Lipidomics and plasma hormone analysis differentiate reproductive and pregnancy statuses in Florida manatees (Trichechus manatus latirostris).

Florida manatees (Trichechus manatus latirostris) are protected as a threatened species, and data are lacking regarding their reproductive physiology. This study aimed to (1) quantify plasma steroid hormones in Florida manatees from two field sites, Crystal River and Indian River Lagoon, at different gestational stages and to (2) identify individual lipids associated with pregnancy status. Ultra-high performance liquid chromatography-tandem mass spectrometric analysis was used to measure plasma steroid hormones and lipids. Pregnant female manatees were morphometrically distinct from male and non-pregnant female manatees, characterized by larger body weight and maximal girth. Progesterone concentrations in manatees were also elevated during early gestation versus late gestation. Cholesterol, an important metabolic lipid, and precursor for reproductive steroids, was not different between groups. Mass spectrometry quantified 949 lipids. Plasma concentrations of glycerophospholipids, glycerolipids, sphingolipids, acylcarnitines, and cholesteryl esters were associated with pregnancy status in the Florida manatee. Most of the lipid species associated with pregnancy were triacylglycerides, phosphatidylethanolamines, and ether-linked phosphatidylethanolamines, which may serve as energy sources for fetal development. This research contributes to improving knowledge of manatee reproductive physiology by providing data on plasma steroid hormones relative to reproductive status and by identifying plasma lipids that may be important for pregnancy. Elucidation of lipid species directly associated with pregnancy has the potential to serve as a diagnostic approach to identify pregnant individuals in fresh and archived samples. These biochemical and morphometric indicators of reproductive status advance the understanding of manatee physiology.

An XAS study of Hg(II) sorption to Al-based drinking water treatment residuals.

Drinking water treatment residuals (DWTRs) are produced from the coagulation and flocculation processes in conventional drinking water treatment. The abundant metal oxide content of these materials resulting from the use of coagulants, like alum and ferric chloride, has driven strong research interest into the reuse of DWTRs as sorptive materials. Using a suite of aluminum-based DWTRs, we provide new insights into Hg(II) sorption mechanisms. Experiments performed at circum-neutral pH show that sorption capacities are related to the amount of organic carbon/matter present in DWTRs. We found that carbon rich samples can scavenge about 9000 mg/kg of Hg, in contrast to 2000 mg/kg for lime based DWTRs. X-ray absorption spectroscopy (XAS) at the Hg L3 edge further characterizes mercury coordination. X-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) results point to a partial association of mercury with sulfur at low mass loadings, transitioning to a full association with oxygen/carbon at higher concentrations of sorbed Hg(II) and in DWTRs with limited sulfur content. These results suggest that sorption of Hg(II) is primarily controlled by the carbon/organic matter fraction of DWTRs, but not by the coagulants.

Trace elements and lipidomic datasets of stranding samples in the blubber of Turpsios truncatus from the Yucatan coast: Association with biological features.

The Yucatan coastal zone is an area that contributes to many anthropogenic activities resulting in substantial contamination (metals, pesticides) in aquatic organisms. The dolphin is an excellent sentinel animal used in studying contamination in this area. Some substances found in dolphins have been identified as toxic causing alterations in the properties of membranes and produce lipid peroxidation especially heavy metals. The dataset presented here is associated with the research article paper entitled "Trace element and lipidomic analysis of bottlenose dolphin blubber from the Yucatan coast: Lipid composition relationships". In this article, we presented the trace element concentrations found in blubber and their comparison with other studies performed in mammal marine organisms. Lipidomic characterization of bottlenose dolphin blubber and their association with trace elements and the differences related to biological characteristics were presented. This data provides a correlation analysis between trace element concentrations, lipid species and body length and the lipid differences related to biological characteristics such as growth stage, stranding code, and the presence of stomach contents. We used Spearman correlation analysis to identify the association with body length, trace elements and lipids. Wilcoxon rank-sum test was used to determine differences in lipids related to stranding code (3: moderate decomposition, 4: advanced decomposition), growth stage (juveniles and adults) and whether they showed presence of stomach contents or not. The data indicates that Cr, Cd and Zn concentrations were higher compared to concentrations found in blubbler of T. truncatus from other studies (See Table 3). Cr, Co, As and Cd were found in higher concentration in larger organisms compared to smaller ones. The results of correlation between lipids and body length showed a decrease in some ceramides (CER, DCER, HCER), sterols (CE), glycerolipids (TAG, DAG) and phosphatidylethanolamines (LPE, PE) in larger dolphins (Table 4). Dolphins with advanced decomposition (code 4) showed lower concentrations of phosphatidylethanolamines (PE) compared with organisms with moderate decomposition (code 3). Organisms with empty stomachs showed higher concentrations of phosphoethanolamines suggesting a preferential metabolism of energy-rich lipids over structural lipids. The information in these datasets may contribute to understanding the potential associations of trace elements, lipids and their associations with biological characteristics.

