Biology subjects

Belcher, S. M.

Publications and source records attributed to Belcher, S. M..

10 recordsLinked to original sources

bioRxiv2026

Human Lactoferrin is a Novel PFAS Target: Implications for Neo-natal Immune Function and Protein Stability

Per-and polyfluoroalkyl substances (PFAS) constitute a diverse class of persistent synthetic chemicals utilized across industrial, medical, and consumer sectors that are pervasive global pollutants. Exposure to PFAS is linked to adverse impacts on both innate and adaptive immune systems. Human lactoferrin (hLF) is a key antimicrobial component of the developing innate immune system present in colostrum and breast milk. We hypothesized that hLF is a potential PFAS binding protein related to PFAS immunotoxicity. The results of thermal stability experiments indicated that all 11 tested PFAS bind and destabilize the structure of hLF. Notably PFBA, PFOS, HFPO-DA, and 6:2 FTSA decreased apo-hLF melting temperatures from 64oC to [≤] 37oC, suggesting that PFAS exposures destabilize the native hLF protein under physiological conditions. Relative binding affinities (Kd) ranged from 0.2-11 mM across tested PFAS. Molecular docking was used to confirm experimental binding affinities and identify molecular interactions involved with PFAS binding. Calculated Gibbs Free Energies of binding ranged from -4.4 to -8.8 kcal/mol. Together, these results demonstrate that PFAS bind hLF at affinities comparable to human serum albumin and other PFAS binding proteins, and that some PFAS can destabilize hLF protein structure at physiologically relevant temperatures and conditions.

pharmacology and toxicology↗

bioRxiv2025

Novel PFAS in Alligator Blood Discovered with Non-Targeted Ion Mobility-Mass Spectrometry

Per- and polyfluoroalkyl substances (PFAS) are a large and growing class of chemicals gaining global attention due to their persistence, mobility, and toxicity. Given the diverse chemical properties of PFAS and their varying distributions in water and tissue, monitoring of different matrices is critical to determine their presence and accumulation. Here, we used a platform combining liquid chromatography, ion mobility spectrometry, and high-resolution mass spectrometry for non-targeted analysis to detect and identify PFAS in alligator plasma from North Carolina (5 years, 2018-2022) and Florida (2021 only). Structures for 12 PFAS were elucidated, including 2 novel structures, and an additional 34 known PFAS were detected. Three of these compounds were previously unreported in environmental media. More PFAS were detected in North Carolina alligators and no novel PFAS were detected in Florida gators. Quantitative analysis of 21 of the known PFAS revealed that plasma concentrations did not change over the 5-year study. HighlightsO_LI46 PFAS were detected in blood plasma from North Carolina and Florida alligators using LC-IMS-HRMS C_LIO_LIStructures for 12 previously unreported PFAS were elucidated, including 2 novel structures C_LIO_LICape Fear River alligators had the most types and highest concentrations of PFAS C_LI

pharmacology and toxicology↗

bioRxiv2023

Quantitative Cross-Species Comparison of Serum Albumin Binding of Per- and Polyfluoroalkyl Substances from Five Structural Classes

Per- and polyfluoroalkyl substances (PFAS) are a class of over 8,000 chemicals that are persistent, bioaccumulative, and toxic to humans, livestock, and wildlife. Serum protein binding affinity is instrumental in understanding PFAS toxicity, yet experimental binding data is limited to only a few PFAS congeners. Previously, we demonstrated the usefulness of a high-throughput, in vitro differential scanning fluorimetry assay for determination of relative binding affinities of human serum albumin for 24 PFAS congeners from 6 chemical classes. In the current study, we used this differential scanning fluorimetry assay to comparatively examine differences in human, bovine, porcine, and rat serum albumin binding of 8 structurally informative PFAS congeners from 5 chemical classes. With the exception of the fluorotelomer alcohol 1H,1H,2H,2H-perfluorooctanol (6:2 FTOH), each PFAS congener bound by human serum albumin was also bound by bovine, porcine, and rat serum albumin. The critical role of the charged functional headgroup in albumin binding was supported by the inability of serum albumin of each species tested to bind 6:2 FTOH. Significant interspecies differences in serum albumin binding affinities were identified for each of the bound PFAS congeners. Relative to human albumin, perfluoroalkyl carboxylic and sulfonic acids were bound with greater affinity by porcine and rat serum albumin, and perfluoroalkyl ether congeners bound with lower affinity to porcine and bovine serum albumin. These comparative affinity data for PFAS binding by serum albumin from human, experimental model and livestock species reduce critical interspecies uncertainty and improve accuracy of predictive toxicity assessments for PFAS.

