Using in vitro data to derive acceptable exposure levels: A case study on PBDE developmental neurotoxicity.

BACKGROUND: Current acceptable chemical exposure levels (e.g., tolerable daily intake) are mainly based on animal experiments, which are costly, time-consuming, considered non-ethical by many, and may poorly predict adverse outcomes in humans. OBJECTIVE: To evaluate a method using human in vitro data and biological modeling to calculate an acceptable exposure level through a case study on 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) developmental neurotoxicity (DNT). METHODS: We reviewed the literature on in vitro assays studying BDE-47-induced DNT. Using the most sensitive endpoint, we derived a point of departure using a mass-balance in vitro disposition model and benchmark dose modeling for a 5% response (BMC05) in cells. We subsequently used a pharmacokinetic model of gestation and lactation to estimate administered equivalent doses leading to four different metrics of child brain concentration (i.e., average prenatal, average postnatal, average overall, and maximum concentration) equal to the point of departure. The administered equivalent doses were translated into tolerable daily intakes using uncertainty factors. Finally, we calculated biomonitoring equivalents for maternal serum and compared them to published epidemiological studies of DNT. RESULTS: We calculated a BMC05 of 164 µg/kg of cells for BDE-47 induced alteration of differentiation in neural progenitor cells. We estimated administered equivalent doses of 0.925-3.767 µg/kg/day in mothers, and tolerable daily intakes of 0.009-0.038 µg/kg/day (composite uncertainty factor: 100). The lowest derived biomonitoring equivalent was 19.75 ng/g lipids, which was consistent with reported median (0.9-23 ng/g lipids) and geometric mean (7.02-26.9 ng/g lipids) maternal serum concentrations from epidemiological studies. CONCLUSION: This case study supports using in vitro data and biological modeling as a viable alternative to animal testing to derive acceptable exposure levels.

Ex vivo exposure to polybrominated diphenyl ether (PBDE) selectively affects the immune response in autistic children.

Children on the autism spectrum have been shown to have immune dysregulation that often correlates with behavioral deficits. The role of the post-natal environment in this dysregulation is an area of active investigation. We examined the association between plasma levels of polybrominated diphenyl ether (PBDE) and immune cell function in age-matched autistic children and non-autistic controls. Plasma from children on the autism spectrum (n = 38) and typically developing controls (TD; n = 60) were analyzed for 14 major PBDE congeners. Cytokine/chemokine production was measured in peripheral blood mononuclear cell (PBMC) supernatants with and without ex vivo BDE-49 exposure. Total plasma concentration (∑PBDE14) and individual congener levels were also correlated with T cell function. ∑PBDE14 did not differ between diagnostic groups but correlated with reduced immune function in children on the autism spectrum. In autistic children, IL-2 and IFN-γ production was reduced in association with several individual BDE congeners, especially BDE-49 (p = 0.001). Furthermore, when PBMCs were exposed ex vivo to BDE-49, cells from autistic children produced elevated levels of IL-6, TNF-α, IL-1ß, MIP-1α and MCP-1 (p < 0.05). Therefore, despite similar plasma levels of PBDE, these data suggest that PBMC function was differentially impacted in the context of several PBDE congeners in autistic children relative to TD children where increased body burden of PBDE significantly correlated with a suppressed immune response in autistic children but not TD controls. Further, acute ex vivo exposure of PBMCs to BDE-49 stimulates an elevated cytokine response in AU cases versus a depressed response in TD controls. These data suggest that exposure to the toxicant BDE-49 differentially impacts immune cell function in autistic children relative to TD children providing evidence for an underlying association between susceptibility to PBDE exposure and immune anomalies in children on the autism spectrum.

Gas chromatography-tandem mass spectrometric analysis of metaldehyde and its metabolite acetaldehyde in initial assessment of hemodialysis abatement of toxicity in live animals.

