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Oxidative DNA damage was studied after exposing two human transformed cell lines (HeLa and Hep G2) and freshly isolated human peripheral lymphocytes to the herbicide, paraquat. We used the alkaline comet assay, modified by incubating nucleoid DNA with endonuclease III to detect oxidised pyrimidines, and with formamidopyrimidine glycosylase to detect 8-oxo-guanine and ring-opened purines. Paraquat induces both strand breaks and oxidised bases, the amounts of each being dependent on the concentration of paraquat and the cell type exposed. Exposure to lower concentrations of paraquat for 1 hour induced dose-dependent DNA damage in Hep G2 cells and in human peripheral lymphocytes. DNA damage was reduced at higher concentrations. Our results support the finding that paraquat induces oxidative stress but, over a certain concentration range, also stimulates antioxidant protection. Reduction of DNA damage was not found in HeLa cells after exposure for 1 hour or 24 hours. Short-term exposure to paraquat induced a moderate amount of oxidative DNA damage (mainly oxidized pyrimidines) in HeLa cells. Exposure for 24 hours induced a high proportion of oxidised bases and strand breaks. Hep G2 cells showed the greatest number of DNA strand breaks, with no sign of base oxidation.

Levels of DNA damage in groups of 10 patients with insulin-dependent diabetes mellitus and 10 matched controls were compared using the comet assay; DNA strand breaks, oxidized pyrimidines (endonuclease III-sensitive sites) and altered purines (sites sensitive to formamidopyrimidine glycosylase) were measured. Mean values of strand breaks and oxidized pyrimidines were significantly higher in diabetics. Strand breaks correlated with body mass index in the diabetic group. A strong correlation was seen between formamidopyrimidine glycosylase-sensitive sites and serum glucose concentrations. When three patients with normal glucose levels were excluded from the statistical analysis, the mean value of formamidopyrimidine glycosylase-sensitive sites was very significantly elevated compared with normal. DNA damage in lymphocytes is thus a useful marker of oxidative stress, and in particular formamidopyrimidine glycosylase-sensitive sites seem to represent changes specifically related to hyperglycemia.

The comet assay (single-cell gel electrophoresis), which measures DNA strand breaks at the level of single cells, is very easily applied to human lymphocytes, and therefore lends itself to human biomonitoring studies. For the examination of DNA base oxidation (a specific marker of oxidative damage), the assay is modified by including a stage at which the DNA is incubated with a suitable lesion-specific endonuclease. Here we report on the reliability and reproducibility of this approach, from the level of comparing results from duplicate gels prepared from the same sample of cells, up to an assessment of the natural intra- and interindividual variability in lymphocyte DNA damage measured in groups of normal, healthy human volunteers. We applied the assay in investigations of human disease and occupational exposure of factory workers.

Glutathione S-transferase genotypes GSTT1, GSTM1, GSTP1 were characterised in 155 middle-aged men and compared with parameters of oxidative stress at the level of DNA and lipids, with antioxidant enzymes, and with plasma antioxidants in smokers and non-smokers. Smokers had on average significantly lower levels of Vitamin C, beta-carotene and beta-cryptoxanthin and higher amounts of oxidised purines and pyrimidines in lymphocyte DNA. The GSTM1 null genotype was associated with elevated glutathione as well as with higher Vitamin C concentration in plasma. Vitamin C was higher in GSTT1+ compared with GSTT1 null--as was glucose-6-phosphate dehydrogenase activity. The homozygous GSTP1 a/a genotype was associated with significantly higher levels of GST activity measured in lymphocytes, in comparison with the b/b genotype. Using multifactorial statistical analysis we found significant associations between smoking, GSTP1 genotype, plasma Vitamin C, and purine base damage in lymphocyte DNA. The difference in Vitamin C plasma levels between smokers and non-smokers was seen only with the GSTP1 b/b genotype. This group accounted also for most of the increase in purine oxidation in smokers. In contrast, the link between smoking and oxidised pyrimidines in DNA was seen only in the GSTT1 null group. It seems that polymorphisms in the phase II metabolising enzyme glutathione S-transferase may be important determinants of commonly measured biomarkers.

Keywords: Ankylosing spondylitis, hyperglycemia, hyperlipidemia, oxidative stress, DNA damage, comet assay, oxidized bases