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The environmental contaminant dibutyl phthalate (DBP) is reported to be hepatotoxic, but the underlying molecular pathways and pathological processes remain unclear. Here we used RNA-sequencing to characterize persistent hepatic transcriptional effects one week after the conclusion of five weeks oral exposure to 10 mg/kg/day or 100 mg/kg/day DBP in adult male mice. The exploratory transcriptome analysis demonstrated five differentially expressed genes (DEGs) in the 10 mg/kg/day group and 13 in the 100 mg/kg/day group. Gene Set Enrichment Analysis (GSEA), which identifies affected biological pathways rather than focusing solely on individual genes, revealed nine significantly enriched Reactome pathways shared by both DBP treatment groups. Additionally, we found 54 upregulated and one downregulated Reactome pathways in the 10 mg/kg/day DBP group, and 29 upregulated and 13 downregulated pathways in the 100 mg/kg/day DBP group. DBP exposure disrupted several key biological processes, including protein translation, protein folding, apoptosis, Hedgehog signaling, degradation of extracellular matrix and alterations in the energy/lipid metabolism. Subsequent liver tissue analysis confirmed that DBP exposure induced tissue disorganization, oxidative stress, lipid accumulation, increased TNF-α, ATP and glucokinase levels, and affected key metabolic proteins, predominantly in a dose-response manner. Overall, the results show that DBP can cause hepatic stress and damage and suggest a potential role for DBP in the development of non-alcoholic fatty liver disease, the most prevalent liver disease worldwide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

DNA methylation plays a pivotal role in cancer. The ubiquitous contaminant perfluorooctanesulfonic acid (PFOS) has been epidemiologically associated with breast cancer, and can induce proliferation and malignant transformation of normal human breast epithelial cells (MCF-10A), but the information about its effect on DNA methylation is sparse. The aim of this study was to characterize the whole-genome methylome effects of PFOS in our breast cell model and compare the findings with previously demonstrated DNA methylation alterations in breast tumor tissues. The DNA methylation profile was assessed at single CpG resolution in MCF-10A cells treated with 1 μM PFOS for 72 h by using Enzymatic Methyl sequencing (EM-seq). We found 12,591 differentially methylated CpG-sites and 13,360 differentially methylated 100 bp tiles in the PFOS exposed breast cells. These differentially methylated regions (DMRs) overlapped with 2406 genes of which 494 were long non-coding RNA and 1841 protein coding genes. We identified 339 affected genes that have been shown to display altered DNA methylation in breast cancer tissue and several other genes related to cancer development. This includes hypermethylation of GACAT3, DELEC1, CASC2, LCIIAR, MUC16, SYNE1 and hypomethylation of TTN and KMT2C. DMRs were also found in estrogen receptor genes (ESR1, ESR2, ESRRG, ESRRB, GREB1) and estrogen responsive genes (GPER1, EEIG1, RERG). The gene ontology analysis revealed pathways related to cancer phenotypes such as cell adhesion and growth. These findings improve the understanding of PFOS's potential role in breast cancer and illustrate the value of whole-genome methylome analysis in uncovering mechanisms of chemical effects, identifying biomarker candidates, and strengthening epidemiological associations, potentially impacting risk assessment., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The unsustainable use of manmade chemicals poses significant threats to biodiversity and human health. Emerging evidence highlights the potential of certain chemicals to cause transgenerational impacts on metabolic health. Here, we investigate male transmitted epigenetic transgenerational effects of the anti-androgenic herbicide linuron in the pancreas of Xenopus tropicalis frogs, and their association with metabolic phenotypes. Reduced representation bisulfite sequencing (RRBS) was used to assess genome-wide DNA methylation patterns in the pancreas of adult male F2 generation ancestrally exposed to environmentally relevant linuron levels (44 ± 4.7 μg/L). We identified 1117 differentially methylated regions (DMRs) distributed across the X. tropicalis genome, revealing potential regulatory mechanisms underlying metabolic disturbances. DMRs were identified in genes crucial for pancreatic function, including calcium signalling (clstn2, cacna1d and cadps2), genes associated with type 2 diabetes (tcf7l2 and adcy5) and a biomarker for pancreatic ductal adenocarcinoma (plec). Correlation analysis revealed associations between DNA methylation levels in these genes and metabolic phenotypes, indicating epigenetic regulation of glucose metabolism. Moreover, differential methylation in genes related to histone modifications suggests alterations in the epigenetic machinery. These findings underscore the long-term consequences of environmental contamination on pancreatic function and raise concerns about the health risks associated with transgenerational effects of pesticides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Cecilia Berg is now employed at The Swedish Medical Products Agency, and Sofie Svanholm is now employed at The Swedish Chemicals Agency, but The Swedish Medical Products Agency or The Swedish Chemicals Agency were not involved in any part of this study. All work was carried out when employed at Uppsala University. The views expressed in this study are that of the authors alone and do not reflect those of The Swedish Medical Products Agency or The Swedish Chemicals Agency., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Chemical risk assessments typically focus on single substances, often overlooking real-world co-exposures to chemical mixtures. Mixture toxicology studies using representative mixtures can reveal potential chemical interactions, but these do not account for the unique chemical profiles that occur in the blood of diverse individuals. Here we used the H295R steroidogenesis assay to screen personalized mixtures of 24 persistent organic pollutants (POPs) for cytotoxicity and endocrine disruption. Each mixture was reconstructed at a human exposure relevant concentration (1×), as well as at 10- and 100-fold higher concentration (10×, 100×) by acoustic liquid handling based on measured blood concentrations in a Swedish cohort. Among the twelve mixtures tested, nine mixtures decreased the cell viability by 4-18%, primarily at the highest concentration. While the median and maximum mixtures based on the whole study population induced no measurable effects on steroidogenesis at any concentration, the personalized mixture from an individual with the lowest total POPs concentration was the only mixture that affected estradiol synthesis (35% increase at the 100× concentration). Mixtures reconstructed from blood levels of three different individuals stimulated testosterone synthesis at the 1× (11-15%) and 10× concentrations (12-16%), but not at the 100× concentration. This proof-of-principle personalized toxicity study illustrates that population-based representative chemical mixtures may not adequately account for the toxicological risks posed to individuals. It highlights the importance of testing a range of real-world mixtures at relevant concentrations to explore potential interactions and non-monotonic effects. Further toxicological studies of personalized contaminant mixtures could improve chemical risk assessment and advance the understanding of human health, as chemical exposome data become increasingly available., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study demonstrated the strengths of in vivo molecular staining coupled with automated imaging analysis in Daphnia magna . A multiwell plate protocol was developed to assess mitochondrial membrane potential using the JC-1 dye. The suitability of five common anesthetics was initially tested, and 5% ethanol performed best in terms of anesthetic effects and healthy recovery. The staining conditions were optimized to 30 min staining with 2 μM JC-1 for best J-aggregate formation. The protocol was validated with the model compound carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and used to measure the effect of four environmental contaminants, 2,4-dinitrophenol, triclosan, n -(1,3-dimethylbutyl)- N '-phenyl-p-phenylenediamine (6PPD), and ibuprofen, on mitochondrial health. Test organisms were imaged using an automated confocal microscope, and fluorescence intensities were automatically quantified. The effect concentrations for CCCP were lower by a factor of 30 compared with the traditional OECD 202 acute toxicity test. Mitochondrial effects were also detected at lower concentrations for all tested environmental contaminants compared to the OCED 202 test. For 2,4-dinitrophenol, mitochondria effects were detectable after 2 h exposure to environmentally relevant concentrations and predicted organism death was observed after 24 h. The high sensitivity and time efficiency of this novel automated imaging method make it a valuable tool for advancing ecotoxicological testing.

