Antonio Planchart

Per-and polyfluoroalkyl substances (PFAS) are ubiquitous environmental contaminants that have been associated with adverse health effects in highly exposed populations. Manufacturers have taken steps to replace toxic long-chain perfluoroalkyl acids (PFAAs) with short-chain PFAAs and perfluoroether acids (PFEAs). There is little to no toxicity data for many of these chemicals. Most of the data that are available are taken from studies that do not account for the pH of highly concentrated PFAS solutions, resulting in highly acidic conditions that do not accurately reflect real-world exposures. The goal of this study was to evaluate the lethality and developmental toxicity of 17 structurally diverse PFAS in zebrafish in a pH-neutral environment. We then compared results to determine the impacts of chain length, head group, and ether linkages on toxicity. The potency of PFAS to induce mortality and developmental toxicity endpoints increased with chain length, and sulfonic acids were more potent than carboxylic acids. The inclusion of ether oxygens was associated with reduced potency relative to PFAAs with equal chain length and head group. Perfluorooctane sulfonic acid (PFOS) was the most potent compound, followed by perfluoroundecanoic acid (PFUnDA) and perfluorododecanoic acid (PFDoDA). Short-chain compounds perfluoro-2-methoxyacetic acid (PFMOAA) and perfluorobutanoic acid (PFBA) were the least potent. These data were used to construct a multiple linear regression model for PFAS potency. Failed swim bladder inflation was the most sensitive developmental toxicity endpoint for all assessed chemicals. Other common phenotypes included spinal curvature, edema, craniofacial malformations, and ocular malformations. Ocular malformations were more common in response to sulfonic acids. No other phenotypes exhibited significant structural specificity. Our study provides new toxicity data for a diverse set of PFAS under environmentally relevant conditions. Future studies should be expanded to include more branched structures and head groups not present in our testing set to allow for improved understanding of how other structural features impact toxicity.

Per-and polyfluoroalkyl substances (PFAS) are ubiquitous contaminants in freshwater ecosystems. Many PFAS are incorporated into food webs, with potential effects on ecological and human health. However, PFAS incorporation into the base of aquatic food webs remains poorly understood. The goal of this study was to quantify the uptake and trophic transfer of both legacy and current use PFAS compounds using a simulated freshwater food chain in a lab setting. Natural periphytic biofilms were placed into trays containing equimolar binary aqueous PFAS mixtures at environmentally relevant concentrations for five days. Following the initial exposure period, newly hatched mayfly larvae were introduced into each tray to feed on periphyton for most of their larval development. The mature larvae were then fed to zebrafish. All water and biota samples contained detectable levels of the tested PFAS. All PFAS were more concentrated in periphyton than in water, and four of six PFAS were further concentrated in mayfly larvae relative to periphyton. PFDA was the most accumulative in all biota. PFAS concentrations in zebrafish were significantly correlated with those in larval mayflies. Assimilation efficiencies in zebrafish were high (>70%) for all compounds. Bioaccumulation of PFAS in periphyton and mayflies was positively correlated with log K _OW and number of carbons.

