Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and inter- as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler) and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters (basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity and proton leak) of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans.

Exercise and caloric restriction improve health, including reducing risk of cardiovascular disease, neurological disease, and cancer. However, molecular mechanisms underlying these protections are poorly understood, partly due to the cost and time investment of mammalian long-term diet and exercise intervention studies. We subjected Caenorhabditis elegans nematodes to a 6-day, twice daily swimming exercise regimen, during which time the animals also experienced brief, transient food deprivation. Accordingly, we included a non-exercise group with the same transient food deprivation, a non-exercise control with ad libitum access to food, and a group that exercised in food-containing medium. Following these regimens, we assessed mitochondrial health and sensitivity to mitochondrial toxicants. Exercise protected against age-related decline in mitochondrial morphology in body-wall muscle. Food deprivation increased organismal basal respiration; however, exercise was the sole intervention that increased spare respiratory capacity and proton leak. We observed increased lifespan in exercised animals compared to both control and transiently food-deprived nematodes. Finally, exercised animals (and to a lesser extent, transiently food-deprived animals) were markedly protected against lethality from acute exposures to the mitotoxicants rotenone and arsenic. Thus, swimming exercise and brief food deprivation provide effective intervention in C. elegans, protecting from age-associated mitochondrial decline and providing resistance to mitotoxicant exposures.

Mitochondrial fission, fusion, and mitophagy are interlinked processes that regulate mitochondrial shape, number, and size, as well as metabolic activity and stress response. The fundamental importance of these processes is evident in the fact that mutations in fission (DRP1), fusion (MFN2, OPA1), and mitophagy (PINK1, PARK2) genes can cause human disease (collectively >1/10,000). Interestingly, however, the age of onset and severity of clinical manifestations varies greatly between patients with these diseases (even those harboring identical mutations), suggesting a role for environmental factors in the development and progression of certain mitochondrial diseases. Using the model organism Caenorhabditis elegans, we screened ten mitochondrial toxicants (2, 4-dinitrophenol, acetaldehyde, acrolein, aflatoxin B1, arsenite, cadmium, cisplatin, doxycycline, paraquat, rotenone) for increased or decreased toxicity in fusion (fzo-1, eat-3)-, fission (drp-1)-, and mitophagy (pdr-1, pink-1)-deficient nematodes using a larval growth assay. In general, fusion-deficient nematodes were the most sensitive to toxicants, including aflatoxin B1, arsenite, cisplatin, paraquat, and rotenone. Because arsenite was particularly potent in fission- and fusion-deficient nematodes, and hundreds of millions of people are chronically exposed to arsenic, we investigated the effects of these genetic deficiencies on arsenic toxicity in more depth. We found that deficiencies in fission and fusion sensitized nematodes to arsenite-induced lethality throughout aging. Furthermore, low-dose arsenite, which acted in a “mitohormetic” fashion by increasing mitochondrial function (in particular, basal and maximal oxygen consumption) in wild-type nematodes by a wide range of measures, exacerbated mitochondrial dysfunction in fusion-deficient nematodes. Analysis of multiple mechanistic changes suggested that disruption of pyruvate metabolism and Krebs cycle activity underlie the observed arsenite-induced mitochondrial deficits, and these disruptions are exacerbated in the absence of mitochondrial fusion. This research demonstrates the importance of mitochondrial dynamics in limiting arsenite toxicity by permitting mitochondrial adaptability. It also suggests that individuals suffering from deficiencies in mitodynamic processes may be more susceptible to the mitochondrial toxicity of arsenic and other toxicants.

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The consequences of damage to the mitochondrial genome (mtDNA) are poorly understood, although mtDNA is more susceptible to damage than nuclear DNA (nucDNA), and many environmental toxicants target the mitochondria. Reports from the toxicological literature suggest that exposure to early-life mitochondrial damage could lead to deleterious consequences later in life (the “Developmental Origins of Health and Disease” paradigm) but reports from other fields often report beneficial (“mitohormetic”) responses to such damage. Here, we test the effects of low (causing no change in lifespan) levels of ultraviolet C (UVC)-induced, irreparable mtDNA damage during early development in Caenorhabditis elegans. This exposure led to life-long reductions in mtDNA copy number and steady-state ATP levels, accompanied by increased oxygen consumption and altered metabolite profiles, suggesting inefficient mitochondrial function. Exposed nematodes were also developmentally delayed, reached smaller adult size, and were rendered more susceptible to subsequent exposure to chemical mitotoxicants. Metabolomic and genetic analysis of key signaling and metabolic pathways supported redox signaling during early development as a mechanism for establishing these persistent alterations. Our results highlight the importance of early-life exposures to environmental pollutants, especially in the context of exposure to chemicals that target mitochondria.

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