Glutathione-dependent Biotransformation Research Articles

Chemical Toxicology of Reactive Intermediates Formed by the Glutathione-Dependent Bioactivation of Halogen-Containing Compounds

The concept that reactive intermediate formation during the biotransformation of drugs and chemicals is an important bioactivation mechanism was proposed in the 1970s and is now accepted as a major mechanism for xenobiotic-induced toxicity. The enzymology of reactive intermediate formation as well as the characterization of the formation and fate of reactive intermediates are now well-established. The mechanism by which reactive intermediates cause cell damage and death is, however, still poorly understood. Although most xenobiotic-metabolizing enzymes catalyze the bioactivation of chemicals, glutathione-dependent biotransformation has been largely associated with detoxication processes, particularly mercapturic acid formation. Abundant evidence now shows that glutathione-dependent biotransformation constitutes an important bioactivation mechanism for halogen-containing drugs and chemicals and has for many compounds been implicated in their organ-selective toxicity and in their mutagenic and carcinogenic potential. The glutathione-dependent biotransformation of haloalkenes is the first step in the cysteine S-conjugate beta-lyase pathway for the bioactivation of nephrotoxic haloalkenes. This pathway has been a rich source of reactive intermediates, including thioacyl halides, alpha-chloroalkenethiolates, 3-halo-alpha-thiolactones, 2,2,3-trihalothiiranes, halothioketenes, and vinylic sulfoxides. Glutathione-dependent bioactivation of gem-dihalomethanes and 1,2-, 1,3-, and 1,4-dihaloalkanes leads to the formation of alpha-chlorosulfides, thiiranium ions, sulfenate esters, and tetrahydrothiophenium ions, respectively, and these reactions lead to reactive intermediate formation.

Glutathione-dependent biotransformation of the fungicide chlorothalonil.

A gene responsible for the chlorothalonil biotransformation was cloned from the chromosomal DNA of Ochrobactrum anthropi SH35B, capable of efficiently dissipating the chlorothalonil. The gene encoding glutathione S-transferase (GST) of O. anthropi SH35B was expressed in Escherichia coli, and the GST was subsequently purified by affinity chromatography. The fungicide chlorothalonil was rapidly transformed by the GST in the presence of glutathione. LC-MS analysis supported the formation of mono-, di-, and triglutathione conjugates of chlorothalonil by the GST. The monoglutathione conjugate was observed as an intermediate in the enzymatic reaction. The triglutathione conjugate has not been previously reported and seems to be the final metabolite in the biotransformation of chlorothalonil. The glutathione-dependent biotransformation of chlorothalonil catalyzed by the bacterial GST is reported.

Alkylation and inactivation of human glutathione transferase zeta (hGSTZ1-1) by maleylacetone and fumarylacetone.

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate or maleylacetone (MA) to fumarylacetoacetate or fumarylacetone (FA), respectively. GSTZ1-1 also catalyzes the glutathione-dependent biotransformation of a range of alpha-haloacids, including dichloroacetic acid. The objective of this study was to investigate the mechanism of inactivation of hGSTZ1-1 by MA and FA and to determine the covalent modification of hGSTZ1-1 by MA and FA in the presence and absence of glutathione. MA and FA (0.01-1 mM) inactivated all hGSTZ1-1 polymorphic variants in a concentration- and time-dependent manner, and this inactivation was blocked by glutathione. The C16A mutant of hGSTZ1c-1c was partially inactivated by MA and FA. Electrospray ionization-tandem mass spectrometry and SALSA (Scoring Algorithm for Spectral Analysis) analyses of tryptic digests of hGSTZ1 polymorphic variants revealed that the active site (SSCSWR) and C-terminal (LLVLEAFQVSHPCR) cysteine residues of hGSTZ1-1 were covalently modified by MA and FA. MA and FA adduction resulted in diagnostic 156-Da shifts in the masses of the modified peptide ions and in their MS-MS fragment ions. Alkylation of the active-site cysteine residues, but not of the C-terminal cysteine, was relatively less intense when hGSTZ1-1 polymorphic variants were incubated with MA or FA in the presence of S-methyl glutathione. These data indicate that MA and FA are substrate and product inactivators of hGSTZ1-1 and covalently modify hGSTZ1-1 at the active-site cysteine residue in the absence of glutathione. The observation that inactivation was blocked by glutathione indicates that binding of glutathione to the active site prevents reaction of MA or FA with the active-site cysteine residue. These data also indicate that MA and FA may covalently modify and inactivate other proteins that have accessible cysteine residues and may, thereby, contribute to dichloroacetic acid-induced or hypertyrosinemia type-I-associated toxicities.

Study on the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene in humans.

To investigate in humans the contribution of the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene (TRI) under controlled repeated exposure in volunteers, and under occupational conditions. Volunteers were exposed to TRI, using repeated 15 min exposures at 50 and 100 ppm. This exposure schedule resulted in internal doses of 1.30 and 2.40 mmol of TRI respectively. Urine samples were collected for a minimum of 45 h. Urine samples were also collected from occupationally exposed workers. The samples were analysed for the known human metabolites of TRI, trichloroethanol (TCE), trichloroacetic acid (TCA) and both regio-isomeric forms of the mercapturic acid N-acetyl-S-(dichlorovinyl)-L-cysteine (DCV-NAC), and for (dichlorovinyl)-L-cysteine (DCVC). In order to further elucidate the metabolism of TRI in humans, we analysed samples for dichloroacetic acid and for the proposed break-down products of 1,2 and 2,2-dichlorovinyl-L-cysteine after deamination: the S-conjugates of 3-mercaptolactic acid, 3-mercaptopyruvic acid and 2-mercaptoacetic acid. None of the glutathione metabolites was found in urine of volunteers. In workers occupationally exposed to TRI at levels between 0.4 and 21 ppm [8-h time-weighted average (TWA)], levels of DCV-NAC in urine samples collected at the end of the 4th working day and also next morning were below detection limit (0.04 mumol/l). This confirms the findings of Bernauer et al. (1996) that these metabolites are excreted at very low levels in humans. Urinary levels of DCVC and six postulated metabolites of dichlorovinyl-S-cysteine conjugates via deamination were also below 0.04 mumol/l, indicating that at most 0.05% of the dose is excreted in the form of these metabolites. These data further strengthen the argument for a very low activity of glutathione-mediated metabolism for chronically exposed workers. This study gives additional data which indicate that glutathione-mediated metabolism is of minor importance in humans exposed to TRI. In spite of indications to the contrary, significant metabolism of the cysteine conjugate via beta-lyase, which could result in a toxic metabolite, cannot be ruled out completely.