Metaldehyde consumption by pets and other mammals constitute medical emergencies ideally requiring rapid poison removal. The purpose of this study was three-fold: 1) development of a sensitive method for metaldehyde quantitation in patient serum samples by gas chromatography combined with tandem quadrupole mass spectrometry (GC/MS/MS); 2) development of a sensitive method for quantitation of the volatile metaldehyde metabolite acetaldehyde by headspace analysis combined with GC/MS/MS; and 3) an initial assessment of the efficacy of combined dialysis and hemoperfusion treatments in diminishing toxin loads in canine victims of metaldehyde poisoning. Both mass spectrometric approaches relied on Multiple Reaction Monitoring (MRM) methodologies. Metaldehyde extracted via liquid-liquid partitioning from serum was detected with a limit of quantitation (LOQ) of 7.3 ± 1.4 ng/mL with linearity in the range 1-250 ng/mL with accuracy improved by inclusion of a deuterated metaldehyde internal standard. Acetaldehyde was determined to have an LOQ of 0.39 µg/mL with linearity in the range 1-1000 µg/mL. The developed methodologies were applied to canine samples taken over various time points during dialysis treatment. Two of three canine patients showed significant abatement of metaldehyde levels by over 50-fold from initial concentrations while a third was shown to be negative with no measureable metaldehyde. The toxic metabolite acetaldehyde was found in one of the metaldehyde-poisoned patients and the detected acetaldehyde was also reduced by roughly 200-fold during the course of treatment. The designed mass spectrometric techniques were thus successful in demonstrating the efficacy of the applied dialysis-hemoperfusion methods which may find wider applicability against other potentially lethal toxins in poisoned patients in future studies.

Common Toxicosis.

Veterinarians are often called upon to diagnose health-related issues on the farm that may be related to trace mineral deficiencies or toxicities. Trace mineral feeding rates are often not available due to the proprietary nature of the trace mineral premixes provided by nutritional consultants. The veterinarian needs to be aware of the common clinical signs of trace mineral deficiencies and toxicities, interactions between trace minerals that may result in deficiencies, clinical samples that are necessary for the proper diagnosis, and the recommended normal ranges of each trace mineral depending on the age of the animal.

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

Developmental Exposure to a Human-Relevant Polychlorinated Biphenyl Mixture Causes Behavioral Phenotypes That Vary by Sex and Genotype in Juvenile Mice Expressing Human Mutations That Modulate Neuronal Calcium.

Polychlorinated biphenyls (PCBs) are putative environmental risks for neurodevelopmental disorders. Here, we tested two hypotheses: (1) developmental exposure to a human-relevant PCB mixture causes behavioral phenotypes relevant to neurodevelopmental disorders; and (2) expression of human mutations that dysregulate neuronal Ca2+ homeostasis influence sensitivity to behavioral effects of developmental PCB exposures. To test these hypotheses, we used mice that expressed a gain-of-function mutation (T4826I) in ryanodine receptor 1 (RYR1), the X-linked fragile X mental retardation 1 (FMR1) CGG repeat expansion or both mutations (double mutant; DM). Transgenic mice and wildtype (WT) mice were exposed to the MARBLES PCB mix at 0, 0.1, 1, and 6 mg/kg/day in the maternal diet throughout gestation and lactation. The MARBLES PCB mix simulates the relative proportions of the 12 most abundant PCB congeners found in the serum of pregnant women at increased risk for having a child with a neurodevelopmental disorder. We assessed ultrasonic vocalizations at postnatal day 7 (P7), spontaneous repetitive behaviors at P25-P30, and sociability at P27-P32. Developmental PCB exposure reduced ultrasonic vocalizations in WT litters in all dose groups, but had no effect on ultrasonic vocalizations in transgenic litters. Developmental PCB exposure significantly increased self-grooming and decreased sociability in WT males in the 0.1 mg/kg dose group, but had no effect on WT females in any dose group. Genotype alone influenced ultrasonic vocalizations, self-grooming and to a lesser extent sociability. Genotype alone also influenced effects of PCBs on sociability. PCB levels in the brain tissue of pups increased in a dose-dependent manner, but within any dose group did not differ between genotypes. In summary, developmental PCB exposure phenocopied social behavior phenotypes observed in mice expressing human mutations that modify intracellular Ca2+ dynamics, and expression of these mutations alleviated PCB effects on ultrasonic vocalizations and repetitive behavior, and modified the dose-response relationships and sex-dependent effects of PCB effects on social behavior. These findings suggest that: (1) developmental PCB exposure causes behavioral phenotypes that vary by sex and genotype; and (2) sex-specific responses to environmental factors may contribute to sex biases in the prevalence and/or severity of neurodevelopmental disorders.

