The Cytotoxicity Of Altenuene On Nih/3t3 Cells And Its Effect On Dna Polymerase β Expression

Background and objective: Alternaria alternate exist extensively in soil and plants, such as grain, vegetables, fruit and crops, and one of the fungus found in contaminated food. It is reported that Alternaria alternate is dominate fungus in food of Lin Xian resident, a district with high incidence of esophageal cancer.Alternaria alternate produces many metabolic toxins, such as alternariol (AOH), alternariol methyl ether (AME), altenuene(ALT), tenuzonic acid (TA) and alter toxinâ… ,â…¡,â…¢,(ATXâ… ,ATXâ…¡,ATXâ…¢). Some of these toxins are intensively researched. Their toxicity and role in tumor etiology are recognized. AOH, one of main toxins, had genotoxicity and cytotoxicity to the mammal cells and can induce DNA polymeraseβ(polβ)overexpression. However, ALT, another metabolic toxins, is seldom researched. Its role on cytotoxicity and its effect on DNA polβexpression have not been reported.DNA polβis an important DNA repair enzymes responsible for base-excision repair (BER), it expresses at low level throughout the cell cycle. The overexpression of polβwill make base mismatch increase when damaged DNA is repaired, which could induce genetic instability. Recently, polβis observed overexpression in many tumor tissues compared with adjacent normal tissues, such as esophageal, prostate, breast, ovarian, and colon cancer tissues. Its role in tumor etiology need to be further elucidated.This research mainly focuses on the cytotoxicity of ALT, the effect on DNA damage, and the expression of polβ. It will provide further evidence that ALT's role in tumoretiology. Methods: AOH was used as positive control, NIH/3T3 cells was treated with ALT of different concentration, the toxicity of which was assessed by the morphological observation; The IC50 and the cell growth curve were measured by MTT colorimetry; Cell cycle distribution was detected by flow cytometry (FCM); DNA damage was detected by single cell gel electrophoresis; The polβexpression level was detected by RT-PCR and western blot. The comparison among groups was performed by simple factor analysis of variance. Took "α=0.05" as test standard.Results:â‘ ALT could inhibit the growth of NIH/3T3 cells and the IC₅₀ was 76.4umol/L to the NIH/3T3 cells. Cytotoxicity was observed after treating NIH/3T3 cells by ALT with 50umol/L and 100μmol/L for 24h. G₂/M phase distribution increased compared to the control group after treat NIH/3T3 cells with ALT of 10.0, 20.0, 50.0μmol/L for 24h, and the difference was statistically significant (p<0.05).â‘¡The tail longth of the cells comet was 19.57±7.74, 30.82±7.38, 37.94±8.77, positive rate was 37%, 75%, 88%, the contont of tail DNA was 19.5±7.74%, 30.82±7.38%, 37.94±8.77%, respectively, after treating with ALT of 10.0, 20.0, 50.0μmol/L, and the difference was statistically significant (p<0.05).â‘¢DNA polβexpression increased after treating NIH/3T3 cells by ALT.Conclusion: ALT has cytotoxicity and DNA damage to NIH/3T3 cells, it can induce the cell cycle arrest and polβexpression overexpression.