Comparative sub-lethal effects of polybrominated diphenyl ethers following simulated maternal transfer and dietary exposure in two species of turtles

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

TMEM63C, a Potential Novel Target for Albuminuria Development, Is Regulated by MicroRNA-564 and Transforming Growth Factor beta in Human Renal Cells

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

MiRNA-29b and miRNA-497 Modulate the Expression of Carboxypeptidase X Member 2, a Candidate Gene Associated with Left Ventricular Hypertrophy

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

Published at Univ. of Calif

AbstrAct: New regulatory restrictions have been placed on the use of some second-generation anticoagulant rodenticides in the United States, and in some situations this action may be offset by expanded use of first-generation compounds. We have recently conducted several studies with captive adult American kestrels and eastern screech-owls examining the toxicity of diphacinone (DPN) using both acute oral and short-term dietary exposure regimens. Diphacinone evoked overt signs of intoxication and lethality in these raptors at exposure doses that were 20 to 30 times lower than reported for traditionally used wildlife test species (mallard and northern bobwhite). Sublethal exposure of kestrels and owls resulted in prolonged clotting time, reduced hematocrit, and/or gross and histological evidence of hemorrhage at daily doses as low as 0.16 mg DPN/kg body weight. Findings also demonstrated that DPN was far more potent in short-term 7-day dietary studies than in single-day acute oral exposure studies. Incorporating these kestrel and owl data into deterministic and probabilistic risk assessments indicated that the risks associated with DPN exposure for raptors are far greater than predicted in analyses using data from mallards and bobwhite. These findings can assist natural resource managers in weighing the costs and benefits of anticoagulant rodenticide use in pest control and eradication programs

Current ecotoxicity testing needs among selected U.S. federal agencies

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

Accumulation and Maternal Transfer of Polychlorinated Biphenyls in Snapping Turtles of the Upper Hudson River, New York, USA

We conducted field studies over three years to assess body burdens and maternal transfer of polychlorinated biphenyls (PCBs) as well as indices of sexual dimorphism in snapping turtles (Chelydra serpentina) of the upper Hudson River (NY, USA.) We collected adult turtles in areas known to be contaminated with PCBs and in nearby reference areas for measurement of body size, precloacal length, and penis size. We analyzed PCB concentrations in eggs collected over three years and in whole blood from adults in one year. Total PCB concentrations (mean +/- standard error) in eggs were 2,800 +/- 520 and 59 +/- 5 ng/g wet weight in the contaminated area and the reference area, respectively. Eggs from the contaminated area were significantly enriched in tri-, penta-, and hepta-PCBs relative to the reference area. Blood from adults in the contaminated area averaged 475 +/- 200 and 125 +/- 34 ng/g wet weight for males and females, respectively. In the reference area, blood PCB concentrations were 7 +/- 3 and 4 +/- 1 ng/g wet weight for males and females, respectively. Significant positive relationships were found between carapace length and blood PCB concentration for both sexes in the contaminated area; however, only a marginal relationship was found between female carapace length and concentration of PCBs in their eggs. Our results suggest that PCB contamination of the upper Hudson River presents risks of establishing high body burdens and of maternal transfer of PCBs to eggs, although our measures of gross morphology revealed no discernable expression of abnormal sexual development or reproduction

Assessment of toxicity and potential risk of the anticoagulant rodenticide diphacinone using Eastern screech-owls (\u3ci\u3eMegascops asio\u3c/i\u3e)

In the United States, new regulatory restrictions have been placed on the use of some second-generation anticoagulant rodenticides. This action may be offset by expanded use of first-generation compounds (e.g., diphacinone; DPN). Single-day acute oral exposure of adult Eastern screech-owls (Megascops asio) to DPN evoked overt signs of intoxication, coagulopathy, histopathological lesions (e.g., hemorrhage, hepatocellular vacuolation), and/ or lethality at doses as low as 130 mg/kg body weight, although there was no dose–response relation. However, this single-day exposure protocol does not mimic the multiple-day field exposures required to cause mortality in rodent pest species and non-target birds and mammals. In 7-day feeding trials, similar toxic effects were observed in owls fed diets containing 2.15, 9.55 or 22.6 ppm DPN, but at a small fraction (\u3c5%) of the acute oral dose. In the dietary trial, the average lowest-observed-adverse-effectlevel for prolonged clotting time was 1.68 mg DPN/kg owl/week (0.24 mg/kg owl/day; 0.049 mg/owl/day) and the lowest lethal dose was 5.75 mg DPN/kg owl/week (0.82 mg/kg owl/day). In this feeding trial, DPN concentration in liver ranged from 0.473 to 2.21 µg/g wet weight, and was directly related to the daily and cumulative dose consumed by each owl. A probabilistic risk assessment indicated that daily exposure to as little as 3–5 g of liver from DPN-poisoned rodents for 7 days could result in prolonged clotting time in the endangered Hawaiian shorteared owl (Asio flammeus sandwichensis) and Hawaiian hawk (Buteo solitarius), and daily exposure to greater quantities (9–13 g of liver) could result in low-level mortality. These findings can assist natural resource managers in weighing the costs and benefits of anticoagulant rodenticide use in pest control and eradication programs

Improving the Environmental Risk Assessment of Substances of Unknown or Variable Composition, Complex Reaction Products, or Biological Materials (UVCBs).

Substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs) pose unique risk assessment challenges to regulators and to product registrants. These substances can contain many constituents, sometimes partially unknown and/or variable, depending on fluctuations in their source material and/or manufacturing process. International regulatory agencies have highlighted the difficulties in characterizing UVCBs and assessing their toxicity and environmental fate. Several industrial sectors have attempted to address these issues by developing frameworks and characterization methods. Based on the output of a 2016 workshop, this critical review examines current practices for UVCB risk assessment and reveals a need for a multipronged and transparent approach integrating whole-substance and constituent-based information. In silico tools or empirical measurements can provide information on discrete and/or blocks of UVCB constituents with similar hazard properties. Read-across and/or whole-substance toxicity and fate testing using adapted emerging methods can provide whole-substance information. Continued collaboration of stakeholders representing government, industry, and academia will facilitate the development of practical testing strategies and guidelines for addressing regulatory requirements for UVCBs. Environ Toxicol Chem 2020;39:2097–2108