Toxicity, recovery, and resilience in a 3D dopaminergic neuronal in vitro model exposed to rotenone

Arch Toxicol. 2018 Aug;92(8):2587-2606. doi: 10.1007/s00204-018-2250-8. Epub 2018 Jun 28.


To date, most in vitro toxicity testing has focused on acute effects of compounds at high concentrations. This testing strategy does not reflect real-life exposures, which might contribute to long-term disease outcome. We used a 3D-human dopaminergic in vitro LUHMES cell line model to determine whether effects of short-term rotenone exposure (100 nM, 24 h) are permanent or reversible. A decrease in complex I activity, ATP, mitochondrial diameter, and neurite outgrowth were observed acutely. After compound removal, complex I activity was still inhibited; however, ATP levels were increased, cells were electrically active and aggregates restored neurite outgrowth integrity and mitochondrial morphology. We identified significant transcriptomic changes after 24 h which were not present 7 days after wash-out. Our results suggest that testing short-term exposures in vitro may capture many acute effects which cells can overcome, missing adaptive processes, and long-term mechanisms. In addition, to study cellular resilience, cells were re-exposed to rotenone after wash-out and recovery period. Pre-exposed cells maintained higher metabolic activity than controls and presented a different expression pattern in genes previously shown to be altered by rotenone. NEF2L2, ATF4, and EAAC1 were downregulated upon single hit on day 14, but unchanged in pre-exposed aggregates. DAT and CASP3 were only altered after re-exposure to rotenone, while TYMS and MLF1IP were downregulated in both single-exposed and pre-exposed aggregates. In summary, our study shows that a human cell-based 3D model can be used to assess cellular adaptation, resilience, and long-term mechanisms relevant to neurodegenerative research.

Keywords: 3D LUHMES; Cellular defence; Gene response; Neurodegeneration; Pesticide; Recovery; Resilience; Rotenone.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Cell Culture Techniques / methods*
  • Dopaminergic Neurons / drug effects*
  • Dopaminergic Neurons / physiology
  • Gene Expression Regulation / drug effects*
  • Humans
  • Insecticides / toxicity
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Neuronal Outgrowth / drug effects
  • Rotenone / toxicity*
  • Toxicity Tests / methods*


  • Insecticides
  • Rotenone
  • Adenosine Triphosphate