0090-9556/97/2502-0196-0105$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 2

Co-oxidative Metabolism of 4-Aminobiphenyl by Lipoxygenase from Soybean and Human Term Placenta

Kaushik Datta, Paul M. Sherblom, and Arun P. Kulkarni

Florida Toxicology Research Center, Department of Environmental and Occupational Health, College of Public Health, University of South Florida

}4-aminobiphenyl (4-ABP) co-oxidation catalyzed by the human term placental lipoxygenase (HTPLO), purified by affinity chromatography, was studied in the presence of linoleic acid (LA). Soybean lipoxygenase (SLO) which is extensively employed as a model lipoxygenase, was used for comparison. Spectral analyses of reaction media containing 4-ABP, LA, and SLO/HTPLO suggested the disappearance of substrate (A at 270 nm) and a gradual appearance of a new peak at 315 nm, indicating a metabolite formation. Under optimal assay conditions, SLO exhibited a specific activity of 350 nmoles of 4-ABP depleted/min/nmole of enzyme. To observe the maximal rate of co-oxidation by the HTPLO (45 nmoles of 4-ABP depleted/min/mg protein), an incubation of 50 µM 4-ABP, 2 mM LA, and 80 µg/ml protein at pH 7.4 was essential. 4-ABP was also oxidized by SLO in the presence of H₂O₂, although at a lower rate. The reversed-phase HPLC analysis of organic extracts of the incubations of 4-ABP with SLO and H₂O₂/LA as well as HTPLO and LA showed the formation of a major peak which was identified by GC-MS as 4,4-azobis(biphenyl). The addition of GSH, BHT, and BHA to the enzymatic incubations decreased the formation of 4-ABP metabolite, suggesting the generation of a free radical as the initial metabolite during 4-ABP oxidation. Both the SLO and HTPLO mediated reactions were significantly inhibited by nordihydroguaiaretic acid, gossypol, and 5,8,11-eicosatriynoic acid. Collectively, these results suggest that the co-oxidation catalyzed by HTPLO may be the underlying biochemical mechanism responsible for the transplacental toxicity of 4-ABP.