Activation of NLRP3 Inflammasome in Liver of Long Evans Lactating Rats and Its Perinatal Effects in the Offspring after Bisphenol F Exposure.

The liver is the organ responsible for the metabolism and detoxification of BPF, the BPA analogue that is replacing it in plastic-based products. It is not known whether BPF can trigger inflammatory responses via the NLRP3 inflammasome, which plays a major role in the development of liver disease. The aim of this study was to evaluate nitrosative stress species (RNS) and NLRP3 inflammasome activation in the liver of lactating dams after BPF exposure. Moreover, it was studied whether this effect could also be observed in the liver of female and male offspring at postnatal day 6 (PND6). 36 Long Evans rats were randomly distributed according to oral treatment into three groups: Control, BPF-low dose (LBPF; 0.0365 mg/kg b.w./day) group and BPF-high dose (HBPF; 3.65 mg/kg b.w./day) group. The levels of nitrosative stress-inducing proteins (eNOS, iNOS, HO-1d), NLRP3 inflammasome components (NLRP3, PyCARD, CASP1) and proinflammatory cytokines (IL-1ß, IL-18, IFN-γ and TNF-α) were measured by gene and protein expression in the liver of lactating dams and in female and male PND6 offspring. Lactating dams treated with LBPF showed a significant increase in iNOS and HO-1d, activation of NLRP3 components (NLRP3, PyCARD, CASP1) and promoted the release of proinflammatory cytokines such as IL-1ß, IL-18, IFN-γ and TNF-α. Similar effects were found in female and male PND6 offspring after perinatal exposure. LBPF oral administration and perinatal exposure caused an increase of nitrosative stress markers and proinflammatory cytokines. Also, NLRP3 inflammasome activation was significantly increased in in the liver of lactating dams and PND6 offspring.

Oxidative stress increases in liver of lactating rats after BPF-low-dose exposure: perinatal effects in the offspring.

Bisphenol F (BPF) is replacing Bisphenol A (BPA) in the manufacture of products due to endocrine-disrupting effects. BPF monomers can also be released into the environment and enter the food chain, resulting in human exposure to low doses. Since bisphenols are primarily metabolized by the liver, this organ is more vulnerable to lower doses of bisphenols than others. Exposure during prenatal development may increase the risk of diseases in adulthood. The aim was to evaluate whether BPF administration could generate oxidative stress in liver of lactating rats, and whether these effects may be also observed in female and male postnatal day 6 (PND6) offspring. Long Evans rats received oral treatment: Control, BPF-low-dose (LBPF) 0.0365 mg/kg b.w./day, and BPF-high-dose (HBPF) 3.65 mg/kg b.w./day. The levels of antioxidant enzymes (CAT, SOD, GR, GPx and GST), glutathione system (GSH, GSSG) and lipid damage markers (MDA, LPO) were measured using colorimetric methods in liver of both lactating dams and in PND6 offspring. Mean values were analyzed using Prism-7. LBPF affected liver defense mechanisms (antioxidant enzymes and glutathione system), increasing ROS levels and producing lipid peroxidation in lactating dams. Similar effects were found in female and male PND6 offspring as a consequence of perinatal exposure.

Low Dose of BPA Induces Liver Injury through Oxidative Stress, Inflammation and Apoptosis in Long-Evans Lactating Rats and Its Perinatal Effect on Female PND6 Offspring.

Bisphenol A (BPA) is a phenolic compound used in plastics elaboration for food protection or packaging. BPA-monomers can be released into the food chain, resulting in continuous and ubiquitous low-dose human exposure. This exposure during prenatal development is especially critical and could lead to alterations in ontogeny of tissues increasing the risk of developing diseases in adulthood. The aim was to evaluate whether BPA administration (0.036 mg/kg b.w./day and 3.42 mg/kg b.w./day) to pregnant rats could induce liver injury by generating oxidative stress, inflammation and apoptosis, and whether these effects may be observed in female postnatal day-6 (PND6) offspring. Antioxidant enzymes (CAT, SOD, GR, GPx and GST), glutathione system (GSH/GSSG) and lipid-DNA damage markers (MDA, LPO, NO, 8-OHdG) were measured using colorimetric methods. Inducers of oxidative stress (HO-1d, iNOS, eNOS), inflammation (IL-1ß) and apoptosis (AIF, BAX, Bcl-2 and BCL-XL) were measured by qRT-PCR and Western blotting in liver of lactating dams and offspring. Hepatic serum markers and histology were performed. Low dose of BPA caused liver injury in lactating dams and had a perinatal effect in female PND6 offspring by increasing oxidative stress levels, triggering an inflammatory response and apoptosis pathways in the organ responsible for detoxification of this endocrine disruptor.

Converging Effects of Three Different Endocrine Disrupters on Sox and Pou Gene Expression in Developing Rat Hippocampus: Possible Role of microRNA in Sex Differences.

Endocrine disrupting chemicals (EDCs) can impair hippocampus-dependent behaviors in rat offspring and in children. In search for key processes underlying this effect, we compared the transcriptomes of rat hippocampus on postnatal day 6 after gestational and lactational exposure to three different EDCs at doses known to impair development of learning and memory. Aroclor 1254, a commercial PCB mixture (5 mg/kg or 0.5 mg/kg), or bisphenol A (5 mg/kg or 0.5 mg/kg) were administered in chow, chlorpyrifos (3 mg/kg or 1 mg/kg) was injected subcutaneously. Male hippocampus exhibited a common effect of all three chemicals on genes involved in cell-autonomous processes, Sox6, Sox11, Pou2f2/Oct2, and Pou3f2/Brn2, all upregulated at the high dose. Additional genes of the Sox and Pou families were affected by only one or two of the chemicals. Real time RT PCR showed a comparable expression change for bisphenol A also at the lower dose. Female hippocampus exhibited much fewer genes with expression changes (almost none with false discovery rate <0.05), and none of the genes of the Sox and Pou families was affected. Since gene network analyses in male hippocampus suggested a link between Sox6 and miR-24, known to be repressed by activation of ER-alpha and to repress Sox6 in other tissues, this microRNA was measured. miR-24 was downregulated by all chemicals at the high dose in males. Values of Sox6 mRNA and miR-24 were inversely correlated in individual male hippocampus samples, supporting the hypothesis that the change in Sox6 expression resulted from an action of miR-24. In contrast, miR-24 levels remained unchanged in hippocampus of females. A sexually dimorphic response of miR-24 may thus be at the basis of the sex difference in Sox6 expression changes following exposure to the three chemicals. ER-alpha expression was also sex-dependent, but the expression changes did not parallel those of potential downstream genes such as Sox6. Sox6 is known to suppress differentiation of Parvalbumin (Pvalb)-expressing interneurons. Individual Sox6 levels (FPKM) were inversely correlated with levels of Pvalb, but not with markers of Sox6-independent interneuron subpopulations, Nos1 and 5HT3aR. Effects on interneuron development are further suggested, in males, by expression changes of Nrg1 and its receptor Erbb4, controlling interneuron migration. Our study disclosed new types of EDC-responsive morphogenetic genes, and illustrated the potential relevance of microRNAs in sexually dimorphic EDC actions.

The ENDpoiNTs Project: Novel Testing Strategies for Endocrine Disruptors Linked to Developmental Neurotoxicity.

Ubiquitous exposure to endocrine-disrupting chemicals (EDCs) has caused serious concerns about the ability of these chemicals to affect neurodevelopment, among others. Since endocrine disruption (ED)-induced developmental neurotoxicity (DNT) is hardly covered by the chemical testing tools that are currently in regulatory use, the Horizon 2020 research and innovation action ENDpoiNTs has been launched to fill the scientific and methodological gaps related to the assessment of this type of chemical toxicity. The ENDpoiNTs project will generate new knowledge about ED-induced DNT and aims to develop and improve in vitro, in vivo, and in silico models pertaining to ED-linked DNT outcomes for chemical testing. This will be achieved by establishing correlative and causal links between known and novel neurodevelopmental endpoints and endocrine pathways through integration of molecular, cellular, and organismal data from in vitro and in vivo models. Based on this knowledge, the project aims to provide adverse outcome pathways (AOPs) for ED-induced DNT and to develop and integrate new testing tools with high relevance for human health into European and international regulatory frameworks.

Organochlorine pesticides in placenta in Kyrgyzstan and the effect on pregnancy, childbirth, and newborn health.

