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Hypobaric hypoxia has been used to induce tumor hypoxia for in vivo comparison of the anti-tumor effects of the bioreductive agents SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide), RSU 1069 (1(2-nitro-1-imidazolyl)-3-aziridino-2-propanol), and Nitromin (methylbis(2-chloroethyl)amine N-oxide). BDF mice bearing the T50/80 mammary carcinoma were treated with these agents over a range of doses under normobaric (oxic) and hypobaric (hypoxic) conditions. The time taken for the tumor to double treatment volume (volume doubling time) was used as a measure of anti-tumor effect. Volume doubling time was plotted against log dose and dose response curves were fitted. A dose enhancement ratio (the ratio of drug doses required to give an equivalent anti-tumor effect under oxic and hypoxic conditions) was determined. The dose enhancement ratios for SR 4233 and RSU 1069 were 8.8 and 8.5, respectively, showing that these agents had an equivalent and substantial enhancement of their cytotoxicity when combined with hypobaric hypoxia. For Nitromin, no significant dose response effect was obtained under oxic conditions precluding the calculation of the dose enhancement ratio. SR 4233 was found to have increased systemic toxicity when combined with hypobaric hypoxia, suggesting that it is more readily activated than the other drugs tested. This in vivo test system will allow determination of the dose enhancement ratio for novel bioreductive agents and facilitate their comparison.

The hepatic microcirculation is believed to cause variable cellular oxygenation within the organ. In this study a marker of cellular hypoxia was used to demonstrate liver oxygen tension gradients in vivo. Covalent binding of misonidazole adducts to cellular macromolecules is enhanced by hypoxia. Autoradiographs of liver from mice treated with radiolabeled misonidazole demonstrated enhanced binding of adducts within hepatocytes surrounding hepatic veins. Livers from both hypoxic and normal mice had characteristic autoradiographic grain patterns reflecting regional oxygen tension variation in vivo. Differential binding of misonidazole adducts formed in hypoxic cells could have an application in studies of liver physiology and biochemistry.