Zyxin is important for the stability and function of podocytes, especially during mechanical stretch.

Podocyte detachment due to mechanical stress is a common issue in hypertension-induced kidney disease. This study highlights the role of zyxin for podocyte stability and function. We have found that zyxin is significantly up-regulated in podocytes after mechanical stretch and relocalizes from focal adhesions to actin filaments. In zyxin knockout podocytes, we found that the loss of zyxin reduced the expression of vinculin and VASP as well as the expression of matrix proteins, such as fibronectin. This suggests that zyxin is a central player in the translation of mechanical forces in podocytes. In vivo, zyxin is highly up-regulated in patients suffering from diabetic nephropathy and in hypertensive DOCA-salt treated mice. Furthermore, zyxin loss in mice resulted in proteinuria and effacement of podocyte foot processes that was measured by super resolution microscopy. This highlights the essential role of zyxin for podocyte maintenance in vitro and in vivo, especially under mechanical stretch.

Genetic ablation of Lmp2 increases the susceptibility for impaired cardiac function.

Proteasome degradation is an integral part of cellular growth and function. Proteasomal intervention may mitigate adverse myocardial remodeling, but is associated with the onset of heart failure. Previously, we have demonstrated that increasing abundance of cardiac Lmp2 and its incorporation into proteasome complexes is an endogenous mechanism for proteasome regulation during hypertrophic remodeling of the heart induced by chronic ß-adrenoreceptor stimulation. Here, we investigated whether Lmp2 is required for myocardial remodeling not driven by inflammation and show that Lmp2 is a tipping element for growth and function in the heart but not for proteasome insufficiency. While it has no apparent impact under unchallenged conditions, myocardial remodeling without Lmp2 exacerbates hypertrophy and restricts cardiac function. Under chronic ß-adrenoreceptor stimulation, as seen in the development of cardiovascular disease and the manifestation of heart failure, genetic ablation of Lmp2 in mice caused augmented concentric hypertrophy of the left ventricle. While the heart rate was similarly elevated as in wildtype, myocardial contractility was not maintained without Lmp2, and apparently uncoupled of the ß-adrenergic response. Normalized to the exacerbated myocardial mass, contractility was reduced by 41% of the pretreatment level, but would appear preserved at absolute level. The lack of Lmp2 interfered with elevated 26S proteasome activities during early cardiac remodeling reported previously, but did not cause bulk proteasome insufficiency, suggesting the Lmp2 containing proteasome subpopulation is required for a selected group of proteins to be degraded. In the myocardial interstitium, augmented collagen deposition suggested matrix stiffening in the absence of Lmp2. Indeed, echocardiography of left ventricular peak relaxation velocity (circumferential strain rate) was reduced in this treatment group. Overall, targeting Lmp2 in a condition mimicking chronic ß-adrenoreceptor stimulation exhibited the onset of heart failure. Anticancer therapy inhibiting proteasome activity, including Lmp2, is associated with adverse cardiac events, in particular heart failure. Sparing Lmp2 may be an avenue to reduce adverse cardiac events when chronic sympathetic nervous system activation cannot be excluded.

Hazard assessment of complex legacy-contaminated groundwater mixtures using a novel approach method in adult fathead minnows.

Traditional risk assessment methods face challenges in the determination of drivers of toxicity for complex mixtures such as those present at legacy-contaminated sites. Bioassay-driven analysis across several levels of biological organization represents an approach to address these obstacles. This study aimed to apply a novel transcriptomics tool, the EcoToxChip, to characterize the effects of complex mixtures of contaminants in adult fathead minnows (FHMs) and to compare molecular response patterns to higher-level biological responses. Adult FHMs were exposed for 4 and 21 days to groundwater mixtures collected from a legacy-contaminated site. Adult FHM showed significant induction of micronuclei in erythrocytes, decrease in reproductive capacities, and some abnormal appearance of liver histology. Parallel EcoToxChip analyses showed a high proportion of upregulated genes and a few downregulated genes characteristic of compensatory responses. The three most enriched pathways included thyroid endocrine processes, transcription and translation cellular processes, and xenobiotics and reactive oxygen species metabolism. Several of the most differentially regulated genes involved in these biological pathways could be linked to the apical outcomes observed in FHMs. We concluded that molecular responses as determined by EcoToxChip analysis show promise for informing of apical outcomes and could support risk assessments of complex contaminated sites.

