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Ischaemia-reperfusion (I/R) injury is a model system of oxidative stress and a potential anti-cancer therapy. Tumour cytotoxicity follows oxygen radical damage to the vasculature which is modulated by tumour production of the vasoactive agent, nitric oxide (NO.). In vivo hydroxylation of salicylate, to 2,3- and 2,5-dihydroxybenzoate (DHBs), was used to measure the generation of hydroxyl radicals (OH.) following temporary vascular occlusion in two murine tumours (with widely differing capacity to produce NO.) and normal skin. Significantly greater OH. generation followed I/R of murine adenocarcinoma CaNT tumours (low NO. production) compared to round cell sarcoma SaS tumours (high NO. production) and normal skin. These data suggest that tumour production of NO. confers resistance to I/R injury, in part by reducing production of oxygen radicals and oxidative stress to the vasculature. Inhibition of NO synthase (NOS), during vascular reperfusion, significantly increased OH. generation in both tumour types, but not skin. This increase in cytotoxicity suggests oxidative injury may be attenuation by tumour production of NO.. Hydroxyl radical generation following I/R injury correlated with vascular damage and response of tumours in vivo, but not skin, which indicates a potential therapeutic benefit from this approach.

We have studied the relationship between extracellular lactate (LACTe) and extracellular pH (pHe) in murine tumours after vascular occlusion (clamping) followed by reperfusion. In tumours occluded at ambient room temperature, LACTe, measured by microdialysis, increased linearly with time and correlated strongly with the acidification of the extracellular compartment (r=0.97, P<0.03, n=4). Significant decrease in LACTe was evident following removal of occlusion at room temperature and is consistent with vascular reperfusion. Occlusion at 35 degrees C, i.e. to maintain tumour temperature during occlusion, resulted in an initial increase in LACTe, which mirrored a rapid reduction in pHe. However further reductions in pHe occurred without increase in LACTe. During vascular occlusion, tumour adenine nucleotide pool decreased and AMP accumulated. AMP subsequently decreased in the 35 degrees C group and this may contribute to the observed differences in accumulation of LACTe, and capacity to recover from vascular occlusion, between the two treatment groups. These data show that extracellular lactate concentration is a good predictor for tumour pH when adequate energy sources are available within the tumour. However, under conditions of more severe stress, resulting in abolition of primary energy stores and cell death, the pHe continues to decline in the absence of a corresponding accumulation of extracellular lactate. This emphasizes the fact that other processes, apart from lactate production, can contribute to reduction in extracellular pH.

Plasma concentrations, after administration of varying doses of nicotinamide, were measured in CBA male mice using a newly-developed high performance liquid chromatography assay. In all dose groups, peak levels were observed within the first 15 min after an i.p. administration of 0.1, 0.2, 0.3 or 0.5 mg g-1 of nicotinamide. There was a clear dose-dependent increase in plasma concentration with increasing dose, with almost a five-fold lower concentration (1.0 vs 4.9 mumol ml-1) achieved with a dose of 0.1 mg g-1 compared with 0.5 mg g-1, respectively. The half-life of nicotinamide increased from 1.4 h to 2.2 h over the dose range (P < 0.01). Comparisons with previous pharmacokinetic data in humans show that clinically-relevant oral doses of 6 and 9 g in humans give plasma levels slightly higher than those achieved at 1 h with doses of 0.1 to 0.2 mg g-1 in mice. Tumour radiosensitisation with carbogen alone, and with carbogen combined with varying doses of nicotinamide (0.05 to 0.5 mg g-1), was investigated using a 10-fraction in 5 days X-ray schedule. Relative to air-breathing mice, a statistically significant increase in sensitisation was observed with both a local tumour control and with an in vivo/in vitro excision assay (P < or = 0.007). With the local control assay, a trend was observed towards lower enhancement ratios (ERs) with decreasing nicotinamide dose (from 1.85 to 1.55); carbogen alone was almost as effective as when combined with 0.1 mg g-1 of nicotinamide. With the excision assay, ERs for carbogen combined with nicotinamide increased with decreased levels of cell survival. At a surviving fraction of 0.02, enhancement ratios of 1.39-1.48 were obtained for carbogen plus 0.1 to 0.3 mg g-1 of nicotinamide. These were lower than those seen with the two higher doses of 0.4 to 0.5 mg g-1 (ERs = 1.63-1.69).