An in vitro-based hazard assessment of liquid smoke food flavourings.

Liquid smoke products are widely used as a food additive to create a desired smoke flavour. These products may contain hazardous chemicals generated during the wood-burning process. However, the toxic effects of these types of hazardous chemicals constituting in the commercially available products are largely unknown. Therefore, a test battery of cell-based in vitro methods, covering different modes of actions of high relevance to human health, was applied to study liquid smoke products. Ten liquid smoke flavourings were tested as non-extracted and extracted. To assess the potential drivers of toxicity, we used two different solvents. The battery of in vitro methods covered estrogenicity, androgenicity, oxidative stress, aryl hydrocarbon receptor activity and genotoxicity. The non-extracted samples were tested at concentrations 0.002 to 1 µL liquid smoke flavouring/mL culture medium, while extracted samples were tested from 0.003 to 200 µL/mL. Genotoxicity was observed for nearly all non-extracted and all hexane-extracted samples, in which the former had higher potency. No genotoxicity was observed for ethyl acetate-extracted samples. Oxidative stress was activated by almost all extracted and non-extracted samples, while approximately half of the samples had aryl hydrocarbon receptor and estrogen receptor activities. This study used effect-based methods to evaluate the complex mixtures of liquid smoke flavourings. The increased bioactivities seen upon extractions indicate that non-polar chemicals are driving the genotoxicity, while polar substances are increasing oxidative stress and cytotoxic responses. The differences in responses indicate that non-extracted products contain chemicals that are able to antagonize toxic effects, and upon extraction, the protective substances are lost.

Assessment of endocrine disruption and oxidative potential of bisphenol-A, triclosan, nonylphenol, diethylhexyl phthalate, galaxolide, and carbamazepine, common contaminants of municipal biosolids.

The use of biosolids as a soil conditioner and fertiliser is hindered by the limited knowledge on the risks of micro-contaminants they contain. This study investigated the binding of six organic contaminants commonly found in biosolids, to the estrogen (ER), androgen (AR), aryl hydrocarbon (AhR), and transthyretin (TTR) receptors and their redox activity. Triclosan (TCS), bisphenol-A (BPA), and technical nonylphenol (TNP) had affinity for the TTR with relative potencies of 0.3, 0.03, and 0.076 respectively. Further, binding to TTR was the only toxicological response observed for carbamazepine, which induced sub-maximal response and relative potency of 0.0017. Estrogenic activity was induced by BPA, galaxolide (HHCB), diethylhexyl phthalate (DEHP) and TNP with BPA having the strongest potency of 5.1 × 10-6 relative to estradiol. Only BPA showed androgenic activity but it was not quantifiable. BPA also showed anti-androgenic activity along with TCS, HHCB, and TNP in the order of TNP > HHCB > TCS ~ BPA (relative potencies 0.126, 0.042, 0.032, 0.03). No compounds exhibited anti-estrogenic or AhR activity, or were redox-active in the dithiothreitol assay. The results highlight the multiple modes of action through which these compounds may impact exposed organisms, and the concentrations at which effects may occur. This allows assessment of the likelihood of effects being observed at environmental concentrations, and the potential contribution of these compounds.

Risk assessment of the endocrine-disrupting effects of nine chiral pesticides.

The increased release of chiral pesticides into the environment has generated interest in the role of enantioselectivity in the environmental fate and ecotoxicological effects of these compounds. However, the information on the endocrine disrupting effects (EDEs) of chiral pesticides is still limited and discrepancies are also usually observed among different assays. In this study, we investigated the enantioselectivity of EDEs via estrogen and thyroid hormone receptors for nine chiral pesticides using in vitro and in silico approaches. The results of the luciferase reporter gene assays showed 7 chiral pesticides possessed enantioselective estrogenic activities and 2 chiral pesticides exerted thyroid hormone antagonistic effects. Proliferation assays in MCF-7 and GH3 cells were also used to verify the results of the dual-luciferase reporter gene assays. At last, the molecular docking results indicated that the enantioselective EDEs of chiral pesticides were partially due to enantiospecific binding affinities with receptors. Our data not only show enantioselective EDEs of nine chiral pesticides, but also would be helpful to better understanding the molecular biological mechanisms of enantioselectivity in EDEs of chiral pesticides.

