Systemic immunological responses are dependent on sex and ovarian hormone presence following acute inhaled woodsmoke exposure.

BACKGROUND: Rural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility. METHODS: This exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode. RESULTS: Metals analysis of WS particulate matter (PM) revealed significantly increased levels of 63Cu, 182W, 208Pb, and 238U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1ß, TNF-α, CXCL-1, CCL-5, TGF-ß, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1ß, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1ß, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche. CONCLUSIONS: In summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.

Biomass smoke inhalation promotes neuroinflammatory and metabolomic temporal changes in the hippocampus of female mice.

Smoke from wildland fires has been shown to produce neuroinflammation in preclinical models, characterized by neural infiltrations of neutrophils and monocytes, as well as altered neurovascular endothelial phenotypes. To address the longevity of such outcomes, the present study examined the temporal dynamics of neuroinflammation and metabolomics after inhalation exposures from biomass-derived smoke. 2-month-old female C57BL/6 J mice were exposed to wood smoke every other day for 2 weeks at an average exposure concentration of 0.5 mg/m3. Subsequent serial euthanasia occurred at 1-, 3-, 7-, 14-, and 28-day post-exposure. Flow cytometry of right hemispheres revealed two endothelial populations of CD31Hi and CD31Med expressors, with wood smoke inhalation causing an increased proportion of CD31Hi. These populations of CD31Hi and CD31Med were associated with an anti-inflammatory and pro-inflammatory response, respectively, and their inflammatory profiles were largely resolved by the 28-day mark. However, activated microglial populations (CD11b+/CD45low) remained higher in wood smoke-exposed mice than controls at day 28. Infiltrating neutrophil populations decreased to levels below controls by day 28. However, the MHC-II expression of the peripheral immune infiltrate remained high, and the population of neutrophils retained an increased expression of CD45, Ly6C, and MHC-II. Utilizing an unbiased approach examining the metabolomic alterations, we observed notable hippocampal perturbations in neurotransmitter and signaling molecules, such as glutamate, quinolinic acid, and 5-α-dihydroprogesterone. Utilizing a targeted panel designed to explore the aging-associated NAD+ metabolic pathway, wood smoke exposure drove fluctuations and compensations across the 28-day time course, ending with decreased hippocampal NAD+ abundance on day 28. Summarily, these results indicate a highly dynamic neuroinflammatory environment, with potential resolution extending past 28 days, the implications of which may include long-term behavioral changes, systemic and neurological sequalae directly associated with wildfire smoke exposure.

Hexavalent Chromium Targets Securin to Drive Numerical Chromosome Instability in Human Lung Cells.

Hexavalent chromium [Cr(VI)] is a known human lung carcinogen with widespread exposure in environmental and occupational settings. Despite well-known cancer risks, the molecular mechanisms of Cr(VI)-induced carcinogenesis are not well understood, but a major driver of Cr(VI) carcinogenesis is chromosome instability. Previously, we reported Cr(VI) induced numerical chromosome instability, premature centriole disengagement, centrosome amplification, premature centromere division, and spindle assembly checkpoint bypass. A key regulator of these events is securin, which acts by regulating the cleavage ability of separase. Thus, in this study we investigated securin disruption by Cr(VI) exposure. We exposed human lung cells to a particulate Cr(VI) compound, zinc chromate, for acute (24 h) and prolonged (120 h) time points. We found prolonged Cr(VI) exposure caused marked decrease in securin levels and function. After prolonged exposure at the highest concentration, securin protein levels were decreased to 15.3% of control cells, while securin mRNA quantification was 7.9% relative to control cells. Additionally, loss of securin function led to increased separase activity manifested as enhanced cleavage of separase substrates; separase, kendrin, and SCC1. These data show securin is targeted by prolonged Cr(VI) exposure in human lung cells. Thus, a new mechanistic model for Cr(VI)-induced carcinogenesis emerges with centrosome and centromere disruption as key components of numerical chromosome instability, a key driver in Cr(VI) carcinogenesis.

Arsenic impairs the lineage commitment of hematopoietic progenitor cells through the attenuation of GATA-2 DNA binding activity.