The Breast Cancer UK-Breast Cancer Prevention Conference addressed risk from environmental pollutants and health behaviour-related breast-cancer risk. Epidemiological studies examining individual chemicals and breast cancer risk have produced inconclusive results including endocrine disrupting chemicals (EDCs) Bisphenol A, per- and polyfluorinated alkyl substances as well as aluminium. However, laboratory studies have shown that multiple EDCs, can work together to exhibit effects, even when combined at levels that alone are ineffective. The TEXB-α/β assay measures total estrogenic load, and studies have provided evidence of a link between multiple-chemical exposures and breast cancer. However, prospective studies using TEXB-α/β are needed to establish a causative link. There is also a need to assess real-life exposure to environmental-chemical mixtures during pregnancy, and their potential involvement in programming adverse foetal health outcomes in later life. Higher rates of breast cancer have occurred alongside increases in potentially-modifiable risk factors such as obesity. Increasing body-mass index is associated with increased risk of developing postmenopausal breast cancer, but with decreased risk of premenopausal breast cancer. In contrast, lower rates of breast cancer in Asian compared to Western populations have been linked to soya/isoflavone consumption. Risk is decreased by breastfeeding, which is in addition to the decrease in risk observed for each birth and a young first-birth. Risk is lower in those with higher levels of self-reported physical activity. Current evidence suggests breast-cancer survivors should also avoid weight gain, be physically active, and eat a healthy diet for overall health. A broad scientific perspective on breast cancer risk requires focus on both environmental exposure to chemicals and health behaviour-related risk. Research into chemical exposure needs to focus on chemical mixtures and prospective epidemiological studies in order to test the effects on breast cancer risk. Behaviour-related research needs to focus on implementation as well as deeper understanding of the mechanisms of cancer prevention., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Many persistent organic pollutants (POPs) are suspected endocrine disruptors and it is important to investigate their effects at low concentrations relevant to human exposure. Here, the OECD test guideline #456 steroidogenesis assay was downscaled to a 96-well microplate format to screen 24 POPs for their effects on viability, and testosterone and estradiol synthesis using the human adrenocortical cell line H295R. The compounds (six polyfluoroalkyl substances, five organochlorine pesticides, ten polychlorinated biphenyls and three polybrominated diphenyl ethers) were tested at human-relevant levels (1 nM to 10 µM). Increased estradiol synthesis, above the OECD guideline threshold of 1.5-fold solvent control, was shown after exposure to 10 µM PCB-156 (153%) and PCB-180 (196%). Interestingly, the base hormone synthesis varied depending on the cell batch. An alternative data analysis using a linear mixed-effects model that include multiple independent experiments and considers batch-dependent variation was therefore applied. This approach revealed small but statistically significant effects on estradiol or testosterone synthesis for 17 compounds. Increased testosterone levels were demonstrated even at 1 nM for PCB-74 (18%), PCB-99 (29%), PCB-118 (16%), PCB-138 (19%), PCB-180 (22%), and PBDE-153 (21%). The MTT assay revealed significant effects on cell viability after exposure to 1 nM of perfluoroundecanoic acid (12%), 3 nM PBDE-153 (9%), and 10 µM of PCB-156 (6%). This shows that some POPs can interfere with endocrine signaling at concentrations found in human blood, highlighting the need for further investigation into the toxicological mechanisms of POPs and their mixtures at low concentrations relevant to human exposure., (© 2024. The Author(s).)

To improve the mechanistic screening of reproductive toxicants in  chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood-testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases., (© 2024. The Author(s).)

The herbicide linuron can cause endocrine disrupting effects in Xenopus tropicalis frogs, including offspring that were never exposed to the contaminant. The mechanisms by which these effects are transmitted across generations need to be further investigated. Here, we examined transgenerational alterations of brain and testis DNA methylation profiles paternally inherited from grandfathers developmentally exposed to an environmentally relevant concentration of linuron. Reduced representation bisulfite sequencing (RRBS) revealed numerous differentially methylated regions (DMRs) in brain (3060 DMRs) and testis (2551 DMRs) of the adult male F2 generation. Key genes in the brain involved in somatotropic (igfbp4) and thyrotropic signaling (dio1 and tg) were differentially methylated and correlated with phenotypical alterations in body size, weight, hind limb length and plasma glucose levels, indicating that these methylation changes could be potential mediators of the transgenerational effects of linuron. Testis DMRs were found in genes essential for spermatogenesis, meiosis and germ cell development (piwil1, spo11 and tdrd9) and their methylation levels were correlated with the number of germ cells nests per seminiferous tubule, an endpoint of disrupted spermatogenesis. DMRs were also identified in several genes central for the machinery that regulates the epigenetic landscape including DNA methylation (dnmt3a and mbd2) and histone acetylation (hdac8, ep300, elp3, kat5 and kat14), which may at least partly drive the linuron-induced transgenerational effects. The results from this genome-wide DNA methylation profiling contribute to better understanding of potential transgenerational epigenetic inheritance mechanisms in amphibians., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All work was carried out when employed at Uppsala University. Sofie Svanholm is now employed at The Swedish Chemicals Agency but The Swedish Chemicals Agency was not involved in any part of this study. The views expressed in this study are that of the author alone and do not reflect those of the Swedish Chemicals Agency., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This work presents a case study in applying a systematic review framework (SYRINA) to the identification of chemicals as endocrine disruptors. The suitability and performance of the framework is tested with regard to the widely accepted World Health Organization definition of an endocrine disruptor (ED). The endocrine disrupting potential of triphenyl phosphate (TPP), a well-studied flame retardant reported to exhibit various endocrine related effects was assessed. We followed the 7 steps of the SYRINA framework, articulating the research objective via Populations, Exposures, Comparators, Outcomes (PECO) statements, performed literature search and screening, conducted study evaluation, performed data extraction and summarized and integrated the evidence. Overall, 66 studies, consisting of in vivo , in vitro and epidemiological data, were included. We concluded that triphenyl phosphate could be identified as an ED based on metabolic disruption and reproductive function. We found that the tools used in this case study and the optimizations performed on the framework were suitable to assess properties of EDs. A number of challenges and areas for methodological development in systematic appraisal of evidence relating to endocrine disrupting potential were identified; significant time and effort were needed for the analysis of in vitro mechanistic data in this case study, thus increasing the workload and time needed to perform the systematic review process. Further research and development of this framework with regards to grey literature (non-peer-reviewed literature) search, harmonization of study evaluation methods, more consistent evidence integration approaches and a pre-defined method to assess links between adverse effect and endocrine activity are recommended. It would also be advantageous to conduct more case studies for a chemical with less data than TPP.

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3β-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Concerns have been raised regarding the potential adverse health effects of the ubiquitous herbicide glyphosate. Here, we investigated long-term effects of developmental exposure to a glyphosate-based herbicide (GBH) by analyzing serum melatonin levels and cellular changes in the striatum of adult male rats (90 days old). Pregnant and lactating rats were exposed to 3% GBH (0.36% glyphosate) through drinking water from gestational day 5 to postnatal day 15. The offspring showed reduced serum melatonin levels (43%) at the adult age compared with the control group. The perinatal exposure to GBH also induced long-term oxidative stress-related changes in the striatum demonstrated by increased lipid peroxidation (45%) and DNA/RNA oxidation (39%) together with increased protein levels of the antioxidant enzymes, superoxide dismutase (SOD1, 24%), glutamate-cysteine ligase (GCLC, 58%), and glutathione peroxidase 1 (GPx1, 31%). Moreover, perinatal GBH exposure significantly increased the total number of neurons (20%) and tyrosine hydroxylase (TH)-positive neurons (38%) in the adult striatum. Mechanistic in vitro studies with primary rat pinealocytes exposed to 50 µM glyphosate demonstrated a decreased melatonin secretion partially through activation of metabotropic glutamate receptor 3 (mGluR3), while higher glyphosate levels (100 or 500 µM) also reduced the pinealocyte viability. Since decreased levels of the important antioxidant and neuroprotector melatonin have been associated with an increased risk of developing neurodegenerative disorders, this demonstrates the need to consider the melatonin hormone system as a central endocrine-related target of glyphosate and other environmental contaminants.

Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 µg flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Müllerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Müllerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 µg/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Müllerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Di-n-butyl phthalate (DBP) is a ubiquitous environmental contaminant linked with various adverse health effects, including immune system dysfunction. Gut microbial dysbiosis can contribute to a wide range of pathogenesis, particularly immune disease. Here, we investigated the impact of DBP on the gut microbiome and examined correlations with immune system changes after five weeks oral exposure (10 or 100 mg/kg/day) in adult male mice. The fecal microbiome composition was characterized using 16S rRNA sequencing. DBP-treated mice displayed a significantly distinct microbial community composition, indicated by Bray-Curtis distance. Numerous amplicon sequence variants (ASVs) at the genus level were altered. Compared to the vehicle control group, the 10 mg/kg/day DBP group had 63 more abundant and 65 less abundant ASVs, while 60 ASVs were increased and 76 ASVs were decreased in the 100 mg/kg/day DBP group. Both DBP treatment groups showed higher abundances of ASVs assigned to Desulfovibrio (Proteobacteria phylum) and Enterorhabdus genera, while ASVs belonging to Parabacteroides, Lachnospiraceae UCG-006 and Lachnoclostridium were less common compared to the control group. Interestingly, an ASV belonging to Rumniniclostridium 6, which was less abundant in DBP-treated mice, demonstrated a negative correlation with the increased number of non-classical monocytes observed in the blood of DBP-treated animals. In addition, an ASV from Lachnospiraceae UCG-001, which was more abundant in the DBP-treated animals, showed a positive correlation with the non-classical monocyte increase. This study shows that DBP exposure greatly modifies the gut bacterial microbiome and indicates a potential contribution of microbial dysbiosis to DBP-induced immune system impairment, illustrating the importance of investigating how interactions between exposome components can affect health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Increased exposure to manmade chemicals may be linked to an increase in immune-related diseases in humans and immune system dysfunction in wildlife. Phthalates are a group of endocrine-disrupting chemicals (EDCs) suspected to influence the immune system. The aim of this study was to characterize the persistent effects on leukocytes in the blood and spleen, as well as plasma cytokine and growth factor levels, one week after the end of five weeks of oral treatment with dibutyl phthalate (DBP; 10 or 100 mg/kg/d) in adult male mice. Flow cytometry analysis of the blood revealed that DBP exposure decreased the total leukocyte count, classical monocyte and T helper (Th) populations, whereas it increased the non-classical monocyte population compared to the vehicle control (corn oil). Immunofluorescence analysis of the spleen showed increased CD11b + Ly6G + (marker of polymorphonuclear myeloid-derived suppressor cells; PMN-MDSCs), and CD43 + staining (marker of non-classical monocytes), whereas CD3 + (marker of total T cells) and CD4 + (marker of Th cells) staining decreased. To investigate the mechanisms of action, levels of plasma cytokines and chemokines were measured using multiplexed immunoassays and other key factors were analyzed using western blotting. The observed increase in M-CSF levels and the activation of STAT3 may promote PMN-MDSC expansion and activity. Increased ARG1, NOX2 (gp91phox), and protein nitrotyrosine levels, as well as GCN2 and phosphor-eIRFα, suggest that oxidative stress and lymphocyte arrest drive the lymphocyte suppression caused by PMN-MDSCs. The plasma levels of IL-21 (promotes the differentiation of Th cells) and MCP-1 (regulates migration and infiltration of monocytes/macrophages) also decreased. These findings show that adult DBP exposure can cause persistent immunosuppressive effects, which may increase susceptibility to infections, cancers, and immune diseases, and decrease vaccine efficacy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Nontarget mass spectrometry has great potential to reveal patterns of water contamination globally through community science, but few studies are conducted in low-income countries, nor with open-source workflows, and few datasets are FAIR (Findable, Accessible, Interoperable, Reusable). Water was collected from urban and rural rivers around Dhaka, Bangladesh, and analyzed by liquid chromatography high-resolution mass spectrometry in four ionization modes (electrospray ionization ±, atmospheric pressure chemical ionization ±) with data-independent MS2 acquisition. The acquisition strategy was complementary: 19,427 and 7365 features were unique to ESI and APCI, respectively. The complexity of water pollution was revealed by >26,000 unique molecular features resolved by MS-DIAL, among which >20,000 correlated with urban sources in Dhaka. A major wastewater treatment plant was not a dominant pollution source, consistent with major contributions from uncontrolled urban drainage, a result that encourages development of further wastewater infrastructures. Matching of deconvoluted MS2 spectra to public libraries resulted in 62 confident annotations ( i.e. , Level 1-2a) and allowed semiquantification of 42 analytes including pharmaceuticals, pesticides, and personal care products. In silico structure prediction for the top 100 unknown molecular features associated with an urban source allowed 15 additional chemicals of anthropogenic origin to be annotated ( i.e. , Level 3). The authentic MS2 spectra were uploaded to MassBank Europe, mass spectral data were openly shared on the MassIVE repository, a tool ( i.e. , MASST) that could be used for community science environmental surveillance was demonstrated, and current limitations were discussed.

Perfluoroalkyl substances (PFAS) have been associated with cancer, but the potential underlying mechanisms need to be further elucidated and include studies of PFAS mixtures. This mechanistic study revealed that very low concentrations (500 pM) of the binary PFOS and PFOA mixture induced synergistic effects on human epithelial breast cell (MCF-10A) proliferation. The cell proliferation was mediated by pregnane X receptor (PXR) activation, an increase in cyclin D1 and CDK6/4 levels, decrease in p21 and p53 levels, and by regulation of phosphor-Akt and β-catenin. The PFAS mixture also altered histone modifications, epigenetic mechanisms implicated in tumorigenesis, and promoted cell migration and invasion by reducing the levels of occludin. High-content screening using the cell painting assay, revealed that hundreds of cell features were affected by the PFAS mixture even at the lowest concentration tested (100 pM). The detailed phenotype profiling further demonstrated that the PFAS mixture altered cell morphology, mostly in parameters related to intensity and texture associated with mitochondria, endoplasmic reticulum, and nucleoli. Exposure to higher concentrations (≥50 µM) of the PFOS and PFOA mixture caused cell death through synergistic interactions that induced oxidative stress, DNA/RNA damage, and lipid peroxidation, illustrating the complexity of mixture toxicology. Increased knowledge about mixture-induced effects is important for better understanding of PFAS' possible role in cancer etiology, and may impact the risk assessment of these and other compounds. This study shows the potential of image-based multiplexed fluorescence assays and high-content screening for development of new approach methodologies in toxicology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The widespread environmental contaminant di-n-butyl phthalate (DBP) has been linked with reduced testosterone levels and adverse reproductive health outcomes in men. However, the underlying mechanisms of these anti-androgenic effects and the potential effects on other classes of steroid hormones remain to be elucidated. Here, we conducted mechanistic studies in human adrenocortical H295R cells exposed to 1-500 µM of DBP or its metabolite, mono-n-butyl phthalate (MBP), for 48 h. Quantification of steroid hormones in the cell medium by liquid chromatography-mass spectrometry revealed that both phthalates significantly decreased testosterone, androstenedione, corticosterone, and progesterone levels, in particular after dibutyryl-cyclic-AMP stimulation of steroidogenesis. Western blot analysis of key steroidogenic proteins showed that DBP induced a dose-dependent decrease of CYP11A1 and HSD3β2 levels, while MBP only significantly decreased CYP17A1 levels, indicating that the compounds affect early steps of the steroidogenesis differently. Both DBP and MBP exposure also lead to a dose-related decrease in HSD17β3, the enzyme which catalyzes the final step in the testosterone biosynthesis pathway, although these effects were not statistically significant. Interestingly, DBP increased the cortisol concentration, which may be due to the non-significant CYP11B1 increase in DBP-exposed cells. In contrast, MBP decreased cortisol concentration. Moreover, the analysis of superoxide generation and quantification of the protein oxidation marker nitrotyrosine demonstrated that DBP induced oxidative stress in H295R cells while MBP reduced protein nitrotyrosine levels. These findings confirm the anti-androgenic effects of DBP and MBP and reveal several differences in their toxicological mechanisms, with possible implications for future research on phthalate toxicity.