In utero and lactational PCB exposure drives anatomic changes in the juvenile mouse bladder.

Bladder dysfunction, including incontinence, difficulty emptying the bladder, or urgency to urinate is a pervasive health and quality of life concern. However, risk factors for developing these symptoms are not completely understood, and the influence of exposure to environmental chemicals, especially during development, on the formation and function of the bladder is understudied. Environmental contaminants such as polychlorinated biphenyls (PCBs) are known to pose a risk to the developing brain; however, their influence on the development of peripheral target organs, such as bladder, are unknown. To address this data gap, C57Bl/6J mouse dams were exposed to an environmentally-relevant PCB mixture at 0, 0.1, 1 or 6 mg/kg daily beginning two weeks prior to mating and continuing through gestation and lactation. Bladders were collected from offspring at postnatal days (P) 28-31. PCB concentrations were detected in bladders in a dose-dependent manner. PCB effects on the bladder were sex- and dose-dependent. Overall, PCB effects were observed in male, but not female, bladders. PCBs increased bladder volume and suburothelial ßIII-tubulin-positive nerve density compared to vehicle control. A subset of these nerves were sensory peptidergic axons indicated by increased calcitonin gene-related protein (CGRP) positive nerve fibers in mice exposed to the highest PCB dose compared to the lowest PCB dose. PCB-induced increased nerve density was also positively correlated with the number of mast cells in the bladder, suggesting inflammation may be involved. There were no detectable changes in epithelial composition or apoptosis as indicated by expression of cleaved caspase 3, suggesting PCBs do not cause overt toxicity. Bladder volume changes were not accompanied by changes in bladder mass or epithelial thickness, indicating that obstruction was not likely involved. Together, these results are the first to suggest that following developmental exposure, PCBs can distribute to the bladder and alter neuroanatomic development and bladder volume in male mice.

Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM. HYPOTHESIS/OBJECTIVES: Based on the association of α-tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α-tocopherol, but not γ-tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM-affected horses. ANIMALS: Vitamin E metabolism: Proof of concept (POC) study; eNAD/EDM-affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM-affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM-affected (n = 12) and age- and sex-matched control (n = 11-12) horses. METHODS: The rates of α-tocopherol/tocotrienol and γ-tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative reverse-transcriptase PCR (qRT-PCR) and droplet digital (dd)-PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog. RESULTS: Metabolic rate of α-tocopherol was increased in eNAD/EDM horses (POC,P < .0001; validation, P = .03), with no difference in the metabolic rate of γ-tocopherol. Horses with eNAD/EDM had increased expression of the CYP4F2 equine orthologue (P = .02) but no differences in copy number. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased α-tocopherol metabolism in eNAD/EDM-affected QHs provides novel insight into alterations in vitamin E processing in eNAD/EDM and highlights the need for high-dose supplementation to prevent the clinical phenotype in genetically susceptible horses.

Quantitation of Methamphetamine and Amphetamine in Postmortem Canine Tissues and Fluids.

The production and use of the highly addictive stimulant methamphetamine are a serious public health problem in the United States and globally. Because of its increased popularity with recreational drug users, accidental or intentional poisoning incidents in companion animals have become an unavoidable scenario in veterinary medicine. We describe a case of methamphetamine poisoning in a 4-year-old female German Shepherd with postmortem analytical quantitation of methamphetamine and its metabolite, amphetamine, in bodily tissues and fluids. Many tissues and bodily fluids can be tested to confirm methamphetamine exposure. More importantly, the higher concentrations found in stomach contents, liver, kidney and heart tissue suggest these are the most useful diagnostic specimens for postmortem confirmation of toxicosis in pets especially in cases in which source material is not available for testing or in cases with no postmortem evaluation.

