Cytotoxic impacts of seven alternative bisphenols on human in vitro cellular models.

Bisphenols (BPs), common in plastics, coatings, and resins, are under scrutiny for potential endocrine disruption. Despite banning bisphenol A (BPA), its perceived safer alternatives may still pose health risks, urging thorough studies on their toxicity mechanisms. This study aimed to investigate the cellular toxicity of the top seven most commonly used BPs, bisphenol S (BPS), bisphenol F (BPF), bisphenol AF (BPAF), bisphenol P (BPP), bisphenol AP (BPAP), bisphenol B (BPB), bisphenol E (BPE) in eight different relevant human in vitro cell models: liver (HepaRG), intestinal (Caco-2), breast (T47D), brain (HMC-3), lungs (MRC-5), kidney (HEK293), endothelial (HMEC-1), and skin (HEK-001) cell lines. BPE manifested the highest cytotoxicity in Caco-2 cells, presenting an EC50 value of roughly 0.2 µM (95% confidence interval). In contrast, HEK293 and HepaRG cells demonstrated significant resilience to BPS (EC50 > 1000 µM). BPAF, BPP, and BPAP had consistently low EC50 values across cell lines (6-27.9 µM, 0.6-134.7 µM, and 3.6-178.8 µM), indicating elevated toxicity. After 24 h, all bisphenols adhered to nominal concentrations except BPAF, BPP, and BPS. BPP's concentration notably decreased (30.82 ± 5.53% of nominal value). The results revealed diverse effects of bisphenol analogs on different cell types. These findings emphasized the considerable cytotoxic potential of specific bisphenol analogs across various human cell models, underlining the necessity for a re-evaluation of their safety and regulatory standards.

An in-depth examination of Per- and Polyfluoroalkyl (PFAS) effects on transporters, with emphasis on the ABC superfamily: A critical review.

Per- and polyfluoroalkyl (PFAS) substances are a type of chemical compound unique for their multiple carbon-fluorine bonds, imbuing them with strength and environmental permanence. While legacy substances have been phased out due to human health risks, short-chain and alternative PFAS remain omnipresent. However, a detailed explanation for the pathways through which PFAS interact on a cellular and molecular level is still largely unknown, and the human health effects remain mechanistically unexplained. Of particular interest when focusing on this topic are the interactions between these exogenous chemicals and plasma and membrane proteins. Such proteins include serum albumin which can transport PFAS throughout the body, solute carrier proteins (SLC) and ATP binding cassette (ABC) transporters which are able to move PFAS into and out of cells, and proteins and nuclear receptors which interact with PFAS intracellularly. ABC transporters as a family have little available human data despite being responsible for the export of endogenous substances and drugs throughout the body. The multifactorial regulation of these crucial transporters is affected directly and indirectly by PFAS. Changes, which can include alterations to membrane transport activity and differences in protein expression, vary greatly depending on the specific PFAS and protein of interest. Together, the myriad of changes caused by understudied PFAS exposure to a class of understudied proteins crucial to cellular function and drug treatments has not been fully explored regarding human health and presents room for further exploration. This critical work aims to provide a novel framework of existing human data on PFAS and ABC transporters, allowing for future advancement and investigation into human transporter activity, mechanisms of regulation, and interactions with emerging contaminants.

Accumulation of Parabens, Their Metabolites, and Halogenated Byproducts in Migratory Birds of Prey: A Comparative Study in Texas and North Carolina, USA.