Studies indicate that phthalates are endocrine disruptors affecting reproductive health. One of the most commonly used phthalates, di-n-butyl phthalate (DBP), has been linked with adverse reproductive health outcomes in men, but the mechanisms behind these effects are still poorly understood. Here, adult male mice were orally exposed to DBP (10 or 100 mg/kg/day) for five weeks, and the testis and adrenal glands were collected one week after the last dose, to examine more persistent effects. Quantification of testosterone, androstenedione, progesterone and corticosterone concentrations by liquid chromatography-mass spectrometry showed that testicular testosterone was significantly decreased in both DBP treatment groups, whereas the other steroids were not significantly altered. Western blot analysis of testis revealed that DBP exposure increased the levels of the steroidogenic enzymes CYP11A1, HSD3β2, and CYP17A1, the oxidative stress marker nitrotyrosine, and the luteinizing hormone receptor (LHR). The analysis further demonstrated increased levels of the germ cell marker DAZL, the Sertoli cell markers vimentin and SOX9, and the Leydig cell marker SULT1E1. Overall, the present work provides more mechanistic understanding of how adult DBP exposure can induce effects on the male reproductive system by affecting several key cells and proteins important for testosterone biosynthesis and spermatogenesis, and for the first time shows that these effects persist at least one week after the last dose. It also demonstrates impairment of testosterone biosynthesis at a lower dose than previously reported.

It is important to identify the factors that influence the prevalence of disinhibitory behaviors, as tobacco and alcohol use in adolescence is a strong predictor of continued use and substance abuse into adulthood. Organochlorine pesticides (OCPs) are persistent organic pollutants that pose a potential risk to the developing fetus and offspring long-term health. We examined associations between prenatal exposure OCPs and their metabolites (i.e., p,p'-DDT, p,p'-DDE, o,p'-DDT, oxychlordane, and hexachlorobenzene (HCB)), both as a mixture and single compounds, and alcohol consumption and smoking at adolescence in a sample (n = 554) from the Child Health and Development Studies prospective birth cohort. Bayesian Kernel Machine Regression demonstrated a trend of higher risk of alcohol use and smoking with higher quartile mixture levels. Single-component analysis showed increased odds of smoking and drinking with increases in lipid-adjusted p,p'-DDE serum levels (aOR = 2.06, 95% CI 0.99-4.31, p = 0.05, per natural log unit increase). We found significant effect modification in these associations by sex with higher p,p'-DDT serum levels (aOR = 0.26, 95% CI 0.09-0.076, p = 0.01, per natural log unit increase) was associated with lower odds of smoking and drinking in female adolescents, while higher p,p'-DDE serum levels (aOR = 2.98, 95% CI 1.04-8.51, p = 0.04, per natural log unit increase) was associated with higher odds of the outcomes. Results of the mutually adjusted model were not significant for male adolescents. Further research to understand reasons for these sex-differences are warranted., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Environmental contaminants including long-chain per- and polyfluoroalkyl substances (PFAS) have been linked to cancer, which is a central cause of mortality in humans and many wildlife species. Today shorter-chain PFAS are extensively used as replacement compounds and commonly found in the environment. Mechanistic studies are important for a better understanding of their toxicological potential and possible role in cancer etiology. Here, we treated normal human breast epithelial cells (MCF-10A) with 500 pM to 500 μM of perfluorohexane sulfonate (PFHxS), undecafluorohexanoic acid (PFHxA), hexafluoropropylene oxide-dimer acid (GenX), perfluoro 3,6 dioxaoctanoic acid (PFO2OA), heptafluorobutyric acid (HFBA) and perfluorobutanesulfonic acid (PFBS) for 72 h to investigate potential effects on cell proliferation and neoplastic transformation. PFHxA, GenX, PFO2OA, HFBA and PFBS induced no alterations compared to controls at any of the concentrations tested. Exposure to 100 μM PFHxS on the other hand was shown to affect important regulatory cell-cycle proteins (cyclin D1, CDK6, p27, p53 and ERK) and induced cell proliferation, at least in part through activation of the constitutive androstane receptor (CAR) and the peroxisome proliferator-activated receptor alpha (PPARα). PFHxS also altered histone modifications and induced cell malignance by reducing the levels of adhesion proteins (E-cadherin and β-integrin) and promoting cell migration and invasion. These results demonstrate that five out of six alternative PFAS tested are clearly less harmful to MCF-10A cells than previously studied PFOS and PFOA, but raise concerns about PFHxS that also has been associated with breast cancer in epidemiological studies., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Per and polifluorinated substances (PFAS) are ubiquitous and persistent contaminants. Studies have indicated that fetuses and infants can be exposed to these chemicals in utero and through breastfeeding. Despite this, limited data about their effects on brain development are available. Here, we compared the effects of perfluoroctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) exposure in rat primary neurons and neural stem cells (NSC). Treatment with 1-250 μM of either of these compounds caused no effects on cell viability or proliferation in primary neurons, while PFOS exposure increased the NSC proliferation already at the lowest concentration tested (1-100 μM). Further analysis showed that both PFOS and PFOA caused morphological alterations of NSC-derived neurons. The neurons derived from NSC treated with either of the PFAS demonstrated a decrease in cell body area. Exposure to 1 and 10 μM PFOA also affected the neurite network and caused an increase in the number of processes and branches per cell. None of the PFAS caused morphological alterations in primary neurons. These data suggest that NSC, mimicking the immature brain, is clearly more susceptible to PFOS and PFOA exposure than the primary neurons. The PFAS-induced alterations in NSC may be related to neurobehavioral alterations observed in rodents developmentally exposed to these compounds, and show the importance to consider the effects of these compounds on human brain development and disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Skin cancer incidence is rapidly increasing. The main risk factor, sun exposure, can be modified. Informational campaigns can be effective in raising skin cancer awareness and target the high-risk population. Still, sun exposure habits in people at high risk of skin cancer are not well-known., Objective: To investigate if and how sun exposure habits differ between low-risk and high-risk individuals., Methods: During the Swedish Euromelanoma campaign of 2018, questionnaires were collected containing information regarding sun exposure habits and risk factors for skin cancer. Data on 4,141 participants was used to investigate the association between risk factors and sun exposure habits., Results: A fair skin type and a previous history of skin cancer were significantly associated with enhanced sun protective behavior. Family history of skin cancer, childhood sunburns and the presence of large/atypical nevi had no effect on sun exposure habits. Going on sunny holidays were particularly unaffected by being at high risk of skin cancer., Conclusion: Individuals at high risk of developing skin cancer showed suboptimal sun exposure habits and harmful traveling behaviors. We suggest that future skin cancer campaigns inform on accurate sun protection behavior during sunny holidays and associated risk factors. Risk factors such as childhood sunburns, numerous common and large/atypical nevi, as well as family history of skin cancer seem to be less recognized by the population., Competing Interests: Competing interests: The Euromelanoma campaign is funded by sponsorship and part of the funding is used for data input and statistical analyses. Oskar Karlsson and Oskar Hagberg were engaged by the campaign to perform these tasks. The sponsors had no impact on the preparation, results, or publication of the study., (©2021 Karlsson et al.)

