Heterogeneous Oxidation Products of Fine Particulate Isoprene Epoxydiol-Derived Methyltetrol Sulfates Increase Oxidative Stress and Inflammatory Gene Responses in Human Lung Cells.

Hydroxyl radical (·OH)-initiated oxidation of isoprene, the most abundant nonmethane hydrocarbon in the atmosphere, is responsible for substantial amounts of secondary organic aerosol (SOA) within ambient fine particles. Fine particulate 2-methyltetrol sulfate diastereoisomers (2-MTSs) are abundant SOA products formed via acid-catalyzed multiphase chemistry of isoprene-derived epoxydiols with inorganic sulfate aerosols under low-nitric oxide conditions. We recently demonstrated that heterogeneous ·OH oxidation of particulate 2-MTSs leads to the particle-phase formation of multifunctional organosulfates (OSs). However, it remains uncertain if atmospheric chemical aging of particulate 2-MTSs induces toxic effects within human lung cells. We show that inhibitory concentration-50 (IC50) values decreased from exposure to fine particulate 2-MTSs that were heterogeneously aged for 0 to 22 days by ·OH, indicating increased particulate toxicity in BEAS-2B lung cells. Lung cells further exhibited concentration-dependent modulation of oxidative stress- and inflammatory-related gene expression. Principal component analysis was carried out on the chemical mixtures and revealed positive correlations between exposure to aged multifunctional OSs and altered expression of targeted genes. Exposure to particulate 2-MTSs alone was associated with an altered expression of antireactive oxygen species (ROS)-related genes (NQO-1, SOD-2, and CAT) indicative of a response to ROS in the cells. Increased aging of particulate 2-MTSs by ·OH exposure was associated with an increased expression of glutathione pathway-related genes (GCLM and GCLC) and an anti-inflammatory gene (IL-10).

CpG methylation patterns in placenta and neonatal blood are differentially associated with neonatal inflammation.

BACKGROUND: Infants born extremely premature are at increased risk for health complications later in life for which neonatal inflammation may be a contributing biological driver. Placental CpG methylation provides mechanistic information regarding the relationship between prenatal epigenetic programming, prematurity, neonatal inflammation, and later-in-life health. METHODS: We contrasted CpG methylation in the placenta and neonatal blood spots in relation to neonatal inflammation in the Extremely Low Gestational Age Newborn (ELGAN) cohort. Neonatal inflammation status was based on the expression of six inflammation-related proteins, assessed as (1) day-one inflammation (DOI) or (2) intermittent or sustained systemic inflammation (ISSI, inflammation on ≥2 days in the first 2 postnatal weeks). Epigenome-wide CpG methylation was assessed in 354 placental samples and 318 neonatal blood samples. RESULTS: Placental CpG methylation displayed the strongest association with ISSI (48 CpG sites) but was not associated with DOI. This was in contrast to CpG methylation in blood spots, which was associated with DOI (111 CpG sites) and not with ISSI (one CpG site). CONCLUSIONS: Placental CpG methylation was strongly associated with ISSI, a measure of inflammation previously linked to later-in-life cognitive impairment, while day-one neonatal blood methylation was associated with DOI. IMPACT: Neonatal inflammation increases the risk of adverse later-life outcomes, especially in infants born extremely preterm. CpG methylation in the placenta and neonatal blood spots were evaluated in relation to neonatal inflammation assessed via circulating proteins as either (i) day-one inflammation (DOI) or (ii) intermittent or sustained systemic inflammation (ISSI, inflammation on ≥2 days in the first 2 weeks). Tissue specificity was observed in epigenetic-inflammatory relationships: placental CpG methylation was associated with ISSI, neonatal blood CpG methylation was associated with DOI. Supporting the placental origins of disease framework, placental epigenetic patterns are associated with a propensity for ISSI in neonates.

Ex vivo exposures to arsenite and its methylated trivalent metabolites alter gene transcription in mouse sperm cells.

