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.

Assessment of lipids and adrenal hormones in the Florida manatee (Trichechus manatus latirostris) from different habitats.

Florida manatees (Trichechus manatus latirostris), a federally protected species, are classified as threatened due to anthropogenic stressors. Manatees inhabit sites that are impacted by human activities that can negatively affect stress physiology and metabolism. Samples collected from healthy manatees (pregnant females, non-pregnant females, and males) at Crystal River and Indian River Lagoon in Florida, were assessed for adrenal hormones, proteins, glucose, and lipid content in plasma. The objective was to determine if healthy manatees sampled between 2010-2014 from the Indian River Lagoon exhibited evidence of stress compared to healthy manatees sampled between 2012-2019 from Crystal River. Plasma cortisol concentrations were not different in male and non-pregnant female manatees between sites but were elevated in pregnant manatees. Plasma aldosterone concentrations were elevated in Indian River Lagoon manatees relative to those at Crystal River, possibly due to differences in salinity and available freshwater between the two environments. Site differences were noted for plasma protein and glucose concentrations in manatees; additionally, differences between the sexes were also observed in glucose concentrations. Fifteen lipid subclasses, including oxidized lysophosphatidylcholines, oxidized phosphatidylcholines, oxidized triacylglycerols, were elevated in manatees from the Indian River Lagoon relative to manatees from Crystal River. Evidence of a stress response in healthy Indian River Lagoon manatees was lacking compared to Crystal River manatees. Differences in metabolites related to energy (glucose, protein, and lipids) may be related to site-specific variables, such as salinity and food availability/quality. This study generates novel data on plasma lipid profiles and provides cortisol, aldosterone, glucose, and protein values from healthy Florida manatees in two disparate sites that can be referenced in future studies. These data contribute to an improved understanding of manatee physiology to better inform population management.

Bisphenol A and bisphenol S disruptions of the mouse placenta and potential effects on the placenta-brain axis.

Placental trophoblast cells are potentially at risk from circulating endocrine-disrupting chemicals, such as bisphenol A (BPA). To understand how BPA and the reputedly more inert bisphenol S (BPS) affect the placenta, C57BL6J mouse dams were fed 200 µg/kg body weight BPA or BPS daily for 2 wk and then bred. They continued to receive these chemicals until embryonic day 12.5, whereupon placental samples were collected and compared with unexposed controls. BPA and BPS altered the expression of an identical set of 13 genes. Both exposures led to a decrease in the area occupied by spongiotrophoblast relative to trophoblast giant cells (GCs) within the junctional zone, markedly reduced placental serotonin (5-HT) concentrations, and lowered 5-HT GC immunoreactivity. Concentrations of dopamine and 5-hydroxyindoleacetic acid, the main metabolite of serotonin, were increased. GC dopamine immunoreactivity was increased in BPA- and BPS-exposed placentas. A strong positive correlation between 5-HT+ GCs and reductions in spongiotrophoblast to GC area suggests that this neurotransmitter is essential for maintaining cells within the junctional zone. In contrast, a negative correlation existed between dopamine+ GCs and reductions in spongiotrophoblast to GC area ratio. These outcomes lead to the following conclusions. First, BPS exposure causes almost identical placental effects as BPA. Second, a major target of BPA/BPS is either spongiotrophoblast or GCs within the junctional zone. Third, imbalances in neurotransmitter-positive GCs and an observed decrease in docosahexaenoic acid and estradiol, also occurring in response to BPA/BPS exposure, likely affect the placental-brain axis of the developing mouse fetus.

Dapagliflozin Treatment Augments Bioactive Phosphatidylethanolamine Concentrations in Kidney Cortex Membrane Fractions of Hypertensive Diabetic db/db Mice and Alters the Density of Lipid Rafts in Mouse Proximal Tubule Cells.