The association of chemotherapy versus hormonal therapy and health outcomes among patients with hormone receptor-positive, HER2-negative metastatic breast cancer: experience from the patient perspective.

This study aimed to characterize the impact of metastatic breast cancer (MBC) and cancer treatments on health-related quality of life, treatment satisfaction, and daily productivity from the patient perspective. This was a cross-sectional survey of patients with MBC (USA, n = 200; EU, n = 160). Post-menopausal women aged ≥50 years with hormone receptor positive (HR+), HER2-negative (HER2-) MBC, currently using hormonal therapy (HT) or using chemotherapy (CT) for ≤1 year were recruited. Fifty three percent (n = 191) reported CT and 47% (n = 169) reported HT use. Adjusting for covariates, HT users reported greater health-related quality of life (p < 0.05), greater satisfaction with treatment and better feelings about side-effects (p < 0.001). HT users reported less bother with treatment side-effects (0-5 scale, p < 0.001) and less activity impairment than CT users (p < 0.001). HT was associated with better patient-reported outcomes than CT in first-line MBC management. These findings should be taken into consideration while making treatment decisions for HR+/HER2- MBC.

Assessment of estrogenic potential of diethyl phthalate in female reproductive system involving both genomic and non-genomic actions.

Phthalates are the diverse group of compounds abundantly present in environment. The present study shows the estrogenic potential of diethyl phthalate (DEP). The data showed that DEP increased the transactivation of ER in CHO and MCF-7 cells suggesting its interaction with ER. In vivo parameters like increased uterine epithelial cell height and up regulation of various steroidogenic genes were also observed in adult female rats. Our uterotrophic assay data from immature female rats suggested that DEP treatment resulted in augmentation of uterine weight as well as luminal epithelial cell heights in both vaginal and uterine tissues. Further, DEP was able to upregulate pS2 gene expression with simultaneous activation of MAPK pathway as demonstrated by increased p-ERK/ERK ratio. Taken together, the present data suggests that DEP acts as an estrogenic compound and based on these data further detailed studies would reveal its mode of action at cellular levels.

Chemical and biological assessment of Angelica herbal decoction: comparison of different preparations during historical applications.

The commonly used Angelica herbal decoction today is Danggui Buxue Tang (DBT), which is a dietary supplement in treating menopausal irregularity in women, i.e. to nourish "Qi" and to enrich "Blood". According to historical record, many herbal decoctions were also named DBT, but the most popular formulation of DBT was written in Jin dynasty (1247 AD) of China, which contained Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) with a weight ratio of 5:1. However, at least two other Angelica herbal decoctions recorded as DBT were prescribed in Song (1155 AD) and Qing dynasties (1687 AD). Although AR and ASR are still the major components in the DBT herbal decoctions, they are slightly varied in the herb composition. In order to reveal the efficiency of different Angelica herbal decoctions, the chemical and biological properties of three DBT herbal extracts were compared. Significantly, the highest amounts of AR-derived astragaloside III, astragaloside IV, calycosin and formononetin and ASR-derived ferulic acid were found in DBT described in 1247 AD: this preparation showed stronger activities in osteogenic, estrogenic and erythropoetic effects than the other two DBT. The current results supported the difference of three DBT in chemical and biological properties, which could be a result of different herbal combinations. For the first time, this study supports the popularity of DBT described in 1247 AD.

Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment.

Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations. Here we discuss a framework designed for this purpose, the adverse outcome pathway (AOP). An AOP is a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment. The practical utility of AOPs for ecological risk assessment of chemicals is illustrated using five case examples. The examples demonstrate how the AOP concept can focus toxicity testing in terms of species and endpoint selection, enhance across-chemical extrapolation, and support prediction of mixture effects. The examples also show how AOPs facilitate use of molecular or biochemical endpoints (sometimes referred to as biomarkers) for forecasting chemical impacts on individuals and populations. In the concluding sections of the paper, we discuss how AOPs can help to guide research that supports chemical risk assessments and advocate for the incorporation of this approach into a broader systems biology framework.

Differential estrogen receptor subtype modulators: assessment of estrogen receptor subtype-binding selectivity and transcription-regulating properties of new cycloalkyl pyrazoles.

Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the estrogen receptor (ER) subtypes, ERalpha and ERbeta, and subtype-specific agonist/antagonist properties were determined. Cyclopentane- and cyclohexane-fused pyrazoles with p-hydroxyphenyl rings at positions 1 and 3 displayed modest ERbeta-binding selectivity and variable agonism through ERalpha, while behaving as full estrogen antagonists through ERbeta in estrogen-responsive element (ERE)-dependent gene expression assays. By contrast, the 2,3-diphenolic derivatives were non-selective and considerably less effective ERbeta antagonists compared to 1,3-diphenolic ones. The cyclohexane-fused 1,3-diphenolic pyrazole 8, in particular, behaved as full ERalpha agonist/ERbeta antagonist in these assays. Molecular modelling revealed the structural determinants possibly accounting for the differential regulation of transcription through the two ERs exhibited by 8. The data also shows that the ER subtype-binding selectivity and agonist/antagonist efficacy of the 1,3-diphenolic pyrazoles is influenced by the cycloalkyl ring fused to the pyrazole core. Using 8 we show that, though the mutant androgen receptor (AR) of LNCaP cells is required for estrogen as well as androgen stimulation of cell growth, estrogen responsiveness of the cells depends on ERbeta and AR but not on ERalpha.

Current and future approach to the pathologist's assessment for targeted therapy in breast cancer.

Breast cancer is a common disease in the population. Contrary to public perception, it is a heterogeneous disease with varying morphology, prognosis and response to therapy. The pathological analysis is at the heart of information provided to surgeons and oncologists to plan further management. The pathologist is increasingly asked to test for biomarkers that provide prognostic and predictive information to direct treatment. Staining cancers for ER, PgR and HER2 has become routine and it is likely that addition of other biomarkers including 'basal markers', VEGF and growth factor receptors such as HER1 (EGFR) will soon follow. Microarray based genomic, transcription and proteomic methods are changing our classification systems and identifying novel targets for the development of new therapeutics. It is important for pathologists to appreciate and embrace the new developments as they will impact on daily clinical practice and require accurate assessment of biomarkers to determine treatment options as part of multidisciplinary teams.

Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment.

There is growing concern that some chemicals can cause endocrine disrupting effects to wild animals and humans. Therefore a rapid and reliable screening assay to assess the activity of endocrine disrupting chemicals (EDCs) is required. These EDCs can act at multiple sites. Most studied mechanism is direct interaction with the hormone receptors, e.g. estrogen receptor. In this study the luciferase reporter gene assay using transgenic human MELN cells was used. Since cytotoxicity of the chemicals can decrease the luminescent signal in the transactivation assays, a cytotoxicity assay must be implemented. Mostly the neutral red (NR) assay is performed in parallel with the estrogenicity assay. To increase the reliability and cost-efficiency of the test, a method to measure estrogenicity and cytotoxicity in the same cell culture plate instead of in parallel plates was developed and evaluated. Therefore the NR-assay was compared with the CytoTox-ONE homogeneous membrane integrity assay. The latter measures LDH (lactate dehydrogenase) leakage based on a fluorometric method. For all compounds tested, the CytoTox-ONE test showed comparable curves and EC50-values to those obtained by the NR-assay. So the CytoTox-ONE kit, which seemed more sensitive than measurements of LDH-leakage based on a colorimetric method, is recommended to test cytotoxicity to MELN cells, with the advantage to use the same cells for ER-transactivation measurements. The chemicals tested in the optimised MELN assay showed estrogenic potencies comparable to those reported for several other transactivation assays.

