Initial intraorgan formation of mercapturic acid.

The disposition of S-benzyl-glutathione (BSG) in male Wistar rats was evaluated by the HPLC method to examine whether the kidney and liver contributed independently to the biosynthesis of S-benzyl-N-acetylcysteine (BNAc), a mercapturic acid (Chart 1). After intravenous injection, BSG was rapidly transported in both the kidney and the liver at a ratio of about 7:3. Simultaneously, a large amount of BNAc was found in both the kidney and the liver. In the kidney, S-benzyl-cysteine (BCys) reached a maximum concentration (Cmax) at 2 min after BSG injection, whereas BNAc reached Cmax within 3 to 5 min. The generation of BNAc was also observed in the liver. While renal BNAc reached Cmax within 3 to 5 min, hepatic BNAc reached Cmax around 5 min after BSG injection. Moreover, the elimination half-life of the BNAc after intravenous injection of the BSG was equivalent to that observed after intravenous injection of the BNAc itself. These results demonstrate that the kidney contributes to the initial intraorgan generation of BNAc and that this mercapturic acid is also synthesized in the liver and preferentially excreted into urine.     

Increased hepatic efflux of glutathione after chronic ethanol feeding

Chronic ethanol feeding increases hepatotoxicity of drugs, such as acetaminophen, which form electrophilic metabolites. Availability of glutathione (GSH) is important in preventing liver damage from reactive metabolites. Chronic ethanol feeding has been reported to increase turnover of hepatic GSH in rats. The results of the present study show that the total hepatic efflux of GSH was increased from 5.95 +/- 0.42 nmoles/min/g liver (control) to 9.96 +/- 0.57 nmoles/min/g (P less than 0.001) in isolated perfused livers from rats 24 hr after withdrawal from chronic ethanol feeding. The increase in total efflux of GSH was due to a significant increase in sinusoidal GSH efflux from 4.76 +/- 0.49 nmoles/min/g liver in control rats to 9.07 +/- 0.47 nmoles/min/g (P less than 0.001) in ethanol-fed rats, while biliary efflux decreased slightly, 1.20 +/- 0.11 (control) vs 0.89 +/- 0.31 (ethanol). The increase in cellular efflux of GSH was similar in magnitude to the increase in hepatic GSH turnover that we reported previously. Biliary GSSG was similar in both groups of animals. Hepatic GGT activity was increased slightly, but not significantly, whereas renal GGT activity was similar in ethanol-fed rats. Hepatic GSH and GSSG levels were also similar. The increase in turnover of hepatic GSH in rats withdrawn from chronic ethanol feeding was most likely due to increased cellular efflux of GSH. This finding suggests that chronic ethanol feeding may increase cellular requirements for GSH, although the mechanism remains unknown. This alteration in GSH turnover may have important consequences for detoxification of xenobiotics or their metabolites by the liver.     

Glutathione conjugation and mercapturic acid formation in the developing rat, in vivo and in vitro.

1. The levels of GSH-S-epoxidetransferase (GSH-S-transferase E, EC 2.5.1.18), gamma-glutamyl transpeptidase (EC 2.3.2.2) and S-substituted cysteine N-acetyltransferase have been measured in the liver and kidney of neonatal to adult rats. 2. GSH-S-epoxidetransferase and S-substituted cysteine N-acetyltransferase activities were less than 10% of the adult values in neonatal rats, rising gradually to reach adult values at about 40 days of age. Renal gamma-glutamyl transpeptidase activity was 27% of the adult value 2 days after birth and increased after 15 days reaching adult levels by 40 days. 3. The percentages of the doses of 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) and of 1,2-epoxybutane, administered at the same dose level to rats aged 4 days to adult, excreted as the corresponding mercapturic acids in 24 h, were not significantly different. 4. Adult and 10 day old rats doses at the same dose level with ENPP excreted N-acetyl-S-[2-hydroxy-3-(p-nitrophenoxy)propyl]-L-cysteine (ENPP-MA) at the same rate. 5. In addition to ENPP-MA, dosed rats under 13 days of age excreted the corresponding substituted cysteine. 6. The correlation between results in vitro and in vivo is discussed.     

