A study of genoprotective effects of functional foods/herbs using various versions of the comet assay

Abstract

Evidence shows that oxidative damage is a key factor in ageing and age-related disease and conceptually increased intake of dietary antioxidants may help to prevent oxidative damage in vivo, maintain DNA integrity, and so promote healthy ageing. DNA can also be damaged by oxidative and non-oxidative process involving ultraviolet (UV) irradiation, and this is a major cause of skin cancer in the elderly. The focus of herbs for health promotion is increasing worldwide, but scientific evidence of benefit is lacking, especially in regard to effects on DNA. The overall aim of this study was to assess the genoprotective and DNA repair effects of selected herbs, including Ganoderma lucidum (Lingzhi), Camellia sinensis (green tea), Vaccinium myrtillus L (bilberry) and Cordyceps sinensis (Cordyceps) by using various versions of the comet assay in human controlled trials and in vitro study. These herbs were selected because of their high reputation for health promotion and on published reports of potential benefit against oxidative stress or on DNA. The main aim of the Lingzhi study was to investigate the acute post ingestion effect of Lingzhi on DNA repair by using the timed repair version of the comet assay. Plasma 'total' antioxidant capacity in plasma pre-and post-ingestion was also assessed by FRAP assay. These were assessed in a cross-over human intervention study, with blood samples collected from 7 healthy volunteers pre and 60 and 180 mins after single dose (3.3g) of Lingzhi. Results indicated that ingestion of Lingzhi did not significantly affect the rate or efficiency of DNA repair of oxidation-induced damage. Lingzhi ingestion was shown to increase plasma antioxidant capacity slightly, however the increase was not statistically significant. The main aims of green tea study was to investigate the genoprotective effect of regular intake of two green teas (Loongjin and Screw-shaped) on oxidative stress (using urine 8-oxodG as the biomarker, measured by LC-MS/MS) and DNA oxidation-induced damage (using the Fpg-assisted version of the comet assay). The relationship between these two biomarkers was also investigated. This trial was a single-blinded, multiple cross-over, placebo-controlled study, and blood and urine samples were collected from 18 healthy volunteers after 4 weeks' supplementation with each tea. Results showed that after supplementation of each tea, Fpg labile sites were ~30% less. However, no significant changes in urine 8-oxodG were seen and no significant correlation was seen between Fpg comet assay results and urine 8-oxodG results. The specific aim of bilberry study was to investigate the effect of supplementation with bilberry (which is rich in anthocyanins) on base excision repair pathway in Type 2 diabetes patients using a lymphocyte extract version of the comet assay. This was assessed by a double-blinded, placebo-controlled cross-over intervention study, blood samples were collected from 20 Type 2 diabetes patients after 4 weeks' supplementation. Results showed no significant changes in hOGG1 activity after bilberry supplementation. The specific aim of Cordyceps study was to investigate the genoprotective effects of polysaccharide-rich Cordyceps mycelial components against UVB-induced damage in normal human fibroblast cells. Cultured fibroblasts were pre-treated for 30 mins and 24 hours with defined concentration of hot water extracts of Cordyceps mycelia or mycelial exopolysaccharides. Fibroblasts were washed, irradiated with UVB, and immediately lysed, after which DNA damage was measured using the T4EV-assisted comet assay that detects predominantly a type of pyrimidine dimer (CPDs) that is a DNA lesion produced specifically by UVB. Fibroblasts showed significant (P<0.01) downward trend in DNA damage with increasing concentrations of each extract for both 30 mins and 24 hrs pre-incubation treatments. Numerous mechanisms are involved in genoprotection, including cytoprotective adaptation, antioxidant property and upregulating DNA repair capacity. Lack of genoprotective effect in particular mechanism does not mean it has no beneficial effect (Lingzhi and bilberry studies). Precaution also should be taken to avoid overstating the genoprotective effect of green tea and Cordyceps, since the extact mechanisms behind the effects are still unclear. Nonetheless, the new data presented here are supportive of green tea against oxidation-induced DNA damage, and are also supportive of Cordyceps polysaccharides against non-oxidation-induced DNA damage caused by UVB.