Time-course metabolomic analysis of manganese toxicity reveals biomarkers of oxidative stress and amino acid metabolism as early cellular targets

Manganese (Mn) is a naturally occurring essential nutrient at low doses that functions as a cofactor in multiple enzymatic reactions, provides structural stability and participates in redox activities. However, high Mn exposure even under federal safety limits for air Mn levels, may cause a dose-dependent progression of Parkinsonism. The molecular mechanisms and critical transitions from normal Mn physiology to low dose toxicity as well as early targets of Mn toxicity nonetheless remains poorly understood. In this temporal study, we used human neuroblastoma SH-SY5Y cells exposed to Mn concentrations varied over a physiologic (5μM MnCl 2 ) and toxicological range (50μM MnCl 2 ) for 0, 0.5, 2, 5 and 10h (n=9 per Mn concentration and time point). Metabolomics analysis was done using liquid chromatography with ultra-high resolution mass spectrometry and cellular Mn accumulation was measured using ICP-MS. Results showed cellular Mn accumulation increased upto 5h in a dose dependent manner, beyond which Mn uptake was stabilized. Pathway enrichment analysis showed that methionine and cysteine metabolism; urea cycle metabolism; glycine, serine, alanine and threonine metabolism; and histidine metabolism were significantly altered within 30min of manganese treatment and persisted upto 10h suggesting these pathways define early targeted mechanisms of Mn toxicity. Discriminatory metabolites involved in methionine and cysteine pathway include methionine, homocysteine, 1,2 dihydroxy-3-keto-5-methylthiopentene and 5’-methylthioadenosine. Discriminatory metabolites involved in glycine, serine, alanine and threonine metabolism include 3-phosphohydroxypyruvate, phosphoserine, serine and creatine. These early responders may reflect rapid transport and redistribution of Mn within the cell. In conclusion, we demonstrate key metabolic biomarkers and pathways that respond dynamically to varying Mn concentration and attempt to provide a chronological fingerprint of Mn dependent toxicity. Time course metabolomics thereby provides a useful tool to predict metabolic status for dynamic systems at the cellular level in response to environmental chemical exposures.