Cysteine conjugate β-lyase of rat kidney cytosol: Characterization, immunocytochemical localization, and correlation with hexachlorobutadiene nephrotoxicity

doi:10.1016/0041-008X(89)90224-X

Toxicology and Applied Pharmacology

Volume 98, Issue 2, April 1989, Pages 185-197

https://doi.org/10.1016/0041-008X(89)90224-X Get rights and content

Abstract

Cysteine conjugate β-lyase (β-lyase) was purified to electrophoretic homogeneity from the kidney cytosol of male Wistar rats. The highly purified enzyme exhibited a monomeric molecular weight of 50,000 Da and was active in the α-β elimination of cysteine conjugates including S-(1,2-dichlorovinyl)-l-cysteine (DCVC), S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC), and S-(2-benzothiazolyl)-l-cysteine, particularly toward DCVC and TFEC. The purified enzyme also exhibited glutamine transaminase K activity with phenylalanine and α-keto-γ-methiolbutyrate as substrates. An antibody was raised to the purified rat protein in sheep and the crude immune serum affinity purified, yielding a specific antibody that recognized only the β-lyase protein in whole kidney homogenates. Immunocytochemical studies on rat kidney sections stained with the purified antibody revealed that the cytosolic β-lyase enzyme was mainly localized in the pars recta of the proximal tubule in untreated rats. This localization is coincident with the site-specific kidney necrosis produced by hexachloro-1,3-butadiene (HCBD). These results indicate that the tissue localization of β-lyase in the proximal tubule plays an important role in determining the specific nephrotoxicity produced by halogenated alkenes such as HCBD.