pharmacology and toxicology↗

bioRxiv2023

Assessing Per- and Polyfluoroalkyl Substances (PFAS) in Fish Fillet Using Non-Targeted Analyses

Per- and polyfluoroalkyl substances (PFAS) are a class of thousands of man-made chemicals that are persistent and highly stable in the environment. Fish consumption has been identified as a key route of PFAS exposure for humans. However, routine fish monitoring targets only a handful of PFAS, and non-targeted analyses have largely only evaluated fish from heavily PFAS-impacted waters. Here, we evaluated PFAS in fish fillets from recreational and drinking water sources in central North Carolina to assess whether PFAS are present in these fillets that would not be detected by conventional targeted methods. We used liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) to collect full scan feature data, performed suspect screening using an in-house library of 100 PFAS for high confidence feature identification, searched for additional PFAS features using non-targeted data analyses, and quantified perfluorooctane sulfonic acid (PFOS) in the fillet samples. A total of 36 PFAS were detected in the fish fillets, including 19 that would not be detected using common targeted methods, with a minimum of 6 and a maximum of 22 in individual fish. Median fillet PFOS levels were concerningly high at 11.6 to 42.3 ppb, and no significant correlation between PFOS levels and number of PFAS per fish was observed. Future PFAS monitoring in this region should target more of these 36 PFAS, and other regions not considered heavily PFAS contaminated should consider incorporating non-targeted analyses into ongoing fish monitoring studies. SYNOPSISConcerningly high PFOS levels were detected in fish fillets from recreational and drinking water sources in central North Carolina. Of the 36 total PFAS identified, 19 would not be detected using routine targeted monitoring methods. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/555938v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b3084org.highwire.dtl.DTLVardef@9e69bborg.highwire.dtl.DTLVardef@2f9628org.highwire.dtl.DTLVardef@2b33c8_HPS_FORMAT_FIGEXP M_FIG C_FIG For use in table of contents only

pharmacology and toxicology↗

bioRxiv2022

Blood Concentrations of Per- and Polyfluoroalkyl Substances are Associated with Lupus-like Autoimmunity in the American Alligator

Surface and ground water of the Cape Fear River basin in central and coastal North Carolina is contaminated with high levels of per- and polyfluoroalkyl substances (PFAS). Elevated levels of PFAS have also been found in blood of fish and wildlife living in and around the Cape Fear River, and in the blood of human populations reliant on contaminated well or surface water from the Cape Fear River basin as a sources of drinking water. While the public and environmental health impacts of long-term PFAS exposures are poorly understood, elevated blood concentrations of some PFAS are linked with immunotoxicity and increased incidence of some chronic autoimmune diseases in human populations. The goal of this study was to evaluate PFAS exposure and biomarkers related to immune health in populations of American alligators (Alligator mississippiensis), a protected and predictive sentinel species of adverse effects caused by persistent toxic pollutants. We found that serum PFAS concentrations in a representative population of alligators from the Cape Fear River were increased compared to a reference population of alligators from the adjoining Lumber River basin. The elevated serum PFAS concentrations in the Cape Fear River alligators were associated with increased innate immune activities, and autoimmune-like phenotypes were observed in this population. In addition to evidence of significantly higher double stranded-DNA binding autoantibodies in adult Cape Fear River alligators, our qRT-PCR analysis found remarkably high induction of Interferon- signature genes implicated in the pathology of human autoimmune disease. We interpret the association of increased PFAS exposure with disrupted immune functions to indicate that PFAS broadly alters immune activities resulting in autoimmune-like pathology in American alligators. This work substantiates and extends evidence from experimental models and human epidemiology studies showing that some PFAS are immune toxicants. HighlightsO_LISerum PFAS concentration were elevated in alligator populations from the Cape Fear River C_LIO_LIHigher PFAS concentrations were associated with disrupted innate immune functions C_LIO_LIInfa-responsive autoimmunity signature genes were induced in Cape Fear River alligators C_LIO_LIElevated PFAS levels were associated with autoimmune-like activity in alligators 26 C_LI

pharmacology and toxicology↗

bioRxiv2021

Gestational Cd Exposure in the CD-1 Mouse Sex-Specifically Disrupts Essential Metal Ion Homeostasis