Simultaneous quantification of vitamin E and vitamin E metabolites in equine plasma and serum using LC-MS/MS.

Vitamin E deficiencies can impact normal growth and development in humans and animals, and assessment of circulating levels of vitamin E and its metabolites may be an important endpoint for evaluation. Development of a sensitive method to detect and quantify low concentrations of vitamin E and metabolites in biological specimens allows for a proper diagnosis for patients and animals that are deficient. We developed a method to simultaneously extract, detect, and quantify the vitamin E compounds alpha-tocopherol (α-TP), gamma-tocopherol (γ-TP), alpha-tocotrienol (α-TT), and gamma-tocotrienol (γ-TT), and the corresponding metabolites formed after ß-oxidation of α-TP and γ-TP, alpha-carboxymethylbutyl hydroxychroman (α-CMBHC) and alpha- or gamma-carboxyethyl hydroxychroman (α- or γ-CEHC), respectively, from equine plasma and serum. Quantification was achieved through liquid chromatography-tandem mass spectrometry. We applied a 96-well high-throughput format using a Phenomenex Phree plate to analyze plasma and serum. Compounds were separated by using a Waters ACQUITY UPLC BEH C18 column with a reverse-phase gradient. The limits of detection for the metabolites and vitamin E compounds were 8-330 pg/mL. To validate the method, intra-day and inter-day accuracy and precision were evaluated along with limits of detection and quantification. The method was then applied to determine concentrations of these analytes in plasma and serum of horses. Alpha-TP levels were 3-6 µg/mL of matrix; the metabolites were found at much lower levels, 0.2-1.0 ng/mL of matrix.

Pharmacokinetics and effects on arachidonic acid metabolism of low doses of cannabidiol following oral administration to horses.

The increasing availability of cannabidiol (CBD) and anecdotal reports of its anti-inflammatory effects has garnered it much interest in the equine industry. The objectives of the current study were to (1) describe the pharmacokinetics of oral CBD in exercising thoroughbreds, (2) characterize select behavioral and physiologic effects, and (3) evaluate effects on biomarkers of inflammation using an ex vivo model. This study was conducted in a randomized balanced 3-way crossover design with a two-week washout period between doses. Horses received a single oral dose (0.5, 1, and 2 mg/kg) of CBD suspended in sesame oil. Blood and urine samples were collected prior to and for 72 hr post drug administration. Additional blood samples collected at select time points were challenged ex vivo with calcium ionophore or lipopolysaccharide to induce eicosanoid production. Drug, metabolite, and eicosanoid concentrations were determined using LC-MS/MS. Cannabidiol was well tolerated with no significant behavioral, gastrointestinal, or cardiac abnormalities observed. CBD was readily absorbed, with parent drug detected in blood at all time points. The carboxylated and hydroxylated metabolites predominated in serum and urine, respectively. The terminal half-life for CBD was 10.7 ± 3.61, 10.6 ± 3.84 and 9.88 ± 3.53 for 0.5, 1, and 2 mg/kg. Although the effects were mixed, results of eicosanoid analysis suggest CBD affects COX-1, COX-2 and LOX at the doses studied here. Results of this study coupled with previous reports in other species, suggest further study of CBD in horses is warranted before its use as an anti-inflammatory can be recommended.

Determination of vitamin E and its metabolites in equine urine using liquid chromatography-mass spectrometry.

Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a hereditary, deteriorating central nervous disease in horses. Currently, the only way to confirm eNAD/EDM is through a postmortem histological evaluation of the central nervous system. Vitamin E, specifically the isoform alpha-tocopherol (α-TP), is known to protect eNAD/EDM susceptible horses from developing the clinical phenotype. While vitamin E is an essential nutrient in the diet of horses, there are no diagnostic tests able to quantitate vitamin E and its metabolites in urine. An ultra-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry (UPLC-APCI-MS/MS) method was developed and validated following acidic hydrolysis and solid phase extraction to quantitate vitamin E and its metabolites in equine urine. A blank control horse urine matrix was used and spiked with different concentrations of analytes to form a standard curve using either alpha-tocopherol-d6 or chlorpropamide as the internal standard. Inter-day and intra-day statistics were performed to evaluate the method for accuracy (90% to 116%) and precision (0.75% to 14%). Matrix effects, percent recovery, and stability were also assessed. The method successfully analyzed alpha-carboxyethyl hydroxychroman (α-CEHC), alpha-carboxymethylbutyl hydroxychromans (α-CMBHC), gamma-carboxyethyl hydroxychroman γ-CEHC, and α-TP concentrations in urine to determine a baseline levels of analytes in healthy horses, and can be used to determine concentrations of vitamin E metabolites in equine urine allowing for its evaluation as a diagnostic approach in the treatment of eNAD/EDM.

Analysis of Covalently Bound Microcystins in Sediments and Clam Tissue in the Sacramento-San Joaquin River Delta, California, USA.

Harmful cyanobacterial blooms compromise human and environmental health, mainly due to the cyanotoxins they often produce. Microcystins (MCs) are the most commonly measured group of cyanotoxins and are hepatotoxic, neurotoxic, and cytotoxic. Due to MCs ability to covalently bind to proteins, quantification in complex matrices is difficult. To analyze bound and unbound MCs, analytical methods were optimized for analysis in sediment and clam tissues. A clean up step was incorporated to remove lipids, improving percent yield. This method was then applied to sediment and clam samples collected from the Sacramento-San Joaquin River Delta (Delta) in the spring and fall of 2017. Water samples were also tested for intracellular and extracellular MCs. These analyses were used to quantify the partitioning of MCs among sediment, clams, and water, and to examine whether MCs persist during non-summer months. Toxin analysis revealed that multiple sediment samples collected in the Delta were positive for MCs, with a majority of the positive samples from sites in the San Joaquin River, even while water samples from the same location were below detection limit. These data highlight the importance of analyzing MCs in complex matrices to accurately evaluate environmental risk.

Severe hypernatremia and transient azotemia in a cat following inadvertent intravenous administration of a commercial polyethylene glycol solution.

OBJECTIVE: To describe the clinical signs, clinicopathologic abnormalities, treatment, and outcome after IV administration of polyethylene glycol 3350 (PEG3350) in a cat. CASE SUMMARY: A cat was inadvertently administered 6 g/kg of PEG3350 in electrolyte solution, IV, resulting in severe hypernatremia (203 mmol/L), diffuse encephalopathy, hemolysis, and moderate azotemia. The hemolysis and acute kidney injury observed immediately following PEG3350 administration resolved with supportive care. Administration of IV and oral electrolyte-free water slowly corrected the hypernatremia and the neurologic signs subsequently improved. Complete resolution of clinical signs was documented one month following hospital discharge. The PEG3350 concentrations in serum, plasma, and urine samples confirmed toxic exposure to PEG3350. Efficacy of treatment was evident by decreasing concentrations of PEG3350 in serum after the first 24 hours of treatment. Renal elimination of PEG3350 was significant and PEG3350 was still detected in the urine 17 days after exposure. NEW INFORMATION PROVIDED: This is the first report to describe the clinical signs and clinicopathologic abnormalities in a cat intoxicated with IV PEG3350. Potential pathophysiologic mechanisms are discussed, and the successful supportive medical treatment is outlined.

Changes in thyroid hormone activity disrupt photomotor behavior of larval zebrafish.