Decreased MARCKS Protein Expression in Kidney Cortex Membrane Fractions of Cathepsin B Knockout Mice Is Associated with Reduced Lysophosphatidylcholine and Protein Kinase C Activity.

Cathpesin B is a multi-functional protease that plays numerous roles in physiology and pathophysiology. We hypothesized that actin cytoskeleton proteins that are substrates of cathepsin B, various lipids, and kinases that are regulated by lipids would be down-regulated in the kidney of cathepsin B knockout mice. Here, we show by Western blot and densitometric analysis that the expression and proteolysis of the actin cytoskeleton proteins myristoylated alanine-rich C-kinase substrate (MARCKS) and spectrin are significantly reduced in kidney cortex membrane fractions of cathepsin B knockout mice compared to C57B6 wild-type control mice. Lipidomic results show that specific lipids are increased while other lipids, including lysophosphatidylcholine (LPC) species LPC (16:0), LPC (18:0), LPC (18:1), and LPC (18:2), are significantly decreased in membrane fractions of the kidney cortex from Cathepsin B null mice. Protein Kinase C (PKC) activity is significantly lower in the kidney cortex of cathepsin B knockout mice compared to wild-type mice, while calcium/calmodulin-dependent protein kinase II (CaMKII) activity and phospholipase D (PLD) activity are comparable between the two groups. Together, these results provide the first evidence of altered actin cytoskeleton organization, membrane lipid composition, and PKC activity in the kidneys of mice lacking cathepsin B.

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.

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.

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.

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.

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.

Putative high-level toxicity pathways based on evidence of brevetoxin immunotoxicity in marine fauna.

Red tide events, caused by a toxin producing dinoflagellate, Karenia brevis, occur annually in Florida and Texas. These events lead to health risks for both humans and wildlife that utilize coastal environments. Brevetoxins, potent lipophilic neurotoxins produced by K. brevis, modulate immune responses in laboratory studies with model organisms and in the natural environment in both humans and wildlife. Studies show that brevetoxins activate immune cells, stimulate production of gamma-globulins, cytokines, and neutrophils, modulate lysozyme activity, induce apoptosis, and modulate lymphocyte proliferation in marine species. The objective of this review was to summarize brevetoxin-induced immunotoxicity in marine animals based on available peer-reviewed literature about K. brevis blooms and associated health concerns and propose putative toxicity pathways. This review identifies knowledge gaps within current brevetoxin induced immunotoxicity research, including assessing the long-term impacts of brevetoxin exposure, elucidating the mechanistic linkages between brevetoxins and immune cells, and evaluating repeated and chronic versus acute brevetoxin exposure implications on overall organismal health. The putative immunotoxicity pathways based on evidence from brevetoxin-exposure in marine fauna described in this review represent a useful tool and resource for researchers, wildlife managers, and policy makers. This review and proposed putative immunotoxicity pathways will inform decisions regarding the risks of algal blooms, as it pertains to marine animal health.

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.