Hepatic Transcriptomic Responses to Ethinylestradiol in Embryonic Japanese Quail and Double-Crested Cormorant.

Understanding species differences in sensitivity to toxicants is a critical issue in ecotoxicology. We recently established that double-crested cormorant (DCCO) embryos are more sensitive than Japanese quail (JQ) to the developmental effects of ethinylestradiol (EE2). We explored how this difference in sensitivity between species is reflected at a transcriptomic level. The EE2 was dissolved in dimethyl sulfoxide and injected into the air cell of eggs prior to incubation at nominal concentrations of 0, 3.33, and 33.3 µg/g egg weight. At midincubation (JQ 9 days; DCCO 16 days), livers were collected from five embryos/treatment group for RNA sequencing. Data were processed and analyzed using EcoOmicsAnalyst and ExpressAnalyst. The EE2 exposure dysregulated 238 and 1,987 genes in JQ and DCCO, respectively, with 78 genes in common between the two species. These included classic biomarkers of estrogen exposure such as vitellogenin and apovitellenin. We also report DCCO-specific dysregulation of Phase I/II enzyme-coding genes and species-specific transcriptional ontogeny of vitellogenin-2. Twelve Kyoto Encyclopedia of Genes and Genomes pathways and two EcoToxModules were dysregulated in common in both species including the peroxisome proliferator-activated receptor (PPAR) signaling pathway and fatty acid metabolism. Similar to previously reported differences at the organismal level, DCCO were more responsive to EE2 exposure than JQ at the gene expression level. Our description of differences in transcriptional responses to EE2 in early life stage birds may contribute to a better understanding of the molecular basis for species differences. Environ Toxicol Chem 2024;43:772-783. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Cross-species apical microinjected selenomethionine toxicity in embryo-larval fishes.

Selenium (Se) is an essential micronutrient that becomes toxic when exposures minimally exceed those that are physiologically required. Studies on Se contaminated aquatic environments have identified that embryo-larval fishes are at particular risk of Se toxicity, primarily due to maternal Se transfer to developing eggs during oogenesis. This study emulated these exposures in embryo-larval fathead minnow (FHM), rainbow trout (RBT), white sucker (WSu), and white sturgeon (WSt) using embryonic selenomethionine (SeMet) microinjections. Adverse Se-outcomes observed across these species included spinal and edematous deformities, total individuals deformed, and reduced survival. Spinal deformity was the most sensitive sublethal endpoint and developed at the lowest concentrations in WSt (10 % effects concentration (EC10) = 12.42 µg (total) Se/g dry weight (d.w.)) followed by WSu (EC10 = 14.49 µg Se/g d.w.) and FHM (EC10 = 18.10 µg Se/g d.w.). High mortality was observed in RBT, but SeMet influences were confounded by the species' innate sensitivity to the microinjections themselves. 5 % hazardous concentrations derived across exposure type data subsets were ∼49 % higher when derived from within-species maternal transfer exclusive data as opposed to all, or within-species microinjection exclusive, data. These results support the current exclusion of SeMet microinjections during regulatory guideline derivation and their inclusion when studying mechanistic Se toxicity across phylogenetically distant fishes.

Interspecies Differences in 6PPD-Quinone Toxicity Across Seven Fish Species: Metabolite Identification and Semiquantification.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire antidegradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations that surpass those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semiquantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkii lewisi), and nonfry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might detoxify 6PPD-Q more effectively. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semiquantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being valuable to environmental monitoring and risk assessment.

Comparing Transcriptomic Responses to Chemicals Across Six Species Using the EcoToxChip RNASeq Database.

