Cardiac Development in the Presence of Cadmium: An in Vitro Study Using Human Embryonic Stem Cells and Cardiac Organoids.

BACKGROUND: Exposure to cadmium (Cd) is associated with cardiovascular diseases. Maternal Cd exposure is a significant risk factor for congenital heart disease. However, mechanisms of Cd on developmental cardiotoxicity are not well defined. OBJECTIVES: We evaluated the effects of Cd on the different stages (mesoderm, cardiac induction, cardiac function) of cardiac development using an early embryo development in vitro model and two- or three-dimensional (2- or 3D) cardiomyocyte and cardiac organoid formation models mimicking early cardiac development. METHODS: Embryonic stem cells (ESCs) form 3D aggregates, called embryoid bodies, that recapitulate events involved with early embryogenesis (e.g., germ layer formation). This model was used for early germ layer formation and signaling pathway identification. The 2D cardiomyocyte differentiation from the NKX2-5eGFP/w human ESCs model was used to explore the effects of Cd exposure on cardiomyocyte formation and to model mesoderm differentiation and cardiac induction, allowing us to explore different developmental windows of Cd toxicity. The 3D cardiac organoid model was used in evaluating the effects of Cd exposure on contractility and cardiac development. RESULTS: Cd (0.6µM; 110 ppb) lowered the differentiation of embryoid bodies to mesoderm via suppression of Wnt/ß-catenin-signaling pathways. During early mesoderm induction, the mesoderm-associated transcription factors MESP1 and EOMES showed a transient up-regulation, which decreased later in the cardiac induction stage. Cd (0.15µM) lowered mesoderm formation and cardiac induction through suppression of the transcription factors and mesoderm marker genes HAND1, SNAI2, HOPX, and the cardiac-specific genes NKX2-5, GATA4, troponin T, and alpha-actinin. In addition, Cd-induced histone modifications for both gene activation (H3K4me3) and repression (H3K27me3), which play vital roles in regulating mesoderm commitment markers. The effects of Cd inhibition on cardiomyocyte differentiation were confirmed in 3D cardiac organoids. DISCUSSION: In conclusion, using a human ESC-derived 2D/3D in vitro differentiation model system and cardiac organoids, we demonstrated that low-dose Cd suppressed mesoderm formation through mesoderm gene histone modification, thus inhibiting cardiomyocyte differentiation and cardiac induction. The studies provide valuable insights into cellular events and molecular mechanisms associated with Cd-induced congenital heart disease. https://doi.org/10.1289/EHP11208.

Calcium/Calmodulin Dependent Protein Kinase Kinase 2 Regulates the Expansion of Tumor-Induced Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are a hetero geneous group of cells, which can suppress the immune response, promote tumor progression and impair the efficacy of immunotherapies. Consequently, the pharmacological targeting of MDSC is emerging as a new immunotherapeutic strategy to stimulate the natural anti-tumor immune response and potentiate the efficacy of immunotherapies. Herein, we leveraged genetically modified models and a small molecule inhibitor to validate Calcium-Calmodulin Kinase Kinase 2 (CaMKK2) as a druggable target to control MDSC accumulation in tumor-bearing mice. The results indicated that deletion of CaMKK2 in the host attenuated the growth of engrafted tumor cells, and this phenomenon was associated with increased antitumor T cell response and decreased accumulation of MDSC. The adoptive transfer of MDSC was sufficient to restore the ability of the tumor to grow in Camkk2-/- mice, confirming the key role of MDSC in the mechanism of tumor rejection. In vitro studies indicated that blocking of CaMKK2 is sufficient to impair the yield of MDSC. Surprisingly, MDSC generated from Camkk2-/- bone marrow cells also showed a higher ability to terminally differentiate toward more immunogenic cell types (e.g inflammatory macrophages and dendritic cells) compared to wild type (WT). Higher intracellular levels of reactive oxygen species (ROS) accumulated in Camkk2-/- MDSC, increasing their susceptibility to apoptosis and promoting their terminal differentiation toward more mature myeloid cells. Mechanistic studies indicated that AMP-activated protein kinase (AMPK), which is a known CaMKK2 proximal target controlling the oxidative stress response, fine-tunes ROS accumulation in MDSC. Accordingly, failure to activate the CaMKK2-AMPK axis can account for the elevated ROS levels in Camkk2-/- MDSC. These results highlight CaMKK2 as an important regulator of the MDSC lifecycle, identifying this kinase as a new druggable target to restrain MDSC expansion and enhance the efficacy of anti-tumor immunotherapy.