Arsenic exposure produces significant hematotoxicity in vitro and in vivo. Our previous work shows that arsenic (in the form of arsenite, AsIII) interacts with the zinc finger domains of GATA-1, inhibiting the function of this critical transcription factor, and resulting in the suppression of erythropoiesis. In addition to GATA-1, GATA-2 also plays a key role in the regulation of hematopoiesis. GATA-1 and GATA-2 have similar zinc finger domains (C4-type) that are structurally favorable for AsIII interactions. Taking this into consideration, we hypothesized that early stages of hematopoietic differentiation that are dependent on the function of GATA-2 may also be disrupted by AsIII exposure. We found that in vitro AsIII exposures disrupt the erythromegakaryocytic lineage commitment and differentiation of erythropoietin-stimulated primary mouse bone marrow hematopoietic progenitor cells (HPCs), producing an aberrant accumulation of cells in early stages of hematopoiesis and subsequent reduction of committed erythro-megakaryocyte progenitor cells. Arsenic significantly accumulated in the GATA-2 protein, causing the loss of zinc, and disruption of GATA-2 function, as measured by chromatin immunoprecipitation and the expression of GATA-2 responsive genes. Our results show that the attenuation of GATA-2 function is an important mechanism contributing to the aberrant lineage commitment and differentiation of early HPCs. Collectively, findings from the present study suggest that the AsIII-induced disruption of erythro-megakaryopoiesis may contribute to the onset and/or exacerbation of hematological disorders, such as anemia.

The immunotoxicity of natural and depleted uranium: From cells to people.

Uranium is a naturally occurring element found in the environment as a mixture of isotopes with differing radioactive properties. Enrichment of mined material results in depleted uranium waste with substantially reduced radioactivity but retains the capacity for chemical toxicity. Uranium mine and milling waste are dispersed by wind and rain leading to environmental exposures through soil, air, and water contamination. Uranium exposure is associated with numerous adverse health outcomes in humans, yet there is limited understanding of the effects of depleted uranium on the immune system. The purpose of this review is to summarize findings on uranium immunotoxicity obtained from cell, rodent and human population studies. We also highlight how each model contributes to an understanding of mechanisms that lead to immunotoxicity and limitations inherent within each system. Information from population, animal, and laboratory studies will be needed to significantly expand our knowledge of the contributions of depleted uranium to immune dysregulation, which may then inform prevention or intervention measures for exposed communities.

Exposures to uranium and arsenic alter intraepithelial and innate immune cells in the small intestine of male and female mice.

Human exposures to environmental metals, including uranium (U) and arsenic (As) are a global public health concern. Chronic exposures to U and As are linked to many adverse health effects including, immune suppression and autoimmunity. The gastrointestinal (GI) tract is home to many immune cells vital in the maintenance of systemic immune health. However, very little is known about the immunotoxicity of U and As at this site. The present study examined the burden of U and As exposure in the GI tract as well as the resultant immunotoxicity to intraepithelial lymphocytes (IELs) and innate immune cells of the small intestine following chronic drinking water exposures of male and female mice to U (in the form of uranyl acetate, UA) and As (in the form of sodium arsenite, As3+). Exposure to U or As3+ resulted in high levels of U or As in the GI tract of male and female mice, respectively. A reduction of small intestinal CD4+ IELs (TCRαß+, CD8αα+) was found following As3+ exposure, whereas U produced widespread suppression of CD4- IEL subsets (TCRαß+ and TCRγδ+). Evaluation of innate immune cell subsets in the small intestinal lamina propria revealed a decrease in mature macrophages, along with a corresponding increase in immature/proinflammatory macrophages following As3+ exposures. These data show that exposures to two prevalent environmental contaminants, U and As produce significant immunotoxicity in the GI tract. Collectively, these findings provide a critical framework for understanding the underlying immune health issues reported in human populations chronically exposed to environmental metals.

Minimal uranium immunotoxicity following a 60-day drinking water exposure to uranyl acetate in male and female C57BL/6J mice.