The EcoToxChip project includes RNA-sequencing data from experiments involving model (Japanese quail, fathead minnow, African clawed frog) and ecological (double-crested cormorant, rainbow trout, northern leopard frog) species at multiple life stages (whole embryo and adult) exposed to eight chemicals of environmental concern known to perturb a wide range of biological systems (ethinyl estradiol, hexabromocyclododecane, lead, selenomethionine, 17ß trenbolone, chlorpyrifos, fluoxetine, and benzo[a]pyrene). The objectives of this short communication were to (1) present and make available this RNA-sequencing database (i.e., 724 samples from 49 experiments) under the FAIR principles (FAIR data are data which meet principles of findability, accessibility, interoperability, and reusability), while also summarizing key meta-data attributes and (2) use ExpressAnalyst (including the Seq2Fun algorithm and EcoOmicsDB) to perform a comparative transcriptomics analysis of this database focusing on baseline and differential transcriptomic changes across species-life stage-chemical combinations. The database is available in NCBI GEO under accession number GSE239776. Across all species, the number of raw reads per sample ranged between 13 and 58 million, with 30% to 79% of clean reads mapped to the "vertebrate" subgroup database in EcoOmicsDB. Principal component analyses of the reads illustrated separation across the three taxonomic groups as well as some between tissue types. The most common differentially expressed gene was CYP1A1 followed by CTSE, FAM20CL, MYC, ST1S3, RIPK4, VTG1, and VIT2. The most common enriched pathways were metabolic pathways, biosynthesis of cofactors and biosynthesis of secondary metabolites, and chemical carcinogenesis, drug metabolism, and metabolism of xenobiotics by cytochrome P450. The RNA-sequencing database in the present study may be used by the research community for multiple purposes, including, for example, cross-species investigations, in-depth analyses of a particular test compound, and transcriptomic meta-analyses. Environ Toxicol Chem 2024;00:1-6. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Characterization of molecular and apical effects of legacy-contaminated groundwater on early life stages of fathead minnows.

Mechanistic toxicology approaches represent a promising alternative to traditional live animal testing; however, the often-noted uncertainties concerning the linkages between effects observed at molecular and apical levels curtails the adoption of such approaches. The objective of this study was to apply a novel transcriptomics tool, EcoToxChips, to characterize the effects of complex mixtures of contaminants in fish and to compare molecular response patterns to higher-level biological responses including swimming behavior, deformities, and mortality. Fathead minnow (FHM) embryos were exposed for seven days to increasing concentrations of groundwater collected from moderate (MIAZ) and high (HIAZ) industrial activity zones of a legacy contaminated site. There was a concentration-dependent disruption of photo-dependent swimming responses associated with avoidance behavior patterns and spinal deformities (HIAZ and MIAZ), and an induction of pericardial edema and mortality (HIAZ-10%). Parallel EcoToxChip analyses showed a shift from a majority of upregulated genes at lower concentrations to a majority of downregulated genes at higher concentrations for both treatment conditions. Many of the significantly differentially regulated genes were involved in biological pathways including induction of oxidative stress, activating of several metabolic processes and growth, cell death, and inhibition of signal transduction signaling processes. Several contaminants present in the groundwater mixtures could have contributed to an exceedance of antioxidant system capacities that possibly led to the deformities, altered swimming behaviours, and mortality observed in FHMs. Therefore, molecular response patterns could be linked to apical outcomes observed in this study. Overall, the results observed in this study demonstrate that transcriptomics approaches such as the EcoToxChip system could be supportive of risk assessment of complex contaminated sites.

Impact of the -1T>C single-nucleotide polymorphism of the CD40 gene on the development of endothelial dysfunction in a pro-diabetic microenvironment.

