Activation of aflatoxin B1 by mouse CYP2A enzymes and cytotoxicity in recombinant yeast cells.
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 microM aflatoxin B1 while CYP2A4 had about 10% and P4507 alpha only 1.5% of this activity. However, Km values were 530 and 10 microM and Vmax 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 microM for CYP2A4, 0.45 +/- 0.10 microM for CYP2A5 and > 320 microM for P4507 alpha 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 microM 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.
Pelkonen
P
P
Department of Pharmacology and Toxicology, University of Kuopio, Finland.
Lang
M A
MA
Wild
C P
CP
Negishi
M
M
Juvonen
R O
RO
D016604:Aflatoxin B1 / Q000096:biosynthesis / Q000493:pharmacokinetics* / Q000633:toxicity*; D000818:Animals; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D018736:DNA Adducts / Q000096:biosynthesis; D004271:DNA, Fungal / Q000187:drug effects / Q000378:metabolism; D004852:Epoxy Compounds / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D010084:Oxidation-Reduction; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics
D018736:DNA Adducts; D004271:DNA, Fungal; D004852:Epoxy Compounds; D007527:Isoenzymes; C072611:aflatoxin B1-DNA adduct; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1