Assessing the cytotoxic effect of hexabromocyclododecane (HBCD) on liver tissue cultures from fathead minnow (Pimephales promelas).

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.