Acivicin-induced alterations in renal and hepatic glutathione concentrations and in gamma-glutamyltransferase activities.

gamma-Glutamyltransferase (gamma-GT) catalyzes the hydrolysis of glutathione, glutathione S-conjugates, and gamma-substituted l-glutamate derivatives. Acivicin is an irreversible inhibitor of gamma-GT that has been used to study the role of gamma-GT in glutathione homeostasis and glutathione-dependent bioactivation reactions. The present studies were undertaken because of reported conflicting effects of acivicin on the nephrotoxicity of some haloalkenes that undergo glutathione-dependent bioactivation. The objective of this study was to test the hypothesis that acivicin may alter renal glutathione concentrations; acivicin-induced changes in renal glutathione concentrations may alter the susceptibility of the kidney to the nephrotoxic effects of haloalkenes. Hence, diurnal and acivicin-induced changes in renal and hepatic glutathione concentrations along with renal and hepatic gamma-GT activities were investigated. The previously observed diurnal variations in hepatic glutathione concentrations in fed rats were confirmed, but no diurnal variations were observed in renal glutathione concentrations or in renal or hepatic gamma-GT activities. Renal and hepatic glutathione concentrations and gamma-GT activities were measured in tissue homogenates from rats given 0, 0.1, or 0.2 mmol acivicin/kg (i.p.) and killed 0, 2, 4, 8, 12, or 24 hr later. Renal glutathione concentrations were increased above control values in acivicin-treated rats, whereas acivicin had no effect on hepatic glutathione concentrations. Renal gamma-GT activities decreased within 2 hr after giving acivicin and remained decreased for 24 hr. Acivicin had no effect on hepatic gamma-GT activities, except at 24 hr after treatment when values in acivicin-treated rats were elevated compared with controls. Although the present studies do not afford an explanation of the mechanism whereby acivicin increases the nephrotoxicity of some haloalkenes, they do indicate that acivicin is not a reliable probe to investigate the role of gamma-GT in haloalkene-induced nephrotoxicity.

Perturbation of maleylacetoacetic acid metabolism in rats with dichloroacetic Acid-induced glutathione transferase zeta deficiency.

Glutathione transferase zeta (GSTZ1-1) catalyzes the isomerization of maleylacetoacetate (MAA) to fumarylacetoacetate, the penultimate step in the tyrosine degradation pathway. GSTZ1-1 is inactivated by dichloroacetic acid (DCA), which is used for the clinical management of congenital lactic acidosis and is a drinking-water contaminant. Metabolic changes associated with chemically induced GSTZ1-1 deficiency are poorly understood. The objective of this study was to investigate the biochemical and toxicological effects of giving 0.3-1.2 mmol DCA/kg/day for 5 days on MAA-metabolism in male Fischer rats. Urine from DCA-treated rats inhibited delta-aminolevulinic acid dehydratase (delta-ALAD) activity, which is used for the diagnosis of hereditary tyrosinemia type I. Mass spectrometric analyses of urine from rats given DCA demonstrated elevated excretion of MAA and its decarboxylation product, maleylacetone (MA); succinylacetone (SA), the reduced analogue of MA, was not detected. DCA-induced changes in MA excretion were dose-dependent and were significantly elevated after day 2 of treatment. MA excretion was reversible after discontinuation of DCA treatment and was enhanced 10-fold by the coadministration of homogentisic acid (HGA). MA was cytotoxic to hepatocytes in vitro (EC50 ~ 350 microM) but morphological changes were not observed in liver, kidney, and brain of rats given both DCA and HGA. These data indicate that DCA-induced inactivation of GSTZ1-1 leads to formation of an MAA-derived intermediate, MA, that may be a mediator and biomarker for DCA-associated toxicities.

Nephrotoxicity of chlorofluoroacetic acid in rats.