Parabens are alkyl esters of p-hydroxybenzoic acid that are commonly used as preservatives in personal care products such as cosmetics. Recent studies have revealed the presence of parabens in surface and tap water because of their use as disinfection products; however, little is known about their occurrence in biological samples and their bioaccumulation potential, particularly in raptor birds known as sentinels for pollutant detection. We examined the occurrence and tissue distribution of parabens, their metabolites, and halogenated byproducts in the liver, kidney, brain, and muscle of birds of prey from Texas and North Carolina (USA). Methylparaben (MeP), propylparaben (PrP), and butylparaben (BuP) were detected in more than 50% of all tissues examined, with the kidney exhibiting the highest concentration of MeP (0.65-6.84 ng/g wet wt). Para-hydroxybenzoic acid (PHBA), a primary metabolite, had the highest detection frequency (>50%) and a high accumulation range in the liver, of 4.64 to 12.55 ng/g. The chlorinated compounds chloromethylparaben and chloroethylparaben were found in over half of the tissues, of which dichloromethylparaben (2.20-3.99 ng/g) and dichloroethylparaben (1.01-5.95 ng/g) in the kidney exhibited the highest concentrations. The dibrominated derivatives dibromideethylparaben (Br2EtP) was detected in more than 50% of samples, particularly in muscle and brain. Concentrations in the range of 0.14 to 17.38 ng/g of Br2EtP were detected in the kidney. Dibromidepropylparaben (Br2PrP) was not frequently detected, but concentrations ranged from 0.09 to 21.70 ng/g in muscle. The accumulations of total amounts (sum) of parent parabens (∑P), metabolites (∑M), and halogenated byproducts (∑H) in different species were not significantly different, but their distribution in tissues differed among the species. Positive correlations were observed among MeP, PrP, BuP, and PHBA in the liver, suggesting similar origins and metabolic pathways. Environ Toxicol Chem 2024;43:2365-2376. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effects of short-chain per- and polyfluoroalkyl substances (PFAS) on toxicologically relevant gene expression profiles in a liver-on-a-chip model.

Short-chain per- and polyfluoroalkyl substances (PFAS) are highly stable and widely used environmental contaminants that pose potential health risks to humans. Aggregating reliable mechanistic information for safety assessments necessitates physiologically relevant high-throughput screening approaches. Here, we demonstrated the utility of a liver-on-a-chip model to investigate the effects of five short-chain PFAS at low (1 nM) and high (1 µM) concentrations on toxicologically-relevant gene expression profiles using the QuantiGene® Plex Assay. We found that the short-chain PFAS tested in this study modulated the expression of ABCG2, a gene encoding for the breast cancer resistance protein (BCRP), with marked and significant upregulation (up to 4-fold) observed for all but one of the short-chain PFAS tested. PFBS and HFPO-DA repressed SLCO1B3 expression, a gene that encodes for an essential liver-specific organic anion transporter. High concentrations of PFBS, PFHxA, and PFHxS upregulated the expression of genes encCYP1A1,CYP2B6 and CYP2C19 with the same treatments resulting in the repression of the expression of the gene encoding CYP1A2. This dysregulation could have consequences for the clearance of endogenous compounds and xenobiotics. However, we acknowledge that increased expression of genes encoding for transporters and biotransformation enzymes may or may not indicate changes to their protein expression or activity. Overall, our study provides important insights into the effects of short-chain PFAS on liver function and their potential implications for human health. The use of the liver-on-a-chip model in combination with the QuantiGene® Plex Assay may be a valuable tool for future high-throughput screening and gene expression profiling in toxicology studies.

Effects of short-chain per- and polyfluoroalkyl substances (PFAS) on human cytochrome P450 (CYP450) enzymes and human hepatocytes: An in vitro study.

Short-chain per- and polyfluoroalkyl substances (PFAS) have been developed as alternatives to legacy long-chain PFAS, but they may still pose risks due to their potential to interact with biomolecules. Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism, and disruptions of these enzymes by PFAS can have significant human health implications. The inhibitory potential of two legacy long-chain (PFOA and PFOS) and five short-chain alternative PFAS (PFBS, PFHxA, HFPO-DA, PFHxS, and 6:2 FTOH) were assessed in recombinant CYP1A2, - 2B6, -2C19, -2E1, and -3A4 enzymes. Most of the short-chain PFAS, except for PFHxS, tested did not result in significant inhibition up to 100 µM. PFOS inhibited recombinant CYP1A2, -2B6, -2C19, and -3A4 enzymes. However, concentrations where inhibition occurred, were all higher than the averages reported in population biomonitoring studies, with IC50 values higher than 10 µM. We also evaluated the activities of CYP1A2 and CYP3A4 in HepaRG monolayers following 48 h exposures of the short-chain PFAS at two concentrations (1 nM or 1 µM) and with or without an inducer (benzo[a]pyrene, BaP, for CYP1A2 and rifampicin for CYP3A4). Our findings suggest that both 1 nM and 1 µM exposures to short-chain PFAS can modulate the CYP1A2 activity induced by BaP. Except for PFHxS, the short-chain PFAS appear to have little effect on CYP3A4 activity. Understanding the effects of PFAS exposure on biotransformation can shed light on the mechanisms of PFAS toxicity and aid in developing effective strategies for managing chemical risks, enabling regulators to make more informed decisions.