We have previously reported that preconception exposure to iAs may contribute to the development of diabetes in mouse offspring by altering gene expressions in paternal sperm. However, the individual contributions of iAs and its methylated metabolites, monomethylated arsenic (MAs) and dimethylated arsenic (DMAs), to changes in the sperm transcriptome could not be determined because all three As species are present in sperm after in vivo iAs exposure. The goal of the present study was to assess As species-specific effects using an ex vivo model. We exposed freshly isolated mouse sperm to either 0.1 or 1 µM arsenite (iAsIII) or the methylated trivalent arsenicals, MAsIII and DMAsIII, and used RNA-sequencing to identify differentially expressed genes, enriched pathways, and associated protein networks. For all arsenicals tested, the exposures to 0.1 µM concentrations had greater effects on gene expression than 1 µM exposures. Transcription factor AP-1 and B cell receptor complexes were the most significantly enriched pathways in sperm exposed to 0.1 µM iAsIII. The Mre11 complex and Antigen processing were top pathways targeted by exposure to 0.1 µM MAsIII and DMAsIII, respectively. While there was no overlap between gene transcripts altered by ex vivo exposures in the present study and those altered by in vivo exposure in our prior work, several pathways were shared, including PI3K-Akt signaling, Focal adhesion, and Extracellular matrix receptor interaction pathways. Notably, the protein networks associated with these pathways included those with known roles in diabetes. This study is the first to assess the As species-specific effects on sperm transcriptome, linking these effects to the diabetogenic effects of iAs exposure.

Differential placental CpG methylation is associated with chronic lung disease of prematurity.

BACKGROUND: Chronic lung disease (CLD) is the most common pulmonary morbidity in extremely preterm infants. It is unclear to what extent prenatal exposures influence the risk of CLD. Epigenetic variation in placenta DNA methylation may be associated with differential risk of CLD, and these associations may be dependent upon sex. METHODS: Data were obtained from a multi-center cohort of infants born extremely preterm (<28 weeks' gestation) and an epigenome-wide approach was used to identify associations between placental DNA methylation and CLD (n = 423). Associations were evaluated using robust linear regression adjusting for covariates, with a false discovery rate of 0.05. Analyses stratified by sex were used to assess differences in methylation-CLD associations. RESULTS: CLD was associated with differential methylation at 49 CpG sites representing 46 genes in the placenta. CLD was associated with differential methylation of probes within genes related to pathways involved in fetal lung development, such as p53 signaling and myo-inositol biosynthesis. Associations between CpG methylation and CLD differed by sex. CONCLUSIONS: Differential placental methylation within genes with key roles in fetal lung development may reflect complex cell signaling between the placenta and fetus which mediate CLD risk. These pathways appear to be distinct based on fetal sex. IMPACT: In extremely preterm infants, differential methylation of CpG sites within placental genes involved in pathways related to cell signaling, oxidative stress, and trophoblast invasion is associated with chronic lung disease of prematurity. DNA methylation patterns associated with chronic lung disease were distinctly based on fetal sex, suggesting a potential mechanism underlying dimorphic phenotypes. Mechanisms related to fetal hypoxia and placental myo-inositol signaling may play a role in fetal lung programming and the developmental origins of chronic lung disease. Continued research of the relationship between the placental epigenome and chronic lung disease could inform efforts to ameliorate or prevent this condition.

Wildfire Variable Toxicity: Identifying Biomass Smoke Exposure Groupings through Transcriptomic Similarity Scoring.

The prevalence of wildfires continues to grow globally with exposures resulting in increased disease risk. Characterizing these health risks remains difficult due to the wide landscape of exposures that can result from different burn conditions and fuel types. This study tested the hypothesis that biomass smoke exposures from variable fuels and combustion conditions group together based on similar transcriptional response profiles, informing which wildfire-relevant exposures may be considered as a group for health risk evaluations. Mice (female CD-1) were exposed via oropharyngeal aspiration to equal mass biomass smoke condensates produced from flaming or smoldering burns of eucalyptus, peat, pine, pine needles, or red oak species. Lung transcriptomic signatures were used to calculate transcriptomic similarity scores across exposures, which informed exposure groupings. Exposures from flaming peat, flaming eucalyptus, and smoldering eucalyptus induced the greatest responses, with flaming peat grouping with the pro-inflammatory agent lipopolysaccharide. Smoldering red oak and smoldering peat induced the least transcriptomic response. Groupings paralleled pulmonary toxicity markers, though they were better substantiated by higher data dimensionality and resolution provided through -omic-based evaluation. Interestingly, groupings based on smoke chemistry signatures differed from transcriptomic/toxicity-based groupings. Wildfire-relevant exposure groupings yield insights into risk assessment strategies to ultimately protect public health.

