Neurotoxicity assessment of QoI strobilurin fungicides azoxystrobin and trifloxystrobin in human SH-SY5Y neuroblastoma cells: Insights from lipidomics and mitochondrial bioenergetics.

Strobilurin fungicides are quinone outside inhibitors (QoI) used to treat fungal pathogens for agricultural and residential use. Here, we compared the potential for neurotoxicity of the widely used strobilurins, azoxystrobin (AZS) and trifloxystrobin (TFS), in differentiated human SH-SY5Y cells. Fungicides did not include cytotoxicity up to 200 µM but both induced loss of cell viability at 48 h, with TFS showing slightly higher toxicity that AZS. Caspase 3/7 activity was induced in SH-SY5Y cells by both fungicides at 48 h (50 µM for AZS and 25 µM for TFS). ATP levels were reduced following a 24-hour exposure to > 25 µM AZS and > 6.25 µM TFS and both fungicides rapidly impaired oxidative respiration (~12.5 µM for AZS and ~3.125 µM TFS) and decreased oligomycin-induced ATP production, maximal respiration, and mitochondrial spare capacity. AZS at 100 µM showed a continual impairment of mitochondrial membrane potential (MMP) between 4 and 48 h while TFS at > 50 µM decreased MMP at 24 h. Taken together, TFS exerted higher mitochondrial toxicity at lower concentrations compared to AZS in SH-SY5Y cells. To discern toxicity mechanisms of strobilurin fungicides, lipidomics was conducted in SH-SY5Y cells following exposure to 6.25 µM and 25 µM AZS, and a total of 1595 lipids were detected, representing 49 different lipid classes. Lipid classes with the largest proportion of lipids detected in SH-SY5Y cells included triglycerides (17%), phosphatidylethanolamines (8%), ether-linked triglycerides (8%), phosphatidylcholines (7%), ether-linked phosphatidylethanolamines (6%), and diacylglycerols (5%). Together, these 5 lipid classes accounted for over 50% of the total lipids measured in SH-SY5Y cells. Lipids that were increased by AZS included acyl carnitine, which plays a role in long chain fatty acid utilization for mitochondrial β-oxidation, as well as non-modified, ether linked, and oxidized triacylglycerols, suggesting compensatory upregulation of triglyceride biosynthesis. The ceramide HexCer-NS, linked to neurodegenerative diseases, was decreased in abundance following AZS exposure. In summary, strobilurin fungicides rapidly inhibit mitochondrial oxidative respiration and alter the abundance of several lipids in neuronal cells, relevant for understanding environmental exposure risks related to their neurotoxicity.