Department of Zoology, University of Ibadan, Ibadan, Nigeria.
Department of Zoology, University of Ibadan, Ibadan, Nigeria; Department of Zoology and Environmental Biology, University of Calabar, Nigeria.
Comp Biochem Physiol C Toxicol Pharmacol. 2018 Nov;213:7-18. doi: 10.1016/j.cbpc.2018.07.001. Epub 2018 Jul 6.
In the present study, we have investigated the endocrine disruptive effects of waterborne di-(2-ethylhexyl) phthalate (DEHP: 0 (control), 10, 100, 200 and 400 μg/L) on juvenile Clarias gariepinus by analyzing transcript patterns for hepatic vitellogenin (vtg), estrogen receptor-α (er-α), aromatase (cyp19a1b) and peroxisome proliferator activated receptor-α (ppar-α) using quantitative real-time PCR after 3, 7 and 14 days exposure period. In addition, we analyzed CYP19 and PPAR protein levels using enzyme-linked immunosorbent assay (ELISA), while cellular testosterone (T) and 17β-estradiol (E2) levels were measured using enzyme immune assay (EIA). Tissue burden of DEHP was measured in the liver using gas chromatography-mass spectroscopy (GC-MS). We observed apparent concentration- and time-dependent increases of vtg, er-α, cyp19a1b transcripts, E2 and T levels after exposure to DEHP. A biphasic pattern of effect was observed for ppar-α, showing a concentration-dependent increase that peaked at 100 μg/L and thereafter, an apparent concentration-dependent decrease at 200 and 400 μg/L at all exposure days. Given that the post-differentiation changes of gonads in C. gariepinus corresponded with the 14-day sampling period, we separated the analyzed data into sexes after histological examination of the gonads, showing that females responded stronger, compared to males, to DEHP exposure at all exposure concentrations. Oocyte atresia, intersex (ovotestis) and karyoplasmic clumping were observed in females while male fish showed distortion and degeneration of seminiferous tubules and condensation of tubular cells in the 400 μg/L exposure group after 14 days. Corresponding canonical analysis (CCA) of all analyzed variables revealed a positive relationship between analyzed biological variables with increasing DEHP concentrations. Overall, molecular, biochemical and physiological responses presented in the present study indicate that exposure of C. gariepinus to waterborne DEHP produced endocrine disruptive responses with potential consequences for overt reproduction, development, physiology and general health of fish populations inhabiting phthalate contaminated aquatic environments. These responses represent valuable and effective biomarkers of exposure and effects, that can be adopted for screening the presence of EDCs in Nigeria and other developing countries.
在本研究中，我们通过分析暴露于水中的邻苯二甲酸二（2-乙基己基）酯（DEHP：0（对照）、10、100、200 和 400μg/L）后 3、7 和 14 天对幼年蓝鳃太阳鱼（Clarias gariepinus）的肝卵黄蛋白原（vtg）、雌激素受体-α（er-α）、芳香酶（cyp19a1b）和过氧化物酶体增殖物激活受体-α（ppar-α）的转录模式，使用定量实时 PCR 来研究内分泌干扰效应。此外，我们使用酶联免疫吸附测定（ELISA）分析 CYP19 和 PPAR 蛋白水平，同时使用酶免疫测定（EIA）测量细胞内睾酮（T）和 17β-雌二醇（E2）水平。使用气相色谱-质谱联用（GC-MS）测量肝脏中的 DEHP 组织负荷。我们观察到暴露于 DEHP 后，vtg、er-α、cyp19a1b 转录物、E2 和 T 水平明显呈现出浓度和时间依赖性增加。ppar-α表现出双相效应模式，在 100μg/L 时呈浓度依赖性增加，随后在 200 和 400μg/L 时明显呈浓度依赖性降低，所有暴露天数均如此。鉴于蓝鳃太阳鱼的性腺在分化后的变化与 14 天的采样期相对应，我们在对性腺进行组织学检查后，将分析数据按性别进行了分离，结果表明，与雄性相比，雌性在所有暴露浓度下对 DEHP 暴露的反应更强。在 14 天的暴露后，雌性观察到卵母细胞闭锁、间性（卵睾）和核质聚集体，而雄性鱼类在 400μg/L 暴露组中表现出精小管扭曲和退化以及管状细胞浓缩。对所有分析变量的对应典型分析（CCA）显示，随着 DEHP 浓度的增加，分析的生物学变量之间存在正相关关系。总的来说，本研究中呈现的分子、生化和生理反应表明，蓝鳃太阳鱼暴露于水中的 DEHP 会产生内分泌干扰反应，这可能对鱼类种群的明显繁殖、发育、生理和整体健康产生潜在影响，这些反应代表了暴露和效应的有价值和有效的生物标志物，可用于筛查尼日利亚和其他发展中国家环境内分泌干扰物的存在。