Dichloroacetic acid (DCA), chlorofluoroacetic acid (CFA), and difluoroacetic acid (DFA) are inhibitors of pyruvate dehydrogenase kinase. DCA is used for the clinical management of congenital lactic acidosis. Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of DCA and CFA, and DCA is a mechanism-based inactivator of GSTZ1-1. In rodents, DCA causes multiorgan toxicities and is hepatocarcinogenic. The toxic effects of CFA, which is an excellent substrate but a poor inactivator of GSTZ1-1, have not been investigated. The objective of this study was to investigate the nephrotoxicity of CFA. Rats given a single dose of 1.5 mmol/kg CFA became anuric and died within 24 h. Urinalysis and light microscopic analysis showed that rats given 0.6-1.2 mmol/kg CFA developed polyuria, glycosuria, and renal proximal tubular damage. Electron microscopic analysis indicated a role for apoptosis in CFA-induced cell death. The nephrotoxicity of CFA was associated with a dose-dependent increase in inorganic fluoride excretion. Treatment of rats with DCA for 5 days to inactivate GSTZ1-1 failed to prevent metabolism of CFA to fluoride and did not block CFA-induced renal damage. A role for GSTZ1-1-catalyzed release of fluoride from CFA is proposed but a role for other enzymes cannot be excluded. DFA, which is not metabolized to fluoride by GSTZ1-1, was given to rats as a control and was also nephrotoxic: rats given 1.2 mmol DFA/kg/day for 5 days had normal urine volumes but showed proximal and distal tubular damage; fluoride excretion was not elevated. The mechanism of DFA-induced nephrotoxicity is not known but appears to differ from that of CFA.

Kinetics of the biotransformation of maleylacetone and chlorofluoroacetic acid by polymorphic variants of human glutathione transferase zeta (hGSTZ1-1).

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate and the biotransformation of a range of alpha-haloacids. The objective of this study was to determine the kinetics of the biotransformation of maleylacetone (MA), an analogue of the natural substrate maleylacetoacetate, and chlorofluoroacetic acid (CFA) by polymorphic variants of recombinant hGSTZ1-1. The k(cat) of the four variants of hGSTZ1-1 with MA as the substrate followed the order: 1c-1c > 1b-1b > 1d-1d > 1a-1a whereas the k(cat) for the biotransformation of CFA followed the order: 1a-1a > 1b-1b approximately 1c-1c approximately 1d-1d. The turnover rates of MA were much higher than those of CFA for each variant and ranged from 22-fold (1a-1a) to 980-fold differences (1c-1c). The catalytic efficiencies of hGSTZ1-1 variants with MA as the substrate were much greater than those with CFA as the substrate, but little difference among the polymorphic variants was observed. MA was a mixed inhibitor of all variants with CFA as substrate: the mean competitive inhibition constant (K(ic)(MA)) for all variants was about 100 microM, and the mean uncompetitive inhibition constant (K(iu)(MA)) was about 201 microM. Hence, MA and alpha-haloacids apparently compete for the same active site on the enzyme. DCA-induced inactivation of the four variants showed that the inactivated enzymes show markedly reduced isomerase activities. The residual activities were different for each variant: 1a-1a (12%) > 1b-1b approximately 1c-1c approximately 1d-1d (<5%). This is the first kinetic analysis of polymorphic variants of hGSTZ1-1, and the similarity of the kinetic constants for hGSTZ1-1 variants with either MA or CFA as substrates indicates that few differences in DCA-induced perturbations of tyrosine metabolism would likely be observed in humans.

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.

Immunohistochemical localization and activity of glutathione transferase zeta (GSTZ1-1) in rat tissues.

Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of alpha-haloacids, including dichloroacetic acid (DCA), and the penultimate step in the tyrosine degradation pathway. DCA is a rodent carcinogen and a common drinking water contaminant. DCA also causes multiorgan toxicity in rodents and dogs. The objective of this study was to determine the expression and activities of GSTZ1-1 in rat tissues with maleylacetone and chlorofluoroacetic acid as substrates. GSTZ1-1 protein was detected in most tissues by immunoblot analysis after immunoprecipitation of GSTZ1-1 and by immunohistochemical analysis; intense staining was observed in the liver, testis, and prostate; moderate staining was observed in the brain, heart, pancreatic islets, adrenal medulla, and the epithelial lining of the gastrointestinal tract, airways, and bladder; and sparse staining was observed in the renal juxtaglomerular regions, skeletal muscle, and peripheral nerve tissue. These patterns of expression corresponded to GSTZ1-1 activities in the different tissues with maleylacetone and chlorofluoroacetic acid as substrates. Specific activities ranged from 258 +/- 17 (liver) to 1.1 +/- 0.4 (muscle) nmol/min/mg of protein with maleylacetone as substrate and from 4.6 +/- 0.89 (liver) to 0.09 +/- 0.01 (kidney) nmol/min/mg of protein with chlorofluoroacetic acid as substrate. Rats given DCA had reduced amounts of immunoreactive GSTZ1-1 protein and activities of GSTZ1-1 in most tissues, especially in the liver. These findings indicate that the DCA-induced inactivation of GSTZ1-1 in different tissues may result in multiorgan disorders that may be associated with perturbed tyrosine metabolism.