In addition to inhibiting renal glucose reabsorption and allowing for glucose excretion, the sodium/glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin may be efficacious in treating various comorbidities associated with type 2 diabetes mellitus (T2DM). The molecular mechanisms by which dapagliflozin exerts its beneficial effects are largely unknown. We hypothesized dapagliflozin treatment in the diabetic kidney alters plasma membrane lipid composition, suppresses extracellular vesicle (EV) release from kidney cells, and disrupts lipid rafts in proximal tubule cells. In order to test this hypothesis, we treated diabetic db/db mice with dapagliflozin (N = 8) or vehicle (N = 8) and performed mass spectrometry-based lipidomics to investigate changes in the concentrations of membrane lipids in the kidney cortex. In addition, we isolated urinary EVs (uEVs) from urine samples collected during the active phase and the inactive phase of the mice and then probed for changes in membrane proteins enriched in the EVs. Multiple triacylglycerols (TAGs) were enriched in the kidney cortex membrane fractions of vehicle-treated diabetic db/db mice, while the levels of multiple phosphatidylethanolamines were significantly higher in similar mice treated with dapagliflozin. EV concentration and size were lesser in the urine samples collected during the inactive phase of dapagliflozin-treated diabetic mice. In cultured mouse proximal tubule cells treated with dapagliflozin, the lipid raft protein caveolin-1 shifted from less dense fractions to more dense sucrose density gradient fractions. Taken together, these results suggest dapagliflozin may regulate lipid-mediated signal transduction in the diabetic kidney.

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.

Increased levels of perfluorooctanesulfonic acid (PFOS) during Hurricane Dorian on the east coast of Florida.

Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic chemicals commonly found in everyday consumer products and are an emerging concern due to their ubiquitous presence in ecosystems around the world. PFAS exposure, which often occurs through contaminated water, has been linked to several adverse health effects in humans and wildlife. PFAS can be transported in surface water and storm runoff in the nearshore environment. Episodic events, such as hurricanes, are projected to increase in frequency and intensity, and a critical unanswered question is: how do episodic events influence the concentrations and distributions of emerging contaminants, such as PFAS, in coastal systems? Here, we investigated the impact of the 2019 Hurricane Dorian on the Florida coast to assess how natural disasters, such as hurricanes, influence the fate and transport of PFAS in surface water. Water samples collected throughout the St. Augustine Intracoastal waterway before, during, and after the storm were analyzed and compared with baseline concentrations. Ultra-high-pressure liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) was used in the detection and quantification of 23 and 17 PFAS, respectively. Perfluorooctane sulfonic acid (PFOS) was the compound with the highest concentration across all sampling sites. Mean PFOS levels showed the highest increase of 177% during the hurricane and returned to baseline levels after two days. Our findings highlight the need for continued research focused on understanding how large storms near all coastlines can impact the transport of environmental pollutants, such as PFOS, that can have adverse effects on human and environmental health. Further monitoring of PFAS in coastal systems is necessary to identify potential PFAS hotspots, investigate the impacts of episodic events on PFAS transport, develop mitigation practices capable of reducing the risk of PFAS exposure.

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)).

C Type Natriuretic Peptide Receptor Activation Inhibits Sodium Channel Activity in Human Aortic Endothelial Cells by Activating the Diacylglycerol-Protein Kinase C Pathway.

The C-type natriuretic peptide receptor (NPRC) is expressed in many cell types and binds all natriuretic peptides with high affinity. Ligand binding results in the activation or inhibition of various intracellular signaling pathways. Although NPRC ligand binding has been shown to regulate various ion channels, the regulation of endothelial sodium channel (EnNaC) activity by NPRC activation has not been studied. The objective of this study was to investigate mechanisms of EnNaC regulation associated with NPRC activation in human aortic endothelial cells (hAoEC). EnNaC protein expression and activity was attenuated after treating hAoEC with the NPRC agonist cANF compared to vehicle, as demonstrated by Western blotting and patch clamping studies, respectively. NPRC knockdown studies using siRNA's corroborated the specificity of EnNaC regulation by NPRC activation mediated by ligand binding. The concentration of multiple diacylglycerols (DAG) and the activity of protein kinase C (PKC) was augmented after treating hAoEC with cANF compared to vehicle, suggesting EnNaC activity is down-regulated upon NPRC ligand binding in a DAG-PKC dependent manner. The reciprocal cross-talk between NPRC activation and EnNaC inhibition represents a feedback mechanism that presumably is involved in the regulation of endothelial function and aortic stiffness.

