Personalized mixture toxicity testing: A proof-of-principle in vitro study evaluating the steroidogenic effects of reconstructed contaminant mixtures measured in blood of individual adults.

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.

Breast cancer risk and prevention in 2024: An overview from the Breast Cancer UK - Breast Cancer Prevention Conference.

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/ß 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/ß 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.

Pesticide-induced transgenerational alterations of genome-wide DNA methylation patterns in the pancreas of Xenopus tropicalis correlate with metabolic phenotypes.

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.

Automated Image-Based Fluorescence Screening of Mitochondrial Membrane Potential in Daphnia magna: An Advanced Ecotoxicological Testing Tool.

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.

Screening persistent organic pollutants for effects on testosterone and estrogen synthesis at human-relevant concentrations using H295R cells in 96-well plates.

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.

Perfluorooctanesulfonic acid (PFOS) induced cancer related DNA methylation alterations in human breast cells: A whole genome methylome study.

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.

Mechanistic screening of reproductive toxicity in a novel 3D testicular co-culture model shows significant impairments following exposure to low-dibutyl phthalate concentrations.

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.

Male-transmitted transgenerational effects of the herbicide linuron on DNA methylation profiles in Xenopus tropicalis brain and testis.

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.

Applying a modified systematic review and integrated assessment framework (SYRINA) - a case study on triphenyl phosphate.

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.

Impaired spermatogenesis and associated endocrine effects of azole fungicides in peripubertal Xenopus tropicalis.

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 3ß-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.

Long-Term Effects of Perinatal Exposure to a Glyphosate-Based Herbicide on Melatonin Levels and Oxidative Brain Damage in Adult Male Rats.

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.

Altered gut microbiota community structure and correlated immune system changes in dibutyl phthalate exposed mice.

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.

Pubertal sexual development and endpoints for disrupted spermatogenesis in the model Xenopus tropicalis.

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.

Nontarget Analysis of Polluted Surface Waters in Bangladesh Using Open Science Workflows.

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.

Persistent immunosuppressive effects of dibutyl phthalate exposure in adult male mice.

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.

High-content analysis shows synergistic effects of low perfluorooctanoic acid (PFOS) and perfluorooctane sulfonic acid (PFOA) mixture concentrations on human breast epithelial cell carcinogenesis.

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 ß-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.

Di-n-Butyl Phthalate and Its Monoester Metabolite Impairs Steroid Hormone Biosynthesis in Human Cells: Mechanistic In Vitro Studies.

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 HSD3ß2 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 HSD17ß3, 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.

Adult Exposure to Di-N-Butyl Phthalate (DBP) Induces Persistent Effects on Testicular Cell Markers and Testosterone Biosynthesis in Mice.

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, HSD3ß2, 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.

Epigenetics in the Anthropocene: an interview with Oskar Karlsson.

In this interview, Oskar Karlsson speaks with Storm Johnson, commissioning editor for Epigenomics, on his work to date in the field of toxicological origins of disease and gene-environment interactions. Oskar Karlsson, is an associate professor at the Science for Life Laboratory (SciLifeLab), Department of Environmental Science, Stockholm University, Sweden. Dr. Karlsson earned a PhD in toxicology at the Department of Pharmaceutical Bioscience, Uppsala University, and has also worked at Centre of Molecular Medicine, Karolinska Institute, as well as Harvard University School of Public Health. His research combines experimental model systems, computational and omics tools, and epidemiological studies to investigate the influence of environmental exposures on wildlife and human health, and underlying molecular mechanisms. In particular, his research focuses on developmental origins of health and disease with an emphasis on environmental exposures and epigenetic mechanisms. The projects concern the effects of exposures such as endocrine disrupting chemicals, flame retardants, pesticides, metals and particulate air pollution, as well as drugs, psycho-social stressors and ethnical disparities. Ongoing efforts include studies of paternal epigenetic inheritance in the ERC-funded project PATER.

Tumorigenic activity of alternative per- and polyfluoroalkyl substances (PFAS): Mechanistic in vitro studies.

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 ß-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.