Biotransformation of the Fungicide Chlorthalonil by Glutathione Conjugation

The biotransformation of the nephrotoxic fungicide chlorthalonil (2,4,5,6-tetrachloroisophthalonitril) has been studied in the rat and in rat liver subcellular fractions. In rat liver cytosol, chlorthalonil was rapidly transformed to 4,6-bis(glutathion-S-yl)-2,5-dichloroisophthalonitril in the presence of glutathione in a reaction catalysed by glutathione S-transferases. 4-(Glutathion-S-yl)-2,5,6-trichloroisiphthalonitril was observed as an intermedite in the glutathione-dependent biotransformation of chlorthalonil. In the bile of rats dosed with chlorthalonil (0.18 mmol/kg, orally) 4,6-bis (glutathion-S-yl)-2,5-dichloroisophthalonitril was recovered as a minor metabolite. In rats, chlorthalonil was transformed to 4,6-bis (N-acetyl-cystein-S-yl)-2,5-dichloroisophthalonitril. This metabolite was excreted in small amounts in the urine of rats dosed orally with chlorthalonil (0.66 and 2.64 mmol/kg). In all experiments, a major part of the dose was recovered as unchanged chlorthalonil in feces. Oral administration of chlorthalonil (0.66 and 2.64 mmol/kg) resulted in a slight increase in the urinary excretion of gamma-glutamyltranspeptidase, indicative of nephrotoxicity. Other parameters indicative for renal proximal tubular damage (urinary glucose and protein excretion) were not changed by acute chlorthalonil administration. Very minor renal lesions were observed in chlorthalonil-dosed animals by histopathological examination.

Biotransformation of the Fungicide Chlorthalonil by Glutathione Conjugation

The biotransformation of the nephrotoxic fungicide chlorthalonil (2,4,5,6-tetrachloroisophthalonitril) has been studied in the rat and in rat liver subcellular fractions. In rat liver cytosol, chlorthalonil was rapidly transformed to 4,6-bis(glutathion-S-yl)-2,5-dichloroisophthalonitril in the presence of glutathione in a reaction catalysed by glutathione S-transferases. 4-(Glutathion-S-yl)-2,5,6-trichloroisiphthalonitril was observed as an intermedite in the glutathione-dependent biotransformation of chlorthalonil. In the bile of rats dosed with chlorthalonil (0.18 mmol/kg, orally) 4,6-bis (glutathion-S-yl)-2,5-dichloroisophthalonitril was recovered as a minor metabolite. In rats, chlorthalonil was transformed to 4,6-bis (N-acetyl-cystein-S-yl)-2,5-dichloroisophthalonitril. This metabolite was excreted in small amounts in the urine of rats dosed orally with chlorthalonil (0.66 and 2.64 mmol/kg). In all experiments, a major part of the dose was recovered as unchanged chlorthalonil in feces. Oral administration of chlorthalonil (0.66 and 2.64 mmol/kg) resulted in a slight increase in the urinary excretion of gamma-glutamyltranspeptidase, indicative of nephrotoxicity. Other parameters indicative for renal proximal tubular damage (urinary glucose and protein excretion) were not changed by acute chlorthalonil administration. Very minor renal lesions were observed in chlorthalonil-dosed animals by histopathological examination.

Glutathione-dependent biotransformation of methyl parathion by mouse liver in vitro

The present study was undertaken in an attempt to reconcile the seemingly conflicting observations that glutathione-dependent biotransformation of methyl parathion ( O,O-dimethyl- O-(4-nitrophenyl) phosphorothionate) by hepatic supernatant or partially purified glutathione S-transferases occurs in vitro, but not to any significant degree in vivo in the mouse. While incubation of 20 μM methyl parathion with glutathione-fortified 100 000 × g hepatic supernatant resulted in biotransformation of this insecticide, addition of the carbon monoxide exposed microsomal fraction (without NADPH) to the supernatant abolished this metabolism. HPLC analyses of the distribution of methyl parathion between 100 000 × g supernatant and carbon monoxide-exposed microsomes revealed that little methyl parathion could be recovered in the 100 000 × g supernatant, and that the bulk of this insecticide was associated with the microsomal fraction. Increasing the concentration of methyl parathion to 1 mM resulted in a greater fraction of methyl parathion found in the supernatant compared to that with 20 μM, although the bulk of methyl parathion remained associated with the microsomal fraction. While this increase in the fraction of substrate located within the supernatant led to limited glutathione-dependent metabolism of methyl parathion, it must be emphasized that a liver concentration of 1 mM methyl parathion is far greater than that which could be achieved in vivo. In conclusion, the results of the present study support the hypothesis that in the mouse, glutathione- dependent metabolism of methyl parathion does not occur to a significant degree in vivo because of its limited access to the soluble glutathione S-transferases.