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Cytotoxicity of 2-bromo-(N-acetylcystein-,S-yl)-hydroquinone isomers in rat renal tubular epithelial cells
143
Toxicology Letters, 53 (1990f 143-145
Elsevier
TOXLET 02401
Cytotoxicity of 2-bromo-(N-acetylcystein-S-yl)hydroquinone isomers in rat renal tubular epithelial cells
Michael Koobl, Andreas KGchling*, Simone Weberl, Serrine S. Lau2, Terrence J. Monks2 and Wolfgang Dekantl lInstitutfiir
Tox~ologie,
of Pharmacology
Universitiit
and Toxicology,
Wiirzburg,
University
Wiirrburg (F.R.G.)
ofTexas,Austin,
and Yoltege
of Pharmacy, Division
TX (U.S.A.1
Key words: Isolated kidney cells; S-Conjugates; Cytotoxicity; Ion transport; Calcium
Glutathione (GSH) S-conjugate formation is frequently associated with the detoxification of reactive intermediates. However, recent evidence suggests that GSH-Sconjugates may be toxic. For example, some nephrotoxic haloalkenes and haloalkanes are bioactivated by GSH-~-conjugate formation [I, 21. The kidney is a frequent target for biosynthetic toxic S-conjugates due to its ability to actively concentrate amino acid derivatives. GSH and cysteine S-conjugates of hydroquinones are another type of nephrotoxic conjugate (3). The mercapturic acids 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone, 2-bromo-5-(N-acetylcystein-S-yl)hydroquinone and 2bromo-6-(~-acetyIcystein-~-yl)hydroquinone are putative metabolites of the nephrotoxic bromoquinone [4]. Their biosynthesis might involve reaction of bromoquinone with GSH to form bromohydroquinone-GSH-S-conjugates, processing by y-glutamyltranspeptidase and dipeptidases to the corresponding cysteine-S-conjugates and, finally, acylation to the mercapturic acid. To study the mechanisms of in vitro nephrotoxicity of these mercapturic acids, the cytotoxicity of synthetic 2-bromo-3-(~-a~tylcystein-~-yl)hydroquinone, 2-bromo-5(N-acetylcystein-S-yi)hydroquinone and 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone was investigated in freshly isolated rat tubular epithelial cells. Cells were
Address for correspondence: W. Dekant, Institut fiir Toxikologie, UniversitIt Strasse 9, D-8700 Wiirzburg, F.R.G.
037&4274/90/S
3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
W~~burg,
Versbacher
144
isolated by collagenase perfusion, and cell viability was determined by the Trypan blue exclusion technique; cellular concentrations of GSH and GSSG were determined by high-performance liquid chromatography by the method of Reed et al. [5]. 2-Bromo-3-(N-acetylcystein-S-yl)hydroquinone, 2-bromo-5-(ZV-acetylcystein-Syl)hydroquinone and 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone, at concentrations of 0.05 and 0.025 mM, caused a dose-dependent reduction of cell viability from 85-90s at 0 min to 30-32s (0.05 mM mercapturate) and 37-39s (0.025 mM), respectively, after 180 min. In control cells, no decrease in viability was observed. The organic ion transport inhibitor probenecid (1 mM) completely protected cells from 2-bromo-3-(N-acetylcystein-9yl)hydroquinone-induced loss of cell viability; the cysteine conjugate /I-lyase inhibitor amino-oxyacetic acid did not offer any protection. Antioxidants, such as a-tocopherol and N,N-diphenylphenylendiamine and the ironchelating agent desferrioxamine partially protected the cells from 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone-induced toxicity. To test the hypothesis that treatment of rat renal tubular epithelial cells results in an elevation of ionized cytosolic calcium prior to cell death and, that activation of catabolic enzymes, such as endonucleases, contributes to cell death, the effect of several agents interfering with this pathway on 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone-induced toxicity was studied. The calcium chelator Quin-2-ethoxyethylester and the endonuclease inhibitor aurintricarboxylic acid protected the cells from 2bromo-3-(N-acetylcystein-S-yl)hydroquinone-induced toxicity. Endonuclease-mediated DNA-damage may lead to cell death through the activation of poly(ADP)ribose)polymerase; thus, 3-aminobenzamide (5 mM), an inhibitor of poly(ADP-ribose)polymerase, also reduced 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone-induced toxicity. 2-Bromo-3-(N-acetylcystein-S-yl)hydroquinone rapidly depleted cellular GSH concentrations to less than 5 % of controls within 5 min of application; no concomitant oxidation of GSH to GSSG was observed. Of the inhibitors used, only probenecid prevented 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone-indu~d GSH depletion. The results suggest that 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone is concentrated in cells by the organic-anion transporter and oxidized to the corresponding quinone; this soft electrophile reacts rapidly with GSH, thus depleting cellular GSH concentrations. As a result of the massive GSH depletion, peroxidative mechanisms then cause an elevation of the cytosolic concentrations of ionized calcium [6], thereby activating catabolic enzymes such as endonucleases. The resulting DNA damage induces poly(ADP-ribose)polymerization followed by depletion by cellular NAD+, finally resulting in cell death [7]. REFERENCES 1 Anders, M.W., Lash, L.H., Dekant, W., Elfarra, A.A. and Dohn, D.R. (1988) Biosynthesis and biotransformation of glutathione S-conjugates to toxic metabolites. CRC Crit. Rev. Toxicol. 18, 31 l-342.
