Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR)

Polychlorinated biphenyls (PCBs) are a family of persistent organic contaminants suspected to cause adverse effects in wildlife and humans. In rodents, PCBs bind to the aryl hydrocarbon (AhR) and pregnane X receptors (PXR) inducing the expression of catabolic cytochrome p450 enzymes of the CYP1A and 3A families. We found that certain highly chlorinated PCBs are potent activators of rodent PXR but antagonize its human ortholog, the steroid and xenobiotic receptor (SXR), inhibiting target gene induction. Thus, exposure to PCBs may blunt the human xenobiotic response, inhibiting the detoxification of steroids, bioactive dietary compounds, and xenobiotics normally mediated by SXR. The antagonistic PCBs are among the most stable and abundant in human tissues. These findings have important implications for understanding the biologic effects of PCB exposure and the use of animal models to predict the attendant risk.     

Binding of estrogenic compounds to recombinant estrogen receptor-alpha: application to environmental analysis

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-alphaligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-alpha and whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-alpha to separate ligands for ER and AhR that are present in river sediments. Immobilized ER-alpha, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.     

Endocrine-Disrupting Effects of Compounds in Danish Streams

Effluents from municipal wastewater-treatment plants and scattered dwellings, as well as runoff from agricultural fields, are sources of endocrine-disrupting compounds (EDCs) in the aquatic environment. The present study investigated the correlation between the occurrence of EDCs in nine Danish streams using passive samplers (polar organic integrative samplers and silicone membranes) and determined their possible biological effects as assessed by mammal cell cultures and the mussel (Unio tumidus). The passive samplers and mussels were exposed simultaneously at the study sites. The extracts from the passive samplers were used to measure the concentrations of EDCs and the biological effects on the estrogen (ER), androgen (AR), and aryl hydrocarbon (AhR)-receptor transactivation. Male mussels were investigated for biomarkers of endocrine effects, such as the levels of vitellogenin-like proteins measured as alkali-labile phosphate (ALP). EDC concentrations, hormone-receptor transactivation (ER, AR, AhR), and level of ALP were greater downstream of wastewater-treatment plants compared with upstream sites and sites supposed to be relatively nonimpacted by wastewater. Furthermore, there was a significant positive correlation between in vitro AhR transactivation and frequency of ALP of male mussels. We conclude that wastewater effluent is an important source of endocrine-disrupting effects in the aquatic environment and that the combination of biological effect measurements and chemical analyses based on passive sampling is useful in the assessment of the ecological state of the aquatic environment.     

Effect of copper complexation on the estrogenic activities of endocrine-disrupting compounds using E-screen bioassay

The complexation reaction between Cu and four targeted endocrine-disrupting compounds (EDCs)-17beta-estradiol (E2), 17alpha-ethynylestradiol (EE2), 4-octylphenol (OP), and bisphenol A (BPA)-was studied using the fluorescence quenching method, with the changed EDC forms evaluated for their estrogenic activities using an E-screen assay. The results of the E-screen showed no estrogenic activity of Cu, whereas results with the EDCs indicated high cell proliferation (decreasing order of induction: E2, EE2, OP, and BPA). The results of the fluorescence quenching response and FTIR (Fourier transformation infrared) strongly support the formation of Cu complexes on the -OH functional group of the targeted EDCs when Cu was spiked at concentrations between 10(-7) and 10(-4) M. The degree of complexation was in the following order: E2, EE2, OP, and BPA. The E-screen result for three of the EDCs, with the exception of BPA, was similar when estimating the effects of Cu on the estrogenic activities of the EDCs, which were significantly reduced at alpha = 0.05 with increasing Cu concentration. Consequently, the results of the complexation reaction and E-screen assay with the Cu-EDC mixtures indicated that Cu plays a significant role in the assessment of the estrogenic activities of EDCs in natural waters.     

Effects of Dicyclohexyl Phthalate Exposure on PXR Activation and Lipid Homeostasis in Mice

Background: Exposure to plastic-associated endocrine disrupting chemicals (EDCs) has been associated with an increased risk of cardiovascular disease (CVD) in humans. However, the underlying mechanisms for this association are unclear. Many EDCs have been shown to function as ligands of the nuclear receptor pregnane X receptor (PXR), which functions as xenobiotic sensor but also has pro-atherogenic effects in vivo.Objective: We sought to investigate the contribution of PXR to the adverse effects dicyclohexyl phthalate (DCHP), a widely used phthalate plasticizer, on lipid homeostasis and CVD risk factors.Methods: Cell-based assays, primary organoid cultures, and PXR conditional knockout and PXR-humanized mouse models were used to investigate the impact of DCHP exposure on PXR activation and lipid homeostasis in vitro and in vivo. Targeted lipidomics were performed to measure circulating ceramides, novel predictors for CVD.Results: DCHP was identified as a potent PXR-selective agonist that led to higher plasma cholesterol levels in wild-type mice. DCHP was then demonstrated to activate intestinal PXR to elicit hyperlipidemia by using tissue-specific PXR-deficient mice. Interestingly, DCHP exposure also led to higher circulating ceramides in a PXR-dependent manner. DCHP-mediated PXR activation stimulated the expression of intestinal genes mediating lipogenesis and ceramide synthesis. Given that PXR exhibits considerable species-specific differences in receptor pharmacology, PXR-humanized mice were also used to replicate these findings.Discussion: Although the adverse health effects of several well-known phthalates have attracted considerable attention, little is known about the potential impact of DCHP on human health. Our studies demonstrate that DCHP activated PXR to induce hypercholesterolemia and ceramide production in mice. These results indicate a potentially important role of PXR in contributing to the deleterious effects of plastic-associated EDCs on cardiovascular health in humans. Testing PXR activation should be considered for risk assessment of phthalates and other EDCs. https://doi.org/10.1289/EHP9262     

A Pilot Study of the Water Quality of the Yarra River, Victoria, Australia, Using In Vitro Techniques

A pilot study was initiated to provide the first information on the recombinant receptor-reporter gene bioassay (hormonal) activity of freshwaters in Victoria. The project involved the collection of water samples from six stations on the main stem of the Yarra River in and upstream of the city of Melbourne, Australia in April 2008 and April 2009. Samples were prepared for measurement of sample toxicity using a modified photobacterium test, genotoxicity using a high-throughput luminescent umu test method, and human and medaka estrogen receptor (hERα and medERα), retinoic acid receptor (RAR), aryl hydrocarbon receptor (AhR) and thyroid receptor (TR) assay activity using the relevant yeast-based bioassays. Most samples were only weakly or moderately toxic, with no relationship observed to location along the river. The data for 2008 suggests that at that time the Yarra River samples contained few compounds that were, in and of themselves, genotoxic. No estrogenic or thyroid, and <1?ng/L retinoic acid receptor activity was observed. AhR activity increased with progressed downstream. AhR activity was higher in April 2009 than at the same time in 2008, perhaps as a result of extensive bush fires in the catchment in the months immediately prior to sampling. About 24% of the total AhR activity observed was associated with suspended solids.     

A toxicity identification evaluation approach to studying estrogenic substances in hog manure and agricultural runoff

Spreading liquid manure on agricultural fields is a routine way of disposing of animal manure and optimizing the use of nutrients for crops. Limited studies suggest that these wastes may contain a variety of endocrine-disrupting compounds (EDCs) that may be released into aquatic environments through runoff. The purpose of this study was to apply a toxicity identification and evaluation approach to isolate and identify estrogenic compounds in hog manure. A recombinant yeast estrogen screen bioassay was used to detect estrogenicity of high-performance liquid chromatography--separated hog manure fractions. Further analytical analyses of the fractions and comparison to authentic standards resulted in the identification of the endogenous estrogens 17 beta-estradiol (E2) and estrone, and the phytoestrogen metabolite, equol. High levels of equol (6.9-16.6 ppm) were found to be present in manure that was stored for several months. The endocrine-disrupting potential of equol was characterized further by using fish hormone estrogen receptor (ER), sex hormone binding protein (SSBP), and goldfish androgen receptor (AR) radioligand binding assays. Equol was found to be approximately 1,000- and 200-fold less potent that E2 in competing for binding sites of the SSBP and ER, respectively. Equol's potency was 2,200-fold less than testosterone for the AR. Additional studies confirmed the presence of compounds with estrogenic activity in tile drain water after application of hog manure to an agriculture field. In this case, the contribution of equol to the total estrogenicity of the tile drain water was minimal relative to that of natural estrogens. Overall, this study indicates that further work is warranted to assess the impact that EDCs that originate from agricultural runoff may have on the ecology or physiology of exposed biota.     