Interactions between environmental factors and genetics underlie the majority of chronic human diseases. Chemical exposures are likely an underestimated contributor, yet gene-environment (GxE) interaction studies rarely assess their modifying effects. Here, we describe a novel method to profile the human genome and identify regions associated with differential population susceptibility to chemical exposures. Single nucleotide polymorphisms (SNPs) implicated in enriched chemical-disease intersections were identified and validated for three chemical classes with expected GxE interaction potential (neuroactive, hepatoactive, and cardioactive compounds). The same approach was then used to characterize consumer product classes with unknown risk for GxE interactions (washing products, cosmetics, and adhesives). Additionally, high-risk variant sets that may confer differential population susceptibility were identified for these consumer product groups through frequent itemset mining and pathway analysis. A dataset of 2454 consumer product chemical-disease linkages, with risk values, SNPs, and pathways for each association was developed, describing the interplay between environmental factors and genetics in human disease progression. We found that genetic hotspots implicated in GxE interactions differ across chemical classes (e.g., washing products had high-risk SNPs implicated in nervous system disease) and illustrate how this approach can discover new associations (e.g., washing product n-butoxyethanol implicated SNPs in the PI3K-Akt signaling pathway for Alzheimer's disease). Hence, our approach can predict high-risk genetic regions for differential population susceptibility to chemical exposures and characterize chemical modifying factors in specific diseases. These methods show promise for describing how chemical exposures can lead to varied health outcomes in a population and for incorporating inter-individual variability into chemical risk assessment., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Underlying drivers of species extinctions need to be better understood for effective conservation of biodiversity. Nearly half of all amphibian species are at risk of extinction, and pollution may be a significant threat as seasonal high-level agrochemical use overlaps with critical windows of larval development. The potential of environmental chemicals to reduce the fitness of future generations may have profound ecological and evolutionary implications. This study characterized effects of male developmental exposure to environmentally relevant concentrations of the anti-androgenic pesticide linuron over two generations of offspring in Xenopus tropicalis frogs. The adult male offspring of pesticide-exposed fathers (F1) showed reduced body size, decreased fertility, and signs of endocrine system disruption. Impacts were further propagated to the grand-offspring (F2), providing evidence of transgenerational effects in amphibians. The adult F2 males demonstrated increased weight and fat body palmitoleic-to-palmitic acid ratio, and decreased plasma glucose levels. The study provides important cross-species evidence of paternal epigenetic inheritance and pollutant-induced transgenerational toxicity, supporting a causal and complex role of environmental contamination in the ongoing species extinctions, particularly of amphibians., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Gene-environment interactions are involved in the development of breast cancer, the tumor type that accounts for the majority of the cancer-related deaths among women. Here, we demonstrate that exposure to PFOS (10 µM) and PFOA (100 µM)-two contaminants ubiquitously found in human blood-for 72 h induced breast epithelial cell (MCF-10A cell line) proliferation and alteration of regulatory cell-cycle proteins (cyclin D1, CDK6, p21, p53, p27, ERK 1/2 and p38) that persisted after a multitude of cell divisions. The contaminants also promoted cell migration and invasion by reducing the levels of E-cadherin, occludin and β-integrin in the unexposed daughter cells. The compounds further induced an increase in global DNA methylation and differentially altered histone modifications, epigenetic mechanisms implicated in tumorigenesis. This mechanistic evidence for PFOS- and PFOA-induced malignant transformation of human breast cells supports a role of these abundant contaminants in the development and progression of breast cancer. Increased knowledge of contaminant-induced effects and their contribution to breast tumorigenesis is important for a better understanding of gene-environment interactions in the etiology of breast cancer.

Developmental exposure to the environmental neurotoxin β-N-methylamino-L-alanine (BMAA), a proposed risk factor for neurodegenerative disease, can induce long-term cognitive impairments and neurodegeneration in rats. While rodent studies have demonstrated a low transfer of BMAA to the adult brain, this toxin is capable to cross the placental barrier and accumulate in the fetal brain. Here, we investigated the differential susceptibility of primary neuronal cells and neural stem cells from fetal rat hippocampus to BMAA toxicity. Exposure to 250 µM BMAA induced cell death in neural stem cells through caspase-independent apoptosis, while the proliferation of primary neurons was reduced only at 3 mM BMAA. At the lowest concentrations tested (50 and 100 µM), BMAA disrupted neural stem cell differentiation and impaired neurite development in neural stem cell-derived neurons (e.g., reduced neurite length, the number of processes and branches per cell). BMAA induced no alterations of the neurite outgrowth in primary neurons. This demonstrates that neural stem cells are more susceptible to BMAA exposure than primary neurons. Importantly, the changes induced by BMAA in neural stem cells were mitotically inherited to daughter cells. The persistent nature of the BMAA-induced effects may be related to epigenetic alterations that interfere with the neural stem cell programming, as BMAA exposure reduced the global DNA methylation in the cells. These findings provide mechanistic understanding of how early-life exposure to BMAA may lead to adverse long-term consequences, and potentially predispose for neurodevelopmental disorders or neurodegenerative disease later in life.

Olfactory dysfunction is implicated in neurodegenerative disorders and typically manifests years before other symptoms. The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) is suggested as a risk factor for neurodegenerative disease. Detection of BMAA in air filters has increased the concern that aerosolization may lead to human BMAA exposure through the air. The aim of this study was to determine if BMAA targets the olfactory system. Autoradiographic imaging showed a distinct localization of radioactivity in the right olfactory mucosa and bulb following a unilateral intranasal instillation of 3 H-BMAA (0.018 µg) in mice, demonstrating a direct transfer of BMAA via the olfactory pathways to the brain circumventing the blood-brain barrier, which was confirmed by liquid scintillation. Treatment of mouse primary olfactory bulb cells with 100 µM BMAA for 24 h caused a disruption of the neurite network, formation of dendritic varicosities and reduced cell viability. The NMDA receptor antagonist MK-801 and the metabotropic glutamate receptor antagonist MCPG protected against the BMAA-induced alterations, demonstrating the importance of glutamatergic mechanisms. The ionotropic non-NMDA receptor antagonist CNQX prevented the BMAA-induced decrease of cell viability in mixed cultures containing both neuronal and glial cells, but not in cultures with neurons only, suggesting a role of neuron-glial interactions and glial AMPA receptors in the BMAA-induced toxicity. The results show that the olfactory region may be a target for BMAA following inhalation exposure. Further studies on the relations between environmental olfactory toxicants and neurodegenerative disorders are warranted.

Polychlorinated biphenyls (PCBs) are chemicals used in a variety of products before they were widely banned due to toxic effects in humans and wildlife. Because of continued persistence and ubiquity of these contaminants, risk of exposure to people living in industrialized countries is still high. Experimental research show that developmental exposure to PCB may alter function of brain pleasure centers and potentially influence disinhibitory behaviors, including tobacco and alcohol use. Yet, the potential effects of developmental PCB exposure on adolescent substance use have not been studied in humans. We used the Child Health and Development Studies (CHDS), a prospective birth cohort study in the Oakland and East Bay areas of California, to investigate associations between prenatal exposure to PCB congeners (66, 74, 99, 118, 138, 153, 170, 180, 187, and 203) and later disinhibitory behaviors in adolescents, specifically alcohol consumption and smoking, in a randomly selected sample (n = 554). Total prenatal PCB exposure was not associated with disinhibitory behaviors, among adolescents. However, the adjusted odds ratio (aOR) for being a current smoker, was higher in subjects within the third quartile of maternal PCB 66 exposure compared to those below the median (aOR = 1.93; 95% CI 1.05, 3.55). The aOR for drinking >2 alcoholic beverages per week, were also higher for adolescents within the third (aOR = 1.46; 95% CI 0.86, 2.47) and fourth quartile of PCB 66 exposure (aOR = 1.39; 95% CI 0.83, 2.35), but the differences did not reach statistical significance. These results suggest that this specific PCB congener may play a role inducing neurodevelopmental alterations that could potentially increase the risk of becoming a long-term user of tobacco and possibly alcohol. There were no notable differences between magnitude or direction of effect between boys and girls. Future replicate analyses with larger longitudinal samples and animal experimental studies of potential underlying mechanisms are warranted., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The widespread environmental contaminant β-methylamino-L-alanine (BMAA) is a developmental neurotoxicant that can induce long-term learning and memory deficits. Studies have shown high transplacental transfer of 3H-BMAA and a significant uptake in fetal brain. Therefore, more information on how BMAA may influence growth and differentiation of neural stem cells is required for assessment of the risk to the developing brain. The aim of this study was to investigate direct and mitotically inherited effects of BMAA exposure using primary striatal neurons and embryonic neural stem cells. The neural stem cells were shown to be clearly more susceptible to BMAA exposure than primary neurons. Exposure to 250 µM BMAA reduced neural stem cell proliferation through apoptosis and G2/M arrest. At lower concentrations (50-100 µM), not affecting cell proliferation, BMAA reduced the differentiation of neural stem cells into astrocytes, oligodendrocytes, and neurons through glutamatergic mechanisms. Neurons that were derived from the BMAA-treated neuronal stem cells demonstrated morphological alterations including reduced neurite length, and decreased number of processes and branches per cell. Interestingly, the BMAA-induced changes were mitotically heritable to daughter cells. The results suggest that early-life exposure to BMAA impairs neuronal stem cell programming, which is vital for development of the nervous system and may result in long-term consequences predisposing for both neurodevelopmental disorders and neurodegenerative disease later in life. More attention should be given to the potential adverse effects of BMAA exposure on brain development.

