Raychoudhury S S, Blake C A, Millette C F
Department of Biological and Physical Sciences, Benedict College, Columbia, South Carolina, USA.
Toxicol Appl Pharmacol. 1999 Jun 15;157(3):192-202. doi: 10.1006/taap.1999.8664.
Alkylphenols, including the estrogenic 4-tert-octylphenol (OP), are environmental pollutants. Because administration of OP to adult male rats impairs spermatogenesis and OP has been shown to be toxic to aquatic animals and to mammalian splenocytes in vitro, we studied whether OP exerts direct toxic effects on cultured spermatogenic cells and Sertoli cells isolated from male rats. Cell viability was assessed with a Live/Dead Eukolight viability/cytotoxicity kit. Culture of mixed spermatogenic cells from adult rats with 10(-8) M OP or Sertoli cells from 19- to 21-day-old rats with 10(-12) M OP, but not with 0.08% EtOH (vehicle) or 10(-6) M 17beta-estradiol (E2) or dexamethasone (DEX), significantly decreased the percentage of viable cells after 24 h of treatment. None of the treatments significantly altered total cell number. Flow cytometric analyses of spermatogenic cells revealed that exposure to 10(-4) or 10(-6) M OP yielded abnormal relative ploidy classes. Four hours of treatment with 10(-6) M OP, but not with 10(-6) M E2 or DEX, caused significant chromatin condensation in Sertoli cells as observed with acridine orange staining. The decreased percentage of viable cells after 24 h of exposure to 10(-6) M OP remained when Sertoli cells were cultured in Ca2+-free medium. Sertoli cells contained nuclei of reduced size and labeled 3'-OH DNA ends as detected by microscopic analyses when the cells had been incubated for 24 h with 10(-6) M OP but not with vehicle or 10(-6) M E2 or DEX. The results demonstrate that OP, but not E2 or DEX, is directly toxic to cultured rat spermatogenic cells and Sertoli cells and suggest that this toxic effect in Sertoli cells is exerted through Ca2+-independent apoptosis.
烷基酚，包括具有雌激素活性的4-叔辛基酚（OP），是环境污染物。由于给成年雄性大鼠施用OP会损害精子发生，并且OP已被证明在体外对水生动物和哺乳动物脾细胞有毒性，因此我们研究了OP是否对从雄性大鼠分离的培养生精细胞和支持细胞产生直接毒性作用。使用活/死真核生物荧光活力/细胞毒性试剂盒评估细胞活力。成年大鼠的混合生精细胞与10^(-8) M OP一起培养，或19至21日龄大鼠的支持细胞与10^(-12) M OP一起培养，但不与0.08%乙醇（溶剂）或10^(-6) M 17β-雌二醇（E2）或地塞米松（DEX）一起培养，在处理24小时后显著降低了活细胞的百分比。所有处理均未显著改变细胞总数。生精细胞的流式细胞术分析显示，暴露于10^(-4)或10^(-6) M OP会产生异常的相对倍性类别。用10^(-6) M OP处理4小时，但不与10^(-6) M E2或DEX处理，会导致支持细胞中出现明显的染色质凝聚，这通过吖啶橙染色观察到。当支持细胞在无钙培养基中培养时，暴露于10^(-6) M OP 24小时后活细胞百分比仍会降低。当细胞与10^(-6) M OP孵育24小时而不是与溶剂或10^(-6) M E2或DEX孵育时，通过显微镜分析检测到支持细胞含有尺寸减小的细胞核并标记有3'-OH DNA末端。结果表明，OP而非E2或DEX对培养的大鼠生精细胞和支持细胞具有直接毒性，并表明支持细胞中的这种毒性作用是通过不依赖钙的凋亡发挥的。
