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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 AFB1 oxidation in human liver microsomes. In the present study, we examine the reaction kinetics of AFB1 oxidation 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 AFB1 oxidation at low substrate concentrations approaching those potentially encountered in the diet. AFBO formation by cDNA-expressed human CYP1A2 followed Michaelis-Menten kinetics (Km = 41 microM, 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 microM, 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 Vmax model 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 AFB1 catalysis and that the average affinity of AFB1 for the two sites was 140-180 microM. Vmax values for AFQ1 formation 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 the in vitro rates 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 microM AFB. The important role of CYP1A2 in the in vitro activation 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 AFB1 to DNA, respectively, indicating that CYP1A2 was responsible for 95% of AFB1-DNA adduct formation at 0.133 microM AFB. These results demonstrate that CYP1A2 dominates the activation of AFB in human liver microsomes in vitro at submicromolar concentrations and support the hypothesis that CYP1A2 is the predominant enzyme responsible for AFBO activation in human liver in vivo at 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 liver in vivo following dietary exposures are uncertain, additional studies in exposed human populations are needed. Quantitative data on the relative rates of AFM1 and AFQ1 excretion (potential biomarkers for CYP1A2 and 3A4 activity, respectively) in humans would be useful to validate the actual contributions of these two enzymes to AFB1 oxidation in vivo.

Although the mechanisms responsible for chemically induced oxidative stress are under intense investigation, little is known about the effects of prooxidant chemicals on the expression of drug-metabolizing enzymes. We examined the effects of diquat (0.1 mmol/kg, ip) and ciprofibrate (0.025% w/w, diet), chemicals which induce oxidative stress via different biochemical mechanisms, on the steady-state messenger RNA (mRNA) levels of six cytochrome P450 enzymes, seven glutathione S-transferase (GST) isoenzymes, UDP-glucuronosyl transferase 1-06 (UGT1*06), gamma-glutamylcysteine synthetase (gamma GCS), NADP(H):quinone oxidoreductase (quinone reductase), Cu/Zn superoxide dismutase (SOD), catalase, and 18S ribosomal RNA in the livers of male Sprague-Dawley rats. Effects of chemical treatments on mRNA levels were compared to changes in catalytic activities for selected enzymes. Ciprofibrate treatment selectively decreased CYP1A2 mRNA expression, whereas both chemicals suppressed CYP3A2 mRNA expression. CYP4A1 mRNA expression and lauric acid hydroxylase activities were induced by ciprofibrate treatment, whereas diquat treatment moderately increased CYP4A1 mRNA levels without affecting lauric acid hydroxylase activities. The steady-state mRNA levels encoding constitutively expressed GST isozymes (Ya1, Ya2, Yb1, Yb2, and Yc1) were decreased by diquat exposure, and the mRNA encoding four of the five constitutively expressed GSTs (Ya1, Ya2, Yb1, and Yc1) were also decreased by ciprofibrate treatment. Nonconstitutively expressed or low constitutively expressed genes (CYP1A1, CYP2B1, CYP2B2, GST Yc2, GST Yf, and UGT1*06) were not induced by exposure to the prooxidants. Changes in isozyme-specific catalytic activities were more consistent with the observed changes in mRNA expression for the GSTs than for the P450s. Both treatments had inhibitory effects on hepatic GSH biosynthesis by decreasing gamma GCS large-subunit mRNA expression, gamma GCS catalytic activities, and hepatic GSH concentrations. Cu/Zn SOD and quinone reductase mRNA levels were increased after ciprofibrate exposure, whereas Cu/Zn SOD mRNA expression was decreased in the diquat-treated animals. The results of this study indicate that diquat and ciprofibrate can decrease the expression profile of a number of phase I, phase II, and antioxidant enzymes and inhibit GSH biosynthesis. These effects may involve the pretranslational loss of hepatic mRNAs, possibly due to accelerated production of reactive oxygen species.