Cysteine Conjugate Β-lyase Research Articles

Renal necrosis and DNA damage caused by selectively deuterated and methylated analogs of 1,2-dibromo-3-chloropropane in the rat

Selectively deuterated and methylated analogs of the nematocide 1,2-dibromo-3-chloropropane (DBCP) were compared to DBCP in causing acute renal damage in rats. All of the six deuterated analogs tested at 340 μmol/kg, including the perdeutero compound, failed to significantly alter the kidney necrosis observed at 48 hr compared to DBCP. Furthermore, when the perdeutero analog was administered at several doses (42.5, 85, 170, and 340 μmol/kg), it caused kidney damage that was not significantly different than that caused by an equivalent molar dose of nondeuterated DBCP. Of the five methylated analogs tested at 170 and 340 μmol/kg, only C 3-methyl-DBCP and 1,2-dibromo-4-chlorobutane caused nephrotoxicity. The C 2-methyl-, C 1-dimethyl-, and C 2-methyl-DBCP analogs failed to cause renal necrosis determined 48 hr after dosing. In distribution studies DBCP, perdeutero-DBCP, and all the methylated analogs were found to concentrate in the kidney approximately 25 times relative to plasma 1 hr after administration. DBCP at doses of 4.3 μmol/kg and higher caused DNA damage in the kidney as early as 10 min after administration, as measured by alkaline elution of DNA from isolated kidney nuclear preparations. Perdeuteration did not decrease the DNA damaging effect of DBCP. The ability of the methylated DBCP analogs to induce renal DNA damage correlated with their necrogenic potential. Experiments using pretreatments that are known to decrease the nephrotoxicity caused by glutathione and cysteine conjugates of several halogenated alkenes were conducted to examine the effect of these pretreatments on DBCP-induced nephrotoxicity. Probenecid, l-(α S,5 S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) and aminooxyacetic acid did not significantly alter renal necrosis or DNA damage induced by DBCP. Based on the absence of any significant isotope effects with the perdeutero-DBCP analog, it appears that breaking of a carbon-hydrogen bond is not the rate-limiting step in DBCP-induced nephrotoxicity. Studies with the methylated DBCP analogs indicate that a vicinal dibromo ethyl group must minimally be present for nephrotoxic potential. Furthermore, it seems unlikely that metabolism by renal cysteine conjugate β-lyase is rate-limiting for DBCP nephrotoxicity.

Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat: Indications for differential mechanisms of bioactivation

1,1-Dichloro-2,2-difluoroethylene (DCDFE) produced marked nephrotoxicity in rats upon an i.p. dose of 150 μmole/kg. At doses higher than 375 μmole/kg, DCDFE also produced hepatotoxicity. Aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase, appeared to be slightly nephrotoxic in Wistar rats. Nevertheless it exerted an inhibitory effect on the nephrotoxicity of DCDFE. The N-acetylcysteine conjugate of DCDFE was identified as a major urinary metabolite of DCDFE. When administered as such, this conjugate appeared to be a potent nephrotoxin, without any effect on the liver, indicating that glutathione conjugation of DCDFE is most likely a bioactivation step for nephrotoxicity. The appearance of traces of chlorodifluoroacetic acid in urine of rats treated with higher doses of DCDFE indicates the existence of an oxidative pathway of metabolism of DCDFE, probably involving epoxidation by hepatic mixed-function oxidases. It is speculated that the latter route might account for the hepatotoxicity at higher doses of DCDFE. The nephro- and hepatotoxicity of DCDFE, therefore, most likely are the result of two different mechanisms of bioactivation.

Role of glutathione in acute N-(3,5-dichlorophenyl) succinimide-induced nephrotoxicity in Sprague-Dawley and Fischer 344 rats

N-(-3,5-Dichlorophenyl)succinimide (NDPS), an experimental agricultural fungicide, has been shown to be a selective nephrotoxin in Sprague-Dawley and Fischer 344 rats. Previous studies have demonstrated that a toxic metabolite contributes to or is responsible for acute NDPS-induced nephrotoxicity. The purpose of this study was to investigate the role of glutathione in NDPS-induced renal effects. In 1 set of experiments, male Sprague-Dawley or Fischer 344 rats received a single intraperitoneal (i.p.) injection of NDPS (0.4 or 1.0 mmol/kg) or sesame oil (2.5 ml/kg). Rats were killed at 1, 3, 6 or 24 h, and reduced (GSH) and oxidized (GSSG) glutathione concentrations determined in liver and renal cortex. In both rat strains NDPS (0.4 or 1.0 mmol/kg) administration produced small decreases in GSH concentrations (1 and 3 h) but moderate increases in GSSG concentrations (1 and 3 h) in liver and kidney. At 24 h both GSH and GSSG concentrations were increased, particularly in kidney. In a second set of experiments, rats were pretreated with the glutathione depletor diethyl maleate (DEM) (0.4 ml/kg, i.p.) 1 h prior to NDPS (0.2, 0.4 or 1.0 mmol/kg, i.p.) or sesame oil (2.5 ml/kg, i.p.) administration, and renal function monitored at 24 and 48 h. DEM pretreatment attenuated the increase in urine volume (24 and 48 h), proteinuria, glucosuria, hematuria and elevated blood urea nitrogen (BUN) concentration by NDPS (0.4 or 1.0 mmol/kg) in both Sprague-Dawley and Fischer 344 rats. NDPS-induced increases in kidney weight also were generally prevented by DEM pretreatment. Proximal tubular necrosis produced by NDPS administration was reduced by DEM but not prevented. Pretreatment with the cysteine conjugate β-lyase inhibitor amino-oxyacetic acid (0.5 mmol/kg, i.p.) 1 h prior to NDPS (0.4 or 1.0 mmol/kg) markedly attenuated all NDPS-induced effects on renal function and morphology. These results suggest that glutathione does not play a protective role against NDPS-induced renal effectc and that a glutathione or cysteine conjugate of NDPS might contribute to NDPS-induced nephrotoxicity.

Biochemistry and physiology of brain ammonia.

Biochemistry and physiology of brain ammonia.

The mechanism of pentachlorobutadienyl-glutathione nephrotoxicity studied with isolated rat renal epithelial cells

Isolated renal epithelial cells were used to study the mechanism of toxicity of pentachlorobutadienyl-glutathione (PCBG), a nephrotoxic glutathione conjugate of hexachlorobutadiene. The cytotoxicity of PCBG displayed a very steep dose-response relationship; at 10 μ m PCBG no toxicity was observed whereas 25, 50, and 100 μM PCBG all resulted in a similar degree of toxicity. In all cases, loss of cell viability was observed only after a 30-min lag period and reached a plateau of 50 to 60% nonviable cells between 90 and 100 min. Toxic doses of PCBG also resulted in the depletion of cellular thiols. Blocking PCBG metabolism by inhibition of γ-glutamyl transpeptidase [1-γ- l-glutamyl-2-(2-carboxyphenyl)hydrazine (anthglutin), 2 m m]or renal cysteine conjugate β-lyase (aminooxyacetic acid, 0.5 m m) resulted in complete protection against PCBG-induced cell damage. Exposure of isolated renal epithelial cells to 100 μ m PCBG resulted in the rapid formation of plasma membrane blebs which appeared to be associated with a loss of Ca 2+ from the mitochondrial compartment and an elevation of cytosolic Ca 2+ concentration as measured by Quin-2. PCBG treatment also resulted in the inhibition of cell respiration and a marked depletion of cellular ATP content, indicating additional mitochondrial effects of the toxin. Our results support a role for renal cysteine conjugate β-lyase in the metabolic activation of PCBG and suggest that PCBG-induced renal cell injury may be the result of selective effects on mitochondrial function.

Mechanism of S-(1,2-dichlorovinyl)glutathione-induced nephrotoxicity

S-(1,2-rmDichlorovinyl)glutathione and S-(1,2- dichlorovinyl)- dl-cysteine are potent nephrotoxins. Agents that inhibit γ-glutamyl transpeptidase, cysteine conjugate β-lyase, and renal organic anion transport systems, namely l -(αS,5S)-α'- amino-3- chloro-4,5- dinydro-5- isoxazoleacetic acid (AT-125), aminooxyacetic acid, and probenecid, respectively, protected against S-conjugate-induced nephrotoxicity. Furthermore, S-(1,2- dichlorovinyl)- dl -α- methylcysteine , which cannot be cleaved by cysteine conjugate β-lyase, was not nephrotoxic. These results strongly support a role for renal γ-glutamyl transpeptidase, cysteine conjugate β-lyase, and organic anion transport systems in S-(1,2-dichlorovinyl)glutathione- and S-(1,2,-dichlorovinyl)cysteine-induced nephrotoxicity.

Cysteine conjugate β-lyase activities in rat kidney cortex: Subcellular localization and relationship to the hepatic enzyme

Kidney cortex cysteine conjugate β-lyase enzymes were characterized using S -(2-benzothiazolyl)- L -cysteine and S -(1,2-dichlorovinyl)- L -cysteine as substrates. The contribution of the hepatic form of cysteine conjugate β-lyase to renal metabolism of these S -cysteine conjugates is not substantial. No cysteine conjugate β-lyase activity was found in kidney cortex brush border membrane vesicles. Two cysteine conjugate β-lyase activities with densities corresponding to the mitochondrial and soluble fractions were separated on Percoll gradients.

