Enrichment of Bioactive Lipids in Urinary Extracellular Vesicles and Evidence of Apoptosis in Kidneys of Hypertensive Diabetic Cathepsin B Knockout Mice after Streptozotocin Treatment.

Cathepsin B (CtsB) is a ubiquitously expressed cysteine protease that plays important roles in health and disease. Urinary extracellular vesicles (uEVs) are released from cells associated with urinary organs. The antibiotic streptozotocin (STZ) is known to induce pancreatic islet beta cell destruction, diabetic nephropathy, and hypertension. We hypothesized that streptozotocin-induced diabetic kidney disease and hypertension result in the release of bioactive lipids from kidney cells that induce oxidative stress and renal cell death. Lipidomics was performed on uEVs isolated from CtsB knockout mice treated with or without STZ, and their kidneys were used to investigate changes in proteins associated with cell death. Lysophosphatidylethanolamine (LPE) (18:1), lysophosphatidylserine (LPS) (22:6), and lysophosphatidylglycerol (LPG) (22:5) were among the bioactive lipids enriched in uEVs from CtsB knockout mice treated with STZ compared to untreated CtsB mice (n = 3 uEV preparations per group). Anti-oxidant programming was activated in the kidneys of the CtsB knockout mice treated with STZ, as indicated by increased expression of glutathione peroxidase 4 (GPX4) and the cystine/glutamate antiporter SLC7A11 (XCT) (n = 4 mice per group), which was supported by a higher reactivity to 4-hydroxy-2-nonenal (4-HNE), a marker for oxidative stress (n = 3 mice per group). Apoptosis but not ferroptosis was the ongoing form of cell death in these kidneys as cleaved caspase-3 levels were significantly elevated in the STZ-treated CtsB knockout mice (n = 4 mice per group). There were no appreciable differences in the pro-ferroptosis enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) or the inflammatory marker CD93 in the kidneys (n = 3 mice per group), which further supports apoptosis as the prevalent mechanism of pathology. These data suggest that STZ treatment leads to oxidative stress, inducing apoptotic injury in the kidneys during the development of diabetic kidney disease and hypertension.

Kidney tubular epithelial cell ferroptosis links glomerular injury to tubulointerstitial pathology in lupus nephritis.

Ferroptosis is a druggable, iron-dependent form of cell death that is characterized by lipid peroxidation but has received little attention in lupus nephritis. Kidneys of lupus nephritis patients and mice showed increased lipid peroxidation mainly in the tubular segments and an increase in Acyl-CoA synthetase long-chain family member 4, a pro-ferroptosis enzyme. Nephritic mice had an attenuated expression of SLC7A11, a cystine importer, an impaired glutathione synthesis pathway, and low expression of glutathione peroxidase 4, a ferroptosis inhibitor. Lipidomics of nephritic kidneys confirmed ferroptosis. Using nephrotoxic serum, we induced immune complex glomerulonephritis in congenic mice and demonstrate that impaired iron sequestration within the proximal tubules exacerbates ferroptosis. Lupus nephritis patient serum rendered human proximal tubular cells susceptibility to ferroptosis which was inhibited by Liproxstatin-2, a novel ferroptosis inhibitor. Collectively, our findings identify intra-renal ferroptosis as a pathological feature and contributor to tubular injury in human and murine lupus nephritis.

Physical Evidence of Oil Uptake and Toxicity Assessment of Amphiphilic Grafted Nanoparticles Used as Oil Dispersants.

Herein, we report the toxicity evaluation of a new prototype dispersant system, silicon dioxide nanoparticles (NPs) functionalized with (3-glycidoxypropyl)triethoxysilane (GPS) and grafted poly(ε-caprolactone)-block-poly[oligo(ethylene glycol)methyl methacrylate mono-methyl ether] (NP-PCL-POEGMA). This serves as a follow up of our previous study where grafted silicon dioxide NPs functionalized with GPS and grafted hyperbranched poly(glycidol) (NP-HPG) were evaluated for reducing the toxicity in embryo, juvenile, and adult fish populations. In this study, the NP-HPG sample is used as a baseline to compare against the new NP-PCL-POEGMA samples. The relative size was established for three NP-PCL-POEGMA samples via cryogenic transmission electron microscopy. A quantitative mortality study determined that these NPs are non-toxic to embryo populations. An ethoxyresorufin-O-deethylase assay was performed on these NP-PCL-POEGMA samples to test for reduced cytochrome P450 1A after the embryos were exposed to the water-accommodated fraction of crude oil. Overall, these NP-PCL-POEGMA NPs better protected the embryo populations than the previous NP-HPG sample (using a protein activity end point), showing a trend in the right direction for prototype dispersants to replace the commercially utilized Corexit.