In CD-1 mice, gestational-only exposure to cadmium (Cd) causes female-specific hepatic insulin resistance, metabolic disruption, and obesity. To evaluate whether sex differences in cadmium uptake and changes in essential metal concentrations contribute to metabolic outcomes, placental and liver cadmium and essential metal concentrations were quantified in male and female offspring perinatally exposed to 500 ppb CdCl2. Exposure resulted in increased maternal liver Cd+2 concentrations (364 g/kg) similar to concentrations found in non-occupationally exposed human liver. At gestational day (GD) 18, placental cadmium and manganese concentrations were significantly increased in exposed males and females, and zinc was significantly decreased in females. Placental efficiency was significantly decreased in GD18 exposed males. Increases in hepatic Cd concentrations and a transient prenatal increase in zinc were observed in exposed female liver. Fetal and adult liver iron concentrations were decreased in both sexes, and decreases in hepatic zinc, iron, and manganese were observed in exposed females. Analysis of GD18 placental and liver metallothionein mRNA expression revealed significant Cd-induced upregulation of placental metallothionein in both sexes, and a significant decrease in fetal hepatic metallothionein in exposed females. In placenta, expression of metal ion transporters responsible for metal ion uptake was increased in exposed females. In liver of exposed adult female offspring, expression of the divalent cation importer (Slc39a14/Zip14) decreased, whereas expression of the primary exporter (Slc30a10) increased. These findings demonstrate that Cd can preferentially cross the female placenta, accumulate in the liver, and cause lifelong dysregulation of metal ion concentrations associated with metabolic disruption.

pharmacology and toxicology↗

bioRxiv2021

Utilizing Pine Needles to Temporally and Spatially Profile Per- and Polyfluoroalkyl Substances

As concerns continue to mount over exposure to per- and polyfluoroalkyl substances (PFAS), novel methods of profiling their presence and modifications are greatly needed as some have known toxic and bioaccumulative characteristics while others have unknown effects. This task however is not simple as over 5000 PFAS of interest have been named by the Environmental Protection Agency and this list continues to grow daily. In this work, we utilized widely available archived and field-sampled pine needles and a novel non-targeted analytical method to evaluate the temporal and spatial presence of numerous PFAS. Over 70 PFAS were detected in the pine needles from this study, providing information from the last six decades related to PFAS exposure, contamination, and reduction.

pharmacology and toxicology↗

bioRxiv2021

Rapid Characterization of Human Serum Albumin Binding for Per- And Polyfluoroalkyl Substances Using Differential Scanning Fluorimetry

Per- and polyfluoroalkyl substances (PFAS) are a diverse class of synthetic chemicals that accumulate in the environment. Many proteins, including the primary human serum transport protein albumin (HSA), bind PFAS. The predictive power of physiologically based pharmacokinetic modeling approaches are currently limited by a lack of experimental data defining albumin binding properties for most PFAS. A novel thermal denaturation assay was optimized to evaluate changes in thermal stability of HSA in the presence of increasing concentrations of known ligands and a structurally diverse set of PFAS. Assay performance was initially evaluated for fatty acids and HSA binding drugs ibuprofen and warfarin. Concentration response relationships were determined and dissociation constants (Kd) for each compound were calculated using regression analysis of the dose-dependent changes in HSA melting temperature. Estimated Kd values for HSA binding of octanoic acid, decanoic acid, hexadecenoic acid, ibuprofen and warfarin agreed with established values. The binding affinities for 24 PFAS that included perfluoroalkyl carboxylic acids (C4-C12), perfluoroalkyl sulfonic acids (C4-C8), mono- and polyether perfluoroalkyl ether acids, and polyfluoroalkyl fluorotelomer substances were determined. These results demonstrate the utility of this differential scanning fluorimetry assay as a rapid high through-put approach for determining the relative protein binding properties and identification of chemical structures involved in binding for large numbers of structurally diverse PFAS.