Historical uranium (U) mining in the Southwestern United States resulted in significant environmental contamination throughout this region and presents a significant risk of chronic metal exposure and toxicity for communities living in close proximity to mine waste sites. Uranium exposure is associated with numerous deleterious health effects including immune dysfunction; however, its effects on the immune system have yet to be fully characterized. We recently published that drinking water exposure to U, in the form of uranyl acetate (UA), results in low overall tissue retention of U (<0.01%), with very little accumulation in immune organs (blood, bone marrow, spleen, and thymus) of male and female mice. In the present study we characterized the immunotoxicity of U, in the form of UA, following a 60-day drinking water exposure to 5 and 50 ppm in male and female C57BL/6J mice. The following immunotoxicity endpoints were evaluated: hematology, immune tissue weights and total cell recoveries, immunophenotying of the spleen and thymus, and immune cell function (lymphocyte mitogenesis and T-dependent antibody response). Uranium exposure had subtle impacts on the immune endpoints evaluated, likely due to low U accumulation at these sites. The only significant alterations were a slight decrease in the percentages of splenic natural killer T-cells and macrophages in exposed male mice. Despite minimal immunological effects, this study highlights the importance of investigating toxicological endpoints in both sexes and developing accurate animal models that model epidemiological exposures in the future.

A whale of a tale: whale cells evade the driving mechanism for hexavalent chromium-induced chromosome instability.

Chromosome instability, a hallmark of lung cancer, is a driving mechanism for hexavalent chromium [Cr(VI)] carcinogenesis in humans. Cr(VI) induces structural and numerical chromosome instability in human lung cells by inducing DNA double-strand breaks and inhibiting homologous recombination repair and causing spindle assembly checkpoint (SAC) bypass and centrosome amplification. Great whales are long-lived species with long-term exposures to Cr(VI) and accumulate Cr in their tissue, but exhibit a low incidence of cancer. Data show Cr(VI) induces fewer chromosome aberrations in whale cells after acute Cr(VI) exposure suggesting whale cells can evade Cr(VI)-induced chromosome instability. However, it is unknown if whales can evade Cr(VI)-induced chromosome instability. Thus, we tested the hypothesis that whale cells resist Cr(VI)-induced loss of homologous recombination repair activity and increased SAC bypass and centrosome amplification. We found Cr(VI) induces similar amounts of DNA double-strand breaks after acute (24 h) and prolonged (120 h) exposures in whale lung cells, but does not inhibit homologous recombination repair, SAC bypass, or centrosome amplification, and does not induce chromosome instability. These data indicate whale lung cells resist Cr(VI)-induced chromosome instability, the major driver for Cr(VI) carcinogenesis at a cellular level, consistent with observations that whales are resistant to cancer.

Tungsten toxicity and carcinogenesis.

Tungsten is an emerging contaminant in the environment. Research has demonstrated that humans are exposed to high levels of tungsten in certain settings, primarily due to increased use of tungsten in industrial applications. However, our understanding of the potential human health risks of tungsten exposure is still limited. An important point we have learned about the toxicity profile of tungsten is that it is complex because tungsten can often augment the effects of other co-exposures or co-stressors, which could result in greater toxicity or more severe disease. This has shaped the tungsten toxicology field and the types of research questions being investigated. This has particularly been true when evaluating the toxicity profile of tungsten metal alloys in combination with cobalt. In this chapter, the current state of the tungsten toxicology field will be discussed focusing on data investigating tungsten carcinogenicity and other major toxicities including pulmonary, cardiometabolic, bone, and immune endpoints, either alone or in combination with other metals. Environmental and human monitoring data will also be discussed to highlight human populations most at risk of exposure to high concentrations of tungsten, the forms of tungsten present in each setting, and exposure levels in each population.

Aging, longevity, and the role of environmental stressors: a focus on wildfire smoke and air quality.

Aging is a complex biological process involving multiple interacting mechanisms and is being increasingly linked to environmental exposures such as wildfire smoke. In this review, we detail the hallmarks of aging, emphasizing the role of telomere attrition, cellular senescence, epigenetic alterations, proteostasis, genomic instability, and mitochondrial dysfunction, while also exploring integrative hallmarks - altered intercellular communication and stem cell exhaustion. Within each hallmark of aging, our review explores how environmental disasters like wildfires, and their resultant inhaled toxicants, interact with these aging mechanisms. The intersection between aging and environmental exposures, especially high-concentration insults from wildfires, remains under-studied. Preliminary evidence, from our group and others, suggests that inhaled wildfire smoke can accelerate markers of neurological aging and reduce learning capabilities. This is likely mediated by the augmentation of circulatory factors that compromise vascular and blood-brain barrier integrity, induce chronic neuroinflammation, and promote age-associated proteinopathy-related outcomes. Moreover, wildfire smoke may induce a reduced metabolic, senescent cellular phenotype. Future interventions could potentially leverage combined anti-inflammatory and NAD + boosting compounds to counter these effects. This review underscores the critical need to study the intricate interplay between environmental factors and the biological mechanisms of aging to pave the way for effective interventions.