145 2 Dekant, W., Lash, L.H. and Anders, M.W. (1988) Fate of ~utathione conjugates and bioactivation of cysteine S-conjugates by cysteine conjugate @-lyase. In: H. Sies and B. Ketterer (Eds.), Glutathione Conjugation: Its Mechanism and Biological Significance. Academic Press, London, pp. 415-447. 3 Lau, S.S., Hill, B.A., Highet, R.J. and Monks, T.J. (1988) Sequential oxidation and glutathione addition to 1,4-benzoquinone: correlation of toxicity with increased glutathione substitution. Mol. Pharmacol. 34,829~836. 4 Monks, T.J., Lau, S.S., Highet, R.J. and Gillette, J.R. (1985) Glutathione conjugates of 2-bromohydroquinone are nephrotoxic. Drug Metab. Disposit. 13,553-559. 5 Reed, D.J., Babson, J.R., Beatty, P.W., Brodie, AX., Ellis, W.W. and Potter, D.W. (1980) High-performance liquid chromatography analysis of nanomole levels of glutathione glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106,55-62. 6 Muehlematter, D., Larsson, R. and Cerutti, P. (1988) Active oxygen induced DNA strand breakage and poty ADP-ribosylation in promotable and non-promotable JB6 mouse epidermal cells. Carcinogenesis 9,239245. 7 Schraufstatter, I.U., Hyslop, P.A., Hinshow, D.B., Spragg, R.G., Sklar, L.A. and Cochrane, C.G. (1986) Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose)polymerase. Proc. Natl. Acad. Sci. USA 83,4908-4912.
Toxicology Letters, 53 (1990f 143-145
Elsevier
TOXLET 02401
Cytotoxicity of 2-bromo-(N-acetylcystein-S-yl)hydroquinone isomers in rat renal tubular epithelial cells
Michael Koobl, Andreas KGchling*, Simone Weberl, Serrine S. Lau2, Terrence J. Monks2 and Wolfgang Dekantl lInstitutfiir
Tox~ologie,
of Pharmacology
Universitiit
and Toxicology,
Wiirzburg,
University
Wiirrburg (F.R.G.)
ofTexas,Austin,
and Yoltege
of Pharmacy, Division
TX (U.S.A.1
Key words: Isolated kidney cells; S-Conjugates; Cytotoxicity; Ion transport; Calcium
Glutathione (GSH) S-conjugate formation is frequently associated with the detoxification of reactive intermediates. However, recent evidence suggests that GSH-Sconjugates may be toxic. For example, some nephrotoxic haloalkenes and haloalkanes are bioactivated by GSH-~-conjugate formation [I, 21. The kidney is a frequent target for biosynthetic toxic S-conjugates due to its ability to actively concentrate amino acid derivatives. GSH and cysteine S-conjugates of hydroquinones are another type of nephrotoxic conjugate (3). The mercapturic acids 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone, 2-bromo-5-(N-acetylcystein-S-yl)hydroquinone and 2bromo-6-(~-acetyIcystein-~-yl)hydroquinone are putative metabolites of the nephrotoxic bromoquinone [4]. Their biosynthesis might involve reaction of bromoquinone with GSH to form bromohydroquinone-GSH-S-conjugates, processing by y-glutamyltranspeptidase and dipeptidases to the corresponding cysteine-S-conjugates and, finally, acylation to the mercapturic acid. To study the mechanisms of in vitro nephrotoxicity of these mercapturic acids, the cytotoxicity of synthetic 2-bromo-3-(~-a~tylcystein-~-yl)hydroquinone, 2-bromo-5(N-acetylcystein-S-yi)hydroquinone and 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone was investigated in freshly isolated rat tubular epithelial cells. Cells were
Address for correspondence: W. Dekant, Institut fiir Toxikologie, UniversitIt Strasse 9, D-8700 Wiirzburg, F.R.G.