BACKGROUND AND AIMS: Hyperglycemia reinforces pro-inflammatory conditions that enhance CD40 expression in endothelial cells (EC). Thymine to cytosine transition (-1T > C) in the promoter of the CD40 gene (rs1883832) further increases the abundance of CD40 protein on the EC surface. This study examines potential associations of the -1T > C SNP of the CD40 gene with type 1 (T1D) or type 2 (T2D) diabetes. Moreover, it investigates the impact of a pro-inflammatory diabetic microenvironment on gene expression in human cultured umbilical vein EC (HUVEC) derived from CC- vs. TT-genotype donors. METHODS: Tetra-ARMS-PCR was used to compare genotype distribution in 252 patients with diabetes. Soluble CD40 ligand (sCD40L) and soluble CD40 receptor (sCD40) plasma levels were monitored using ELISA. RNA-sequencing was performed with sCD40L-stimulated CC- and TT-genotype HUVEC. Quantitative PCR, Western blot, multiplex-sandwich ELISA array, and immunocytochemistry were used to analyse changes in gene expression in these cells. RESULTS: Homozygosity for the C-allele was associated with a significant 4.3-fold higher odds of developing T2D as compared to individuals homozygous for the T-allele. Inflammation and endothelial-to-mesenchymal transition (EndMT) driving genes were upregulated in CC-genotype but downregulated in TT-genotype HUVEC when exposed to sCD40L. Expression of EndMT markers significantly increased while that of endothelial markers decreased in HUVEC following exposure to hyperglycemia, tumour necrosis factor-α and sCD40L. CONCLUSIONS: The -1T > C SNP of the CD40 gene is a risk factor for T2D. Depending on the genotype, it differentially affects gene expression in human cultured EC. CC-genotype HUVEC adopt a pro-inflammatory and intermediate EndMT-like phenotype in a pro-diabetic microenvironment.

Characterisation of Lipoma-Preferred Partner as a Novel Mechanotransducer in Vascular Smooth Muscle Cells.

In arteries and arterioles, a chronic increase in blood pressure raises wall tension. This continuous biomechanical strain causes a change in gene expression in vascular smooth muscle cells (VSMCs) that may lead to pathological changes. Here we have characterised the functional properties of lipoma-preferred partner (LPP), a Lin11-Isl1-Mec3 (LIM)-domain protein, which is most closely related to the mechanotransducer zyxin but selectively expressed by smooth muscle cells, including VSMCs in adult mice. VSMCs isolated from the aorta of LPP knockout (LPP-KO) mice displayed a higher rate of proliferation than their wildtype (WT) counterparts, and when cultured as three-dimensional spheroids, they revealed a higher expression of the proliferation marker Ki 67 and showed greater invasion into a collagen gel. Accordingly, the gelatinase activity was increased in LPP-KO but not WT spheroids. The LPP-KO spheroids adhering to the collagen gel responded with decreased contraction to potassium chloride. The relaxation response to caffeine and norepinephrine was also smaller in the LPP-KO spheroids than in their WT counterparts. The overexpression of zyxin in LPP-KO VSMCs resulted in a reversal to a more quiescent differentiated phenotype. In native VSMCs, i.e., in isolated perfused segments of the mesenteric artery (MA), the contractile responses of LPP-KO segments to potassium chloride, phenylephrine or endothelin-1 did not vary from those in isolated perfused WT segments. In contrast, the myogenic response of LPP-KO MA segments was significantly attenuated while zyxin-deficient MA segments displayed a normal myogenic response. We propose that LPP, which we found to be expressed solely in the medial layer of different arteries from adult mice, may play an important role in controlling the quiescent contractile phenotype of VSMCs.

Ectodomain Shedding by ADAM17 Increases the Release of Soluble CD40 from Human Endothelial Cells under Pro-Inflammatory Conditions.