Placental programming, perinatal inflammation, and neurodevelopment impairment among those born extremely preterm.

Individuals born extremely preterm are at significant risk for impaired neurodevelopment. After discharge from the neonatal intensive care, associations between the child's well-being and factors in the home and social environment become increasingly apparent. Mothers' prenatal health and socioeconomic status are associated with neurodevelopmental outcomes, and emotional and behavioral problems. Research on early life risk factors and on mechanisms underlying inter-individual differences in neurodevelopment later in life can inform the design of personalized approaches to prevention. Here, we review early life predictors of inter-individual differences in later life neurodevelopment among those born extremely preterm. Among biological mechanisms that mediate relationships between early life predictors and later neurodevelopmental outcomes, we highlight evidence for disrupted placental processes and regulated at least in part via epigenetic mechanisms, as well as perinatal inflammation. In relation to these mechanisms, we focus on four prenatal antecedents of impaired neurodevelopment, namely, (1) fetal growth restriction, (2) maternal obesity, (3) placental microorganisms, and (4) socioeconomic adversity. In the future, this knowledge may inform efforts to detect and prevent adverse outcomes in infants born extremely preterm. IMPACT: This review highlights early life risk factors and mechanisms underlying inter-individual differences in neurodevelopment later in life. The review emphasizes research on early life risk factors (fetal growth restriction, maternal obesity, placental microorganisms, and socioeconomic adversity) and on mechanisms (disrupted placental processes and perinatal inflammation) underlying inter-individual differences in neurodevelopment later in life. The findings highlighted here may inform efforts to detect and prevent adverse outcomes in infants born extremely preterm.

Isoprene-Derived Secondary Organic Aerosol Induces the Expression of MicroRNAs Associated with Inflammatory/Oxidative Stress Response in Lung Cells.

Exposure to fine particulate matter (PM2.5), of which secondary organic aerosol (SOA) is a major constituent, is linked to adverse health outcomes, including cardiovascular disease, lung cancer, and preterm birth. Atmospheric oxidation of isoprene, the most abundant nonmethane hydrocarbon emitted into Earth's atmosphere primarily from vegetation, contributes to SOA formation. Isoprene-derived SOA has previously been found to alter inflammatory/oxidative stress genes. MicroRNAs (miRNAs) are epigenetic regulators that serve as post-transcriptional modifiers and key mediators of gene expression. To assess whether isoprene-derived SOA alters miRNA expression, BEAS-2B lung cells were exposed to laboratory-generated isoprene-derived SOA constituents derived from the acid-driven multiphase chemistry of authentic methacrylic acid epoxide (MAE) or isomeric isoprene epoxydiols (IEPOX) with acidic sulfate aerosol particles. These IEPOX- and MAE-derived SOA constituents have been shown to be measured in large quantities within PM2.5 collected from isoprene-rich areas affected by acidic sulfate aerosol particles derived from human activities. A total of 29 miRNAs were identified as differentially expressed when exposed to IEPOX-derived SOA and 2 when exposed to MAE-derived SOA, a number of which are inflammatory/oxidative stress associated. These results suggest that miRNAs may modulate the inflammatory/oxidative stress response to SOA exposure, thereby advancing the understanding of airway cell epigenetic response to SOA.

Evaluation of inhaled low-dose formaldehyde-induced DNA adducts and DNA-protein cross-links by liquid chromatography-tandem mass spectrometry.

