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Comparative Analysis of Microcystin Toxicity in 2D and 3D Cultured Primary Human Hepatocytes
Citation:
Richardson, V. AND J. McGehee. Comparative Analysis of Microcystin Toxicity in 2D and 3D Cultured Primary Human Hepatocytes. Presented at International Society for the Study of Xenobiotics, Boston, MA, September 10 - 13, 2023. https://doi.org/10.23645/epacomptox.24164310
Impact/Purpose:
Poster presented to the International Society for the Study of Xenobiotics (ISSX) Conference September 2023. Microcystins are hepatotoxic algal toxins that pose a risk to humans and animals due to their presence in drinking and recreational waters. There are over 200 microcystin congeners whose physical and chemical characteristics differ by amino acid composition and hydrophobicity. Multiple data gaps exist in the understanding of the specific effects of microcystins on human cells; however, most toxicological data focuses on the effects of the moderately hydrophilic, microcystin-LR. Many of the microcystin congeners are hydrophilic and require active transport into cells to cause toxic effects. Found exclusively in the liver, organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 have been shown to be the most efficient transporters of microcystin. Primary hepatocytes in standard two-dimensional (2D) cultures are widely used in liver toxicity screenings, but they fail to recapitulate the morphology and functionality of the liver. Hepatocytes in three-dimensional (3D) cultures are stable and functionally active model systems, with sustained cellular function over many weeks. This study attempts to narrow the data gaps by investigating the toxic potential of hydrophilic and hydrophobic microcystin congeners and compare the toxic effects of microcystins on human hepatocytes cultured in 2D and 3D. The results of this study show: (1) More hydrophobic congeners caused a greater decrease in cell viability, compared to the less hydrophobic congeners in both culture models; (2) 3D cultures have greater sensitivity to microcystin toxicity, compared to 2D cultures; and (3) OATP1B1 mRNA expression was significantly increased in the 3D model relative to the 2D model, however no significant difference was seen in OATP1B3 mRNA expression. This study would be of interest to the Office of Water and other researchers, because it fills data gaps on the effects of multiple microcystin congeners of various degrees of hydrophobicity and shows the utility of primary human hepatocytes culture in 3D for studying microcystin toxicity.
Description:
Microcystin (MC) are a class of cyanobacterial hepatotoxins produced by numerous species of freshwater cyanobacteria. Over 200 microcystin congeners have been identified, each distinguished by a unique amino acid composition. The amino acid composition is thought to affect the hydrophobicity, bioavailability, and ultimately the toxicity of each congener. MC uptake, facilitated by organic anion transporting polypeptides (OATPs), is congener-dependent and the differences in toxic potency between more hydrophobic and less hydrophobic MC congeners can be attributed to differences in OATP-mediated uptake. Primary human hepatocytes (PHH) in conventional monolayer cultures (2D) are known as the ‘gold standard’ for evaluating xenobiotic metabolism and toxicity; however, hepatocytes cultivated into spheroids (3D) maintain a more in vivo-like morphology and functionality. In this study, we hypothesize that 3D PHH spheroid cultures are more sensitive to MC congeners than 2D monolayer cultures, regardless of hydrophobicity. PHH, from a single donor lot, were cultivated in 2D and 3D and treated with more hydrophobic MC congeners (LA, LW, or LY) or less hydrophobic MC congeners (LR, YR, or RR) at concentrations ranging from 0.0 to 10.0 μM for 24 hours. After treatment, the EC50s from cell viability experiments were used to determine the toxicity of each MC congener. Total RNA was isolated from untreated cells from each culture type and subsequently used to analyze OATP1B1 and OATP1B3 mRNA expression using RT-qPCR. In the 2D and 3D cultures, the rank order of the EC50 measurements from least to most toxic was: LW>LY>LA>LR>YR>RR. The 3D cultures were 2- to 8-times more sensitive to microcystins compared to the 2D cultures. OATP1B1 mRNA expression was significantly higher (3.6-fold) in the untreated 3D model relative to the untreated 2D model, however no significant difference was observed in OATP1B3 mRNA expression. The results indicate that the hydrophobic congeners are more toxic in 2D and 3D cultures compared to hydrophilic congeners. The results also suggests that the increased mRNA expression of OATP1B1 in 3D cultures could be partially responsible for the increases in microcystin toxicity. Overall, this study shows the utility of 3D cultivated PHH in testing microcystin toxicity and suggest a potential strategy for predicting toxicity in vivo. This abstract of a proposed presentation does not necessarily reflect the policies of the US EPA.
URLs/Downloads:
DOI: Comparative Analysis of Microcystin Toxicity in 2D and 3D Cultured Primary Human Hepatocytes
RICHARDSON_ISSX POSTER 090523_CLEARANCE.PDF (PDF, NA pp, 793.34 KB, about PDF)