BACKGROUND: Homozygosity for the C allele of the -1T>C single nucleotide polymorphism (SNP) of the CD40 gene (rs1883832) is associated with susceptibility to coronary heart disease (CHD), enhanced CD40 expression, and shedding. The disintegrin metalloprotease ADAM17 can cleave various cell surface proteins. This study investigates an association between ADAM17-mediated CD40 shedding and inflammation in CC genotype human endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVEC) carrying the CC genotype were stimulated with soluble CD40 ligand (sCD40L) or tumor necrosis factor-α (TNFα). Messenger RNA and protein expression were determined with standard methods. Levels of high sensitive c-reactive protein (hs-CRP), interleukin-6 (IL-6), and sCD40 in plasma samples from patients with CHD were assessed using ELISA. RESULTS: ADAM17 surface abundance was elevated following stimulation with CD40L and TNFα just as its regulator iRhom2. Inhibition of ADAM17 prevented TNFα-induced sCD40 and soluble vascular cell adhesion molecule-1 release into the conditioned medium and reinforced CD40 surface abundance. Secondary to inhibition of ADAM17, stimulation with CD40L or TNFα upregulated monocyte chemoattractant protein-1 mRNA and protein. Levels of sCD40 and the inflammatory biomarkers hs-CRP and IL-6 were positively correlated in the plasma of patients with CHD. CONCLUSIONS: We provide a mechanism by which membrane-bound CD40 is shed from the endothelial cell surface by ADAM17, boosting sCD40 formation and limiting downstream CD40 signaling. Soluble CD40 may represent a robust biomarker for CHD, especially in conjunction with homozygosity for the C allele of the -1T>C SNP of the CD40 gene.

Apical and mechanistic effects of 6PPD-quinone on different life-stages of the fathead minnow (Pimephales promelas).

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone) is an emerging contaminant of concern that is generated through the environmental oxidation of the rubber tire anti-degradant 6PPD. Since the initial report of 6PPD-quinone being the cause of urban runoff mortality syndrome of Coho salmon, numerous species have been identified as either sensitive or insensitive to acute lethality caused by 6PPD-quinone. In sensitive species, acute lethality might be caused by uncoupling of mitochondrial respiration in gills. However, little is known about effects of 6PPD-quinone on insensitive species. Here we demonstrate that embryos of fathead minnows (Pimephales promelas) are insensitive to exposure to concentrations as great as 39.97 µg/L for 168 h, and adult fathead minnows are insensitive to exposure to concentrations as great as 9.4 µg/L for 96 h. A multi-omics approach using a targeted transcriptomics array, (EcoToxChips), and proton nuclear magnetic resonance (1H NMR) was used to assess responses of the transcriptomes and metabolomes of gills and livers from adult fathead minnows exposed to 6PPD-quinone for 96 h to begin to identify sublethal effects of 6PPD-quinone. There was little agreement between results of the EcoToxChip and metabolomics analyses, likely because genes present on the EcoToxChip were not representative of pathways suggested to be perturbed by metabolomic analysis. Changes in abundances of transcripts and metabolites in livers and gills suggest that disruption of one­carbon metabolism and induction of oxidative stress might be occurring in gills and livers, but that tissues differ in their sensitivity or responsiveness to 6PPD-quinone. Overall, several pathways impacted by 6PPD-quinone were identified as candidates for future studies of potential sublethal effects of this chemical.

Wastewater discharges alter microbial community composition in surface waters of the canadian prairies.

Microbial communities are an important component of freshwater biodiversity that is threatened by anthropogenic impacts. Wastewater discharges pose a particular concern by being major sources of anthropogenic contaminants and microorganisms that may influence the composition of natural microbial communities. Nevertheless, the effects of wastewater treatment plant (WWTP) effluents on microbial communities remain largely unexplored. In this study, the effects of wastewater discharges on microbial communities from five different WWTPs in Southern Saskatchewan were investigated using rRNA gene metabarcoding. In parallel, nutrient levels and the presence of environmentally relevant organic pollutants were analyzed. Higher nutrient loads and pollutant concentrations resulted in significant changes in microbial community composition. The greatest changes were observed in Wascana Creek (Regina), which was found to be heavily polluted by wastewater discharges. Several taxa occurred in greater relative abundance in the wastewater-influenced stream segments, indicating anthropogenic pollution and eutrophication, especially taxa belonging to Proteobacteria, Bacteroidota, and Chlorophyta. Strong decreases were measured within the taxa Ciliphora, Diatomea, Dinoflagellata, Nematozoa, Ochrophyta, Protalveolata, and Rotifera. Across all sample types, a significant decline in sulfur bacteria was measured, implying changes in functional biodiversity. In addition, downstream of the Regina WWTP, an increase in cyanotoxins was detected which was correlated with a significant change in cyanobacterial community composition. Overall, these data suggest a causal relationship between anthropogenic pollution and changes in microbial communities, possibly reflecting an impairment of ecosystem health.