The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) is a glutamate receptor agonist that can induce oxidative stress and has been implicated as a possible risk factor for neurodegenerative disease. Detection of BMAA in mussels, crustaceans, and fish illustrates that the sources of human exposure to this toxin are more abundant than previously anticipated. The aim of this study was to determine uptake of BMAA in the pineal gland and subsequent effects on melatonin production in primary pinealocyte cultures and a rat model. Autoradiographic imaging of 10-day-old male rats revealed a high and selective uptake in the pineal gland at 30 minutes to 24 hours after 14 C-L-BMAA administration (0.68 mg/kg). Primary pinealocyte cultures exposed to 0.05-3 mmol/L BMAA showed a 57%-93% decrease in melatonin synthesis in vitro. Both the metabotropic glutamate receptor 3 (mGluR3) antagonist Ly341495 and the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate prevented the decrease in melatonin secretion, suggesting that BMAA inhibits melatonin synthesis by mGluR3 activation and PKC inhibition. Serum analysis revealed a 45% decrease in melatonin concentration in neonatal rats assessed 2 weeks after BMAA administration (460 mg/kg) and confirmed an inhibition of melatonin synthesis in vivo. Given that melatonin is a most important neuroprotective molecule in the brain, the etiology of BMAA-induced neurodegeneration may include mechanisms beyond direct excitotoxicity and oxidative stress., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Some studies document racial disparities in self-reported health associated with alcohol use and abuse. However, few studies examined biomarkers that underlie the onset of alcohol-related chronic diseases. We investigated whether the association between alcohol abuse and five biomarkers of inflammation (CRP, IL-6, fibrinogen, E-selectin, sICAM-1) vary between Black and White Americans aged 35 to 84 (n = 1173) from the Midlife in the United States Biomarker Study. Multivariable Ordinary Least Squares regressions were used to assess Black-White differences in the association between alcohol abuse and the biomarkers. Race moderated the association between alcohol abuse and CRP (b = 0.56, SE = 0.28, p = 0.048), IL-6 (b = 0.65, SE = 0.22, p = 0.004), and a composite inflammation score (b = 0.014, SE = 0.07, p = 0.041). These findings potentially shed light for why alcohol has a stronger negative association with poorer health for Blacks compared to Whites. Analysis should be replicated in larger prospective cohorts.

Phthalates are endocrine-disrupting chemicals that can cross the placenta and affect the fetal epigenome. Among various epigenetic regulators of gene expression, long noncoding RNAs (lncRNAs) are important players that may also be involved in the manifestation of endocrine-disrupting chemical toxicity. We sought to explore the association between maternal urinary phthalate metabolite concentrations and lncRNA expression in human placenta to better understand potential mechanisms through which lncRNAs participate in mediating phthalate toxicity. Ten patients with uncomplicated dichorionic diamniotic twin pregnancies at term were included in this study. Urinary (n = 10) and placenta samples (n = 20) were collected for all participants. Urinary samples were analyzed for 15 phthalate metabolites and 2 phthalate alternative metabolites. Real-time PCR arrays were used to identify and quantify 87 lncRNAs from the placental samples. We tested the Spearman correlation matrix to compare prenatal phthalate measures against placental lncRNA levels. lncRNA levels showed large variations across samples, with no significant differences in lncRNA expression within twin pairs. Mono-(carboxynonyl) phthalate demonstrated consistently strong correlations with most lncRNAs. The strongest correlation was observed between mono-hydroxyisobutyl phthalate and LOC91450 (Rspearman = 0.88, p < .001). This correlation remained significant after Bonferroni adjustment. Other strong correlations were observed between mono-isobutyl phthalate, DPP10 and HOTTIP (Rspearman = -0.91, p < .001). AIRN, DACT3.AS1, DLX6, DPP10, HOTTIP, LOC143666, and LOC91450 were strongly correlated with the greatest number of phthalate metabolites. Further studies are needed to validate these results and understand if the altered expression of lncRNAs in human placenta has clinical significance.

Despite significant advances in early detection and treatment, breast cancer remains a major cause of morbidity and mortality. Perfluorooctanoic acid (PFOA) is a suspected endocrine disruptor and a common environmental pollutant associated with various diseases including cancer. However, the effects of PFOA and its mechanisms of action on hormone-responsive cells remain unclear. Here, we explored the potential tumorigenic activity of PFOA (100 nM-1 mM) in human breast epithelial cells (MCF-10A). MCF-10A cells exposed to 50 and 100 µM PFOA demonstrated a higher growth rate compared to controls. The compound promoted MCF-10A proliferation by accelerating G 0 /G 1 to S phase transition of the cell cycle. PFOA increased cyclin D1 and CDK4/6 levels, concomitant with a decrease in p27. In contrast to previous studies of perfluorooctane sulfate (PFOS), the estrogen receptor antagonist ICI 182,780 had no effect on PFOA-induced cell proliferation, whereas the PPARα antagonist GW 6471 was able to prevent the MCF-10A proliferation, indicating that the underlying mechanisms involve PPARα-dependent pathways. Interestingly, we also showed that PFOA is able to stimulate cell migration and invasion, demonstrating its potential to induce neoplastic transformation of human breast epithelial cells. These results suggest that more attention should be paid to the roles of PFOA in the development and progression of breast cancer.

Perfluorooctanesulfonic acid (PFOS) is a synthetic fluorosurfactant widely used in the industry and a prominent environmental toxicant. PFOS is persistent, bioaccumulative, and toxic to mammalian species. Growing evidence suggests that PFOS has the potential to interfere with estrogen homeostasis, posing a risk of endocrine-disrupting effects. Recently, concerns about a potential link between PFOS and breast cancer have been raised, but the mechanisms underlying its actions as a potential carcinogen are unknown. By utilizing cell proliferation assays, flow cytometry, immunocytochemistry, and cell migration/invasion assays, we examined the potentially tumorigenic activity of PFOS (100 nM-1 mM) in MCF-10A breast cell line. The results showed that the growth of MCF-10A cells exposed to 1 and 10 µM PFOS was higher compared to that of the control. Mechanistic studies using 10 µM PFOS demonstrated that the compound promotes MCF-10A proliferation through accelerating G 0 /G 1- to-S phase transition of the cell cycle after 24, 48, and 72 h of treatment. In addition, PFOS exposure increased CDK4 and decreased p27, p21, and p53 levels in the cells. Importantly, treatment with 10 µM PFOS for 72 h also stimulated MCF-10A cell migration and invasion, illustrating its capability to induce neoplastic transformation of human breast epithelial cells. Our experimental results suggest that exposure to low levels of PFOS might be a potential risk factor in human breast cancer initiation and development.

