Performance of Three-Dimensional Rainbow Trout (Oncorhynchus mykiss) Hepatocyte Spheroids for Evaluating Biotransformation of Pyrene.

The aquatic bioconcentration of a chemical is typically determined using conventional fish tests. To foster the approach of alternatives to animal testing, a combination of computational models and in vitro substrate depletion bioassays (e.g., primary hepatocytes) can be used. One recently developed in vitro assay is the three-dimensional (3D) hepatic spheroid model from rainbow trout (Oncorhynchus mykiss). The aim of the present study was to evaluate the metabolic competence of the 3D spheroids from rainbow trout when exposed to pyrene, using 2 different sampling procedures (SP1 and SP2). The results were compared with previously published intrinsic clearance (CL) results from S9 fractions and primary hepatocyte assays. Extraction of pyrene using SP1 suggested that the spheroids had depleted 33% of the pyrene within 4 h of exposure, reducing to 91% after 30 h. However, when applying SP2 a substantial amount (36%) of the pyrene was bound to the exposure vial within 2 h, decreasing after 6 h of exposure. Formation of hydroxypyrene-glucuronide (OH-PYR-Glu) was obtained throughout the study, displaying the metabolic competence of the 3D spheroids. The 2 sampling procedures yielded different CLin vitro , where pyrene depletion using SP2 was very similar to published studies using primary hepatocytes. The 3D spheroids demonstrated reproducibile, log-linear biotransformation of pyrene and displayed formation of OH-PYR-Glu, indicating their metabolic competence for 30 h or more. Environ Toxicol Chem 2019;38:1738-1747. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Development of a screening system for the detection of chemically induced DNA methylation alterations in a zebrafish liver cell line.

Early molecular events with correlation to disease, such as aberrant DNA methylation, emphasize the importance of DNA methylation as a potential environmental biomarker. Currently, little is known regarding how various environmental contaminants and mixtures alter DNA methylation in aquatic organisms, and testing is both time- and labor-consuming. Therefore, the potential of an in vitro screening method was evaluated by exposing zebrafish liver cells (ZF-L) for 96 h to the nonmutagenic model substance 5'-azacytidine (AZA), as well as a selection of environmental pollutants such as sodium arsenite (NAS), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 17α-ethinylestradiol (EE2), and diethylstilbestrol (DES). Six single genes with reported and anticipated importance in cancer were selected for analysis. Methylation of gene promoter areas was monitored by bisulfite conversion and high-resolution melt (HRM) analysis after exposure to sublethal concentrations of the test compounds. Subsequently, results were validated with direct bisulfite sequencing. Exposure of ZF-L cells to 0.5 µM AZA for 96 h led to hypomethylation of genes with both low and high basal methylation indicating similarity to mechanism of action in mammals. Further, NAS, EE2, and DES were shown to induce significant alterations in methylation, whereas TCDD did not. It was concluded that cell line exposure in combination with HRM may provide an initial contaminant screening assay by quantifying DNA methylation alterations with high throughput capacity. In addition, the rapid determination of effects following contaminant exposure with this in vitro system points to the possibility for new in vivo applications to be useful for environmental monitoring.