037&4274/90/S
3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
W~~burg,
Versbacher
144
isolated by collagenase perfusion, and cell viability was determined by the Trypan blue exclusion technique; cellular concentrations of GSH and GSSG were determined by high-performance liquid chromatography by the method of Reed et al. [5]. 2-Bromo-3-(N-acetylcystein-S-yl)hydroquinone, 2-bromo-5-(ZV-acetylcystein-Syl)hydroquinone and 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone, at concentrations of 0.05 and 0.025 mM, caused a dose-dependent reduction of cell viability from 85-90s at 0 min to 30-32s (0.05 mM mercapturate) and 37-39s (0.025 mM), respectively, after 180 min. In control cells, no decrease in viability was observed. The organic ion transport inhibitor probenecid (1 mM) completely protected cells from 2-bromo-3-(N-acetylcystein-9yl)hydroquinone-induced loss of cell viability; the cysteine conjugate /I-lyase inhibitor amino-oxyacetic acid did not offer any protection. Antioxidants, such as a-tocopherol and N,N-diphenylphenylendiamine and the ironchelating agent desferrioxamine partially protected the cells from 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone-induced toxicity. To test the hypothesis that treatment of rat renal tubular epithelial cells results in an elevation of ionized cytosolic calcium prior to cell death and, that activation of catabolic enzymes, such as endonucleases, contributes to cell death, the effect of several agents interfering with this pathway on 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone-induced toxicity was studied. The calcium chelator Quin-2-ethoxyethylester and the endonuclease inhibitor aurintricarboxylic acid protected the cells from 2bromo-3-(N-acetylcystein-S-yl)hydroquinone-induced toxicity. Endonuclease-mediated DNA-damage may lead to cell death through the activation of poly(ADP)ribose)polymerase; thus, 3-aminobenzamide (5 mM), an inhibitor of poly(ADP-ribose)polymerase, also reduced 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone-induced toxicity. 2-Bromo-3-(N-acetylcystein-S-yl)hydroquinone rapidly depleted cellular GSH concentrations to less than 5 % of controls within 5 min of application; no concomitant oxidation of GSH to GSSG was observed. Of the inhibitors used, only probenecid prevented 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone-indu~d GSH depletion. The results suggest that 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone is concentrated in cells by the organic-anion transporter and oxidized to the corresponding quinone; this soft electrophile reacts rapidly with GSH, thus depleting cellular GSH concentrations. As a result of the massive GSH depletion, peroxidative mechanisms then cause an elevation of the cytosolic concentrations of ionized calcium [6], thereby activating catabolic enzymes such as endonucleases. The resulting DNA damage induces poly(ADP-ribose)polymerization followed by depletion by cellular NAD+, finally resulting in cell death [7]. REFERENCES 1 Anders, M.W., Lash, L.H., Dekant, W., Elfarra, A.A. and Dohn, D.R. (1988) Biosynthesis and biotransformation of glutathione S-conjugates to toxic metabolites. CRC Crit. Rev. Toxicol. 18, 31 l-342.
145 2 Dekant, W., Lash, L.H. and Anders, M.W. (1988) Fate of ~utathione conjugates and bioactivation of cysteine S-conjugates by cysteine conjugate @-lyase. In: H. Sies and B. Ketterer (Eds.), Glutathione Conjugation: Its Mechanism and Biological Significance. Academic Press, London, pp. 415-447. 3 Lau, S.S., Hill, B.A., Highet, R.J. and Monks, T.J. (1988) Sequential oxidation and glutathione addition to 1,4-benzoquinone: correlation of toxicity with increased glutathione substitution. Mol. Pharmacol. 34,829~836. 4 Monks, T.J., Lau, S.S., Highet, R.J. and Gillette, J.R. (1985) Glutathione conjugates of 2-bromohydroquinone are nephrotoxic. Drug Metab. Disposit. 13,553-559. 5 Reed, D.J., Babson, J.R., Beatty, P.W., Brodie, AX., Ellis, W.W. and Potter, D.W. (1980) High-performance liquid chromatography analysis of nanomole levels of glutathione glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106,55-62. 6 Muehlematter, D., Larsson, R. and Cerutti, P. (1988) Active oxygen induced DNA strand breakage and poty ADP-ribosylation in promotable and non-promotable JB6 mouse epidermal cells. Carcinogenesis 9,239245. 7 Schraufstatter, I.U., Hyslop, P.A., Hinshow, D.B., Spragg, R.G., Sklar, L.A. and Cochrane, C.G. (1986) Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose)polymerase. Proc. Natl. Acad. Sci. USA 83,4908-4912.