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M W Anders

Publications and source records attributed to M W Anders.

At least 37 records · Page 2Linked to original sources
PubMed2002

Toxic, halogenated cysteine S-conjugates and targeting of mitochondrial enzymes of energy metabolism.

Several haloalkenes are metabolized in part to nephrotoxic cysteine S-conjugates; for example, trichloroethylene and tetrafluoroethylene are converted to S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), respectively. Although DCVC-induced toxicity has been investigated since the 1950s, the toxicity of TFEC and other haloalkene-derived cysteine S-conjugates has been studied more recently. Some segments of the US population are exposed to haloalkenes either through drinking water or in the workplace. Therefore, it is important to define the toxicological consequences of such exposures. Most halogenated cysteine S-conjugates are metabolized by cysteine S-conjugate beta-lyases to pyruvate, ammonia, and an alpha-chloroenethiolate (with DCVC) or an alpha-difluoroalkylthiolate (with TFEC) that may eliminate halide to give a thioacyl halide, which reacts with epsilon-amino groups of lysine residues in proteins. Nine mammalian pyridoxal 5'-phosphate (PLP)-containing enzymes catalyze cysteine S-conjugate beta-lyase reactions, including mitochondrial aspartate aminotransferase (mitAspAT), and mitochondrial branched-chain amino acid aminotransferase (BCAT(m)). Most of the cysteine S-conjugate beta-lyases are syncatalytically inactivated. TFEC-induced toxicity is associated with covalent modification of several mitochondrial enzymes of energy metabolism. Interestingly, the alpha-ketoglutarate- and branched-chain alpha-keto acid dehydrogenase complexes (KGDHC and BCDHC), but not the pyruvate dehydrogenase complex (PDHC), are susceptible to inactivation. mitAspAT and BCAT(m) may form metabolons with KGDHC and BCDHC, respectively, but no PLP enzyme is known to associate with PDHC. Consequently, we hypothesize that not only do these metabolons facilitate substrate channeling, but they also facilitate toxicant channeling, thereby promoting the inactivation of proximate mitochondrial enzymes and the induction of mitochondrial dysfunction.

Animals↗
PubMed2002

Molecular identification of a novel carnitine transporter specific to human testis. Insights into the mechanism of carnitine recognition.

l-Carnitine is an essential component of mitochondrial fatty acid beta-oxidation and plays a pivotal role in the maturation of spermatozoa within the male reproductive tract. Epididymal plasma contains the highest levels of l-carnitine found in the human body, and initiation of sperm motility occurs in parallel to l-carnitine increase in the epididymal lumen. Using a specific carrier, epididymal epithelium secretes l-carnitine into the lumen by an active transport mechanism; however, the structure-activity relationship comprising the carnitine-permeation pathway is poorly understood. We discovered a novel carnitine transporter (CT2) specifically located in human testis. Analyzing the primary structure of CT2 revealed that it is phylogenetically located between the organic cation transporter (OCT/OCTN) and anion transporter (OAT) families. Hence, CT2 represents a novel transporter family. When expressed in Xenopus oocytes, CT2 mediates the high affinity transport of l-carnitine but does not accept mainstream OCT/OCTN cationic or OAT anionic substrates. We synthesized and tested various carnitine-related compounds and investigated the physicochemical properties of substrate recognition by semi-empirical computational chemistry. The data suggest that the quaternary ammonium cation bulkiness and relative hydrophobicity be the most important factors that trigger CT2-substrate interactions. Immunohistochemistry showed that the CT2 protein is located in the luminal membrane of epididymal epithelium and within the Sertoli cells of the testis. The identification of CT2 represents an interesting evolutionary link between OCT/OCTNs and OATs, as well as provides us with an important insight into the maturation of human spermatozoa.

Amino Acid Sequence↗
PubMed2002

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.

Acetates↗
PubMed2002

Reactive intermediate formation from the 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A)-derived cysteine S-conjugate S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine in pyridoxal model systems.

