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.
Highlights
The molecular mechanism of toxicity is still being debated it has been proposed that a free radical formed by a one-electron reduction of SR 4233 is the toxic species (Baker et al, 1988; Laderoute et al, 1988) as the two-electron and four-electron reduction metabolites, SR 4317 and SR 4330 did not prevent colony formation (Baker et al, 1988)
SR 4233 was cytotoxic under an aerobic environment only when the cell's defence system against oxidative stress was compromised by inactivating hepatocyte catalase with azide or glutathione reductase with BCNU beforehand
SR 4233 is shown to be metabolised in isolated hepatocytes by NADPH:cytochrome P-450 reductase resulting in cellular toxicity in the absence of oxygen which can probably be attributed to the SR 4233 radical
Summary
The molecular mechanism of toxicity is still being debated it has been proposed that a free radical formed by a one-electron reduction of SR 4233 is the toxic species (Baker et al, 1988; Laderoute et al, 1988) as the two-electron and four-electron reduction metabolites, SR 4317 and SR 4330 did not prevent colony formation (Baker et al, 1988). Toxicity has been postulated to occur via hydrogen abstraction from DNA and other cell constituents by the one-electron reduced SR 4233 free radical (Zeman et al, 1986; Baker et al, 1988) which was presumably detoxified by oxygen. DTdiaphorase isolated from Walker 256 rat tumour cells, was shown to detoxify SR 4233 by catalysing two- and fourelectron reduction of SR 4233 (Riley & Workman, 1992)
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