Application of SCGE-FPG in the study of arsenic-induced oxidative DNA damage in PHA-stimulated and unstimulated human lymphocytes / 中华预防医学杂志

<p><b>OBJECTIVE</b>To confirm that arsenic (As) induces oxidative DNA damage in phytohemagglutinin (PHA)-stimulated and unstimulated human lymphocytes.</p><p><b>METHODS</b>The alkaline comet assay combined with specific enzyme (Formamidopyrimidine-DNA glycosylase, FPG) digestion was used to measure As-induced base damage.</p><p><b>RESULTS</b>The enzyme-sensitive sites were readily detected with the alkaline comet assay after the cells were treated with 10 micromol As for 2 hours. The repair patterns observed for FPG-created DNA single strand breaks (SSBs) in As-treated cells were comparable to those in hydrogen peroxide (H(2)O(2))-treated cells. The enzyme-created SSBs, As-induced base damage, were more significantly revealed in PHA-stimulated lymphocytes. About 63% and 68% of SSBs induced by As and H(2)O(2), respectively, were repaired in PHA-stimulated lymphocytes by 2-hour repair incubation, but about 34% and 43%, respectively, were repaired in unstimulated cells. About 40% and 49% of base damage induced by As and H(2)O(2), respectively, were repaired in PHA-stimulated lymphocytes, but about 19% and 21 %, respectively, were repaired in unstimulated cells.</p><p><b>CONCLUSIONS</b>As induces oxidative DNA damage in human lymphocytes within micromolar concentrations. Like the damage induced by H(2)O(2), As-induced DNA damage was more slowly repaired in unstimulated lymphocytes.</p>

Arsenic Induces DNA Damage via Reactive Oxygen Species in Human Cells

To elucidate arsenic-induced oxidative DNA damage, the genotoxicity of arsenic in human cells was comparatively studied with single cell gel electrophoresis (SCGE) assay in combination with the observation of the protective effects of dimethyl sulfoxide (DMSO) and catalase. Arsenic, at the concentration of 2.4 μM by coincubation for 24 hours, significantly induced DNA damage in HL60, a human promyelocytic leukemia cell line. In contrast, significant DNA damage was found in human mononucleocytes at the concentration of 4.8 μM or above. The cells were incubated separately with DMSO (12 mM/l), a well-known hydroxyl radical (OH-) scavenger, and catalase (1,300 U/ml), a hydrogen peroxide (H2O2) scavenger, for 6 hours and then further coincubated with various concentrations of arsenic for 24 hours at 37°C and 5% CO2. The findings showed that both DMSO and catalase significantly reduced the arsenic-induced tail moment, a parameter of total damaged DNA, in HL60 and mononucleocytes. Hence our findings indicate that arsenic, with micromolar concentrations, induces typical and various extents of DNA damage in human cells via reactive oxygen species in a dose-dependent manner.

Effects of (-)-Epigallocatechin Gallate (EGCG) on DNA Strand Breaks as Evaluated by Single-cell Gel Electrophoresis (SCG) in Human Lymphocytes

(-)-Epigallocatechin gallate (EGCG), a catechin polyphenol component, is the main ingredient of green tea extract. Although the anti-carcinogenic and cancer inhibitory effects of EGCG have been widely reported, its genotoxicity is not clear and seldom reported. In this study, we examined the effects of EGCG on DNA strand breaks in the isolated lymphocytes and whole blood lymphocytes obtained from two smoking subjects and a nonsmoking healthy subject using a single-cell gel electrophoresis (SCG) assay. The results showed that after 2 hrs of treating the isolated lymphocytes from the smokers, EGCG induced a significant increase in DNA strand breaks at concentrations from 2.5 × 10-5 M to 2.0 × 10-4 M, while after 2 hrs of treating the whole blood obtained from the same smokers, EGCG suppressed the DNA strand breaks in the lymphocytes at concentrations of 1.0 × 10-4 M and 2.0 × 10-4 M. A similar suppressive result was also shown in the whole blood lymphocytes from the nonsmoker at nearly the same concentrations, while at concentrations of 1.0 × 10-3 M or 2.0 × 10-3 M, EGCG induced a significant increase in DNA strand breaks in the whole blood lymphocytes from the nonsmoker. This result suggests that EGCG is not only inhibitory against DNA strand breaks in whole blood, but also genotoxic to the isolated or whole blood lymphocytes at high concentrations. Thus, more research is needed to comprehensively assess the effects of EGCG on genetic materials.