Multiple metabolic changes mediate the response of Caenorhabditis elegans to the complex I inhibitor rotenone.

Rotenone, a mitochondrial complex I inhibitor, has been widely used to study the effects of mitochondrial dysfunction on dopaminergic neurons in the context of Parkinson's disease. Although the deleterious effects of rotenone are well documented, we found that young adult Caenorhabditis elegans showed resistance to 24 and 48 h rotenone exposures. To better understand the response to rotenone in C. elegans, we evaluated mitochondrial bioenergetic parameters after 24 and 48 h exposures to 1 µM or 5 µM rotenone. Results suggested upregulation of mitochondrial complexes II and V following rotenone exposure, without major changes in oxygen consumption or steady-state ATP levels after rotenone treatment at the tested concentrations. We found evidence that the glyoxylate pathway (an alternate pathway not present in higher metazoans) was induced by rotenone exposure; gene expression measurements showed increases in mRNA levels for two complex II subunits and for isocitrate lyase, the key glyoxylate pathway enzyme. Targeted metabolomics analyses showed alterations in the levels of organic acids, amino acids, and acylcarnitines, consistent with the metabolic restructuring of cellular bioenergetic pathways including activation of complex II, the glyoxylate pathway, glycolysis, and fatty acid oxidation. This expanded understanding of how C. elegans responds metabolically to complex I inhibition via multiple bioenergetic adaptations, including the glyoxylate pathway, will be useful in interrogating the effects of mitochondrial and bioenergetic stressors and toxicants.

Application of a Framework for Grouping and Mixtures Toxicity Assessment of PFAS: A Closer Examination of Dose-Additivity Approaches.

Environmental occurrence and biomonitoring data for per- and polyfluoroalkyl substances (PFAS) demonstrate that humans are exposed to mixtures of PFAS. This article presents a new and systematic analysis of available PFAS toxicity study data using a tiered mixtures risk assessment framework consistent with United States and international mixtures guidance. The lines of evidence presented herein include a critique of whole mixture toxicity studies and analysis of dose-response models based on data from subchronic oral toxicity studies in rats. Based on available data to-date, concentration addition and relative potency factor methods are found to be inappropriate due to differences among sensitive effects and target organ potencies and noncongruent dose-response curves for the same effect endpoints from studies using the same species and protocols. Perfluorooctanoic acid and perfluorooctane sulfonic acid lack a single mode of action or molecular initiating event and our evaluation herein shows they also have noncongruent dose-response curves. Dose-response curves for long-chain perfluoroalkyl sulfonic acids (PFSAs) also significantly differ in shapes of the curves from short-chain PFSAs and perfluoroalkyl carboxylic acids evaluated, and additional differences are apparent when curves are evaluated based on internal or administered dose. Following well-established guidance, the hazard index method applied to perfluoroalkyl carboxylic acids and PFSAs grouped separately is the most appropriate approach for conducting a screening level risk assessment for nonpolymeric PFAS mixtures, given the current state-of-the science. A clear presentation of assumptions, uncertainties, and data gaps is needed before dose-additivity methods, including hazard index , are used to support risk management decisions. Adverse outcome pathway(s) and mode(s) of action information for perfluorooctanoic acid and perfluorooctane sulfonic acid and for other nonpolymer PFAS are key data gaps precluding more robust mixtures methods. These findings can guide the prioritization of future studies on single chemical and whole mixture toxicity studies.

Human health hazard assessment of quaternary ammonium compounds: Didecyl dimethyl ammonium chloride and alkyl (C12-C16) dimethyl benzyl ammonium chloride.