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The kidney possess most of the common drug-metabolising enzymes, and thus is able to make an important contribution to the body’s metabolism of drugs and other xenobiotics. The catalytic activity of the various enzymes is lower than in the liver, however enzymes involved in the processing of GSH conjugates to their mercapturic acids primarily involves the kidney. Xenobiotic-metabolising enzymes are not evenly distributed along the nephron: cytochromes P450 and enzymes involved in conjugation, glucuronidation or sulfation are present in higher concentrations in proximal tubular cells then other regions. There are however exception where some isoenzymes of P450 and GST’s are selectively localized in cells of the thick ascending limb. Most of the major form of cytochrome P450 are detected in the kidneys of experimental animals and humans. However, members of the CYP1 family, CYP2D6 and CYP2E1 appear to be, absent in human kidney. GST’s have been most extensively characterised in rat and humans kidney. The renal enzyme cysteine conjugate β-lyase involved in the bioactivation of haloalkane and haloalkene S-conjugates have been cloned and sequenced from both rat and human kidney and shown to be identical to glutamine transaminase K and KAT. Cloning approaches have also demonstrated the presence in the kidney of multiple forms of UGT, flavin monooxygenases, CES and SULTS. Bioactivation of certain chemicals by SULTS can generate reactive metabolites that modify DNA and leads to renal cancer. All of the enzymes discussed in addition to detoxification of foreign chemicals, and in a few case causing bioactivation have endogenous functions in the body. Such as regulation of salt and water balance, the control of blood pressure and synthesis of vitamin D and there are many more physiological roles for these enzymes awaiting discovery.
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The kidney is recognized as one of the most common target organs of toxicity of drugs and environmental chemicals. It is particularly susceptible because of the high blood flow to this organ relative to its mass and the unique property of renal tubular epithelium in concentrating urine and its constituents, including drugs and chemicals. The gold standard method for identification of renal toxicity potential of new products is light microscopic examination of the kidney, supplemented with renal function tests and biochemical biomarkers of renal cell injury in blood and urine. Purposely designed time-course studies that utilize ultrastructural pathology play a pivotal role in continued development of products exhibiting nephrotoxic potential. To refine the risk assessment, establishment of the cellular and subcellular organelle targets of xenobiotic injury by the pathologist forms a cornerstone in expanding the mechanistic understanding of the injury process. Demonstration of the pathogenesis of the full spectrum of toxic lesions enables the definition of primary or secondary changes and reversible, irreversible, or progressive processes. Through identification of cellular or subcellular organelle target sites, the most sensitive biomarkers can be identified to non-invasively monitor for the presence of a specific xenobiotic insult in laboratory animals and eventually in humans. This chapter emphasizes the structural and biochemical changes that occur in nephrotoxicity and the potential mechanisms involved. The rat is the species of focus, since it is the most important model in renal toxicologic pathology. Gross, microscopic, and ultrastructural anatomy is presented, followed by physiologic considerations, notable species, sex- and age-related differences in susceptibility to nephrotoxicity, and occurrence of common spontaneous renal diseases in laboratory animals. Screening strategies for new chemicals, potential mechanisms of renal injury, and the methods to characterize these mechanisms are reviewed on a sub-topographical basis using specific drug and chemical examples for illustration.
Kidney
2013, Haschek and Rousseaux's Handbook of Toxicologic Pathology, Third Edition: Volume 1-3
The kidney is recognized as one of the most common target organs of toxicity of drugs and environmental chemicals. It is particularly susceptible because of the high blood flow to this organ relative to its mass and the unique property of renal tubular epithelium in concentrating urine and its constituents, including drugs and chemicals. The gold standard method for identification of renal toxicity potential of new products is light microscopic examination of the kidney, supplemented with renal function tests and biochemical biomarkers of renal cell injury in blood and urine. Purposely designed time-course studies that utilize ultrastructural pathology play a pivotal role in continued development of products exhibiting nephrotoxic potential. To refine the risk assessment, establishment of the cellular and subcellular organelle targets of xenobiotic injury by the pathologist forms a cornerstone in expanding the mechanistic understanding of the injury process. Demonstration of the pathogenesis of the full spectrum of toxic lesions enables the definition of primary or secondary changes and reversible, irreversible, or progressive processes. Through identification of cellular or subcellular organelle target sites, the most sensitive biomarkers can be identified to non-invasively monitor for the presence of a specific xenobiotic insult in laboratory animals and eventually in humans. This chapter emphasizes the structural and biochemical changes that occur in nephrotoxicity and the potential mechanisms involved. The rat is the species of focus, since it is the most important model in renal toxicologic pathology. Gross, microscopic, and ultrastructural anatomy is presented, followed by physiologic considerations, notable species, sex- and age-related differences in susceptibility to nephrotoxicity, and occurrence of common spontaneous renal diseases in laboratory animals. Screening strategies for new chemicals, potential mechanisms of renal injury, and the methods to characterize these mechanisms are reviewed on a sub-topographical basis using specific drug and chemical examples for illustration.
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All of the common drug-metabolizing enzymes involved in phase 1 and phase 2 metabolism of xenobiotics are present in the kidney, although the catalytic activity of the various enzymes in the whole kidney is lower than that in the liver. Renal drug-metabolizing enzymes are, however, not evenly distributed along the nephron; cytochrome’s P450 and enzymes involved in conjugation with glutathione (GSH), glucuronic acid, or sulfate are present at much higher concentrations in cells of the proximal tubule than in other parts of the nephron, and thus comparison on a whole organ basis underestimates renal cell activity. Hence, the proximal tubule is the principal site of xenobiotic biotransformation and is particularly susceptible to chemical insult, while the localization of prostaglandin H-synthase (PHS) to the inner medulla and papilla may be a contributory factor to the toxicity produced by chemicals in this part of the nephron.
