Effect of Humic Acid on the Growth Arrest in Sertoli Cell Line, TM4

• Main Authors: Chen Shu Chun, 陳淑君
• Other Authors: 呂 鋒 洲
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/26969008819387026941

碩士 === 國立臺灣大學 === 生化學研究所 === 88 === Abstract Humic acid (HA) is a fluorescent deep brown organic, polymeric compound composed of phenolic acids, which is ubiquitous in terrestrial and aquatic environments. Nuclear magnetic resonance (NMR) and infrared spectroscopic analyses revealed that HA consisted of aromatic rings, phenolic hydroxyl, ketone carbonyl, quinone carbonyl, carboxyl, and alkoxyl groups. HA was implicated as a causal factor of goiter, cancer, "blackfoot" disease, an endemic peripheral vascular disease prevailing in the southwest coast of Taiwan, and Keshin-Beck disease, a chronic osteoarthritic disorder with necrosis of chondrocytes, prevailing in mainland China. Resently, we found that intraperitoneal injection of HA in rats induced testicular morphological changes'' including degeneration of seminiferous tubes, reduction in the number of Sertoli cells and spermatogonia, and loss of spermatids. Although the final stage of the atrophy was germinal cell depletion, the primary damage site was found to be in Sertoli cells. Phthalic acid, one of the chemical decomposition products of HA, was found to reduce fertility and induce testicular atrophy in rats by disrupting normal zinc metabolism. And the Sertoli cell was the primary site of phthalate-induced testicular toxicity. The actual mechanism of testicular atrophy induced by HA is unclear. In order to disclose the mechanism of HA induced testicular atrophy of rats, we selected the Sertoli cell line, TM4, to investigate the effect of HA on Sertoli cells and try to understand the mechanism of testicular atrophy. After 4 days of treatment with HA, the numbers of TM4 cells were reduced dose-dependently. FACScan analysis of DNA content of HA-treated TM4 cells revealed that a large proportion of TM4 cells were arrested at the G1 phase. The percentage of TM4 cells at the G1 phase increased from 36% to 84% after HA treatment. The protein expressions of cyclin D1, cdk4 and E2F1 were significantly decreased after 4 days of treatment with HA while the expression of the cdk inhibitor p27KIP1 was significantly increased. Alought the amount of cyclin E and cdk2 protein did not change, the immunocomplex kinase experiments showed that HA inhibited the activity of cyclin-dependent kinase 2 (cdk2) due to increasing the protein expression of p27KIP1 after 2 days of treatment with HA and became more obvious after 4 days. The DNA binding activity of E2F was determined by EMSA (electrophoretic mobility shift assay); the activity was not changed at 2 days of treatment, but was sighificantly reduced at 4 days. Besides these, HA could totally inhibit the DNA binding activity of E2F when nuclear extract was exposed to 50μg HA. HA also interfered the mitochondrial transmembrane potential dose-dependently due to reducing the mitochondrial mass. Because of losing mitochondrial activity of TM4 cells, the cell energy was depleted and the cell cycle progression was also retarded. These results suggest that HA exerts its growth retardation effects through the reduction in the expression of the key G1 regulatory proteins such as cyclin D1, cdk4 and E2F1, modulation of the kinase activity of cdk2 and also induction of cdk inhibitor p27KIP1. HA-induced testicular atrophy might be linked in part to its growth arrest of Sertoli cells.