Background: There are more than 55 million people with dementia worldwide, and it is estimated that currently 6.5 million people are living with Alzheimer’s disease (AD) in the United States. AD exhibits racial disparities whereby the prevalence is 2‐fold higher in Non‐Hispanic Blacks (NHB) compared to Non‐Hispanic Whites (NHW). It is hypothesized that epigenetics and aberrant imprinting caused by early life exposure to adverse environmental agents may contribute to AD risk later in life, although to date a comprehensive assessment of the imprinting state of the genome in AD‐affected populations has not been reported.Methods: To address this hypothesis, we investigated cytosine methylation at candidate imprint control regions (ICRs) in post‐mortem tissues (brain) of nine AD cases (5NHB, 4NHW) and eight controls (4NHB, 4NHW), using whole genome bisulfite sequencing. Furthermore, we performed a limited clinical study in which blood samples from six individuals, three AD patients (2NHB, 1NHW) and three controls (all NHB), were subjected to methyl‐sequencing. These data were analyzed against an expanded set of candidate ICRs previously described by our group (i.e., human imprintome) to identify regions of the genome exhibiting aberrant methylation in AD patients compared to controls.Results: We found differential methylation in DNA derived from post‐mortem tissues of AD cases compared to controls overlapping with the expanded set of ICRs in the human imprintome (79 in NHBs and 27 in NHWs), including candidate ICR on chromosome 17 near the NLRP1 inflammasome gene (5771207‐5771575; Hg38). When extended to blood from extant controls and patients, our analysis identified a single nucleotide polymorphism (CA → CG; rs12451480) within the candidate ICR on chromosome 17, that distinguishes controls (CA) from AD patients (CG), and in which the cytosine is methylated.Conclusion: We demonstrate concordance between the methylation state of post‐mortem brain‐ and blood‐derived DNA distinguishing controls from AD patients. Furthermore, the discovery of a SNP differentiating controls from AD patients, within a candidate ICR that alters its methylation state, is consistent with an aberrant imprinting phenomenon. Our results support the hypothesis that aberrant imprinting, as can result from adverse exposures during early development, may contribute to later risk of AD.

β-N-Methylamino-l-alanine (BMAA) is a non-proteinogenic amino acid produced by cyanobacteria, which has been implicated in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). It is postulated that chronic exposure to BMAA can lead to formation of protein aggregates, oxidative stress, and/or excitotoxicity, which are mechanisms involved in the etiology of ALS. While specific genetic mutations are identified in some instances of ALS, it is likely that a combination of genetic and environmental factors, such as exposure to the neurotoxin BMAA, contributes to disease. We used a transgenic zebrafish with an ALS-associated mutation, compared with wild-type fish to explore the potential neurotoxic effects of BMAA through chronic long-term exposures. While our results revealed low concentrations of BMAA in the brains of exposed fish, we found no evidence of decreased swim performance or behavioral differences that might be reflective of neurodegenerative disease. Further research is needed to determine if chronic BMAA exposure in adult zebrafish is a suitable model to study neurodegenerative disease initiation and/or progression.

The molecular mechanisms connecting environmental exposures to adverse endpoints are often unknown, reflecting knowledge gaps. At the Comparative Toxicogenomics Database (CTD), we developed a bioinformatics approach that integrates manually curated, literature-based interactions from CTD to generate a “CGPD-tetramer”: a four-unit block of information organized as a step-wise molecular mechanism linking an initiating Chemical, an interacting Gene, a Phenotype, and a Disease outcome. Here, we describe a novel, user-friendly tool called CTD Tetramers that generates these evidence-based CGPD-tetramers for any curated chemical, gene, phenotype, or disease of interest. Tetramers offer potential solutions for the unknown underlying mechanisms and intermediary phenotypes connecting a chemical exposure to a disease. Additionally, multiple tetramers can be assembled to construct detailed modes-of-action for chemical-induced disease pathways. As well, tetramers can help inform environmental influences on adverse outcome pathways (AOPs). We demonstrate the tool’s utility with relevant use cases for a variety of environmental chemicals (e.g., perfluoroalkyl substances, bisphenol A), phenotypes (e.g., apoptosis, spermatogenesis, inflammatory response), and diseases (e.g., asthma, obesity, male infertility). Finally, we map AOP adverse outcome terms to corresponding CTD terms, allowing users to query for tetramers that can help augment AOP pathways with additional stressors, genes, and phenotypes, as well as formulate potential AOP disease networks (e.g., liver cirrhosis and prostate cancer). This novel tool, as part of the complete suite of tools offered at CTD, provides users with computational data sets and their supporting evidence to potentially fill exposure knowledge gaps and develop testable hypotheses about environmental health.

There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in aca­demic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Liter­ature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure medium (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.