The number of epigenetic studies on brain functions and diseases are dramatically increasing, but little is known about the impact of post-mortem intervals and post-sampling effects on DNA modifications such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we examined post-mortem-induced changes in global brain 5mC and 5hmC levels at post-mortem intervals up to 540 min., and studied effects of tissue heat stabilization, using LUMA and ELISA. The global 5mC and 5hmC levels were generally higher in the cerebellum of adult rats than neonates. When measured by ELISA, the global 5mC content in adults, but not neonates, decreased with the post-mortem interval reaching a significantly lower level in cerebellum tissue at the post-mortem interval 540 min. (2.9 ± 0.7%; mean ± S.E.M.) compared to control (3.7 ± 0.6%). The global 5hmC levels increased with post-mortem interval reaching a significantly higher level at 540 min. (0.29 ± 0.06%) compared to control (0.19 ± 0.03%). This suggests that the post-mortem interval may confound 5mC and 5hmC analysis in human brain tissues as the post-mortem handling could vary substantially. The reactive oxygen species (ROS) level in cerebellum also increased over time, in particular in adults, and may be part of the mechanism that causes the observed post-mortem changes in 5mC and 5hmC. The global 5mC and 5hmC states were unaffected by heat stabilization, allowing analysis of tissues that are stabilized to preserve more labile analytes. Further studies in human samples are needed to confirm post-mortem effects on DNA methylation/hydroxymethylation and elucidate details of the underlying mechanisms., (© 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disorders. BMAA is also a known developmental neurotoxin and research indicates that the sources of human and wildlife exposure may be more diverse than previously anticipated. The aim of the present study was therefore to examine whether BMAA can be transferred into birds' eggs. Egg laying quail were dosed with 14 C-labeled BMAA. The distribution of radioactivity in the birds and their laid eggs was then examined at different time points by autoradiography and phosphoimaging analysis. To evaluate the metabolic stability of the BMAA molecule, the distribution of 14 C-methyl- and 14 C-carboxyl-labeled BMAA were compared. The results revealed a pronounced incorporation of radioactivity in the eggs, predominantly in the yolk but also in the albumen. Imaging analysis showed that the concentrations of radioactivity in the liver decreased about seven times between the 24h and the 72h time points, while the concentrations in egg yolk remained largely unchanged. At 72h the egg yolk contained about five times the concentration of radioactivity in the liver. Both BMAA preparations gave rise to similar distribution pattern in the bird tissues and in the eggs, indicating metabolic stability of the labeled groups. The demonstrated deposition into eggs warrants studies of BMAAs effects on bird development. Moreover, birds' eggs may be a source of human BMAA exposure, provided that the laying birds are exposed to BMAA via their diet., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Health outcomes, including chronic disease and mortality, attributed to or associated with alcohol abuse are discrepant between African Americans and Whites. To date, the topic is not fully understood and few studies conducted have used biomarker indicators of health. We investigated whether the association between alcohol abuse and biomarkers of the neuroendocrine system vary between black or African American and White respondents aged 34-84 from the Midlife in the United States Study (MIDUS) II (2004-2006) (n=1129). Alcohol abuse was assessed with a modified version of the Michigan Alcohol Screening Test. Ordinary least squared (OLS) regression was used to evaluate whether race moderated the associations between alcohol abuse and four biomarkers-urinary cortisol and serum dehydroepiandrosterone sulfate (DHEA-S), epinephrine and norepinephrine-and two composite summary scores, each consisting of two components that characterize the hypothalamic pituitary adrenal (HPA)-axis and sympathetic nervous systems (SNS), respectively. Covariates included age, sex, education, income, current drinking, smoking, exercise, fast food consumption, heart disease, blood pressure, diabetes, body mass index, medication use, anxiety/depression, sleep duration, and cholesterol markers. Race significantly moderated the associations between alcohol abuse and norepinephrine concentration (χ 2 [1]=4.48, p=0.034) and the SNS composite score (χ 2 [1]=5.83, p=0.016). Alcohol abuse was associated with higher mean norepinephrine levels (b=0.26, standard error (SE)=0.12, p=0.034) and SNS composite score (b=0.23, SE=0.11, p=0.016) for African Americans compared to Whites. Interestingly, for Whites a paradoxical association between alcohol abuse, norepinephrine and SNS levels was observed; those who abused alcohol had lower mean norepinephrine levels than non-abusers. Race differences in neuroendocrine response could be biological pathways that contribute the excess risk of chronic disease and mortality attributed to alcohol abuse among African Americans compared to Whites. Replication of these analyses in larger cohorts are warranted in addition to further studies of underlying mechanisms among Blacks and Whites separately., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The impact of euthanasia methods on endocrine and metabolic parameters in rodent tissues and biological fluids is highly relevant for the accuracy and reliability of the data collected. However, few studies concerning this issue are found in the literature. We compared the effects of three euthanasia methods currently used in animal experimentation (i.e. decapitation, CO 2 inhalation and pentobarbital injection) on the serum levels of corticosterone, insulin, glucose, triglycerides, cholesterol and a range of free fatty acids in rats. The corticosterone and insulin levels were not significantly affected by the euthanasia protocol used. However, euthanasia by an overdose of pentobarbital (120 mg/kg intraperitoneal injection) increased the serum levels of glucose, and decreased cholesterol, stearic and arachidonic acids levels compared with euthanasia by CO 2 inhalation and decapitation. CO 2 inhalation appears to increase the serum levels of triglycerides, while euthanasia by decapitation induced no individual discrepant biomarker level. We conclude that choice of the euthanasia methods is critical for the reliability of serum biomarkers and indicate the importance of selecting adequate euthanasia methods for metabolic analysis in rodents. Decapitation without anaesthesia may be the most adequate method of euthanasia when taking both animal welfare and data quality in consideration., (© 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The environmental toxin β-N-methylamino-L-alanine (BMAA) has been proposed to contribute to neurodegenerative diseases. We have previously shown that neonatal exposure to BMAA results in dose-dependent cognitive impairments, proteomic alterations and progressive neurodegeneration in the hippocampus of adult rats. A high BMAA dose (460mg/kg) also induced intracellular fibril formation, increased protein ubiquitination and enrichment of proteins important for lipid transport and metabolism. The aim of this study was therefore to elucidate the role of neuronal lipids in BMAA-induced neurodegeneration. By using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS), we characterized the spatial lipid profile in the hippocampus of six month-old rats that were treated neonatally (postnatal days 9-10) with 460mg/kg BMAA. Multivariate statistical analysis revealed long-term changes in distinct ganglioside species (GM, GD, GT) in the dentate gyrus. These changes could be a consequence of direct effects on ganglioside biosynthesis through the b-series (GM3-GD3-GD2-GD1b-GT1b) and may be linked to astrogliosis. Complementary immunohistochemistry experiments towards GFAP and S100β further verified the role of increased astrocyte activity in BMAA-induced brain damage. This highlights the potential of imaging MS for probing chemical changes associated with neuropathological mechanisms in situ. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann., (Copyright © 2016 Elsevier B.V. All rights reserved.)

During the last decades, imaging mass spectrometry has gained significant relevance in biomedical research. Recent advances in imaging mass spectrometry have paved the way for in situ studies on drug development, metabolism and toxicology. In contrast to whole-body autoradiography that images the localization of radiolabeled compounds, imaging mass spectrometry provides the possibility to simultaneously determine the discrete tissue distribution of the parent compound and its metabolites. In addition, imaging mass spectrometry features high molecular specificity and allows comprehensive, multiplexed detection and localization of hundreds of proteins, peptides and lipids directly in tissues. Toxicologists traditionally screen for adverse findings by histopathological examination. However, studies of the molecular and cellular processes underpinning toxicological and pathologic findings induced by candidate drugs or toxins are important to reach a mechanistic understanding and an effective risk assessment strategy. One of IMS strengths is the ability to directly overlay the molecular information from the mass spectrometric analysis with the tissue section and allow correlative comparisons of molecular and histologic information. Imaging mass spectrometry could therefore be a powerful tool for omics profiling of pharmacological/toxicological effects of drug candidates and toxicants in discrete tissue regions. The aim of the present review is to provide an overview of imaging mass spectrometry, with particular focus on MALDI imaging mass spectrometry, and its use in drug development and toxicology in general.