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) is a degradation product of the anesthetic sevoflurane and undergoes cysteine conjugate beta-lyase-dependent bioactivation to nephrotoxic metabolites in rats. The present experiments were designed to identify reactive intermediates formed from S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine, a compound A-derived cysteine S-conjugate, in two pyridoxal model systems, namely Cu2+/pyridoxal and N-dodecylpyridoxal in cetyltrimethylammonium micelles. S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine was incubated in the model systems with benzyl bromide, pentafluorobenzyl bromide, aniline, and o-phenylenediamine as trapping agents. The products were purified by TLC and identified by 19F and 1H NMR spectroscopy and by GC/MS. In the absence of trapping agents, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid, which have been identified previously in biotransformation studies, were formed. With the chemical models, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropanethiolate, the expected first intermediate, was not trapped with benzyl bromide. Rather, the dehydrofluorination product 2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenylthiolate was trapped with benzyl bromide to give benzyl 2-(fluoromethoxy)-3,3,3-trifluoropropanethioate, which was formed in both chemical models. When pentafluorobenzyl bromide was used as a trapping agent, GC/MS analysis showed that the expected thiolate was trapped to give pentafluorobenzyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide in the N-dodecylpyridoxal model. In both chemical models, 2-(fluoromethoxy)-3,3,3-trifluorothioacyl fluoride was trapped with aniline to give N-phenyl 2-(fluoromethoxyl)-3,3,3-trifluoropropanethioamide, which cyclized to give 3-phenyl-4-thiono-5-(trifluoromethyl)-1,3-oxazolane. The results demonstrate that most of the reactive intermediates and products formed by the beta-lyase-catalyzed biotransformation of compound A-derived cysteine S-conjugates are also formed in the two chemical systems studied. Some products were, however, formed in chemical systems that have not been observed in previous in vivo and in vitro studies; it is not known whether these products are formed in biological systems and whether they contribute to the observed nephrotoxicity of cysteine S-conjugates.

Anesthetics, Inhalation↗
PubMed2002

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.

Acetone↗
PubMed2002

Biotransformation of 1,1,1,3,3-Pentafluoropropane (HFC-245fa).

1,1,1,3,3-Pentafluoropropane (HFC-245fa) is being developed as a CFC substitute. 1,1,1,3,3-Pentafluoropropane has a low potential for toxicity: the only remarkable toxic effect seen in rats after inhalation exposure to 1,1,1,3,3-pentafluoropropane in concentrations of up to 50,000 ppm for 90 days was an increased incidence of diffuse myocarditis. To elucidate the possible role of biotransformation in 1,1,1,3,3-pentafluoropropane-induced cardiotoxicity, the biotransformation of 1,1,1,3,3-pentafluoropropane was investigated in rats after inhalation exposure and in rat and human liver microsomes. Male and female rats were exposed by inhalation to 50 000, 10 000, and 2000 ppm 1,1,1,3,3-pentafluoropropane for 6 h, urine was collected for 72 h, and metabolites excreted were identified by 19F NMR spectroscopy and quantified by GC/MS. Trifluoroacetic acid and inorganic fluoride were identified as major urinary metabolites of 1,1,1,3,3-pentafluoropropane; 3,3,3-trifluoropropanoic acid and 1,1,1,3,3-pentafluoropropane-2-ol were minor metabolites. The extent of 1,1,1,3,3-pentafluoropropane biotransformation after inhalation was dependent on exposure concentrations. Neither 3,3,3-trifluoropropanoic acid nor 3,3,3-trifluoropyruvic acid were metabolized to trifluoroacetic acid in vitro or in rats. In rat and human liver microsomes, 1,1,1,3,3-pentafluoropropane was biotransformed by a cytochrome P450-dependent reaction to trifluoroacetic acid and 3,3,3-trifluoropropanoic acid. Rates of trifluoroacetic acid formation were 99.2 +/- 20.5 pmol (mg of protein)(-)(1) min(-)(1) and of 3,3,3-trifluoropropanoic acid formation were 17.5 +/- 4.0 pmol (mg of protein)(-)(1) min(-)(1) in liver microsomes from male rats. In human liver microsomes, rates of trifluoroacetic acid formation ranged from 0 to 11.6 pmol (mg of protein)(-)(1) min(-)(1), and rates of 3,3,3-trifluoropropanoic acid formation ranged from 0.7 to 7.6 pmol (mg of protein)(-)(1) min(-)(1). The results show that 1,1,1,3,3-pentafluoropropane is metabolized at low rates in vivo and in vitro. The toxic effects of 1,1,1,3,3-pentafluoropropane may be associated with the formation of the minor metabolite 3,3,3-trifluoropropanoic acid, which is highly toxic in rats.