Quaternary ammonium compounds (Quats) are a large class of permanently charged cationic chemicals that are used in a variety of consumer and industrial products for their antimicrobial properties. Didecyl dimethyl ammonium chloride (DDAC) and alkyl (C12, C14, C16) dimethyl benzyl ammonium chloride (C12-C16 ADBAC) are frequently used as active ingredients in antimicrobials and are the focus of the current hazard assessment. Robust toxicology databases exist for both DDAC and C12-C16 ADBAC; however, the majority of available studies for DDAC and C12-C16 ADBAC are unpublished, but have been submitted to and reviewed by regulatory agencies (i.e., EPA and European Chemicals Agency) to support antimicrobial product registration. With the objective of contributing to public understanding of the robust and complete toxicology database available for DDAC and C12-C16 ADBAC, a comprehensive review was conducted using available peer-reviewed literature and unpublished data submitted to and summarized by regulatory agencies. A review of available literature indicates that DDAC and C12-C16 ADBAC have similar hazard profiles. Both DDAC and C12-C16 ADBAC are poorly absorbed via the oral and dermal exposure routes (≤10%), are not systemically distributed, and are primarily excreted in feces. DDAC and C12-C16 ADBAC are not dermal sensitizers, are not specific developmental or reproductive toxicants, are not carcinogenic or genotoxic, and do not cause systemic toxicity. DDAC and C12-C16 ADBAC are irritating/corrosive to skin at high concentrations, and are acutely toxic via the oral, dermal (C12-C16 ADBAC only), and inhalation exposure routes; however, both DDAC and C12-C16 ADBAC are considered non-volatile and are not readily aerosolized. Both DDAC and C12-C16 ADBAC can cause toxicity in repeated dose oral toxicity studies with no-observed-adverse-effect levels ranging from 10 to 93.1 mg/kg-day for DDAC and 3.7-188 mg/kg-day for C12-C16 ADBAC in subchronic and chronic studies conducted with beagles, mice, and rats. The toxicological effects associated with reported lowest-observed-adverse-effect levels for both DDAC and C12-C16 ADBAC are consistently characterized by reduced food consumption, reduced mean body weight, reduced body weight gain, and local irritation. These effects are consistent with the mode of action of an irritating/corrosive chemical. Based upon currently available data, the main concern associated with exposure to DDAC and C12-C16 ADBAC is local effects through irritation.

Zebrafish CYP1A expression in transgenic Caenorhabditis elegans protects from exposures to benzo[a]pyrene and a complex polycyclic aromatic hydrocarbon mixture.

Polycyclic aromatic hydrocarbons (PAHs) are environmental toxicants primarily produced during incomplete combustion; some are carcinogens. PAHs can be safely metabolized or, paradoxically, bioactivated via specific cytochrome P450 (CYP) enzymes to more reactive metabolites, some of which can damage DNA and proteins. Among the CYP isoforms implicated in PAH metabolism, CYP1A enzymes have been reported to both sensitize and protect from PAH toxicity. To clarify the role of CYP1A in PAH toxicity, we generated transgenic Caenorhabditis elegans that express CYP1A at a basal (but not inducible) level. Because this species does not normally express any CYP1 family enzyme, this approach permitted a test of the role of basally expressed CYP1A in PAH toxicity. We exposed C. elegans at different life stages to either the PAH benzo[a]pyrene (BaP) alone, or a real-world mixture dominated by PAHs extracted from the sediment of a highly contaminated site on the Elizabeth River (VA, USA). This site, the former Atlantic Wood Industries, was declared a Superfund site due to coal tar creosote contamination that caused very high levels (in the [mg/mL] range) of high molecular weight PAHs within the sediments. We demonstrate that CYP1A protects against BaP-induced growth delay, reproductive toxicity, and reduction of steady state ATP levels. Lack of sensitivity of a DNA repair (Nucleotide Excision Repair)-deficient strain suggested that CYP1A did not produce significant levels of DNA-reactive metabolites from BaP. The protective effects of CYP1A in Elizabeth River sediment extract (ERSE)-exposed nematodes were less pronounced than those seen in BaP-exposed nematodes; CYP1A expression protected against ERSE-induced reduction of steady-state ATP levels, but not other outcomes of exposure to sediment extracts. Overall, we find that in C. elegans, a basal level of CYP1A activity is protective against the examined PAH exposures.

A Mitochondrial Progesterone Receptor Increases Cardiac Beta-Oxidation and Remodeling.

