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. 1993 Sep;68(3):484–491. doi: 10.1038/bjc.1993.374

Molecular mechanisms of SR 4233-induced hepatocyte toxicity under aerobic versus hypoxic conditions.

J M Silva 1, P J O'Brien 1
PMCID: PMC1968404  PMID: 8394729

Abstract

SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is the lead compound of the benzotriazene-di-N oxides which are selectively toxic to tumour cells under hypoxic conditions. However much higher concentrations given to rats caused bone marrow toxicity and necrosis of the low oxygen Zone 3 part of the liver. In the following effects of SR 4233 on hepatocytes under hypoxic vs aerobic conditions have been compared. (1) SR 4233 did not affect hepatocyte viability (as determined by plasma membrane disruption) or glutathione levels under aerobic conditions. SR 4233 however induced cyanide-resistant respiration, an indicator of redox cycling mediated oxidative stress and became cytotoxic if hepatocyte catalase or glutathione reductase was inactivated. Glutathione oxidation occurred well before cytotoxicity ensued. Addition of ascorbate markedly enhanced SR 4233 cytotoxicity to these compromised hepatocytes. (2) In contrast, SR 4233 was highly toxic to hypoxic hepatocytes. Addition of ascorbate to enhance SR 4233 reduction also caused a marked increase in hepatocyte toxicity and an SR 4233 radical was detected with ESR spectroscopy. SR 4233 cellular reduction and toxicity was prevented with fructose or inhibitors of NADPH:cytochrome P-450 reductase. Inactivation of catalase or glutathione reductase had no effect on SR 4233 toxicity and hepatocyte GSH was not oxidised indicating oxidative stress did not occur during hypoxic SR 4233 hepatocyte toxicity. (3) The lack of SR 4233 cytotoxicity under aerobic conditions could probably be attributed to the detoxification of the SR 4233 radical by mitochondrial oxidation as SR 4233, but not its metabolite SR 4317 markedly increased state III and IV mitochondrial respiration in the presence of NADH. The increased respiration was inhibited by the respiratory inhibitors KCN and antimycin A but not by rotenone. Furthermore SR 4233 cytotoxicity under aerobic conditions was markedly increased by partially inhibiting hepatocytes respiration with cyanide but not rotenone.

