Phenacetin is well known to cause
hemolytic anemia and
methemoglobinemia in humans. Early mechanistic studies clearly established a causal role for active/reactive
drug metabolites in the process but did not unequivocally identify these metabolite(s) or resolve the question of whether these two hemotoxicities are mechanistically linked. As part of ongoing studies on the mechanism underlying arylamine-induced hemotoxicities, we have recently shown that the arylhydroxylamine metabolites of
aniline and
dapsone mediate the hemolytic activity of
aniline and
dapsone, respectively. The present study was undertaken to determine if
N-hydroxyphenetidine (PNOH), the known arylhydroxylamine metabolite of
phenacetin, is responsible for
phenacetin-induced
hemolytic anemia. As measured by decreased survival of 51Cr-labeled erythrocytes in rats,
phenacetin, p-
phenetidine, and PNOH were all hemolytic in vivo, with PNOH being significantly the most potent of the three. In vitro exposure of 51Cr-tagged erythrocytes to PNOH, followed by transfusion into isologous rats, resulted in a concentration-dependent reduction in erythrocyte survival, indicating that PNOH is a direct-acting hemolytic agent.
Phenacetin and p-
phenetidine were inactive.
Phenacetin, p-
phenetidine, and PNOH all produced dose-dependent
methemoglobinemia in rats. In parallel in vitro studies, PNOH elevated
methemoglobin levels, p-
phenetidine and
phenacetin did not. However, attempts to identify PNOH in the blood of
phenacetin- and p-
phenetidine-treated rats were unsuccessful, despite the use of a highly sensitive analytical method. Hemotoxic concentrations of PNOH were found to be highly unstable in the presence of red cells, though relatively stable in the
buffer vehicle alone. Inhibitors of acetylation (
p-aminobenzoic acid [
PABA]) and deacetylation (
bis-[p-nitrophenyl]phosphate [BNPP]), used to alter the cyclic interconversion of
phenacetin and p-
phenetidine, caused changes in
phenacetin hemotoxicity that indicated the
hemotoxin was a deacetylated metabolite distal to p-
phenetidine. These data are consistent with the hypothesis that PNOH, formed during the metabolic clearance of
phenacetin, mediates
phenacetin-induced
hemolytic anemia and
methemoglobinemia through direct
toxic actions in the erythrocyte.