Lipid Profiles of Urinary Extracellular Vesicles Released during the Inactive and Active Phases of Aged Male Mice with Spontaneous Hypertension.

Hypertension remains a major problem, especially in the elderly, as it increases the risk for cardiovascular, coronary artery, cerebrovascular, and kidney diseases. Extracellular vesicles (EVs) play a role in the aging process and contribute to pathophysiology. Our goal was to examine differences in lipid profiles of urinary EVs (uEVs) collected during the inactive and active phases of aged mice and investigate whether these EVs regulate the density of lipid rafts in mouse cortical collecting duct (mpkCCD) principal cells. Here, we demonstrate the epithelial sodium channel (ENaC) inhibitor benzyl amiloride reduced systolic blood pressure in aged male mice during the inactive and active phases. Lipidomics data demonstrate differential enrichment of lipids between the two groups. For example, there are more phosphatidylethanolamine plasmalogens, particularly in the form of alkyl phosphatidylethanolamines, that are enriched in active phase uEVs compared to inactive phase uEVs from the same mice. Amiloride-sensitive transepithelial current increased more in mpkCCD cells challenged with uEVs from the active phase group. Moreover, more ENaC alpha protein was distributed to lipid raft fractions of mpkCCD cells challenged with active phase uEVs. Taken together, the identification of bioactive lipids associated with lipid rafts that are enriched in EVs released during the active phase of aged mice may offer clues to help understand lipid raft organization in recipient principal cells after EV uptake and increased renal ENaC activity, leading to a time-of-day dependent regulation of blood pressure in an aging model.

Increased endothelial sodium channel activity by extracellular vesicles in human aortic endothelial cells: putative role of MLP1 and bioactive lipids.

Extracellular vesicles (EVs) contain biological molecules and are secreted by cells into the extracellular milieu. The endothelial sodium channel (EnNaC) plays an important role in modulating endothelial cell stiffness. We hypothesized EVs secreted from human aortic endothelial cells (hAoECs) positively regulate EnNaC in an autocrine-dependent manner. A comprehensive lipidomic analysis using targeted mass spectrometry was performed on multiple preparations of EVs isolated from the conditioned media of hAoECs or complete growth media of these cells. Cultured hAoECs challenged with EVs isolated from the conditioned media of these cells resulted in an increase in EnNaC activity when compared with the same concentration of media-derived EVs or vehicle alone. EVs isolated from the conditioned media of hAoECs but not human fibroblast cells were enriched in MARCKS-like protein 1 (MLP1). The pharmacological inhibition of the negative regulator of MLP1, protein kinase C, in cultured hAoECs resulted in an increase in EV size and release compared with vehicle or pharmacological inhibition of protein kinase D. The MLP1-enriched EVs increased the density of actin filaments in cultured hAoECs compared with EVs isolated from human fibroblast cells lacking MLP1. We quantified 141 lipids from glycerolipids, glycerophospholipids, and sphingolipids in conditioned media EVs that represented twice the number found in control media EVs. The concentrations of sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine were higher in conditioned media EVs. These results provide the first evidence for EnNaC regulation in hAoECs by EVs and provide insight into a possible mechanism involving MLP1, unsaturated lipids, and bioactive lipids.

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.

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.

Novel effective mosquito larvicide DL-methionine: Lack of toxicity to non-target aquatic organisms.