As a widespread industrial chemical, formaldehyde carcinogenicity has been highly controversial. Meanwhile, formaldehyde is an essential metabolite in all living cells. Previously, we have demonstrated exogenous formaldehyde causes DNA adducts in a nonlinear manner between 0.7 and 15.2 ppm using [13CD2]-formaldehyde for exposure coupled with the use of sensitive mass spectrometry. However, the responses from exposure to low doses of formaldehyde are still unknown. In this study, rats were exposed to 1, 30, and 300 ppb [13CD2]-formaldehyde for 28 days (6 h/day) by nose-only inhalation, followed by measuring DNA mono-adduct (N2-HOMe-dG) and DNA-protein crosslinks (dG-Me-Cys) as formaldehyde specific biomarkers. Both exogenous and endogenous DNA mono-adducts and dG-Me-Cys were examined with ultrasensitive nano-liquid chromatography-tandem mass spectrometry. Our data clearly show that endogenous adducts are present in all tissues analyzed, but exogenous adducts were not detectable in any tissue samples, including the most susceptible nasal epithelium. Moreover, formaldehyde exposure at 1, 30 and 300 ppb did not alter the levels of endogenous formaldehyde-induced DNA adducts or DNA-protein crosslinks. The novel findings from this study provide new data for risk assessment of exposure to low doses of formaldehyde.

Accurate Measurement of Formaldehyde-Induced DNA-Protein Cross-Links by High-Resolution Orbitrap Mass Spectrometry.

Genomic instability caused by DNA-protein cross-link (DPCs)-induced DNA damage is implicated in disease pathogenesis, aging, and cancer development. The covalent linkages between DNA and protein are induced by chemical reactions catalyzed by the endogenous metabolic intermediates and exogenous agents, such as aldehydes, chemotherapeutic agents, and ionizing radiation. Formaldehyde has been classified as a genotoxic carcinogen. In addition, endogenous formaldehyde-induced DPCs may increase the risks of bone marrow toxicity and leukemia. There is a need to develop an effective detection method for DPC analysis, including the structural differentiation of endogenous and exogenous formaldehyde-induced DPCs. To this end, our group previously reported a useful liquid chromatography-selected reaction monitoring (LC-SRM) approach coupled with stable isotope labeling and low mass resolution-triple quadrupole mass spectrometry. In the present work, we further demonstrate an accurate quantification method using a high-resolution, accurate-mass Orbitrap mass spectrometer for the measurement of the covalent linkage between 2'-deoxyguanosine (dG) and cysteine (Cys), specifically termed dG-Me-Cys, one kind of linkages derived from the formaldehyde-induced DPCs. This quantification method with a wide dynamic range of at least 3 orders generates an interference-free spectrum for unbiased and unambiguous quantification, resulting in good intra- and interday precisions and accuracies with less than 10% variations. The endogenous and exogenous amounts of dG-Me-Cys in a human cell line treated with formaldehyde are analyzed by our new methodology. The quantification strategy demonstrated in this study can be widely applied to characterize and quantify other DPC linkages induced by formaldehyde or other chemical agents.

DNA Product Formation in Female Sprague-Dawley Rats Following Polyhalogenated Aromatic Hydrocarbon (PHAH) Exposure.

