The studies of DNA single strand breaks and apoptosis in the aflatoxin B1-induced cytotoxicity in rat peritoneal lavage cells and hepatocytes

• Main Authors: ying-ling sun, 孫英玲
• Other Authors: Jiann-Gwu Lee
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/36820781191728445434

碩士 === 國立臺灣大學 === 獸醫學研究所 === 88 === Aflatoxin B1（AFB1）, a well-known feed contaminant produced by certain strains of Aspergillus flavus and Aspergillus parasiticus, has been shown to exhibit hepatic toxicity and immunological suppression of both humoral and cellular immunity in many animals. The objectives of the studies were to investigate the mechanisms of DNA single strand breaks and apoptosis in AFB1-induced cytotoxicities in rat peritoneal lavage cells and hepatocytes. The results of trypan blue dye exclusion assay indicated that significant cytotoxic effects were obvious when peritoneal lavage cells were exposed to 10, 50 and 100 μg/ml AFB1 for 1 and up to 48 hours, and the effects were in dose and time-dependent manners as well. These cells also exhibited significant increases in DNA single strand breaks by SCGE assay in similar patterns. The same concentration of AFB1 exposure induced 185-200 bp DNA ladder fragmentation after 18 hours of incubation. Electron microscopic analysis confirmed that cells treated with 50 μg/ml AFB1 for 12 and 24 hours lead to both necrosis and apoptosis simultaneously. However western blot analysis of total cellular protein revealed that the peritoneal lavage cells did not express p53 and Bcl-2 proteins after exposed to 50 μg/ml AFB1 for 0, 6, 12 and 24 hours. In addition, AFB1 given intraperitonealy caused 100﹪fatality in rats treated at 5 mg/kg but not other concentrations. When hepatocytes were isolated from rats with 0.25, 0.5 and 1 mg/kg AFB1 treatments, DNA single strand breaks increased significantly in a dose-dependent manner. 185-200 bp ladder of DNA fragments were only shown in genomic DNA of hepatocytes collected from rats treated with 5 mg/kg AFB1. Histopathologically, liver tissue sections prepared from rats received 2 mg/kg AFB1 treatment showed bile ducts heperplasia and acute-subacute hepatitis with a few hepatocytes underwent apoptotic changes. Hemorrhage, massive hepatic necrosis, and acute hepatitis were observed in the rats after 5 mg/kg AFB1 treatment. In addition to necrotic cells, large proportions of apoptotic cells able to be confirmed by in the in situ TUNEL staining experiment. Western blot analysis of cellular proteins revealed that the liver did not express p53 and Bcl-2 proteins. In conclusion, AFB1 induced cellular toxicities including apoptosis and necrosis in hepatocytes and/or peritoneal lavage cells, could be the consequences of nucleic DNA damage resulting from AFB1 exposure. The role of Bcl-2 and p53 in cellular toxicities will remain to be determined in the future studies.