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Selected References

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  1. Babson J. R., Reed D. J. Inactivation of glutathione reductase by 2-chloroethyl nitrosourea-derived isocyanates. Biochem Biophys Res Commun. 1978 Jul 28;83(2):754–762. doi: 10.1016/0006-291x(78)91053-7. [DOI] [PubMed] [Google Scholar]
  2. Baker M. A., Zeman E. M., Hirst V. K., Brown J. M. Metabolism of SR 4233 by Chinese hamster ovary cells: basis of selective hypoxic cytotoxicity. Cancer Res. 1988 Nov 1;48(21):5947–5952. [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Costa A. K., Baker M. A., Brown J. M., Trudell J. R. In vitro hepatotoxicity of SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide), a hypoxic cytotoxin and potential antitumor agent. Cancer Res. 1989 Feb 15;49(4):925–929. [PubMed] [Google Scholar]
  5. Doussière J., Vignais P. V. Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation. Eur J Biochem. 1992 Aug 15;208(1):61–71. doi: 10.1111/j.1432-1033.1992.tb17159.x. [DOI] [PubMed] [Google Scholar]
  6. Gores G. J., Nieminen A. L., Wray B. E., Herman B., Lemasters J. J. Intracellular pH during "chemical hypoxia" in cultured rat hepatocytes. Protection by intracellular acidosis against the onset of cell death. J Clin Invest. 1989 Feb;83(2):386–396. doi: 10.1172/JCI113896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Iles R. A., Griffiths J. R., Stevens A. N., Gadian D. G., Porteous R. Effects of fructose on the energy metabolism and acid-base status of the perfused starved-rat liver. A 31phosphorus nuclear magnetic resonance study. Biochem J. 1980 Oct 15;192(1):191–202. doi: 10.1042/bj1920191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Laderoute K., Wardman P., Rauth A. M. Molecular mechanisms for the hypoxia-dependent activation of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233). Biochem Pharmacol. 1988 Apr 15;37(8):1487–1495. doi: 10.1016/0006-2952(88)90010-x. [DOI] [PubMed] [Google Scholar]
  9. Lloyd R. V., Duling D. R., Rumyantseva G. V., Mason R. P., Bridson P. K. Microsomal reduction of 3-amino-1,2,4-benzotriazine 1,4-dioxide to a free radical. Mol Pharmacol. 1991 Sep;40(3):440–445. [PubMed] [Google Scholar]
  10. Mason R. P. Assay of in situ radicals by electron spin resonance. Methods Enzymol. 1984;105:416–422. doi: 10.1016/s0076-6879(84)05058-8. [DOI] [PubMed] [Google Scholar]
  11. Moldéus P., Högberg J., Orrenius S. Isolation and use of liver cells. Methods Enzymol. 1978;52:60–71. doi: 10.1016/s0076-6879(78)52006-5. [DOI] [PubMed] [Google Scholar]
  12. Moulder J. E., Rockwell S. Tumor hypoxia: its impact on cancer therapy. Cancer Metastasis Rev. 1987;5(4):313–341. doi: 10.1007/BF00055376. [DOI] [PubMed] [Google Scholar]
  13. O'Brien P. J. Molecular mechanisms of quinone cytotoxicity. Chem Biol Interact. 1991;80(1):1–41. doi: 10.1016/0009-2797(91)90029-7. [DOI] [PubMed] [Google Scholar]
  14. Patel J. M., Ortiz E., Kolmstetter C., Leibman K. C. Selective inactivation of rat lung and liver microsomal NADPH-cytochrome c reductase by acrolein. Drug Metab Dispos. 1984 Jul-Aug;12(4):460–463. [PubMed] [Google Scholar]
  15. Reed D. J., Babson J. R., Beatty P. W., Brodie A. E., Ellis W. W., Potter D. W. High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal Biochem. 1980 Jul 15;106(1):55–62. doi: 10.1016/0003-2697(80)90118-9. [DOI] [PubMed] [Google Scholar]
  16. Riley R. J., Workman P. Enzymology of the reduction of the potent benzotriazine-di-N-oxide hypoxic cell cytotoxin SR 4233 (WIN 59075) by NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2) purified from Walker 256 rat tumour cells. Biochem Pharmacol. 1992 Jan 22;43(2):167–174. doi: 10.1016/0006-2952(92)90274-m. [DOI] [PubMed] [Google Scholar]
  17. Rossi L., Silva J. M., McGirr L. G., O'Brien P. J. Nitrofurantoin-mediated oxidative stress cytotoxicity in isolated rat hepatocytes. Biochem Pharmacol. 1988 Aug 15;37(16):3109–3117. doi: 10.1016/0006-2952(88)90308-5. [DOI] [PubMed] [Google Scholar]
  18. Rush G. F., Alberts D. tert.-Butyl hydroperoxide metabolism and stimulation of the pentose phosphate pathway in isolated rat hepatocytes. Toxicol Appl Pharmacol. 1986 Sep 30;85(3):324–331. doi: 10.1016/0041-008x(86)90339-x. [DOI] [PubMed] [Google Scholar]
  19. Sies H., Noack G. Proton movement accompanying monocarboxylate permeation in hemoglobin-free perfused rat liver. FEBS Lett. 1972 May 1;22(2):193–196. doi: 10.1016/0014-5793(72)80042-5. [DOI] [PubMed] [Google Scholar]
  20. Silva J. M., O'Brien P. J. Diaziquone-induced cytotoxicity in isolated rat hepatocytes. Cancer Res. 1989 Oct 15;49(20):5550–5554. [PubMed] [Google Scholar]
  21. Stocchi V., Cucchiarini L., Magnani M., Chiarantini L., Palma P., Crescentini G. Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells. Anal Biochem. 1985 Apr;146(1):118–124. doi: 10.1016/0003-2697(85)90405-1. [DOI] [PubMed] [Google Scholar]
  22. Walton M. I., Wolf C. R., Workman P. Molecular enzymology of the reductive bioactivation of hypoxic cell cytotoxins. Int J Radiat Oncol Biol Phys. 1989 Apr;16(4):983–986. doi: 10.1016/0360-3016(89)90900-0. [DOI] [PubMed] [Google Scholar]
  23. Walton M. I., Workman P. Enzymology of the reductive bioactivation of SR 4233. A novel benzotriazine di-N-oxide hypoxic cell cytotoxin. Biochem Pharmacol. 1990 Jun 1;39(11):1735–1742. doi: 10.1016/0006-2952(90)90119-6. [DOI] [PubMed] [Google Scholar]
  24. Wang J., Biedermann K. A., Brown J. M. Repair of DNA and chromosome breaks in cells exposed to SR 4233 under hypoxia or to ionizing radiation. Cancer Res. 1992 Aug 15;52(16):4473–4477. [PubMed] [Google Scholar]
  25. White I. N., Cahill A., Davies A., Carthew P. Acute lesions in rats caused by 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) or nitromin: a comparison with rates of reduction in microsomal systems from target organs. Arch Toxicol. 1992;66(2):100–106. doi: 10.1007/BF02342502. [DOI] [PubMed] [Google Scholar]
  26. Winterbourn C. C. Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase. Arch Biochem Biophys. 1981 Jun;209(1):159–167. doi: 10.1016/0003-9861(81)90268-x. [DOI] [PubMed] [Google Scholar]
  27. Zeman E. M., Baker M. A., Lemmon M. J., Pearson C. I., Adams J. A., Brown J. M., Lee W. W., Tracy M. Structure-activity relationships for benzotriazine di-N-oxides. Int J Radiat Oncol Biol Phys. 1989 Apr;16(4):977–981. doi: 10.1016/0360-3016(89)90899-7. [DOI] [PubMed] [Google Scholar]
  28. Zeman E. M., Brown J. M., Lemmon M. J., Hirst V. K., Lee W. W. SR-4233: a new bioreductive agent with high selective toxicity for hypoxic mammalian cells. Int J Radiat Oncol Biol Phys. 1986 Jul;12(7):1239–1242. doi: 10.1016/0360-3016(86)90267-1. [DOI] [PubMed] [Google Scholar]

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