Acetaminophen and analogs as cosubstrates and inhibitors of prostaglandin H synthase.

Previous studies have shown that acetaminophen (APAP) is converted by prostaglandin H synthase (PGHS) to both one-electron oxidized products and the two-electron oxidized product, N-acetyl-p-benzoquinone imine (NAPQI). The present study further characterizes this reaction and shows that relatively low concentrations (20-200 microM) of APAP stimulate PGHS activity in ram seminal vesicle microsomes, whereas high concentrations (greater than 10 mM) inhibit the conversion of arachidonic acid (AA) to 15-hydroperoxy-9,11-peroxidoprosta-5,13-dienoic acid (PGG2). Stimulatory and inhibitory activities apparently involve the reduction of oxidized complexes of PGHS, and stimulatory and inhibitory activities roughly correlate with the electrochemical half-wave oxidation potentials of a series of hydroxyacetanilides. Using APAP as a probe, it was found that at low concentrations, APAP is converted in a cooxidation reaction with arachidonic acid to a dimer, 4'4"'-dihydroxy-3', 3"'-biacetanilide (bi-APAP), and other polymeric products. Moreover, an electrophilic metabolite of acetaminophen, NAPQI, was detected directly and also detected indirectly by its reaction with glutathione (GSH) to form 3'-(S-glutathionyl)acetaminophen (GS-APAP). The formation of all products was inhibited by indomethacin and the reductants, ascorbic acid and butylated hydroxyanisole (BHA). However, in the presence of GSH, ascorbic acid only partially inhibited the formation of GS-APAP while almost completely inhibiting the formation of bi-APAP. The same products of APAP (bi-APAP and NAPQI) were formed by PGHS and hydrogen peroxide in reactions that were not inhibited by indomethacin. At high concentrations of APAP that inhibit PGHS, the formation of products in the presence of arachidonic acid but not H2O2 was inhibited. These findings are generally consistent with a mechanism of acetaminophen oxidation by PGHS that involves common intermediate enzyme forms for both cyclooxygenase- and hydroperoxidase-catalyzed reactions. At least one of the intermediate complexes is reduced by relatively low concentrations of APAP and stimulates PGHS, whereas another intermediate complex is reduced by APAP at higher concentrations to inhibit the enzyme.