Effect Of Tea Polyphenols On The Soybean Lipoxygenase-mediated Interaction Of Phenothiazines With Benzidine And Other Xenobiotics

[Objectives] This study is to research the effect of natural polyphenol antioxidants such as tea poly phenols on the interaction between xenobiotics mediated by lipoxygenase, and to raise a new mechanism of anti-toxic and anti-cancer effect of this kind of antioxidants by inhibiting lipoxygenase-catalyzed stimulation of xenobiotics oxidation. At the same time, further proof would be provided that lipoxygenase is the alternative pathway of xenobiotics oxidation mediated by cytochrome P450 and other oxidases .[Methods] Under in vitro reaction conditions, soybean lipoxygenase (SLO), and/or modifiers , one or two substrates and H2O2 were added into reaction medium,and followed by recording the different spectrum using spectrophotometer or detecting the free radical intermediate in the reaction medium using electron spin resonance spectrometer (ESR).On the foundation of measuring the oxidative rate of benzidine and other chemicals with the benzene ring under varying conditions, calculating other indexes including enzyme specific activity Jnhibitory rate, 50% inhibitive concentration (ICso) of these antioxidants for the enzyme oxidation .[Results} SLO-generated chlorpromazine cation free radical triggered a rapid oxidation of benzidine to benzidine diimine in the presence of H2O2 .The metabolic interaction resulted in the maximum amplificaton of 24-fold in the rate of benzidine oxidation .The magnitude of stimulation of benzidine oxidation displayed a dependence on the pH of the reaction medium, concentration of the enzyme , clilorpromazine , benzidine and H2O2 .All other phenothiazines were also found to increase benzidine oxidation ,with a lesser degree.The chlorpromazine cation free radical enhanced the oxidation of three other chemicals possessing the benzene ring such as o-dianisidine , p-phenylenediamine and guaiacol tested .Classical lipoxygenase inhibitor gossypol, recluctants reducedIVglutathion(GSH) and dithiothreitol(DTT), free radical scavenger butylated hydroxyanisole (BHA)and butylated hydroxy toluene( BHT), and tea polyphenol and its EGCG monomer ,all exhibited a dose-response relationship with the above enzymatic reaction of oxidation enhancement. Under the optimal oxidation stimulation conditions , EGCG exhibited the lowest value of ICso .which indicated that the inhibitive effect of EGCG was more significant potent than five other modifiers of lipoxygenase activity .In addition to, two species of cation free radical, originated from the two interactive substrates mediated by lipoxygenase, were detected with ESR.[Conclusions) Chlorpromazine and other phenothiazines cation free radical generated by SLO stimulated the oxidation of benzidine and other chemicals with the benzene ring in the presence of HjOi .Tea polyphenols and its EGCG monomer .contrasted by gossypol, DTT, GSH, BHA,and BHT, displayed a more significant inhibitory effect ,which seems to be the concentration-dependent manner in specific range.The mechanism of antioxidation of tea polyphenols and EGCG is very closely related to inhibiting enzyme activity and scavenging free radical and so forth.The inhibitory effect of natural antioxicanrs on lipoxygenanse-mediated co-oxidation and activation of xenobiotics may be one of the mechanisms of their anti-toxic and anti-cancer effect.