Organochlorine pesticides (OCPs) were determined by gas chromatography in 241 placentas from cotton-growing regions, 121 placentas from an urban area (city of Osh), and 146 placentas from unpolluted mountain regions of Kyrgyzstan. Manifestations of disease were recorded in the mothers during pregnancy and parturition and in their newborns during the first 6 days of life. OCPs were detected in 240 out of 508 placentas (47.2%), with increased incidence in the two polluted regions (65%), particularly in placentas from women living near former pesticide storehouses and agro air-strips (99%), but only in 2.7% of placentas from the unpolluted region. α-, ß-, and γ-hexachlorocyclohexane (HCH); DDT; DDE; aldrin; and heptachlor were detected. The sum of concentrations of all OCPs (total OCPs) was calculated for each of the 240 placentas with detectable OCPs (median 9.5 µg/kg placenta, mean 88.3 µg/kg, range 0.1-3070 µg/kg). The incidence of health problems in four subgroups of this data set, with increasing levels of total OCPs, was compared with the incidence of health problems in the group of 268 placentas, where OCPs were undetectable. Relative risk of health problems in both, mothers and newborns, increased significantly, in a concentration-dependent manner, with increasing levels of total OCPs (p < 0.0001). Health complications with increased incidence in OCP-exposed newborns included, i.a., low birth weight, congenital malformations, infections, and stillbirths, in OCP-exposed mothers preterm delivery, (pre-)eclampsia/gestosis, and frequency of hospitalizations after delivery (infections). Women living near former pesticide storehouses and agro airstrips should be considered as being at risk. Reduction of exposure is urgently needed.

Efficiency control of dietary pesticide intake reduction by human biomonitoring.

In spite of food safety controls for pesticide residues, a conventional diet still leads to a noticeable exposure of the general population to several pesticides. In a pilot study the response of exposure reduction by organic diet intervention on the urinary levels of pesticide metabolites was investigated. In the study two adult individuals were kept on a conventional diet for 11days and morning urine voids were collected at the last four days of the period. Afterwards, the participants switched to exclusively organic food intake for 18days and likewise morning urine samples were collected at the last four days of this period. In the urine samples six pyrethroid metabolites, six dialkylphosphates, four phenolic parameter for organophosphate pesticides and carbamates, 6-chloronicotinic acid (ClNA) as parameter for neonicotinoid insecticides, seven phenoxy herbicides, glyphosate and its metabolite AMPA were quantified using gas chromatographic mass spectrometric methods. Generally, the comparative analyses revealed greater shares as well as higher levels of the parameters in the samples taken during the common diet period compared to the organic diet period. Considerable decrease of the levels was found for almost all pyrethroid metabolites, dialkyphosphates and phenoxy herbicids, as well as for the phenolic metabolites 4-nitrophenol and 3,5,6-trichloropyridinol. In contrast, higher values were found for the organic diet period for ClNA and the metabolite of coumaphos in one of the volunteers. The present study confirms the results of former studies which indicated that an organic diet intervention results in considerable lower exposure to organophosphate pesticides and pyrethroids. It also verifies the former experience that monitoring of urinary parameters for non-persistent pesticides permits a reliable efficiency control of short-time effects by dietary interventions. Additionally to former studies, the results of the present study highlight the need of an extension of the parameter spectrum to all prominent pesticide groups.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes.

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Differential gene expression patterns in developing sexually dimorphic rat brain regions exposed to antiandrogenic, estrogenic, or complex endocrine disruptor mixtures: glutamatergic synapses as target.

The study addressed the question whether gene expression patterns induced by different mixtures of endocrine disrupting chemicals (EDCs) administered in a higher dose range, corresponding to 450×, 200×, and 100× high-end human exposure levels, could be characterized in developing brain with respect to endocrine activity of mixture components, and which developmental processes were preferentially targeted. Three EDC mixtures, A-Mix (anti-androgenic mixture) with 8 antiandrogenic chemicals (di-n-butylphthalate, diethylhexylphthalate, vinclozolin, prochloraz, procymidone, linuron, epoxiconazole, and DDE), E-Mix (estrogenic mixture) with 4 estrogenic chemicals (bisphenol A, 4-methylbenzylidene camphor, 2-ethylhexyl 4-methoxycinnamate, and butylparaben), a complex mixture, AEP-Mix, containing the components of A-Mix and E-Mix plus paracetamol, and paracetamol alone, were administered by oral gavage to rat dams from gestation day 7 until weaning. General developmental endpoints were not affected by EDC mixtures or paracetamol. Gene expression was analyzed on postnatal day 6, during sexual brain differentiation, by exon microarray in medial preoptic area in the high-dose group, and by real-time RT-PCR in medial preoptic area and ventromedial hypothalamus in all dose groups. Expression patterns were mixture, sex, and region specific. Effects of the analgesic drug paracetamol, which exhibits antiandrogenic activity in peripheral systems, differed from those of A-Mix. All mixtures had a strong, mixture-specific impact on genes encoding for components of excitatory glutamatergic synapses and genes controlling migration and pathfinding of glutamatergic and GABAergic neurons, as well as genes linked with increased risk of autism spectrum disorders. Because development of glutamatergic synapses is regulated by sex steroids also in hippocampus, this may represent a general target of ECD mixtures.