1,2-Dichloropropane: investigation of the mechanism of mercapturic acid formation in the rat

1. Three mercapturic acid metabolites were identified in the urine of male and female Fischer 344 rats given 1,2-dichloropropane (DCP) orally (100 mg/kg) or by inhalation exposure (100 ppm, 6 h). 2. These compounds (N-acetyl-S-(2-hydroxypropyl)-L-cysteine, N-acetyl-S-(2-oxopropyl)-L-cysteine and N-acetyl-S-(1-carboxyethyl)-L-cysteine) were isolated from the urine following acidification and extraction with ethyl acetate. The extracts were derivatized with diazomethane and N,O-bis(trimethylsilyl)trifluoroacetamide and analysed by chemical ionization g.l.c.-mass spectrometry. 3. Further mechanistic studies were carried out with the stable isotope-labelled analogue, D6-DCP (105 mg/kg, orally). Analysis of the resulting mass spectra indicated retention of primarily three deuterium atoms in the 2-hydroxypropyl-mercapturic acid formed from D6-DCP. Similar isotope retention was observed for the 2-oxopropyl-mercapturic acid metabolite. 4. These results do not support a sulphonium ion intermediate in the formation of the 2-hydroxypropyl-mercapturic acid metabolite of DCP. Instead, this metabolite is thought to arise via direct oxidation of DCP, either prior to or following conjugation with glutathione.     

1,2-Dichloropropane: investigation of the mechanism of mercapturic acid formation in the rat

1. Three mercapturic acid metabolites were identified in the urine of male and female Fischer 344 rats given 1,2-dichloropropane (DCP) orally (100mg/kg) or by inhalation exposure (100 ppm, 6h).

2. These compounds (N-acetyl-S-(2-hydroxypropyl)-L-cysteine, N-acetyl-S-(2-oxopropyl)-L-cysteine and N-acetyl-S-(1-carboxyethyl)-L-cysteine) were isolated from the urine following acidification and extraction with ethyl acetate. The extracts were derivatized with diazomethane and N,O-bis(trimethylsilyl)trifluoroacetamide and analysed by chemical ionization g.l.c.-mass spectrometry.

3. Further mechanistic studies were carried out with the stable isotope-labelled analogue, D₆-DCP (105mg/kg, orally). Analysis of the resulting mass spectra indicated retention of primarily three deuterium atoms in the 2-hydroxypropyl-mercapturic acid formed from D₆-DCP. Similar isotope retention was observed for the 2-oxopropyl-mercapturic acid metabolite.

4. These results do not support a sulphonium ion intermediate in the formation of the 2-hydroxypropyl-mercapturic acid metabolite of DCP. Instead, this metabolite is thought to arise via direct oxidation of DCP, either prior to or following conjugation with glutathione.     

Caffeic acid, chlorogenic acid, and dihydrocaffeic acid metabolism: glutathione conjugate formation.

The antioxidant properties of the dietary dihydroxycinnamic acids [caffeic (CA), dihydrocaffeic (DHCA), and chlorogenic (CGA) acids] have been well studied but little is known about their metabolism. In this article, evidence is presented showing that CA, DHCA, and CGA form quinoids and hydroxylated products when oxidized by peroxidase/H(2)O(2) or tyrosinase/O(2). Mass spectrometry analyses of the metabolites formed with peroxidase/H(2)O(2)/glutathione (GSH) revealed that mono- and bi-glutathione conjugates were formed for all three compounds except CGA, which formed a bi-glutathione conjugate only when GSH was present. In contrast, the metabolism of the dihydroxycinnamic acids by tyrosinase/O(2)/GSH resulted in the formation of only mono-glutathione conjugates. In the absence of GSH, hydroxylated products and p-quinones of CA or CGA were formed by peroxidase/H(2)O(2). DHCA formed a hydroxylated adduct (even though GSH was present), as well as the corresponding p-quinone and dihydroesculetin, an intramolecular cyclization product. NADPH also supported rat liver microsomal-catalyzed CA-, CGA-, and DHCA-glutathione conjugate formation, which was prevented by benzylimidazole, a cytochrome P450 inhibitor. Furthermore, the cytotoxicity of CA, CGA, and DHCA toward isolated rat hepatocytes was markedly enhanced by hydrogen peroxide or cumene hydroperoxide-supported cytochrome P450 and was prevented by benzylimidazole. Cytotoxicity was also markedly enhanced by dicumarol, an NADPH/oxidoreductase inhibitor. These results suggest that dihydroxycinnamic acids were metabolically activated by P450 peroxidase activity to form cytotoxic quinoid metabolites.     

