Anthony J. Streeter

The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro

Covalent binding of the reactive metabolite of acetaminophen has been investigated in hepatic microsomal preparations from phenobarbital-pretreated mice. Low molecular weight thiols (cysteine and glutathione) were found to inhibit this binding, whereas several other amino acids which were tested did not. Bovine serum albumin (BSA), which contains a single free sulfhydryl group per molecule and which thus represents a macromolecular thiol compound, inhibited covalent binding of the reactive acetaminophen metabolite to microsomal protein in a concentration-dependent manner. The acetaminophen metabolite also became irreversibly bound to BSA in these experiments, although this binding was reduced by approx. 47% when the thiol function of BSA was selectively blocked prior to incubation. Covalent binding of the acetaminophen metabolite to bovine alpha s1-casein, a soluble protein which does not contain any cysteine residues, was found to occur to an extent of 37% of that which became bound to native BSA. These results were taken to indicate that protein thiol groups are major sites of covalent binding of the reactive metabolite of acetaminophen in vitro. The covalent binding characteristics of synthetic N-acetyl-p-benzoquinoneimine (NAPQI), the putative electrophilic intermediate produced during oxidative metabolism of acetaminophen, paralleled closely those of the reactive species generated metabolically. These findings support the contention that NAPQI is indeed the reactive arylating metabolite of acetaminophen which binds irreversibly to protein.

Structural characterization of the major covalent adduct formed in vitro between acetaminophen and bovine serum albumin

The structure of the covalent adduct formed in vitro between [14C]-acetaminophen ([14C]APAP) and bovine serum albumin (BSA) has been investigated with the aid of new analytical methodology. The APAP-BSA adduct, isolated from mouse liver microsomal incubations to which the radiolabeled drug and BSA had been added, was cleaved using a combination of specific (cyanogen bromide) and non-specific (acid hydrolysis) procedures, following which the mixture of amino acids obtained was derivatized, in aqueous solution, with ethyl chloroformate. The resulting ethoxycarbonyl derivatives were recovered by extraction into ethylacetate, methylated and subjected to profile analysis using both reverse-phase and normal-phase HPLC techniques. In each HPLC step, one major radioactive amino acid adduct was detected and was identified by mass spectrometry as the derivative of 3-cystein-S-yl-4-hydroxyaniline. Based on this finding, and with a knowledge of the behavior under acidic hydrolysis conditions of the 3-cysteinyl conjugate of APAP, it could be concluded that the major APAP-BSA adduct is one in which the drug is bound, via a thioether linkage at the C-3 position, to a sulfhydryl group on the protein. Furthermore, it could be established that this -SH function almost certainly is that associated with the cys-34 residue of BSA.

Covalent binding of acetaminophen to mouse hemoglobin. Identification of major and minor adducts formed in vivo and implications for the nature of the arylating metabolites

When hepatotoxic doses of [ring-U-14C]acetaminophen ([ring-U-14C]APAP) were administered to mice, radioactivity became bound irreversibly to hemoglobin as well as to proteins in the liver and kidney. The covalent binding to hemoglobin was dose-dependent, and in phenobarbital-pretreated mice occurred to the extent of approximately 8% of the corresponding binding to liver proteins. Degradation of the modified globin by acid hydrolysis yielded 3-cystein-S-yl-4-hydroxyacetanilide as the major radioactive product, accounting for approximately 70% of protein-bound drug residues. This finding is consistent with the view that the majority of covalent binding of APAP to proteins is mediated by N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite which preferentially arylates cysteinyl thiol residues. However, after administration of [acetyl-3H]APAP to mice, it was found that approximately 20% of the drug bound to hemoglobin had lost the N-acetyl side-chain, indicating the existence of a second type of APAP-protein adduct. One minor component of the globin hydrolysate was identified as S-(2,5-dihydroxyphenyl)-cysteine, which most likely arises from binding to hemoglobin of p-benzoquinone, a hydrolysis product of NAPQI. The two adducts reported represent the first identified examples of arylating drugs binding to hemoglobin. Experiments on the influence of different cytochrome P-450 inducing agents on the ratio of drug bound to hemoglobin versus hepatic proteins suggested that the reactive metabolites of APAP are formed in the liver and migrate to the erythrocyte, rather than being produced by hemoglobin-catalyzed oxidation of APAP. These findings imply that the reactive metabolites of APAP escape from hepatocytes in some latent forms, which then participate in the arylation of protein thiols in red blood cells and, possibly, at other remote sites.

Cross-linking of protein molecules by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinone imine, and related quinoid compounds.

Acetaminophen (4’-hydroxyacetanilide; 4HAA; Fig. 1) is a widely used analgesic and antipyretic agent which, while considered to be safe at therapeutic dose levels, has been found to cause acute hepatic centrilobular necrosis in both humans and experimental animals when consumed in large doses (Prescott et al., 1971; Boyd and Bereczky, 1966). Evidence from a variety of animal studies (Mitchell et al., 1975; Dahlin et al., 1984) has indicated that cytochrome P-450 plays an important role in the oxidation of acetaminophen to a chemically-reactive and potentially toxic electrophilic metabolite, N-acetyl-p-benzoquinone imine (NAPQI; Fig. 1), which binds covalently to hepatic protein. Recently, we have shown that the major covalent adduct formed between 4HAA and proteins is a 3’-cystein-S-yl conjugate of the drug (Streeter et al., 1984b; Hoffmann et al., 1985a,b). This finding supports the contention that 4HAA is first metabolized to NAPQI, which then arylates proteins by selective reaction with cysteinyl thiol residues.

Ion-pairing systems for separation of N-nitrosodimethylamine and its metabolites in reversed-phase high-performance liquid chromatography

Abstract Separation of N-nitrosodimethylamine and certain of its metabolites on a C18 column is made possible by the inclusion of ion-pairing reagent into a mobile phase consisting of 7 mM ammonium phosphate, pH 3.0. The use of alkanesulfonates (5 mM) of varying alkyl chain length to afford retention of the putative metabolites, monomethylamine and dimethylamine, is discussed. The incorporation of sodium 1-heptane- or 1-octanesulfonate into the mobile phase appears to provide optimal separation under conditions in which the parent nitrosamine is present in the samples at relatively low or high concentrations, respectively.

Denitrosation of N-Nitrosodimethylamine in the Rat in Vivo

The potent hepatocarcinogen N-nitrosodimethylamine (NDMA) has been shown to be metabolized by liver microsomes from acetone-induced rats via two pathways (Figure 1). While the denitrosation pathway has been studied fairly extensively in vitro (Keefer et al., 1987b; Wade et al., 1987; Amelizad et al., 1988), only a few tentative observations about its course in the intact animal have so far appeared in the literature (Heath and Dutton, 1958; Keefer et al., 1987a; Magee et al., 1988). The easiest and most direct assessment of denitrosation in vivo should come from measuring the extent of conversion of NDMA to MA, if the NDMA is labeled with carbon-14 so that the metabolically produced MA can be differentiated from the endogenous material that is known to be present (Davis and de Ropp, 1961).