Developmental effects of perinatal exposure to PBDE and PCB on gene expression in sexually dimorphic rat brain regions and female sexual behavior.

The developing nervous system is a potential target of environmental contaminants such as polybrominated diphenylethers (PBDE), which accumulate in the biosphere. We compared effects of 2,2',4,4',5-pentabromo-BDE (PBDE99), a PBDE congener present in environmental samples, and PCB on brain development. Time-pregnant rats were subcutaneously injected with PBDE99 (1 or 10mg/kg), the PCB mixture Aroclor 1254 (10mg/kg), or vehicle from gestational day 10-18. mRNA levels of genes involved in central control of reproductive functions and sexual behavior were analyzed by real time RT PCR in two sexually dimorphic brain regions, medial preoptic area (MPO) and ventromedial hypothalamus (VMH) of adult offspring of both sexes. Exposure to PBDE99 or the PCB mixture during pre- and postnatal development affected mRNA expression levels in a treatment-, region- and sex-specific manner, and changed the sensitivity of target genes to estradiol. The sex difference in progesterone receptor mRNA levels of VMH normally seen in untreated controls was abolished by both, PBDE99 and PCB. Estrous cycles were significantly affected, and preliminary experiments suggest an impairment of female sexual behavior. Our data indicate that developmental exposure to PBDE99 at doses below signs of general toxicity affects the regulation of estrogen target genes in rat brain. Since PBDE99 was detected in blood and adipose tissue of adult offspring, these effects may result from interactions with developmental processes, with adult functions, or a combination of both.

Exposure patterns of UV filters, fragrances, parabens, phthalates, organochlor pesticides, PBDEs, and PCBs in human milk: correlation of UV filters with use of cosmetics.

In order to assess potential risks of exposure to environmental chemicals, more information on concomitant exposure to different chemicals is needed. We present data on chemicals in human milk of a cohort study (2004, 2005, 2006) of 54 mother/child pairs, where for the first time, cosmetic UV filters, synthetic musks, parabens and phthalate metabolites were analyzed in the same sample along with persistent organochlor pollutants (POPs), i.e., organochlor pesticides and metabolites, polybrominated diphenylethers and polychlorinated biphenyls (PCBs). The two groups of chemicals exhibited different exposure patterns. Six out of seven PCB congeners and a majority of pesticides were present in all milk samples, with significant correlations between certain PCB congener and pesticide levels, whereas the cosmetic-derived compounds, UV filters, parabens and synthetic musks, exhibited a more variable exposure pattern with inter-individual differences. UV filters were present in 85.2% of milk samples, in the range of PCB levels. Comparison with a questionnaire revealed a significant correlation between use of products containing UV filters and their presence in milk for two frequently used and detected UV filters, 4-methylbenzylidene camphor and octocrylene, and for the whole group of UV filters. Concentrations of PCBs and organochlor pesticides were within ranges seen in Western and Southern European countries. For several POPs, mean and/or maximum daily intake calculated from individual concentrations was above recent US EPA reference dose values. Our data emphasize the need for analyses of complex mixtures to obtain more information on inter-individual and temporal variability of human exposure to different types of chemicals.

Fundamental questions to sun protection: A continuous education symposium on vitamin D, immune system and sun protection at the University of Zürich.

