Citation:

McGehee, J. AND V. Richardson. Toxicity of Hydrophobic and Hydrophilic Microcystin Congeners Using 2D and 3D Models of Primary Human Hepatocytes. Gordon Research Conference Titled: Mycotoxins and Phycotoxins: The Impact of Biotoxins on our Society: From Detection to Mitigation, Easton, MA, June 18 - 23, 2023. https://doi.org/10.23645/epacomptox.23818737

Impact/Purpose:

Poster presented to the Gordon Research Conference Titled: Mycotoxins and Phycotoxins: The Impact of Biotoxins on our Society: From Detection to Mitigation June 2023. Under the right conditions (i.e., temperature, nutrients), cyanobacteria can rapidly proliferate and produce dense blooms. Because of the toxins they release, the increased occurrence and persistence of cyanobacterial blooms in the US are of great interest to the Office of Water. Microcystins are among the most detected toxins associated with cyanobacterial blooms worldwide and have been reported in surface waters in most of the U.S. and Europe. This study investigates the cytotoxic effects of three hydrophobic and three hydrophilic microcystin congeners on primary human hepatocytes in 2D or 3D cultures. The data show that hydrophobic microcystin congeners are more toxic than hydrophillic congeners and the congeners are more toxic in hepatocytes cultured in 3D in comparison to 2D cultures. These results show the utility of 3D hepatic cultures when determining the toxicity of microcystin congeners, which can be beneficial when assessing human health effects. 

Description:

Microcystins (MC) are a class of naturally occurring cyanotoxins produced by several species of freshwater cyanobacteria. These cyclic peptides are highly toxic to a wide range of organisms, including humans, and their occurrence in freshwater systems has become a major environmental and public health concern. Over 200 microcystin congeners have been identified, each with a distinct amino acid composition. This composition is believed to influence the kinetics of the hepatotoxin, ultimately contributing to the toxicity of each congener. While microcystin LR (MCLR) has been extensively studied, there is limited toxicity data available for other microcystin congeners. As a known hepatotoxin, microcystin congeners with higher hydrophobicity have been shown to decrease hepatocyte viability compared to more hydrophilic congeners. Primary human hepatocytes (PHH), a gold standard for evaluating xenobiotic toxicity, are often cultured using the conventional 2D methodology; however, 3D culture models have demonstrated superior capabilities in recapitulating in vivo-like liver functionality. The ability of 3D cultures to mimic the in vivo microenvironment and sustain the stable metabolic enzyme expression allows for more accurate responses to chemical stimuli. The objective of this study is to assess and compare the impact of microcystin exposure on the viability of PHH cultured in both 2D and 3D models. PHH were cultured in collagen I-coated plates in 2D (monolayer) or in ultra-low attachment plates to support 3D (spheroid) development. Cells were treated for 24 hours with more hydrophobic microcystin congeners (LA, LW, or LY) or less hydrophobic microcystin congeners (LR, YR, or RR) in concentrations ranging from 0.0 to 10.0 μM. The cytotoxic potency of each MC congener was determined by using EC50s obtained from cell viability experiments. The order of toxicity for the 2D and 3D cultures, from most toxic to least toxic, was LW>LY>LA>LR>YR>RR. The results show that the more hydrophobic congeners caused a greater decrease in cell viability, compared to the less hydrophobic congeners in both culture models. While the toxicity ranking remained consistent across both models, the 3D cultures were more sensitivity to microcystin exposure relative to the 2D cultures. This led to notably lower EC50 values for the 3D cultures, with reductions ranging from 2- to 8-fold compared to the 2D cultures. Understanding the physiochemical properties and mechanisms of toxicity is essential for understanding the health effects related to microcystin exposure. These results demonstrate the utility of 3D cultures in predicting the toxic effects of microcystin congeners and reveal an effective strategy in evaluating risks to human health. (This abstract does not represent EPA policy)

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