Progesterone is primarily a pregnancy-related hormone, produced in substantial quantities after ovulation and during gestation. Traditionally known to function via nuclear receptors for transcriptional regulation, there is also evidence of nonnuclear action. A previously identified mitochondrial progesterone receptor (PR-M) increases cellular respiration in cell models. In these studies, we demonstrated that expression of PR-M in rat H9c2 cardiomyocytes resulted in a ligand-dependent increase in oxidative cellular respiration and beta-oxidation. Cardiac expression in a TET-On transgenic mouse resulted in gene expression of myofibril proteins for remodeling and proteins involved in oxidative phosphorylation and fatty acid metabolism. In a model of increased afterload from constant transverse aortic constriction, mice expressing PR-M showed a ligand-dependent preservation of cardiac function. From these observations, we propose that PR-M is responsible for progesterone-induced increases in cellular energy production and cardiac remodeling to meet the physiological demands of pregnancy.

Perfluorohexanoic acid toxicity, part II: Application of human health toxicity value for risk characterization.

Perfluorohexanoic acid (PFHxA) is a short-chain, six-carbon PFAA and is a primary impurity, degradant, and metabolite associated with the short-chain fluorotelomer-based chemistry used in the United States, Europe and Japan today. With the shift towards short-chain PFAA chemistry, uncertainties remain regarding human health risks associated with current exposure levels. Here, we present a critical review and assessment of data relevant to human health risk assessment to today's short-chain PFAA chemistry. Human biomonitoring surveys indicate that PFHxA is infrequently detected in the environment as well as in human serum and urine; however, human health concerns may persist in locations where PFHxA is detected. In a companion paper (Luz et al., 2019) we comprehensively evaluate the available toxicity data for PFHxA, and derive a chronic human health-based reference dose (RfD) for PFHxA of 0.25 mg/kg-day based on benchmark dose modeling of renal papillary necrosis in chronically exposed female rats. In this paper, we apply this RfD in human health-based screening levels calculations, and derive a drinking water lifetime health advisory of 1400 µg/L and a residential groundwater screening level for children of 4000 µg/L. Compared to environmental concentration data, even sites with more elevated concentrations of PFHxA in the environment are at least an order of magnitude lower than these screening levels. Available PFHxA human serum and urine biomonitoring data, used as a biomarker for general population exposure, demonstrates that the general human population exposures to PFHxA are low. Previous estimates of daily intake rates for infants exposed to PFHxA through breast milk, formula, and baby foods (Lorenzo et al., 2016) combined with the most conservative PFHxA peer-reviewed toxicity value (Luz et al., 2019) demonstrate that the margin of safety for PFHxA is high. Therefore, PFHxA and related fluorotelomer precursors currently appear to present negligible human health risk to the general population and are not likely to drive or substantially contribute to risk at sites contaminated with PFAS mixtures. PFHxA may also represent a suitable marker for the safety of fluorotelomer replacement chemistry used today.

Perfluorohexanoic acid toxicity, part I: Development of a chronic human health toxicity value for use in risk assessment.

Perfluorohexanoic acid (PFHxA) is a short-chain, six-carbon perfluoroalkyl acid (PFAA) and is a primary impurity, degradant, and metabolite associated with the short-chain fluorotelomer-based chemistry used globally today. The transition to short-chain fluorotelomer-based products as a cornerstone in replacement fluorochemistry has raised questions regarding potential human health risks associated with exposure to fluorotelomer-based substances and therefore, PFHxA. Here, we present a critical review of data relevant to such a risk assessment, including epidemiological studies and in vivo and in vitro toxicity studies that examined PFHxA acute, subchronic, and chronic toxicity. Key findings from toxicokinetic and mode-of-action studies are also evaluated. Sufficient data exist to conclude that PFHxA is not carcinogenic, is not a selective reproductive or developmental toxicant, and does not disrupt endocrine activity. Collectively, effects caused by PFHxA exposure are largely limited to potential kidney effects, are mild and/or reversible, and occur at much higher doses than observed for perfluorooctanoic acid (PFOA). A chronic human-health-based oral reference dose (RfD) for PFHxA of 0.25 mg/kg-day was calculated using benchmark dose modeling of renal papillary necrosis from a chronic rat bioassay. This RfD is four orders of magnitude greater than the chronic oral RfD calculated by the U.S. Environmental Protection Agency for PFOA. The PFHxA RfD can be used to inform public health decisions related to PFHxA and fluorotelomer precursors for which PFHxA is a terminal degradant. These findings clearly demonstrate that PFHxA is less hazardous to human health than PFOA. The analyses presented support site-specific risk assessments as well as product stewardship initiatives for current and future short-chain fluorotelomer-based products.