Towards regulation of Endocrine Disrupting chemicals (EDCs) in water resources using bioassays - A guide to developing a testing strategy.

Endocrine disrupting chemicals (EDCs) are found in every environmental medium and are chemically diverse. Their presence in water resources can negatively impact the health of both human and wildlife. Currently, there are no mandatory screening mandates or regulations for EDC levels in complex water samples globally. Bioassays, which allow quantifying in vivo or in vitro biological effects of chemicals are used commonly to assess acute toxicity in water. The existing OECD framework to identify single-compound EDCs offers a set of bioassays that are validated for the Estrogen-, Androgen-, and Thyroid hormones, and for Steroidogenesis pathways (EATS). In this review, we discussed bioassays that could be potentially used to screen EDCs in water resources, including in vivo and in vitro bioassays using invertebrates, fish, amphibians, and/or mammalians species. Strengths and weaknesses of samples preparation for complex water samples are discussed. We also review how to calculate the Effect-Based Trigger values, which could serve as thresholds to determine if a given water sample poses a risk based on existing quality standards. This work aims to assist governments and regulatory agencies in developing a testing strategy towards regulation of EDCs in water resources worldwide. The main recommendations include 1) opting for internationally validated cell reporter in vitro bioassays to reduce animal use & cost; 2) testing for cell viability (a critical parameter) when using in vitro bioassays; and 3) evaluating the recovery of the water sample preparation method selected. This review also highlights future research avenues for the EDC screening revolution (e.g., 3D tissue culture, transgenic animals, OMICs, and Adverse Outcome Pathways (AOPs)).

Impact of bisphenol-A and synthetic estradiol on brain, behavior, gonads and sex hormones in a sexually labile coral reef fish.

Endocrine disrupting chemicals, such as bisphenol A (BPA) and ethinylestradiol (EE2), are detected in the marine environment from plastic waste and wastewater effluent. However, their impact on reproduction in sexually labile coral reef fish is unknown. The objective of this study was to determine impacts of environmentally relevant concentrations of BPA and EE2 on behavior, brain gene expression, gonadal histology, sex hormone profile, and plasma vitellogenin (Vtg) levels in the anemonefish, Amphiprion ocellaris. A. ocellaris display post-maturational sex change from male to female in nature. Sexually immature, male fish were paired together and fed twice daily with normal food (control), food containing BPA (100 µg/kg), or EE2 (0.02 µg/kg) (n = 9 pairs/group). Aggression toward an intruder male was measured at 1, 3, and 6 months. Blood was collected at 3 and 6 months to measure estradiol (E2), 11-ketotestosterone (11-KT), and Vtg. At the end of the study, fish were euthanized to assess gonad morphology and to measure expression of known sexually dimorphic genes in the brain. Relative to control, BPA decreased aggression, altered brain transcript levels, increased non-vitellogenic and vitellogenic eggs in the gonad, reduced 11-KT, and increased plasma Vtg. In two BPA-treated pairs, both individuals had vitellogenic eggs, which does not naturally occur. EE2 reduced 11-KT in subordinate individuals and altered expression of one transcript in the brain toward the female profile. Results suggest BPA, and to a lesser extent EE2, pollution in coral reef ecosystems could interfere with normal reproductive physiology and behavior of the iconic sexually labile anemonefish.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Untargeted lipidomics reveals the toxicity of bisphenol A bis(3-chloro-2- hydroxypropyl) ether and bisphenols A and F in zebrafish liver cells.