DNA oxidation damage has been regarded as one of the possible mechanisms for the hepatic carcinogenesis of dioxin-like compounds (DLCs). In this study, we evaluated the toxic equivalency factor (TEF) from the standpoint of induced DNA oxidation products and their relationship to toxicity and carcinogenicity. Nine DNA oxidation products were analyzed in the liver of female Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) alone or the tertiary mixture of TCDD, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) by gavage for 14, 31, and 53 weeks (5 days/week) by LC-MS/MS: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo); 1,N6-etheno-2'-deoxyadenosine (1,N6-εdAdo); N2,3-ethenoguanine (N2,3-εG); 7-(2-oxoethly)guanine (7-OEG); 1,N2-etheno-2'-deoxyguanosine (1,N2-εdGuo); malondialdehyde (M1dGuo); acrolein (AcrdGuo); crotonaldehyde (CrdGuo); and 4-hydroxynonenal (HNEdGuo) derived 2'-deoxyguanosine adducts. Exposure to TCDD (100 ng/kg/day) significantly induced 1,N6-εdAdo at 31 and 53 weeks, while no increase of 8-oxo-dGuo was observed. Significant increases were observed for 8-oxo-dGuo and 1,N6-εdAdo at all time points following exposure to the tertiary mixture (TEQ 100 ng/kg/day). Exposure to TCDD for 53 weeks only significantly increased 1,N6-εdAdo, while increases of N2,3-εG and 7-OEG were only found in the highest dose group (100 ng/kg/day). Exposure to the tertiary mixture for 53 weeks had no effect on N2,3-εG in any exposure group (TEQ 0, 22, 46, or 100 ng/kg/day), while significant increases were observed for 1,N6-εdAdo (all dose groups), 8-oxo-dGuo (46 and 100 ng/kg/day), and 7-OEG (100 ng/kg/day). While no significant increase was observed at 53 weeks for 1,N2-εdGuo, M1dGuo, AcrdGuo, or CrdGuo following exposure to TCDD (100 ng/kg/day), all of them were significantly induced in animals exposed to the tertiary mixture (TEQ 100 ng/kg/day). This oxidation DNA product data suggest that the simple TEF methodology cannot be applied to evaluate the diverse patterns of toxic effects induced by DLCs.

Prolactin prevents hepatocellular carcinoma by restricting innate immune activation of c-Myc in mice.

Women are more resistant to hepatocellular carcinoma (HCC) than men despite equal exposure to major risk factors, such as hepatitis B or C virus infection. Female resistance is hormone-dependent, as evidenced by the sharp increase in HCC incidence in postmenopausal women who do not take hormone replacement therapy. In rodent models sex-dimorphic HCC phenotypes are pituitary-dependent, suggesting that sex hormones act via the gonadal-hypophyseal axis. We found that the estrogen-responsive pituitary hormone prolactin (PRL), signaling through hepatocyte-predominant short-form prolactin receptors (PRLR-S), constrained TNF receptor-associated factor (TRAF)-dependent innate immune responses invoked by IL-1ß, TNF-α, and LPS/Toll-like receptor 4 (TLR4), but not TRIF-dependent poly(I:C)/TLR3. PRL ubiquitinated and accelerated poststimulatory decay of a "trafasome" comprised of IRAK1, TRAF6, and MAP3K proteins, abrogating downstream activation of c-Myc-interacting pathways, including PI3K/AKT, mTORC1, p38 MAPK, and NF-κB. Consistent with this finding, we documented exaggerated male liver responses to immune stimuli in mice and humans. Tumor promotion through, but regulation above, the level of c-Myc was demonstrated by sex-independent HCC eruption in Alb-Myc transgenic mice. PRL deficiency accelerated liver carcinogenesis in Prl(-/-) mice of both sexes. Conversely, pharmacologic PRL mobilization using the dopamine D2 receptor antagonist domperidone prevented HCC in tumor-prone C3H/HeN males. Viewed together, our results demonstrate that PRL constrains tumor-promoting liver inflammation by inhibiting MAP3K-dependent activation of c-Myc at the level of the trafasome. PRL-targeted therapy may hold promise for reducing the burden of liver cancer in high-risk men and women.

Polychlorinated Biphenyls Induce Oxidative DNA Adducts in Female Sprague-Dawley Rats.