Administration, Inhalation↗
PubMed2002

Structure-activity relationship for the biotransformation of haloalkenes by rat liver microsomal glutathione transferase 1.

Many haloalkenes are nephrotoxic in rodents, and experimental evidence supports a glutathione-dependent bioactivation pathway that leads to nephrotoxicity or nephrocarcinogenicity, or both. The reaction of glutathione with haloalkenes is catalyzed by cytosolic glutathione transferases (cGST) and microsomal glutathione transferase 1 (MGST1). The aim of this study was to develop a computational approach to predict the competency of cGST and MGST1 to catalyze the reaction of glutathione with a range of haloalkenes. The hypothesis tested was that the semiempirically computed energy of the lowest unoccupied molecular orbital (E(LUMO)) of a haloalkene may be used to predict the competency of cGST and MGST1 to catalyze its reaction with glutathione. The MGST1- and cGST-catalyzed reaction of glutathione with nine haloalkenes with E(LUMO) values ranging from -1.14 to 0.38 eV was determined experimentally. The data indicated that the E(LUMO) values for haloalkenes were inversely related to the specific activity of the MGST1-catalyzed reaction but not the cGST-catalyzed reaction. These data also demonstrated that MGST1 catalyzed the reaction of glutathione with haloalkenes with E(LUMO) values equal to or more negative than -0.73 eV and that cGST catalyzed the reaction of glutathione with haloalkenes with E(LUMO) values more negative than -0.06 eV.

Animals↗
PubMed2002

Mass spectral characterization of dichloroacetic acid-modified human glutathione transferase zeta.

Glutathione transferase zeta (GSTZ1-1) is widely expressed in eukaryotic species, and four human allelic variants of hGSTZ1-1 have been described. GSTZ1-1 catalyzes the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate and the biotransformation of a range of alpha-haloalkanoic acids. GSTZ1-1-catalyzed biotransformation of fluorine-lacking alpha,alpha-dihaloalkanoic acids, including dichloroacetic acid (DCA), results in the mechanism-based inactivation and covalent modification of the enzyme. The objective of this study was to investigate further the DCA-induced inactivation of hGSTZ1c-1c and to explore the mechanism of inactivation by characterization of the sites and types of DCA-induced covalent modifications. The partition ratio for the DCA-induced, mechanism-based inactivation of hGSTZ1c-1c was (5.7 +/- 0.5) x 10(2), and the k(cat) for the biotransformation of DCA was 39 min(-)(1). Inactivation of hGSTZ1c-1c in vitro was limited at high enzyme concentrations and was inhibited by glyoxylate. The stoichiometry of DCA binding to hGSTZ1c-1c was approximately 0.5 mol of DCA/mol of enzyme monomer. A single DCA-derived adduct was observed and was assigned to cysteine-16 by a combination of matrix-assisted laser-desorption-ionization time-of-flight and electrospray-ionization quadrupole ion-trap mass spectrometry and by analysis of [1-(14)C]DCA binding to C16A hGSTZ1c-1c. The DCA-derived adduct contained both glutathione and the carbon skeleton of DCA, presumably in a dithioacetal linkage. Also, cysteine-16 formed a mixed disulfide bond with glutathione. These data support a mechanism of inactivation whereby glutathione displaces a chlorine atom from DCA, and cysteine-16 in the enzyme active site displaces the second chlorine atom to result in a covalently modified and inactivated enzyme. These findings explain the DCA-induced inactivation of GSTZ1-1 observed in humans and rats.

Binding Sites↗
PubMed2002

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.

Acetates↗
PubMed2002

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.

Acetates↗
PubMed2002

Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1): insights into the mechanisms of substrate recognition.

