m-Cresol affects the lipid bilayer in membrane models and living neurons

Abstract

m-Cresol is a preservative used in pharmaceutical formulations, including insulin and vaccines, which contain up to 30 millimolar concentrations of this excipient. We studied the effects of m-cresol (from 1 : 1 to 1 : 10⁴ dilution relative to the formulations) on membrane model systems containing liquid ordered domains, alone or in an insulin formulation. These studies allowed to understand the effects of this compound on cell membranes when present in pharmaceutical formulations, and to explain its cytotoxic effects. Changes in the intrinsic fluorescence of m-cresol showed a clear interaction with liposomes that depends on their lipid composition. Liposomes labeled with several membrane probes revealed the superficial location of m-cresol, alone and within insulin formulation, with an unusual preference for liquid ordered cholesterol–sphingolipid-enriched domains due to their high membrane dipole potential. Membrane probes detected the effects of m-cresol at a concentration of 1 : 10 000 relative to the original formulations. Atomic force microscopy imaging of ternary supported lipid bilayers allowed the observation that addition of m-cresol affects precisely the ordered raft-like nanodomains, with a fast (5 min) effect at 1 : 10 dilution that became massive after 1 h. When contained in insulin formulation, similar m-cresol effects were observed. To evaluate the importance of the direct interactions of m-cresol with membrane lipids at the cellular level, membrane leakage was monitored in three neuronal cell lines. m-Cresol was applied during constant superfusion and leak current alterations were followed. The leak conductance in living neurons underwent a dose-dependent increase. The effects reported here are likely to underlie the mechanisms of m-cresol toxicity for cells and tissues at or near the injection site.