Lithium nickel manganese cobalt oxide particles cause developmental neurotoxicity in Caenorhabditis elegans

Abstract

Lithium is increasingly used in rechargeable batteries for mobile devices, electric vehicles, and energy storage, among other applications. One of the common formulations of lithium batteries is lithium nickel manganese cobalt oxide (LiNMC) particles. Increasing utilization of LiNMC batteries would require adequate disposal and/or recycling, and yet the potential disposal of lithium batteries as waste either in or outside of landfills might lead to toxic effects to people and wildlife. However, understanding of the potential toxicity of LiNMC particles is limited. Based on previous literature investigating the mechanisms of toxicity of the constituent metals, as well as lithium cobalt oxide (LCO) nanoparticles, we hypothesized that LiNMCs would cause toxicity via mitochondrial impairment and oxidative stress. We further hypothesized that LiNMC toxicity would be exacerbated by knockdown of frh-1 and gas-1, Caenorhabditis elegans orthologs of human mitochondrial disease genes frataxin and NDUFS2. Finally, we predicted that LiNMC exposure would cause developmental neurotoxicity. We tested these predictions by carrying out LiNMC exposures, and found these did not significantly impact the redox state, steady-state ATP levels, mitochondrial:nuclear DNA ratio, or oxygen consumption in worms exposed developmentally to amounts of LiNMC that caused mild growth inhibition. We discuss possible reasons for the difference between our results and previous publications, including particle size. Furthermore, while knockdown of frh-1 and gas-1 altered several parameters, knockdown of these genes did not increase or decrease the effects of LiNMCs. However, we did find that exposure to LiNMC caused degeneration of dopaminergic, cholinergic, glutamatergic, and GABAergic neurons, but not serotonergic neurons or glial cells. Interestingly, it appears that the developmental neurotoxicity was driven either by a particle-specific effect, or a component other than lithium, because exposure to lithium chloride at the same concentration had no effect.

Fig. 1. Experimental concentrations and design. (A) Sources of LiNMC and potential introduction in the environment through landfill leaching. (B) Concentration-response of length measured at the L4 stage of worms exposed to LiNMC during development. (C) Schematic of experimental design. One-way ANOVA with Dunnet's test, error bars are SEM, n = 3 biological replicates (100 worms per treatment), (**) p < 0.01, (***) p < 0.001.

Fig. 2. Chemical uptake of LiNMC. Internal concentrations in C. elegans of (A) lithium, (B) nickel, (C) manganese, and (D) cobalt in worms that were exposed to 0, 8, or 32 mM LiNMC, and EV, frh-1, or gas-1 RNAi bacteria. Cobalt was not detected in unexposed worms. (E) Summary of the main effect and interaction p-value terms for RNAi and LiNMC concentration effects on chemical uptake in worms. Two-way ANOVA with Tukey-HSD test, n = 5 biological replicates (1050–2400 worms per treatment), asterisks in panels (A–D) indicate (*) p < 0.05 or (***) p < 0.001.

Fig. 3. LiNMC exposure does not affect redox state or steady-state ATP levels in wild-type worms or after gas-1 or frh-1 knockdown. (A) JV2 length normalized to 0 mM LiNMC (B) redox state ratio (oxidized : reduced roGFP). (C) PE255 normalized length to 0 mM LiNMC. (D) ATP in percentage of control. (E) Summary of the main effects of RNAi and LiNMC concentration in chemical uptake in worms. Two-way ANOVA with Tukey-HSD test, n = 3 biological replicates for roGFP and Perceval quantification (12–18 technical replicates per 3 biological replicates, 300–1000 worms per technical replicate), n = 3 biological replicates for length determination (100 worms per treatment), error bars are SEM, (**) p < 0.01, (***) p < 0.001.

Fig. 4. OCR and mtDNA copy number are unaltered after exposure to LiNMC. Comparison of (A) basal OCR, (B) maximal OCR, (C) spare OCR capacity, (D) mitochondrial basal OCR, (E) proton leak, and (F) non-mitochondrial respiration between groups exposed to either 0, 8, or 32 mM LiNMC, and fed empty vector (EV), frh-1 or gas-1 RNAi bacteria. (G) Mitochondrial to nuclear DNA copy number ratio comparison for all groups. Summary of main effects of LiNMC and RNAi on (H) OCR and (I) mtDNA to nDNA copy number ratio. Two-way ANOVA with Tukey-HSD; error bars are SEM, n = 3 biological replicates (5 technical replicates per biological replicate, 20–30 L4 worms per technical replicate).

Fig. 5. Parental exposure to LiNMC causes neurodegeneration in progeny. (A) Parental (P0) worms were exposed to LiNMC for 24 hours after reaching the L4 larval stage, their progeny was recovered, and neurodegeneration in this F1 generation quantified at the L4 larval stage. Neurodegeneration scored for neuronal types (defined by neurotransmitter) after parental exposure to LiNMC: (B) dopaminergic, (C) glutamatergic, (D) cholinergic, (E) GABAergic, (F) serotonergic, and (G) glial cells. Neurodegeneration scored for neuronal types (defined by neurotransmitter) after parental exposure to lithium chloride: (H) dopaminergic, (I) glutamatergic, (J) cholinergic, and (K) GABAergic. Chi-square with Bonferroni correction, n = 3 biological replicates (20 worms per treatment), (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.