OXIDATIVE-METABOLISM OF AFLATOXIN B-1 BY LIPOXYGENASE PURIFIED FROM HUMAN TERM PLACENTA AND INTRAUTERINE CONCEPTAL TISSUES

Aflatoxin B-1 (AFB(1)) is a teratogen in rodents and may be a human tra nsplacental carcinogen. Although the presence of DNA adducts of AFB(1) in the human placentas has been noted, the enzyme(s) responsible for the bioactivation was not identified. in this investigation, the linoleic acid (LA)-dependent cooxidation of AFB, catalyzed by lipoxygenase (LO) purified by Con A affinity chromatography from the term placentas of nonsmokers was studied. HPLC chromatograms detected the presence of 5- and 15-HETE as the major metabolites and 12-H ETE as a minor metabolite upon incubation of arachidonic acid (AA) with affinity purified human term placental LO. These results svggest that a mixture of LO isozymes is present in the affinity-purified enzyme preparations of term placentas. The optimal essay conditions io observe maximum rate of epoxidation included incubation of 250 mu M AFB(1) with 80 mu g LO and 3.5 mM LA at pH 7.2. AFB(1)-8,9-tris-diol produced in the reaction was estimated specirofluorimetrically. A V-max of 432+/- 26 pmol of AFB(1)-8,9-tris diol produced/min/mg protein and a K-m of 77 mu M for AFB(1) were observed. The AFB,-8,9-tris-diol formation was dependent on the incubation lime, concentration of enzyme protein, AFB(1), and LA. LO catalyzed epoxidation of AFB(1) was inhibited by NDGA, BHT, BHA, ETI, and gossypol. The evidence presented here clearly demonstrates that placental LO is capable of epoxidation of AFB(1). Similar results were observed with LO preparations of human intrauterine conceptal tissues at 8-10 weeks of gestation. (C) 1994 Wiley-Liss, Inc.