Acute inhalation of tungsten particles results in early signs of cardiac injury.

Epidemiological studies have established that exposure to tungsten increases the risk of developing cardiovascular diseases. However, no studies have investigated how tungsten affects cardiac function or the development of cardiovascular disease. Inhalation of tungsten particulates is relevant in occupational settings, and inhalation of particulate matter has a known causative role in driving cardiovascular disease. This study examined if acute inhalation to tungsten particulates affects cardiac function and leads to heart tissue alterations. Female BALB/c mice were exposed to Filtered Air or 1.5 ± 0.23 mg/m3 tungsten particles, using a whole-body inhalation chamber, 4 times over the course of two weeks. Inhalation exposure resulted in mild pulmonary inflammation characterized by an increased percentage and number of macrophages and metabolomic changes in the lungs. Cardiac output was significantly decreased in the tungsten-exposed group. Additionally, A', an indicator of the amount of work required by the atria to fill the heart was elevated. Cardiac gene expression analysis revealed, tungsten exposure increased expression of pro-inflammatory cytokines, markers of remodeling and fibrosis, and oxidative stress genes. These data strongly suggest exposure to tungsten results in cardiac injury characterized by early signs of diastolic dysfunction. Functional findings are in parallel, demonstrating cardiac oxidative stress, inflammation, and early fibrotic changes. Tungsten accumulation data would suggest these cardiac changes are driven by systemic consequences of pulmonary damage.

Arsenic causes distinct gene expression changes in macrophages polarized in vitro with either interferon-γ or interleukin-4.

Arsenic exposure is correlated with atherosclerosis in epidemiological studies and in animal models. We have previously shown that arsenic exposure enhanced the atherosclerotic plaque size, increased the plaque lipid content, and decreased the plaque smooth muscle cell and collagen contents in the apolipoprotein E knockout (apoE-/-) mice. However, the percentage of plaque-resident macrophages, the primary drivers of atherosclerosis remained unchanged. Therefore, we hypothesized that although arsenic does not change the quantity of macrophages, it alters the macrophage transcriptome towards a proatherogenic state. To test this hypothesis, we used bone marrow-derived macrophages, polarized them to either interferon-γ (IFN-É£) stimulated, proinflammatory or interleukin-4 (IL-4) stimulated, alternatively activated macrophages in the presence or absence of 0.67 µM (50 ppb) arsenic and performed RNA sequencing. Arsenic exposure altered the gene expression of the macrophages in a subtype-specific manner. Most differentially expressed genes (88%) were altered specifically in either IFN-É£- or IL-4-stimulated macrophages, whereas in the remaining 12% of genes that changed in both cell types, did so in opposite directions. In IL-4-stimulated macrophages, arsenic significantly downregulated the genes involved in cholesterol biosynthesis and the chemokines CCL17/CCL22, whereas in IFN-É£-stimulated macrophages, the genes associated with the liver X receptor (LXR) pathway were downregulated by arsenic. Using a bone marrow transplant experiment, we validated that the deletion of LXRα from the hematopoietic compartment rescued arsenic-enhanced atherosclerosis in the apoE-/- mouse model. Together, these data suggest that arsenic modulates subtype-specific transcriptomic changes in macrophages and further emphasize the need to define macrophage heterogeneity in atherosclerotic plaques in order to evaluate the proatherogenic role of arsenic.

Arsenite Methyltransferase Is an Important Mediator of Hematotoxicity Induced by Arsenic in Drinking Water.

