The clinical agent PR-104 is converted systemically to PR-104A, a nitrogen mustard prodrug designed to target tumor hypoxia. Reductive activation of PR-104A is initiated by oneelectron oxidoreductases in a process reversed by oxygen. The identity of these oxidoreductases is unknown, with the exception of cytochrome P450 reductase (POR). To identify other hypoxia-selective PR-104A reductases, nine candidate oxidoreductases were expressed in HCT116 cells. Increased PR-104A-cytotoxicity was observed in cells expressing methionine synthase reductase (MTRR), novel diflavin oxidoreductase 1 (NDOR1), and inducible nitric-oxide synthase (NOS2A), in addition to POR. Plasmid-based expression of these diflavin oxidoreductases also enhanced bioreductive metabolism of PR-104A in an anoxia-specific manner. Diflavin oxidoreductase-dependent PR-104A metabolism was suppressed Ͼ90% by pan-flavoenzyme inhibition with diphenyliodonium chloride. Antibodies were used to quantify endogenous POR, MTRR, NDOR1, and NOS2A expression in 23 human tumor cell lines; however, only POR protein was detectable and its expression correlated with anoxic PR-104A reduction (r 2 ϭ 0.712). An anti-POR monoclonal antibody was used to probe expression using human tissue microarrays; 13 of 19 cancer types expressed detectable POR with 21% of cores (185 of 874) staining positive; this heterogeneity suggests that POR is a useful biomarker for PR-104A activation. Immunostaining for carbonic anhydrase 9 (CAIX), reportedly an endogenous marker of hypoxia, revealed only moderate coexpression (9.6%) of both CAIX and POR across a subset of five cancer types.

The radical species underlying the activity of the bioreductive anticancer prodrug, SN30000, have been identified by electron paramagnetic resonance and pulse radiolysis techniques. Spin-trapping experiments indicate both an aryl-type radical and an oxidising radical, trapped as a carbon-centred radical, are formed from the protonated radical anion of SN30000. The carbon-centred radical, produced upon the one-electron oxidation of the 2-electron reduced metabolite of SN30000, oxidises 2-deoxyribose, a model for the site of damage on DNA which leads to double strand breaks. Calculations using density functional theory support the assignments made.

Epidemiological studies have associated coffee consumption with an inverse risk of developing Parkinson's disease, hepatocellular carcinoma and cirrhosis. The molecular mechanisms by which low concentrations of the constituents of coffee measured in human plasma can reduce the incidence of such diseases are not clear. Using an in vitro plasmid DNA system and radiolytically generated reactive oxygen species under constant radical scavenging conditions, we have shown that coffee chlorogenic acid, its derivatives and certain metabolites of caffeine reduce some of the free radical damage sustained to the DNA. A reduction in the amount of prompt DNA single-strand breaks (SSBs) was observed for all compounds whose radical one-electron reduction potential is < 1.0 V. However, except for chlorogenic acid, the compounds were found to be inactive in reducing the amount of radical damage to the DNA bases. These results support a limited antioxidant role for such compounds in their interaction with DNA radicals.