Assessment of prediction confidence and domain extrapolation of two structure-activity relationship models for predicting estrogen receptor binding activity.

Quantitative structure-activity relationship (QSAR) methods have been widely applied in drug discovery, lead optimization, toxicity prediction, and regulatory decisions. Despite major advances in algorithms and software, QSAR models have inherent limitations associated with a size and chemical-structure diversity of the training set, experimental error, and many characteristics of structure representation and correlation algorithms. Whereas excellent fit to the training data may be readily attainable, often models fail to predict accurately chemicals that are outside their domain of applicability. A QSAR's utility and, in the case of regulatory decisions, justification for usage increasingly depend on the ability to quantify a model's potential for predicting unknown chemicals with some known degree of certainty. It is never possible to predict an unknown chemical with absolute certainty. Here we report on two QSAR models based on different data sets for classification of chemicals according to their ability to bind to the estrogen receptor. The models were developed by using a novel QSAR method, Decision Forest, which combines the results of multiple heterogeneous but comparable Decision Tree models to produce a consensus prediction. We used an extensive cross-validation process to define an applicability domain for model predictions based on two quantitative measures: prediction confidence and domain extrapolation. Together, these measures quantify the accuracy of each prediction within and outside of the training domain. Despite being based on large and diverse training sets, both QSAR models had poor accuracy for chemicals within the domain of low confidence, whereas good accuracy was obtained for those within the domain of high confidence. For prediction in the high confidence domain, accuracy was inversely proportional to the degree of domain extrapolation. The model with a larger training set of 1,092, compared with 232 for the other, was more accurate in predicting chemicals at larger domain extrapolation, and could be particularly useful for rapidly prioritizing potential endocrine disruptors from large chemical universe.

Aromatase inhibitors: current indications and future prospects for treatment of postmenopausal breast cancer.

This article provides a comprehensive review of aromatase inhibitors (AIs) for geriatricians, because it appears that more elderly women will be using these drugs in the near future. Computerized literature searches of Medline were conducted through May 2003. Key words/phrases included in the literature searches were aromatase inhibitors, estrogen, and breast cancer. Limits included English language, age 65 and older, and female sex. All relevant articles were selected and reviewed. AIs suppress intratumoral and plasma estrogen levels significantly. Third-generation AIs have excellent pharmacological profiles, with no significant drug interactions and better tolerability. These drugs have shown superiority compared with conventional therapies. The results of anastrozole, tamoxifen, and combination trials favors anastrozole over tamoxifen for adjuvant treatment, but further follow-up is required. AIs are approved for the treatment of advanced metastatic breast cancer (BC) in postmenopausal women whose disease has progressed during tamoxifen therapy. Recent trials have shown that the highly selective third-generation AIs are effective when used as first-line therapy in metastatic BC. Their possible use in preventive, adjuvant, and neoadjuvant settings is also being explored.

Assessment of the estrogenicity of isoflavonoids, using MCF-7-ERE-Luc cells.

In the current study, our research focused on the estrogenic activity of isoflavonoids, mainly genistein, biochanin A and daidzein. Genistein enhanced the reporter gene expression of MCF-7-ERE-Luc cells, at a concentration as low as 10 nM, with a concentration of 100 nM the achieved gene expression effects were similar to those of 10 pM 17beta-estradiol, Based on the estrogenic activities of biochanin A and daidzein, hydroxyl groups at the 4' and 5 positions are needed for the maximal effect of the genistein. The estrogenic effects of these isoflavonoids were inhibited by the concomitant treatment with tamoxifen. The data showed that the estrogenic effects of isoflavonoids were mediated through estrogen receptors. When the isoflavonoids were tested as mixtures, the estrogenic effects were lower than the arithmetic sum of those induced by each individual isoflavonoid. The estrogenic potency of each isoflavonoid was presented at EC50 levels with a 17beta-estradiol equivalent concentration (EEQ) based on the dose response of each chemical. The EC50s and EEQs of genistein, biochanin A and daidzein were 4.15, 0.89 and 0.18 microM, and 15.0, 5.12 and 1.83 microM/M, respectively. Our data clearly demonstrated that the pERE-luciferase reporter gene assay was suited for the sensitive and quantitative measurement, and large scale screening, of the estrogenicity of chemicals in vitro.

