The effects of two progestins and 17α-ethinylestradiol on cultured hepatocyte spheroids of brown trout (salmo trutta)

Abstract

Progestins are classified as emerging micropollutants in freshwater ecosystems. The concentrations of this class of contaminants in water have been increasing, since they are of anthropogenic origin and their use in human and veterinary medicine is in expansion. Apart from well-studied effects of progestins on fish, such as alterations of behavioral processes, impaired reproduction and involvement in intersex, the effects on structure and metabolism of liver are understudied; namely when progestins and estrogens are concurrent. Thus, this study is exploring sub-chronic effects of in vitro exposure hepatocyte spheroids to progestins and 17α-ethinylestradiol. Primary hepatocytes were isolated from brown trout (n = 3) and cultured for 12 days in non-adhesive plates which were constantly agitated with frequency of 60 times per minute at 18 °C. Shortly after plating, hepatocytes started to spontaneously form spheroids (3D cellular aggregates), which increased their size over time. At day 12, spheroids were exposed to either 17α-ethinylestradiol (0.3 μM), levonorgestrel and megestrol acetate in two concentrations (0.3 and 0.6 μM) or binary mixtures of these chemicals in 0.1% ethanol as a solvent. After a 6 days exposure, spheroids were measured and sampled for biochemical (lactate dehydrogenase and resazurin assays), biometrical, immunohistochemical and gene expression analyses. Both biochemical assays did not show differences between exposed groups or between exposed and control and solvent control groups, which supported that viability of exposed spheroids was not compromised. For biometry, no significant differences in sphericity, area and diameter of spheroids were found between groups. However, when anti-vitellogenin antibody was applied on histological sections, spheroids from all exposed groups had increased expression comparing with controls, proving all three chemicals could modify the hepatocyte function. Besides estrogenic targets, genes related with lipid-metabolism pathways are under study. As a perspective, analysis of the modeling effect of temperature in the same targets is being considered.