Many xenobiotic-biotransformation enzymes have been cloned and sequenced, and their presence in the kidney detected. Most of the major families of cytochrome P450 involved in xenobiotic metabolism have been detected in the kidneys of experimental animals and humans. Members of the CYP2B subfamily appear to be absent from rat and mouse kidney but present in the rabbit and hamster. The CYP4A subfamily involved in ω and ω–1 oxidation of fatty acids and arachidonic acid (AA) are expressed at particularly high levels in the kidney and are involved in the side chain metabolism of many drugs and xenobiotics. Glutathione S-transferases (GSTs) have been most extensively characterized in rat and human kidney, this tissue having a different subunit profile from that of the liver, with a predominance of A (alpha) and P1-1 (pi) classes. Cloning has identified a number of relatively new forms of GSTs, and although these enzymes are mainly located in the cytosol, some are associated with microsomes and mitochondria. The kidney plays a pivotal role in the processing of GSH conjugates to their cysteine conjugates and then mercapturic acids for excretion in urine. The renal enzyme cysteine conjugate β-lyase involved in the bioactivation of haloalkane and haloalkene S-cysteine conjugates has been cloned and sequenced from both rat and human kidney, and is shown to be identical to glutamine transaminase K and kynurenine aminotransferase. Cloning approaches have also demonstrated the presence in the kidney of multiple forms of UDP-glucuronosyltransferase, flavin monooxygenase, carboxylesterase, and sulfotransferase
The presence and localization of xenobiotic biotransformation enzymes in human kidney are still limited, although information on embryonic and fetal tissue is beginning to emerge. Many of the enzymes discussed, in addition to their ability to metabolize drugs or foreign compounds, have endogenous functions in the kidney, for instance, in the regulation of salt and water balance, the control of blood pressure and inflammation, and the synthesis of vitamin D.
Analysis of renal cell transformation following exposure to trichloroethene in vivo and its metabolite S-(dichlorovinyl)-l-cysteine in vitro
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Trichloroethene (TCE) is classified as a potential human carcinogen although there is a significant debate regarding the mechanism of TCE induced renal tumor formation. This controversy stems in part from the extremely high doses of TCE required to induce renal tumors and the potential contribution of the associated nephrotoxicity to tumorigenesis. We have used Eker rats, which are uniquely susceptible to renal carcinogens, to determine if exposures to TCE in vivo or exposure to its metabolite S-(dichlorovinyl)-l-cysteine (DCVC) in vitro can transform kidney epithelial cells in the absence of cytotoxicity. Treatment with TCE (0, 100, 250, 500, 1000 mg/kg bw by gavage, 5 days a week) for 13 weeks resulted in a significant increase in cell proliferation in kidney tubule cells, but did not enhance formation of preneoplastic lesions or tumor incidence in Eker rat kidneys as compared to controls. In vitro, concentrations of DCVC, which reduced cell survival to 50%, were able to transform rat kidney epithelial cells. However, no carcinogen-specific mutations were identified in the VHL or Tsc-2 tumor suppressor genes in the transformants. Taken together, the inability of TCE to enhance formation of preneoplastic changes or neoplasia and the absence of carcinogen-specific alteration of genes accepted to be critical for renal tumor development suggest that TCE mediated carcinogenicity may occur secondary to continuous toxic injury and sustained regenerative cell proliferation.
The role of glutamine transaminase K (GTK) in sulfur and α-keto acid metabolism in the brain, and in the possible bioactivation of neurotoxicants
2004, Neurochemistry International
Citation Excerpt :
As discussed in Section 3, a cysteine S-conjugate β-lyase of kidney cytosol has been identified as GTK (Stevens et al., 1986; Perry et al., 1993, 1995; MacFarlane et al., 1989; Goldfarb et al., 1996). It has generally been assumed that GTK is a major culprit in bioactivating DCVC and TFEC in rat kidney (e.g., Perry et al., 1993, 1995; MacFarlane et al., 1989; Goldfarb et al., 1996). However, at least nine mammalian PLP-containing enzymes, including kynureninase (Stevens, 1985), alanine-glyoxylate aminotransferase II, and several glutamate-utilizing aminotransferases have also been shown to catalyze cysteine S-conjugate β-lyase reactions (reviewed by Cooper et al., 2002a).
Glutamine transaminase K (GTK), which is a freely reversible glutamine (methionine) aromatic amino acid aminotransferase, is present in most mammalian tissues, including brain. Quantitatively, the most important amine donor in vivo is glutamine. The product of glutamine transamination (i.e., α-ketoglutaramate; αKGM) is rapidly removed by cyclization and/or conversion to α-ketoglutarate. Transamination is therefore “pulled” in the direction of glutamine utilization. Major biological roles of GTK are to maintain low levels of phenylpyruvate and to close the methionine salvage pathway. GTK also catalyzes the transamination of cystathionine, lanthionine, and thialysine to the corresponding α-keto acids, which cyclize to ketimines. The cyclic ketimines and several metabolites derived therefrom are found in brain. It is not clear whether these compounds have a biological function or are metabolic dead-ends. However, high-affinity binding of lanthionine ketimine (LK) to brain membranes has been reported.
Mammalian tissues possess several enzymes capable of catalyzing transamination of kynurenine in vitro. Two of these kynurenine aminotransferases (KATs), namely KAT I and KAT II, are present in brain and have been extensively studied. KAT I and KAT II are identical to GTK and α-aminoadipate aminotransferase, respectively. GTK/KAT I is largely cytosolic in kidney, but mostly mitochondrial in brain. The same gene codes for both forms, but alternative splicing dictates whether a 32-amino acid mitochondrial-targeting sequence is present in the expressed protein. The activity of KAT I is altered by a missense mutation (E61G) in the spontaneously hypertensive rat. The symptoms may be due in part to alteration of kynurenine transamination. However, owing to strong competition from other amino acid substrates, the turnover of kynurenine to kynurenate by GTK/KAT I in nervous tissue must be slow unless kynurenine and GTK are sequestered in a compartment distinct from the major amino acid pools. The possibility is discussed that the spontaneous hypertension in rats carrying the GTK/KAT I mutation may be due in part to disruption of glutamine transamination.
GTK is one of several pyridoxal 5′-phosphate (PLP)-containing enzymes that can catalyze non-physiological β-elimination reactions with cysteine S-conjugates containing a good leaving group attached at the sulfur. These elimination reactions may contribute to the bioactivation of certain electrophiles, resulting in toxicity to kidney, liver, brain, and possibly other organs. On the other hand, the β-lyase reaction catalyzed by GTK may be useful in the conversion of some cysteine S-conjugate prodrugs to active components in vivo.
The roles of GTK in (a) brain nitrogen, sulfur, and aromatic amino acid/kynurenine metabolism, (b) brain α-keto acid metabolism, (c) bioactivation of certain electrophiles in brain, (d) prodrug targeting, and (e) maintenance of normal blood pressure deserve further study.

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Cysteine conjugate β-lyase of rat kidney cytosol: Characterization, immunocytochemical localization, and correlation with hexachlorobutadiene nephrotoxicity

Abstract

Anal. Biochem.

Anal. Biochem.

Biochem. Pharmacol.

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