Metabolic activation by human arylacetamide deacetylase, CYP2E1, and CYP1A2 causes phenacetin-induced methemoglobinemia
Graphical abstract
Introduction
Phenacetin has been widely used as an analgesic antipyretic. Although phenacetin had been developed as a prodrug of acetaminophen (APAP), it was withdrawn from the market as it caused methemoglobinemia and renal failure [1], [2]. Phenacetin is primarily metabolized to APAP through O-deethylation by human cytochrome (CYP) 1A2 as well as partially by CYP2E1 and is also metabolized to p-phenetidine through a hydrolysis reaction [3], [4], [5]. p-Phenetidine is considered to be further metabolized to N-hydroxy-p-phenetidine, a metabolite thought to contribute adverse effects [1], [6].
Methemoglobin (Met-Hb) is formed when the iron of hemoglobin is oxidized from the ferrous (Fe2+) to the ferric state (Fe3+). Met-Hb cannot bind and transport oxygen; consequently, the increased levels of Met-Hb are associated with clinically severe symptoms [7]. The normal level of Met-Hb is 1.5–2% of the total hemoglobin. As the Met-Hb level rises above 10% of the total hemoglobin levels, cyanosis usually develops. This is followed by anxiety, fatigue, and tachycardia at Met-Hb levels between 20% and 50%. Finally, under conditions of Met-Hb levels exceeding 50–70% of the total hemoglobin, coma and death may occur [8].
The metabolic pathway of phenacetin hydrolysis and subsequent metabolism is thought to contribute to phenacetin-induced methemoglobinemia. However, this involvement has not been experimentally shown. Contributing to this paucity in experimental evidence is the fact that the enzymes responsible for phenacetin-induced methemoglobinemia have not been identified. Previously, we have shown that human arylacetamide deacetylase (AADAC) is the principal enzyme catalyzing the hydrolysis of phenacetin [9]. AADAC is one of the major serine esterases expressed in human liver and the gastrointestinal tract [10]. AADAC is capable of hydrolyzing the antiandrogen drug flutamide, the antituberculosis drug rifampicin [10], [11], and phenacetin. Nakayama and Masuda reported that an NADPH-dependent enzyme(s) is associated with an increase in the formation of Met-Hb formation induced by p-phenetidine [12]. Therefore, representative NADPH-dependent enzymes such as the CYP enzymes may play an important role in the metabolism of p-phenetidine and formation of Met-Hb. CYP enzymes are widely involved in drug metabolism (approximately 75% of all clinically used drugs) and are sometimes involved in the development of toxicity. Building on this background information, we investigated with in vivo and in vitro studies whether AADAC and CYP enzymes are responsible for phenacetin hydrolysis and the subsequent metabolism, respectively, resulting in phenacetin-induced methemoglobinemia.
Section snippets
Chemicals and reagents
APAP, phenacetin, potassium cyanide, potassium hexacyanoferrate (III), p-nitrophenol, and eserine were purchased from Wako Pure Chemical Industries (Osaka, Japan). p-Phenetidine, p-nitrophenyl acetate (PNPA), chlorzoxazone, 6-hydroxychlorzoxazone, and bis-(p-nitrophenyl)phosphate (BNPP) were obtained from Sigma–Aldrich (St. Louis, MO). Tri-o-tolyl phosphate (TOTP) was obtained from Kanto Chemical (Tokyo, Japan). Human liver microsomes (HLM) (pooled donors, n = 50), recombinant human CYP1A2,
Changes of Met-Hb levels upon phenacetin administration in mice and plasma concentrations of phenacetin and its metabolites
After administration of phenacetin (250 mg/kg, p.o.) to male C57BL/6 mice, the levels of Met-Hb and the plasma concentrations of phenacetin, p-phenetidine, and APAP were measured at various time points over 0–6 h (Fig. 1). In preliminary work, we could detect the formation of Met-Hb 1 h after the administration of either 250 mg/kg or 500 mg/kg phenacetin (16.7 ± 2.1% and 32.3 ± 3.5%, respectively), but not following the administration of 100 mg/kg phenacetin (1.7 ± 0.6%). Because high levels of Met-Hb
Discussion
Phenacetin, an analgesic antipyretic, was withdrawn from the market because it caused methemoglobinemia and renal failure [1], [2]. It was suggested that phenacetin-induced methemoglobinemia was associated with hydrolysis and subsequent N-hydroxylation of phenacetin [1], [22]. Previously, our studies showed that AADAC was the principal enzyme catalyzing the hydrolysis of phenacetin [9]. However, it has not been experimentally shown which enzymes play essential roles in phenacetin-induced
Acknowledgement
The Japan Society for the Promotion of Science supported this study [Grant-in-Aid for Young Scientists (B) 21790148].
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