On seven undescribed leaf insect species revealed within the recent "Tree of Leaves" (Phasmatodea, Phylliidae).

With the recent advance in molecular phylogenetics focused on the leaf insects (Phasmatodea, Phylliidae), gaps in knowledge are beginning to be filled. Yet, shortcomings are also being highlighted, for instance, the unveiling of numerous undescribed phylliid species. Here, some of these taxa are described, including Phylliumiyadaonsp. nov. from Mindoro Island, Philippines; Phylliumsamarensesp. nov. from Samar Island, Philippines; Phylliumortizisp. nov. from Mindanao Island, Philippines; Pulchriphylliumheraclessp. nov. from Vietnam; Pulchriphylliumdelisleisp. nov. from South Kalimantan, Indonesia; and Pulchriphylliumbhaskaraisp. nov. from Java, Indonesia. Several additional specimens of these species together with a seventh species described herein, Pulchriphylliumanangusp. nov. from southwestern India, were incorporated into a newly constructed phylogenetic tree. Additionally, two taxa that were originally described as species, but in recent decades have been treated as subspecies, are elevated back to species status to reflect their unique morphology and geographic isolation, creating the following new combinations: Pulchriphylliumscythe (Gray, 1843) stat. rev., comb. nov. from Bangladesh and northeastern India, and Pulchriphylliumcrurifolium (Audinet-Serville, 1838) stat. rev., comb. nov. from the Seychelles islands. Lectotype specimens are also designated for Pulchriphylliumscythe (Gray, 1843) stat. rev., comb. nov. and Pulchriphylliumcrurifolium (Audinet-Serville, 1838) stat. rev., comb. nov. from original type material.

Evaluating the effect of acute diesel exhaust particle exposure on P-glycoprotein efflux transporter in the blood-brain barrier co-cultured with microglia.

A growing public health concern, chronic Diesel Exhaust Particle (DEP) exposure is a heavy risk factor for the development of neurodegenerative diseases like Alzheimer's (AD). Considered the brain's first line of defense, the Blood-Brain Barrier (BBB) and perivascular microglia work in tandem to protect the brain from circulating neurotoxic molecules like DEP. Importantly, there is a strong association between AD and BBB dysfunction, particularly in the Aß transporter and multidrug resistant pump, P-glycoprotein (P-gp). However, the response of this efflux transporter is not well understood in the context of environmental exposures, such as to DEP. Moreover, microglia are seldom included in in vitro BBB models, despite their significance in neurovascular health and disease. Therefore, the goal of this study was to evaluate the effect of acute (24 hr.) DEP exposure (2000 µg/ml) on P-gp expression and function, paracellular permeability, and inflammation profiles of the human in vitro BBB model (hCMEC/D3) with and without microglia (hMC3). Our results suggested that DEP exposure can decrease both the expression and function of P-gp in the BBB, and corroborated that DEP exposure impairs BBB integrity (i.e. increased permeability), a response that was significantly worsened by the influence of microglia in co-culture. Interestingly, DEP exposure seemed to produce atypical inflammation profiles and an unexpected general downregulation in inflammatory markers in both the monoculture and co-culture, which differentially expressed IL-1ß and GM-CSF. Interestingly, the microglia in co-culture did not appear to influence the response of the BBB, save in the permeability assay, where it worsened the BBB's response. Overall, our study is important because it is the first (to our knowledge) to investigate the effect of acute DEP exposure on P-gp in the in vitro human BBB, while also investigating the influence of microglia on the BBB's responses to this environmental chemical.