Chronic arsenic exposures via the consumption of contaminated drinking water are clearly associated with many deleterious health outcomes, including anemia. Following exposure, trivalent inorganic arsenic (AsIII) is methylated through a series of arsenic (+III oxidation state) methyltransferase (As3MT)-dependent reactions, resulting in the production of several intermediates with greater toxicity than the parent inorganic arsenicals. The extent to which inorganic vs. methylated arsenicals contribute to AsIII-induced hematotoxicity remains unknown. In this study, the contribution of As3MT-dependent biotransformation to the development of anemia was evaluated in male As3mt-knockout (KO) and wild-type, C57BL/6J, mice following 60-day drinking water exposures to 1 mg/L (ppm) AsIII. The evaluation of hematological indicators of anemia revealed significant reductions in red blood cell counts, hemoglobin levels, and hematocrit in AsIII-exposed wild-type mice as compared to unexposed controls. No such changes in the blood of As3mt-KO mice were detected. Compared with unexposed controls, the percentages of mature RBCs in the bone marrow and spleen (measured by flow cytometry) were significantly reduced in the bone marrow of AsIII-exposed wild-type, but not As3mt-KO mice. This was accompanied by increased levels of mature RBCS in the spleen and elevated levels of circulating erythropoietin in the serum of AsIII-exposed wild-type, but not As3mt-KO mice. Taken together, the findings from the present study suggest that As3MT-dependent biotransformation has an essential role in mediating the hematotoxicity of AsIII following drinking water exposures.

Neuroinflammatory and Metabolomic Temporal Dynamics Following Wood Smoke Inhalation.

Smoke from wildland fires has been shown to produce neuroinflammation in preclinical models, characterized by neural infiltrations of neutrophils and monocytes, as well as altered neurovascular endothelial phenotypes. To address the longevity of such outcomes, the present study examined the neuroinflammatory and metabolomic temporal dynamics after inhalation exposures from biomass-derived smoke. 2-month-old female C57BL/6J mice were exposed to wood smoke every other day for two weeks at an average exposure concentration of 0.5mg/m 3 . Subsequent serial euthanasia occurred at 1-, 3-, 7-, 14-, and 28-days post-exposure. Flow cytometry of right hemispheres revealed two endothelial populations of PECAM (CD31), high and medium expressors, with wood smoke inhalation causing an increased proportion of PECAM Hi . These populations of PECAM Hi and PECAM Med were associated with an anti-inflammatory and pro-inflammatory response, respectively, and their inflammatory profiles were largely resolved by the 28-day mark. However, activated microglial populations (CD11b + /CD45 low ) remained higher in wood smoke-exposed mice than controls at day 28. Infiltrating neutrophil populations decreased to levels below controls by day 28. However, the MHC-II expression of the peripheral immune infiltrate remained high, and the population of neutrophils retained an increased expression of CD45, Ly6C, and MHC-II. Utilizing an unbiased approach examining the metabolomic alterations, we observed notable hippocampal perturbations in neurotransmitter and signaling molecules like glutamate, quinolinic acid, and 5-α-dihydroprogesterone. Utilizing a targeted panel designed to explore the aging-associated NAD + metabolic pathway, wood smoke exposure drove fluctuations and compensations across the 28-day time course, ending with decreased hippocampal NAD + abundance at day 28. Summarily, these results indicate a highly dynamic neuroinflammatory environment, with potential resolution extending past 28 days, the implications of which may include long-term behavioral changes, systemic and neurological sequalae directly associated wtith wildfire smoke exposure.

Arsenite-induced autophagy is associated with proteotoxicity in human lymphoblastoid cells.

Epidemiological studies of arsenic-exposed populations have provided evidence that arsenic exposure in humans is associated with immunosuppression. Previously, we have reported that arsenite-induced toxicity is associated with the induction of autophagy in human lymphoblastoid cell lines (LCL). Autophagy is a cellular process that functions in the degradation of damaged cellular components, including protein aggregates formed by misfolded or damaged proteins. Accumulation of misfolded or damaged proteins in the endoplasmic reticulum (ER) lumen causes ER stress and activates the unfolded protein response (UPR). In an effort to investigate the mechanism of autophagy induction by arsenite in the LCL model, we examined the potential contribution of ER stress and activation of the UPR. LCL exposed to sodium arsenite for 8-days induced expression of UPR-activated genes, including CHOP and GRP78, at the RNA and the protein level. Evidence for activation of the three arms of the UPR was observed. The arsenite-induced activation of the UPR was associated with an accumulation of protein aggregates containing p62 and LC3, proteins with established roles in the sequestration and autophagic clearance of protein aggregates. Taken together, these data provide evidence that arsenite-induced autophagy is associated with the generation of ER stress, activation of the UPR, and formation of protein aggregates that may be targeted to the lysosome for degradation.

Autophagy in toxicology: self-consumption in times of stress and plenty.

Autophagy is a critical cellular process orchestrating the lysosomal degradation of cellular components in order to maintain cellular homeostasis and respond to cellular stress. A growing research effort over the last decade has proven autophagy to be essential for constitutive protein and organelle turnover, for embryonic/neonatal survival and for cell survival during conditions of environmental stress. Emphasizing its biological importance, dysfunctional autophagy contributes to a diverse set of human diseases. Cellular stress induced by xenobiotic exposure typifies environmental stress, and can result in the induction of autophagy as a cytoprotective mechanism. An increasing number of xenobiotics are notable for their ability to modulate the induction or the rate of autophagy. The role of autophagy in normal cellular homeostasis, the intricate relationship between cellular stress and the induction of autophagy, and the identification of specific xenobiotics capable of modulating autophagy, point to the importance of the autophagic process in toxicology. This review will summarize the importance of autophagy and its role in cellular response to stress, including examples in which consideration of autophagy has contributed to a more complete understanding of toxicant-perturbed systems.

Microneedle Array Technique for the Longitudinal Extraction of Interstitial Fluid without Hair Removal.

Interstitial fluid (ISF) bathes the cells and tissues and is in constant exchange with blood. As an exchange medium for waste, nutrients, exosomes, and signaling molecules, ISF is recognized as a plentiful source of biomolecules. Many basic and pre-clinical small animal studies could benefit from an inexpensive and efficient technique that allows for the in vivo extraction of ISF for the subsequent quantification of molecules in the interstitial space. We have previously reported on a minimally invasive technique for the extraction of ISF using a 3D-printed microneedle array (MA) platform for comprehensive biomedical applications. Previously, hairless animal models were utilized, and euthanasia was performed immediately following the procedure. Here, we demonstrate the technique in Sprague Dawley rats, without the need for hair removal, over multiple extractions and weeks. As an example of this technique, we report simultaneous quantification of the heavy metals Copper (Cu), Lead (Pb), Lithium (Li), and Nickel (Ni) within the ISF, compared with whole blood. These results demonstrate the MA technique applicability to a broader range of species and studies and the reuse of animals, leading to a reduction in number of animals needed to successfully complete ISF extraction experiments.

Neuroinflammatory and Neurometabolomic Consequences From Inhaled Wildfire Smoke-Derived Particulate Matter in the Western United States.

Utilizing a mobile laboratory located >300 km away from wildfire smoke (WFS) sources, this study examined the systemic immune response profile, with a focus on neuroinflammatory and neurometabolomic consequences, resulting from inhalation exposure to naturally occurring wildfires in California, Arizona, and Washington in 2020. After a 20-day (4 h/day) exposure period in a mobile laboratory stationed in New Mexico, WFS-derived particulate matter (WFPM) inhalation resulted in significant neuroinflammation while immune activity in the peripheral (lung, bone marrow) appeared to be resolved in C57BL/6 mice. Importantly, WFPM exposure increased cerebrovascular endothelial cell activation and expression of adhesion molecules (VCAM-1 and ICAM-1) in addition to increased glial activation and peripheral immune cell infiltration into the brain. Flow cytometry analysis revealed proinflammatory phenotypes of microglia and peripheral immune subsets in the brain of WFPM-exposed mice. Interestingly, endothelial cell neuroimmune activity was differentially associated with levels of PECAM-1 expression, suggesting that subsets of cerebrovascular endothelial cells were transitioning to resolution of inflammation following the 20-day exposure. Neurometabolites related to protection against aging, such as NAD+ and taurine, were decreased by WFPM exposure. Additionally, increased pathological amyloid-beta protein accumulation, a hallmark of neurodegeneration, was observed. Neuroinflammation, together with decreased levels of key neurometabolites, reflect a cluster of outcomes with important implications in priming inflammaging and aging-related neurodegenerative phenotypes.

Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance.

Promoting the macroautophagy/autophagy-mediated degradation of specific proteins and organelles can potentially be utilized to induce apoptosis in cancer cells or sensitize tumor cells to therapy. To examine this concept, we enriched for autophagosomes from histone deacetylase inhibitor (HDACi)-sensitive U937 lymphoma cells and isogenic HDACi-resistant cells. Mass spectrometry on autophagosome-enriched fractions revealed that HDACi-resistant cells undergo elevated pexophagy, or autophagy of the peroxisome, an organelle that supports tumor growth. To disturb peroxisome homeostasis, we enhanced pexophagy in HDACi-resistant cells via genetic silencing of peroxisome exportomer complex components (PEX1, PEX6, or PEX26). This consequently sensitized resistant cells to HDACi-mediated apoptosis, which was rescued by inhibiting ATM/ataxia-telangiectasia mutated (ATM serine/threonine kinase), a mediator of pexophagy. We subsequently engineered melanoma cells to stably repress PEX26 using CRISPR interference (CRISPRi). Melanoma cells with repressed PEX26 expression showed evidence of both increased pexophagy and peroxisomal matrix protein import defects versus single guide scrambled (sgSCR) controls. In vivo studies showed that sgPEX26 melanoma xenografts recurred less compared to sgSCR xenografts, following the development of resistance to mitogen-activated protein kinase (MAPK)-targeted therapy. Finally, prognostic analysis of publicly available datasets showed that low expression levels of PEX26, PEX6 and MTOR, were significantly associated with prolonged patient survival in lymphoma, lung cancer and melanoma cohorts. Our work highlighted that drugs designed to disrupt peroxisome homeostasis may serve as unconventional therapies to combat therapy resistance in cancer.Abbreviations: ABCD3/PMP70: ATP binding cassette subfamily D member 3; ACOX1: acyl-CoA oxidase 1; AP: autophagosome; COX: cytochrome c oxidase; CQ: chloroquine; CRISPRi: clustered regularly interspaced short palindromic repeats interference; DLBCL: diffuse large B-cell lymphoma; GO: gene ontology; dCas9: Cas9 endonuclease dead, or dead Cas9; HDACi: histone deacetylase inhibitors; IHC: Immunohistochemistry; LAMP2: lysosomal associated membrane protein 2; LCFAs: long-chain fatty acids; LFQ-MS: label-free quantitation mass spectrometry; LPC: lysophoshatidylcholine; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PBD: peroxisome biogenesis disorders; PTS1: peroxisomal targeting signal 1; ROS: reactive oxygen species; sgRNA: single guide RNA; VLCFAs: very-long chain fatty acids; Vor: vorinostat; WO: wash-off.

Arsenite and monomethylarsonous acid disrupt erythropoiesis through combined effects on differentiation and survival pathways in early erythroid progenitors.

Strong epidemiological evidence demonstrates an association between chronic arsenic exposure and anemia. We recently found that As+3 impairs erythropoiesis by disrupting the function of GATA-1; however the downstream pathways impacted by the loss of GATA-1 function have not been evaluated. Additionally, our previous findings indicate that the predominant arsenical in the bone marrow of mice exposed to As+3 in their drinking water for 30 days was MMA+3, but the impacts of this arsenical on erythorpoisis also remain largely unknown. The goal of this study was to address these critical knowledge gaps by evaluating the comparative effects of arsenite (As+3) and the As+3 metabolite, monomethyarsonous acid (MMA+3) on two critical regulatory pathways that control the differentiation and survival of early erythroid progenitor cells. We found that 500 nM As+3 and 100 and 500 nM MMA+3 suppress erythropoiesis by impairing the differentiation of early stage erythroid progenitors. The suppression of early erythroid progenitor cell development was attributed to combined effects on differentiation and survival pathways mediated by disruption of GATA-1 and STAT5. Our results show that As+3 primarily disrupted GATA-1 function; whereas, MMA+3 suppressed both GATA-1 and STAT5 activity. Collectively, these findings provide novel mechanistic insights into arsenic-induced dyserythropoiesis and suggest that MMA+3 may be more toxic than As+3 to early developing erythroid cells.