Pluripotent Stem Cells in Developmental Toxicity Testing: A Review of Methodological Advances.

Millions of children are born each year with a birth defect. Many of these defects are caused by environmental factors, although the underlying etiology is often unknown. In vivo mammalian models are frequently used to determine if a chemical poses a risk to the developing fetus. However, there are over 80 000 chemicals registered for use in the United States, many of which have undergone little safety testing, necessitating the need for higher-throughput methods to assess developmental toxicity. Pluripotent stem cells (PSCs) are an ideal in vitro model to investigate developmental toxicity as they possess the capacity to differentiate into nearly any cell type in the human body. Indeed, a burst of research has occurred in the field of stem cell toxicology over the past decade, which has resulted in numerous methodological advances that utilize both mouse and human PSCs, as well as cutting-edge technology in the fields of metabolomics, transcriptomics, transgenics, and high-throughput imaging. Here, we review the wide array of approaches used to detect developmental toxicants, suggest areas for further research, and highlight critical aspects of stem cell biology that should be considered when utilizing PSCs in developmental toxicity testing.

Arsenic Alters Exosome Quantity and Cargo to Mediate Stem Cell Recruitment Into a Cancer Stem Cell-Like Phenotype.

Inorganic arsenic is a human carcinogen that can target the prostate. Accumulating evidence suggests arsenic can disrupt stem cell (SC) dynamics during the carcinogenic process. Previous work demonstrated arsenic-transformed prostate epithelial (CAsE-PE) cells can recruit prostate SCs into rapidly acquiring a cancer SC (CSC) phenotype via the secretion of soluble factors. Exosomes are small, membrane-derived vesicles that contain lipids, RNA, and proteins, and actively contribute to cancer initiation and progression when taken up by target cells. Here we hypothesized that CAsE-PE cells are recruiting SCs to a CSC-like phenotype via exosomal signaling. CAsE-PE cells secreted 700% more exosomes than parental RWPE-1 cells. CAsE-PE exosomes were enriched with oncogenic factors, including oncogenes (KRAS, NRAS, VEFGA, MYB, and EGFR), inflammation-related (cyclooxygenase-2, interleukin 1B (IL1B), IL6, transforming growth factor-ß, and tumor necrosis factor-A), and apoptosis-related (CASP7, CASP9, and BCL2) transcripts, and oncogenesis-associated microRNAs. When compared with SCs cultured in exosome-depleted conditioned medium (CM), SCs cultured in CM containing CAsE-PE-derived exosomes showed increased (198%) matrix metalloproteinase activity and underwent an epithelial-to-mesenchymal transition in morphology, suggesting an exosome-mediated transformation. KRAS plays an important role in arsenic carcinogenesis. Although KRAS transcript (>24 000%) and protein (866%) levels were elevated in CAsE-PE exosomes, knock-down of KRAS in these cells only partially mitigated the CSC-like phenotype in cocultured SCs. Collectively, these results suggest arsenic impacts both exosomal quantity and cargo. Exosomal KRAS is only minimally involved in this recruitment, and additional factors (eg, cancer-associated miRNAs) likely also play a role. This work furthers our mechanistic understanding of how arsenic disrupts SC dynamics and influences the tumor microenvironment during carcinogenesis.

The high-production volume fungicide pyraclostrobin induces triglyceride accumulation associated with mitochondrial dysfunction, and promotes adipocyte differentiation independent of PPARγ activation, in 3T3-L1 cells.