Short-chain per- and polyfluoralkyl substances (PFAS) effects on oxidative stress biomarkers in human liver, kidney, muscle, and microglia cell lines.

Long-chain per- and polyfluoralkyl substances (PFAS) are ubiquitous contaminants implicated in the induction of intracellular reactive oxygen species (ROS), compromising antioxidant defense mechanisms in vitro and in vivo. While a handful of studies have assessed oxidative stress effects by PFAS, few specifically address short-chain PFAS. We conducted an evaluation of oxidative stress biomarkers in vitro following exposures to low (1 nM) and high (1 µM) concentrations of five short-chain PFAS compounds: perfluorobutanesulfonic acid (PFBS), perfluorohexanoic acid (PFHxA), [undecafluoro-2-methyl-3-oxahexanoic acid (HFPO-DA)], 6:2 fluorotelomer alcohol (6:2 FTOH) and perfluorohexanesulfonic acid (PFHxS). We conducted experiments in human kidney (HEK293-hTLR2), liver (HepaRG), microglia (HMC-3), and muscle (RMS-13) cell lines. Fluorescence microscopy measurements in HepaRG cells indicated ROS generation in cells exposed to PFBS and PFHxA for 24 h. Antioxidant enzyme activities were determined following 24 h short-chain PFAS exposures in HepaRG, HEK293-hTLR2, HMC-3, and RMS-13. Notably, exposure to PFBS for 24 h increased the activity of GPX in all four cell types at 1 µM and 1 nM in HepaRG and RMS-13 cells. Every short-chain PFAS evaluated, except for PFHxS, increased the activity of at least one antioxidant enzyme. To our knowledge, this is the first study of its kind to explore antioxidant defense alterations to microglia and muscle cell lines by PFAS. The findings of this study hold great potential to contribute to the limited understanding of short-chain PFAS mechanisms of toxicity and provide data necessary to inform the human health risk assessment process.

Comparative cytotoxicity induced by parabens and their halogenated byproducts in human and fish cell lines.

Parabens are a group of para-hydroxybenzoic acid (p-HBA) esters widely used in pharmaceutical industries. Their safety is well documented in mammalian models, but little is known about their toxicity in non-mammal species. In addition, chlorinated and brominated parabens resulting from wastewater treatment have been identified in effluents. In the present study, we explored the cytotoxic effects (EC50) of five parabens: methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BuP), and benzylparaben (BeP); the primary metabolite, 4-hydroxybenzoic acid (4-HBA), and three of the wastewater chlorinated/brominated byproducts on fish and human cell lines. In general, higher cytotoxicity was observed with increased paraben chain length. The tested compounds induced toxicity in the order of 4-HBA < MP < EP < PP < BuP < BeP. The halogenated byproducts led to higher toxicity with the addition of second chlorine. The longer chain-parabens (BuP and BeP) caused a concentration-dependent decrease in cell viability in fish cell lines. Intriguingly, the main paraben metabolite, 4-HBA, proved to be more toxic to fish hepatocytes than human hepatocytes by 100-fold. Our study demonstrated that the cytotoxicity of some of these compounds appears to be tissue-dependent. These observations provide valuable information for early cellular responses in human and non-mammalian models upon exposure to paraben congeners.

Comparative cytotoxicity of seven per- and polyfluoroalkyl substances (PFAS) in six human cell lines.