Acute exposure to fine particle (PM 2.5 ) induces DNA methylation changes implicated in inflammation and oxidative stress. We conducted a crossover trial to determine whether B-vitamin supplementation averts such changes. Ten healthy adults blindly received a 2-h, controlled-exposure experiment to sham under placebo, PM 2.5 (250 μg/m 3 ) under placebo, and PM 2.5 (250 μg/m 3 ) under B-vitamin supplementation (2.5 mg/d folic acid, 50 mg/d vitamin B 6 , and 1 mg/d vitamin B 12 ), respectively. We profiled epigenome-wide methylation before and after each experiment using the Infinium HumanMethylation450 BeadChip in peripheral CD4 + T-helper cells. PM 2.5 induced methylation changes in genes involved in mitochondrial oxidative energy metabolism. B-vitamin supplementation prevented these changes. Likewise, PM 2.5 depleted 11.1% [95% confidence interval (CI), 0.4%, 21.7%; P = 0.04] of mitochondrial DNA content compared with sham, and B-vitamin supplementation attenuated the PM 2.5 effect by 102% ( P interaction = 0.01). Our study indicates that individual-level prevention may be used to complement regulations and control potential mechanistic pathways underlying the adverse PM 2.5 effects, with possible significant public health benefit in areas with frequent PM 2.5 peaks.

Breastmilk has many documented beneficial effects on the developing human infant, but the components of breastmilk that influence these developmental pathways have not been fully elucidated. Increasing evidence suggests that non-coding RNAs encapsulated in extracellular vesicles (EVs) represent an important mechanism of communication between the mother and child. Long non-coding RNAs (lncRNAs) are of particular interest given their key role in gene expression and development. However, it is not known whether breastmilk EVs contain lncRNAs. We used qRT-PCR to determine whether EVs isolated from human breastmilk contain lncRNAs previously reported to be important for developmental processes. We detected 55 of the 87 screened lncRNAs in EVs from the 30 analyzed breastmilk samples, and CRNDE, DANCR, GAS5, SRA1 and ZFAS1 were detected in >90% of the samples. GAS5, SNHG8 and ZFAS1 levels were highly correlated (Spearman's rho > 0.9; P < 0.0001), which may indicate that the loading of these lncRNAs into breastmilk EVs is regulated by the same pathways. The detected lncRNAs are important epigenetic regulators involved in processes such as immune cell regulation and metabolism. They may target a repertoire of recipient cells in offspring and could be essential for child development and health. Further experimental and epidemiological studies are warranted to determine the impact of breastmilk EV-encapsulated lnRNAs in mother to child signaling.

The cyanobacterial non-proteinogenic amino acid β-N-methylamino-l-alanine (BMAA) is proposed to be involved in the etiology of amyotrophic lateral sclerosis/parkinsonism dementia complex. When administered as single doses to neonatal rats, BMAA gives rise to cognitive and neurodegenerative impairments in the adult animal. Here, we employed mass spectrometry (LC-MS/MS) and autoradiographic imaging to examine the mother-to-pup transfer of BMAA in rats. The results show that unchanged BMAA was secreted into the milk and distributed to the suckling pups. The concentration of BMAA in pup stomach milk and the neonatal liver peaked after 8h, while the concentration in the pup brain increased throughout the study period. About 1 and 6% of the BMAA recovered from adult liver and brain were released following hydrolysis, suggesting that this fraction was associated with protein. No association to milk protein was observed. Injection of rat pups with [methyl-(14)C]-l-BMAA or [carboxyl-(14)C]-l-BMAA resulted in highly similar distribution patterns, indicating no or low metabolic elimination of the methylamino- or carboxyl groups. In conclusion, BMAA is transported as a free amino acid to rat milk and suckling pups. The results strengthen the proposal that mothers' milk could be a source of exposure for BMAA in human infants., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

An individual's risk of developing a common disease typically depends on an interaction of genetic and environmental factors. Epigenetic research is uncovering novel ways through which environmental factors such as diet, air pollution, and chemical exposure can affect our genes. DNA methylation and histone modifications are the most commonly studied epigenetic mechanisms. The role of long non-coding RNAs (lncRNAs) in epigenetic processes has been more recently highlighted. LncRNAs are defined as transcribed RNA molecules greater than 200 nucleotides in length with little or no protein-coding capability. While few functional lncRNAs have been well characterized to date, they have been demonstrated to control gene regulation at every level, including transcriptional gene silencing via regulation of the chromatin structure and DNA methylation. This review aims to provide a general overview of lncRNA function with a focus on their role as key regulators of health and disease and as biomarkers of environmental exposure.

Melanin is a polyanionic pigment that colors, e.g., the hair, skin and eyes. The pigment neuromelanin is closely related to melanin and is mainly produced in specific neurons of the substantia nigra. Certain drugs and chemicals bind to melanin/neuromelanin and are retained in pigment cells for long periods. This specific retention is thought to protect the cells but also to serve as a depot that slowly releases accumulated compounds and may cause toxicity in the eye and skin. Moreover, neuromelanin and compounds with high neuromelanin affinity have been suggested to be implicated in the development of adverse drug reactions in the central nervous system (CNS) as well as in the etiology of Parkinson's disease (PD). Epidemiologic studies implicate the exposure to pesticides, metals, solvents and other chemicals as risk factors for PD. Neuromelanin interacts with several of these toxicants which may play a significant part in both the initiation and the progression of neurodegeneration. MPTP/MPP(+) that has been casually linked with parkinsonism has high affinity for neuromelanin, and the induced dopaminergic denervation correlates with the neuromelanin content in the cells. Recent studies have also reported that neuromelanin may interact with α-synuclein as well as activate microglia and dendritic cells. This review aims to provide an overview of melanin binding of drugs and other compounds, and possible toxicological implications, with particular focus on the CNS and its potential involvement in neurodegenerative disorders.

Neonatal porcine diarrhoea of uncertain aetiology has been reported from a number of European countries. The aim of the present study was to use viral metagenomics to examine a potential viral involvement in this diarrhoea and to describe the intestinal virome with focus on eukaryotic viruses. Samples from the distal jejunum of 50 diarrhoeic and 19 healthy piglets from 10 affected herds were analysed. The viral fraction of the samples was isolated and nucleic acids (RNA and DNA fractions) were subjected to sequence independent amplification. Samples from diarrhoeic piglets from the same herds were pooled whereas samples from healthy piglets were analysed individually. In total, 29 clinical samples, plus two negative controls and one positive control consisting of a mock metagenome were sequenced using the Ion Torrent platform. The resulting sequence data was subjected to taxonomic classification using Kraken, Diamond and HMMER. In the healthy specimens, eight different mammalian virus families were detected (Adenoviridae, Anelloviridae, Astroviridae, Caliciviridae, Circoviridae, Parvoviridae, Picornaviridae, and Reoviridae) compared to four in the pooled diarrhoeic samples (Anelloviridae, Circoviridae, Picornaviridae, and Reoviridae). It was not possible to associate a particular virus family with the investigated diarrhoea. In conclusion, this study does not support the hypothesis that the investigated diarrhoea was caused by known mammalian viruses. The results do, however, indicate that known mammalian viruses were present in the intestine as early as 24-48 hours after birth, indicating immediate infection post-partum or possibly transplacental infection.