65] Cysteine conjugate β-lyase

The cysteine conjugate β-lyase enzyme is effective with thioethers of cysteine and with derivatives of alanine bearing a good leaving group at the β-carbon. The β-1yase represents the initial enzyme in a shunt pathway from mercapturic acid synthesis; that also includes thiol S -methyltransferase. Two assay methods for this enzyme are discussed in this chapter. Both methods allow continuous spectrophotometric estimation of the reaction with time. In one of these methods, the formation of 2-mercaptobenzothiazole at 316 nm provides a sensitive assay in both crude and purified systems. The disadvantage of the method is the need to synthesize the substrate. The other method is presented for β-chloroalanine, a substrate that is commercially available, but the assay can be adapted for use with any of the substrates of the enzyme that produce pyruvate. This method is ineffective for crude extracts because of the presence of interfering activities.

Metabolism of 2-chloro-N-isopropylacetanilide in chickens

2-Chloro- N -isopropylacetanilide was quantitatively metabolized in chickens in the mercapturic acid pathway (MAP). The MAP metabolites (cysteine conjugate, S -carboxymethyl- N -acetyl cysteine, N -acetyl cysteine conjugate, and N -acetyl cysteine sulfoxide conjugate) were excreted mainly with the urine with minor amounts (less than 7% of the dose) excreted with bile. The cysteine conjugate was precursor for all the MAP metabolites, as well as, for N -isopropyloxanilic acid and N -isopropylacetanilide. Cysteine conjugate β-lyase activity was detected in the intestinal contents in vitro but was not manifested in vivo when the cysteine conjugate was injected intracecally.

Cysteine conjugate beta-lyase in the gastrointestinal bacterium Fusobacterium necrophorum.

A cysteine conjugate beta-lyase from the anaerobic gastrointestinal bacterium Fusobacterium necrophorum was purified 51-fold by heat treatment, ammonium sulphate fractionation, gel-filtration chromatography, and anion-exchange chromatography. This enzyme catalyses the cleavage of the thioether linkage in cysteine conjugates of the following S-alkyl- or S-aryl-linked compounds: cysteine conjugate of propachlor (2-S-cysteinyl-N-isopropylacetanilide); 1,2-dihydro-1-hydroxy-2-S-cysteinylnaphthalene and S-(2-benzothiazolyl)cysteine. 2-Mercapto-N-isopropylacetanilide, pyruvic acid and ammonia were produced from the beta-lyase cleavage of the cysteine conjugate of propachlor in equimolar ratios. The apparent Km values for the cysteine conjugate of propachlor and S-(benzothiazolyl)cysteine were 1.1 and 1.0 mM, respectively. Pyridoxal phosphate was required for enzymic activity. Ammonium ion activated enzymic activity, while hydroxylamine completely inhibited the enzyme. Dithiothreitol and bovine serum albumin had no effect on enzymic activity.

Assay of cysteine conjugate β-lyase activity with S-(2-benzothiazolyl)cysteine as the substrate

Cysteine conjugate β-lyases convert S-substituted cysteine conjugates to pyruvate, ammonia, and thiols. A simple assay for cysteine conjugate β-lyase activity was developed with S-(2-benzothiazolyl)cysteine as the substrate. The production of 2-mercaptobenzothiazole was measured by its intense absorbance at 321 nm in trichloroacetic acid-quenched reaction mixtures. The formation of 2-mercaptobenzothiazole was directly proportional to protein concentrations of 0.17 to 1.2 mg/ml with rat liver cytosol as the source of β-lyase activity. Production of 2-mercaptobenzothiazole was stoichiometric with pyruvate and was increased by addition of pyridoxal phosphate only at reaction times of 5 min or longer. The simplicity, sensitivity, and specificity of this procedure offer significant advantages over other methods for the assay of cysteine conjugate β-lyase activity.

31] Cysteine conjugate β-lyase

This chapter presents a procedure for the preparation of cysteine conjugate β-lyase. The Pprinciple of assay method states that cysteine conjugates of aromatic compounds (R) serve as the substrate of β-lyase. Utilizing a substrate labeled with 35 S, the product of the reaction, R-[ 35 S]H, can be measured after converting to R-[ 35 S]CH 3 with methyl iodide. The derivatized product, thus, can be purified by thin-layer chromatography and its radioactivity can be determined. Purification procedure involves the extraction of supernatant fluid, followed by heat treatment and the addition of ammonium sulfate. After thisthe enzyme solution is concentrated to 20 ml with a Diaflo PM-10 membrane filter and is applied to a column of DEAE-cellulose equilibrated with the same buffer; further, the combined enzyme preparation is concentrated to approximately 15 ml with a Diaflo PM-10 membrane and is applied to a Sephadex G-200 column. Overall, 1100-fold purification is achieved through the Sephadex step.