Metabolomic Responses to Manganese Dose in SH-SY5Y Human Neuroblastoma Cells

Abstract

Manganese (Mn)-associated neurotoxicity has been well recognized. However, Mn is also an essential nutrient to maintain physiological function. Our previous study of human neuroblastoma SH-SY5Y cells showed that Mn treatment comparable to physiological and toxicological concentrations in human brain resulted in different mitochondrial responses, yet cellular metabolic responses associated with such different outcomes remain uncharacterized. Herein, SH-SY5Y cells were examined for metabolic responses discriminated by physiological and toxicological levels of Mn using high-resolution metabolomics (HRM). Before performing HRM, we examined Mn dose (from 0 to100 lM) and time effects on cell death. Although we did not observe any immediate cell death after 5 h exposure to any of the Mn concentrations assessed (0-100 lM), cell loss was present after a 24-h recovery period in cultures treated with Mn ! 50 lM. Exposure to Mn for 5 h resulted in a wide range of changes in cellular metabolism including amino acids (AA), neurotransmitters, energy, and fatty acids metabolism. Adaptive responses at 10 lM showed increases in neuroprotective AA metabolites (creatine, phosphocreatine, phosphoserine). A 5-h exposure to 100 mM Mn, a time before any cell death occurred, resulted in decreases in energy and fatty acid metabolites (hexose-1,6 bisphosphate, acyl carnitines). The results show that adjustments in AA metabolism occur in response to Mn that does not cause cell death while disruption in energy and fatty acid metabolism occur in response to Mn that results in subsequent cell death. The present study establishes utility for metabolomics analyses to discriminate adaptive and toxic molecular responses in a human in vitro cellular model that could be exploited in evaluation of Mn toxicity.

Transcriptome Analysis Reveals Distinct Responses to Physiologic versus Toxic Manganese Exposure in Human Neuroblastoma Cells

“…SH-SY5Y, human neuroblastoma cell line, obtained from the American Type Culture Collection (ATCC-Manassas, VA) was cultured and exposed to Mn dose as previously described ( Supplementary Datasheet 1 ) (Fernandes et al, 2017; Fernandes et al, 2018; Fernandes et al, 2019). Briefly, cells were cultured in Dulbecco’s modified Eagle medium/Ham’s F12 medium and, at 80% confluency, were treated with Mn (0, 1, 5, 10, 50, 100 μM as MnCl 2 ; Sigma-Aldrich, St. Louis, MO) for 5 h. The 5-h time point was chosen to correlate transcriptomic changes with cellular Mn concentration comprising physiological (≤10 μM Mn treatment) to pathophysiological Mn levels (≥50 μM Mn treatment) that was optimized previously (Fernandes et al, 2017) based on the recommended dosage of treatment and accumulation of cellular Mn relevant to in vivo levels from human brain and in vitro studies (Csaszma et al, 2003; Bowman and Aschner, 2014; Kumar et al, 2015; Fernandes et al, 2017).…”

“…As Mn dose was increased, increased oxidative stress was represented by decreased cellular thiols, increased mitochondrial H 2 O 2 production, and biphasic change in mitochondrial respiration (Fernandes et al, 2017). Respiration increased to a maximum at the highest dose supporting cell survival (10 μM MnCl 2 ) after 24 h and decreased at concentrations (≥50 μM MnCl 2 ) resulting in cell death after 24 h (Fernandes et al, 2017; Fernandes et al, 2019). In these studies, no cell death was detected at 5 h for any Mn concentration studied.…”

“…In these studies, no cell death was detected at 5 h for any Mn concentration studied. However, substantial cell death was observed after a 24-h recovery period in cells treated with 50 or 100 μM Mn for 5 h while no significant cell death was observed after 24 h recovery in Mn concentrations ≤ 10, thus demonstrating ≥ 50 μM Mn as a toxic dose (Fernandes et al, 2019). Subsequent metabolomics studies using this model further allowed discrimination of adaptive responses to physiologic variation of Mn from toxic responses resulting in subsequent cell death after 24 h (Fernandes et al, 2019).…”

“…However, substantial cell death was observed after a 24-h recovery period in cells treated with 50 or 100 μM Mn for 5 h while no significant cell death was observed after 24 h recovery in Mn concentrations ≤ 10, thus demonstrating ≥ 50 μM Mn as a toxic dose (Fernandes et al, 2019). Subsequent metabolomics studies using this model further allowed discrimination of adaptive responses to physiologic variation of Mn from toxic responses resulting in subsequent cell death after 24 h (Fernandes et al, 2019). Metabolomics further showed increases in neuroprotective amino acid metabolites (creatine, phosphocreatine, phosphoserine) after 5 h at a non-toxic 10 μM Mn while 5-h exposure to a concentration that subsequently caused cell death resulted in decreases in energy and fatty acid metabolites (hexose-1,6 bisphosphate, acyl carnitines).…”

ZIP14 is degraded in response to manganese exposure

“…Mn has been shown to be toxic to a variety of cell types at lM concentrations (Choi et al 2019;Fernandes et al 2019;Porte Alcon et al 2018;Roth et al 2002).…”