Effects Of Endocrine Disrupting Compounds Research Articles

17α-Ethynylestradiol and Levonorgestrel Exposure of Rainbow Trout RTL-W1 Cells at 18 °C and 21 °C Mainly Reveals Thermal Tolerance, Absence of Estrogenic Effects, and Progestin-Induced Upregulation of Detoxification Genes

Fish are exposed to increased water temperatures and aquatic pollutants, including endocrine-disrupting compounds (EDCs). Although each stressor can disturb fish liver metabolism independently, combined effects may exist. To unveil the molecular mechanisms behind the effects of EDCs and temperature, fish liver cell lines are potential models needing better characterisation. Accordingly, we exposed the rainbow trout RTL-W1 cells (72 h), at 18 °C and 21 °C, to ethynylestradiol (EE2), levonorgestrel (LNG), and a mixture of both hormones (MIX) at 10 µM. The gene expression of a selection of targets related to detoxification (CYP1A, CYP3A27, GST, UGT, CAT, and MRP2), estrogen exposure (ERα, VtgA), lipid metabolism (FAS, FABP1, FATP1), and temperature stress (HSP70b) was analysed by RT-qPCR. GST expression was higher after LNG exposure at 21 °C than at 18 °C. LNG further enhanced the expression of CAT, while both LNG and MIX increased the expressions of CYP3A27 and MRP2. In contrast, FAS expression only increased in MIX, compared to the control. ERα, VtgA, UGT, CYP1A, HSP70b, FABP1, and FATP1 expressions were not influenced by the temperature or the tested EDCs. The RTL-W1 model was unresponsive to EE2 alone, sensitive to LNG (in detoxification pathway genes), and mainly insensitive to the temperature range but had the potential to unveil specific interactions.

Endocrine-disrupting compounds and their impact on human placental function: evidence from placenta organ-on-chip studies.

The effects of endocrine-disrupting compounds (EDCs) on the placenta, a critical gestational organ for xenobiotic protection, are well reported; however, models to determine the role of EDCs in placental disruption are limited. An advanced 2nd-trimester human placenta organ-on-chip model (2TPLA-OOC) was developed and validated, with six representative cells of the maternal and the fetal interface interconnected with microchannels. Various EDCs (150 ng mL-1 each of bisphenol A, bisphenol S, and polybrominated diphenyl ethers-47 and -99) were gradually propagated across the chip for 72 hours, and their various effects were determined. Cigarette smoke extract (CSE), an environmental risk factor, was used as a positive control. EDCs produced overall oxidative stress in the placental/decidual cells, induced cell-specific endocrine effects, caused limited (<10%) apoptosis/necrosis in trophoblasts and mesenchymal cells, induced localized inflammation but an overall anti-inflammatory shift, did not change immune cell migration from stroma to decidua, and did not affect placental nutrient transport. Overall, (1) the humanized 2TPLA-OOC recreated the placental organ and generated data distinct from the trophoblast and other cells studied in isolation, and (2) at doses associated with adverse pregnancies, EDCs produced limited and localized insults, and the whole organ compensated for the exposure.

Environmental Pollution to Blame for Depressive Disorder?

Public concern has emerged about the effects of endocrine-disrupting compounds (EDCs) on neuropsychiatric disorders. Preclinical evidence suggests that exposure to EDCs is associated with the development of major depressive disorder (MDD) and could result in neural degeneration. The interaction of EDCs with hormonal receptors is the best-described mechanism of their biological activity. However, the dysregulation of the hypothalamic-pituitary-gonadal adrenal axis has been reported and linked to neurological disorders. At a worldwide level and in Mexico, the incidence of MDD has recently been increasing. Of note, in Mexico, there are no clinical associations on blood levels of EDCs and the incidence of the MDD. Methodology: Thus, we quantified for the first time the serum levels of parent compounds of two bisphenols and four phthalates in patients with MDD. The levels of di-ethyl-hexyl-phthalate (DEHP), butyl-benzyl-phthalate (BBP), di-n-butyl phthalate (DBP), and di-ethyl-phthalate (DEP), bisphenol A (BPA), and bisphenol S (BPS) in men and women with or without MDD were determined with a gas chromatograph-mass spectrometer. Results/conclusion: We found significant differences between concentrations of BBP between controls and patients with MDD. Interestingly, the serum levels of this compound have a dysmorphic behavior, being much higher in women (~500 ng/mL) than in men (≤10 ng/mL). We did not observe significant changes in the serum concentrations of the other phthalates or bisphenols tested, neither when comparing healthy and sick subjects nor when they were compared by gender. The results point out that BBP has a critical impact on the etiology of MDD disorder in Mexican patients, specifically in women.

