Browsing by Subject "Cadmium"
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Item Open Access Allosteric effects of external K+ ions mediated by the aspartate of the GYGD signature sequence in the Kv2.1 K+ channel.(Pflugers Archiv : European journal of physiology, 2006-03) Chapman, Mark L; Blanke, Marie L; Krovetz, Howard S; VanDongen, Antonius MJK+ channels achieve exquisite ion selectivity without jeopardizing efficient permeation by employing multiple, interacting K+-binding sites. Introduction ofa cadmium (Cd2+)-binding site in the external vestibule of Kv2.1 (drk1), allowed us to functionally characterize a binding site for external monovalent cations. Permeant ions displayed higher affinity for this site than non-permeant monovalent cations, although the selectivity profile was different from that of the channel. Point mutations identified the highly conserved aspartate residue immediately following the selectivity filter as a critical determinant of the antagonism between external K+ and Cd2+ ions. A conservative mutation at this position (D378E) significantly affected the open-state stability. Moreover, the mean open time was found to be modulated by external K+ concentration, suggesting a coupling between channel closing and the permeation process. Reducing the Rb+ conductance by mutating the selectivity filter to the sequence found inKv4.1, also significantly reduced the effectiveness ofRb+ ions to antagonize Cd2+ inhibition, thereby implicating the selectivity filter as the site at which K+ions exert their antagonistic effect on Cd2+ block. The equivalent of D378 in KcsA, D80, takes part in an inter-subunit hydrogen-bond network that allows D80to functionally interact with the selectivity filter. The results suggest that external K+ ions antagonize Cd2+inhibition (in I379C) and modulate the mean open time(in the wild-type Kv2.1) by altering the occupancy profile of the K+-binding sites in the selectivity filter.Item Open Access Effects of chemical exposures on mitochondrial mutagenesis across species(2022) Leuthner, Tess CMitochondria 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 (GT/CA), 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.
Item Open Access Embryonic exposures to cadmium and PAHs cause long-term and interacting neurobehavioral effects in zebrafish.(Neurotoxicology and teratology, 2024-03) Stickler, Alexandra; Hawkey, Andrew B; Gondal, Anas; Natarajan, Sarabesh; Mead, Mikayla; Levin, Edward DDevelopmental exposure to either polycyclic aromatic hydrocarbons (PAHs) or heavy metals has been shown to cause persisting and overlapping neurobehavioral effects in animal models. However, interactions between these compounds have not been well characterized, despite their co-occurrence in a variety of environmental media. In two companion studies, we examined the effects of developmental exposure to cadmium (Cd) with or without co-exposure to prototypic PAHs benzo[a]pyrene (BaP, Exp. 1) or fluoranthene (FA, Exp. 2) using a developing zebrafish model. Zebrafish embryos were exposed to Cd (0-0.3 μM), BaP (0-3 μM), FA (0-1.0 μM), or binary Cd-PAH mixtures from 5 to 122 h post fertilization (hpf). In Exp. 1, Cd and BaP produced independent effects on an array of outcomes and interacting effects on specific outcomes. Notably, Cd-induced deficits in dark-induced locomotor stimulation were attenuated by BaP co-exposure in the larval motility test and BaP-induced hyperactivity was attenuated by Cd co-exposure in the adolescent novel tank test. Likewise, in Exp. 2, Cd and FA produced both independent and interacting effects. FA-induced increases on adult post-tap activity in the tap startle test were attenuated by co-exposure with Cd. On the predator avoidance test, FA- and 0.3 μM Cd-induced hyperactivity effects were attenuated by their co-exposure. Taken together, these data indicate that while the effects of Cd and these representative PAHs on zebrafish behavior were largely independent of one another, binary mixtures can produce sub-additive effects for some neurobehavioral outcomes and at certain ages. This research emphasizes the need for detailed risk assessments of mixtures containing contaminants of differing classes, and for clarity on the mechanisms which allow cross-class toxicant interactions to occur.Item Open Access Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice.(American journal of physiology. Heart and circulatory physiology, 2014-11) Wang, W; Landstrom, AP; Wang, Q; Munro, ML; Beavers, D; Ackerman, MJ; Soeller, C; Wehrens, XHTExpression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca(2+) leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca(2+) leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca(2+) release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca(2+) imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca(2+) leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na(+)/Ca(2+)-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd(2+) administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca(2+) leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca(2+). This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca(2+) leak and heart failure development.Item Open Access The use of comparative genomics to investigate mechanisms of cadmium induced transcription(2009) Tvermoes, Brooke ErinCadmium is a human carcinogen and a persistent environmental pollutant of increasing concern. Yet, the exact molecular targets of cadmium toxicity and the molecular mechanisms by which cadmium influences gene expression have not been fully elucidated. Therefore, the characterization of cadmium-inducible genes will provide a better understanding of the underlying mechanism involved in sensing cadmium-stress and the subsequent signaling pathways important for cellular defense against cadmium toxicity. To this end, we characterized two cadmium-responsive genes of no known biological function from the nematode Caenorhabditis elegans (C. elegans), numr-1 and numr-2.
