Activation and Metabolism of P-Nitrosophenoland 2,2'-Thiobis-Ethanol by Horse Liver and Human Alcohol Dehydrogenase: Toxicological Implications.

Abstract

C-nitroso compounds are reactive intermediates in the toxic sequelae of nitroarene metabolism. Alcohol dehydrogenase (ADH) contributes significantly to NADH-dependent C-nitroso reduction by cytosol relative to NAD(P)H: quinone oxidoreductase (NQOR). NADPH-dependent pNSP reduction by liver cytosol of ADH+ animals is mostly dicumarol-sensitive which implicates NQOR as the major NADPH-dependent activity. Although, extensive structural homology between horse and different isozymes of human ADH exist, significant differences occur within the active site, which account for great variability among substrate specificities, kinetic constants, and response to inhibitors. We have studied the metabolic activity of horse liver alcohol dehydrogenase (HLADH), purified class I alphaalpha, beta 1beta1 and beta2beta2, class II pi, class III chi and class IV sigma human isozymes, and human liver cytosol (HLC) towards the C-nitroso substrate pNSP. Significant differences in the reaction rates towards pNSP were observed between HLADH and the human isozymes. The relative order of the rates of pNSP reduction by ADH was HLADH > alphaalpha > pi > sigma > chi > beta2beta2 > beta1beta 1. pNSP reduction catalyzed by HLADH, purified class I alphaalpha, beta1beta1, and beta2beta 2, class III chi and class IV sigma human isozymes show pH dependence with maxima greater than or equal to pH 6. This pH dependence seemingly reflects a protein moiety involved in the proton relay system. Thiodiglycol (TDG) can undergo oxidation catalyzed by ADH. We have compared the catalytic activity of purified human liver class I alphaalpha, beta 1beta1, beta2beta2 and gamma 1gamma1 ADH, class II pi ADH, class III chi ADH, and class IV sigma ADH with respect to TDG oxidation. Specific activities with respect to TDG were 123, 79, 347, 647, and 12 nmol/min/mg for the alphaalpha, beta 1beta1, beta2beta2, gamma 1gamma1, and pi ADH, respectively. Class III chi ADH did not exhibit activity with this substrate. The specific activity of class IV sigma ADH was estimated at about 1632 nmol/min/mg. The activities of class IV sigma and class I gamma1 ADH are of significant interest because of their prevalence in the eyes, lungs, stomach and skin, all potential target organs of sulfur mustard toxicity.