Assessment of the sensitivity of North American fish species to endocrine disrupting chemicals in vitro

There is concern regarding exposure of aquatic organisms to chemicals that interfere with the endocrine system. Disruption of the endocrine system can lead to impacts on sexual development, altered hormone levels, intersex, and ultimately reproductive failure. While effects of endocrine disrupting chemicals (EDCs) on standard laboratory species have been subject of intense study, to this day there is a large gap in knowledge and a high degree of uncertainty regarding the sensitivity of wild fish species to these compounds. One of the main concerns with current toxicity testing approaches is that they require the use of a large number of live animals, particularly when working with native species. Therefore, the aim of this study was to develop in vitro tissue explant assays that would enable the assessment of the sensitivity of different wild fish species native to North America to the exposure with EDCs. Specifically, two in vitro assays were developed: 1) A liver explant assay to assess effects of EDCs that can interact with the estrogen receptor (environmental estrogens), and 2) a gonadal explant assay to assess effects of EDCs on sex-steroid production. The test species selected were northern pike (Esox lucius), walleye (Sander vitreus), and white sucker (Catostomus commersoni) that were sampled from Lake Diefenbaker, Saskatchewan, Canada, and white sturgeon (Acipenser transmontanus) that were randomly selected from an in house stock reared from eggs. Liver tissue was excised from male fishes and exposed for 24 h to a synthetic estrogen, 17α- ethinylestradiol (EE2). Transcript abundance of vitellogenin (VTG), estrogen receptor (ER) α and β in liver tissue were quantified using qPCR. Gonad tissue from both male and female were excised and exposed for 24 h to a model inducer (forskolin) and inhibitor (prochloraz) of steroidogenesis. 11-ketotestosterone (11-KT) and estradiol (E2) were quantified in media by use of ELISA. Exposure to EE2 resulted in a concentration dependent increase in VTG in all species, and an increase in ERα in northern pike. Walleye males showed the greatest sensitivity to EE2. Gonad tissues exposed to forskolin showed a concentration dependent increase in 11-KT and E2. Exposure to prochloraz resulted in a decrease of 11-KTand E2. Male and female white sucker showed greatest sensitivity to forskolin, while male and female walleye showed greatest sensitivity to prochloraz. The seasonal time point during which gonad explants were excised and exposed had an impact on the potency and magnitude of response, resulting in a seasonal effect on sensitivity. Also, gonad explants from these species were found to have greater sensitivity than responses previously reported for in vitro explants of other fish species such as the fathead minnow (Pimephales promelas), and stable cell lines currently used as screening applications to detect chemicals that might disrupt the endocrine system. Therefore, current approaches that use stable cell lines or tissue explants from standardized small bodied laboratory species might not be protective of some wild fish species. These tissue explants represent a promising approach to help understand species sensitivity to EDCs, and if appropriately validated, could be a powerful tool for chemical screening

Repeat measures of DNA methylation in an inception cohort of firefighters

Objectives: Firefighters face exposures associated with adverse health outcomes including risk for multiple cancers. DNA methylation, one type of epigenetic regulation, provides a potential mechanism linking occupational hazards to adverse health outcomes. We hypothesised that DNA methylation profiles would change in firefighters after starting their service and that these patterns would be associated with occupational exposures (cumulative fire-hours and fire-runs). Methods: We profiled DNA methylation with the Infinium MethylationEPIC in blood leucocytes at two time points in non-smoking new recruits: prior to live fire training and 20-37 months later. Linear mixed effects models adjusted for potential confounders were used to identify differentially methylated CpG sites over time using data from 50 individuals passing all quality control. Results: We report 680 CpG sites with altered methylation (q value <0.05) including 60 with at least a 5% methylation difference at follow-up. Genes with differentially methylated CpG sites were enriched in biological pathways related to cancers, neurological function, cell signalling and transcription regulation. Next, linear mixed effects models were used to determine associations between occupational exposures with methylation at the 680 loci. Of these, more CpG sites were associated with fire-runs (108 for all and 78 for structure-fires only, q<0.05) than with fire-hours (27 for all fires and 1 for structure fires). These associations were independent of time since most recent fire, suggesting an impact of cumulative exposures. Conclusions: Overall, this study provides evidence that DNA methylation may be altered by fireground exposures, and the impact of this change on disease development should be evaluated.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Use of ascorbic and thioglycollic acids to eliminate interference from iron in the aluminon method for determining aluminium

The use of ascorbic and thioglycollic acids as inhibitors for the interference of iron in the aluminon method of Hsu have been examined. The use of ascorbic acid, as proposed by Jayman and Sivasubramaniam, has been found to change iron interference from positive to negative causing aluminium to be underestimated. However, the addition of 0.2 ml of a 1% V/V solution of thioglycollic acid to solutions containing aluminium in amounts ranging from 10 to 50 μg has been proved to suppress the interference from up to 900 μg of iron.Peer Reviewe

