Among several heavy metallic elements, arsenic, cadmium and mercury are most toxic in the environment. These metals are acquired by humans through food and water, which result in severe health related issues. One of the most toxic metals is arsenic and it is derived from the natural environment. The main source of arsenic toxicity is due to contamination of drinking water from natural geological sources rather than from mining, smelting, or agricultural sources. Another naturally occurring highly toxic heavy metal is cadmium and possesses considerable toxicity with destructive impact on most organ systems. To date cadmium has no physiological function in the human body. Mercury is another important toxic metal found in nature and noted for inducing public health disasters. The current study has been considered to investigate individual and combined toxic effects in HepG2 hepatocarcinoma and SH-SY5Y neuroblastoma cell lines as a measure of hepatotoxicity and neurotoxicity, respectively. The aim was achieved through investigation of individual metals and their combinations on cell density, mitochondrial membrane potential, adenosine triphosphate levels, reactive oxygen species generation, glutathione levels and caspase-3/7 activity.
Arsenic displayed gradual, dose-dependent cytotoxicity in both cell lines. In the HepG2 cells, IC50 of arsenic was determined as 6.71 mg/L and in the SH-SY5Y cells, IC50 of 1.19 mg/L. Arsenic had minimal effects on HepG2 mitochondrial membrane potential at IC50 (~12%). A gradual, tapered reduction (25% to 62%) was observed in the SH-SY5Y cell line from 0.94 mg/L to 2.78 mg/L. This decline parallels the reduction in cell density in the SH-SY5Y cell line. Arsenic profoundly decreased adenosine triphosphate levels at IC75 (9.61 mg/L for HepG2 and 2.78 mg/L for SH-SY5Y cells) for both cells indicating mitochondrial toxicity. A decline in reactive oxygen species level was noted for both cells after 24 h incubation with arsenic. The glutathione was reduced by 35%, 64% and 94% in HepG2 cells and 47%, 67% and 96% in SH-SY5Y cells which paralleled the results obtained for cell density, adenosine triphosphate levels and mitochondrial membrane potential. Mitochondrial toxicity lead to an increase in caspase-3/7 in both cell lines. Greater cytotoxicity was displayed in the HepG2 cells after cadmium exposure (IC50 = 0.43 mg/L) than the SH-SY5Y cells (IC50 = 1.47 mg/L). The dose dependent mitochondrial depolarization leads to mitochondrial toxicity and there is a direct correlation to the reduction in cell density. Cadmium decreased adenosine triphosphate levels when exposed to the IC25 (0.22 mg/L) by 39%, by 62% at IC50 (0.43 mg/L) and by 88% at IC75 (1.11 mg/L). The trend was similar to that detected for arsenic, albeit being less toxic. In SH-SY5Y cells, there was complete reduction in adenosine triphosphate levels as was found by arsenic. A gradual decrease in reactive oxygen species was noted in both cells after exposure to cadmium. The reduction in glutathione levels related to cell density as well as mitochondrial membrane potential reduction. The increase in caspase 3/7 activity in SH-SY5Y cells was nearly double that observed in HepG2 cells at the same concentrations tested indicating apoptosis.
Mercury was more cytotoxic towards SH-SY5Y cells (IC50 = 11.99 mg/L) than HepG2 cells (IC50 = 26.23 mg/L). Mercury was found to be the least toxic among all three metals tested. Mercury decreased mitochondrial membrane potential and reactive oxygen species in both cells in a dose dependent manner. The adenosine triphosphate concentration was almost completely inhibited in HepG2 cells while a more gradual decrease in the SH-SY5Y cells of 30%, 77% and 99% when exposed to IC25 (8.45 mg/L), IC50 (11.99 mg/L) and IC75 (14.36 mg/L) concentrations of mercury, was noted.
The glutathione reduction in both cell lines was dose dependent, with virtually total inhibition (99%) of glutathione when treated with the IC75 concentration of mercury. This reduction in glutathione levels correlates with the reduction in cell density, reduction in adenosine triphosphate level as well as the loss in mitochondrial membrane potential. Mercury decreased caspase-3/7 in HepG2 cells from basal level at all concentrations tested. In contrast caspase-3/7 activity was increased by 320%, 181% and 327% in SH-SY5Y cells when exposed to IC25 (8.45 mg/L), IC50 (11.99 mg/L) and IC75 (14.36 mg/L) concentrations of mercury.
After exposure to the IC and EPA mixtures, the reduction in cell density was far greater than that observed for any of the single metals alone in HepG2 cells, which may imply additive or synergistic activity. Combinations of metal mixtures displayed greater mitochondrial toxicity than the metals alone. This was most prominent in the HepG2 cell line. The combination mixtures in both the cell lines almost completely inhibited adenosine triphosphate levels indicating cell death from apoptotic to necrotic pathways.
Reactive oxygen species was reduced in a dose dependent manner in both cell lines. The IC combinations abolished glutathione levels completely in hepatoma and neuronal cells. All the EPA combinations reduced glutathione levels in a dose dependent manner which paralleled what was noted for cell density, mitochondrial membrane potential and adenosine triphosphate levels. An increase in caspase 3/7 activity was noted when the cells were exposed to 4mg/L EPA mixture concentration with respect to arsenic (505%) indicating additive or synergistic effect.
Understanding how metal mixtures affect health is critical to decide on treatment strategies. The results indicate the potential mechanistic routes of cytotoxicity incurred by the heavy metals and their combinations. Cytotoxicity of metal mixtures was more pronounced than when cells were exposed to individual metals.