Assessment of putative endocrine disrupters in an in vivo crustacean assay and an in vitro insect assay.

Concern over endocrine disrupters in coastal ecosystems has stimulated global efforts to understand their potential impacts on fish and invertebrate communities. Given that marine crustaceans are sensitive to the effects of alkylphenols and other xenobiotics, we are currently investigating whether these responses may be caused via an endocrine mechanism. Tisbe battagliai (Copepoda: Harpacticoida) is representative of an ecologically important group of meiofauna, is sensitive to toxicants and is used as an international test species in marine ecotoxicology. A 21-day life-cycle test that incorporates assessment of survival, development, reproduction and sex ratios, has shown that populations of T. battagliai are not significantly affected by environmentally relevant levels of steroidal oestrogen agonists, or by related synthetic receptor agonists. The absence of in vivo effects due to these steroid agonists and antagonists prompted in vitro studies of ecdysteroid receptor activity of a range of reference compounds and environmental contaminants with an ecdysteroid receptor (EcR/USP)-based screening assay derived from the BII haemocyte line of Drosophila melanogaster. The implications for environmental monitoring of endocrine disrupters are discussed.

Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents.

This study reports on the development and application of a fish-specific estrogen-responsive reporter gene assay. The assay is based on the rainbow trout (Oncorhynchus mykiss) gonad cell line RTG-2 in which an acute estrogenic response is created by cotransfecting cultures with an expression vector containing rainbow trout estrogen receptor a complementary DNA (rtERalpha cDNA) in the presence of an estrogen-dependent reporter plasmid and an estrogen receptor (ER) agonist. In a further approach, RTG-2 cells were stably transfected with the rtERalpha cDNA expression vector, and clones responsive to 17beta-estradiol (E2) were selected. The estrogenic activity of E2, 17alpha-ethinylestradiol, 4-nonylphenol, nonylphenoxy acetic acid, 4-tert-octylphenol, bisphenol A, o,p'-DDT, p,p'-DDT, o,p'-2,2-bis(chlorophenyl)-1,1-dichloroethylene (o,p'-DDE), p,p'-DDE, o,p'-2,2-bis(chlorophenyl)-1,1-di-chloroethane (o,p'-DDD), p,p'-DDD, and p,p'-2,2-bis(chlorophenyl)acetic acid (p,p'-DDA) was assessed at increasing concentrations. All compounds except o,p'-DDT, p,p'-DDE, and p,p'-DDA showed logistic dose-response curves, which allowed the calculation of lowest-observed-effect concentrations and the concentrations at which half-maximal reporter gene activities were reached. To check whether estrogen-responsive RTG-2 cells may be used to detect the estrogenic activity of environmental samples, an extract from a sewage treatment plant (STP) effluent was assessed and found to have estrogenic activity corresponding to the transcriptional activity elicited by 0.05 nM of E2. Dose-response curves of nonylphenol, octylphenol, bisphenol A, and o,p'-DDD revealed that the RTG-2 reporter gene assay is more sensitive for these compounds when compared to transfection systems recombinant for mammalian ERs. These differences may have an effect on the calculation of E2 equivalents when estrogenic mixtures of known constitution, or environmental samples, such as STP effluents, are assessed.

Assessment of estrogenic activity of natural compounds using improved E-screen assay.

