Recovery of 1-chloro-2,4-dinitrobenzene detoxification by N-acetyl-L-cysteine in glutathione predepleted human erythrocytes

Glutathione is an important thiol-containing compound involved in the detoxification process in erythrocytes. Its thiol group reacts with a variety of xenobiotics in a glutathione S-transferase catalyzed reaction to form conjugates that are effluxed from the erythrocytes by an ATP dependent transport mechanism. A well studied experimental system is the transport of the conjugate of glutathione and 1-chloro-2,4-dinitrobenzene. We investigated whether N-acetyl-L-cysteine protects the free-SH content and restores 1-chloro-2,4-dinitrobenzene detoxification in erythrocytes or replaces glutathione in detoxification process in glutathione predepleted erythrocytes. Our results indicate that N-acetyl-L-cysteine restores the intracellular free-SH content following depletion by 1-chloro-2,4-dinitrobenzene and N-ethylmaleimide. N-acetyl-L-cysteine (10 mM) increased the intraerythrocyte free-SH level to 14 ± 1 µmol/ml erythrocytes in 10 min in erythrocytes treated with N-ethylmaleimide. The control level was 5 ± 0.1 µmol/ml RBC. Results showed that N-acetyl-L-cysteine, in the presence and absence of L-buthioninesulfoximine, significantly recovered the 1-cloro-2,4-dinitrobenzene detoxification process in erythrocytes. The rate of conjugate transport in glutathione predepleted and N-acetyl-L-cysteine treated erythrocytes was 449 ± 38 nmol/ml erythrocytes. In the absence of N-acetyl-L-cysteine the rate of transport was 214 ± 21 nmol/ml erythrocytes which remained similar to the control. Our results suggest that N-acetyl-L-cysteine recovers the dinitrophenyl-glutathione transport and also replaces glutathione in the detoxification of 1-cloro-2,4-dinitrobenzene in glutathione predepleted erythrocytes.

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1. Sies H. Glutathione and its role in cellular functions. Free Radical Biol Med 27: 916-921, 1999. 2. Hinhman C A, Matsumoto H, Simmons T W, et al. Intrahepatic conversion of glutathione conjugate to its mercapturic acid. J Biol Chem 266: 22179-22185, 1991. 3. Cossum P A. Role of the red blood cell in drug metabolism. Biopharm Drug Dispos. 9: 321-336, 1988. 4. Flora S J S. Arsenic-induced oxidative stress and its reversibility following combined administration of N-acetylcysteine and meso 2,3-dimercaptosuccinic acid in rats. Clin Exp Pharmacol P 26: 865-869, 1999. 5. Rebbeor J F, Wang W, Clifton D, et al. Glutathione S-conjugate formation and metabolism in HepG2 cells: a cell model of mercapturic acid biosynthesis. J Toxicol Env Heal 53: 651-663, 1998. 6. Lunn G, Dale G L, Beutler E. Transport accounts for glutathione turnover in human erythrocytes. Blood 54: 239-244, 1979. 7. Eckert K G, Eyer P. Formation and transport of xenobiotic glutathione S-conjugates in red cells. Biochem Pharmacol 35: 325-329, 1986. 8. Strange R C, Jones P W, Fryer A A. Glutathione S-transferase: genetics and role in toxicology. Toxicol Lett 112: 357-363, 2000. 9. Board P G. Transport of glutathione S-conjugate from human erythrocytes. FEBS Lett 124: 163-165, 1981. 10. Keppler D. Export pumps for glutathione S-conjugates. Free Radical Biol Med 27: 985-991, 1999. 11. Awasthi Y C, Garg H S, Dao D D. Enzymatic conjugation of erythrocyte glutathione with 1-chloro-2,4-dinitrobenzene: the fate of glutathione conjugate in erythrocytes and the effect of glutathione depletion on hemoglobin. Blood 4: 733-738, 1981. 12. Vries N D, Flora S D. N-aceytl-L-cysteine. J Cell Biochem 17F: 270-277, 1993. 13. Flora S D, Bennicelli C, Camoirano A, et al. In vivo effects of Nacetylcysteine on glutathione metabolism and on the biotransformation carcinogenic and/or mutagenic compounds. Carcinogenesis 6: 1735-1745, 1985. 14. Mazor D, Golan E, Philip V, et al. Red blood cell permeability to thiol compounds following oxidative stress. Eur J Haematol 57: 241-246, 1996. 15. Sedlak J, Lindsay R H. Determination of sulfhydryl groups in biological samples. Anal Biochem 25: 192-205, 1968. 16. Traub A, Margulis S B, Stricker R B. Topical immune modulation with diclorobenzene in HIV disease: a controlled trial from Brazil. Dermatology 195(4): 369-73, 1997. 17. Griffith O W. Biologic and pharmacological regulation of mammalian glutathione synthesis. Free Radical Biol Med 27: 922- 935, 1999.
18. Libermann J E, Hahn S M, Cook J A, et al. Glutathione depletion by L- buthionine sulfoximine antagonizes taxol toxicity. Cancer Res 53: 2066-2070, 1993. 19. Hercbergs A, Simoni F B, Holtzman F, et al. Erythrocyte glutathine and tumor response to chemotherapy. Lancet 339: 1074-1076, 1992. 20. Myers S R, Pinorini-Godly M T, Reddy T V, et al. Gas chromatographic and mass spectrometric determination of hemoglobin adducts of 1,3-dinitronbenzene and 1,3,5- trinitrobenzene in shrew. Toxicol Appl. Pharmacol. (Submitted) 21. Weinander R, Anderson C, Morgenstern R. Identification of Nacetylcysteine as a new substrate for rat liver microsomal glutathione transferase. A study of thiol ligands. J Biol Chem 269(1): 71-6, 1994. 22. Sugimoto M, Kuhlenkamp J, Ookhtens M, et al. Gammaglutamylcysteine: a substrate for glutathione-S-transferase. Biochem Pharmacol 34(20): 3643-7, 1985.