Human exposures to perfluoroalkyl and polyfluoroalkyl substances (PFAS) have been linked to several diseases associated with adverse health outcomes. Animal studies have been conducted, though these may not be sufficient due to the inherent differences in metabolic processes between humans and rodents. Acquiring relevant data on the health effects of short-chain PFAS can be achieved through methods supported by in vitro human cell-based models. Specifically, cytotoxicity assays are the crucial first step to providing meaningful information used for determining safety and providing baseline information for further testing. To this end, we exposed human cell lines representative of six different tissue types, including colon (CaCo-2), liver (HepaRG), kidney (HEK293), brain (HMC-3), lung (MRC-5), and muscle (RMS-13) to five short-chain PFAS and two legacy PFAS. The exposure of the individual PFAS was assessed using a range of concentrations starting from a low concentration (10-11 M) to a high concentration of (10-4 M). Our results indicated that CaCo-2 and HEK293 cells were the least sensitive to PFAS exposure, while HMC-3, HepaRG, MRC-5, and RMS-13 demonstrated significant decreases in viability in a relatively narrow range (EC50 ranging from 1 to 70 µM). The most sensitive cell line was the neural HMC-3 for all short- and long-chain PFAS (with EC50 ranging from 1.34 to 2.73 µM). Our data suggest that PFAS do not exert toxicity on all cell types equally, and the cytotoxicity estimates we obtained varied from previously reported values. Overall, this study is novel because it uses human cell lines that have not been widely used to understand human health outcomes associated with PFAS exposure.

Effects of perfluoroalkyl substances (PFASs) and benzo[a]pyrene (BaP) co-exposure on phase I biotransformation in rainbow trout (Oncorhynchus mykiss).

The presence of perfluoroalkyl substances (PFASs) in the environment, especially in aquatic ecosystems, continues to be a significant concern for human and environmental health. Previous studies have suggested that several PFASs do not undergo biotransformation due to their chemical stability, yet perfluorooctanesulfonic acid (PFOS)- and perfluorooctanoic acid (PFOA)-exposed organisms have presented altered activity of important biotransformation pathways. Given the fundamental role of biotransformation in biological organisms and the significant distribution of PFAS in aquatic environments, the present study investigated the influence of PFOA and PFOS on phase I biotransformation enzymes in vitro using the rainbow trout liver RTL-W1 cell line and in vivo using juvenile rainbow trout. Cells and fish were exposed and co-exposed to environmentally relevant concentrations of PFOA, PFOS, and benzo[a]pyrene (BaP), for 72 h and 10 days, respectively, prior to measurements of cytotoxicity and biotransformation ability through measurements of CYP1A1-, CYP1A2-, and CYP3A4-like activities. Our results indicate that exposure to PFAS-BaP binary mixtures altered CYP1A-like activity in vivo; however, those alterations were not observed in vitro. Similarly, while BaP did not significantly induce CYP3A4 in vivo, exposure to the PFAS led to significantly lower enzymatic activity relative to basal levels. These observations may have implications for organisms simultaneously exposed to PFASs and other environmental pollutants for which biotransformation is necessary, especially in detoxification mechanisms. Furthermore, the interference with biotransformation pathways could potentially predispose exposed organisms to a compromised physiology, which may increase their vulnerability to other stressors and erode their survival fitness.

Metabolomics of peripheral artery disease.

The science of metabolomics has emerged as a novel tool for studying changes in metabolism that accompany different disease states. Several studies have applied this evolving field to the study of various cardiovascular disease states, which has led to improved understanding of metabolic changes that underlie heart failure and ischemic heart disease. A significant amount of progress has also been made in the identification of novel biomarkers of cardiovascular disease. Another common atherosclerotic disease, peripheral artery disease (PAD) affects arteries of the lower extremities. Although certain aspects of the disease pathophysiology overlap with other cardiovascular diseases in general, PAD patients suffer unique manifestations that lead to significant morbidity and mortality as well as severe functional limitations. Furthermore, because over half of PAD patients are asymptomatic, there is a need for improved diagnostic and screening methods. Identification of metabolites associated with the disease may thus be a promising approach for PAD. However, PAD remains highly understudied. In this chapter, we discuss the application of metabolomics to the study of PAD.

The use of in vitro methods in assessing human health risks associated with short-chain perfluoroalkyl and polyfluoroalkyl substances (PFAS).

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a large class of industrial chemicals with a ubiquitous and persistent presence in the environment. Of the thousands of PFAS used by consumers and industry, very few have been thoroughly characterized for potential adverse effects. This is especially true for the novel short-chain (C < 8) alternatives that replaced legacy PFAS. Perfluoroalkyl and polyfluoroalkyl substances have revealed inconsistencies in the toxicokinetics predicted by animal models and empirical findings in humans. To adequately assess the possible health effects of short-chain PFAS, there is a need for robust aggregated data sets on the mechanistic underpinnings and physiochemical properties of these alternatives. Acquiring relevant data on the health effects of short-chain PFAS can be achieved through high-throughput methods supported by in vitro human cell-based models. This review briefly summarizes some of the toxicity data obtained using human cells in vitro, discusses the advantages and limitations of cell-based models, and provides insights on potential solutions to challenges presented with the use of these methods for use in safety assessments.