Given the opposing responses reported for bisphenol A (BPA) in terms of induction of obesogenic effects and impaired lipid metabolism, the increasing use of bisphenol F (BPF), and the relatively low information available regarding the effects of bisphenol A bis(3-chloro-2- hydroxypropyl) ether (BADGE·2HCl) in aquatic organisms, this work aims to use the zebrafish liver cell line (ZFL) as an alternative model to characterize the toxicity and the lipid metabolism disruptive potential of the selected compounds in fish. All three bisphenols increased intracellular levels of dihydroceramides and ether-triacylglycerides (ether-TGs), suggestive of inhibited cell growth. However, while BPA and BADGE·2HCl caused an increase of saturated and lower unsaturated TGs, BPF caused oxidative stress and the decrease of TGs containing polyunsaturated fatty acids (PUFAs). Analysis by qPCR highlighted the up-regulation of the lipogenic genes scd and elovl6 by BPA and BPF in line with an increase of lipids containing saturated and monounsaturated FA and a decrease of lipids containing PUFAs. This study shows that BPA, BPF and BADGE·2HCl target lipid homeostasis in ZFL cells through different mechanisms, and highlights the higher lipotoxicity of BADGE·2HCl compared to BPA and BPF.

Toxicity assessment of a novel oil dispersant based on silica nanoparticles using Fathead minnow.

Oil spill accidents are a major concern for aquatic organisms. In recent history, the Deepwater Horizon blowout spilled 500 million liters of crude oil into the Gulf of Mexico. Corexit 9500A was used to disperse the oil since it was the method approved at that time, despite safety concerns about its use. A better solution is necessary for dispersing oil from spills that reduces the toxicity to exposed aquatic organisms. To address this challenge, novel engineered nanoparticles were designed using silica cores grafted with hyperbranched poly(glycidol) branches. Because the silica core and polymers are known to be biocompatible, we hypothesized that these particles are nontoxic to fathead minnows (Pimephales promelas) and would decrease their exposure to oil polyaromatic hydrocarbons. Fathead minnow embryos, juveniles and adult stages were exposed to the particles alone or in combination with a water-accommodated fraction of oil. Acute toxicity of nanoparticles to fish was tested by measuring mortality. Sub-lethal effects were also measured including gene expression of cytochrome P450 1a (cyp1a) mRNA and heart rate in embryos. In addition, a mixture of particles plus the water-accommodated fraction was directly introduced to adult female fathead minnows by gavage. Three different nanoparticle concentrations were used (2, 10, and 50 mg/L) in either artificial fresh water or the water-accommodated fraction of the oil. In addition, nanoparticle-free controls were carried out in the two solutions. No significant mortality was observed for any age group or nanoparticle concentration, suggesting the safety of the nanoparticles. In the presence of the water-accommodated fraction alone, juvenile and adult fathead minnows responded by increasing expression of cyp1a. The addition of nanoparticles to the water-accommodated fraction reduced cyp1a gene expression in treatments. Heart rate was also restored to normal parameters in embryos co-exposed to nanoparticles and to the water-accommodated fraction. Measurement of polyaromatic hydrocarbons confirmed their presence in the tested solutions and the reduction of available PAH in WAF treated with the nanoparticles. Our findings suggest the engineered nanoparticles may be protecting the fish by sequestering polyaromatic hydrocarbons from oil, measured indirectly by the induction of cypa1 mRNAs. Furthermore, chemical analysis showed a reduction in PAH content in the water accommodated fraction with the presence of nanoparticles.

Transcriptome and physiological effects of toxaphene on the liver-gonad reproductive axis in male and female largemouth bass (Micropterus salmoides).