Killing two birds with one stone: Pregnancy is a sensitive window for endocrine effects on both the mother and the fetus

Pregnancy is a complex process requiring tremendous physiological changes in the mother in order to fulfill the needs of the growing fetus, and to give birth, expel the placenta and nurse the newborn. These physiological modifications are accompanied with psychological changes, as well as with variations in habits and behaviors. As a result, this period of life is considered as a sensitive window as impaired functional and physiological changes in the mother can have short- and long-term impacts on her health. In addition, dysregulation of the placenta and of mechanisms governing placentation have been linked to chronic diseases later-on in life for the fetus, in a concept known as the Developmental Origin of Health and Diseases (DOHaD). This concept stipulates that any change in the environment during the pre-conception and perinatal (in utero life and neonatal) period to puberty, can be “imprinted” in the organism, thereby impacting the health and risk of chronic diseases later in life. Pregnancy is a succession of events that is regulated, in large part, by hormones and growth factors. Therefore, small changes in hormonal balance can have important effects on both the mother and the developing fetus. An increasing number of studies demonstrate that exposure to endocrine disrupting compounds (EDCs) affect both the mother and the fetus giving rise to growing concerns surrounding these exposures. This review will give an overview of changes that happen during pregnancy with respect to the mother, the placenta, and the fetus, and of the current literature regarding the effects of EDCs during this specific sensitive window of exposure.

Endocrine Disruptor Compounds-A Cause of Impaired Immune Tolerance Driving Inflammatory Disorders of Pregnancy?

Endocrine disrupting compounds (EDCs) are prevalent and ubiquitous in our environment and have substantial potential to compromise human and animal health. Amongst the chronic health conditions associated with EDC exposure, dysregulation of reproductive function in both females and males is prominent. Human epidemiological studies demonstrate links between EDC exposure and infertility, as well as gestational disorders including miscarriage, fetal growth restriction, preeclampsia, and preterm birth. Animal experiments show EDCs administered during gestation, or to either parent prior to conception, can interfere with gamete quality, embryo implantation, and placental and fetal development, with consequences for offspring viability and health. It has been presumed that EDCs operate principally through disrupting hormone-regulated events in reproduction and fetal development, but EDC effects on maternal immune receptivity to pregnancy are also implicated. EDCs can modulate both the innate and adaptive arms of the immune system, to alter inflammatory responses, and interfere with generation of regulatory T (Treg) cells that are critical for pregnancy tolerance. Effects of EDCs on immune cells are complex and likely exerted by both steroid hormone-dependent and hormone-independent pathways. Thus, to better understand how EDCs impact reproduction and pregnancy, it is imperative to consider how immune-mediated mechanisms are affected by EDCs. This review will describe evidence that several EDCs modify elements of the immune response relevant to pregnancy, and will discuss the potential for EDCs to disrupt immune tolerance required for robust placentation and optimal fetal development.

Developing and applying a classification system for ranking the biological effects of endocrine disrupting chemicals on male rockfish Sebastiscus marmoratus in the Maowei Sea, China

Endocrine disrupting compounds (EDCs) in marine environments has become a major environmental concern. Nonetheless, the biological effects of EDCs on organisms in coastal environments remain poorly characterized. In this study, biomonitoring of EDCs in male fish Sebastiscus marmoratus was carried out in the Maowei Sea, China. The results showed that the concentration of 4-nonylphenol (4-NP) was below the detection limit, the concentrations of 4-tert-octylphenol (4-t-OP) and bisphenol A (BPA) in seawater were moderate compared with those in other global regions, and the possible sources are the municipal wastewater discharge. Nested ANOVA analyses suggest significant differences of the brain aromatase activities and plasma vitellogenin (VTG) expression between the port area and the oyster farming area. A new fish expert system (FES) was developed for evaluating the biological effects of EDCs on fish. Our findings show that the FES is a potential tool to evaluate the biological effects of marine pollutants.

In vivo assessment of gonad status, secondary sex characteristics and spawning in transparent Casper zebrafish

Important aspects of vertebrate reproduction, such as gametogenesis, involve changes in organs found deep internally and thus not easily studied directly in most living vertebrates due to obscuring pigment and overlying tissues. Transparent lines of zebrafish, especially the Casper double mutant, allow direct observation and analysis of reproductive events in the gonads in vivo. The natural production of fertilized eggs in zebrafish is a complex process involving oogenesis, spermatogenesis, mating behavior, endocrine and neurological processes with inputs from the environment including light, temperature and nutrition. While these factors play important roles, the hypothalamic-pituitary-gonadal axis (HPGA) is central in the regulation of embryo output. Endocrine disrupting compounds (EDCs) include a variety of pollutants often present in the environment. EDCs may have direct effects on the HPGA or indirect effects through toxic action on supporting organs such as the liver or kidney. Estrogenic compounds such as diethylstilbestrol (DES) have been reported to affect reproduction in a variety of species including man. In this study, the effects of DES on reproduction were determined in a novel way by using transparent Casper zebrafish that allow direct visualization of gonad status over time. Changes in gonad status with DES treatment were correlated with effects on secondary sex characteristics (i.e., genital vent size and breeding tubercles) spawning and embryo production. The results suggest that the Casper zebrafish is a useful model for studying dynamics of reproductive events in vertebrate gonads in vivo and for determining effects of EDCs on zebrafish reproduction.

Autism spectrum disorders, endocrine disrupting compounds, and heavy metals in amniotic fluid: a case-control study

BackgroundEvidence has indicated that some non-inherited factors such as exposure to environmental pollutants are associated with neurodevelopment disorders like autism spectrum disorder (ASD). Studies report that endocrine disrupting compounds (EDCs), including polychlorinated biphenyls, organochlorine pesticides, perfluoroalkyl substances (PFAS), and some metals, have adverse effects on the fetal neurodevelopment. The aim of this study was to measure the amniotic fluid (AF) levels of EDCs and metals as well as the receptor transactivities induced by AF and investigate the possible link between prenatal exposure to EDCs and heavy metals and ASD risk.MethodsIn this case-control study, we included AF samples of 75 ASD cases and 135 frequency-matched controls and measured the levels of the endogenous sex hormones, PFAS, and elements including heavy metals. The combined effect of endogenous hormones and EDCs on the receptor of estrogen (ER), androgen (AR), aryl hydrocarbon (AhR), and thyroid hormone-like activity were also determined and expressed as receptor ligand equivalents. We assessed the associations of AF levels of chemicals, sex hormones, and receptor activities with ASD risk using unconditional logistical regression analyses. To control for multiple comparisons, the false discovery rate (FDR) was used and q values less than 0.25 were designated as statistical significance.ResultsPFAS and metals were detectable in AF samples. The ASD cases had significantly lower AF levels of PFAS than controls, and the adjusted odds ratio (OR) was 0.410 (95% CI 0.174, 0.967; p = 0.042; FDR qvalue = 0.437) for perfluorooctane sulfonate (PFOS). The principal component, including PFAS congeners, copper, iron, and estrogenic activity, was significantly inversely associated with ASD risk (adjusted OR = 0.100; 95% CI 0.016, 0.630; p = 0.014; FDR qvalue = 0.098).Testosterone level in AF weakly associated with ASD risk (adjusted OR = 1.002; 95% CI 1.000, 1.004; p = 0.05). However, after multiple comparison correction, the association was not significant (FDR qvalue = 0.437). No significant associations between AF-induced receptor transactivities and ASD risk were observed. The adjusted OR was 2.176 (95%CI 0.115, 41.153) for the ratio of the combined androgenic activity to combined estrogenic activity.ConclusionsThe presence of PFAS and heavy metals in AF indicates that they can cross the placenta. The inverse association between levels of PFAS congeners in AF and ASD risk might relate to the weak estrogenic activities and anti-androgenic activities of PFAS.The observed tendency of positive association between the ratio of combined androgenic effect to the combined estrogenic effect and ASD risk needs further studies to explore whether EDCs together with endogenous hormones play a role in the development of ASD.

Direct and indirect parental exposure to endocrine disruptors and elevated temperature influences gene expression across generations in a euryhaline model fish.

Aquatic organisms inhabiting polluted waterways face numerous adverse effects, including physiological disruption by endocrine disrupting compounds (EDCs). Little is known about how the temperatures associated with global climate change may influence the response of organisms exposed to EDCs, and the effects that these combined stressors may have on molecular endpoints such as gene expression. We exposed Menidia beryllina (inland silversides) to environmentally relevant concentrations (1 ng/L) of two estrogenic EDCs (bifenthrin and 17α-ethinylestradiol; EE2) at 22 °C and 28 °C. We conducted this experiment over multiple generations to better understand the potential effects to chronically exposed populations in the wild. We exposed adult parental fish (F0) for 14 days prior to spawning of the next generation. F1 larvae were then exposed from fertilization until 21 days post hatch (dph) before being transferred to clean water tanks. F1 larvae were reared to adulthood, then spawned in clean water to test for further effects of parental exposure on offspring (F2 generation). Gene expression was quantified by performing qPCR on F0 and F1 gonads, as well as F1 and F2 larvae. We did not detect any significant differences in the expression of genes measured in the parental or F1 adult gonads. We found that the 28 °C EE2 treatment significantly decreased the expression of nearly all genes measured in the F1 larvae. This pattern was transferred to the F2 generation for expression of the follicle-stimulating hormone receptor (FSHR) gene. Expression of 17β-hydroxysteroid dehydrogenase (17β-HSD) and G protein-coupled receptor 30 (GPR30) revealed changes not measured in the previous generation. Effects of the bifenthrin treatments were not observed until the F2 generation, which were exposed to the chemicals indirectly as germ cells. Our results indicate that effects of EDCs and their interactions with abiotic factors, may not be adequately represented by singular generation testing. These findings will contribute to the determination of the risk of EDC contamination to organisms inhabiting contaminated waterways under changing temperature regimes.

The Role of Uron and Chlorobenzene Derivatives, as Potential Endocrine Disrupting Compounds, in the Secretion of ACTH and PRL.

Uron herbicides polluting the environment represent a serious concern for environmental health and may be regarded as endocrine-disrupting compounds (EDCs), which influence the regulation of human homeostasis. We aimed to investigate the effect of EDC urons (phenuron: PU, monuron: MU, and diuron: DU) and chlorobenzenes on the basal release of the adrenocorticotropic hormone (ACTH), which is a part of the adenohypophysis-adrenocortical axis. Hormone secretion in the presence of EDC was studied in two cell types: normal adenohypophysis cells (AdH) and cells of prolactinomas (PRLOMA). PRLOMA was induced in female Wistar rats by subcutaneously injecting them with estrone acetate for 6 months. AdH and PRLOMA were separated from treated and untreated experimental animals, dissociated enzymatically and mechanically in order to create monolayer cell cultures, which served as an experimental in vitro model. We investigated the effects of ED agents separately and in combination on ACTH and prolactin (PRL) release through the hypophyseal-adrenal axis. Hormone determination was carried out by the luminescent immunoassay and the radioimmunoassay methods. Our results showed that (1) uron agents separately did not change ACTH and PRL release in AdH culture; (2) ACTH secretion in arginine vasopressin- (AVP-) activated AdH cells was significantly increased by EDC treatment; (3) ED agents increased the basal hormone release (ACTH, PRL) in PRLOMA cells; and (4) EDC exposure increased ACTH release in AVP-activated PRLOMA cells. We conclude that the herbicides PU, MU, and DU carry EDC effects and show human toxicity potential.

Endocrine disrupters and pubertal timing.

The current review summarizes recent epidemiologic data demonstrating the effects of endocrine disrupting compounds (EDCs) on the timing of puberty and highlights the complexity of understanding the interplay of environmental and genetic factors on pubertal timing. In girls, there have been mixed results, with some exposures being associated with earlier timing of puberty, and some with later puberty. In boys, prepubertal exposures to nondioxin-like polychlorinated biphenyls accelerate puberty, whereas levels of insecticides, dioxin-like compounds, organochlorine pesticides, and lead delay puberty. The effects of EDCs on pubertal timing are sexually dimorphic, compound specific, and varies according to the window of exposure. These studies confirm that low-level exposures to a mix of environmental compounds may mask the effects of individual compounds and complicate our ability to translate data from animal studies to human health and to fully understand the clinical implications of environmental epidemiology studies.

Evaluation of performance with small and scale-up rotating and flat reactors; photocatalytic degradation of bisphenol A, 17β–estradiol, and 17α–ethynyl estradiol under solar irradiation

In this study, the performances of photocatalytic reactors of the small and scale-up rotating and flat types were evaluated to investigate the treatment of new emerging contaminants such as bisphenol A (BPA), 17α-ethynyl estradiol (EE2), and 17β-estradiol (E2) that are known as endocrine disrupting compounds (EDCs). In the laboratory tests with the small-scale rotating and flat reactors, the degradation efficiencies of the mixed EDCs were significantly influenced by the change of the hydraulic retention time (HRT). In particular, considering the effective two-dimensional reaction area with light and nanotubular TiO2 (NTT) on a Ti substrate, the rotating reactors showed the more effective performance than the flat reactor because the degradation efficiencies are similar in the small effective area. In addition, the major parameters affecting the photocatalytic activities of the NTT were evaluated for the rotating reactors according to the effects of single and mixed EDCs, the initial concentrations of the EDCs, the UV intensity, and dissolved oxygen. In the extended outdoor tests with the scale-up photocatalytic reactors and NTT, it was confirmed from the four representative demonstrations that an excellent rotating-reactor performance is consistently shown in terms of the degradation of the target pollutants under solar irradiation.

Scaling Up Endocrine Disruption Effects from Individuals to Populations: Outcomes Depend on How Many Males a Population Needs.

Assessing how endocrine disrupting compounds (EDCs) affect population dynamics requires tracking males and females (and sex-reversed individuals) separately. A key component in any sex-specific model is the "mating function" (the relationship between sex ratio and reproductive success) but this relationship is not known for any fish species. Using a model, we found that EDC effects on fish populations strongly depend upon the shape of the mating function. Additionally, masculinization is generally more detrimental to populations than feminization. We then quantified the mating function for the inland silverside (Menidia beryllina), and used those results and the model to assess the status of wild silverside populations. Contrary to the expectation that a few males can spawn with many females, silversides exhibited a nearly linear mating function. This implies that small changes in the sex ratio will reduce reproductive success. Four out of five wild silverside populations exhibited sex ratios far from 50:50 and thus are predicted to be experiencing population declines. Our results suggest that managers should place more emphasis on mitigating masculinizing rather than feminizing EDC effects. However, for species with a nearly linear mating function, such as Menidia, feminization and masculinization are equally detrimental.

Regulation of arcuate genes by developmental exposures to endocrine-disrupting compounds in female rats.

Developmental exposure to endocrine-disrupting compounds (EDCs) alters reproduction and energy homeostasis, both of which are regulated by the arcuate nucleus (ARC). Little is known about the effects of EDC on ARC gene expression. In Experiment #1, pregnant dams were treated with either two doses of bisphenol A (BPA) or oil from embryonic day (E)18-21. Neonates were injected from postnatal day (PND)0-7. Vaginal opening, body weights, and ARC gene expression were measured. Chrm3 (muscarinic receptor 3) and Adipor1 (adiponectin receptor 1) were decreased by BPA. Bdnf (brain-derived neurotropic factor), Igf1 (insulin-like growth factor 1), Htr2c (5-hydroxytryptamine receptor), and Cck2r (cholescystokinin 2 receptor) were impacted. In Experiment #2, females were exposed to BPA, diethylstilbestrol (DES), di(2-ethylhexyl)phthalate, or methoxychlor (MXC) during E11-PND7. MXC and DES advanced the age of vaginal opening and ARC gene expression was impacted. These data indicate that EDCs alter ARC genes involved in reproduction and energy homeostasis in females.

Preliminary study on diminution level of RNA/DNA ratio in tissue of Labeo rohita by exposure to some endocrine disrupting compounds (EDCs)

Effects of EDCs particularly on RNA/DNA ratio are yet to be investigated to manage the effluents in natural waters. We investigated exposure effects of endocrine disrupting compounds (EDCs) phthalic acid ester (PAE) and hexachlorocyclohexane (HCH) on the RNA/DNA ratio in tissue of an Indian major carp Labeo rohita. Fish were exposed to pre-determined sublethal concentrations of phthalic acid ester (Di-methyl phthalate (DMP), di-butyl phthalate (DBP), and di-(2- ethylhexyl) phthalate (DEHP) and also HCH for determining the tissue RNA/DNA ratio after 30, 60 and 90 days of exposure in the doses of 0.2 mg L-1, 0.3 mg L-1, and 0.5 mg L-1 respectively. All these tested chemicals significantly (P0.05) inhibited RNA/DNA ratio. The ratio gradually significantly (P0.05) decreased after DEHP where it was 1.9±0.51 F1, 18=15.8 P=0.014 n=19; in case of DBP it was 1.92±0.62 F1, 20=6.5 P=0.012 n=19 and for HCH it was 0.94±0.21 F1, 18=18.08 P=0.0012 n=19 at treatments concentrations of 0.3 mg L-1 and 0.5 mg L-1, compared to control (2.9±0.2) after 90 days. However, there was no statistical significance (P0.05) in RNA/DNA ratio after the DMP (F1, 20=2.4 P=0.15n=21) treatment.

Thyroid endocrine status of wild European eels (Anguilla anguilla) in the Loire (France). Relationships with organic contaminant body burdens

In teleost fish, thyroid function is involved in various critical physiological processes. Given the complexity of the hypothalamo–pituitary–thyroid (HPT) axis, a large number of genes and proteins can be the potential target of endocrine-disrupting compounds (EDCs). The aim of this study was to evaluate, in yellow and silver European eels (Anguilla anguilla), potential effects of EDCs on thyroid status by analyzing the associations between EDC body burdens and thyroid hormones (THs). In yellow individuals, greater free T3/T4 ratios (FT3/FT4) and lower plasma FT4 levels were associated with greater concentrations of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), as highlighted by significant correlations with many congeners. Few positive relationships with alkylphenols were noticed. In contrast, silver eels usually exhibited less significant correlations between THs and contaminant loads. Expression of a series of genes involved in the HPT axis was also investigated in the silver individuals. Concerning mRNA expression in silver females, some main correlations were noticed: thyroid-stimulating hormone (TSHβ) gene expression was significantly correlated to numerous PCBs, and hepatic mRNA levels of deiodinase 2 (Deio 2) were negatively correlated to 2-hydroxyfluorene (2-OHF) and 2-naphtol (2-OHNa). Thyroid receptor (TRα and TRβ) mRNA levels exhibited weak negative correlations with some PBDEs in silver females and males. Hepatic vitellogenin (Vtg) mRNA levels were detected in all silver males but at lower levels than in silver females. In males, Vtg mRNA levels were positively associated to FT4/TT4. In silver females, strong positive correlations were found between congeners of PCBs, PBDEs and PFAS suggesting potential estrogenic effects. Overall, the observed results indicate that several organic contaminants, mainly dl-, ndl-PCBs and PBDEs, could be associated with changes in thyroid homeostasis in these fish, via direct or indirect interactions with peripheral deiodination, metabolism of T4 and mechanisms involved in TSHβ, Deio 2 and Vtg gene transcription.

