The MT1G gene in LUHMES Neurons is a Sensitive Biomarker of Neurotoxicity

Identification of toxicants that underlie neurological diseases is a neglected area awaiting a valid strategy to identify such toxicants. We sought biomarkers that respond to known neurotoxicants in LUHMES immortalized neurons and evaluated these biomarkers for use in screening libraries of environmental toxicants. LUHMES immortalized human dopaminergic neurons were surveyed by RNA sequencing following challenge with Parkinsonian toxicants rotenone, 6-hydroxydopamine, MPP+, and ziram (zinc dimethyldithiocarbamate; Zn2+DDC2), as well as additional toxicants paraquat, MS275, and methylmercury. The metallothionein gene MT1G was the most dynamic gene expression response to all seven toxicants. Multiple toxicants also increased transcripts for SLC30A1 and SLC30A2 zinc secretion transporters, the SLC7A11 xCT cystine/glutamate antiporter important for glutathione synthesis, DNA damage Inducible Transcript 3 (DDIT3), and secreted growth factors FIBIN and CXCL12, whereas several toxicants decreased expression of the Apelin growth factor (APLN). These biomarker genes showed advantages in sensitivity by responding to many toxicants at sub-cytotoxic concentrations. Since several of these biomarker genes and prior neurological disease studies implicated disruption of metal distribution, we tested metal chelator thiram (dimethyldithiocarbamate, DDC), Zn2+DDC2, and several other metals and metal chelates for cytoxicity and induction of MT1G expression. Metals and chelators that caused dynamic increases in MT1G expression caused cytotoxicity, whereas Ni2+DDC2 caused MT1G increase, but no cytotoxicity. These results bolster prior work suggesting that neurons are characteristically sensitive to depletion of glutathione or to disruption of cellular metal distribution and provide biomarkers to search for such neurotoxicants in chemical libraries. Overall design: Dopaminergic neurons differentiated from LUHMES cells were exposed to a vehicle control or one of seven different toxicants. The vehicle control and 7 toxicants were tested on cultured cells in triplicate for a total of 24 biological samples (3 control, 21 toxicant treated samples). Cells were treated for 6 hours prior to initiation of sample prep and RNA extraction.