Since exposure to sunlight is a main factor in the development of non-melanoma skin cancer and there are associations between malignant melanoma and short-term intense ultraviolet (UV) exposure, particularly burning in childhood, strict protection from UV-radiation is recommended. However, up to 90% of all requisite vitamin D has to be formed within the skin through the action of the sun-a serious problem, for a connection between vitamin D deficiency, demonstrated in epidemiological studies, and various types of cancer and other diseases has been confirmed. A UVB-triggered skin autonomous vitamin D(3) synthesis pathway has recently been described, producing the active Vitamin D metabolite calcitriol. This cutaneous vitamin D(3) pathway is unique. Keratinocytes and dendritic cells can convert vitamin D to calcitriol. Cutaneous T cells activated in the presence of calcitriol express the chemokine receptor CCR10 attracting them to the chemokine CCL27 that keratinocytes express selectively in the epidermis, and migrate from dermal layers of the skin to the epidermis under UV radiation. Thus, calcitriol has endocrine roles beyond its calciotropic action, including cell growth and cancer prevention. Therefore, strict sun protection procedures to prevent skin cancer may induce the risk of vitamin D deficiency. As there is evidence that the protective effect of less intense solar radiation can outweigh its mutagenic effect, better balanced approaches to sun protection should be sought.

Female sexual behavior, estrous cycle and gene expression in sexually dimorphic brain regions after pre- and postnatal exposure to endocrine active UV filters.

The developing female brain represents a potential target for estrogenic environmental chemicals because it depends on estrogen but is exposed to low endogenous estrogen levels, thus facilitating competition by exogenous estrogen receptor (ER) agonists. We investigated effects of two estrogenic UV filters, 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor (3-BC). 4-MBC has been detected in human milk, indicating potential exposure of fetus and infant. The two chemicals were administered in chow to rats of the parent generation before mating, during pregnancy and lactation, and to their offspring until adulthood. Female sexual behavior was recorded on videotape in adult female offspring on proestrus evening at the beginning of the dark phase. 4-MBC (7 and 24mg/kg bw/day) and 3-BC (2.4 and 7mg/kg bw/day) reduced proceptive behavior (jump and ear wiggling) and receptive behavior (lordosis quotient), and increased rejection behavior towards the male. Estrous cycles were not affected by 4-MBC but disturbed by 3-BC. mRNAs encoding for genes involved in female sexual behavior, ERalpha, ERbeta, progesterone receptor (PR) and steroid receptor coactivator-1 (SRC-1), were measured by real-time RT-PCR in ventromedial hypothalamic nucleus (VMH) and medial preoptic area of adult male and female offspring (studied in diestrus) after pre- and postnatal exposure to 3-BC (0.24, 0.7, 2.4 and 7mg/kg bw/day). Gene expression was affected in a sex- and region-specific manner. PR mRNA in female VMH was reduced to male levels at dose levels of 2.4 and 7mg/kg bw/day 3-BC. Our data demonstrate that female sexual behavior represents a sensitive target of endocrine disrupters and point to an involvement of PR in VMH.

Region-specific growth effects in the developing rat prostate following fetal exposure to estrogenic ultraviolet filters.

BACKGROUND AND OBJECTIVES: Exposure to environmental endocrine disruptors is a potential risk factor for humans. Many of these chemicals have been shown to exhibit disruption of normal cellular and developmental processes in animal models. Ultraviolet (UV) filters used as sunscreens in cosmetics have previously been shown to exhibit estrogenic activity in in vitro and in vivo assays. We examined the effects of two UV filters, 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor (3-BC), in the developing prostate of the fetal rat. METHODS: Pregnant Long Evans rats were fed diets containing doses of 4-MBC and 3-BC that resulted in average daily intakes of these chemicals corresponding to the lowest observed adverse effects level (LOAEL) and the no observed adverse effects level (NOAEL) doses in prior developmental toxicity studies. Using digital photographs of serial sections from postnatal day 1 animals, we identified, contoured, and aligned the epithelial ducts from specific regions of the developing prostate, plus the accessory sex glands and calculated the total volume for each region from three-dimensional, surface-rendered models. RESULTS: Fetal exposure to 4-MBC (7.0 mg/kg body weight/day) resulted in a significant increase (p < 0.05) in tissue volume in the prostate and accessory sex glands. Treated males exhibited a 62% increase in the number of ducts in the caudal dorsal prostate. Increased distal branching morphogenesis appears to be a consequence of exposure in the ventral region, resulting in a 106% increase in ductal volume. CONCLUSIONS: 4-MBC exposure during development of the male reproductive accessory sex glands exhibited classical growth effects associated with estrogenic endocrine disruptors. The different regional responses suggest that the two developmental processes of ductal outgrowth and branching morphogenesis are affected independently by exposure to the environmental chemicals.