Toxaphene is an organochlorine pesticide and environmental contaminant that is concerning due to its atmospheric transport and persistence in soil. In Florida, toxaphene and other organochlorine pesticides were used heavily in agriculture on the north shore of Lake Apopka and they are still detectable in soil. Wild largemouth bass that inhabit the lake and the marshes along the north shore have been exposed to a variety of organochlorine pesticides including dieldrin, methoxychlor, and p,p'-DDE, among others. While these other organochlorine pesticides have been studied for their endocrine disrupting effects in largemouth bass, there is little information for toxaphene. In this study, male and female largemouth bass were given food containing 50 mg/kg toxaphene for almost 3 months, to achieve tissue levels similar to those found in fish at Lake Apopka. Sex-specific toxicity was then evaluated by measuring various reproductive endpoints and transcriptomic changes. In females, gonadosomatic index showed a trend towards reduction (p = 0.051) and plasma vitellogenin was reduced by ~40% relative to controls. However plasma levels of 17ß-estradiol and testosterone were not perturbed by toxaphene exposure. These data suggest that toxaphene does not act as a weak estrogen as many other organochlorine pesticides do, but rather appears to be acting as an antiestrogen in female fish. There were no obvious changes in the gonadosomatic index and plasma hormones in male bass. However, ex vivo explant experiments revealed that toxaphene prevented human chorionic gonadotropin-stimulated testosterone production in the testis. This suggested that toxaphene had anti-androgenic effects in males. Subsequent transcriptomic analyses of the testis revealed that androgen receptor/beta-2-microglobulin signaling was up-regulated while insulin-related pathways were suppressed with toxaphene, which could be interpreted as a compensatory response to androgen suppression. In the male liver, the transcriptome analysis revealed an overwhelming suppression in immune-related signaling cascades (e.g. lectin-like receptor and ITSM-Containing Receptor signaling, CD16/CD14 Proinflammatory Monocyte Activation, and CD38/CD3-JUN/FOS/NF-kB Signaling in T-cell Proliferation). Overall, this study showed that toxaphene induced sex-specific effects. The transcriptomic and physiological responses observed can contribute to the development of adverse outcome pathways for toxaphene exposure in fish.

Steroid hormones and estrogenic activity in the wastewater outfall and receiving waters of the Chascomús chained shallow lakes system (Argentina).

Natural and synthetic steroid hormones, excreted by humans and farmed animals, have been considered as important sources of environmental endocrine disruptors. A suite of estrogens, androgens and progestogens was measured in the wastewater treatment plant outfall (WWTPO) of Chascomús city (Buenos Aires province, Argentina), and receiving waters located downstream and upstream from the WWTPO, using solid phase extraction and high-performance liquid chromatography mass spectrometry. The following natural hormones were measured: 17ß-estradiol (E2), estrone (E1), estriol (E3), testosterone (T), 5α-dihydrotestosterone (DHT), progesterone (P), 17-hydroxyprogesterone (17OHP) and the synthetic estrogen 17α-ethinylestradiol (EE2). Also, in order to complement the analytical method, the estrogenic activity in these surface water samples was evaluated using the in vitro transactivation bioassay that measures the estrogen receptor (ER) activity using mammalian cells. All-natural steroid hormones measured, except 17OHP, were detected in all analyzed water samples. E3, E1, EE2 and DHT were the most abundant and frequently detected. Downstream of the WWTPO, the concentration levels of all compounds decreased reaching low levels at 4500 m from the WWTPO. Upstream, 1500 m from the WWTPO, six out of eight steroid hormones analyzed were detected: DHT, T, P, 17OHP, E3 and E2. Moreover, water samples from the WWTPO and 200 m downstream from it showed estrogenic activity exceeding that of the EC50 of the E2 standard curve. In sum, this work demonstrates the presence of sex steroid hormones and estrogenic activity, as measured by an in vitro assay, in superficial waters of the Pampas region. It also suggests the possibility of an unidentified source upstream of the wastewater outfall.

Antineoplastic Agents: Environmental Prevalence and Adverse Outcomes in Aquatic Organisms.