Ecdysone-Related Biomarkers of Toxicity in the Model Organism Chironomus riparius: Stage and Sex-Dependent Variations in Gene Expression Profiles

Despite being considered a model organism in toxicity studies, particularly in assessing the environmental impact of endocrine disrupting compounds (EDCs) and other chemicals, the molecular basis of development is largely unknown in Chironomus riparius. We have characterized the expression patterns of important genes involved in the ecdysone pathway from embryos to pupa, but specially during the different phases of C. riparius fourth larval instar, according to the development of genital and thoracic imaginal discs. Real-Time PCR was used to analyze: EcR and usp, two genes encoding the two dimerizing partners of the functional ecdysone receptor; E74, an early response gene induced by ecdysteroids; vg (vitellogenin), an effector gene; hsp70 and hsc70, two heat-shock genes involved in the correct folding of the ecdysone receptor; and rpL13, as a part of the ribosomal machinery. Our results show for the first time stage and sex-dependent variations in ecdysone-responsive genes, specially during the late larval stage of C. riparius. The induction in the expression of EcR and usp during the VII-VIII phase of the fourth instar is concomitant with a coordinated response in the activity of the other genes analyzed, suggesting the moment where larvae prepare for pupation. This work is particularly relevant given that most of the analyzed genes have been proposed previously in this species as sensitive biomarkers for the toxicological evaluation of aquatic ecosystems. Identifying the natural regulation of these molecular endpoints throughout the Chironomus development will contribute to a more in-depth and accurate evaluation of the disrupting effects of EDCs in ecotoxicological studies.

Xenoestrogens disrupt type 2 collagen expression in chondrocytes in vitro.

Purpose: In recent years chemicals with endocrine properties, called Endocrine Disrupting Compounds (EDC), have become a topic of scientific and public discussion. In the field of osteoarthritic pathologies, EDC compounds that mimic estrogenic agonists or antagonists are of major concern, because of the central role of estrogens in skeletal developmental processes (ref). EDC are acting on their target genes upon binding the aryl hydrocarbon receptor (AhR). During the ten past years, interactions between estrogen receptor (ER) and some xenobiotic-activated AhR have been well demonstrated. Very few studies have been reported on EDC effects on bone or cartilage. Our laboratory recently studied the effects of low doses and cocktail xenoestrogens on rat cartilage development in vivo, by using a rodent gestational/lactational model (T.A. Auxietre, et al, 2014). We observed transient modifications of caudal vertebral body associated with a decrease in growth plate cartilage thickness with greater impact on the hypertrophic chondrocyte zone. We inferred from these data that the tested compounds could interfere with the dynamic of chondrogenic differentiation and/or maturation processes, possibly by impacting type 2 collagen (Col2), one of the the major components of cartilage that assures cartilage function. The present work was aimed to study the in vitro effects of two xenoestrogens (genistein, G; bisphenol A, BPA) and one anti-androgen (vinclozolin, V) and its metabolite M2 on Col2 expression in two different models of cultured chondrocytes: during the process of chondrogenic induction and in mature chondrocytes. Methods: A murine stem cell line inducible towards chondrogenesis (C1) was used for dynamic studies of differentiation using chondrogenic and non-chondrogenic markers. In parallel, post-natal murine chondrocytes were used for steady-state investigations in differentiated chondrocytes in primary culture (P0) or after dedifferentiation by passages (P3) or FGF2 treatment. Biological markers were studied at the mRNA (qPCR) and protein (western blot) levels. COL2A protein expression was evaluated by western blot and immunocytochemistry by using an antibody which only recognizes the specific COL2A N-terminal propeptide. Fulvestrant and SB 203580 were used respectively as specific inhibitors of ER or p38MEK pathways. Implication of Tia1-splicing was shown after cell transfection with specific anti-Tia1 siRNAs. Results: Vinclozolin was practically inactive. M2, alone or combined with G or BPA, modified the dynamic of Col2A immature isoform of COL2 during chondrogenic induction. These compounds extended the basal expression of COL2A and delayed its replacement by the mature isoform COL2B in C1 cells. This effect was dose dependent with maximum at 10-6M. In post natal chondrocytes, COL2A expression increased with cell dedifferentiation or FGF2 treatment. EDC were inactive on differentiated chondrocytes while COL2A doubled upon M2, G and BPA addition in dedifferentiated cells. Estrogen receptor (ER) and the p38-MEK pathway were involved. As these effects were only partly transcriptional, we investigated and found an effect of FGF2 on Tia-1 splice protein expression. EDC showed no effects on other chondrogenic markers (SOX9, Aggrecan, Col10α1) nor non-chondrogenic markers. Conclusions: These data showed that xenosestrogens as well as the vinclozolin metabolite M2 modified the rate of early chondrogenic differentiation by transiently maintaining COL2A expression. Our hypothesis is that COL2A induced persistence, might provoke matrix weakening in the long term and/or sequester growth factors, eventually modifying the course of cartilage degenerative diseases such as osteoarthritis. EDC also modified COL2A expression in dedifferentiated chondrocytes such as found in aging or osteoarthritic tissues. These data are in accordance with the strong Col2A expression reported in OA cartilage but its role remains to be studied.

