Activation of aflatoxin B1 by mouse CYP2A enzymes and cytotoxicity in recombinant yeast cells

Abstract

The ability of three highly homologous mouse liver CYP2A enzymes to activate aflatoxin B₁ was studied by expressing them in recombinant AH22 Saccharomyces cerevisiae yeast cells. The reconstituted monooxygenase complex with CYP2A5 purified from yeast cell microsomes produced epoxide at a rate of 17.2 nmol/min per nmol P450 in the presence of 50 μM aflatoxin B₁ while CYP2A4 had about 10% and P4507α only 1.5% of this activity. However, K_m values were 530 and 10 μM and V_max values 12.5 and 14.3 nmol/min per nmol P450 for CYP2A4 and CYP2A5, respectively. When recombinant yeast cells were exposed to aflatoxin B₁ LC₅₀ concentrations were 7.5 ± 5.5 μM for CYP2A4, 0.45 ± 0.10 μM for CYP2A5 and > 320 μM for P4507α expressing yeast cells. Aflatoxin B₁-DNA adduct levels in the same yeast cells were 50, 890 pmol/mg DNA and below detection limit when 3.0 μM aflatoxin B₁ was used in the incubation mixture. Coumarin an inhibitor for CYP2A4 and a substrate for CYP2A5 diminished the toxicity of aflatoxin B₁ in a dose-dependent manner for these recombinant yeast cells. These data demonstrate that (1) highly homologous mouse CYP2A enzymes activate aflatoxin B₁ in a different manner and (2) that recombinant yeast cells expressing mammalian CYP enzymes are a useful and inexpensive system to test the role of different enzymes in aflatoxin B₁ toxicity. The data also indicate that mouse CYP2A5 and its counterpart in other species could have a significant role in aflatoxin B₁ toxicity in organs where it is expressed at high levels.