High throughput in vitro, in silico, and computational approaches have identified numerous environmental chemicals that interfere with thyroid hormone (TH) activity, and it is posited that human exposures to such chemicals are a contributing factor to neurodevelopmental disorders. However, whether hits in screens of TH activity are predictive of developmental neurotoxicity (DNT) has yet to be systematically addressed. The zebrafish has been proposed as a second tier model for assessing the in vivo DNT potential of TH active chemicals. As an initial evaluation of the feasibility of this proposal, we determined whether an endpoint often used to assess DNT in larval zebrafish, specifically photomotor behavior, is altered by experimentally induced hyper- and hypothyroidism. Developmental hyperthyroidism was simulated by static waterborne exposure of zebrafish to varying concentrations (3-300 nM) of thyroxine (T4) or triiodothyronine (T3) beginning at 6 h post-fertilization (hpf) and continuing through 5 days post-fertilization (dpf). Teratogenic effects and lethality were observed at 4 and 5 dpf in fish exposed to T4 or T3 at concentrations >30 nM. However, as early as 3 dpf, T4 (> 3 nM) and T3 (> 10 nM) significantly increased swimming activity triggered by sudden changes from light to dark, particularly during the second dark period (Dark 2). Conversely, developmental hypothyroidism, which was induced by treatment with 6-propyl-2-thiouracil (PTU), morpholino knockdown of the TH transporter mct8, or ablation of thyroid follicles in adult females prior to spawning, generally decreased swimming activity during dark periods, although effects did vary across test days. All effects of developmental hypothyroidism on photomotor behavior occurred independent of teratogenic effects and were most robust during Dark 2. Treatment with the T4 analog, Tetrac, restored photomotor response in mct8 morphants to control levels. Collectively, these findings suggest that while the sensitivity of photomotor behavior in larval zebrafish to detect TH disruption is influenced by test parameters, this test can distinguish between TH promoting and TH blocking activity and may be useful for assessing the DNT potential of TH-active chemicals.

Determination of Total Mercury and Methylmercury Concentrations in Commercial Canine Diets.

Methylmercury is an organic form of mercury that is well recognized for its bioaccumulation in aquatic species. Consumption of fish contaminated with methylmercury poses a toxicological health risk to both humans and animals. Salmon is an increasingly common ingredient in commercial pet foods because of manufacturers' interest in unconventional protein sources and inclusion of omega-3 polyunsaturated fatty acids. Previous studies have measured total mercury, but not methylmercury, in commercial pet foods. The purpose of this study was to evaluate total mercury and methylmercury concentrations in commercially available salmon-containing and nonsalmon-containing canine diets and to estimate risk of chronic exposure in dogs fed these diets long term. Total mercury was detected in 3 of 24 diets evaluated (12.5%), 2 of which did not contain any ingredients from fish. The single salmon-containing diet that contained total mercury was the lowest of the 3 but was also the only sample positive for methylmercury. None of the 3 mercury-containing diets contained fish oil. Concentrations of total mercury were similar to most data previously reported for pet foods. Using expected calorie intake for dogs of 2 body weights, the mercury concentrations determined in this study were applied to theoretical chronic exposure calculations to assess risk of toxicosis to dogs. Total mercury and methylmercury were uncommonly identified in the commercially available canine diets sampled in this study and were found in concentrations unlikely to pose risk to healthy adult dogs. Common sources of mercury in pet foods remain unknown and are not reliably of seafood origin.

Tissue steroid levels in response to reduced testicular estrogen synthesis in the male pig, Sus scrofa.

Production of steroid hormones is complex and dependent upon steroidogenic enzymes, cofactors, receptors, and transporters expressed within a tissue. Collectively, these factors create an environment for tissue-specific steroid hormone profiles and potentially tissue-specific responses to drug administration. Our objective was to assess steroid production, including sulfated steroid metabolites in the boar testis, prostate, and liver following inhibition of aromatase, the enzyme that converts androgen precursors to estrogens. Boars were treated with the aromatase inhibitor, letrozole from 11 to 16 weeks of age and littermate boars received the canola oil vehicle. Steroid profiles were evaluated in testes, prostate, and livers of 16, 20, and 40 week old boars using liquid chromatography/mass spectrometry. Testis, prostate, and liver had unique steroid profiles in vehicle-treated animals. Only C18 steroid hormones were altered by treatment with the aromatase inhibitor, letrozole; no significant differences were detected in any of the C19 or C21 steroids evaluated. Testis was the only tissue with significantly decreased free estrogens following treatment with the aromatase inhibitor; estrone and estradiol concentrations were lower (p < 0.05) in testes from 16, 20, and 40 week letrozole-treated boars. However, concentrations of the sulfated conjugates, estrone-sulfate and estradiol-sulfate, were significantly decreased (p<0.05) in 16 and 20 week boar testes, prostates, and livers from letrozole-treated boars. Hence, the distribution of estrogens between the free and conjugated forms was altered in a tissue-specific manner following inhibition of aromatase. The results suggest sulfated testicular estrogens are important estrogen precursors for the prostate, potentially enabling peripheral target tissues to synthesize free estrogens in the male pig.