Development and Initial Testing of EcoToxChip, a Novel Toxicogenomics Tool for Environmental Management and Chemical Risk Assessment.

New approach methods (NAMs) are increasingly important to help accelerate the pace of ecological risk assessment and offer more ethical, affordable, and efficient alternatives to traditional toxicity tests. In the present study, we describe the development, technical characterization, and initial testing of a toxicogenomics tool, EcoToxChip (384-well quantitative polymerase chain reaction [qPCR] array), to support chemical management and environmental monitoring for three laboratory model species-fathead minnow (Pimephales promelas), African clawed frog (Xenopus laevis), and Japanese quail (Coturnix japonica). Chip design, including gene selection, was informed by a diverse end-user group and quality control metrics (e.g., primer assay, reverse transcription, and PCR efficiency) performed well based on a priori established criteria. Correlation with RNA sequencing (seq) data provided additional confidence in this novel toxicogenomics tool. Although the present study represents an initial testing of only 24 EcoToxChips for each of the model species, the results provide increased confidence in the robustness/reproducibility of EcoToxChips for evaluating perturbations in gene expression associated with chemical exposure and thus, this NAM, combined with early-life stage toxicity testing, could augment current efforts for chemical prioritization and environmental management. Environ Toxicol Chem 2023;42:1763-1771. © 2023 SETAC.

Membrane remodelling triggers maturation of excitation-contraction coupling in 3D-shaped human-induced pluripotent stem cell-derived cardiomyocytes.

The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca2+-dependent excitation-contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca2+ channels critical for EC coupling in close proximity, the L-type Ca2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca2+-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches.

New Approach Methodologies for the Endocrine Activity Toolbox: Environmental Assessment for Fish and Amphibians.

Multiple in vivo test guidelines focusing on the estrogen, androgen, thyroid, and steroidogenesis pathways have been developed and validated for mammals, amphibians, or fish. However, these tests are resource-intensive and often use a large number of laboratory animals. Developing alternatives for in vivo tests is consistent with the replacement, reduction, and refinement principles for animal welfare considerations, which are supported by increasing mandates to move toward an "animal-free" testing paradigm worldwide. New approach methodologies (NAMs) hold great promise to identify molecular, cellular, and tissue changes that can be used to predict effects reliably and more efficiently at the individual level (and potentially on populations) while reducing the number of animals used in (eco)toxicological testing for endocrine disruption. In a collaborative effort, experts from government, academia, and industry met in 2020 to discuss the current challenges of testing for endocrine activity assessment for fish and amphibians. Continuing this cross-sector initiative, our review focuses on the current state of the science regarding the use of NAMs to identify chemical-induced endocrine effects. The present study highlights the challenges of using NAMs for safety assessment and what work is needed to reduce their uncertainties and increase their acceptance in regulatory processes. We have reviewed the current NAMs available for endocrine activity assessment including in silico, in vitro, and eleutheroembryo models. New approach methodologies can be integrated as part of a weight-of-evidence approach for hazard or risk assessment using the adverse outcome pathway framework. The development and utilization of NAMs not only allows for replacement, reduction, and refinement of animal testing but can also provide robust and fit-for-purpose methods to identify chemicals acting via endocrine mechanisms. Environ Toxicol Chem 2023;42:757-777. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Maternal Transfer and Apical and Physiological Effects of Dietary Hexabromocyclododecane Exposure in Parental Fathead Minnows (Pimephales promelas).