pharmacology and toxicology↗

bioRxiv2020

Heterozygous mutation of Sonic Hedgehog receptor (Ptch) drives cerebellar overgrowth and sex-specifically alters hippocampal and cortical layer structure, activity, and social behavior in female mice

Sonic hedgehog (SHH) signaling is essential for the differentiation and migration of early stem cell populations during cerebellar development. Dysregulation of SHH-signaling can result in cerebellar overgrowth and the formation of the brain tumor medulloblastoma. Treatment for medulloblastoma is extremely aggressive and patients suffer life-long side effects including behavioral deficits. Considering that other behavioral disorders including autism spectrum disorders, holoprosencephaly, and basal cell nevus syndrome are known to present with cerebellar abnormalities, it is proposed that some behavioral abnormalities could be inherent to the medulloblastoma sequalae rather than treatment. Using a haploinsufficient SHH receptor knockout mouse model (Ptch1+/-), a partner preference task was used to explore activity, social behavior and neuroanatomical changes resulting from dysregulated SHH signaling. Compared to wild-type, Ptch1+/- females displayed increased activity by traveling a greater distance in both open-field and partner preference tasks. Social behavior was also sex-specifically modified in Ptch1+/- females that interacted more with both novel and familiar animals in the partner preference task compared to same-sex wild-type controls. Haploinsufficency of PTCH resulted in cerebellar overgrowth in lobules IV/V and IX of both sexes, and female-specific decreases in hippocampal size and isocortical layer thickness. Taken together, neuroanatomical changes related to deficient SHH signaling may alter social behavior.

neuroscience↗

bioRxiv2017

Estrogen and Soy Isoflavonoids Decrease Sensitivity of Medulloblastoma and Central Nervous System Primitive Neuroectodermal Tumor Cells to Chemotherapeutic Cytotoxicity

BackgroundOur previous studies demonstrated that growth and migration of medulloblastoma (MB), the most common malignant brain tumor in children, are stimulated by 17{beta}-estradiol. The growth stimulating effects of estrogens are mediated through ER{beta} and insulin-like growth factor 1 signaling to inhibit caspase 3 activity and reduce tumor cell apoptosis. The objective of this study was to determine whether estrogens decreased sensitivity of MB cells to cytotoxic actions of chemotherapeutic drugs.\n\nMethodsUsing in vitro cell viability and clonogenic survival assays, concentration response analysis was used to determine whether the cytoprotective effects of estradiol protected human D283 Med MB cells from the cytotoxic actions of the MB chemotherapeutic drugs cisplatin, vincristine, or lomustine. Additional experiments were done to determine whether the ER antagonist fulvestrant or the selective ER modulator tamoxifen blocked the cytoprotective actions of estradiol. ER-selective agonists and antagonists were used to define receptor specificity, and the impacts of the soy-derived phytoestrogens genistein, daidzein, and s-equol on chemosensitivity were evaluated.\n\nResultsIn D283 Med cells the presence of 10 nM estradiol increased the IC50 for cisplatin-induced inhibition of viability 2-fold from ~5 M to >10 M. In clonogenic survival assays estradiol decreased the chemosensitivity of D283 Med exposed to cisplatin, lomustine and vincristine. The ER{beta} selective agonist DPN and low physiological concentrations of the soy-derived phytoestrogens genistein, daidzein, and s-equol also decreased sensitivity of D283 Med cells to cisplatin. The protective effects of estradiol were blocked by the antiestrogens 4-hydroxytamoxifen, fulvestrant (ICI 182,780) and the ER{beta} selective antagonist PPHTP. Whereas estradiol also decreased chemosensitivity of PFSK1 cells, estradiol increased sensitivity of Daoy cell to cisplatin, suggesting that ER{beta} mediated effects may vary in different subtypes of MB.\n\nConclusionsThese findings demonstrate that E2 and environmental estrogens decrease sensitivity of MB to cytotoxic chemotherapeutics, and that ER{beta} selective and non-selective inhibition of estrogen receptor activity blocks these cytoprotective actions. These findings support the therapeutic potential of antiestrogen adjuvant therapies for MB, and findings that soy phytoestrogens also decrease sensitivity of MB cells to cytotoxic chemotherapeutics suggest that decreased exposure to environmental estrogens may benefit MB patient responses to chemotherapy.

pharmacology and toxicology↗