Expression analysis of numr-1 and numr-2 revealed the same temporal and spatial expression patterns of both genes in the absence and presence of metal treatment. In the absence of metal, constitutive expression of numr-1/-2 was developmentally regulated. When adult animals were exposed to metal, numr-1/-2 expression dramatically increased. We show that worms overexpressing numr-1/-2 were more resistant to metal stress and longer lived than control animals; whereas reducing numr-1/-2 activity resulted in increased sensitivity to metal exposure. Furthermore, in the absence of metal, the two numr-1 mutant alleles, tm2775 and ok2239, exhibited decreased muscular functions. The molecular characterization of numr-1 and numr-2 also revealed that the expression of these two genes, at least in part, was regulated by changes in intracellular calcium concentrations ([Ca2+]i). This finding lead us to reevaluate the role of calcium mobilization in cadmium-induced transcription.
While several studies have indicated that exposure to cadmium resulted in increased [Ca2+]i, the mechanism by which cadmium can effect [Ca2+]i and concurrent effects on gene expression remain poorly understood. Therefore, we investigated the effects of low-level cadmium exposure, sufficient to induce transcription of cadmium-responsive genes, on the regulation of [Ca2+]i. In these studies, we utilized the protein-based calcium sensor YC 3.60 stably expressed in a HEK293 cell line. YC 3.60 is insensitive to cadmium ions, and thus is useful to monitor changes in [Ca2+]i following cadmium treatment. Exposing HEK293 cells to 1-30 µM cadmium was sufficient to induce transcription of cadmium-responsive genes such as metallothionein. Cadmium exposure from 1-10 µM had no effect on cell viability, [Ca2+]i mobilization, or increased transcriptional activity of calcium-responsive genes. In contrast, exposure to 30 µM cadmium significantly decreased cell viability, reduced intracellular calcium stores, and significantly altered the transcriptional activity of calcium-responsive genes. Taken together, these data indicate that low-level cadmium exposures (1-10 µM) can induce transcription of cadmium-responsive genes such as metallothionein independent of [Ca2+]i mobilization.
To gain further insight into the mechanistic relationship between cadmium and calcium we investigated the effects of cadmium exposure on the defecation cycle of C. elegans. Defecation is a highly rhythmic behavior that is regulated by calcium oscillations. We found that low-level cadmium exposures, sufficient to induce expression of cadmium-responsive genes such as numr-1/-2, significantly shortened the defecation cycle but did not alter the rhythm of the cycle or the magnitude of the intestinal calcium oscillations. Modulation of lipid metabolism in C. elegans results in a similar shortened defecation cycle, whereas modulation of [Ca2+]i results in lengthened and arrhythmic defection cycles, suggesting that the mechanism by which cadmium alters defecation is independent of [Ca2+]i mobilization.
In summary, the data in this work demonstrates that low-level cadmium exposure induces expression of cadmium-responsive genes independent of calcium mobilization. Thus, modulation of intracellular calcium is unlikely the primary mechanism by which cadmium regulates transcription at low-levels of exposure.