Effects of Glycidamide on Activity and Progesterone Biosynthesis of Rat R2C Leydig Cells

Objective] This study aimed to investigate the effects of glycidamide （GA） on growth and progesterone biosynthesis of rat R2C Leydig cel s cultured in vitro. [Method] The R2C Leydig cel s were treated with GA with concentrations of 0.25, 0.5, 0.75, 1, 2, 4 and 6 mmol/L respectively for 48 h. The IC25, IC50 and IC75 values of GA were al detected by MTT assay. The Leydig cel s were treated with GA at concentrations of IC25, IC50 and IC75 respectively for 48 h, and then the morphology of Leydig cel s was observed. After the Leydig cel s were treated with GA for 4 h, the cel ular DNA damage was measured by the comet assay technique; and after the Leydig cel s were with treated with GA for 24 h, the progesterone biosynthesis amount was detected by radioimmunoassay （RIA）. [Result] GA could inhibit the via-bility of R2C Leydig cel s, and its IC25, IC50 and IC75 were 0.635, 0.872 and 1.198 mmol/L, respectively. The GA at concentrations of IC25, IC50 and IC75 affected the growth and morphology of rat R2C Leydig cel s in varying degrees. The 4-h treat-ment of GA could significantly damage the DNA of R2C Leydig cel s, and the 24-h treatment of GA at concentrations of IC25, IC50 and IC75 al reduced the progesterone biosynthesis amount. [Conclusion] GA could inhibit the growth and progesterone biosynthesis of rat R2C Leydig cel s.

Cytotoxicity of acrylamide and its epoxide glycidamide in CHO cells expressing human cytochrome P450 2E1

Objective： To investigate whether CYP2E1 is responsible for the acrylamide metabolic activation in FIp-In CHO cell system. Methods： CYP2E1 cDNA was subcloned from the human liver full-length cDNA library and subsequently transfected into the FIp-In CHO cells to generate the stable transfectant of CYP2E1. The CYP2E1 mRNA expression was determined by RT-PCR. Acrylamide and its epoxide glycidamide induced cytotoxicity and cell cycle arrest in G2/M were conducted using MTS assay and flow cytometry, respectively. Results： In the CHO cell stably expressing CYP2E1 （CHO-2E1）, a -1.5 kb size of band was detected from the mRNA in the cells while no corresponding band in the CHO-vector cells, which indicated that CYP2E1 was successfully transfected in the CHO cells. Compared with the CHO-vector cells, acrylamide showed a concentration dependent loss of viability in the CHO-2E1 cells but no significant change of G2/M arrest was found. As expected, glycidamide induced similar profile of cytotoxicity in both of the cells, and G2/M arrest presented a concentration-dependent increased in the CHO-2E1 cells. Conclusion： The result suggested that CYP2E1 might be responsible for the acrylamide metabolism, and its metabolite glycidamide was a direct cytotoxic and genotoxic agent. It should be further considered whether acrylamide-induced toxicity is through its epoxide glycidamide in the presence of CYP2E1.