Neurotoxicity assessment of triazole fungicides on mitochondrial oxidative respiration and lipids in differentiated human SH-SY5Y neuroblastoma cells

Abstract

Indiscriminate overuse or occupational exposure to agricultural chemicals can lead to neurotoxicity. Many pesticides act to impair mitochondrial function which can lead to exacerbation of neurodegeneration. Triazole fungicides are applied to grain, fruit, and vegetable crops to combat mold and fungi and their use is increasing worldwide. Here, we assessed the in vitro toxicity of two widely used triazole fungicides, propiconazole and tebuconazole, to mitochondria using differentiated SH-SY5Y neuroblastoma cells as an in vitro cell model used in Parkinson’s disease research. Cell viability (based on ATP levels), mitochondrial membrane potential, oxidative respiration, and reactive oxygen species (ROS) were measured following fungicide treatments. Cell viability was decreased with 100 μM propiconazole after 24 and 48 h, while tebuconazole required higher doses to affect viability (−200 μM at 24 h). Mitochondrial membrane potential (MMP) was reduced with 50 μM propiconazole after 24 h while 200 μM tebuconazole reduced MMP. Oxidative respiration of SH-SY5Y cells was then measured using a XFe24 Flux analyzer and 100 μM propiconazole reduced basal respiration, oligomycin-induced ATP production, and FCCP-induced maximum respiration by −40−50%, while tebuconazole did not affect mitochondrial bioenergetics at the concentrations tested. Acute exposure to 100 μM propiconazole over 4 h did not immediately affect oxidative respiration in SH-SY5Y cells. ROS were not induced by propiconazole and tebuconazole up to 100 and 300 μM respectively. Based on these results, we focused our lipidomics investigations on SH-SY5Y exposed only to propiconazole, as lipid dysregulation is associated with mitochondrial dysfunction. Both 50 and 100 μM propiconazole altered the abundance of some ceramides, specifically reducing glucosylceramide non-hydroxyfatty acid-sphingosine (HexCer-NS) and increasing N-stearoyl-phytosphingosine (CerNP). Moreover, a recently discovered bioactive lipid called fatty acid ester of hydroxy fatty acid (FAHFA) was increased 5-fold, hypothesized to be a neuroprotective mechanism that has been demonstrated in other studies of human diseases. Additional lipids reduced in abundance included oxidized phosphatidylcholine (OxPC) and oxidized phosphatidylethanolamine (OxPE). There were no changes in cellular triacylglycerols nor total lipids with exposure to propiconazole. Taken together, this study provides insight into the toxicity of triazole fungicides in neuronal cells, which has implications for neurodegenerative diseases that involve the mitochondria such as Parkinson’s disease.