Comparative Analyses of the 12 Most Abundant PCB Congeners Detected in Human Maternal Serum for Activity at the Thyroid Hormone Receptor and Ryanodine Receptor.

Polychlorinated biphenyls (PCBs) pose significant risk to the developing human brain; however, mechanisms of PCB developmental neurotoxicity (DNT) remain controversial. Two widely posited mechanisms are tested here using PCBs identified in pregnant women in the MARBLES cohort who are at increased risk for having a child with a neurodevelopmental disorder (NDD). As determined by gas chromatography-triple quadruple mass spectrometry, the mean PCB level in maternal serum was 2.22 ng/mL. The 12 most abundant PCBs were tested singly and as a mixture mimicking the congener profile in maternal serum for activity at the thyroid hormone receptor (THR) and ryanodine receptor (RyR). Neither the mixture nor the individual congeners (2 fM to 2 µM) exhibited agonistic or antagonistic activity in a THR reporter cell line. However, as determined by equilibrium binding of [3H]ryanodine to RyR1-enriched microsomes, the mixture and the individual congeners (50 nM to 50 µM) increased RyR activity by 2.4-19.2-fold. 4-Hydroxy (OH) and 4-sulfate metabolites of PCBs 11 and 52 had no TH activity; but 4-OH PCB 52 had higher potency than the parent congener toward RyR. These data support evidence implicating RyRs as targets in environmentally triggered NDDs and suggest that PCB effects on the THR are not a predominant mechanism driving PCB DNT. These findings provide scientific rationale regarding a point of departure for quantitative risk assessment of PCB DNT, and identify in vitro assays for screening other environmental pollutants for DNT potential.

Polychlorinated biphenyls influence on autism spectrum disorder risk in the MARBLES cohort.

BACKGROUND: Autism spectrum disorder (ASD) is suspected to have environmental and genetic contributions. Polychlorinated biphenyls (PCBs) are environmental risk factors of interest due to their potential as neurodevelopmental toxicants and environmental persistence despite a US production ban in the 1970s. METHODS: Participants were mother-child pairs from MARBLES, a high-risk pregnancy cohort that enrolls families who have one child diagnosed with ASD and are planning to have another child. PCB concentrations were measured in maternal blood at each trimester of pregnancy using gas chromatography coupled with triple quadruple mass spectrometry. Concentrations were summed into total PCB and two categories based on function/mechanisms of action: dioxin-like (DL), and ryanodine receptor (RyR)-activating PCBs. Multinomial logistic regression assessed risk of clinical outcome classification of ASD and non-typical development (Non-TD) compared to typically developing (TD) in the children at 3 years old. RESULTS: A total of 104 mother-child pairs were included. There were no significant associations for total PCB; however, there were borderline significant associations between DL-PCBs and decreased risk for Non-TD outcome classification (adjusted OR: 0.41 (95% CI 0.15-1.14)) and between RyR-activating PCBs and increased risk for ASD outcome classification (adjusted OR: 2.63 (95% CI 0.87-7.97)). CONCLUSION: This study does not provide strong supporting evidence that PCBs are risk factors for ASD or Non-TD. However, these analyses suggest the need to explore more deeply into subsets of PCBs as risk factors based on their function and structure in larger cohort studies where non-monotonic dose-response patterns can be better evaluated.