Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response.
Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response.
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DOI：
10.1289/ehp.5458
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发表时间：
2003-01
影响因子：
10.4
通讯作者：
Kubinski, Dana
中科院分区：
文献类型：
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作者：
Raychoudhury, Samir S;Kubinski, Dana
多环芳烃（PAHs）是一种普遍存在的持久性环境污染物。一些多环芳烃是致癌物，可能会影响男性生殖系统。因此，我们将培养的大鼠支持细胞暴露于多种多环芳烃中，以确定对精子上皮细胞可能的直接毒性作用。选择支持细胞是因为它们支持生殖细胞发育和维持精子发生。从19-21日龄雄性大鼠中分离支持细胞，在含0.08%二甲亚砜的培养基中或在多种多环芳烃的存在下培养。在第一组实验中，培养的Sertoli细胞在10(-4)M、10(-6)M、10(-8)M、10(-12)M和10(-16)M荧光蒽（FL）中孵育24小时。24小时后，细胞活力测定显示，10(4)、10（-6）和10(-8)M的FL杀死了大量的支持细胞。在10(-6)M和10(-8)M FL存在下培养的支持细胞表现出形态变化。细胞蛋白水平降低，乳酸产量在培养基中以浓度依赖的方式增加。此外，与未处理的对照组相比，暴露于10(6)M和10(-8)M FL的支持细胞表现出f -肌动蛋白和α -微管蛋白分布的改变。由于FL在10(-4)M（100微g/mL）下杀死约62%的细胞，在10(-6)M（1微g/mL）下杀死48%的细胞，在两种浓度下乳酸产量增加约3倍，在10(-4)M（100微g/mL）下细胞蛋白减少一半，我们决定在10至100微g/mL的浓度范围内使用苯并[a]蒽（BaA），苯并[a]芘（BaP）或苯并[b]荧光蒽（BbF）进行第二组实验。24小时后，BaA（100微g/mL）、BaP（50和100微g/mL）和BbF（100微g/mL）显著提高培养基中乳酸水平，且呈浓度依赖性。在第三组实验中，细胞在仅10微g/mL的FL、BaA、baap或BbF中均匀培养24小时。这些多环芳烃的细胞毒性作用导致不同的细胞凋亡反应，这是原位荧光染色的特征。显微镜下观察凋亡细胞发现，当Sertoli细胞与10微g BaP或BbF孵育24小时时，细胞核缩小，3 -OH DNA末端标记，而与载药、培养基、FL或BaA孵育24小时则没有。因此，我们的研究结果表明，BaA和BbF对支持细胞的毒性作用是通过凋亡发挥的，而FL和BaA不会引起凋亡反应。
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and persistent environmental contaminants. Some PAHs are carcinogens and may affect the male reproductive system. Therefore, we exposed cultured rat Sertoli cells to a variety of PAHs to determine possible direct toxic effects on the cells of the seminiferous epithelium. Sertoli cells were chosen because they support germ cell development and maintain spermatogenesis. Sertoli cells were isolated from 19-21-day-old male rats and cultured in medium containing 0.08% dimethylsulfoxide as vehicle or in the presence of a variety of PAHs. In the first set of experiments, cultured Sertoli cells were incubated in the presence of 10(-4) M, 10(-6 )M, 10(-8) M, 10(-12) M, and 10(-16) M fluoranthene (FL) for 24 hr. After 24 hr, FL at 10(4), 10(-6), and 10(-8) M killed significant numbers of Sertoli cells as revealed by cell viability determinations. Sertoli cells cultured in the presence of 10(-6) M and 10(-8) M FL showed morphologic changes. Cell protein levels were decreased and lactate production in the medium increased in a concentration-dependent manner. In addition, Sertoli cells exposed to 10(6) M and 10(-8) M FL exhibited altered F-actin and alpha-tubulin distributions compared with untreated controls. Because FL killed about 62% of cells at 10(-4) M (100 micro g/mL) and 48% of cells at 10(-6) M (1 micro g/mL), increased lactate production about 3-fold at both concentrations, and decreased cell protein by half at 10(-4) M (100 micro g/mL), we decided to use a range of concentrations between 10 and 100 micro g/mL for the second set of experiments using benz[a]anthracene (BaA), benzo[a]pyrene (BaP), or benzo[b]fluoranthene (BbF). After 24 hr, BaA (100 micro g/mL), BaP (50 and 100 micro g/mL), and BbF (100 micro g/mL) significantly increased lactate level in the medium in a concentration-dependent manner. In a third set of experiments, cells were treated in culture uniformly with only 10 micro g/mL FL, BaA, BaP, or BbF for 24 hr. The cytotoxic effects exerted by these PAHs tested resulted in different apoptotic responses as characterized by in situ fluorescence staining. Microscopic analysis of apoptotic cells demonstrated nuclei of reduced size and labeled 3 -OH DNA ends when Sertoli cells had been incubated for 24 hr with 10 micro g BaP or BbF, but not with vehicle, media, FL, or BaA. Thus, our results demonstrate that the toxic effects of BaA and BbF on Sertoli cells are exerted through apoptosis, whereas FL and BaA do not elicit the apoptotic response.