In vitro-in vivo biotransformation and phase I metabolite profiling of benzo[a]pyrene in Gulf killifish (Fundulus grandis) populations with different exposure histories.

Chronic exposure to pollution may lead populations to display evolutionary adaptations associated with cellular and physiological mechanisms of defense against xenobiotics. This could result in differences in the way individuals of the same species, but inhabiting different areas, cope with chemical exposure. In the present study, we explore two Gulf killifish (Fundulus grandis) populations with different exposure histories for potential differences in the biotransformation of benzo[a]pyrene (BaP), and conduct a comparative evaluation of in vitro and in vivo approaches to describe the applicability of new approach methodologies (NAMs) for biotransformation assessments. Pollution-adapted and non-adapted F. grandis were subjected to intraperitoneal (IP) injections of BaP in time-course exposures, prior to measurements of CYP biotransformation activity, BaP liver concentrations, and the identification and quantification of phase I metabolites. Additionally, substrate depletion bioassays using liver S9 fractions were employed for measurements of intrinsic hepatic clearance and to evaluate the production of metabolites in vitro. Pollution-adapted F. grandis presented significantly lower CYP1A activity and intrinsic clearance rates that were 3 to 4 times lower than non-adapted fish. The metabolite profiling of BaP showed the presence of 1­hydroxy-benzo[a]pyrene in both the in vitro and in vivo approaches but with no significant population differences. Contrarily, 9­hydroxy-benzo[a]pyrene and benzo[a]pyrene-4,5-dihydrodiol, only identified through the in vivo approach, presented higher concentrations in the bile of pollution-adapted fish relative to non-adapted individuals. These observations further the understanding of the evolutionary adaptation of F. grandis inhabiting heavily polluted environments in the Houston Ship Channel, TX, USA, and highlight the need to consider the evolutionary history of populations of interest during the implementation of NAMs.

Reduced biotransformation of polycyclic aromatic hydrocarbons (PAHs) in pollution-adapted Gulf killifish (Fundulus grandis).

Anthropogenic pollution represents a significant source of selection, potentially leading to the emergence of evolutionary adaptations in chronically exposed organisms. A recent example of this scenario corresponds to Gulf killifish (Fundulus grandis) populations inhabiting the Houston Ship Channel (HSC), Texas, USA, which have been documented to have adapted to this heavily contaminated environment. Although not fully elucidated, one particularly important aspect of their adaptation involves the reduced inducibility of the aryl hydrocarbon receptor (AhR) and, potentially, the alteration of major biotransformation pathways. In the present study, we employed a modified Organization for Economic Cooperation and Development (OECD) 319-B test guideline to explore population and sex-related differences in the hepatic biotransformation of six polycyclic aromatic hydrocarbons (PAHs) in F. grandis populations with different exposure histories. Pollution-adapted F. grandis showed significantly lower hepatic clearance of PAHs than non-adapted fish, especially for high molecular weight PAHs (chrysene, benzo[k]fluoranthene, and benzo[a]pyrene), with pollution-adapted females presenting the lowest clearance. The characterization of different phase I biotransformation enzymes revealed that the basal activity of CYP1A, fundamental in the biotransformation of PAHs, was significantly lower in pollution-adapted fish, especially in females, which showed the lowest activity. Contrarily, basal CYP2C9-like activity was significantly higher in pollution-adapted fish. These results demonstrate the importance of exposure and evolutionary histories in shaping organisms' responses to pollution and provide significant evidence of sex-specific biotransformation differences in F. grandis populations.

Plasma Vitellogenin Reveals Potential Seasonal Estrogenicity in Fish from On-Site Wastewater Treatment Systems in Semi-Arid Streams Influenced by Snowmelt.