The L-type amino acid transporter 1 (LAT1) is an Na(+)-independent neutral amino acid transporter subserving the amino acid transport system L. Because of its broad substrate selectivity, system L has been proposed to be responsible for the permeation of amino acid-related drugs through the plasma membrane. To understand the mechanisms of substrate recognition, we have examined the LAT1-mediated transport using a Xenopus laevis oocyte expression system. LAT1-mediated [(14)C]phenylalanine uptake was strongly inhibited in a competitive manner by aromatic-amino acid derivatives including L-dopa, alpha-methyldopa, melphalan, triiodothyronine, and thyroxine, whereas phenylalanine methyl ester, N-methyl phenylalanine, dopamine, tyramine, carbidopa, and droxidopa did not inhibit [(14)C]phenylalanine uptake. Gabapentin, a gamma-amino acid, also exerted a competitive inhibition on LAT1-mediated [(14)C]phenylalanine uptake. Although most of the compounds that inhibited LAT1-mediated uptake were able to induce the efflux of [(14)C]phenylalanine preloaded to the oocytes expressing LAT1 through the obligatory exchange mechanism, melphalan, triiodothyronine, and thyroxine did not induce the significant efflux. Based on the experimental and semiempirical computational analyses, it is proposed that, for an aromatic amino acid to be a LAT1 substrate, it must have a free carboxyl and an amino group. The carbonyl oxygen closer to the amino group needs a computed charge of -0.55 approximately -0.56 and must not participate in hydrogen bonding. In addition, the hydrophobic interaction between the substrate side chain and the substrate binding site of LAT1 seems to be crucial for the substrate binding. A substrate, however, becomes a blocker once Connolly accessible areas become large and/or the molecule has a high calculated logP value, such as those for melphalan, triiodothyronine, and thyroxine.

Amino Acids↗
PubMed2001

Role of mitochondrial dysfunction in S-(1,2-dichlorovinyl)-l-cysteine-induced apoptosis.

The nephrotoxicity of trichloroethylene and dichloroacetylene has previously been linked to mitochondrial dysfunction induced by the metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC). In this study, we examined whether key biochemical steps associated with mitochondria occur in DCVC-induced apoptosis in cultured porcine proximal tubular LLC-PK1 cells. DCVC caused a decrease in mitochondrial membrane potential (mt Delta Psi) beginning at 4 h and a release of cytochrome c into the cytoplasm at 6 h. Caspase-3-like activity was detected at 6 h and extensive DNA fragmentation was observed at 8 h. Decreases in cellular ATP were not evident until 8 h and later, even though electron microscopy showed that the mitochondria were extensively swollen. Aminooxyacetic acid (AOAA), an inhibitor of cysteine-conjugate beta-lyase, protected against mitochondrial changes and apoptosis. Overexpression of the antiapoptotic Bcl-2 protein desensitized LLC-PK1 cells to DCVC-induced apoptosis. These results support the interpretation that mitochondrial release of cyt c and cyt c-dependent activation of caspase-3 could have a central role in nephrotoxicity due to haloalkene-derived cysteine S-conjugates.

Aminooxyacetic Acid↗
PubMed2001

GSTZ1d: a new allele of glutathione transferase zeta and maleylacetoacetate isomerase.

The zeta class glutathione transferases (GSTs) are known to catalyse the isomerization of maleylacetoacetate (MAA) to fumarylacetoacetate (FAA), and the biotransformation of dichloroacetic acid to glyoxylate. A new allele of human GSTZ1, characterized by a Thr82Met substitution and termed GSTZ1d, has been identified by analysis of the expressed sequence tag (EST) database. In European Australians, GSTZ1d occurs with a frequency of 0.16. Like GSTZ1b-1b and GSTZ1c-1c, the new isoform has low activity with dichloroacetic acid compared with GSTZ1a-1a. The low activity appears to be due to a high sensitivity to substrate inhibition. The maleylacetoacetate isomerase (MAAI) activity of all known variants was compared using maleylacetone as a substrate. Significant differences in activity were noted, with GSTZ1a-1a having a notably lower catalytic efficiency. The unusual catalytic properties of GSTZ1a-1a in both reactions suggest that its characteristic arginine at position 42 plays a significant role in the regulation of substrate access and/or product release. The different amino acid substitutions have been mapped on to the recently determined crystal structure of GSTZ1-1 to evaluate and explain their influence on function.

