Protective effects of olive oil antioxidant phenols on mercury-induced phosphatidylserine externalization in erythrocyte membrane: Insights into scramblase and flippase activity

In several physiopathological processes, phosphatidylserine (PS), normally sequestered to the inner leaflet of the plasma membrane, becomes exposed to the cell surface. In erythrocytes (RBC), PS externalization is a crucial event for the removal of aged/damaged cells but can also be associated with increased prothrombotic activity. Structurally related olive oil antioxidants, including hydroxytyrosol (HT), are able to significantly reduce the percentage of PS-exposing RBC, when cells are exposed to toxic compounds such as the heavy metal mercury (Hg). The aim of the present study was to identify the molecular mechanisms underlying the protective effect, with a focus on two different phospholipid translocases, the ATP-dependent flippase ATP11C and the calcium-dependent scramblase PLSCR1, which are responsible for PS internalization and exposure, respectively. In addition to HT, its monophenol analogue, tyrosol, and its in vivo metabolite, homovanillic alcohol, were also tested. Our investigation revealed that exposure of human intact RBC to HgCl₂ induced a decrease in flippase activity and an increase in scramblase activity, and that all the selected phenols restored the control activity, regardless of their different scavenging properties. Interestingly, all phenols restored the ATP level of control cells, which were significantly reduced by HgCl₂ treatment. Conversely, no variation in intracellular calcium was observed under our experimental conditions. Additionally, all phenols restored the glutathione levels, significantly reduced in the presence of HgCl₂. In line with the data on the enzymatic activity, Western blotting analysis indicated changes in the membrane expression of the two enzymes, alterations prevented by antioxidant pre-treatment. Finally, molecular docking analysis suggests that the tested antioxidants may be able to directly interact with ATP11C. Our findings provide an experimental basis for the use of olive oil bioactive compounds in nutritional/nutraceutical strategies for the prevention of Hg-related toxicity, particularly in relation to the cardiovascular tissues.