Cr(vi) permanently binds to the lipid bilayer in an inverted hexagonal phase throughout the reduction process

Abstract

Cr(vi) is a harmful, carcinogenic agent with a high permeability rate throughout the lipid membranes. In an intracellular environment and during interactions with cellular membranes, it undergoes an instant reduction to lower oxidation states throughout radical states, recognized as the most dangerous factor for cells. The cellular membrane is the most visible cellular organelle in the interior and exterior of a cell. In this study, liposomes and non-lamellar inverted hexagonal phase lipid structures based on phosphoethanolamine (PE) were used as model cellular bilayers because of their simple composition, preparation procedure, and the many other properties of natural systems. The lipid membranes were subjected to 0.075 mM Cr(vi) for 15 min, after which the Cr content was removed via dialysis. This way, the remaining Cr content could be studied qualitatively and quantitatively. Using the combined XRF/XAS/EPR approach, we revealed that some Cr content (Cr(iii) and Cr(vi)) was still present in the samples even after long-term dialysis at a temperature significantly above the phase transition for the chosen liposome. The amount of bound Cr increased with increasing PE and -C[double bond, length as m-dash]C- bond content in lipid mixtures. Internal membrane order decreased in less fluid membranes, while in more liquified ones, internal order was only slightly changed after subjecting them to the Cr(vi) agent. The results suggest that the inverted hexagonal phase of lipid structures is much more sensitive to oxidation than the lamellar lipid phase, which can play an important role in the strong cytotoxicity of Cr(vi).

Fig. 1. X-ray powder diffractograms of two of liposomal sample groups that were both treated and not treated with Cr(vi): (A) – small DOPE fraction liposomes; (B) – large DOPE fraction liposomes. Grey areas represents regions described in the text.

Fig. 2. Data obtained in the XAS experiment. (A) – Cr K-edge XANES region; (B) – pre-edge region of XANES spectra. Arrow indicates an edge shift; (C) – calibration curve of oxidation state versus Cr K edge position.

Fig. 3. Data obtained in XRF experiment for Kapton foil (background, black line), DMPC + Cr liposomes (red line) and DMPC/DOPE 5 : 1 + Cr (blue line) liposomes.

Fig. 4. Top: a relation between Cr(vi) and reduced fraction of Cr in function of the DOPE concentration; bottom: a total Cr concentration (yellow) and each components: Cr(vi) (red) Cr(iii) (blue) in respect to the DOPE concentration. Results are plotted as mean ± total uncertainty. The * symbol indicates statistically significant difference for t-student test at p-0.05.

Fig. 5. EPR spectra of liposomes of different composition (pure DMPC, DMPC : DOPE 1 : 1, DMPC : DOPC 1 : 1, DMPC : DMPE 1 : 1) incubated with 0.075 mM solution of K₂Cr₂O₇. The spectra were aquired at 37 °C.