Effects of chemical exposures on mitochondrial mutagenesis across species

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2022

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Mitochondria are essential organelles required for all eukaryotic life on earth. Each organelle contains multiple copies of the mitochondrial genome (mtDNA) that encodes genes essential for energy production. Mutations in the mitochondrial genome are associated with mitochondrial diseases and diseases of aging, particularly neurodegenerative diseases, such as Parkinson’s Disease, and cancer. mtDNA mutation rates are often higher than nuclear DNA mutation rates. However, the origin of mtDNA mutations is poorly understood. Mitochondria lack many of the basic DNA repair mechanisms that are in the nucleus, potentially rendering mtDNA vulnerable to DNA damage-induced mtDNA mutations. Very few studies have investigated the impact of chemical exposures, in particular pollutants, on mtDNA mutagenesis, as reviewed in Chapter 4 of this dissertation (published as Leuthner and Meyer, 2021). Therefore, the two research aims of this thesis were designed to investigate the role of chemical exposures on mtDNA damage and mutagenesis in two species, chosen based on particular research strengths that each offered. The overarching hypothesis of this dissertation was that exposure to known nuclear genotoxicants and mutagens would result in the accumulation of mtDNA damage, which would ultimately lead to mtDNA mutations. The first aim of this thesis was to investigate the impact of the ubiquitous pollutant, cadmium (Cd), on mtDNA mutagenesis in the aquatic keystone species, Daphnia pulex. Cd is a known nuclear mutagen and carcinogen, yet the effects of Cd exposure on mtDNA mutations remain unknown. D. pulex offers a number of major advantages for this investigation, as discussed in Chapter 2. A unique aspect for this thesis was the use of a wild, Cd-tolerant population of D. pulex. Remarkably, this population of D. pulex sampled from Simon Lake in Sudbury, Ontario, Canada has adapted to high levels of Cd due to over a century of exposure to pollution from mining and smelting processes. Thousands of generations of experimental evolution were performed under laboratory conditions (an approach termed Mutation Accumulation, or MA, lines) in a Simon Lake isolate and an isolate collected from pristine Buck Lake (Dorset, Ontario, Canada) in both the presence and absence of Cd. This allowed investigation of the effects of Cd on mtDNA mutagenesis in D. pulex populations with very different evolutionary histories. Whole genome sequencing was conducted and mtDNA reads were extracted for analysis of mtDNA mutation frequencies, rates, and signatures. Hundreds of single nucleotide mutates were detected after >2,000 and >12,000 total generations of mutation accumulation, or about 40 to 250 fold more mutations than previous Daphnia mtDNA MA line studies. This afforded the resolution to determine the mechanism of endogenous mtDNA mutagenesis in Daphnia for the first time; these results indicate that endogenous mtDNA mutagenesis is likely mostly driven by polymerase γ error at sites of oxidized and deaminated cytosines (G  A/C T). At the earlier timepoint, Cd exposure further increased the rate of this mutation in the Cd-sensitive Buke Lake Daphnia compared to the Cd-tolerant Simon Lake Daphnia by about 3.6-fold. The results of this research aim suggest that Cd has a small effect on mtDNA mutagenesis, and that the adapted population is resistant to Cd-induced mtDNA mutations. However, after an additional >10,000 generations of mutation accumulation, there was no effect of Cd on this mutation spectrum, and the number of mtDNA mutations that were present at very low frequency increased significantly compared to the earlier timepoint. A small number of mutations did reach fixation or near-fixation however, and these mutations are discussed individually in Chapter 2. We propose that this result is consistent with natural selection acting on germline mtDNA mutation rates and heteroplasmy. Mitochondria harbor various quality control mechanisms that act in response to stress and mitochondrial dysfunction, such as mitophagy, fission, and fusion. Previous studies indicate that mitophagy may be involved in purifying selection against deleterious mtDNA mutations, in addition to targeted degradation of organelles that contain damaged mtDNA. Therefore, the next research aim of this thesis was to investigate the role of mitophagy on the accumulation of mtDNA damage and mutagenesis after exposure to Cd and another genotoxin, Aflatoxin B1 (AfB1), in the organism Caenorhabditis elegans. C. elegans are often used for MA studies to investigate mutational processes in both the nuclear and mitochondrial genomes, and offer a variety of strengths for such studies, as discussed in Chapter 3. A particular strength for the purposes of this thesis was the ability to work with strains carrying mutations in mitophagy genes. A MA experiment was conducted in wild-type C. elegans and two mitophagy-deficient strains, dct-1 and pink-1, in control, 50µM Cd, and 10µM AfB1 conditions. AfB1 was selected because it causes mtDNA damage that is not expected to be efficiently repaired in mtDNA. After an average of 50 generations of MA, about 10 MA lines were selected for each strain/treatment combination for Duplex Sequencing. Duplex Sequencing is an ultra-sensitive, error-corrected sequencing approach that allows for detection of mutations as low as 1 in 10,000 base pairs. Until the preparation of these samples, no study had yet conducted targeted mtDNA Duplex Sequencing in C. elegans. Wild-type and mitophagy-deficient strains all had mutation spectra indicative of oxidative damage driving mtDNA mutagenesis (GT/CA), contrary to what was observed in Daphnia in Aim 1, and contrary to what has been reported in other organisms. However, this confirmed results from a very recent study that also used mtDNA targeted Duplex Sequencing of wild-type C. elegans. Surprisingly, even though more mtDNA mutations were detected and at a lower frequency than ever previously reported, there was no clear effect of either Cd of AfB1 exposure on mtDNA mutations in any strain, despite a marginally significant increase in G:C  A:T mutations in pink-1 AfB1 MA lines compared to wild-type AfB1 MA lines. Overall, this suggests that mitochondria are resistant to exogenous damage-induced point mutations in C. elegans. Further investigations into what mechanisms are responsible for maintaining mtDNA homeostasis that are independent of mitophagy are an exciting future next step. Understanding the impact of chemicals on mtDNA mutations is critical for human and environmental health, as addressed in the Chapter 4 “Mitochondrial DNA mutagenesis: A feature of and biomarker for environmental health,” which has been published as a review. The primary research chapters of this dissertation (Chapters 2 and 3) further contribute to understanding how chemicals impact mitochondrial genome quality and integrity. Collectively, it will be critical to continue to use improved sequencing technologies to continue to investigate the origin and mechanisms of mtDNA mutagenesis resulting from both endogenous and exogenous factors.

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Leuthner, Tess C (2022). Effects of chemical exposures on mitochondrial mutagenesis across species. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/25139.

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