Investigating the effect of the heavy metals cadmium, chromium and lead, alone and in combination on an endothelial cell line

Abstract

Heavy metals are natural elements characterized by their relatively large atomic mass as well as their high density. Heavy metal poisoning has occurred in numerous countries in the past, with the United States having the highest prevalence. Heavy metals can also be introduced into the ecosystem by the mining of heavy metals from deep within the earth’s crust thereby exposing them into air and water systems. Another common source of heavy metal exposure is cigarette smoke. Cigarette smoke has been shown to have carcinogenic, toxic and genotoxic properties. Although all the toxic elements in cigarette smoking have not yet been identified, several heavy metals have been found to contribute to the pathophysiological consequences associated with smoking. A study conducted by Yaprak et al., in 2019 indicated that cadmium, lead and chromium are the most abundant metals found in cigarette smoke. The current study therefore focused on cadmium, lead and chromium, alone and as part of metal mixtures to determine the role of these heavy metals on endothelial cells. The EA.hy926 endothelial cell line was exposed to different concentrations of each of these metal and their combinations and analysed at different time points to determine the effect of the heavy metals on endothelial cell function. The cytotoxicity of the metals was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Crystal Violet assays, reactive oxygen species productions was studied using the 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) assay, and cell viability was determined using flow cytometry. Finally, morphological changes caused by these metals was studied using scanning electron microscopy. Exposure of endothelial cells to cadmium, lead and chromium alone and in combination at three different concentrations (X0.1, X1 and X2) showed no significant cytotoxicity as indicated by the MTT and Crystal Violet assay results. At 24 h exposure, cadmium alone showed an increase in percentage free radical formation whereas lead alone showed the greatest percentage radical formation for the X1 concentration group. At 48 h exposure, chromium alone as well as the triple combination group showed an increase in the percentage radical formation between 0.1 and X2 concentrations. Cadmium caused the highest percentage radical formation in the X1 concentration group and lead at the X2 concentration group. After 72 h, both the cadmium and lead as single metals showed a gradual increase in percentage radical formation between the 0.1 and X2 concentrations, with cadmium showing the highest increase in the X1 concentration. Flow cytometric analyses with the Annexin V and the Propidium Iodide assay, showed an increase in early apoptotic and necrotic cells with higher concentrations of the lead and chromium combination as well as in the triple combination group. Increased necrosis was also evident in the cadmium and chromium combination, the lead and chromium as well as the triple combination groups. Morphological changes were also seen with scanning electron microscopy, with almost all of the X2 concentrations of metals showing either damage to the cell membrane, cell blebs present or a combination of the two. As endothelial cells have a protective function in the human body, cigarette smoke diminishes its protective ability through both disruption of cellular processes and morphology. Extensive research has been done on pulmonary endothelial cells and the link that exists between excessive cigarette smoke exposure and necrotic endothelial cells. With this taken into account, although no cytotoxicity was observed at the concentrations tested, changes on ultrastructural level are present and should be further investigated.