Mosquito larvicides are an effective tool for reducing numbers of adult females that bite and potentially spread pathogenic organisms. Methionine, an essential amino acid in humans, has been previously demonstrated to be a highly effective larvicide against four (4) mosquito species in three (3) genera, Anopheles, Culex and Aedes. The aim of the present study was to determine the potential impact on non-target aquatic organisms of methionine applied as a mosquito larvicide. DL-methionine concentrations ranging from 0.06% to 1.00% were used; wherein the highest concentration of 1.00% would result in 100% mortality within 48 h in mosquitoes. Acute toxicity assays were conducted in accordance with the US Environmental Protection Agency (US EPA) guidelines for the water flea (Daphnia magna Straus; Cladocera: Daphniidae) and the fathead minnow (Pimephales promelas Rafinesque; Cypriniformes: Cyprinidae). Water fleas and fish were placed directly into the solutions in glass containers and tanks for 48-hours and 96-hours, respectively. When applied within the above-mentioned range of effective mosquito larvicide concentrations, DL-methionine meets US EPA criteria as a "practically non-toxic" pesticide for both species. These results suggest that methionine is a viable alternative to current mosquito larvicide options, which are typically classified as moderately to highly toxic and may be a valuable addition to a mosquito integrated pest management program.

Quantification of steroid hormones in low volume plasma and tissue homogenates of fish using LC-MS/MS.

Quantification of steroid hormones in fish is an important step for toxicology and endocrinology studies. Among the hormone analysis techniques, liquid chromatography tandem mass spectrometry (LC-MS/MS) has widely been used for measuring hormones in various biological samples. Despite all improvements in the technique, detection of several hormones in a low volume of serum or plasma is still challenging. We developed a robust method for simultaneous quantification of 14 steroid hormones including corticosterone, cortisol, 11-ketotestosterone, progesterone, testosterone, 17OH-progesterone, aldosterone, dihydrotestosterone, estrone, 17ß-estradiol, estriol, ethinylestradiol, levonorgestrel and equilin from volumes as low as 10 µL serum or plasma in a short run by LC-MS/MS. The lowest limit of detection in 10 µL serum was 0.012 ng/mL measured for cortisol, progesterone, testosterone, 17OH-progesterone and estrone. Use of high (25 times more) serum volume improved detection limit of hormones by 2-40 times. The method was compared with the radioimmunoassay technique in which testosterone and 17ß-estradiol were highly correlated with R2 of 0.95 and 0.96, respectively. We validated the method by measuring four selected hormones, in low and high plasma volumes of largemouth bass (Micropterus salmoides). In addition, we developed a method to quantify hormones in whole body fish homogenates of small fish and compared the values to plasma concentrations, using fathead minnow (Pimephales promelas). Calculated concentrations of the hormones in plasma were consistent with those in the homogenate and 11-ketotestosterone and 17ß-estradiol were significantly different in males and females. The ability to measure hormones from whole body homogenates was further evaluated in two model small fish species, zebrafish (Danio rerio) and juvenile silverside (Menidia beryllina). These results suggest that whole tissue homogenate is a reliable alternative for hormone quantification when sufficient plasma is not available.

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.

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.

Computational in Vitro Toxicology Uncovers Chemical Structures Impairing Mitochondrial Membrane Potential.

