Fresco, Paula Maria Façanha da Cruz; (1996) The role of ascorbate in the activation of chromium(V) to its ultimate carcinogenic form. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Chromium(V) compounds are established human carcinogens. It is now well accepted that chromium(V) is activated by a number of intracellular reducing agents to its ultimate carcinogenic form. However, despite intensive research efforts during the last ten years, both the nature of the ultimate DNA damaging species and the relative contribution of the various possible reductive pathways for the activation of chromium(V) remain obscure. Prompted by recent reports on the role of ascorbate as an important reductant of chromium(V) in vivo this study has investigated the potential of ascorbate to activate chromium(V) to DNA damaging species. Single-strand breaks (SSB) and apurinic/apyrimidinic sites (AP-sites) were readily observed in isolated DNA of the bacteriophage PM2 which was treated with chromate in the presence of ascorbate levels similar to those observed in mammalian cells. The number of both SSB and AP-sites rose with increasing concentrations of chromate. An as yet unidentified intermediate formed during the reduction of chromate by ascorbate was found to be responsible for the DNA damage. Molecular oxygen was essential for the process leading to SSB and AP-sites. Both these lesions occurred with equal probability and their formation followed a very similar time course. Attempts were made to probe the nature of the DNA damaging species arising from chromate/ascorbate. Modified DNA bases resulting from attack by hydroxyl radicals were not observed using Gas Chromatography-Mass Spectrometry in selective ion monitoring mode, ruling out hydroxyl radicals as the species causing SSB and AP-sites. Finally, the potential of the DNA lesions formed by chromium(V)/ascorbate to cause mutations was assessed. Treatment of plasmid DNA (pUC19) with chromate and ascorbate gave rise to elevated mutation frequencies in the lacZ' gene, detected after transformation of E. coli TG1, indicating that the reductive conversion of chromium(V) by ascorbate may be an important pathway in the causation of mutations.

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