Effluents from on-site wastewater treatment systems can influence surface water quality, particularly when infrastructure is aging, malfunctioning, and improperly installed. Municipal wastewater often contains chemical compounds that can lead to adverse biological effects, such as reproductive impairment, in organisms that are chronically exposed. A significant number of these compounds are endocrine-disrupting chemicals. Water quality influences of on-site systems are poorly studied in semi-arid regions where instream flows are seasonally dependent on snowmelt, and when instream dilution of wastewater effluents is minimal during other times of the year. Here we examined surface water estrogenicity in low order tributaries of two unique semi-arid streams with on-site wastewater treatment systems, for which seasonal instream flow fluctuations occur in Park City, UT, USA. Water samples were collected from a total of five locations along two lotic systems downstream from active on-site treatment systems. Samples were extracted for targeted chemical analyses and to perform in vivo and in vitro bioassays with juvenile rainbow trout. Estrogenic activity was measured by quantifying the concentration and expression of vitellogenin (VTG) in plasma and liver, respectively. Plasma VTG presented elevated levels in fish exposed to water samples collected at the two sites in close proximity to on-site systems and during seasons with low stream discharge, though the levels observed did not suggest severe endocrine disruption. However, long-term exposure to these surface water could compromise the fish populations. While the sensitivity of in vitro bioassays was low and targeted chemical analyses did not identify causative compounds, the use of complementary lines of evidence (e.g., in vivo biological models) was advantageous in identifying estrogenic activity in waters influenced by effluents from on-site wastewater systems.

Multi-approach assessment for the evaluation of spatio-temporal estrogenicity in fish from effluent-dominated surface waters under low instream flow.

Current practices employed by most wastewater treatment plants (WWTP) are unable to completely remove endocrine disrupting compounds (EDCs) from reclaimed waters, and consistently discharge these substances to receiving systems. Effluent-dominated and dependent surface waters, especially during low instream flows, can increase exposure and risks to aquatic organisms due to adverse biological effects associated with EDCs. Given the ecological implications that may arise from exposure to such compounds, the present a multi-approach study examined spatio-temporal estrogenic potential of wastewater effluent to fish in East Canyon Creek (ECC), Utah, USA, a unique urban river with instream flows seasonally influenced by snowmelt. Juvenile rainbow trout (Oncorhynchus mykiss) were caged at different upstream and downstream sites from an effluent discharge during the summer and fall seasons. In the summer, where approximately 50% of the streamflow was dominated by effluent, fish from the upstream and a downstream site, located 13 miles away from the effluent discharge, presented significantly elevated concentrations of plasma vitellogenin (VTG). Similarly, significantly high 17ß-estradiol to 11-ketotestosterone ratios were measured in the summer across all sites and time points, compared to the fall. In the laboratory, juvenile fish and primary hepatocytes were exposed to concentrated effluent and surface water samples. Quantification of VTG, although in significantly lower levels, resembled response patterns observed in fish from the field study. Furthermore, analytical quantification of common EDCs in wastewater revealed the presence of estriol and estrone, though these did not appear to be related to the observed biological responses, as these were more significant in sites were no EDCs were detected. These combined observations suggest potential estrogenicity for fish in ECC under continuous exposures and highlight the advantages of following weight-of-evidence (WoE) approaches for environmental monitoring, as targeted analytically-based assessments may or may not support the identification of causative contaminants for adverse biological effects.

The Nitric Oxide System in Peripheral Artery Disease: Connection with Oxidative Stress and Biopterins.

