Oxidative metabolism of aflatoxin B1 by lipoxygenase purified from human term placenta and intrauterine conceptal tissues.
Aflatoxin B1 (AFB1) is a teratogen in rodents and may be a human transplacental carcinogen. Although the presence of DNA adducts of AFB1 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 AFB1 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-HETE as a minor metabolite upon incubation of arachidonic acid (AA) with affinity purified human term placental LO. These results suggest that a mixture of LO isozymes is present in the affinity-purified enzyme preparations of term placentas. The optimal assay conditions to observe maximum rate of epoxidation included incubation of 250 microM AFB1 with 80 micrograms LO and 3.5 mM LA at pH 7.2. AFB1-8,9-tris-diol produced in the reaction was estimated spectrofluorimetrically. A Vmax of 432 +/- 26 pmol of AFB1-8,9-tris diol produced/min/mg protein and a Km of 77 microM for AFB1 were observed. The AFB1-8,9-tris-diol formation was dependent on the incubation time, concentration of enzyme protein, AFB1, and LA. LO catalyzed epoxidation of AFB1 was inhibited by NDGA, BHT, BHA, ETI, and gossypol. The evidence presented here clearly demonstrates that placental LO is capable of epoxidation of AFB1. Similar results were observed with LO preparations of human intrauterine conceptal tissues at 8-10 weeks of gestation.
Datta
K
K
Florida Toxicology Research Center, Department of Environmental and Occupational Health, College of Public Health, University of South Florida, Tampa 33612.
Kulkarni
A P
AP
D016604:Aflatoxin B1 / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005321:Extraembryonic Membranes / Q000201:enzymology*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005260:Female; D006801:Humans; D008084:Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D010084:Oxidation-Reduction; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011263:Pregnancy Trimester, Third; D013050:Spectrometry, Fluorescence
D005231:Fatty Acids, Unsaturated; D016859:Lipoxygenase Inhibitors; C092991:aflatoxin B1-8,9-dihydrodiol-Tris buffer complex; D016718:Arachidonic Acid; D016604:Aflatoxin B1; D008084:Lipoxygenase