Polychlorinated biphenyls (PCBs) are organic chemicals that were traditionally produced and widely used in industry as mixtures and are presently formed as byproducts of pigment and dye manufacturing. They are known to persist and bioaccumulate in the environment. Some have been shown to induce liver cancer in rodents. Although the mechanism of the toxicity of PCBs is unknown, it has been shown that they increase oxidative stress, including lipid peroxidation. We hypothesized that oxidative stress-induced DNA damage could be a contributor for PCB carcinogenesis and analyzed several DNA adducts in female Sprague-Dawley rats exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and a binary mixture (PCB 126 + 153) for 14, 31, and 53 wks. Eight adducts were measured to profile oxidative DNA lesions, including 8-oxo-deoxyguanosine (8-oxo-dG), 1,N(6)-ethenodeoxyadenosine (1,N(6)-εdA), N(2),3-ethenoguanine (N(2),3-εG), 1,N(2)-ethenodeoxyguanosine (1,N(2)-εdG), as well as malondialdehyde (M1dG), acrolein (AcrdG), crotonaldehyde (CrdG), and 4-hydroxynonenal-derived dG adducts (HNEdG) by LC-MS/MS analysis. Statistically significant increases were observed for 8-oxo-dG and 1,N(6)-εdA concentrations in hepatic DNA of female rats exposed to the binary mixture (1000 ng/kg/day + 1000 µg/kg/day) but not in rats exposed to PCB 126 (1000 ng/kg/day) or PCB 153 (1000 µg/kg/day) for 14 and 31 wks. However, exposure to PCB 126 (1000 ng/kg/day) for 53 wks significantly increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, and M1dG. Exposure to PCB 153 (1000 µg/kg/day) for 53 wks increased 8-oxo-dG, and 1,N(6)-εdA. Exposure to the binary mixture for 53 wks increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, 1,N(2)-εdG, and N(2),3-εG significantly above control groups. Increased hepatic oxidative DNA adducts following exposure to PCB 126, PCB 153, or the binary mixture shows that an increase in DNA damage may play an important role in hepatic toxicity and carcinogenesis in female Sprague-Dawley rats.

Identifying and Responding to Lead in Drinking Water in a University Setting.

Lead is an established neurotoxicant, and it has known associations with adverse neurodevelopmental and reproductive outcomes. Exposure to lead at any level is unsafe, and the United States (US) has enacted various federal and state legislations to regulate lead levels in drinking water in K-12 schools and childcare facilities; however, no regulations exist for higher education settings. Upon the discovery of lead in drinking water fixtures in the University of North Carolina at Chapel Hill (UNC-CH) campus, a cross-campus water testing network and sampling plan was developed and deployed. The campaign was based on the US Environmental Protection Agency's (EPA) 3Ts (Training, Testing, and Taking Action) guidance. The seven-month campaign involved 5954 tests on 3825 drinking water fixtures across 265 buildings. A total of 502 (8.43%) tests showed lead above the limit of detection (1 part per billion, ppb), which represented 422 (11.03%) fixtures. Fewer than 1.5% of the tests were above the EPA action level for public water systems (15 ppb). In conclusion, systematic testing of all the fixtures across campus was required to identify localized contamination, and each entity in the cross-campus network undertook necessary roles to generate a successful testing campaign. UNC-CH established preventative measures to test drinking water fixtures every three years, which provide a framework for other higher education institutions in responding to lead contamination.

Extracellular Vesicles altered by a Per- and Polyfluoroalkyl Substance Mixture: In Vitro Dose-Dependent Release, Chemical Content, and MicroRNA Signatures involved in Liver Health.

Per- and polyfluoroalkyl substances (PFAS) have emerged as high priority contaminants due to their ubiquity and pervasiveness in the environment. Numerous PFAS co-occur across sources of drinking water, including areas of North Carolina (NC) with some detected concentrations above the Environmental Protection Agency's health advisory levels. While evidence demonstrates PFAS exposure induces harmful effects in the liver, the involvement of extracellular vesicles (EVs) as potential mediators of these effects has yet to be evaluated. This study set out to evaluate the hypothesis that PFAS mixtures induce dose-dependent release of EVs from liver cells, with exposures causing differential loading of microRNAs (miRNAs) and PFAS chemical signatures. To test this hypothesis, a defined PFAS mixture was prioritized utilizing data collected by the NC PFAS Testing Network. This mixture contained three substances, PFOS, PFOA, and PFHxA, selected based upon co-occurrence patterns and the inclusion of both short-chain (PFHxA) and long-chain (PFOA and PFOS) substances. HepG2 liver cells were exposed to equimolar PFAS, and secreted EVs were isolated from conditioned media and characterized for count and molecular content. Exposures induced a dose-dependent release of EVs carrying miRNAs that were differentially loaded upon exposure. These altered miRNA signatures were predicted to target mRNA pathways involved in hepatic fibrosis and cancer. Chemical concentrations of PFOS, PFOA, and PFHxA were also detected in both parent HepG2 cells and their released EVs, specifically within a 15-fold range after normalizing for protein content. This study therefore established EVs as novel biological responders and measurable endpoints for evaluating PFAS-induced toxicity.