Longitudinal evaluation of whole blood miRNA expression in firefighters

Background: Dysregulated microRNA (miRNA) expression could provide a mechanism linking firefighter exposure to increased cancer risk. Objective: To determine if changes in longitudinal miRNA expression in firefighters are associated with occupational exposures. Methods: Whole blood MiRNA was evaluated in 52 new recruits prior to live-fire training and 20–37 months later. Linear mixed effects models adjusted for age, ethnicity, BMI, and batch effects were used to determine associations separately for all fires and structure fires only between employment duration, cumulative fire-hours and fire-runs, and time since most recent fire with (1) nine a priori and (2) the full array of 799 miRNAs. Results: For multivariable models including all fires, two a priori miRNAs were associated with employment duration and four with time since most recent fire. For multivariable models restricted to structure fires, three a priori miRNAs were associated with employment duration and one with fire-runs. Additional miRNAs from the full array were associated with employment duration for all fires and/or structure fires. In general, tumor suppressive miRNAs decreased and oncogenic miRNAs increased with exposure. Significance: Changes in miRNAs may serve as biomarkers of exposure effects and a mechanism for increased cancer risk in firefighters. © 2021, The Author(s).Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Assessment of the toxicity of firefighter exposures using the PAH CALUX bioassay

Firefighters can be exposed to a complex set of contaminants while at a fire scene. Identifying new ways to monitor and assess exposure, particularly relating to toxicity is essential to determine the effectiveness of intervention techniques to reduce exposure. This study investigated the use of the polycyclic aromatic hydrocarbon (PAH) CALUX® bioassay for the assessment of exposure and associated toxicity firefighters might encounter. This was done through analysis of extracts of dermal wipes and urine samples collected from firefighters before and after a controlled fire. An increased bioassay response was observed from post-fire neck and calf samples, indicating a greater concentration of PAH-like compounds on the skin. The use of a baby wipe to clean the face and neck during rehab resulted in the attenuation of the observed bioassay response from the neck post-fire. Though a correlation was observed between the bioassay response and hydroxylated PAH concentrations found in the urine, the increased bioassay response from the post-fire urine samples was likely due to unknown compounds other than the hydroxylated PAHs tested. Our results suggest that this bioassay provides a useful measure of firefighter exposure, particularly relating to the potential toxicity of contaminants.Published versionhis study was funded by the Federal Emergency ManagementAgency,grantnumberEMW-2014-FP-00200

Differences in Activation of Aryl Hydrocarbon Receptors of White Sturgeon Relative to Lake Sturgeon Are Predicted by Identities of Key Amino Acids in the Ligand Binding Domain

Dioxin-like compounds (DLCs) are pollutants of global environmental concern. DLCs elicit their adverse outcomes through activation of the aryl hydrocarbon receptor (AhR). However, there is limited understanding of the mechanisms that result in differences in sensitivity to DLCs among different species of fishes. Understanding these mechanisms is critical for protection of the diversity of fishes exposed to DLCs, including endangered species. This study investigated specific mechanisms that drive responses of two endangered fishes, white sturgeon (<i>Acipenser transmontanus</i>) and lake sturgeon (<i>Acipenser fulvescens</i>) to DLCs. It determined whether differences in sensitivity to activation of AhRs (AhR1 and AhR2) can be predicted based on identities of key amino acids in the ligand binding domain (LBD). White sturgeon were 3- to 30-fold more sensitive than lake sturgeon to exposure to 5 different DLCs based on activation of AhR2. There were no differences in sensitivity between white sturgeon and lake sturgeon based on activation of AhR1. Adverse outcomes as a result of exposure to DLCs have been shown to be mediated through activation of AhR2, but not AhR1, in all fishes studied to date. This indicates that white sturgeon are likely to have greater sensitivity <i>in vivo</i> relative to lake sturgeon. Homology modeling and <i>in silico</i> mutagenesis suggests that differences in sensitivity to activation of AhR2 result from differences in key amino acids at position 388 in the LBD of AhR2 of white sturgeon (Ala-388) and lake sturgeon (Thr-388). This indicates that identities of key amino acids in the LBD of AhR2 could be predictive of both <i>in vitro</i> activation by DLCs and <i>in vivo</i> sensitivity to DLCs in these, and potentially other, fishes

Evaluating changes in firefighter urinary metabolomes after structural fires: an untargeted, high resolution approach

