Aflatoxin B1-induced DNA Damage Research Articles

African Nightshade (Solanum scabrum Mill.): Impact of Cultivation and Plant Processing on Its Health Promoting Potential as Determined in a Human Liver Cell Model.

Plant cultivation and processing may impact nutrient and phytochemical content of vegetables. The present study aimed at determining the influence of cultivation and processing on the health promoting capacity of African nightshade (Solanum scabrum Mill.) leaves, an indigenous vegetable, rich in nutrients and phytochemicals. Anti-genotoxicity against the human liver carcinogen aflatoxin B1 (AFB1) as determined by the comet assay and radical oxygen species (ROS) scavenging capacity of ethanolic and aqueous extracts were investigated in human derived liver (HepG2) cells. ROS scavenging activity was assessed using electron paramagnetic spin resonance and quantification of ARE/Nrf2 mediated gene expression. The cultivation was done under different environmental conditions. The processing included fermentation and cooking; postharvest ultraviolet irradiation (UV-C) treatment was also investigated. Overall, S. scabrum extracts showed strong health promoting potential, the highest potential was observed with the fermented extract, which showed a 60% reduction of AFB1 induced DNA damage and a 38% reduction in FeSO4 induced oxidative stress. The content of total polyphenols, carotenoids and chlorophylls was indeed affected by cultivation and processing. Based on the present in vitro findings consumption of S. scabrum leaves could be further encouraged, preferentially after cooking or fermentation of the plant.

Aflatoxin B₁-Induced Developmental and DNA Damage in Caenorhabditis elegans.

Aflatoxin B1 (AFB1) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB1 has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB1 is also a potent genotoxic mutagen that causes DNA damage in vitro and in vivo. However, the link between DNA damage and abnormal development and reproduction is unclear. To address this issue, we examined the DNA damage, germline apoptosis, growth, and reproductive toxicity following exposure to AFB1, using Caenorhabditis elegans as a study model. Results found that AFB1 induced DNA damage and germline apoptosis, and significantly inhibited growth and reproduction of the nematodes in a concentration-dependent manner. Exposure to AFB1 inhibited growth or reproduction more potently in the DNA repair-deficient xpa-1 nematodes than the wild-type N2 strain. According to the relative expression level of pathway-related genes measured by real-time PCR, the DNA damage response (DDR) pathway was found to be associated with AFB1-induced germline apoptosis, which further played an essential role in the dysfunction of growth and reproduction in C. elegans.

Cytoprotective and genoprotective effects of β-glucans against aflatoxin B1-induced DNA damage in broiler chicken lymphocytes

The polysaccharide β-glucan presents beneficial effects on the immune system, although the mechanisms of the immunomodulatory effect remain poorly understood. The potential cytoprotective and genoprotective effects of β-glucans were evaluated in broiler chicken lymphocytes exposed to increasing concentrations of aflatoxin B1 (AFB1) and/or β-glucans. AFB1 significantly decreased cell viability at the concentrations of 10 and 20μg/ml at 72h of incubation (p<0.01 and p<0.001, respectively). Moreover, the AFB1 concentrations of 1, 10 and 20μg/ml increased DNA fragmentation levels at 24h (p<0.001). Conversely, lymphocyte death was prevented by β-glucans at the concentrations of 1% and 10%, indicating a cytoprotective effect. Reactive oxygen species levels were increased in the cells treated with 20μg/ml AFB1 at 24h (p<0.05) and 10% β-glucans with or without AFB1 at 24, 48 and 72h of incubation (p<0.001). DNA damage increased by more than 100% in AFB1-treated lymphocytes when compared to control group. β-glucans at 1% was able to fully revert the AFB1-induced lymphocyte DNA damage, indicating a genoprotective effect and maintaining DNA integrity. In conclusion, β-glucans showed in vitro dose-dependent cytoprotective and genoprotective effects in broiler chicken lymphocytes exposed to AFB1.

Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100μM nicotine (a substrate of CYP2A13) or 1μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system.

Ochratoxin A reduces aflatoxin B1 induced DNA damage detected by the comet assay in Hep G2 cells

Mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) can be present together in food commodities. These food contaminants are considered to be genotoxins, acting by different mechanisms. The aim of this work was to characterize combined genotoxic in vitro effects of both mycotoxins in Hep G2 cells. For this purpose, cytotoxicity was first determined in isolated and combined treatments in order to determine the dose range of genotoxicity studies. Co-exposure of cells to OTA+AFB1 for 24h resulted in additive effects. Genotoxicity was determined in Hep G2 cells by the modified comet assay with restriction enzymes (endo III and FPG). Significant reactive oxygen species formation was detected in both single and combined treatments. AFB1 was genotoxic after 3h with external metabolic activation (S9 mix) and after 24h without metabolic activation. Co-exposure to OTA significantly decreased DNA damage induced by AFB1, not only in breaks and apurinic sites but also in FPG-sensitive sites. The apparent contradiction between additive cytotoxic effects and antagonic genotoxic effects may be explained if AFB1 and OTA compete for the same CYPs, yielding more ROS but less AFB1 adducts.

Aflatoxin B1‐Induced DNA Damage in Labeo rohita: Protective Effect of an Antioxidant Supplement, Amrita Bindu

The present study was undertaken to investigate the effect of potent hepatocarcinogen aflatoxin B1 in adduct formation and DNA damage in Labeo rohita. Also, the salubrious efficacy of an antioxidant supplement Amrita Bindu (based on Indian system of Medicine) was investigated. Fish weighing 175-250 g were administered intraperitoneally a single dose of 100 microg aflatoxin B1/100 g body wt. and another group was given 20% solution of Amrita Bindu along with aflatoxin B1 at 100 microg/100 g body wt. On the 3rd and 6th day, the liver tissue was analyzed for aflatoxin concentration, aflatoxin-DNA adduct formation and DNA damage measured in terms of single strand breaks. The fishes administered with aflatoxin B1 showed elevated concentration of aflatoxin along with a parallel increase in the DNA adduct when compared with the controls. While the fish co-administered with Amrita Bindu showed 34% and 24% reduction in aflatoxin deposition (accumulation) and aflatoxin-DNA adduct formation respectively on the 3rd day, a further reduction by around 41% and 33% in aflatoxin deposition and DNA adduct formation respectively was observed on the 6th day. Furthermore, the increased single strand breaks (measured by alkaline single cell gel assay) and fragmentation observed in agarose gel electrophoresis in aflatoxin B1 administered fish were significantly reduced by Amrita Bindu co-administration. In conclusion, this is the first report to show aflatoxin B1-induced DNA adduct formation and DNA damage in one of the major Indian culturable fish, Labeo rohita. Also, our observations show that the antioxidant supplement, Amrita Bindu, has a potential role in ameliorating the aflatoxin B1-induced DNA damage thus suggesting its applicability in preventing the vital macromolecule DNA.

発がん物質による標的臓器のＤＮＡ損傷におけるグレープフルーツジュース飲用の影響‐食物成分の相互作用‐

Co-intake of grapefruit juice with drugs results in a substantial increase in oral drug bioavailability. In contrast, DNA damage in target organ induced by a food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was reduced in rats by grapefruit juice intake. Aflatoxin B1-induced DNA damage was also suppressed in rats treated with grapefruit juice and an ethyl acetate extract of grapefruit juice. A significant decrease in hepatic CYP3A content, but not in CYP1A, CYP2C, glutathione S-transferase and microsomal epoxide hydrolase contents was observed in rats after grapefruit juice intake. No significant differences in the portal blood and liver concentrations of aflatoxin B1, nor in blood concentration of PhIP, were observed between control rats and rats ingesting grapefruit juice. Thus, grapefruit juice intake causes suppression of carcinogen-induced DNA damage at least in part through decreased metabolic activation in rat liver.

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40–400 μM AFB1, were 0.07–0.74 adducts per kilobase (kb). In vitro modification with 0.1–4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03–0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.