A recent Canada goose (Branta canadensis) die-off at a petroleum refinery fly ash pond in Delaware was attributed to vanadium (V) toxicity. Because of the paucity of V toxicity data for wild birds, a ...series of studies was undertaken using the forms of V believed to have resulted in this incident. In 7-d single oral dose trials with mallard drakes (Anas platyrhynchos), the estimated median lethal dose (LD50) for vanadium pentoxide was 113 mg/kg body weight, while the LD50 for sodium metavanadate was 75.5 mg/kg. Sodium metavanadate was found to be even more potent (LD50 = 37.2 mg/kg) in male Canada geese. The most distinctive histopathological lesion of both forms of V was lympho-granulocytic enteritis with hemorrhage into the intestinal lumen. Vanadium accumulation in liver and kidney was proportional to the administered dose, and predictive analyses based on these data suggest that V concentrations of 10 μg/g dry weight (dw) in liver and 25 μg/g dw in kidney are associated with mortality (>90% confidence that exposure is >LD50) in mallards acutely exposed to sodium metavanadate. Chronic exposure to increasing dietary concentrations of sodium metavanadate (38.5 to 2651 ppm) over 67 d resulted in V accumulation in liver and kidney (25.2 and 13.6 μg/g dw, respectively), mild intestinal hemorrhage, blood chemistry changes, and evidence of hepatic oxidative stress in mallards, although some of these responses may have been confounded by food avoidance and weight loss. Dietary exposure of mallards to 250 ppm sodium metavanadate for 4 wk resulted in modest accumulation of V in liver and kidney (<5 μg/g dw) and mild intestinal hemorrhage. Based on these data and other observations, it is unlikely that chronic low-level dietary exposure to V poses a direct lethal hazard to wildlife. However, point sources, such as the V-laden fly ash pond encountered by geese at the petroleum refinery in Delaware, may pose a significant hazard to water birds.
U.S. regulatory and research agencies use ecotoxicity test data to assess the hazards associated with substances that may be released into the environment, including but not limited to industrial ...chemicals, pharmaceuticals, pesticides, food additives, and color additives. These data are used to conduct hazard assessments and evaluate potential risks to aquatic life (e.g., invertebrates, fish), birds, wildlife species, or the environment. To identify opportunities for regulatory uses of non-animal replacements for ecotoxicity tests, the needs and uses for data from tests utilizing animals must first be clarified. Accordingly, the objective of this review was to identify the ecotoxicity test data relied upon by U.S. federal agencies. The standards, test guidelines, guidance documents, and/or endpoints that are used to address each of the agencies’ regulatory and research needs regarding ecotoxicity testing are described in the context of their application to decision-making. Testing and information use, needs, and/or requirements relevant to the regulatory or programmatic mandates of the agencies taking part in the Interagency Coordinating Committee on the Validation of Alternative Methods Ecotoxicology Workgroup are captured. This information will be useful for coordinating efforts to develop and implement alternative test methods to reduce, refine, or replace animal use in chemical safety evaluations.
•This review summarizes the ecotoxicity data needs of six U.S. federal agencies.•It identifies 87 tests utilizing vertebrate and invertebrate species.•It identifies challenges related to cross-taxa and interspecies extrapolation.•This review will inform development and implementation of non-animal methods.
Polybrominated diphenyl ethers (PBDEs) are contaminants of concern as their concentrations have been increasing in the environment in recent years. This project sought to determine the effects of ...embryonic and dietary exposure to two PBDE congeners (BDE-47 and BDE-99) on a suite of endpoints including development, growth, metabolic rate, behavior and thyroid function of embryonic, hatchling and juvenile red-eared slider turtles (Trachemys scripta elegans) and snapping turtles (Chelydra serpentina). Topical egg dosing was employed for embryonic exposures; transfer efficiencies across the red-eared slider eggshell were 25.82 % and 9.87 % for BDE-47 and -99 respectively whereas they were 31.30 % and 12.53 % across the snapping turtle eggshell. These transfer efficiencies were taken into account when topically dosing eggs in a subsequent exposure-response study of embryonic exposure to BDE-47. Sodium perchlorate was included as a positive control for thyroid disruption in the embryonic exposure study. Embryonic exposure to five concentrations of BDE-47 (target exposure range from 40 ng/g–1000 ng/g ww) led to patterns of elevated standard metabolic rate in hatchlings of both species and increased liver weights in snapping turtles. No impacts were found on incubation time, hatching success or total glandular thyroxine (T4) of the hatchlings. Embryonically exposed red-eared slider juveniles displayed delayed righting response behavior and both species showed patterns of reduced thyroid size and T4 following exposure. Sodium perchlorate had significant impacts on survival, incubation time, volume of the external yolk and T4 in the red-eared slider hatchlings. In snapping turtles, sodium perchlorate exposures led to impacts on hatching success, standard metabolic rate, liver and thyroid sizes, and T4. A separate study of dietary exposure to BDE-47 and BDE-99 (2055 ng/g and 1425 ng/g respectively) over a six month period in both species revealed altered behavior and decreased T4 in red-eared sliders and elevated standard metabolic rate in snapping turtles. Embryonic and dietary exposures to BDE-47 and -99 can elicit a suite of impacts potentially related to thyroid system function and are cause for concern, but the observed species specific differences in response require further investigation.
Human podocytes (hPC) play an important role in the pathogenesis of renal diseases. In this context, angiotensin II (Ang II) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) ...play a crucial role in podocyte injury. Recently, transmembrane protein (Tmem) 63c, a member of the Tmem-family was found to be expressed in kidney and associated with podocyte function. In this study, we analysed the expression regulation and functional impact of Tmem63c on cell viability and apoptosis in hPC in the context of Ang II activation.
Expression of Tmem63c in response to Ang II and the NFκB inhibitor Bay 11–7082 was analysed by Real-Time PCR and Western blotting. Cellular functions were determined by functional assays.
We found Ang II to induce Tmem63c expression in hPC in a concentration-dependent manner. Inhibition of NFκB by Bay 11–7082 reduced basal as well as Ang II-induced Tmem63c expression. SiRNA-mediated down-regulation of Tmem63c diminished cell viability and protein kinase B (Akt) signaling and increased cell apoptosis of resting as well as Ang II-activated hPC.
These data show that Ang II induced the expression of Tmem63c in hPC, possibly via NFκB-dependent mechanisms. Moreover, down-regulation of Tmem63c was associated with reduced cell viability, indicating Tmem63c to be a potential pro-survival factor in hPC.
•Angiotensin II induced Tmem63c expression in human podocytes•NFκB Inhibition affects Tmem63c expression in human podocytes•Tmem63c down-regulation reduced viability and increased apoptosis of podocytes.•Tmem63c is a potential pro-survival factor in human podocytes.
Left ventricular hypertrophy (LVH) is a major risk factor for adverse cardiovascular events. Recently, a novel candidate gene encoding the carboxypeptidase X member 2 (CPXM2) was found to be ...associated with hypertension-induced LVH. CPXM2 belongs to the M14 family of metallocarboxypeptidases, yet it lacks detectable enzyme activity, and its function remains unknown. Here, we investigated the impact of micro (mi)RNA-29b, miRNA-195, and miRNA-497 on the posttranscriptional expression control of CPXM2. Candidate miRNAs for CPXM2 expression control were identified in silico. CPXM2 expression in rat cardiomyocytes (H9C2) was characterized via real-time PCR, Western blotting, and immunofluorescence. Direct miRNA/target mRNA interaction was analysed by dual luciferase assay. CPXM2 was expressed in H9C2 and co-localised with z-disc associated protein PDZ and LIM domain 3 (Pdlim3). Transfection of H9C2 with miRNA-29b, miRNA-195, and miRNA-497 led to decreased levels of CPXM2 mRNA and protein, respectively. Results of dual luciferase assays revealed that miRNA-29b and miRNA-497, but not miRNA-195, directly regulated CPXM2 expression on a posttranscriptional level via binding to the 3'UTR of CPXM2 mRNA. We identified two miRNAs capable of the direct posttranscriptional expression control of CPXM2 expression in rat cardiomyocytes. This novel data may help to shed more light on the-so far-widely unexplored expression control of CPXM2 and its potential role in LVH.
Introduction: Transmembrane protein (TMEM) 63C is a member of the TMEM gene family and was recently linked to glomerular filtration barrier function and albuminuria. Its molecular function and ...expression regulation are largely unknown. Objective: In this study, we set out to characterize the regulating impact of microRNAs (miRNAs) such as miRNA-564 (miR-564) on TMEM63C expression in renal cells. Also, we examined the influence of transforming growth factor beta (TGF-ß) on TMEM63C expression and the potential impact of TMEM63C inhibition on epithelial-mesenchymal transition (EMT) in renal cells and on cell viability in human embryonic kidney 293 cells (HEK 293). Methods: Expression analyses were done using real-time PCR and Western blot. Dual luciferase assay was performed to determine the miRNA-mediated expression control. Cell viability was assessed via trypan blue exclusion staining. Results and Conclusions: MiR-564 reduced TMEM63C expression in HEK 293 and human podocytes (hPC). The treatment of renal cells with TGF-ß led to an increased expression of TMEM63C. Moreover, a reduced TMEM63C expression was associated with a changed ratio of EMT marker proteins such as α-smooth muscle actin versus E-cadherin in HEK 293 and decreased nephrin expression in hPC. In addition, cell viability was reduced upon inhibition of TMEM63C expression in HEK 293. This study demonstrates first mechanisms involved in TMEM63C expression regulation and a link to EMT in renal cells.
Human podocytes (hPC) are essential for maintaining normal kidney function and dysfunction or loss of hPC play a pivotal role in the manifestation and progression of chronic kidney diseases including ...diabetic nephropathy. Previously, α-Lipoic acid (α-LA), a licensed drug for treatment of diabetic neuropathy, was shown to exhibit protective effects on diabetic nephropathy in vivo. However, the effect of α-LA on hPC under non-diabetic conditions is unknown. Therefore, we analyzed the impact of α-LA on cell viability and expression of nephrin and zinc finger protein 580 (ZNF580) in normal hPC in vitro.
Protein analyses were done via Western blot techniques. Cell viability was determined using a functional assay. hPC viability was dynamically modulated via α-LA stimulation in a concentration-dependent manner. This was associated with reduced nephrin and ZNF580 expression and increased nephrin phosphorylation in normal hPC. Moreover, α-LA reduced nephrin and ZNF580 protein expression via 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) inhibition. These data demonstrate that low α-LA had no negative influence on hPC viability, whereas, high α-LA concentrations induced cytotoxic effects on normal hPC and reduced nephrin and ZNF580 expression via NF-κB inhibition. These data provide first novel information about potential cytotoxic effects of α-LA on hPC under non-diabetic conditions.
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