Chromium(VI)-mediated DNA damage: oxidative pathways resulting in the formation of DNA breaks and abasic sites.

Inside cells chromium(VI) is activated to its ultimate carcinogenic form by reducing agents including glutathione (GSH) and ascorbate (AsA). The precise mechanism by which DNA damaging species are formed is unclear. In earlier in vitro work with isolated DNA we have shown that chromium(VI) in combination with GSH or AsA is able to induce similar numbers of single strand breaks and apurinic/apyrimidinic sites (AP-sites). Moreover, the formation of both lesions followed a similar temporal pattern. It is conceivable that the two forms of DNA damage arise from a common precursor lesion (e.g. hydrogen abstraction at C4' of the DNA sugar moiety) with a partitioning along two pathways, one yielding an AP-site, the other a single strand break (SSB) and a base propenal. The present study is intended to test this hypothesis by analysing whether oxidation products of deoxyribose can be formed in the presence of chromium(VI) and GSH or AsA. It was found that mixtures of chromium(VI) and GSH or AsA were able to oxidise 2-deoxyribose to yield malondialdehyde, which was detected by reaction with thiobarbituric acid. The characteristic pink chromogen, which forms upon reaction with thiobarbituric acid, was also observed with calf thymus DNA as the substrate. In both experimental systems the addition of catalase prevented the formation of deoxyribose breakdown products. Hydroxyl radicals did not seem to be important for the generation of DNA damage as the characteristic modified DNA bases could not be detected by using gas chromatography-mass spectrometry. These results lead us to conclude that the formation of SSB during the reductive conversion of chromium(VI) proceeds primarily via hydrogen abstraction from C4'. The observation that Fenton chemistry is not involved in these processes is intriguing and necessitates further research into the ways in which chromium can activate molecular oxygen to form DNA damaging species.

Taurine release in the rat vas deferens is modulated by Ca2+ and is independent of contractions.

Electrical field stimulation induces taurine release in rat vas deferens. In the present study, it was investigated if this release is secondary to contraction. The influence of Ca2+ and of the stimulation conditions was also studied. Contractions evoked by electrical field stimulation (5 Hz/270 pulses, transverse or longitudinal) were recorded and released taurine was quantified by high performance liquid chromatography with fluorimetric detection. Ca2+ removal abolished contractions, but not the overflow of taurine. Overflow elicited by longitudinal electrical field stimulation was higher than that elicited by transverse electrical field stimulation. Increasing the current strength also increased taurine overflow. In Ca2+-free medium, taurine overflow was decreased by caffeine (5 mM) or ryanodine (10 microM) but increased by dantrolene (50 microM). The results indicate that taurine release evoked by electrical field stimulation is (i) independent of contraction, (ii) modulated by Ca2+, (iii) potential dependent, and may be due to a decrease in taurine affinity for the plasma membrane and/or to an increase of Na+-dependent outward transport.

The reductive conversion of the carcinogen chromium (VI) and its role in the formation of DNA lesions.

The potential of Cr(VI), in combination with glutathione (GSH) or ascorbate (AsA) to induce apurinic/apyrimidinic sites (AP-sites) and single strand breaks (SSB) in isolated deoxyribonucleic acid (DNA) was investigated. The observation that both lesions were formed with equal probability and followed a similar time course suggests that they might arise from attack of a reactive species at C4' of the DNA sugar moeity. This idea is further substantiated by the finding that malondialdehyde-like products are released in chromate/GSH- and chromate/AsA-treated DNA. The generation of AP-sites and SSB was dependent on molecular oxygen and could be suppressed by the addition of catalase. Our results rule out hydroxyl radicals as the DNA damaging species. Furthermore, Cr(V), an intermediate formed during reaction with GSH or AsA, is not directly involved in the generation of DNA damage, unless activated by molecular oxygen. Our findings indicate that a superoxo- or peroxo-complex involving Cr(V) or Cr(IV) might be the species responsible for DNA damage. Evidence is presented that the DNA lesions arising from chromate/AsA have the potential to cause gene mutations.

The reductive conversion of chromium (VI) by ascorbate gives rise to apurinic/apyrimidinic sites in isolated DNA.

The formation of apurinic/apyrimidinic sites (AP-sites) and single strand breaks (SSB) by chromate and ascorbate (AsA) in isolated DNA was investigated using a number of agents that cleave DNA at AP-sites (putrescine, exonuclease III, the tripeptide Lys-Trp-Lys, and an AP-endonuclease containing fraction isolated from human fibroblasts). Relative to the number of SSB caused by chromate and AsA alone, all these agents induced additional nicking, indicating the induction of AP-sites. Chromate/AsA-induced AP-sites contain aldehyde groups, as cleavage by putrescine could be prevented by treatment with borohydride which reduced the aldehyde. The time course for the formation of both DNA lesions was very similar, and there was a 1:1 ratio of the number of SSB to the number of AP-sites. The addition of catalase to incubation mixtures containing chromate/AsA led to an almost complete suppression of AP-sites and SSB. In systems containing lower concentrations of chromate/AsA, the exclusion of oxygen inhibited the formation of both lesions. It is suggested that AP-sites and SSB arise from attack by reactive species deriving from chromate/AsA on one single site at DNA, probably the sugar moiety. In view of the known mutagenicity of AP-sites, these results could aid an understanding of the mechanisms underlying chromium(VI) carcinogenicity.

The formation of DNA cleaving species during the reduction of chromate by ascorbate.

A detailed study of the ability of chromate in combination with ascorbate to induce DNA single-strand breaks in the absence of iron(II) and copper(II) has been carried out. In solutions containing 1 mM ascorbate and chromate in the range 0.1-1 mM extensive DNA cleavage occurred. Chromate alone or the final product of the chromate/ascorbate reaction were not responsible for the cleavage observed. Evidence is presented that an intermediate generated during the reduction of chromate is the reactive species. No strand breaks occurred upon addition of catalase, pointing to a role for peroxidic species in the steps leading to the generation of the cleaving species. The exclusion of oxygen led to a substantial decrease in the number of strand breaks. Furthermore, the formation of strand breaks declined with decreasing concentrations of phosphate in the phosphate buffers used as the incubation medium. No DNA strand breaks were induced in medium containing HEPES. These observations rule out chromium(V) as the agent directly responsible for the DNA degradation, as chromium(V) is formed during the reduction of chromate by ascorbate in HEPES buffer. Our results lead us to suggest that the DNA-damaging ability of chromate upon reduction by ascorbate arises from the activation of oxygen exacerbated by phosphate and points to a peroxo or superoxo complex involving chromium(V) or chromium(IV) as a possible candidate.