism-based inhibitor of CYP1A2, also followed Michaelis–Menten kinetics (Km= 32-47 μM, Vmax= 0.36-0.69 nmol/min/nmol P450). The formation of AFBO (activation product) and AFQ1(detoxification product) in cDNA-expressed human CYP3A4 microsomes was sigmoidal and consistent with the kinetics of substrate activation. Accordingly, application of a sigmoid Vmaxmodel equivalent to the Hill equation produced excellent fits to the cDNA-expressed CYP3A4 data and also to the data from HLMs pretreated with furafylline to remove CYP1A2. The Hill model predicted that two substrate binding sites are involved in CYP3A4-mediated AFB1catalysis and that the average affinity of AFB1for the two sites was 140–180 μM. Vmaxvalues for AFQ1formation were 10-fold greater than those for AFBO, and total substrate turnover to both was 67 nmol/min/nmol CYP3A4. Using the derived kinetic parameters for CYP1A2 and 3A4 to model thein vitrorates of AFB activation at low substrate concentrations, it was predicted that CYP1A2 contributes to over 95% of AFB activation in human liver microsomes at 0.1 μMAFB. The important role of CYP1A2 in thein vitroactivation of AFB at low substrate concentrations was supported by DNA binding studies. AFB1–DNA binding in control HLMs (reflecting the contribution of CYP1A2 and CYP3A4) and furafylline-pretreated microsomes (reflecting the contribution of CYP3A4 only) catalyzed the binding of 1.71 and 0.085 pmol equivalents of AFB1to DNA, respectively, indicating that CYP1A2 was responsible for 95% of AFB1–DNA adduct formation at 0.133 μMAFB. These results demonstrate that CYP1A2 dominates the activation of AFB in human liver microsomesin vitroat submicromolar concentrations and support the hypothesis that CYP1A2 is the predominant enzyme responsible for AFBO activation in human liverin vivoat the relatively low dietary concentrations encountered in the human diet, even in high AFB exposure regions of the world. However, because the actual concentrations of AFB in liverin vivofollowing dietary exposures are uncertain, additional studies in exposed human populations are needed. Quantitative data on the relative rates of AFM1and AFQ1excretion (potential biomarkers for CYP1A2 and 3A4 activity, respectively) in humans would be useful to validate the actual contributions of these two enzymes to AFB1oxidationin vivo.