Pyraclostrobin is one of the most heavily used fungicides, and has been detected on a variety of produce, suggesting human exposure occurs regularly. Recently, pyraclostrobin exposure has been linked to a variety of toxic effects, including neurodegeneration and triglyceride (TG) accumulation. As pyraclostrobin inhibits electron transport chain complex III, and as mitochondrial dysfunction is associated with metabolic syndrome (cardiovascular disease, type II diabetes, obesity), we designed experiments to test the hypothesis that mitochondrial dysfunction underlies its adipogenic activity. 3T3-L1 cells were differentiated according to standard protocols in the presence of pyraclostrobin, resulting in TG accumulation. However, TG accumulation occurred without activation of the peroxisome proliferator activated nuclear receptor gamma (PPARγ), the canonical pathway mediating adipogenesis. Furthermore, cells failed to express many markers of adipogenesis (PPARγ, lpl, CEBPα), while co-exposure to pyraclostrobin and two different PPARγ antagonists (GW9662, T0070907) failed to mitigate TG accumulation, suggesting TG accumulation occurred through a PPARγ-independent mechanism. Instead, pyraclostrobin reduced steady-state ATP, mitochondrial membrane potential, basal mitochondrial respiration, ATP-linked respiration, and spare respiratory capacity, demonstrating mitochondrial dysfunction, while reduced expression of genes involved in glucose transport (Glut-4), glycolysis (Pkm, Pfkl, Pfkm), fatty acid oxidation (Cpt-1b), and lipogenesis (Fasn, Acacα, Acacß) further suggested a disruption of metabolism. Finally, inhibition of cAMP responsive element binding protein (CREB), a PPARγ coactivator, partially mitigated pyraclostrobin-induced TG accumulation, suggesting TG accumulation is occurring through a CREB-driven mechanism. In contrast, rosiglitazone, a known PPARγ agonist, induced TG accumulation in a PPARγ-dependent manner and enhanced mitochondrial function. Collectively, these results suggest pyraclostrobin-induced mitochondrial dysfunction inhibits lipid homeostasis, resulting in TG accumulation. Exposures that disrupt mitochondrial function may have the potential to contribute to the rising incidence of metabolic syndrome, and thus more research is needed to understand the human health impact of pyraclostrobin exposure.

Mitochondrial fusion, fission, and mitochondrial toxicity.

Mitochondrial dynamics are regulated by two sets of opposed processes: mitochondrial fusion and fission, and mitochondrial biogenesis and degradation (including mitophagy), as well as processes such as intracellular transport. These processes maintain mitochondrial homeostasis, regulate mitochondrial form, volume and function, and are increasingly understood to be critical components of the cellular stress response. Mitochondrial dynamics vary based on developmental stage and age, cell type, environmental factors, and genetic background. Indeed, many mitochondrial homeostasis genes are human disease genes. Emerging evidence indicates that deficiencies in these genes often sensitize to environmental exposures, yet can also be protective under certain circumstances. Inhibition of mitochondrial dynamics also affects elimination of irreparable mitochondrial DNA (mtDNA) damage and transmission of mtDNA mutations. We briefly review the basic biology of mitodynamic processes with a focus on mitochondrial fusion and fission, discuss what is known and unknown regarding how these processes respond to chemical and other stressors, and review the literature on interactions between mitochondrial toxicity and genetic variation in mitochondrial fusion and fission genes. Finally, we suggest areas for future research, including elucidating the full range of mitodynamic responses from low to high-level exposures, and from acute to chronic exposures; detailed examination of the physiological consequences of mitodynamic alterations in different cell types; mechanism-based testing of mitotoxicant interactions with interindividual variability in mitodynamics processes; and incorporating other environmental variables that affect mitochondria, such as diet and exercise.

Deficiencies in mitochondrial dynamics sensitize Caenorhabditis elegans to arsenite and other mitochondrial toxicants by reducing mitochondrial adaptability.

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.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans.

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Reported impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H2O2), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl2, low-level DNA damage (∼0.25 lesions/10kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H2O2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H2O2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H2O2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion.

Correction: Mitochondrial Morphology and Fundamental Parameters of the Mitochondrial Respiratory Chain Are Altered in Caenorhabditis elegans Strains Deficient in Mitochondrial Dynamics and Homeostasis Processes.

[This corrects the article DOI: 10.1371/journal.pone.0130940.].

In Vivo Determination of Mitochondrial Function Using Luciferase-Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced Dysfunction.

Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase-expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady-state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short-term inhibitor exposure indicate toxicant-induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant-induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc.