Per- and polyfluoroalkyl substances (PFAS) and male reproductive function in young adulthood; a cross-sectional study.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are a large family of persistent industrial chemicals with endocrine disrupting properties. OBJECTIVES: To examine biomarkers of reproductive function in young adult males according to current environmental exposure to single and combined PFAS. METHODS: The study population consisted of young men (n = 1041, age 18-21) from the Fetal Programming of Semen Quality (FEPOS) cohort. These men were recruited from pregnancies included in the Danish National Birth Cohort (DNBC) between 1996 and 2002. From 2017 to 2019, participants answered an online questionnaire, completed a clinical examination and provided a blood and a semen sample. Exposure to 15 PFAS was measured in plasma. Six compounds were quantified above the limit of detection in at least 80% of the participants. We applied negative binomial regression and weighted quantile sum (WQS) regression models to assess associations between single and combined exposure to PFAS and measures of semen quality, testicular volume and reproductive hormones among the young men. RESULTS: We found no consistent associations between plasma concentrations of PFAS, semen quality and testicular volume. Higher levels of single and combined PFAS were associated with slightly higher levels of follicle-stimulating hormone (FSH) (WQS 4% difference, 95% confidence interval: 0, 9). Perfluorooctanoic acid (PFOA) was the main contributor to this finding with positive signals also from perfluorodecanoic acid (PFDA) and perfluorohexane sulfonic acid (PFHxS). DISCUSSION: We examined exposure to a range of common PFAS in relation to biomarkers of male reproductive function and found an association with higher levels of FSH among young men from the general population in Denmark. Further studies on especially combined exposure to PFAS are needed to expand our understanding of potential endocrine disruption from both legacy and emerging compounds in relation to male reproductive function.

Perfluorooctane sulfonate (PFOS) exposure of bovine oocytes affects early embryonic development at human-relevant levels in an in vitro model.

Perfluorooctane sulfonate (PFOS) has been added to Stockholm Convention for global phase out, but will continue to contribute to the chemical burden in humans for a long time to come due to extreme persistence in the environment. In the body, PFOS is transferred into to the ovarian follicular fluid that surrounds the maturing oocyte. In the present study, bovine cumulus oocyte complexes were exposed to PFOS during 22 h in vitro maturation. Concentrations of 2 ng g-1 (PFOS-02) representing average human exposure and 53 ng g-1 (PFOS-53) relevant to highly exposed groups were used. After exposure, developmental competence was recorded until day 8 after fertilisation. Blastocysts were fixed and either stained to evaluate blastomere number and lipid distribution using confocal microscopy or frozen and pooled for microarray-based gene expression and DNA methylation analyses. PFOS-53 delayed the first cleavage to two-cell stage and beyond at 44 h after fertilisation (p < .01). No reduction of proportion blastocysts were seen at day 8 in either of the groups, but PFOS-53 exposure resulted in delayed development into more advanced stages of blastocysts seen as both reduced developmental stage (p = .001) and reduced number of blastomeres (p = .04). Blastocysts showed an altered lipid distribution that was more pronounced after exposure to PFOS-53 (increased total lipid volume, p=.0003, lipid volume/cell p < .0001) than PFOS-02, where only decreased average lipid droplet size (p=.02) was observed. Gene expression analyses revealed pathways differently regulated in the PFOS-treated groups compared to the controls, which were related to cell death and survival through e.g., P38 mitogen-activated protein kinases and signal transducer and activator of transcription 3, which in turn activates tumour protein 53 (TP53). Transcriptomic changes were also associated with metabolic stress response, differentiation and proliferation, which could help to explain the phenotypic changes seen in the blastocysts. The gene expression changes were more pronounced after exposure to PFOS-53 compared to PFOS-02. DNA-methylation changes were associated with similar biological functions as the transcriptomic data, with the most significantly associated pathway being TP53. Collectively, these results reveal that brief PFOS exposure during oocyte maturation alters the early embryo development at concentrations relevant to humans. This study adds to the evidence that PFOS has the potential to affect female fertility.

Transcriptional effects of binary combinations of PFAS in FaO cells.

Per- and polyfluoroalkyl substances (PFAS) represent a large class of structurally diverse chemicals of increasing public concern, mostly due to their chemical stability and undetermined toxicity profiles. In laboratory animals, adverse effects implicated for certain PFAS, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in particular, include liver toxicity and the associated metabolic dysregulation, immune and thyroid alterations, reproductive toxicity, and selected tumors. The broad commercialization and environmental distribution of PFAS has drawn attention to the need for understanding risks associated with combined exposure to multiple PFAS in complex mixtures. The purpose of this investigation is to determine whether binary combinations of PFAS elicit a molecular response that is either greater than or less than the sum of the individual responses. Exposure of FaO rat hepatoma cells for 24 h to 25 µM-200 µM of the 4- and 8-carbon perfluorocarboxylic acids (PFBA and PFOA) or the 4, 6, and 8-carbon perfluorosulfonic acids (PFBS, PFHxS, and PFOS, respectively) individually caused a dose-dependent increase in PPARα-regulated expression of peroxisomal bifunctional enzyme (Ehhadh). Potency increased with carbon number, with the carboxylates eliciting a greater transcriptional response than the corresponding sulfonates. Combined exposure to PFOA and PFBA produced an effect that was significantly less than the sum of the individual responses. The response to the combination of PFOA and PFOS produced a summative effect at concentrations that were not cytotoxic. Combined exposures to PFOS and either PFBS or PFHxS at low noncytotoxic concentrations produced a transcriptional effect that was significantly less than the sum of the individual effects. The results demonstrate that among the five structurally related perfluoroalkyl acids included in this investigation, PPARα transcriptional activation in response to combined binary exposures is consistently at or below that predicted by the sum of the individual effects.

Plasma, liver, and kidney exposures in rats after oral doses of industrial chemicals predicted using physiologically based pharmacokinetic models: A case study of perfluorooctane sulfonic acid.

A simplified physiologically based pharmacokinetic (PBPK) model consisting of chemical receptor, metabolizing and/or excreting, and central compartments was recently proposed. In the current study, this type of PBPK model was set up for perfluorooctane sulfonate, an environmental toxicant with liver effects, as a model compound; the model was then used to estimate tissue concentrations. The pharmacokinetic parameter input values for the model were calculated to give the best fit to reported/measured blood substrate concentrations in rats. The maximum concentrations and areas under the concentration versus time curves in plasma, liver, and kidney extrapolated using PBPK models for perfluorobutane sulfonic acid, perfluorohexane sulfonic acid, and perfluorooctane sulfonic acid were consistent with the reported mean values in rats. Using the rat models and scaled-up human PBPK models, some accumulation of perfluorooctane sulfonic acid in plasma and liver was seen after repeated doses. The reported 50th and 95th percentile concentrations of perfluorooctane sulfonic acid in human blood (0.0048 and 0.0183 ng/mL, respectively) in the general population underwent reverse dosimetry analysis using our PBPK models. These human blood concentrations potentially imply exposures of 0.041 and 0.16 µg/kg/day, respectively, for 90 days, values that are roughly similar to the reference dose (0.02 µg/kg/day) with an uncertainty factor of 30. These results indicate the relatively good estimates for tissue and blood exposures of chemical substrates after oral doses generated using the latest PBPK models.

Perfluoroalkyl acids potentiate glutamate excitotoxicity in rat cerebellar granule neurons.

Perfluoroalkyl acids (PFAAs) are persistent man-made chemicals, ubiquitous in nature and present in human samples. Although restrictions are being introduced, they are still used in industrial processes as well as in consumer products. PFAAs cross the blood-brain-barrier and have been observed to induce adverse neurobehavioural effects in humans and animals as well as adverse effects in neuronal in vitro studies. The sulfonated PFAA perfluorooctane sulfonic acid (PFOS), has been shown to induce excitotoxicity via the N-methyl-D-aspartate receptor (NMDA-R) in cultures of rat cerebellar granule neurons (CGNs). In the present study the aim was to further characterise PFOS-induced toxicity (1-60 µM) in rat CGNs, by examining interactions between PFOS and elements of glutamatergic signalling and excitotoxicity. Effects of the carboxylated PFAA, perfluorooctanoic acid (PFOA, 300-500 µM) on the same endpoints were also examined. During experiments in immature cultures at days in vitro (DIV) 8, PFOS increased both the potency and efficacy of glutamate, whereas in mature cultures at DIV 14 only increased potency was observed. PFOA also increased potency at DIV 14. PFOS-enhanced glutamate toxicity was further antagonised by the competitive NMDA-R antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at DIV 8. At DIV 8, PFOS also induced glutamate release (9-13 fold increase vs DMSO control) after 1-3 and 24 h exposure, whereas for PFOA a large (80 fold) increase was observed, but only after 24 h. PFOS and PFOA both also increased alanine and decreased serine levels after 24 h exposure. In conclusion, our results indicate that PFOS at concentrations relevant in an occupational setting, may be inducing excitotoxicity, and potentiation of glutamate signalling, via an allosteric action on the NMDA-R or by actions on other elements regulating glutamate release or NMDA-R function. Our results further support our previous findings that PFOS and PFOA at equipotent concentrations induce toxicity via different mechanisms of action.

Humanized Anti-Tumor-Associated Glycoprotein-72 for Submillimeter Near-Infrared Detection of Colon Cancer in Metastatic Mouse Models.

BACKGROUND: Tumor-associated glycoprotein (TAG)-72 is a pancarcinoma antigen that is overexpressed in greater than 80% of colorectal adenocarcinomas. CC49 is a TAG-72-specific antibody. The aim of the present study was to demonstrate selective imaging of colon tumors and metastases with the humanized TAG-72 antibody (anti-huCC49) conjugated to a near-infrared fluorophore in orthotopic mouse models. METHODS: Anti-huCC49 was conjugated to near-infrared dye IR800CW. Mouse imaging was performed with the Pearl Trilogy Small Animal and FLARE Imaging Systems. Subcutaneous mouse models of colon cancer cell line LS174T were used to determine the optimal dose of administration and timing of imaging. Orthotopic mouse models of LS174T were established by surgical orthotopic implantation of LS174T tumors onto the serosa of the cecum. Peritoneal carcinomatosis models were established by injection of LS174T cells into the peritoneum of nude mice. Mice were administered anti-huCC49-IR800 via tail vein injection. Mice were euthanized 72 h later and imaged after laparotomy. RESULTS: Subcutaneous LS174T xenografts demonstrated optimal tumor detection 72 h after administration with 50 µg anti-huCC49-IR800CW. Tumors were visualized with fluorescence imaging with a mean tumor-to-liver ratio of 7.39 (standard deviation: 2.76). In the orthotopic model, metastases smaller than 1 mm were fluorescently visualized that were invisible with bright light. CONCLUSIONS: Anti-huCC49-IR800CW provides sensitive and specific imaging of colon cancer and metastases at a submillimeter resolution in metastatic nude mice models. This provides a promising near-infrared probe for the imaging of colon cancer and metastases for preoperative diagnosis and fluorescence-guided surgery.

Exposure to perfluorooctane sulfonate based on circadian rhythm changes the fecundity and expression of certain genes on the hypothalamic-pituitary-gonadal-liver axis of female zebrafish.

Exposure of a variety of experimental animals to perfluorooctane sulfonate (PFOS) has shown that it is a potent endocrine-disrupting chemical. However, its interaction with the circadian rhythm on responses along the hypothalamic - pituitary - gonadal - liver (HPGL) axis should be of significant value but has not been adequately investigated. In present study, the effects of PFOS on fecundity, levels of estradiol (E2) and expression of certain genes on the HPGL axis at two time points (8:00 AM and 7:00 PM) were compared after female zebrafish were exposed to 0, 2, 20 and 200 µg/L PFOS for 21 days. In brain, expressions of gonadotropin-releasing hormone (GnRH), gonadotropin-releasing hormone receptor (GnRHr), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were significantly different after the exposure when sampled at 8:00 AM and at 7:00 PM (P < .05). In liver, significant down-regulation of vitellogenin1 (VTG1) and estrogenic receptor α (ERα) were observed at 7:00 PM compared with 8:00 AM (P < .05). In ovary, the level of CYP19 was significantly different at the two time points (P < .05). The increase of E2 after exposure to 20 µg/L PFOS at 8:00 AM caused compensatory down-regulation of GnRHr and up-regulation of VTG1 and ERα, but not at 7:00 PM. Profiles of concentrations of E2 and several gene expressions alongside the HPGL axis were different between two times points. The change of E2 and gene expressions were more perturbed by PFOS at 8:00 AM than at 7:00 PM with circadian rhythm.

Neurodevelopmental and Metabolomic Responses from Prenatal Coexposure to Perfluorooctanesulfonate (PFOS) and Methylmercury (MeHg) in Sprague-Dawley Rats.

Methylmercury (MeHg) and perfluorooctanesulfonate (PFOS) are major contaminants of human blood that are both common in dietary fish, thereby raising questions about their combined impact on human development. Here, pregnant Sprague-Dawley rats ingested a daily dose, from gestational day 1 through to weaning, of either 1 mg/kg bw PFOS (PFOS-only), 1 mg/kg MeHg (MeHg-only), a mixture of 0.1 mg/kg PFOS and 1 mg/kg MeHg (Low-Mix), or of 1 mg/kg of PFOS and 1 mg/kg MeHg (High-Mix). Newborns were monitored for physical milestones and reflexive developmental responses, and in juveniles the spontaneous activity, anxiety, memory, and cognition were assessed. Targeted metabolomics of 199 analytes was applied to sectioned brain regions of juvenile offspring. Newborns in the High-Mix group had decreased weight gain as well as delayed reflexes and innate behavioral responses compared to controls and individual chemical groups indicating a toxicological interaction on early development. In juveniles, cumulative mixture effects increased in a dose-dependent manner in tests of anxiety-like behavior. However, other developmental test results suggested antagonism, as PFOS-only and MeHg-only juveniles had increased hyperactivity and thigmotaxic behavior, respectively, but fewer effects in Low-Mix and High-Mix groups. Consistent with these behavioral observations, a pattern of antagonism was also observed in neurochemicals measured in rat cortex, as PFOS-only and MeHg-only juveniles had altered concentrations of metabolites (e.g., lipids, amino acids, and biogenic amines), while no changes were evident in the combined exposures. The cortical metabolites altered in PFOS-only and MeHg-only exposed groups are involved in inhibitory and excitatory neurotransmission. These proof-of-principle findings at relatively high doses indicate the potential for toxicological interaction between PFOS and MeHg, with developmental-stage specific effects. Future mixture studies at lower doses are warranted, and prospective human birth cohorts should consider possible confounding effects from PFOS and mercury exposure on neurodevelopment.

Long-term storage of PEGylated liposomal oxaliplatin with improved stability and long circulation times in vivo.

Liposomal anticancer drugs have been developed with improved clinical effects and reduced side effects. We have developed a PEGylated liposomal formulation of oxaliplatin that has anticancer effects in animal models of colorectal cancer with a favorable toxicity profile. To move this formulation into clinical development, a formulation that is stable during long term storage is needed, which has similar pharmacokinetics and therapeutic activity against solid tumors to the original formulation. In this study, we found that PEGylated liposomal oxaliplatin showed no changes in particle size after long term storage (12 months at 2-8 °C), but phospholipid degradation had occurred. Hence, the stored formulation had compromised membrane integrity, resulting in decreased in vivo circulation times of the liposomes. To improve the stability during long-term storage, a screening study to obtain an appropriate stabilizer was carried out. The buffer 2-morpholinoethansulfonic acid (MES) attenuated not only phospholipid degradation but also oxaliplatin degradation, unlike most other excipients. After 12 months storage at 2-8 °C in the presence of MES only slight degradation of phospholipids in PEGylated liposomal oxaliplatin occurred, resulting in similar in vivo pharmacokinetic profiles of the encapsulated oxaliplatin to the original formulation. Long term stability of PEGylated liposomal oxaliplatin was achieved by addition of MES, resulting in long circulation half-lives in vivo, a property which would be expected to lead to increased suppression of tumor growth and reduced side effects. Our formulation may now be suitable for clinical development.

Implementation and benchmarking of a novel analytical framework to clinically evaluate tumor-specific fluorescent tracers.

During the last decade, the emerging field of molecular fluorescence imaging has led to the development of tumor-specific fluorescent tracers and an increase in early-phase clinical trials without having consensus on a standard methodology for evaluating an optical tracer. By combining multiple complementary state-of-the-art clinical optical imaging techniques, we propose a novel analytical framework for the clinical translation and evaluation of tumor-targeted fluorescent tracers for molecular fluorescence imaging which can be used for a range of tumor types and with different optical tracers. Here we report the implementation of this analytical framework and demonstrate the tumor-specific targeting of escalating doses of the near-infrared fluorescent tracer bevacizumab-800CW on a macroscopic and microscopic level. We subsequently demonstrate an 88% increase in the intraoperative detection rate of tumor-involved margins in primary breast cancer patients, indicating the clinical feasibility and support of future studies to evaluate the definitive clinical impact of fluorescence-guided surgery.

Paradoxical Protective Effect of Perfluorooctanesulfonic Acid Against High-Fat Diet-Induced Hepatic Steatosis in Mice.

Perfluorooctanesulfonic acid (PFOS) is a persistent organic pollutant with worldwide bioaccumulation due to a very long half-life. Perfluorooctanesulfonic acid exposure results in significant hepatic effects including steatosis, proliferation, hepatomegaly, and in rodents, carcinogenesis. The objective of this study was to determine whether PFOS exposure exacerbates nonalcoholic fatty liver disease and nonalcoholic steatohepatitis pathogenesis. Eight-week-old male C57BL/6 J mice (n = 5 per group) were fed ad libitum normal chow diet (ND) alone, 60% high-fat diet (HFD) alone, ND + PFOS, and HFD + PFOS (0.0001% w/w (1 mg/kg) of PFOS) for 6 weeks. Both HFD alone and the ND + PFOS treatment induced significant adiposity and hepatomegaly, but the HFD + PFOS treatment showed a marked protection. Oil Red O staining and quantitative analysis of hepatic lipid content revealed increased hepatic steatosis in ND + PFOS and in HFD alone fed mice, which was prevented in HFD + PFOS treatment. Further studies revealed that ND + PFOS treatment significantly affected expression of lipid trafficking genes to favor steatosis, but these changes were absent in HFD + PFOS group. Specifically, expression of CD36, the major lipid importer in the cells, and peroxisome proliferator-activated receptor gamma (PPARγ), its major regulator, were induced in HFD + no treatment (NT) and ND + PFOS-fed mice but remained unchanged in HFD + PFOS mice. In conclusion, these data indicate that coadministration of PFOS with HFD mitigates steatosis and hepatomegaly induced by HFD and that by PFOS fed in ND diet via regulation of cellular lipid import machinery. These findings suggest dietary lipid content be considered when performing risk management of PFOS in humans and the elucidation of PFOS-induced hepatotoxicity.

High concentrations of perfluorooctane sulfonate in mucus of tiger puffer fish Takifugu rubripes: a laboratory exposure study.

Distribution of perfluorooctane sulfonate (PFOS) was investigated in tissues (plasma, blood clot, mucus, skin, liver, muscle, and gonad) of tiger puffer fish Takifugu rubripes. A single dose of PFOS was intraperitoneally injected at 0.1 mg/kg body weight with samples taken over a 14-day period. The highest concentration of PFOS was found in the plasma, 861 ng/mL at 14 days, followed by the mucus, liver, blood clot, gonads, muscles, and skin of fish. A gradual upward trend in PFOS concentration was observed in the mucus and liver whereas there was no change in the plasma, blood clot, gonad, muscle, and skin after the initial increase in PFOS concentrations following injection. No significant trend for estimated total PFOS content in whole body was observed during the experimental period. Relatively high concentrations of PFOS (690 ng/g ww after 14 days) were detected in body surface mucus that continuously oozes from the skin. These results may suggest that mucus is one of the elimination pathways of PFOS in tiger puffer fish.

Perfluorooctane sulphonate induces oxidative hepatic damage via mitochondria-dependent and NF-κB/TNF-α-mediated pathway.

Perfluorooctane sulphonate (PFOS) has been reported to accumulate in liver and cause damage. The molecular mechanism of the PFOS-induced hepatotoxicity has not been completely elucidated. The aim of the present study was to investigate whether PFOS-induced oxidative stress plays an important role in liver damage, and if so, what pathway it undergoes for the mechanism of its toxicological action. Male Sprague-Dawley (SD) rats were orally administrated with PFOS at single dose of 1 or 10 mg/kg body weight for 28 consecutive days. Increased serum levels of liver enzymes and abnormal ultra structural changes were observed in the PFOS-exposed rats. Particularly, PFOS exposure significantly increased intracellular reactive oxygen species (ROS) and nitric oxide (NO) production, but weakened intracellular antioxidant defence by inhibiting catalase and superoxide dismutase activities. Signal transduction studies showed that PFOS exposure significantly elevated inducible nitric oxide synthase (iNOS), Bax, cytochrome c, cleaved caspase-9 and cleaved caspase-3, indicating the mitochondria-dependent apoptotic pathway was activated. On the other hand, significant alterations of the PFOS-induced protein expression of NF-κB and IκBα in association with an enhanced level of TNF-α were observed. Taken together, these results indicate that mitochondria play an important role in PFOS-induced hepatotoxicity.

Tissue toxicokinetics of perfluoro compounds with single and chronic low doses in male rats.

To examine the kinetics of low doses of perfluoro compounds (PFCs), we administered perfluorohexanoic acid (C6A), perfluorooctanoic acid (C8A), perfluorononanoic acid (C9A) and perfluorooctane sulfonate (C8S) with a single oral dose (50-100 µg/kg BW), and in drinking water at 1, 5, and 25 µg/L for one and three months to male rats; and examined the distribution in the brain, heart, liver, spleen, kidney, whole blood and serum. C6A was very rapidly absorbed, distributed and eliminated from the tissues with nearly the same tissue t1/2 of 2-3 hr. Considering serum Vd, and the tissue delivery, C6A was mainly in the serum with the lowest delivery to the brain; and no tissue accumulation was observed in the chronic studies as estimated from the single dose study. For the other PFCs, the body seemed to be an assortment of independent one-compartments with a longer elimination t1/2 for the liver than the serum. The concentration ratio of liver/serum increased gradually from C0 to a steady state. The high binding capacity of plasma protein may be the reason for the unusual kinetics, with only a very small fraction of free PFCs moving gradually to the liver. Although the tissue specific distribution was time dependent and different among the PFCs, the Vd and ke of each tissue were constant throughout the study. The possibility of extremely high C6A accumulation in the human brain and liver was suggested, by comparing the steady state tissue concentration of this study with the human data reported by Pérez et al. (2013).

Perfluorooctane sulfonate induces neuronal vulnerability by decreasing GluR2 expression.

Perfluorooctane sulfonate (PFOS) is a persistent environmental contaminant. Although studies have described PFOS-induced neurotoxicity in animal brains and neuronal cells, the molecular mechanisms of PFOS-induced neurotoxicity based on the distribution properties, especially during developmental periods, have not been clarified. To clarify the mechanisms of PFOS-induced neuronal vulnerability during developmental periods, we examined changes in glutamate receptor 2 (GluR2) expression and related neurotoxicity in PFOS-treated primary cortical neurons and neonatal rat brains. Exposure of cortical neurons to 1 µM PFOS for 9 days resulted in decreased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR2 expression, which subsequently enhanced vulnerability to glutamate by increasing intracellular Ca2+ concentrations. The brain-plasma ratio of PFOS in pups was approximately five times higher than that in dams, although there were no differences in liver-plasma ratio between dams and pups. GluR2 expression in pup cerebral cortex decreased after exposure to 2.0 mg/kg PFOS, and kainic acid induced histopathological abnormalities in PFOS-exposed pups. Our findings suggest that decreased neuronal GluR2 expression is involved in PFOS-induced neurotoxicity, especially during the fetal and neonatal periods.

Oral perfluorooctane sulfonate (PFOS) lessens tumor development in the APCmin mouse model of spontaneous familial adenomatous polyposis.

BACKGROUND: Colorectal cancer is the second most common cause of cancer deaths for both men and women, and the third most common cause of cancer in the U.S. Toxicity of current chemotherapeutic agents for colorectal cancer, and emergence of drug resistance underscore the need to develop new, potentially less toxic alternatives. Our recent cross-sectional study in a large Appalachian population, showed a strong, inverse, dose-response association of serum perfluorooctane sulfonate (PFOS) levels to prevalent colorectal cancer, suggesting PFOS may have therapeutic potential in the prevention and/or treatment of colorectal cancer. In these preliminary studies using a mouse model of familial colorectal cancer, the APCmin mouse, and exposures comparable to those reported in human populations, we assess the efficacy of PFOS for reducing tumor burden, and evaluate potential dose-response effects. METHODS: At 5-6 weeks of age, APCmin mice were randomized to receive 0, 20, 250 mg PFOS/kg (females) or 0, 10, 50 and 200 mg PFOS/kg (males) via their drinking water. At 15 weeks of age, gastrointestinal tumors were counted and scored and blood PFOS levels measured. RESULTS: PFOS exposure was associated with a significant, dose-response reduction in total tumor number in both male and female mice. This inverse dose-response effect of PFOS exposure was particularly pronounced for larger tumors (r2 for linear trend = 0.44 for males, p's <0.001). CONCLUSIONS: The current study in a mouse model of familial adenomatous polyposis offers the first experimental evidence that chronic exposure to PFOS in drinking water can reduce formation of gastrointestinal tumors, and that these reductions are both significant and dose-dependent. If confirmed in further studies, these promising findings could lead to new therapeutic strategies for familial colorectal cancer, and suggest that PFOS testing in both preventive and therapeutic models for human colorectal cancer is warranted.

Testosterone-Mediated Endocrine Function and TH1/TH2 Cytokine Balance after Prenatal Exposure to Perfluorooctane Sulfonate: By Sex Status.

Little information exists about the evaluation of potential developmental immunotoxicity induced by perfluorooctane sulfonate (PFOS), a synthetic persistent and increasingly ubiquitous environmental contaminant. To assess potential sex-specific impacts of PFOS on immunological health in the offspring, using male and female C57BL/6 mice, pups were evaluated for developmental immunotoxic effects after maternal oral exposure to PFOS (0.1, 1.0 and 5.0 mg PFOS/kg/day) during Gestational Days 1-17. Spontaneous TH1/TH2-type cytokines, serum levels of testosterone and estradiol were evaluated in F1 pups at four and eight weeks of age. The study showed that male pups were more sensitive to the effects of PFOS than female pups. At eight weeks of age, an imbalance in TH1/TH2-type cytokines with excess TH2 cytokines (IL-4) was found only in male pups. As for hormone levels, PFOS treatment in utero significantly decreased serum testosterone levels and increased estradiol levels only in male pups, and a significant interaction between sex and PFOS was observed for serum testosterone at both four weeks of age (pinteraction = 0.0049) and eight weeks of age (pinteraction = 0.0227) and for estradiol alternation at four weeks of age (pinteraction = 0.0351). In conclusion, testosterone-mediated endocrine function may be partially involved in the TH1/TH2 imbalance induced by PFOS, and these deficits are detectable among both young and adult mice and may affect males more than females.

Chronic perfluorooctanesulphonic acid (PFOS) exposure produces estrogenic effects in zebrafish.

Perfluorooctanesulphonic acid (PFOS) is a ubiquitous contaminant in the aquatic environment and our earlier studies demonstrated that chronic PFOS exposures lead to a female-biased sex ratio and decreased sperm quality in male zebrafish. The underlying mechanism for these reproductive effects is unknown. In the present study, 8 h post-fertilization (hpf) zebrafish were exposed to PFOS at 250 µg/L for 5 months, and the levels of sex hormones, expression of sex determination related genes, and histological and ultrastructural changes of gonads were fully characterized. During the sex differentiation period, we observed elevated estradiol (E2) and decreased testosterone (T) levels in whole tissue homogenates from PFOS exposed juveniles. In fully mature adult male fish, serum E2 levels were slightly increased, however, the estrogen receptor alpha (esr1) was significantly elevated in PFOS treated male gonads. Histological and electron microscopic examinations revealed structural changes in the gonads of PFOS exposed male and female adult zebrafish. In summary, chronic PFOS exposure disrupts sex hormone level and related gene expression and impairs gonadal development, which may contribute to the previously reported PFOS reproductive toxicity.

Perfluorooctane sulfonate-induced insulin resistance is mediated by protein kinase B pathway.

Perfluorooctane sulfonate (PFOS), a persistent organic pollutant, is blamed to be associated with the incidence of insulin resistance in the general human population. In this study, we found that PFOS inhibited the phosphorylation and activation of protein kinase B (AKT), a key mediator of cellular insulin sensitivity, in human hepatoma HepG2 cells. The mRNA level of the gluconeogenic gene PEPCK, a downstream target gene of AKT, was increased in PFOS-treated cells. Due to stimulated gluconeogenesis, insulin-stimulated glucose uptake was decreased in HepG2 cells. In our previous study, we found that PFOS disturbed autophagy in HepG2 cells. We proposed that PFOS could inhibit the activation of AKT through inhibiting mTORC2, a key regulator of autophagy. In this study, we found that the levels of triglyceride were increased in HepG2 cells. PFOS-induced accumulation of hepatic lipids also contributed to the inhibition of AKT. Eventually, the inhibition of AKT led to insulin resistance in PFOS-treated cells. Our data would provide new mechanistic insights into PFOS-induced hepatic insulin resistance.

Ovarian responses of dairy buffalo cows to timed artificial insemination protocol, using new or used progesterone devices, during the breeding season (autumn-winter).

This study evaluated the effect of new or used P4 devices on the ovarian responses of dairy buffalo that were administered an estradiol (E2) plus progesterone (P4)-based timed artificial insemination (TAI) protocol during the breeding season. On the first day of the TAI protocol, 142 cows were randomly assigned to receive one of the following: a new device (New; 1.0 g of P4; n = 48); a device that had previously been used for 9 days (Used1x, n = 47); or a device that had previously been used for 18 days (Used2x, n = 47). Ultrasound was used to evaluate the following: the presence of a corpus luteum (CL); the diameter of the dominant follicle (ØDF) during protocol; ovulatory response; and pregnancies per AI (P/AI). Despite similar responses among the treatments, there was a significant positive association of the ØDF during TAI protocol with ovulatory responses and number of pregnancies. In conclusion, satisfactory ovarian responses and a satisfactory pregnancy rate were achieved when grazing dairy buffalo were subjected to the TAI protocol in breeding season, independent of whether a new or used P4 device was used. Furthermore, the presence of the larger follicle was associated with a higher ovulation rate and higher P/AI following TAI.