Insulin resistance and metabolic hepatocarcinogenesis with parent-of-origin effects in A×B mice.

Insulin resistance is a defining feature of metabolic syndrome and type 2 diabetes mellitus but also may occur independently of these conditions. Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of these disorders, increases the risk of hepatocellular carcinoma (HCC). However, mechanisms linking hyperinsulinemia to NAFLD and HCC require clarification. We describe a novel model of primary insulin resistance and HCC with strong parent-of-origin effects. Male AB6F1 (A/JCr dam × C57BL/6 sire) but not B6AF1 (B6 dam × A/J sire) mice developed spontaneous insulin resistance, NAFLD, and HCC without obesity or diabetes. A survey of mitochondrial, imprinted, and sex-linked traits revealed modest associations with X-linked genes. However, a diet-induced obesity study, including B6.A chromosome substitution-strain (consomic) mice, showed no segregation by sex chromosome. Thus, parent-of-origin effects were specified within the autosomal genome. Next, we interrogated mechanisms of insulin-associated hepatocarcinogenesis. Steatotic hepatocytes exhibited adipogenic transition characterized by vacuolar metaplasia and up-regulation of vimentin, adipsin, fatty acid translocase (CD36), peroxisome proliferator-activated receptor-γ, and related products. This profile was largely recapitulated in insulin-supplemented primary mouse hepatocyte cultures. Importantly, pyruvate kinase M2, a fetal anabolic enzyme implicated in the Warburg effect, was activated by insulin in vivo and in vitro. Thus, our study reveals parent-of-origin effects in heritable insulin resistance, implicating adipogenic transition with acquired anabolic metabolism in the progression from NAFLD to HCC.

Chemical Mixtures in Household Environments: In Silico Predictions and In Vitro Testing of Potential Joint Action on PPARγ in Human Liver Cells.

There are thousands of chemicals that humans can be exposed to in their everyday environments, the majority of which are currently understudied and lack substantial testing for potential exposure and toxicity. This study aimed to implement in silico methods to characterize the chemicals that co-occur across chemical and product uses in our everyday household environments that also target a common molecular mediator, thus representing understudied mixtures that may exacerbate toxicity in humans. To detail, the Chemical and Products Database (CPDat) was queried to identify which chemicals co-occur across common exposure sources. Chemicals were preselected to include those that target an important mediator of cell health and toxicity, the peroxisome proliferator activated receptor gamma (PPARγ), in liver cells that were identified through query of the ToxCast/Tox21 database. These co-occurring chemicals were thus hypothesized to exert potential joint effects on PPARγ. To test this hypothesis, five commonly co-occurring chemicals (namely, benzyl cinnamate, butyl paraben, decanoic acid, eugenol, and sodium dodecyl sulfate) were tested individually and in combination for changes in the expression of PPARγ and its downstream target, insulin receptor (INSR), in human liver HepG2 cells. Results showed that these likely co-occurring chemicals in household environments increased both PPARγ and INSR expression more significantly when the exposures occurred as mixtures vs. as individual chemicals. Future studies will evaluate such chemical combinations across more doses, allowing for further quantification of the types of joint action while leveraging this method of chemical combination prioritization. This study demonstrates the utility of in silico-based methods to identify chemicals that co-occur in the environment for mixtures toxicity testing and highlights relationships between understudied chemicals and changes in PPARγ-associated signaling.

Wildfires and extracellular vesicles: Exosomal MicroRNAs as mediators of cross-tissue cardiopulmonary responses to biomass smoke.