Inhibition of rat liver glutathione S-transferases by glutathione conjugates and corresponding L-cysteines and mercapturic acids

Glutathione S-transferases from rat liver were partially purified by ion exchange chromatography. Active peaks, tentatively identified as containing the 1-2, 2-2, 3-3, 3-4, 4-4 and 5-5 isoenzymes were kept for study. The glutathione conjugates, S-hexyl-, S-benzyl- and S-(2,4-dinitrophenyl) L-glutathione were tested as inhibitors of the enzymes. The 1-2, 2-2, 3-3 and 3-4 fractions were inhibited to similar extents by these conjugates. For all enzymes the hexyl conjugate at 0.1 mM concentration was strongly inhibitory, the benzyl conjugate moderately so and the dinitrophenyl compound was only weakly inhibitory. In contrast, the epoxide conjugating activity in the 4-4 and 5-5 peak was barely affected by the substituted glutathiones at 0.1 mM concentrations. Studies on a purified ligandin (isoenzyme 1-2) from rat liver showed that further metabolism of the glutathione conjugates, to the corresponding cysteines or mercapturic acids, resulted in products with inhibitory properties approximately three orders of magnitude less potent than those of the parent S-substituted glutathiones.     

Metabolism of 2,3,4',6-tetrachlorobiphenyl: formation and tissue localization of mercapturic acid pathway metabolites in mice

2,3,4',6-Tetrachlorobiphenyl (tetraCB) and the corresponding 14C-labelled compound (14C-tetraCB) were synthesized. Two reference compounds, 4-methylthio- and 4-methylsulphonyl-2,3,4',6-tetrachlorobiphenyl were also prepared and characterized. TetraCB and 35S-cysteine were given to groups of female mice. Formation of methyl[35S]sulphonyl-tetraCB was indicated by the presence of extractable sulphuric acid-soluble radioactivity in lung, liver, kidney and fat of the tetraCB-treated mice. As demonstrated by gel permeation chromatography followed by gas chromatography-mass spectrometry, the tissues of the tetraCB-treated mice contained mainly methylsulphonyl-tetraCB, minor amounts of tetraCB and traces of methylthiotetraCB. The major compound present in lung was 4-methylsulphonyl-tetraCB, indicating the presence of specific binding sites for this metabolite in lung tissue. According to autoradiography of mice injected with 14C-tetraCB, these binding sites were present mainly in the tracheo-bronchial mucosa.     

The formation of a novel mercapturic acid during the metabolism of an N-methyl aromatic amine, 4-cyano-N,N-dimethylaniline

As part of a collaborative study on the genotoxicity of aromatic amines¹ we have recently investigated the metabolism of 4-cyano-N,N-dimethylaniline (CDA) in rats and mice. We now wish to report the formation of a sulphur containing metabolite, which indicates an important role of glutathione in the metabolism of an aromatic amine N-methyl group.     