Developmental toxicity of UV filters and environmental exposure: a review.

Several ultraviolet (UV) filters exhibit estrogenic, some also anti-androgenic activity. They are present in waste water treatment plants, surface waters and biosphere including human milk, suggesting potential exposure during development. Developmental toxicity was studied in rats for the UV filters 4-methylbenzylidene camphor (4-MBC, 0.7, 7, 24, 47 mg/kg/day) and 3-benzylidene camphor (3-BC, 0.07, 0.24, 0.7, 2.4, 7 mg/kg/day) administered in chow to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. Neonates exhibited enhanced prostate growth after 4-MBC and altered uterine gene expression after both chemicals. 4-MBC and 3-BC delayed male puberty and affected reproductive organ weights of adult offspring. Effects on the thyroid axis were also noted. Expression and oestrogen sensitivity of oestrogen-regulated genes and nuclear receptor coregulator levels were altered at mRNA and protein levels in adult uterus, prostate and brain regions involved in gonadal control and sexual behaviour. Female sexual behaviour was impaired by both filters; 3-benzylidene camphor caused irregular cycles. Classical endpoints exhibited lowest observed adverse effect levels (LOAELs) and no observed adverse effect levels (NOAELs) of 7/0.7 mg/kg for 4-MBC and 0.24/0.07 mg/kg for 3-BC. Molecular endpoints were affected by the lowest doses studied. Our data indicate that the potential risk posed by endocrine active UV filters warrants further investigations.

Salbutamol exhibits androgenic activity in vitro.

BACKGROUND: Salbutamol has been shown to mediate anabolic effects after intravenous administration. However, the mechanism responsible for the anabolic actions of salbutamol remains unknown. AIM: To investigate the potential mechanism by which salbutamol mediates anabolic effects in vitro. METHODS: The potential androgenic activity of salbutamol was investigated in vitro by the A-Screen assay that measures androgen-dependent inhibition of proliferation of the androgen receptor (AR)-positive human mammary carcinoma cell line, MCF7-AR1. RESULTS: The assay was validated with three known androgens; methyltrienolone (R1881), 5alpha-dihydrotestosterone (DHT) and danazol. IC50 values of R1881, DHT and danazol, 4.41x10(-11), 4.44x10(-11) and 1.08x10(-8) M, respectively, were in the ranges known from earlier studies. Our results demonstrate that salbutamol exhibits androgenic activity, with an IC50 value of 8.93x10(-6) M. Anti-estrogenic or cytotoxic effects, which might have interfered with the assay, were excluded by additional experiments on wild-type MCF7 and MCF7-AR1 cells, respectively. CONCLUSION: These data indicate that salbutamol exerts anabolic effects through androgen receptor agonistic activity in vitro.

Sexually dimorphic gene regulation in brain as a target for endocrine disrupters: developmental exposure of rats to 4-methylbenzylidene camphor.

The developing neuroendocrine brain represents a potential target for endocrine active chemicals. The UV filter 4-methylbenzylidene camphor (4-MBC) exhibits estrogenic activity, but also interferes with the thyroid axis. We investigated effects of pre- and postnatal exposure to 4-MBC in the same rat offspring at brain and reproductive organ levels. 4-MBC (7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to the offspring until adulthood. mRNA of estrogen target genes involved in control of sexual behavior and gonadal functions was measured by real-time RT-PCR in ventromedial hypothalamic nucleus (VMH) and medial preoptic area (MPO) of adult offspring. 4-MBC exposure affected mRNA levels of ER alpha, progesterone receptor (PR), preproenkephalin (PPE) and insulin-like growth factor-I (IGF-I) in a sex- and region-specific manner. In order to assess possible changes in sensitivity of target genes to estrogens, offspring were gonadectomized on day 70, injected with estradiol (E2, 10 or 50 microg/kg s.c.) or vehicle on day 84, and sacrificed 6 h later. The acute induction of PR mRNA, and repression (at 6 h) of PPE mRNA by E2 was enhanced by 4-MBC in male and female VMH and female MPO, whereas male MPO exhibited reduced responsiveness of both genes. Steroid receptor coactivator SRC-1 mRNA levels were increased in female VMH and MPO. The data indicate profound sex- and region-specific alterations in the regulation of estrogen target genes at brain level. Effect patterns in baseline and E2-induced gene expression differ from those in uterus and prostate.

