Joel Meyer

Collagen mutations are commonly used in the creation of <i>Caenorhabditis elegans</i> transgenic strains, but their secondary effects are not fully characterized <i>.</i> We compared the mitochondrial function of N2, <i>dpy-10, rol-6,</i> and PE255 <i>C. elegans</i> . N2 worms exhibited ~2-fold greater volume, mitochondrial DNA copy number, and nuclear DNA copy number than collagen mutants (p<0.05). Whole-worm respirometry and ATP levels were higher in N2 worms, but differences in respirometry largely disappeared after normalization to mitochondrial DNA copy number. This data suggests that <i>rol-6</i> and <i>dpy-10</i> mutants are developmentally delayed but have comparable mitochondrial function to N2 worms once the data is normalized to developmental stage.

<h4>Aims</h4>Mitochondrial dysfunction is implicated in several diseases, including neurological disorders such as Parkinson's disease. However, there is uncertainty about which of the many mechanisms by which mitochondrial function can be disrupted may lead to neurodegeneration. Pentachlorophenol (PCP) is an organic pollutant reported to cause mitochondrial dysfunction including oxidative stress and mitochondrial uncoupling. We investigated the effects of PCP exposure in <i>Caenorhabditis elegans</i>, including effects on mitochondria and dopaminergic neurons. We hypothesized that mild mitochondrial uncoupling by PCP would impair bioenergetics while decreasing oxidative stress, and therefore would not cause dopaminergic neurodegeneration.<h4>Results</h4>A 48-hour developmental exposure to PCP causing mild growth delay (∼10 % decrease in growth during 48 h, covering all larval stages) reduced whole-organism ATP content > 50 %, and spare respiratory capacity ∼ 30 %. Proton leak was also markedly increased. These findings suggest a main toxic mechanism of mitochondrial uncoupling rather than oxidative stress, which was further supported by a concomitant shift toward a more reduced cellular redox state measured at the whole organism level. However, exposure to PCP did not cause dopaminergic neurodegeneration, nor did it sensitize animals to a neurotoxic challenge with 6-hydroxydopamine. Whole-organism uptake and PCP metabolism measurements revealed low overall uptake of PCP in our experimental conditions (50 μM PCP in the liquid exposure medium resulted in organismal concentrations of < 0.25 μM), and no measurable production of the oxidative metabolites tetra-1,4-benzoquinone and tetrachloro-p-hydroquinone.<h4>Innovation</h4>This study provides new insights into the mechanistic interplay between mitochondrial uncoupling, oxidative stress, and neurodegeneration in <i>C. elegans.</i> These findings support the premise of mild uncoupling-mediated neuroprotection, but are inconsistent with proposed broad "mitochondrial dysfunction"-mediated neurodegeneration models, and highlight the utility of the <i>C. elegans</i> model for studying mitochondrial and neurotoxicity.<h4>Conclusions</h4>Developmental exposure to pentachlorophenol causes gross toxicological effects (growth delay and arrest) at high levels. At a lower level of exposure, still causing mild growth delay, we observed mitochondrial dysfunction including uncoupling and decreased ATP levels. However, this was associated with a more-reduced cellular redox tone and did not exacerbate dopaminergic neurotoxicity of 6-hydroxydopamine, instead trending toward protection. These findings may be informative of efforts to define nuanced mitochondrial dysfunction-related adverse outcome pathways that will differ depending on the form of initial mitochondrial toxicity.

Silver nanoparticles (AgNPs) are well-proven antimicrobial nanomaterials, yet little is elucidated regarding the mechanism underlying cytotoxicity induced by these nanoparticles. Here, we tested the hypothesis that mitochondria are primary intracellular targets of two AgNPs and silver ions in mouse hepatocytes (AML12) cultured in glucose- and galactose-based media. AML12 cells were more sensitive to mitochondrial uncoupling when grown with galactose rather than glucose. However, 24 h treatments with 15 nm AgNPs and 6 nm GA-AgNPs (5 and 10 μg/mL) and AgNO₃ (1 and 3 μg/mL), concentrations that resulted in either 10 or 30% cytotoxicity, failed to cause more toxicity to AML12 cells grown on galactose than glucose. Furthermore, colocalization analysis and subcellular Ag quantification did not show any enrichment of silver content in mitochondria in either medium. Finally, the effects of the same exposures on mitochondrial respiration were mild or undetectable, a result inconsistent with mitochondrial toxicity causing cell death. Our results suggest that neither ionic Ag nor the AgNPs that we tested specifically target mitochondria and are inconsistent with mitochondrial dysfunction being the primary cause of cell death after Ag exposure under these conditions.

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.