Parental effects of endocrine disrupting compounds in aquatic wildlife: Is there evidence of transgenerational inheritance?

The effects of endocrine disrupting compounds (EDCs) on aquatic wildlife are increasingly being recognized for their complexity. Investigators have detected alterations at multiple levels of biological organization in offspring exposed to EDCs through the blood or germ line of the parents, suggesting that generational consequences of EDCs are evident. Exposure to EDCs through the parents is concerning because if the resulting phenotype of the offspring is heritable and affects fitness, then evolutionary consequences may be evident. This review summarizes the evidence for transgenerational effects of EDCs in aquatic wildlife and illustrates cases where alterations appear to be transmitted maternally, paternally, or parentally. The literature indicates that EDC exposure to the parents induces developmental, physiological, endocrinological, and behavioral changes as well as increased mortality of offspring raised in clean environments. What is lacking, however, is a clear demonstration of heritable transgenerational effects in aquatic wildlife. Therefore, it is not known if the parental effects are the result of developmental or phenotypic plasticity or if the altered phenotypes are durably passed to subsequent generations. Epigenetic changes to gene regulation are discussed as a possible mechanism responsible for EDC induced parental effects. Additional research is needed to evaluate if heritable effects of EDCs are evident in aquatic wildlife, as has been demonstrated for terrestrial mammals.

Detection of immunotoxic effects of estrogenic and androgenic endocrine disrupting compounds using splenic immune cells of the female three-spined stickleback, Gasterosteus aculeatus (L.)

Today, the list of endocrine disrupting compounds (EDCs) in freshwater and marine environments that mimic or block endogenous hormones is expanding at an alarming rate. As immune and reproductive systems may interact in a bidirectional way, some authors proposed the immune capacities as attractive markers to evaluate the hormonal potential of environmental samples. Thus, the present work proposed to gain more knowledge on direct biological effects of natural and EDCs on female fish splenic leucocyte non-specific immune activities by using ex vivo assays. After determining the optimal required conditions to analyze splenic immune responses, seven different EDCs were tested ex vivo at 0.01, 1 and 100nM over 12h on the leucocyte functions of female three-spined stickleback, Gasterosteus aculeatus. In summary, we found that natural hormones acted as immunostimulants, whilst EDCs were immunosuppressive.