Role of lipoxygenase in metabolism of hepatocarcinogens

The importance of cytochrome P-450, flavin-containing mono-oxygenase and prostaglandin synthetase in biotransformation of xenobiotics is widely accepted. The presence of cytochrome P-450 in liver adequately explains hepatocarcinogenesis due to certain chemicals. However, extrahepatic tissues where cytochrome P-450 and flavin-containing monooxygenase occur only to a negligible extent have also been shown to be susceptible to toxicity induced by these chemicals (e.g. 2-aminofluorene). Interestingly, certain chemicals (e.g. benzidine) inspite of being potent hepatocarcinogens, are not metabolized by liver cytochrome P-450. The inhibitory effect of polyunsaturated fatty acids on the mixed function oxidase catalyzed oxidation of benzo(a)pyrene and certain food carcinogens (e.g. aflatoxin B{dollar}sb1{dollar}), has raised questions regarding the relative importance of hepatic cytochrome P-450 in vivo. Thus, the identity of pathway(s) responsible for the bioactivation of these xenobiotics remains unclear and a need for further research exists. The goal of this study was to test the hypothesis that lipoxygenase plays a significant role in the metabolism of hepatocarcinogens in the liver.; Co-oxidation of 2-aminofluorene, a putative hepatocarcinogen and teratogen, was studied using soybean lipoxygenase as a model. The reaction displayed a K{dollar}sb{lcub}rm m{rcub}{dollar} of 0.29 mM and specific activity of 521.7 nmol/min/nmol of enzyme. Significant covalent binding to protein and calf thymus DNA clearly suggested bioactivation of 2-aminofluorene. The reaction was blocked by lipoxygenase inhibitors. Subcellular distribution of the dioxygenase activity of the rat liver lipoxygenase was investigated. The oxygen uptake data suggested that the dioxygenase activity primarily resides in the cytosol. Cytosolic dioxygenase activity in the presence of 3.5 mM linoleic acid at pH 7.2 was 74.07 {dollar}pm{dollar} 1.43 nmoles/min/mg protein. Benzidine was oxidized at the rate of 3.18 {dollar}pm{dollar} 0.13 nmoles/min/mg cytosolic protein to benzidine diimine. Partially purified preparations of rat liver lipoxygenase, free of hemoglobin, exhibited a dioxygenase activity of 223.1 {dollar}pm{dollar} 65.9 nmoles/min/mg protein and hydroperoxidase activity of 6.1 {dollar}pm{dollar} 0.5 nmoles/min/mg protein towards benzidine. Benzidine was oxidized by partially purified human liver lipoxygenase from cytosol by affinity chromatography using concanavalin A-sepharose Cl-4B. A marked stimulation of partially purified lipoxygenase by CaCl{dollar}sb2,{dollar} ATP, H{dollar}sb2{dollar}O{dollar}sb2{dollar} and KCl was observed. Xenobiotics viz. N,N,N{dollar}spprime{dollar},N{dollar}spprime{dollar}-tetramethyl-p-phenylenediamine, 3,3{dollar}spprime,{dollar}5,5{dollar}spprime{dollar}-tetramethylbenzidine, o-dianisidine, pyrogallol and 2,2{dollar}spprime{dollar}-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) were also co-oxidized. Aflatoxin B{dollar}sb1{dollar} was co-oxidized to the reactive metabolite aflatoxin B{dollar}sb1{dollar} 8,9-epoxide as evidenced by Tris-diol formation. In a separate study, the ability of lipoxygenase in rat embryos was evaluated for xenobiotic co-oxidation. Significant dioxygenase activity (7.5 {dollar}mu{dollar}mol/min/mg protein) towards linoleic acid was found in rat embryos in different developmental stages on day 9 and 10 of gestation. Benzidine, N,N,N{dollar}spprime{dollar},N{dollar}spprime{dollar}-tetramethyl-p-phenylenediamine, 3,3{dollar}spprime{dollar},5,5{dollar}spprime{dollar}-tetramethylbenzidine and o-dianisidine, were oxidized at significant rate. All the co-oxidation reactions in this study were inhibited by {dollar}mu{dollar}molar concentrations of lipoxygenase inhibitors suggesting clearly the involvement of lipoxygenase.