Adolescent↗
PubMed2001

Mercapturic acids (N-acetylcysteine S-conjugates) as endogenous substrates for the renal organic anion transporter-1.

Mercapturic acids are N-acetyl-L-cysteine S-conjugates that are formed from a range of endogenous and exogenous chemicals. Although the kidney is a major site for elimination of mercapturic acids, the transport mechanisms involved have not been identified. The present study examined whether mercapturic acids are substrates for the renal basolateral organic anion transporter-1 (Oat1) from rat kidney. This carrier mediates uptake of organic anions from the bloodstream in exchange for intracellular alpha-ketoglutarate. Uptake of [(3)H]p-aminohippuric acid (PAH) in Oat1-expressing Xenopus laevis oocytes was strongly inhibited by S-(2,4-dinitrophenyl)-N-acetyl-L-cysteine (DNP-NAC) and by all other mercapturic acids tested, including the endogenous mercapturic acid N-acetyl-leukotriene E(4). Inhibition by the mercapturic acids was competitive, which is consistent with the hypothesis that these compounds are substrates for Oat1. This conclusion was supported by the direct demonstration of saturable [(35)S]DNP-NAC uptake in Oat1-expressing oocytes. [(35)S]DNP-NAC uptake was inhibited by PAH and other mercapturic acids and was stimulated in oocytes preloaded with glutarate. The apparent K(m) value for DNP-NAC uptake was only 2 microM, indicating that this mercapturic acid is a high affinity substrate for Oat1. Together, these data indicate that clearance of endogenous mercapturic acids is an important function of the renal organic anion transporter.

Acetylcysteine↗
PubMed2001

Identification of novel glutathione transferases and polymorphic variants by expressed sequence tag database analysis.

The human expressed sequence tag (EST) database can be searched by different sequence alignment strategies to identify new members of gene families and allelic variants. To illustrate the value of database analysis for gene discovery, we have focused on the glutathione S-transferase (GST) super family, an approach that has led to the identification of the Zeta class. The Zeta class GSTs catalyze the glutathione-dependent biotransformation of alpha-haloacids and the isomerization of maleylacetoacetic acid to fumarylacetoacetic acid, an essential step in the catabolism of tyrosine. Allelic variants of the GST Z1 and GST A2 genes have also been identified by EST database analysis. One GST Z1 variant (GST Z1A) has significantly higher activity with dichloroacetic acid as a substrate than other GST Z1 isoforms. This variant may be important in the clinical treatment of lactic acidosis where dichloroacetic acid is prescribed. Our experience with the application of EST database searching methods suggests that it may be productively applied to other gene families of pharmacogenetic interest.

Amino Acid Sequence↗
PubMed2001

Efflux of glutathione conjugate of monochlorobimane from striatal and cortical neurons.

Evidence for the presence of a novel transporter in primary cultures of rat striatal neurons and mouse cortical neurons similar in function to the multidrug resistance-associated protein (MRP1) is presented. Functional activity was assessed by efflux studies with the glutathione conjugate of monochlorobimane (B-SG). The glutathione transferase-catalyzed formation of B-SG in rat striatal neurons and mouse cortical neurons was inhibited by ethacrynic acid. The efflux of B-SG from rat striatal neurons and mouse cortical neurons was lower at 20 degrees C than at 37 degrees C and was lower in cells with reduced ATP concentrations compared with cells with constitutive ATP concentrations. In addition, the efflux of B-SG was inhibited by MK-571 in both rat striatal and mouse cortical neurons and by probenecid in rat striatal neurons, but not in mouse cortical neurons. Verapamil did not inhibit B-SG efflux in either rat striatal or mouse cortical neurons. Although functionally similar to MRP1, Western blot analysis with commercially available antibodies directed against human and mouse MRP1 failed to show MRP1-like protein in either whole-cell homogenates of rat striatal neurons or mouse cortical neurons, indicating that the described neuronal transporter differs in structure from human or mouse MRP1 or lacks epitopes in common with MRP1.

ATP-Binding Cassette Transporters↗