Cancer is the second leading cause of death worldwide, with 9.6 million cancer-related deaths in 2018. Cancer incidence has increased over time, and so has the prescription rate of chemotherapeutic drugs. These pharmaceuticals, known as antineoplastic agents, enter the aquatic environment via human excretion and wastewater. The objectives of the present critical review were to investigate the risk of antineoplastics to aquatic species and to summarize the current state of knowledge regarding their levels in the environment, because many antineoplastics are not adequately removed during wastewater treatment. We conducted 2 separate literature reviews to synthesize data on the global environmental prevalence and toxicity of antineoplastics. The antineoplastics most frequently detected in the environment included cyclophosphamide, ifosfamide, tamoxifen, methotrexate, and 5-fluorouracil; all were detectable in multiple water sources, including effluent and surface waters. These antineoplastics span 3 different mechanistic classes, with cyclophosphamide and ifosfamide classified as alkylating agents, tamoxifen as a hormonal agent, and methotrexate and 5-fluorouracil as antimetabolites. Studies that characterize the risk of antineoplastics released into aquatic environments are scarce. We summarize the biological impacts of the most environmentally prevalent antineoplastics on aquatic organisms and propose an adverse outcome pathway for cyclophosphamide and ifosfamide, 2 widely prescribed drugs with a similar immunotoxic mode of action. Acute and chronic ecotoxicity studies using aquatic models are needed for risk characterization of antineoplastics. Environ Toxicol Chem 2020;39:967-985. © 2020 SETAC.

Organochlorine pesticides: Agrochemicals with potent endocrine-disrupting properties in fish.

Organochlorine pesticides (OCPs) are persistent environmental contaminants that act as endocrine disruptors and organ system toxicants. These pesticides (e.g. dichlorodiphenyltrichloroethane (DDT), dieldrin, toxaphene, among others) are ranked as some of the most concerning chemicals for human health. These pesticides (1) act as teratogens, (2) are neuroendocrine disruptors, (3) suppress the immune and reproductive systems, and (4) dysregulate lipids and metabolism. Using a computational approach, we revealed enriched endocrine-related pathways in the Comparative Toxicogenomics Database sensitive to this chemical class, and these included reproduction (gonadotropins, estradiol, androgen, steroid biosynthesis, oxytocin), thyroid hormone, and insulin. Insight from the Tox21 and ToxCast programs confirm that these agrochemicals activate estrogen receptors, androgen receptors, and retinoic acid receptors with relatively high affinity, although differences exist in their potency. We propose an adverse outcome pathway for OCPs toxicity in the fish testis as a novel contribution to further understanding of OCP-induced toxicity. Organochlorine pesticides, due to their persistence and high toxicity to aquatic and terrestrial wildlife as well as humans, remain significant agrochemicals of concern.

Steroidogenic acute regulatory protein transcription is regulated by estrogen receptor signaling in largemouth bass ovary.

Estrogenic contaminants in the environment are linked to the occurrence of reproductive abnormalities in many aquatic species, including largemouth bass (Micropterus salmoides; LMB). Previous work has shown that many different types of xenoestrogens regulate expression of the Steroidogenic Acute Regulatory protein (StAR), a cholesterol-transporting protein vital to steroid hormone biosynthesis; however, the regulatory mechanisms of StAR are incompletely characterized in fish. To learn more about endogenous expression patterns of StAR in the ovary, LMB were collected from the St. John's River (Florida, USA) over an entire breeding season to investigate StAR expression. Plasma 17ß-estradiol (E2) and StAR mRNA levels were positively correlated in females, and StAR mRNA levels displayed ~ 100-fold increase between primary oocyte growth stages and final maturation. To further study the regulation of StAR, female LMB in the laboratory were fed at ≃2% of their weight on a diet laden with 17α-ethinylestradiol (EE2, 70 or 200 ng EE2 per gram feed). Diets were designed to achieve a physiologically-relevant exposure to EE2, and StAR expression was assessed in vivo. We observed a dose-dependent suppression of StAR mRNA levels, however both diets led to high, pharmacological levels in the blood and do not represent normal physiological ranges of estrogens. In the 200 ng EE2/gm feed group, ovarian StAR mRNA levels were suppressed to approximately 5% of that of the LMB control group. These investigations suggest that LMB StAR increases in expression during oocyte maturation and that it is suppressed by E2 feedback when estrogen levels are high, through the HPG axis. A 2.9 kb segment of the LMB StAR promoter was examined for putative E2 response elements using in silico software, and a putative estrogen receptor binding element (ERE/-1745) was predicted in the promoter. The functionality of the ERE was examined using MA-10 mouse Leydig cells transfected with the LMB StAR promoter. Estrogen receptor (ER) interaction with ERE/-1745 was evaluated under basal and human chorionic gonadotropin (hCG)-treated conditions in the presence and absence of E2. Chromatin immunoprecipitation (ChIP) experiments revealed that ESR1 binding to the promoter was enriched under basal conditions and E2 exposure elicited an increase in enrichment (4-fold) above that observed under basal conditions. ESR2 was not strongly enriched at the ERE/-1745 site, suggesting that StAR may be preferentially regulated by LMB estrogen receptor 1 (esr1). Taken together, these different experiments provide evidence that LMB StAR is under the control of estrogens and that ESR1 binds directly to the LMB StAR promoter in an E2-responsive manner.

