Use of Cellular and Molecular Based Assays to Evaluate the Toxicity of Manganese (II) to RTgill‐W1 Cell Cultures

Manganese (Mn) is an essential nutrient to most complex life. In high concentrations, manganese (II) chloride (MnCl‐ 2 ) (a common pollutant from several anthropological sources) proves to be damaging, if not lethal organisms with chronic exposure. Available mammalian research indicates manganese acts as a neurotoxin when in high concentrations and gives rise to parkinsonian‐like symptoms by way of heavy methylation in the PINK1‐PARK2 region. While the effects and mechanisms of manganese toxicity are known in mammals, the mechanisms by which manganese affects aquatic life are still poorly characterized. Additionally, recent findings that manganese may have negative effects at concentrations lower than those previously believed to cause impairment makes it prudent to further evaluate. Acute viability testing indicates a 20% increase in cellular death in cultures exposed to 0.198 uM, and a 50% increase in death in cultures exposed to 3.97 uM. The objective of this study is to determine the level of manganese found to create impairment in a rainbow trout gill cell line and to elucidate the mechanism of impairment. Analyses of multiple response endpoints with fluorescent dyes and toxicity assays indicate a similar linear trend in reactive oxygen species generation, caspase‐3 generation, and cell membrane viability. Rainbow trout gill cells were exposed to varying manganese chloride concentrations ranging from. 198 uM to 31.8 uM, over a period of 24 hours. Further evaluation of MT1A (metallothionein‐1A) and PARP1 (Poly (ADP‐ribose) polymerase‐1) mRNA and expression via qPCR and immunoblotting is underway. Additionally, apoptosis, necrosis, mitochondrial damage, and comet formation is also being evaluated to identify further response proteins for qPCR. The use of qPCR presents a faster, cheaper, and more sensitive alternative to whole‐organism testing in the future. Support or Funding Information Research has been funded by National Science Foundation (NSF) Award OIA‐1458952, the Marshall University SURE Fellowship, the Marshall research scholar's award, and the WV NASA Consortium Affiliate Undergraduate Fellowship Program.Viability notated by amount of fluorescence from calcein activated by esterase cleavage of calcein AM in vitro . Viability tests indicate dose relationship in cell death with MnCl 2 in a 24 hour period (p<0.05). (mean, standard error, 8 replicates.)Reactive Oxygen Species (ROS) detection assays indicate a substantial increase in production of ROS in cells treated with MnCl 2 . Concentration of DCF (dichlorofluorescein) is proportional to amount of reactive oxygen species in vitro over a 24 hour period (p<0.05). (mean, standard error, 8 replicates)Caspase 3 CaspSelect assays indicate increased caspase‐3 activity in all treated cultures, with variance in higher concentration MnCl 2 cultures due to short exposure time and MnCl 2 precipitation (p<0.05). (means, standard error, 8 replicates)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .