Biochemical analysis of toxic effects of 5-hydroxymethyl-2'-deoxyuridine in mammalian cells

Cellular and biochemical analyses of the toxic effects of the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine (hmdUrd), were carried out using V79.5 Chinese hamster cells. It was found that the toxic effects of hmdUrd could be totally suppressed by the addition of thymidine at 1/10th the concentration of hmdUrd. When other pyrimidines were tested, deoxyuridine was found to also suppress toxicity, although not as well as thymidine, while orotate, uridine, cytidine, and deoxycytidine did not have significant effect. Biochemical analyses of the metabolic fate of hmdUrd demonstrated low but significant levels of hmdUrd triphosphate and the incorporation of 5-hydroxymethyluracil (hmUra) residues into DNA. Surprisingly, in addition to these metabolites, relatively high levels of free hmUra were also detected in the acid-soluble cell extracts. Further analysis demonstrated that when V79.5 cells were exposed to hmdUrd significant amounts of hmUra were released into the culture medium. In vitro assays provided evidence that hmdUrd was first phosphorylated to its monophosphate and then degraded to hmUra, possibly via the action of a new enzyme, hydroxymethyldeoxyuridylate phosphorylase. Exposure of cells to hmUra alone, at concentrations as high as 3 mM, had no effect on viability. However, when V79.5 cells were simultaneously exposed to low, nontoxic concentrations of hmdUrd and high, nontoxic concentrations of hmUra, a synergistic reduction in viability was observed. This synergistic effect was found to correlate with increased incorporation of hmUra into DNA, possibly via end-product inhibition of an hmUra-DNA glycosylase.