Purification and characterization of 3-mercaptopyruvic acid S-conjugate reductases

Three kinds of 3-mercaptopyruvic acid S-conjugate reductase (MPR-I, MPR-II and MPR-III) were purified from rat liver cytosol. These enzymes reduced 3-mercaptopyruvic acid S-conjugates derived from cysteine conjugates and some endogenous alpha-keto acids to the corresponding alpha-hydroxy acids in the presence of either NADH (for MPR-I and MPR-II) or NADPH (MPR-III), while simple aldehydes or ketones did not significantly induce substrate activity. The molecular weight of the present enzymes was about 80 kDa composed of two subunits of the same molecular weight. Km values of MPR-I, MPR-II and MPR-III were 0.38, 0.06 and 0.29 mM for S-(4-bromophenyl)-3-thiopyruvic acid, respectively, and 0.15 mM for NADH (MPR-I, MPR-II) and NADPH (MPR-III). Vmax values of MPR-I, MPR-II and MPR-III for this substrate were 5.3, 20 and 13 nmol/min/mg, respectively. The sulphydryl-modifying agents inhibited the enzyme activities of all the three reductases. Based on the properties including substrate selectivity for alpha-keto acids derived from aromatic amino acids, we assumed that MPR-II and aromatic alpha-keto acid reductase are the same enzyme, while enzymes similar to MPR-I and MPR-III have not been reported. From the viewpoints of metabolism of xenobiotics, these enzymes are likely to be important in biotransformation of cysteine conjugates to 3-mercaptolactic acid S-conjugates.     

Differences in the localization and extent of the renal proximal tubular necrosis caused by mercapturic acid and glutathione conjugates of 1,4-naphthoquinone and menadione

We have previously demonstrated that administration of various benzoquinol-glutathione (GSH) conjugates to rats causes renal proximal tubular necrosis and the initial lesion appears to lie within that portion of the S3 segment within the outer stripe of the outer medulla (OSOM). The toxicity may be a consequence of oxidation of the quinol conjugate to the quinone followed by covalent binding to tissue macromolecules. We have therefore synthesized the GSH and N-acetylcysteine conjugates of 2-methyl-1,4-naphthoquinone (menadione) and 1,4-naphthoquinone. The resulting conjugates have certain similarities to the benzoquinol-GSH conjugates, but the main difference is that reaction with the thiol yields a conjugate which remains in the quinone form. 2-Methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone caused a dose-dependent (50-200 mumol/kg) necrosis of the proximal tubular epithelium. The lesion involved the terminal portion of the S2 segment and the S3 segment within the medullary ray. At the lower doses, that portion of the S3 segment in the outer stripe of the outer medulla displayed no evidence of necrosis. In contrast, 2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) caused no apparent histological alterations to the kidney. 2-(Glutathion-S-yl)-1,4-naphthoquinone and 2,3-(diglutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) were relatively weak proximal tubular toxicants and the lesion involved the S3 segment at the junction of the medullary ray and the OSOM. A possible reason(s) for the striking difference in the toxicity of the N-acetylcysteine conjugate of menadione, as opposed to the lack of toxicity of the GSH conjugate of menadione, is discussed. The basis for the localization of the lesion caused by 2-methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone requires further study.     

Binding of ethacrynic acid to hepatic glutathione S-transferases in vivo in the rat

Ethacrynic acid is a substrate for rat glutathione $\text{S}$-transferases $\text{in}$$\text{vitro}$ (1,2). In their study defining the importance of the glutathione $\text{S}$-transferases in the hepatic metabolism and biliary excretion of ethacrynic acid, Wallin $\text{et}$$\text{al}$. (3) suggested that a small but consistent portion of the administered drug was bound selectively and covalently to the transferases. Such an association would represent a unique example of covalent binding of a drug to the metabolizing enzyme without prior microsomal enzyme activation. We have undertaken these studies, therefore, to characterize the binding of ethacrynic acid to the glutathione $\text{S}$-transferases and to delineate the selectivity of this binding.     

Metabolism of the mercapturic acid of 2,4',5-trichlorobiphenyl in rats and mice

2,4',5-Trichloro[14C]biphenyl mercapturic acids (triCB-MA) were metabolized in part to methylsulphide, methylsulphoxide and methylsulphone metabolites after i.p., i.v. (hepatic portal vein) and p.o. administration to rats. Methylthio-triCB and triCB-MA were present in faeces and 3- and 4- methylsulphonyl-triCB (triCB-SO2CH3) were present in carcasses of all rats. Only triCB-SO2CH3 was present in lung tissue. As shown by autoradiography, radioactivity accumulated in the tracheo-bronchial mucosa of mice given either intracaecal or tail-vein injections of triCB-MA. Radioactivity extracted from the lungs of mice was shown to be present as triCB-SO2CH3.     