AIM: To improve E-screen assay and make it more accurate to screen estrogenic compounds. METHODS: Estrogen receptor antisense RNA expression plasmid (pCASER) was constructed and introduced into MCF-7 with lipofectAMINE(TM), and positive clones were screened out with G418. PCR amplification was employed to identify whether estrogen receptor (ER) cDNA fragment had been inserted into MCF-7 cell genomes. Western blot was applied to detect the expression of ER. Cell growth was determined by MTT assay. RESULTS: One ER antisense clone (MTASER) had been screened out. The effects of 17beta-estradiol, genistein, droloxifen, miyabenol C, and kobophenol A on MCF-7 were stronger than those effects on MTASER. Epidermal growth factor (EGF) had equivalent stimulatory effects on the proliferation of MCF-7 and MTASER. CONCLUSION: The improved E-screen assay could screen estrogenic compounds more accurately than original E-screen assay did.

Assessment of the estrogenic activity of flue gases from burning processes by means of the yeast based human estrogen receptor (hER) bioassay.

Combustion processes are known to produce organic micro-pollutants in the flue gas at concentrations ranging over several orders of magnitude. Some organic micro-pollutants are suspected of being pseudo-estrogens and as such they can affect the public health. In this study, the possible application of the yeast based human estrogen receptor (hER) bioassay to screen flue gas streams for the presence of estrogenic active micro-pollutants was explored. Specifically, the protocol was modified to allow the detection and quantification of the potential estrogenic active non-polar organic micro-pollutants contained in the flue gas matrix. The modified assay was calibrated using a model estrogenic compound (17-alpha-ethinylestradiol (EE2)) dissolved in methylene chloride at concentrations ranging from 3 ng l(-1) to 3000 ng l(-1). The effective concentration to elucidate a 50% response (EC50) was 87 ng l(-1) of equivalent dissolved in methylene chloride. Samples of methylene chloride used to trap non-polar micro-pollutants in flue gas from combustion of pine wood were found to clearly register estrogenic activity by the bioassay under certain conditions. The combustion tests were performed with pinewood alone and with pine wood in the presence of both Copper-naphthenate and copper(II)chloride at 600 degrees C and 1000 degrees C. These conditions must be considered as experimental rather than practical. Overall, the results suggest that, by means of this modified assay, it is possible and warranted to screen systematically for estrogens in flue gas combustion processes.

Problems for risk assessment of endocrine-active estrogenic compounds.

Estrogenic industrial compounds such as bisphenol A (BPA) and nonylphenol typically bind estrogen receptor (ER) alpha and ERBeta and induce transactivation of estrogen-responsive genes/reporter genes, but their potencies are usually greater than or equal to 1,000-fold lower than observed for 17Beta-estradiol. Risk assessment of estrogenic compounds on the basis of their potencies in simple reporter gene or binding assays may be inappropriate. For example, selective ER modulators (SERMs) represent another class of synthetic estrogens being developed for treatment of hormone-dependent problems. SERMs differentially activate wild-type ERalpha and variant forms expressing activation function 1 (ER-AF1) and AF2 (ER-AF2) in human HepG2 hepatoma cells transfected with an estrogen-responsive complement C3 promoter-luciferase construct, and these in vitro differences reflect their unique in vivo biologies. The HepG2 cell assay has also been used in our laboratories to investigate the estrogenic activities of the following structurally diverse synthetic and phytoestrogens: 4 -hydroxytamoxifen; BPA; 2 ,4 ,6 -trichloro-4-biphenylol; 2 ,3 ,4 ,5 -tetrachloro-4-biphenylol; p-t-octylphenol; p-nonylphenol; naringenin; kepone; resveratrol; and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane. The results show that synthetic and phytoestrogens are weakly estrogenic but induce distinct patterns of ER agonist/antagonist activities that are cell context- and promoter-dependent, suggesting that these compounds will induce tissue-specific (in vivo(ER agonist or antagonist activities. These results suggest that other receptors, such as the aryl hydrocarbon receptor, that also bind structurally diverse ligands may exhibit unique responses in vivo that are not predicted by standard in vitro bioassays.