Endocrine, immune and renal toxicity in male largemouth bass after chronic exposure to glyphosate and Rodeo®.

Glyphosate is the most used herbicide worldwide, with no historical comparison. It is used for genetically modified crops, and particularly in Florida, it is used as a sugar cane ripener. An aquatic formulation (Rodeo®) is used to treat aquatic weeds in waterbodies and drainage canals. Because of its extended use, glyphosate can run off or be sprayed directly into waterbodies, and chronically expose aquatic wildlife. Exposure in animal models has been associated with kidney and liver damage and glyphosate has been suggested as an endocrine disruptor. We exposed adult male largemouth bass for 21 days to two doses of glyphosate and Rodeo® (chemically equivalent concentration of glyphosate) at 0.5 mg L-1 and 10 mg L-1 and to a clean water control (n=4 fish/tank in quadruplicate). Concentrations during the experiment were corroborated with UHPLC-MS/MS. Total RNA was isolated from the trunk kidney and head kidney. RNA-seq was performed for the high doses compared to controls. Transcripts were analyzed with fish and mammalian pathway analyses software. Transcripts mapped to Zebrafish metabolic pathways using PaintOmics showed steroid hormone biosynthesis in the trunk kidney as the most significantly enriched pathway. Steroid hormones were measured in plasma by UHPLC-MS/MS. Total androgens were significantly reduced at 0.5 mg L-1 of glyphosate and at equivalent concentrations in Rodeo® compared to controls. 11-ketotestosterone and estrone concentrations were significantly reduced in all doses. A gene involved in the conversion of testosterone to 11-ketotestosterone was down-regulated by glyphosate. Using the mammalian pathway analysis algorithm, cellular processes associated with T-cell activation/development and intracellular pH were significantly enriched in the trunk kidney by glyphosate and Rodeo® exposure. Endocrine disruption was corroborated at the hormone and gene expression levels. Rodeo® and glyphosate share gene expression pathways, however, Rodeo® had more pronounced effects in largemouth bass.

Trace element and lipidomic analysis of bottlenose dolphin blubber from the Yucatan coast: Lipid composition relationships.

Bottlenose dolphins (Tursiops truncatus) are found in coastal and estuarine ecosystems where they are in continuous contact with multiple abiotic and biotic stressors in the environment. Due to their role as predators, they can bioaccumulate contaminants and are considered sentinel organisms for monitoring the health of coastal marine ecosystems. The northern zonal coast of the Yucatan peninsula of Mexico has a high incidence of anthropogenic activities. The principal objectives of this study were two-fold: 1) to determine the presence of trace metals and their correlation with lipids in bottlenose dolphin blubber, and 2) to use a lipidomics approach to characterize their biological responses. Levels of trace elements (Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Cd, Pb) were analyzed using ICP-MS and lipids were measured using a targeted lipidomics approach with LC-MS/MS. Spearman correlation analysis was used to identify associations between lipids and trace elements. The influences of gender, stranding codes, presence of stomach content, growth stages and body length were also analyzed. Blubber lipid composition was dominated by triacylglycerols (TAG). Our results demonstrated the presence of heavy-metal elements such as Cd and As, which were correlated with different lipid species, mainly the ceramides and glycerophospholipids, respectively. Organisms with Cd showed lower concentrations of ceramides (CER, HCER and DCER), TAG and cholesteryl esters (CE). Trace elements Cr, Co, As and Cd increased proportionately with body length. This study provides a novel insight of lipidomic characterization and correlations with trace elements in the bottlenose dolphin which might contribute to having a better understanding of the physiological functions and the risks that anthropogenic activities can bring to sentinel organisms from coastal regions.

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.

Human Alpha-1 Antitrypsin Attenuates ENaC and MARCKS and Lowers Blood Pressure in Hypertensive Diabetic db/db Mice.