Purification and characterization of 3-mercaptolactic acid S-conjugate oxidases.

Two enzymes catalysing the oxidative formation of 3-mercaptopyruvic acid S-conjugates from L-3-mercaptolactic acid S-conjugates were purified to apparent homogeneity from rat liver cytosol. The two enzymes, tentatively designated MLO-I and MLO-II, showed a molecular mass of 160 and 250 kDa and were composed of four and six subunits of 41 and 39 kDa, respectively. Both enzymes possessed flavin mononucleotide as prosthetic group and oxidized several aromatic and aliphatic S-substituted L-3-mercaptolactic acids as well as alpha-hydroxy acids such as L-3-phenyllactic acid and L-2-hydroxyisocaproic acid. Glycolic acid and 3-(4-hydroxyphenyl)-lactic acid were the specific substrates for MLO-I and MLO-II, respectively. Neither of the enzymes oxidized beta- and gamma-hydroxy acids such as 3- and 4-hydroxybutyric acid. 2-Hydroxyisobutyric acid, ethyl-2-hydroxybutyrate, malic acid, 1-butanol, benzyl alcohol and L-leucine did not act as substrates for the enzymes. MLO-I and MLO-II exerted their maximum activities around pH 5.5 with Km of 0.5 and 0.25 mM and Vmax of 0.9 and 0.2 mumol/min/mg, respectively, when S-(4-bromophenyl)-3-thiolactic acid was used as substrate. MLO-I was inhibited by sulphydryl-modifying agents, while MLO-II was not. Both enzymes were strongly inhibited by divalent metal ions. These results indicate that MLO-I and MLO-II are different from L-amino acid oxidase (EC 1.4.3.2), malate oxidase (EC 1.1.3.3), L-alpha-hydroxy acid oxidase (EC 1.1.3.15) and glycolate oxidase (EC 1.1.3.1). The present enzymes are likely to be involved in the formation of cysteine conjugates from L-3-mercaptolactic acid S-conjugates in conjunction with cysteine conjugate aminotransferases.     

Coumarin mercapturic acid isolated from rat urine indicates metabolic formation of coumarin 3,4-epoxide.

A coumarin mercapturic acid, N-acetyl-S-(3-coumarinyl)cysteine, has been identified in the urine of coumarin-treated rats. [14C]Coumarin was applied by gavage as a single dose to male Wistar rats (10-150 mg/kg body weight). Twenty-four-hour urine was collected, and the deproteinized concentrate was analyzed for radiolabeled metabolites by HPLC. The new mercapturic acid metabolite is supposed to result from oxidative biotransformation of coumarin to its 3,4-epoxide and subsequent coupling with glutathione.     

Dichloroacetic acid up-regulates hepatic glutathione synthesis via the induction of glutamate-cysteine ligase

Dichloroacetic acid (DCA) has potential for use in cancer therapy and the treatment of metabolic acidosis. However, DCA can create a deficiency of glutathione transferase Zeta (GSTZ1-1). Gstz1 knockout mice have elevated oxidative stress and low glutathione levels that increases their sensitivity to acetaminophen toxicity. As it is highly likely that patients that are treated with DCA will develop drug induced GSTZ1-1 deficiency we considered they could be at risk of elevated toxicity if they are exposed to other drugs that cause oxidative stress or consume glutathione (GSH). To test this hypothesis we treated mice with DCA and acetaminophen (APAP). Surprisingly, the mice pre-treated with DCA suffered less APAP-mediated hepatotoxicity than untreated mice. This protection is most likely due to an increased capacity for the liver to synthesize GSH, since DCA increased the expression and activity of glutamate-cysteine ligase GCL, the rate-limiting enzyme of GSH synthesis. Other pathways for acetaminophen disposal were unchanged or diminished by DCA. Pre-treatment with DCA may be of use in other settings where the maintenance of protective levels of GSH are required. However, DCA may lower the efficacy of drugs that rely on oxidative stress and the depletion of GSH to enhance their cytotoxicity or of drugs that are detoxified by GSH conjugation. Consequently, as the use of DCA in the clinic is likely to increase, it will be critical to evaluate the interactions of DCA with other drugs to ensure the combinations retain their efficacy and do not cause enhanced toxicity.     

