Cytotoxicity in Primary Human Hepatocytes Exposed to Various Microcystin Congeners
Citation:
Richardson, V., J. Strasser, AND E Hilborn. Cytotoxicity in Primary Human Hepatocytes Exposed to Various Microcystin Congeners. ISSX, Montreal, Quebec, CANADA, July 16 - 20, 2018.
Impact/Purpose:
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 100 microcystin congeners whose physical and chemical characteristic 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 hydrophilic, microcystin-LR. This study attempts to narrow the data gaps through the investigation of the cytotoxic effects of hydrophilic and hydrophobic microcystin congeners on primary human hepatocytes.
Description:
Microcystins (MCs) are monocyclic heptapeptide algal toxins produced by cyanobacterial species. MCs are the most common cyanotoxin found in the environment and are found in most surface waters worldwide. MCs pose a risk to humans and animals due to their presence in drinking and recreational waters. Over 100 MC congeners have been identified with variable amino acid compositions. The amino acid composition determines physical/chemical properties including hydrophilicity and is also thought to determine the chemicals’ kinetics that will in part determine the toxicity of each congener. Microcystin-LR (MCLR), the most commonly studied congener, is considered highly toxic and induces hepatic apoptosis and an accumulation of reactive oxygen species (ROS) when administered to laboratory animals. In contrast to MCLR, toxicity data for other MCs are limited. In this study, the effects of the more hydrophobic (MCLF, -LW, -LA, -LR) and the more hydrophilic (MCWR, -YR) congeners on cell viability and ROS production were determined using primary human hepatocytes (HH). HH were plated in 96-well microtiter plates at a density of 50,000 cells/well. 24 hours after plating, the HH were exposed to MCLR, -LW, -LA, -LF, -WR, or -YR at 10 different concentrations (0 to 20 µM) for 24 hours. After 24 hours, the HH were assayed for viability (CellTiter Glo 2.0; Promega Corp.). The EC50 was then determined for each congener. Based on the EC50, the rank order of cytotoxicity to HH was MCLW>LA>LF>LR>WR>YR. To analyze ROS production in response to MC exposure, HH were plated as previously described. 24 hours after plating, HH were exposed to MCLR, -LW, -LA, -LF, -WR, or -YR at respective EC50 concentrations (as determined in viability studies) for 6 hours. After the exposure period, the HH were assayed for ROS (ROS-Glo H2O2; Promega Corp.). MCLW, one of the most hydrophobic congeners, was the only congener that displayed a statistically significant increase in ROS in HH. These results indicate that the more hydrophobic congeners caused a greater decrease in cell viability in HH, compared to the more hydrophilic congeners. These results also suggest that the cytotoxic effects of each congener do not necessarily involve ROS. When coupled with kinetic information on uptake and distribution, our results can be important in understanding and predicting toxic effects produced by these congeners in vivo. [This abstract of a proposed presentation does not necessarily reflect the policies of the US EPA.]