Oxidation of Acetaminophen to Its Toxic Quinone Imine and Nontoxic Catechol Metabolites by Baculovirus-Expressed and Purified Human Cytochromes P450 2E1 and 2A6

Abstract

Acetaminophen (APAP), a widely used analgesic and antipyretic agent, is bioactivated by cytochromes P450 to cause severe hepatotoxicity. APAP is oxidized by two pathways to form a toxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI), and a nontoxic catechol metabolite, 3-hydroxy-APAP (3-OH-APAP). We investigated the role of P450 2E1 and 2A6 in APAP oxidation by using baculovirus-expressed and highly purified forms of human P450 2E1 and 2A6. An electrochemical HPLC assay was developed to quantify both oxidative metabolites simultaneously. For the first time, it was demonstrated that human P450 2E1 selectively oxidized APAP to NAPQI (assayed as its glutathione conjugate, GS-APAP), whereas human P450 2A6 selectively oxidized APAP to 3-OH-APAP. At 1 mM APAP, the relative ratio for the formation of GS-APAP vs 3-OH-APAP with human P450 2E1 was approximately 6:1, whereas the ratio with human P450 2A6 was 1:3. Apparent Km and Vmax values for the formation of GS-APAP by human P450 2E1 were 1.3 mM and 6.9 nmol/min/nmol of P450, respectively, whereas they were 4.6 mM and 7.9 nmol/min/nmol of P450 for P450 2A6. Apparent Km and Vmax values for the formation of 3-OH-APAP by human P450 2E1 were 4.0 mM and 2.5 nmol/min/nmol of P450, respectively, whereas they were 2.2 mM and 14.2 nmol/min/nmol of P450, respectively, for P450 2A6. Thus, although at toxic doses of APAP P450 2E1 is the more efficient catalyst for the formation of the toxic metabolite NAPQI, P450 2A6 also can contribute significantly to NAPQI production.

Advantages of in vitro cytotoxicity testing by using primary rat hepatocytes in comparison with established cell lines.

“…With regard to acetaminophen, the incubation time of 24 hr may be too short to reflect the 2-4 days' delay that occurs in the production of clinic liver injury by acetaminophen. At a toxic dose, CYP2E1 is found to be the most efficient catalyst for the bioactivation of acetaminophen to NAPQI (N-acetyl-p-benzoquinone imine) (Chen et al, 1998), which causes depletion of GSH and protein thiols and arylation of the latter, but in rat hepatocytes the contents of CYP2E1 are relatively lower. Furthermore, the significant formation of non-toxic metabolite 3-hydroxyacetaminophen in rats by various cytochrome P450 enzymes involved in the oxidation of acetaminophen may have an important role for the lower sensitivity of rat hepatocytes compared to that of the mouse (To and Wells, 1985;Harvison et al,1986;Patten et al,1993;Zand et al, 1998).…”

Methemoglobin—It's not just blue: A concise review

“…The 5-hydroxycompounds can be spontaneously oxidized to the quinine-imine, probably producing hydrogen peroxide [83]. This reaction is analogous to the formation of toxic metabolites form acetaminophen [84]. But which is the more important in the production of hemolytic anemia and methemoglobinemia in vivo is not known, nor is the mechanism of hemoglobin oxidation by the phenolic compound.…”

Investigation of a Hepatotoxicity Screening System in Primary Cell Cultures-"What Biomarkers Would Need to Be Addressed to Estimate Toxicity in Conventional and New Approaches?"-

“…Therefore, we can evaluate hepatotoxicity in an environment closely resembling an in vivo system by utilizing a primary cell culture system (Paillard et al, 1999;Fry et al, 1990;Wang et al, 2002). Acetaminophen, amiodarone and carbon tetrachloride have been reported to induce hepatotoxicity via their active metabolites after transformation by CYP (Holme et al, 1984;Chen W. et al, 1998;Hart et al, 1982;Patten et al, 1993;Albano et al, 1985;Mitchell et al, 1973;Somani et al, 1990;Recknagel et al, 1989), and we were able to detect and quantify the toxicity of the 4 reference compounds including active metabolite inducing toxicity because hepatocytes functions were still intact. It has been reported that CYP activities last for about 24-48 hr in a monolayer primary culture system (Begue et al, 1984).…”