Abstract Firefighters have elevated rates of urinary tract cancers and other adverse health outcomes, which may be attributable to environmental occupational exposures. Untargeted metabolomics was applied to characterize this suite of environmental exposures and biological changes in response to occupational firefighting. 200 urine samples from 100 firefighters collected at baseline and two to four hours post-fire were analyzed using untargeted liquid-chromatography and high-resolution mass spectrometry. Changes in metabolite abundance after a fire were estimated with fixed effects linear regression, with false discovery rate (FDR) adjustment. Partial least squares discriminant analysis (PLS-DA) was also used, and variable important projection (VIP) scores were extracted. Systemic changes were evaluated using pathway enrichment for highly discriminating metabolites. Metabolome-wide-association-study (MWAS) identified 268 metabolites associated with firefighting activity at FDR q < 0.05. Of these, 20 were annotated with high confidence, including the amino acids taurine, proline, and betaine; the indoles kynurenic acid and indole-3-acetic acid; the known uremic toxins trimethylamine n-oxide and hippuric acid; and the hormone 7a-hydroxytestosterone. Partial least squares discriminant analysis (PLS-DA) additionally implicated choline, cortisol, and other hormones. Significant pathways included metabolism of urea cycle/amino group, alanine and aspartate, aspartate and asparagine, vitamin b3 (nicotinate and nicotinamide), and arginine and proline. Firefighters show a broad metabolic response to fires, including altered excretion of indole compounds and uremic toxins. Implicated pathways and features, particularly uremic toxins, may be important regulators of firefighter’s increased risk for urinary tract cancers

Associations Between Epigenetic Age Acceleration and microRNA Expression Among U.S. Firefighters.

Epigenetic changes may be biomarkers of health. Epigenetic age acceleration (EAA), the discrepancy between epigenetic age measured via epigenetic clocks and chronological age, is associated with morbidity and mortality. However, the intersection of epigenetic clocks with microRNAs (miRNAs) and corresponding miRNA-based health implications have not been evaluated. We analyzed DNA methylation and miRNA profiles from blood sampled among 332 individuals enrolled across 2 U.S.-based firefighter occupational studies (2015-2018 and 2018-2020). We considered 7 measures of EAA in leukocytes (PhenoAge, GrimAge, Horvath, skin-blood, and Hannum epigenetic clocks, and extrinsic and intrinsic epigenetic age acceleration). We identified miRNAs associated with EAA using individual linear regression models, adjusted for sex, race/ethnicity, chronological age, and cell type estimates, and investigated downstream effects of associated miRNAs with miRNA enrichment analyses and genomic annotations. On average, participants were 38 years old, 88% male, and 75% non-Hispanic white. We identified 183 of 798 miRNAs associated with EAA (FDR q &lt; 0.05); 126 with PhenoAge, 59 with GrimAge, 1 with Horvath, and 1 with the skin-blood clock. Among miRNAs associated with Horvath and GrimAge, there were 61 significantly enriched disease annotations including age-related metabolic and cardiovascular conditions and several cancers. Enriched pathways included those related to proteins and protein modification. We identified miRNAs associated with EAA of multiple epigenetic clocks. PhenoAge had more associations with individual miRNAs, but GrimAge and Horvath had greater implications for miRNA-associated pathways. Understanding the relationship between these epigenetic markers could contribute to our understanding of the molecular underpinnings of aging and aging-related diseases

Linking Oxidative Stress and Magnitude of Compensatory Responses with Life-Stage Specific Differences in Sensitivity of White Sturgeon (<i>Acipenser transmontanus</i>) to Copper or Cadmium

Sensitivity of white sturgeon (<i>Acipenser transmontanus</i>) to copper (Cu) or cadmium (Cd) has been shown to significantly differ as a function of life-stage. This study investigated oxidative stress, metal homeostasis, and associated compensatory responses as potential mechanisms of this sensitivity pattern in three early life-stages. Sturgeon were most sensitive to Cu at 15 days post hatch (dph), which was accompanied by a significant increase in lipid peroxidation (LPO). Genes involved with amelioration of oxidative stress were significantly less inducible at this stage than in older, less sensitive fry. At 48 dph, acute lethality of sturgeon exposed to Cd was greatest and body LPO was significantly induced by 3.5-fold at 5 μg Cd/L. Moreover, there was a small but significant increase in antioxidative responses. At 139 dph, sturgeon were most tolerant to Cu and Cd and accumulation of these metals was least. Also, expression of metallothionein (MT) and apoptotic genes were greatest while expression of metal transporters was reduced and concentration of LPO was not different from controls. Our results suggest that life-stage specific sensitivity of white sturgeon to metals is complex, encompassing differences in the ability to mount compensatory responses important for metal homeostasis and combating oxidative stress and concomitant damages