Nalini Mistry

Vitamin C exhibits pro-oxidant properties

Vitamin C is marketed as a dietary supplement, partly because of its ‘antioxidant’ properties. However, we report here that vitamin C administered as a dietary supplement to healthy humans exhibits a pro-oxidant, as well as an antioxidant, effect in vivo.

Novel repair action of vitamin C upon in vivo oxidative DNA damage

There appears to be a paucity of data examining the effect of dietary antioxidants on levels of oxidative DNA damage in vivo, limiting evidence‐based assessment of antioxidant efficacy, mechanisms and recommendation for optimal intake. We have examined levels of 8‐oxo‐2′‐deoxyguanosine (8‐oxodG) in mononuclear cell DNA, serum and urine from subjects undergoing supplementation with 500 mg/day vitamin C. Significant decreases in DNA levels of 8‐oxodG were seen, correlating strongly with increases in plasma vitamin C concentration. Furthermore we established a timecourse for sequential, significant increases in serum and urinary 8‐oxodG levels. These results illustrate, for the first time in humans, the kinetics of 8‐oxodG removal and processing in vivo, suggesting a role for vitamin C in the regulation of DNA repair enzymes and thereby demonstrating a non‐scavenging antioxidant effect.

Immunogenicity of DNA damaged by reactive oxygen species--implications for anti-DNA antibodies in lupus.

Reactive oxygen species (ROS) are implicated in the inflammatory, autoimmune, connective tissue disease, systemic lupus erythematosus (SLE), particularly in respect of processes leading to the formation of pathological anti-DNA antibodies. Exposure to ROS increases the antigenicity of DNA for SLE antibodies, but data on the immunogenicity of ROS-DNA are not conclusive. In this study, we have examined the immunogenicity in rabbits, of DNA modified by three hydroxyl radical generating systems. Additionally, we investigated the antigenicity of UVA, UVB, and UVC irradiated DNA for lupus anti-DNA antibodies. Modification of DNA by both ROS and far UV dramatically increased its immunogenicity; the Fe2+ and H2O2 system resulted in antibodies that recognized both native and modified DNA. In our ELISA system, none of the UV antigens showed any antigenicity above native DNA for SLE sera. The data suggested that different profiles of antigenicity and immunogenicity arise dependent on the method of ROS production, but also that ROS-DNA may be a factor in antigen-driven immune complex formation in SLE.

A novel HPLC procedure for the analysis of 8-oxoguanine in DNA

The chromatographic quantitation of 8-oxoguanine adducts in DNA is widespread in the literature, although results obtained by HPLC of 8-oxodeoxyguanosine do not always agree with levels determined by GC-MS. To help explain this discrepancy, here we describe a novel procedure for the analysis of 8-oxoguanine adducts in DNA. Although it proved difficult to directly quantitate 8-oxoguanine in the presence of high levels of endogenous guanine using conventional reversed-phase HPLC, a simple preincubation of DNA acid hydrolysates with guanase allowed such analyses. The assay relied on our observation that 8-oxoguanine was not a substrate for guanase, and on sensitive electrochemical detection. The limit of detection for 8-oxoguanine was 5 nM or 250 fmol on column. Using this procedure, the background level of 8-oxoguanine in commercially available calf thymus DNA was 0.4 nmol/mg DNA or 3.2 mol/10(5) mol guanine.

Role of dietary antioxidants in the prevention of in vivo oxidative DNA damage.

Epidemiological evidence consistently shows that diets high in fresh fruit and vegetables significantly lower cancer risk. Given the postulated role of oxidative DNA damage in carcinogenesis, the assumption has been made that it is the antioxidant properties of food constituents, such as vitamin C, E and carotenoids, which confer protection. However, epidemiological studies with specific antioxidants, either singly or in combination, have not, on the whole, supported this hypothesis. In contrast, studies examining the in vitro effect of antioxidants upon oxidative DNA damage have generally been supportive, in terms of preventing damage induction. The same, however, cannot be said for the in vivo intervention studies where overall the results have been equivocal. Nevertheless, recent work has suggested that some dietary antioxidants may confer protective properties through a novel mechanism, unrelated to their conventional free-radical scavenging abilities. Upregulation of antioxidant defence, xenobiotic metabolism, or DNA-repair genes may all limit cellular damage and hence promote maintenance of cell integrity. However, until further work has clarified whether dietary supplementation with antioxidants confers a reduced risk of cancer and the mechanism by which this effect is exerted, the recommendation for a diet rich in fruit and vegetables remains valid empirically.

Immunochemical detection of UV-induced DNA damage and repair.

The application of an antiserum to ultraviolet radiation (UVR)-damaged DNA is presented. A novel experimental system was employed to ascertain the limits of detection for this antiserum. Using a DNA standard containing a known amount of dimer, the limits of detection were found to be 0.9 fmol of dimer. This was compared to a limit of 20-50 fmol dimer using gas chromatography-mass spectrometry (GC-MS). Induction of thymine dimers in DNA following UVR exposure, as assessed using this antiserum in an enzyme-linked immunosorbent assay (ELISA), was compared with GC-MS measurements. The ELISA method successfully demonstrated the induction of lesions in DNA irradiated either with UVC or UVB, although despite high sensitivity, no discernible binding was seen to UVA-irradiated DNA. The antiserum was also shown to be applicable to immunocytochemistry, localising damage in the nuclei of UVR exposed keratinocytes in culture. The ability of the antiserum to detect DNA damage in skin biopsies of individuals exposed to sub-erythemal doses of UVR was also demonstrated. Moreover, the subsequent removal of this damage, as evidenced by a reduction in antiserum staining, was noted in sections of biopsies taken in the hours following irradiation.

Immunochemical quantitation of UV-induced oxidative and dimeric DNA damage to human keratinocytes

There is growing evidence to suggest that solar radiation-induced, oxidative DNA damage may play an important role in skin carcinogenesis. Numerous methods have been developed to sensitively quantitate 8-oxo-2′deoxyguanosine (8-oxodG), a recognised biomarker of oxidative DNA damage. Immunoassays may represent a means by which the limitations of many techniques, principally derived from DNA extraction and sample workup, may be overcome. We report the evaluation of probes to thymine dimers and oxidative damage in UV-irradiated cells and the DNA derived therefrom. Thymine dimers were most readily recognised, irrespective of whether in situ in cells or in extracted DNA. However, using antibody-based detection the more subtle oxidative modifications required extraction and, in the case of 8-oxodG, denaturation of the DNA prior to successful recognition. In contrast, a recently described novel probe for 8-oxodG detection showed strong recognition in cells, although appearing unsuitable for use with extracted DNA. The probes were subsequently applied to examine the relative induction of lesions in cells following UV irradiation. Guanine-glyoxal lesions predominated over thymine dimers subsequent to UVB irradiation, whereas whilst oxidative lesions increased significantly following UVA irradiation, no induction of thymine dimers was seen. These data support the emerging importance of oxidative DNA damage in UV-induced carcinogenesis.

Novel monoclonal antibody recognition of oxidative DNA damage adduct, deoxycytidine-glyoxal.

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p > 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC50 values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2′-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.

Deoxycytidine glyoxal: lesion induction and evidence of repair following vitamin C supplementation in vivo.

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p =.001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p =.0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected.

Dose-dependent modulation of the T cell proteome by ascorbic acid

To investigate the hypothesis that the micronutrient ascorbic acid can modulate the functional genome, T cells (CCRF-HSB2) were treated with ascorbic acid (up to 150 microM) for up to 24 h. Protein expression changes were assessed by two-dimensional electrophoresis. Forty-one protein spots which showed greater than two-fold expression changes were subject to identification by matrix-assisted laser desorption ionisation time of flight MS. The confirmed protein identifications were clustered into five groups; proteins were associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of phosphatidylinositol transfer protein (promotes intracellular signalling) within 5 min of ascorbic acid treatment was confirmed by Western blotting. Together, these observations suggest that ascorbic acid modulates the T cell proteome in a time- and dose-dependent manner and identify molecular targets for study following antioxidant supplementation in vivo.