Peripheral artery disease (PAD) pathophysiology extends beyond hemodynamics to include other operating mechanisms, including endothelial dysfunction. Oxidative stress may be linked to endothelial dysfunction by reducing nitric oxide (NO) bioavailability. We aimed to investigate whether the NO system and its regulators are altered in the setting of PAD and to assess the relationship between NO bioavailability and oxidative stress. Sera from 35 patients with intermittent claudication (IC), 26 patients with critical limb ischemia (CLI), and 35 non-PAD controls were analyzed to determine levels of tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), nitrate/nitrite (nitric oxides, or NOx), arginine, citrulline, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and the oxidative stress markers 8-Oxo-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), advanced glycation end products (AGEs), and protein carbonyls. NOx was significantly lower in IC and CLI patients compared to controls in association with elevated oxidative stress, with the greatest NOx reductions observed in CLI. Compared with controls, IC and CLI patients had reduced BH4, elevated BH2, and a reduced BH4/BH2 ratio. SDMA, the arginine/SDMA ratio, and the arginine/ADMA ratio were significantly higher in CLI patients. The NO system and its regulators are significantly compromised in PAD. This dysregulation appears to be driven by increased oxidative stress and worsens as the disease progresses from claudication to CLI.

Pharmaceutical uptake kinetics in rainbow trout: In situ bioaccumulation in an effluent-dominated river influenced by snowmelt.

Whether seasonal instream flow dynamics influence bioaccumulation of pharmaceuticals by fish is not well understood, specifically for urban lotic systems in semi-arid regions when flows are influenced by snowmelt. We examined uptake of select pharmaceuticals in rainbow trout (Oncorhynchus mykiss) caged in situ upstream and at incremental distances downstream (0.1, 1.4, 13 miles) from a municipal effluent discharge to East Canyon Creek in Park City, Utah, USA during summer and fall of 2018. Fish were sampled over 7-d to examine if uptake occurred, and to define uptake kinetics. Water and fish tissues were analyzed via isotope dilution liquid chromatography tandem mass spectrometry. Several pharmaceuticals were consistently detected in water, fish tissue and plasma, including carbamazepine, diphenhydramine, diltiazem, and fluoxetine. Pharmaceutical levels in water ranged up to 151 ng/L for carbamazepine, whereas the effluent tracer sucralose was consistently observed at low µg/L levels. During both summer and fall experiments at each of three downstream locations from effluent discharge, rainbow trout rapidly accumulated these pharmaceuticals; tissue levels reached steady state conditions within 24-96 h. Spatial and temporal differences for pharmaceutical levels in rainbow trout directly corresponded with surface water exposure concentrations, and uptake kinetics for individual pharmaceuticals did not vary among sites or seasons. Such observations are consistent with recent laboratory bioconcentration studies, which collectively indicate inhalational exposure from water governs rapid accumulation of ionizable base pharmaceuticals by fish in inland surface waters.

Metabolomic-based assessment reveals dysregulation of lipid profiles in human liver cells exposed to environmental obesogens.

Significant attention has been given to the potential of environmental chemicals to disrupt lipid homeostasis at the cellular level. These chemicals, classified as obesogens, are abundantly used in a wide variety of consumer products. However, there is a significant lack of information regarding the mechanisms by which environmental exposure can contribute to the onset of obesity and non-alcoholic fatty liver disease (NAFLD). Several studies have described the interaction of potential obesogens with lipid-related peroxisome proliferator-activated receptors (PPAR). However, no studies have quantified the degree of modification to lipidomic profiles in relevant human models, making it difficult to directly link PPAR agonists to the onset of lipid-related diseases. A quantitative metabolomic approach was used to examine the dysregulation of lipid metabolism in human liver cells upon exposure to potential obesogenic compounds. The chemicals rosiglitazone, perfluorooctanoic acid, di-2-ethylexylphthalate, and tributyltin significantly increased total lipids in liver cells, being diglycerides, triglycerides and phosphatidylcholines the most prominent. Contrarily, perfluorooctane sulfonic acid and the pharmaceutical fenofibrate appeared to lower total lipid concentrations, especially those belonging to the acylcarnitine, ceramide, triglyceride, and phosphatidylcholine groups. Fluorescence microscopy analysis for cellular neutral lipids revealed significant lipid bioaccumulation upon exposure to obesogens at environmentally relevant concentrations. This integrated omics analysis provides unique mechanistic insight into the potential of these environmental pollutants to promote diseases like obesity and NAFLD. Furthermore, this study provides a significant contribution to advance the understanding of molecular signatures related to obesogenic chemicals and to the development of alternatives to in vivo experimentation.