Glutathione, gamma-glutamyl transpeptidase, and the mercapturic acid pathway as modulators of 2-bromohydroquinone oxidation

Glutathione (GSH) conjugates of 2-bromohydroquinone are more difficult to oxidize than the parent hydroquinone. Hydrolysis catalyzed by gamma-glutamyl transpeptidase (gamma-GT), however, results in the formation of the corresponding cysteine conjugate which is more readily oxidized than the parent hydroquinone. N-Acetylation of the cysteine conjugate to yield the mercapturate regenerates a compound that is more stable to oxidation than either the parent quinol or its cysteine conjugate. Thus, 2-bromohydroquinone oxidation is exquisitely modulated via GSH conjugation and its metabolism through the mercapturic acid pathway. The ability of gamma-GT to facilitate quinol oxidation by catalyzing the formation of the labile cysteine conjugate may have important biological consequences. Cells exhibiting high gamma-GT activity may be predisposed to the potentially toxic effects of quinol-thioethers.     

The effect of chronic ethanol ingestion on hepatic lipid peroxide, glutathione, glutathione peroxidase and glutathione transferase in rats

Water containing 20% ethanol was given for a period of 3, 6 and 9 weeks to rats, and changes in hepatic lipid peroxide, glutathione, glutathione peroxidase and glutathione transferases were investigated. Lipid peroxide levels and glutathione peroxidase activities remained unchanged after 3 weeks and started to increase thereafter. Glutathione levels and glutathione transferase activities were significantly increased following ethanol consumption. These results show that chronic ethanol consumption stimulates hepatic lipid peroxidation in rats. This stimulation is not dependent on glutathione depletion and the increased glutathione peroxidase and glutathione transferase activities may reflect an adaptive change against ethanol-induced lipid peroxide toxicity.     

Enterohepatic circulation of the mercapturic acid and cysteine conjugates of propachlor

Germfree (GF) rats and antibiotic-treated rats with cannulated bile ducts (ABC) were given single oral doses of 2-(S-(N-[2H3]acetyl)cysteine)-N-isopropyl[1-14C]acetanilide. The GF rats excreted the dose in about equal quantities in the urine and feces; and the ABC rats excreted the dose in about equal quantities in the urine, feces, and bile. The mercapturate (60-75% of the dose in both cases) was isolated and the amount of exchange of N-acetyl deuterium to N-acetyl hydrogen was determined for all samples by mass spectrometry. The percentages of exchange were: ABC rats, bile 6.5%, urine 13%, feces 0.0%; GF rats, urine 15%, feces 4.7%. The remainder of the doses was present as the mercapturic acid sulfoxide. ABC rats dosed with 2-(S-[3,3-3H]cysteine)-N-isopropyl[1-14C]acetanilide (14C/3H = 0.50) excreted 42% of the dose in the urine and bile as the mercapturic acid that had a 14C/3H ratio the same as the original cysteine conjugate. The results of these studies show that a mercapturic acid and a cysteine conjugate can be absorbed from the gastrointestinal tract and resecreted in the bile or excreted by the kidney without having undergone metabolism other than acetylation of the cysteine nitrogen atom and oxidation of the sulfur to a sulfoxide. ABC rats were also dosed with 2-methylthio-N-isopropyl[1-14C]acetanilide, a suspected metabolic intermediate in the metabolism of propachlor (2-chloro-N-isopropylacetanilide). The dose was excreted in about equal quantities in the bile (48%) and urine (46%) as metabolites with a methylsulfonyl group in the 2-position. All are known metabolites of propachlor in conventional rats.