Hexabromocyclododecane (HBCD) is a persistent organic pollutant that has been characterized as an endocrine disruptor, undergoes maternal transfer, and hinders development and growth in oviparous organisms. The present study examined the apical effects of dietary HBCD (11.5, 36.4, 106 mg/kg, wet wt) on adult fathead minnow exposed for 49 days and the subsequent accumulation and maternal transfer kinetics in adult tissue and eggs, respectively. Exposed adults displayed a significant increase in egg production in the medium treatment group, but no other significant effects were noted. Maternal transfer of dietary HBCD had a similar egg-to-muscle ratios (EMR) in the low and medium treatment groups (1.65 and 1.27 [wet wt], respectively). However, the high treatment group deviated from other treatments with an EMR of 4.2 (wet wt), potentially due to differences in total lipid content in food and/or reaching diffusion/lipid saturation limits in adult tissue, resulting in lower accumulation in the adult muscle tissue. A positive correlation was observed between egg HBCD concentration and time of exposure, which indicates that maternal transfer of HBCD is of concern in fish, and further studies should be conducted to fully elucidate the potential adverse effects that may be observed in the early life stage of oviparous organisms. Environ Toxicol Chem 2023;42:143-153. © 2022 SETAC.

Correlation between in vitro toxicity of pesticides and in vivo risk guidelines in support of complex operating site risk management: A meta-analysis.

In vitro cell systems can support hazard characterization and identify mechanisms involved in toxicity; however, using in vitro data for risk assessment still is challenging. As part of an effort to develop approaches for a complex operating site used for biocide packaging and distribution, we evaluated in vitro assays that could be used in a site management format. Across 66 studies, 108 pesticides were assessed on ten human-derived cell types at four endpoints. In vitro IC50s were compared to in vivo guidelines, NOEL/NOAELs, and ADIs using Spearman correlation and linear regression models. While human neuroblastoma cells (SH-SY5Y) were the most sensitive, HepG2 was the most used cell line in evaluating the toxicity of pesticides. Amongst the ten human cell lines, the IC50s derived from SH-SY5Y cells, using MTT-24 & 48 h (the most used assay) correlated (rho = 0.56-0.79; p < 0.05) with ADIs and NOEL/NOAELs. Although in vitro cell systems have some limitations, the correlation between in vitro data derived from SH-SY5Y cells and in vivo safety guidelines can provide site investigators with a tool to survey and prioritize areas and media of concern at complex operating sites impacted by pesticide mixtures.

Association between liver enzymes and metabolic syndrome in Canadian adults: results from the Canadian health measures survey - cycles 3 &4.

Background: The relationship between liver enzymes and Metabolic Syndrome (MetS) in different populations, including Canadians, is not consistent and well understood. We used the Canadian Health Measures Survey data (Cycles 3 and 4) to examine the cross-sectional relationships between select liver biomarkers and MetS in the adult Canadian population. The biomarkers selected were gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), and alkaline phosphatase (ALKP). Methods: Fasting blood samples (FBS) were collected from adults above the age of 20 years for Cycle 3 and Cycle 4 (n = 3003). MetS was diagnosed if the subjects had three or more risk determinants according to the Joint Interim Statement criteria. Primary risk factors included quartile cut-offs for each of the biomarkers ALKP, AST, GGT for males and females separately. A multivariable logistic regression technique based on a maximum likelihood approach was used to evaluate the association between quartiles of ALKP, AST, and GGT, other individual and contextual factors, and the prevalence of MetS. Results: MetS was prevalent in 32.3% of subjects. BMI was an effect modifier in the relationship between GGT and MetS prevalence, while sex was an effect modifier in the relationship between ALKP and MetS prevalence; and age was an effect modifier in the relationship between AST and MetS prevalence. Conclusions: Since the mechanisms to underpin the associations between the liver enzymes activity and MetS are unknown, further epidemiologic investigations using longitudinal designs are necessary to understand these associations.