Hypertension may develop before or after the onset of diabetes and it is known to increase the risk of developing diabetic nephropathy. Alpha-1 antitrypsin (AAT) is a multi-functional protein with beneficial effects in various diseases but its role in reducing blood pressure in the diabetic kidney has not been thoroughly studied. Like blood pressure, epithelial sodium channels (ENaC) and its adaptor protein myristoylated alanine-rich C-kinase substrate (MARCKS) are regulated by circadian rhythms. Our hypothesis is that administration of human AAT (hAAT) reduces blood pressure in hypertensive diabetic mice by attenuating membrane expression of ENaC and its association with the actin cytoskeleton. First, we show hAAT administration results in reduced blood pressure in diabetic db/db mice compared to vehicle treatment in both the inactive and active cycles. Western blotting and immunohistochemistry analyses showed a reduction of ENaC and the actin cytoskeleton protein, MARCKS in the kidneys of diabetic db/db mice treated with hAAT compared to vehicle. hAAT treatment resulted in elevated amounts of extracellular vesicles present in the urine of diabetic db/db mice compared to vehicle treatment both in the inactive and active cycles. Multiple hexosylceramides, among other lipid classes increased in urinary EVs released from hAAT treated hypertensive diabetic mice compared to vehicle treated mice. Taken together, these data suggest hAAT treatment could normalize blood pressure in the diabetic kidney in a mechanism involving attenuation of renal ENaC and MARCKS protein expression and possibly ceramide metabolism to hexosylceramide in kidney cells.

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)).

Tempol Alters Urinary Extracellular Vesicle Lipid Content and Release While Reducing Blood Pressure during the Development of Salt-Sensitive Hypertension.

Salt-sensitive hypertension resulting from an increase in blood pressure after high dietary salt intake is associated with an increase in the production of reactive oxygen species (ROS). ROS are known to increase the activity of the epithelial sodium channel (ENaC), and therefore, they have an indirect effect on sodium retention and increasing blood pressure. Extracellular vesicles (EVs) carry various molecules including proteins, microRNAs, and lipids and play a role in intercellular communication and intracellular signaling in health and disease. We investigated changes in EV lipids, urinary electrolytes, osmolality, blood pressure, and expression of renal ENaC and its adaptor protein, MARCKS/MARCKS Like Protein 1 (MLP1) after administration of the antioxidant Tempol in salt-sensitive hypertensive 129Sv mice. Our results show Tempol infusion reduces systolic blood pressure and protein expression of the alpha subunit of ENaC and MARCKS in the kidney cortex of hypertensive 129Sv mice. Our lipidomic data show an enrichment of diacylglycerols and monoacylglycerols and reduction in ceramides, dihydroceramides, and triacylglycerols in urinary EVs from these mice after Tempol treatment. These data will provide insight into our understanding of mechanisms involving strategies aimed to inhibit ROS to alleviate salt-sensitive hypertension.

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.

Practical Euthanasia Method for Common Sea Stars (Asterias rubens) That Allows for High-Quality RNA Sampling.

Sea stars in research are often lethally sampled without available methodology to render them insensible prior to sampling due to concerns over sufficient sample quality for applied molecular techniques. The objectives of this study were to describe an inexpensive and effective two-step euthanasia method for adult common sea stars (Asterias rubens) and to demonstrate that high-quality RNA samples for further use in downstream molecular analyses can be obtained from pyloric ceca of MgCl2-immersed sea stars. Adult common sea stars (n = 15) were immersed in a 75 g/L magnesium chloride solution until they were no longer reactive to having their oral surface tapped with forceps (mean: 4 min, range 2-7 min), left immersed for an additional minute, and then sampled with sharp scissors. RNA from pyloric ceca (n = 10) was isolated using a liquid-liquid method, then samples were treated with DNase and analyzed for evaluation of RNA integrity number (RIN) for assessment of the quantity and purity of intact RNA. Aversive reactions to magnesium chloride solution were not observed and no sea stars regained spontaneous movement or reacted to sampling. The calculated RIN ranged from 7.3-9.8, demonstrating that the combination of animal welfare via the use of anesthesia and sampling for advanced molecular techniques is possible using this low-cost technique.

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.