Technological advances in molecular biology have enabled high-throughput screening (HTS) of large chemical libraries. These approaches have provided valuable toxicity data for many physiological responses, including mitochondrial dysfunction. While several quantitative structure-activity relationship (QSAR) models have been developed for mitochondrial dysfunction, there remains a need to identify specific chemical features associated with this response. Thus, the objective of this study was to identify chemical structures associated with altered mitochondrial membrane potential (MMP). To achieve this, we developed computational models to examine the relationship between specific chemotypes (e.g., ToxPrints) and bioactivity in ToxCast/Tox21 HTS assays for altered MMP. The analysis revealed that the "bond:COH_alcohol_aromatic", "bond:COH_alcohol_aromatic_phenol", and "ring:aromatic_benzene" ToxPrints had the highest average correlations (phi coefficient) with ToxCast/Tox21 assay component endpoints for decreased MMP. These structures also constituted a "core" group of ToxPrints for decreased MMP in a force-directed network model and were the most important chemotypes in a random forest (RF) classification model for the "TOX21_MMP_ratio_down" assay component endpoint. Based on multiple lines of evidence, these structures, which are present in numerous chemicals (e.g., aromatic hydrocarbons, pesticides, and industrial chemicals) are likely involved in mitochondrial dysfunction. Because of the hierarchical structure of ToxPrints, these chemotypes were highly convergent and, when excluded from training data, had limited effects on the classification performance as related structures compensated for predictor loss. These results highlight the flexibility of the RF algorithm and ToxPrints for QSAR modeling, which is useful to identify chemicals affecting mitochondrial function.

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.

Hydrogen Peroxide Stimulates Exosomal Cathepsin B Regulation of the Receptor for Advanced Glycation End-Products (RAGE).

Exosomes are nano-sized vesicles that are secreted into the extracellular environment. These vesicles contain various biological effector molecules that can regulate intracellular signaling pathways in recipient cells. The aim of this study was to examine a correlation between exosomal cathepsin B activity and the receptor for advanced glycation end-products (RAGE). Type 1 alveolar epithelial (R3/1) cells were treated with or without hydrogen peroxide and exosomes isolated from the cell conditioned media were characterized by NanoSight analysis. Lipidomic and proteomic analysis showed exosomes released from R3/1 cells exposed to oxidative stress induced by hydrogen peroxide or vehicle differ in their lipid and protein content, respectively. Cathepsin B activity was detected in exosomes isolated from hydrogen peroxide treated cells. The mRNA and protein expression of RAGE increased in cultured R3/1 cells treated with exosomes containing active cathepsin B while depletion of exosomal cathepsin B attenuated RAGE mRNA and protein expression. These results suggest exosomal cathepsin B regulates RAGE in type 1 alveolar cells under conditions of oxidative stress. J. Cell. Biochem. 119: 599-606, 2018. © 2017 Wiley Periodicals, Inc.

Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells.

Exosomes are endosome-derived nanovesicles that are involved in cellular communication and signaling. Exosomes are produced by epithelial cells and are found in biologic fluids including blood and urine. The packaged material within exosomes includes proteins and lipids, but the molecular comparison within exosome subtypes is largely unknown. The purpose of this study was to investigate differences between exosomes derived from the apical plasma membrane and basolateral plasma membrane of polarized murine cortical collecting duct principal cells. Nanoparticle tracking analysis showed that the size and concentration of apical and basolateral exosomes remained relatively stable across 3 different temperatures (23, 37, and 42°C). Liquid chromatography-tandem mass spectrometry analysis revealed marked differences between the proteins packaged within the two types of exosomes from the same cells. Several proteins expressed at the inner leaflet of the plasma membrane, including α-actinin-1, moesin, 14-3-3 protein ζ/δ, annexin A1/A3/A4/A5/A6, clathrin heavy chain 1, glyceraldehyde-3-phosphate dehydrogenase, α-enolase, filamin-A, and heat shock protein 90, were identified in samples of apical plasma membrane-derived exosomes, but not in basolateral plasma membrane exosomes from mouse cortical collecting duct cells. In addition to differences at the protein level, mass spectrometry-based shotgun lipidomics analysis showed significant differences in the lipid classes and fatty acid composition of the two types of exosomes. We found higher levels of sphingomyelin and lower levels of cardiolipin, among other phospholipids in the apical plasma membrane compared to the basolateral plasma membrane exosomes. The molecular analyses of exosome subtypes presented herein will contribute to our understanding of exosome biogenesis, and the results may have potential implications for biomarker discovery.-Dang, V. D., Jella, K. K., Ragheb, R. R. T., Denslow, N. D., Alli, A. A. Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells.