Study On The DNA Damage Induced By Aflatoxin B₁ In Duckling Hapetic Cells

Aflatoxins(AFs) are one of the most potent naturally occurring toxic substances.AFs are widely encountered contaminants of cereal crops and other foodstuffs in regions of the world with hot and humid climates.Aflatoxin B₁(AFB₁) is among the most potent naturally occurring carcinogens and classified as a groupâ… carcinogen by International Agency for Research on Cancer.AFB₁ is metabolized in mammalian liver through cytochromes P450 to the ultimate carcinogen aflatoxin B₁-8,9- epoxid (AFBO).AFBO can readily bind to DNA forming AFB₁-DNA adduct,which is correlated with relevant DNA lesions caused by AFB₁.This damage may be assessed by the Comet assay,which is a simple and sensitive test for investigation of DNA damage,including double- and single- strand breaks,incomplete repair sites,alkali-labile sites and crosslinks at individual cell level.There were three parts in this study. (1) The aim of the first experiment was to improve a modified Comet assay to detect DNA damage induced by AFB₁.Ducklings were administered orally with AFB₁ and the samples of liver cells and blood cells were isolated at 2 h post treatment.The modified comet assay was performed and the DNA damage was determined by an increase in the comet parameters in tested animals.The results showed that AFB₁ expousure could significantly increase tail length,the percentage of tail DNA(tail DNA%), tail moment and Olive tail moment compared with blank and solvent control.Almost all of blood cells comet image of every group appeared severious tailing and could not be analysised by Comet Assay Software Project.The result indicated that the modified comet assay performed liver cells was a valuable genotoxic marker for exposure to AFB₁ in duckling.Duckling blood cells have nucleated red blood cells that are unstable for the comet assay.(2) The aim of the secend experiment was to study the dynamic changes of DNA damage in duckling hepatic cells induced by AFB₁ at different dose levels (3μg,30μg,300μg/kg BW).96 male Beijing ducklings were randomly divided into 16 groups with 6 ducklings in each group.The modified Comet assay was performed at 1h,2 h,8 h,24 h and 48 h on hepatic cells after AFB₁ administration.The results showed that ducklings were sensitive to DNA damage induced by AFB₁,and the level of DNA damage was associated with concentration and time after AFB₁ intake.DNA strand breaks reached peak value at 2 h of AFB₁ exposure,and all different doses of AFB₁ could induce significantly higher tail length,tail DNA%,tail moment,Olive tail moment than in control group(P＜0.05).With AFB₁ concentration increasing,the duration of DNA damage prolonged,and under the condition of the same treatment time,the DNA damage was more serious.The result indicated that the level of DNA damage reached the peak at 2 h after a single oral administration of toxin,and at this time the dose-effect relationship was obviously.Low level(1/100 LD₅₀) exposure of AFB₁ could induce significantly hepatic DNA damage in ducklings.These results also suggested that duckling was an ideal animal model for study of AFB₁ genotoxicity,and Comet assay was an useful tool for monitoring the hepatic DNA damage induced by AFB₁ in ducklings.(3) Two experiments were conducted to evaluate the potential of three kinds of adsorbents in reduction of AFB₁-induced DNA damage.In test one,20 male Beijing ducklings were randomly assigned to five experimental groups: control(without AFB₁ and adsorbent),AFB₁ + A(2.5μg AFB₁/ml lml + 30 mg adsorbent A),AFB₁ + B (2.5μg AFB₁/ml 1ml + 30 mg adsorbent B),AFB₁ + C(2.5μg AFB₁/ml 1ml + 30 mg adsorbent C).In test two,20 male Beijing ducklings were randomly assigned to five experimental groups:control (without AFB₁ and adsorbent),AFB₁ + A(25μg AFB₁/ml 1ml + 30 mg adsorbent A),AFB₁ + B(25μg AFB₁/ml 1ml + 30 mg adsorbent B),AFB₁ + C(25μg AFB₁/ml 1ml + 30 mg adsorbent C).After ducklings were administered orally two hours,Comet assay was performed on liver cells.The result show that two doses of AFB₁ could induce significantly higher tail length,tail DNA%,tail moment, Olive tail moment than in control group(P＜0.05).Three kinds of adsorbents had a certain tend to ruduce AFB₁-induced DNA damage.The dose of AFB₁ and the kind of adsorbents could influence the efficacy of adsorbrnt.The result indicated that the adsorbents could suppresse AFB₁-induced liver DNA damage,and compared with A and C,adsorbent B could alleviate the genotoxic effect of AFB₁ more effectively.The adsorbents could reduce AFB₁-induced DNA damage more effectively at lower dose of AFB₁ than higher dose.