Investigating the Structure‐Toxicity Relationship of Methylimidazolium Ionic Liquids in Isolated Primary Human Hepatocytes

Abstract

Ionic liquids are man‐made non‐volatile chemicals proposed to be “environmentally‐friendly” alternatives to current volatile solvents. However, they persist in the environment. In this respect, an 8 carbon alkyl chain derivative methylimidazolium ionic liquid (M8OI) has recently been found to be present at high levels in soils near a landfill waste site in the UK in an area characterized by a high incidence of the autoimmune liver disease “primary biliary cholangitis” (PBC). Recent work by this laboratory also demonstrated that M8OI was toxic to the liver progenitor (B‐13) cell line (Probert et al., J. Hepatol 69:1123–1135). To gain insight into its mechanism(s) of toxicity, the effects of several structurally‐related methylimidazolium liquids (differing in their alkyl chain length from 2C to 10C) have been examined in primary human hepatocytes. The ionic liquids inhibited human hepatocytes oxygen consumption within minutes of exposure, with potency increased with increasing alkyl chain length, an effect associated primarily with inhibition of mitochondrial respiration and ATP production. These effects were accompanied by an increase in extracellular acidification rate indicative of compensatory enhanced glycolysis, confirmed by dose‐dependent increases in glucose consumption, lactate production and AMPK phosphorylation. These metabolic changes preceded induction of caspase 3/7 activities and genomic DNA fragmentation, indicative of an initial apoptotic mode of cell death. Time‐ and dose‐dependent loss of MTT reduction capacities reflected these effects. Similar mechanistic changes were seen with B‐13 cells although sensitivities were increased (with regards to MTT reduction capacity). Despite their effects on mitochondrial oxygen consumption, none of the ionic solvents directly inhibited the function of the electron transport chain complexes at concentrations relevant to toxicity. However, chemical reduction and electron paramagnetic resonance (EPR) spectroscopy studies suggest that all the ionic solvents function as electron acceptors under biologically‐relevant reducing environments, suggesting that the methylimidazolium ionic liquids have the potential capacity to accept electrons from the mitochondrial electron transport chain. However, nile red fluorescence measurements indicated that only longer chain ionic solvents form a non‐aqueous phase or micelle under aqueous physiological conditions. These data, therefore, suggest that the longer chain methylimidazolium solvents are most toxic because they both readily integrate within the inner mitochondrial membrane and accept electrons from the electron chain. This reduction leads to an inhibition of oxidative phosphorylation and a compensatory increase in glycolysis before the induction of apoptosis.Support or Funding InformationThis work was funded by the Newton‐Mosharafa Fund (in the form of a studentship supporting T.M.A) and by a research grant from the LIVErNORTH charity (to A.C.L and M.C.W.).