INTRODUCTION: Wildfires are a threat to public health world-wide that are growing in intensity and prevalence. The biological mechanisms that elicit wildfire-associated toxicity remain largely unknown. The potential involvement of cross-tissue communication via extracellular vesicles (EVs) is a new mechanism that has yet to be evaluated. METHODS: Female CD-1 mice were exposed to smoke condensate samples collected from the following biomass burn scenarios: flaming peat; smoldering peat; flaming red oak; and smoldering red oak, representing lab-based simulations of wildfire scenarios. Lung tissue, bronchoalveolar lavage fluid (BALF) samples, peripheral blood, and heart tissues were collected 4 and 24 h post-exposure. Exosome-enriched EVs were isolated from plasma, physically characterized, and profiled for microRNA (miRNA) expression. Pathway-level responses in the lung and heart were evaluated through RNA sequencing and pathway analyses. RESULTS: Markers of cardiopulmonary tissue injury and inflammation from BALF samples were significantly altered in response to exposures, with the greatest changes occurring from flaming biomass conditions. Plasma EV miRNAs relevant to cardiovascular disease showed exposure-induced expression alterations, including miR-150, miR-183, miR-223-3p, miR-30b, and miR-378a. Lung and heart mRNAs were identified with differential expression enriched for hypoxia and cell stress-related pathways. Flaming red oak exposure induced the greatest transcriptional response in the heart, a large portion of which were predicted as regulated by plasma EV miRNAs, including miRNAs known to regulate hypoxia-induced cardiovascular injury. Many of these miRNAs had published evidence supporting their transfer across tissues. A follow-up analysis of miR-30b showed that it was increased in expression in the heart of exposed mice in the absence of changes to its precursor molecular, pri-miR-30b, suggesting potential transfer from external sources (e.g., plasma). DISCUSSION: This study posits a potential mechanism through which wildfire exposures induce cardiopulmonary responses, highlighting the role of circulating plasma EVs in intercellular and systems-level communication between tissues.

Placental genomics mediates genetic associations with complex health traits and disease.

As the master regulator in utero, the placenta is core to the Developmental Origins of Health and Disease (DOHaD) hypothesis but is historically understudied. To identify placental gene-trait associations (GTAs) across the life course, we perform distal mediator-enriched transcriptome-wide association studies (TWAS) for 40 traits, integrating placental multi-omics from the Extremely Low Gestational Age Newborn Study. At [Formula: see text], we detect 248 GTAs, mostly for neonatal and metabolic traits, across 176 genes, enriched for cell growth and immunological pathways. In aggregate, genetic effects mediated by placental expression significantly explain 4 early-life traits but no later-in-life traits. 89 GTAs show significant mediation through distal genetic variants, identifying hypotheses for distal regulation of GTAs. Investigation of one hypothesis in human placenta-derived choriocarcinoma cells reveal that knockdown of mediator gene EPS15 upregulates predicted targets SPATA13 and FAM214A, both associated with waist-hip ratio in TWAS, and multiple genes involved in metabolic pathways. These results suggest profound health impacts of placental genomic regulation in developmental programming across the life course.

Measurement of mitochondrial DNA copy number in dried blood spots: A pilot study.

BACKGROUND: We evaluated the feasibility of mitochondrial DNA (mtDNA) copy number measurement in dried blood spots (DBS), its comparability with measurement in whole blood samples, and stability of mtDNA copy number from DBS over time. METHODS: Women in this pilot study were participants in the Sister Study, a large prospective cohort. Sister Study participants provided a whole blood sample and DBS at enrollment. A second DBS sample was collected 5-10 years later from a subcohort of women with and without an incident breast cancer diagnosis between collections. Among 54 women (27 with breast cancer, 27 without) we measured mtDNA copy number from whole blood at enrollment and from DBS at both time points. RESULTS: The average age at enrollment was 58.7 years (range:50-69). Values of mtDNA copy number measured in whole blood samples and DBS from enrollment were moderately correlated (Spearman R = 0.45; p = 0.005). Stability of mtDNA copy number in DBS from the two time points was moderate overall (ICC = 0.50) and similar between women with (ICC = 0.50) and without (ICC = 0.51) a breast cancer diagnosis between the two collections. CONCLUSIONS: Our results suggest that measurement of mtDNA copy number in DBS is feasible and may be a valid alternative to measurement in whole blood samples.