Twenty years of transcriptomics, 17alpha-ethinylestradiol, and fish.

In aquatic toxicology, perhaps no pharmaceutical has been investigated more intensely than 17alpha-ethinylestradiol (EE2), the active ingredient of the birth control pill. At the turn of the century, the fields of comparative endocrinology and endocrine disruption research witnessed the emergence of omics technologies, which were rapidly adapted to characterize potential hazards associated with exposures to environmental estrogens, such as EE2. Since then, significant advances have been made by the scientific community, and as a result, much has been learned about estrogen receptor signaling in fish from environmental xenoestrogens. Vitellogenin, the egg yolk precursor protein, was identified as a major estrogen-responsive gene, establishing itself as the premier biomarker for estrogenic exposures. Omics studies have identified a plethora of estrogen responsive genes, contributing to a wealth of knowledge on estrogen-mediated regulatory networks in teleosts. There have been ~40 studies that report on transcriptome responses to EE2 in a variety of fish species (e.g., zebrafish, fathead minnows, rainbow trout, pipefish, mummichog, stickleback, cod, and others). Data on the liver and testis transcriptomes dominate in the literature and have been the subject of many EE2 studies, yet there remain knowledge gaps for other tissues, such as the spleen, kidney, and pituitary. Inter-laboratory genomics studies have revealed transcriptional networks altered by EE2 treatment in the liver; networks related to amino acid activation and protein folding are increased by EE2 while those related to xenobiotic metabolism, immune system, circulation, and triglyceride storage are suppressed. EE2-responsive networks in other tissues are not as comprehensively defined which is a knowledge gap as regulated networks are expected to be tissue-specific. On the horizon, omics studies for estrogen-mediated effects in fish include: (1) Establishing conceptual frameworks for incorporating estrogen-responsive networks into environmental monitoring programs; (2) Leveraging in vitro and computational toxicology approaches to identify chemicals associated with estrogen receptor-mediated effects in fish (e.g., male vitellogenin production); (3) Discovering new tissue-specific estrogen receptor signaling pathways in fish; and (4) Developing quantitative adverse outcome pathway predictive models for estrogen signaling. As we look ahead, research into EE2 over the past several decades can serve as a template for the array of hormones and endocrine active substances yet to be fully characterized or discovered.

Effects of Chlorpyrifos on Cholinesterase and Serine Lipase Activities and Lipid Metabolism in Brains of Rainbow Trout (Oncorhynchus mykiss).

Chlorpyrifos is an organophosphorus insecticide that elicits acute toxicity through inhibition of acetylcholinesterase (AChE), leading to acetylcholine accumulation and prolonged stimulation of cholinergic receptors throughout the central and peripheral nervous systems. Previous studies have indicated that neurodevelopment may also be impaired through alternative pathways, including reduction of cyclic adenosine monophosphate (cAMP)-catalyzed downstream events. The upstream initiating events that underlie noncholinergic neurological actions of chlorpyrifos and other organophosphorus compounds remain unclear. To investigate the potential role of fatty acid signaling disruption as a mechanism of toxicity, lipid metabolism and fatty acid profiles were examined to identify alterations that may play a critical role in upstream signaling in the central nervous system (CNS). Juvenile rainbow trout were treated for 7 days with nominal chlorpyrifos concentrations previously reported to diminish olfactory responses (10, 20, and 40 µg/l). Although lethality was noted higher in doses, measured chlorpyrifos concentrations of 1.38 µg/l (nominal concentration 10 µg/l) significantly reduced the activity of AChE and two serine lipases, monoacylglycerol lipase, and fatty acid amide hydrolase in the brain. Reductions in lysophosphatidylethanolamines (16:0, 18:0, 18:1, and 22:6) derived from the phosphatidylethanolamines and free fatty acids (palmitic acid 16:0, linolenic acid 18:3, eicosadienoic acid 20:2, arachidonic acid 20:4, and docosahexaenoic acid 22:6) were also noted, suggesting that chlorpyrifos inhibited the metabolism of select phospholipid signaling precursors at sublethal concentrations. These results indicate that in addition to AChE inhibition, environmentally relevant chlorpyrifos exposure alters serine lipase activity and lipid metabolites in the trout brain, which may compromise neuronal signaling and impact neurobehavioral responses in aquatic animals.

Blood Transcriptomics Analysis of Fish Exposed to Perfluoro Alkyls Substances: Assessment of a Non-Lethal Sampling Technique for Advancing Aquatic Toxicology Research.

In contrast to mammals, the blood from other vertebrates such as fish contains nucleated red cells. Using a fathead minnow ( Pimephales promelas) oligonucleotide microarray, we compared altered transcripts in the liver and whole blood after exposure to environmentally relevant concentrations of perfluorooctanesulfonic acid (PFOS) and a mixture of seven types of perfluoro alkyl substances (PFAS), including perfluorooctanoic acid (PFOA). We used quantitative polymerase chain reactions and cell-based assays to confirm the main effects and found that blood responded with a greater number of altered genes than the liver. The exposure to PFAS altered similar genes with central roles in a cellular pathway in both tissues, including estrogen receptor α and peroxisome proliferator activator ß and γ, indicating that the genes previously associated with PFAS exposure are differentially expressed in blood and liver. The altered transcripts are involved with cholesterol metabolism and mitochondrial function. Our data confirmed that PFAS are weak xenoestrogens and exert effects on DNA integrity. Gene expression profiling from blood samples not related with the immune system, including very-low-density lipoprotein, vitellogenin, estrogen receptor, and thyroid hormone receptor, demonstrated that blood is a useful tissue for assessing endocrine disruption in non-mammalian vertebrates. We conclude that the use of blood for non-lethal sampling in genomics studies is informative and particularly useful for assessing the effects of pollution in endangered species. Further, using blood will reduce animal use and widen the experimental design options for studying the effects of contaminant exposure on wildlife.

Human exposure to polycyclic aromatic hydrocarbons: Metabolomics perspective.

Polycyclic aromatic hydrocarbons (PAHs) are organic contaminants exhibiting carcinogenic toxicity. They are widespread in the environment, especially in urban areas. Humans are exposed to PAHs via inhalation, ingestion and dermal contact. Though much research has investigated their toxicity, little is known regarding the metabolic responses in humans after exposing to PAHs. However, those studies are important since PAHs become carcinogenic after metabolic activation by humans as indirect-acting carcinogens. As such, it is important to study their metabolism in humans based on metabolomics analysis. The goal of metabolomics study is to obtain a comprehensive view of metabolic reactions in humans after exposing to PAHs to better control the underlying metabolisms and reduce their genotoxicity. This article reviewed the biomarkers, analytical techniques including nuclear magnetic resonance and mass spectrometry, big data multivariate statistical analysis, and animal models that have been utilized to better understand the biological effects of PAHs, PAH-derivatives, and their metabolites and biotransformation products on humans.

In Silico Computational Transcriptomics Reveals Novel Endocrine Disruptors in Largemouth Bass ( Micropterus salmoides).

In recent years, decreases in fish populations have been attributed, in part, to the effect of environmental chemicals on ovarian development. To understand the underlying molecular events we developed a dynamic model of ovary development linking gene transcription to key physiological end points, such as gonadosomatic index (GSI), plasma levels of estradiol (E2) and vitellogenin (VTG), in largemouth bass ( Micropterus salmoides). We were able to identify specific clusters of genes, which are affected at different stages of ovarian development. A subnetwork was identified that closely linked gene expression and physiological end points and by interrogating the Comparative Toxicogenomic Database (CTD), quercetin and tretinoin (ATRA) were identified as two potential candidates that may perturb this system. Predictions were validated by investigation of reproductive associated transcripts using qPCR in ovary and in the liver of both male and female largemouth bass treated after a single injection of quercetin and tretinoin (10 and 100 µg/kg). Both compounds were found to significantly alter the expression of some of these genes. Our findings support the use of omics and online repositories for identification of novel, yet untested, compounds. This is the first study of a dynamic model that links gene expression patterns across stages of ovarian development.

Toward an adverse outcome pathway for impaired growth: Mitochondrial dysfunction impairs growth in early life stages of the fathead minnow (Pimephales promelas).

Chemical contaminants present in the environment can affect mitochondrial bioenergetics in aquatic organisms and can have substantial effects on individual fitness. As early life stages of fish are particularly vulnerable to environmental contaminants, they are ideal models for examining the relationship between impaired mitochondrial bioenergetics (ATP-dependent respiration, basal oxidative respiration) and apical endpoints such as growth. Here, early life stages of the fathead minnow (Pimephales promelas), an ecologically relevant North American species, were used to investigate the relationship between mitochondrial bioenergetics and growth following perturbation with model mitochondrial toxicants 2,4-dinitrophenol and octylamine. Fathead minnows were exposed to 2,4-dinitrophenol and octylamine at 3 concentrations for 24 h and endpoints related to mitochondrial bioenergetics were measured with the Agilent Seahorse XFe24 Bioanalyzer. In order to link changes in mitochondrial bioenergetics to growth, fathead minnows were exposed to the same chemical contaminants for 7-14 days and growth was measured by measuring total length on a weekly basis. There was a significant correlation between decrease in average length at 14 days and basal respiration (r = 0.997, p = 0.050, n = 3), as well as maximal respiration (r = 0.998, p-value = 0.043, n = 3) for embryos exposed to 2,4 dinitrophenol. For octylamine, ATP production was highly correlated with average length at 7 days (p-value = 0.1) and spare respiratory capacity and average length at 14 days were highly correlated (p-value = 0.1). These data improve understanding of how mitochondrial toxicants impair growth in fish larvae and may be useful for developing an adverse outcome pathway for growth.

Activated carbon as a means of limiting bioaccumulation of organochlorine pesticides, triclosan, triclocarban, and fipronil from sediments rich in organic matter.

Addition of activated carbon to contaminated sediment is an established means of remediation but its applicability to sediments high in organic carbon is presently unknown. We evaluated the effects of adding either granular activated carbon (GAC) or pelletized fine-grained activated carbon (PfAC, containing ∼ 50% AC) to contaminated sediments from Lake Apopka featuring a very high total organic carbon content (∼39% w/w dry). Sediments showing background levels of legacy pesticides were spiked with a mixture of 5 chemicals (p,p'-DDE, dieldrin, triclosan, triclocarban, and fipronil) to a nominal concentration of 2 µg/g sediment for each chemical. Following incubation of spiked sediments with the addition of activated carbon for 30 days, we assessed the success on limiting bioaccumulation using Lumbriculus variegatus (blackworm). In contaminant-spiked sediments amended with PfAC, blackworm body burdens of triclosan, triclocarban, and fipronil decreased by >50% and those of p,p'-DDE and dieldrin decreased by <30%. GAC addition to spiked sediments was less impactful, and yielded notable benefits in worm body burden reduction only for fipronil (40%). Fipronil achieved high treatment efficiency within the 30 day amendment with both GAC and PfAC. This is the first study to examine AC treatment in artificially contaminated sediments intrinsically very rich in organic matter content. PfAC exhibited superior performance over GAC for mitigating the uptake of certain organochlorines by aquatic organisms. These results indicate that further studies focusing on additional types of sediments and a broader spectrum of hydrophobic pollutants are warranted.