Hexabromocyclododecane (HBCD) is a ubiquitous environmental contaminant of current concern despite its global ban in 2013 due to its characteristics as a persistent organic pollutant. While the toxicity of HBDC in vertebrates has been extensively studied, the specific molecular mechanisms underlying its toxicity in fish are not fully understood to date. Therefore, the aim of this work was to determine the in vitro cytotoxicity of HBCD in the fathead minnow (Pimephales promelas) using liver explants, and to investigate the molecular mechanisms underlying these effects. Explants were incubated with nine different concentrations of HBCD (0.00032, 0.0016, 0.008, 0.04, 0.2, 1, 5, 25 and 125 mg HBCD/L) for 6 and 24 h, and cytotoxicity was tested by using the Lactate Dehydrogenase (LDH) assay. The expression of genes with a key role in the regulation of apoptosis, oxidative stress, cryoprotective responses to reactive oxygen species (ROS), and xenobiotic metabolism was also measured in liver explants after exposure to 0.00032, 0.0016, 0.008, 0.2, and 25 mg HBCD/L. After 6 h, a concentration-dependent significant increase in cytotoxicity was found between 0.008 and 1 mg/L HBCD, followed by a decrease between 1 and 25 mg/L. Cytotoxicity reached 100 % at a concentration of 125 mg/L HBCD. After 24 h, HBCD showed a biphasic response with a concentration-dependent decrease in cytotoxicity between 0.0016 and 1 mg/L that returned to baseline levels at 5 mg/L. Then, cytotoxicity increased at concentrations greater than 5 mg/L to reach a maximum value at 125 mg/L. Changes in the expression of genes related to apoptosis (apoEn, apoIn, caspase2, caspase9 and bax) were also time- and concentration-dependent. Genes related to antioxidant responses such as gst and catalase were generally decreased after 6 h of incubation and increased after 24 h. The same pattern was observed for cyp1a and cyp3a, both related to xenobiotic metabolism. The expression of genes related to cryoprotective responses anti ROS (akt and pi3k) decreased at almost all HBCD concentrations tested after 6 h but remained unaltered after 24 h. Overall, we demonstrated that the cytotoxic effect of HBCD in fathead minnow liver explant was not proportional to its concentration in the culture media. Cytotoxicity was highly dynamic and did not follow a typical concentration-response pattern, complicating its toxicological characterization.

尽管六溴环十二烷（HBCD）具有持久性有机污染物的特性，但由于其在2013年被全球禁止，因此是目前引起普遍关注的一种普遍存在的环境污染物。尽管已经广泛研究了HBDC在脊椎动物中的毒性，但迄今为止，尚未完全了解其在鱼类中毒性的具体分子机制。因此，这项工作的目的是确定在黑头fat鱼（Pimephales promelas）中六溴环十二烷的体外细胞毒性）使用肝脏外植体，并研究这些作用的分子机制。将外植体与九种不同浓度的HBCD（0.00032、0.0016、0.008、0.04、0.2、1、5、25和125 mg HBCD / L）孵育6和24小时，并使用乳酸脱氢酶（LDH）测试细胞毒性分析。在暴露于0.00032、0.0016、0.008、0.2和25 mg的肝脏外植体中，还测量了在调节细胞凋亡，氧化应激，对活性氧（ROS）的冷冻保护反应以及异源生物代谢中起关键作用的基因的表达。六溴环十二烷/ L。6小时后，发现细胞毒性的浓度依赖性显着增加是在0.008和1 mg / L六溴环十二烷之间，然后是在1和25毫克/升之间。当浓度为125 mg / L六溴环十二烷时，细胞毒性达到100％。24小时后，六溴环十二烷显示出双相反应，其细胞毒性在0.0016至1 mg / L之间呈浓度依赖性降低，并在5 mg / L时恢复至基线水平。然后，浓度大于5 mg / L时细胞毒性增加，达到125 mg / L的最大值。与凋亡相关的基因表达的变化（apoEn，apoIn，caspase2，caspase9和bax的）也是时间和浓度依赖性的。与抗氧化剂反应有关的基因，例如gst和过氧化氢酶，通常在孵育6小时后减少，而在24 h后增加。对于cyp1a和cyp3a观察到相同的模式，两者均与异源生物代谢有关。与ROS（akt和pi3k）的低温保护反应相关的基因表达。）在6小时后几乎所有测试的HBCD浓度下均下降，但在24小时后保持不变。总的来说，我们证明六溴环十二烷在黑头head鱼肝移植物中的细胞毒性作用与其在培养基中的浓度不成比例。细胞毒性是高度动态的，没有遵循典型的浓度-反应模式，使其毒理学特征复杂化。