Genetic Sensitivity to Mitochondrial Toxicity
Mitochondria are the main cellular producers of ATP, and play key roles in cellular signaling and apoptosis. Mitochondria also contain their own genomes (mtDNA), which encode 13 subunits of the electron transport chain (ETC), 22 tRNAs, and 2 rRNAs, making mtDNA integrity critical to both mitochondrial and organismal health. Mitochondria are dynamic organelles that fuse and divide to maintain mitochondrial shape, number, and size. However, mitochondrial fission and fusion also play a major role in the mitochondrial stress response. For example, mildly damaged mitochondria can fuse with healthy mitochondria allowing contents to mix, resulting in the generation of healthy mitochondria, which is known as functional complementation. Alternatively, when mitochondria become damaged beyond repair, they are targeted for autophagosomal degradation, or mitophagy. The overall importance of fission, fusion, mitophagy, and mtDNA is demonstrated by the fact that deficiencies in these processes and mtDNA content cause human disease. Interestingly, the age of onset, and severity of clinical manifestations of mitochondrial disease vary from patient to patient, even in individuals harboring identical mutations. These observations suggest a role for the environment in the development and progression of certain mitochondrial diseases; however, the relationship remains poorly understood.
To investigate the role of environmental toxicants in the development, progression, and exacerbation of mitochondrial disease I have taken two approaches using the in vivo model organism Caenorhabditis elegans. First, ten known and suspected mitochondrial toxicants (2,4-dinitrophenol (DNP), acetaldehyde, acrolein, aflatoxin B1 (AfB1), arsenite, cadmium, cisplatin, doxycycline, paraquat, rotenone) were screened for exacerbation of larval growth delay in wild-type, fission-, fusion-, and mitophagy-deficient nematodes using the COPAS Biosort. Second, a C. elegans model of mtDNA depletion was developed using chronic low-dose ethidium bromide exposure. Five toxicants (AfB1, arsenite, paraquat, rotenone, ultraviolet C radiation (UVC)) were tested for exacerbation of mitochondrial function (assessed via changes in steady-state ATP levels) in nematodes with reduced mtDNA content. Mitochondrial health was then further assessed for some of the identified gene-environment interactions. Mitochondrial respiration was measured using the Seahorse XFe24 Extracellular Flux Analyzer, while steady-state ATP levels were assessed using transgenic luciferase expression nematodes and traditional extraction protocols. Gene expression, mtDNA, and nuclear DNA copy number were assessed using real-time PCR, while enzyme activity was assessed using microplate reader-based assays.
Results from the fission, fusion, and mitophagy toxicant screen revealed that fusion-deficient nematodes were sensitive to a variety of toxicants (DNP, AfB1, arsenite, cisplatin, paraquat, rotenone), while pink-1 mitophagy-deficient nematodes were sensitive to rotenone, and fission- and pdr-1 mitophagy-deficient nematodes were only mildly sensitive to paraquat, and rotenone, respectively. As mitochondrial disease is rare, but chronic arsenite exposure is widespread, we further investigated the mechanisms underlying arsenite sensitivity in fission- and fusion-deficient nematodes. Although not sensitive in the larval growth assay, fission-deficient nematodes were sensitive to arsenite later in life in both reproduction and lethality assays. Seahorse and ATP analysis revealed that arsenite disrupts mitochondrial function in fusion-deficient nematodes at multiple life stages (L4, 8- and 12-days of age), while enhancing mitochondrial function in 8-day old wild-type nematodes, and has minimal effect on mitochondrial function in fission-deficient nematodes. Lastly, arsenite inhibited both pyruvate and isocitrate dehydrogenase activity in fusion-deficient nematodes, suggesting a disruption of pyruvate metabolism and Krebs cycle activity underlie the observed mitochondrial dysfunction. These results suggest that deficiencies in mitochondrial fusion may sensitive individuals to arsenite toxicity.
Lastly, I have found that reducing mtDNA content 35-55% only mildly sensitized nematodes to certain secondary toxicant exposures, including UVC and arsenite. Alternatively, reduced mtDNA content did not sensitize nematodes to acute or chronic paraquat or AfB1 exposure, and provided resistance to rotenone. However, we also found that EtBr can induce cytochrome P450s (CYPs), which play a major role in rotenone metabolism; thus, it is likely that induction of CYPs and not reduced mtDNA content is responsible for rotenone resistance. These results suggest that individuals with reduced mtDNA content may be sensitive to certain toxicant exposures, but also highlight the robust mechanism that exist to maintain the integrity of mitochondria and mtDNA.
Collectively, these results suggest individuals suffering from mitochondrial disease caused by mutations in mitochondrial fission, fusion, or mitophagy genes, or by depletion of mtDNA, may be especially sensitive to certain environmental toxicant exposures, including arsenic. Arsenic’s pervasive contamination of drinking water results in chronic exposure for over 100 million people worldwide; thus, dramatically increasing the probability of exposure for individuals suffering from mitochondrial disease, and warrants further investigation in the human populous.
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