Estrogen sensitivity of target genes and expression of nuclear receptor co-regulators in rat prostate after pre- and postnatal exposure to the ultraviolet filter 4-methylbenzylidene camphor.

BACKGROUND AND OBJECTIVES: In previous studies, we found that the ultraviolet filter 4-methyl-benzylidene camphor (4-MBC) exhibits estrogenic activity, is a preferential estrogen receptor (ER)-beta ligand, and interferes with development of female reproductive organs and brain of both sexes in rats. Here, we report effects on male development. METHODS: 4-MBC (0.7, 7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to offspring until adulthood. mRNA was determined in prostate lobes by real-time reverse transcription-polymerase chain reaction and protein was determined by Western blot analysis. RESULTS: 4-MBC delayed male puberty, decreased adult prostate weight, and slightly increased testis weight. Androgen receptor (AR), insulin-like growth factor-1 (IGF-1), ER-alpha, and ER-beta expression in prostate were altered at mRNA and protein levels, with stronger effects in dorsolateral than ventral prostate. To assess sensitivity of target genes to estrogens, offspring were castrated on postnatal day 70, injected with 17beta-estradiol (E(2); 10 or 50 microg/kg, sc) or vehicle on postnatal day 84, and sacrificed 6 hr later. Acute repression of AR and IGF-1 mRNAs by E(2), studied in ventral prostate, was reduced by 4-MBC exposure. This was accompanied by reduced co-repressor N-CoR (nuclear receptor co-repressor) protein in ventral and dorsolateral prostate, whereas steroid receptor coactivator-1 (SRC-1) protein levels were unaffected. CONCLUSIONS: Our data indicate that 4-MBC affects development of male reproductive functions and organs, with a lowest observed adverse effect level of 0.7 mg/kg. Nuclear receptor coregulators were revealed as targets for endocrine disruptors, as shown for N-CoR in prostate and SRC-1 in uterus. This may have widespread effects on gene regulation.

Gene expression and estrogen sensitivity in rat uterus after developmental exposure to the polybrominated diphenylether PBDE 99 and PCB.

Considering the presence of polybrominated diphenylethers (PBDEs) in human milk and cord blood, and the estrogenic activity of some congeners, it is conceivable that PBDEs may interact with developing neuroendocrine systems. We investigated effects of 2,2',4,4',5-pentabromo-DE (PBDE 99), a major congener in human milk, on development of brain and reproductive organs, with focus on estrogen target gene expression. Time-pregnant Long Evans rats were subcutaneously injected with PBDE 99 (1 or 10 mg/kg/day), the PCB mixture Aroclor 1254 (10 mg/kg/day), known to interfere with sexual development, or vehicle, from gestational day (GD) 10 to GD 18. In female offspring, anogenital distance was unaffected by PBDE 99 but increased by Aroclor; puberty (vaginal opening) was not significantly changed. Adult PBDE 99-exposed offspring exhibited unchanged uterine weight but increased ovarian weight. Uterine mRNA levels of estrogen target genes were determined by real-time PCR. Progesterone receptor (PR) mRNA was down-regulated at both PBDE 99 doses, estrogen receptor alpha (ER alpha), ER beta and insulin-like growth factor-I (IGF-I) were up-regulated at the lower dose. Aroclor induced different effect patterns. In order to investigate possible changes in sensitivity of target genes to estrogen, some offspring were ovariectomized at 10 weeks of age, s.c. injected with estradiol-17beta (E2, 10 microg/kg) or vehicle at 12 weeks, and sacrificed 6 h later. PBDE 99 dose-dependently reduced the magnitude of IGF-I mRNA induction by E2, and increased the magnitude of ER beta repression. PBDE 99 also influenced baseline levels of PR, IGF-I and ER beta mRNAs in ovariectomized, vehicle-injected controls. These data indicate that developmental exposure to PBDE 99 at doses devoid of general toxicity, affects the regulation of estrogen target genes in uterus. Since PBDE 99 was detected in blood and adipose tissue of adult offspring, these effects may result from interactions with developmental processes, adult functions, or a combination of both.