Detection of retinoic acid receptor agonistic activity and identification of causative compounds in municipal wastewater treatment plants in Japan

Retinoic acid (RA) receptor (RAR) agonists are potential toxicants that can cause teratogenesis in vertebrates. To determine the occurrence of RAR agonists in municipal wastewater treatment plants (WWTPs), we examined the RARα agonistic activities of influent and effluent samples from several municipal WWTPs in Osaka, Japan, using a yeast two-hybrid assay. Significant RARα agonistic activity was detected in all the influent samples investigated, suggesting that municipal wastewater consistently contains RAR agonists. Fractionations using high-performance liquid chromatography, directed by the bioassay, found several bioactive peaks from influent samples. The RAR agonists, all-trans RA (atRA), 13-cis RA (13cRA), 4-oxo-atRA, and 4-oxo-13cRA, possibly arising from human urine, were identified by liquid chromatography ion trap time-of-flight mass spectrometry. Quantification of the identified compounds in municipal WWTPs confirmed that they were responsible for the majority of RARα agonistic activity in WWTP influents, and also revealed they were readily removed from wastewater by activated sludge treatment. Simultaneous measurement of the RARα agonistic activity revealed that although total activity typically declined concomitant with the reduction of the four identified compounds, it remained high after the decline of RAs and 4-oxo-RAs in one WWTP, suggesting the occurrence of unidentified RAR agonists during the activated sludge treatment.     

Rapid screening of environmental chemicals for estrogen receptor binding capacity.

Over the last few years, an increased awareness of endocrine disrupting chemicals (EDCs) and their potential to affect wildlife and humans has produced a demand for practical screening methods to identify endocrine activity in a wide range of environmental and industrial chemicals. While it is clear that in vivo methods will be required to identify adverse effects produced by these chemicals, in vitro assays can define particular mechanisms of action and have the potential to be employed as rapid and low-cost screens for use in large scale EDC screening programs. Traditional estrogen receptor (ER) binding assays are useful for characterizing a chemical's potential to be an estrogen-acting EDC, but they involve displacement of a radioactive ligand from crude receptor preparations at low temperatures. The usefulness of these assays for realistically determining the ER binding interactions of weakly estrogenic environmental and industrial compounds that have low aqueous solubility is unclear. In this report, we present a novel fluorescence polarization (FP) method that measures the capacity of a competitor chemical to displace a high affinity fluorescent ligand from purified, recombinant human ER-[alpha] at room temperature. The ER-[alpha] binding interactions generated for 15 natural and synthetic compounds were found to be similar to those determined with traditional receptor binding assays. We also discuss the potential to employ this FP technology to binding studies involving ER-ss and other receptors. Thus, the assay introduced in this study is a nonradioactive receptor binding method that shows promise as a high throughput screening method for large-scale testing of environmental and industrial chemicals for ER binding interactions.     

Differential estrogenic actions of endocrine-disrupting chemicals bisphenol A, bisphenol AF, and zearalenone through estrogen receptor α and β in vitro

Background: Endocrine-disrupting chemicals (EDCs) are widely found in the environment. Estrogen-like activity is attributed to EDCs, such as bisphenol A (BPA), bisphenol AF (BPAF), and zearalenone (Zea), but mechanisms of action and diversity of effects are poorly understood.Objectives: We used in vitro models to evaluate the mechanistic actions of BPA, BPAF, and Zea on estrogen receptor (ER) α and ERβ.Methods: We used three human cell lines (Ishikawa, HeLa, and HepG2) representing three cell types to evaluate the estrogen promoter activity of BPA, BPAF, and Zea on ERα and ERβ. Ishikawa/ERα stable cells were used to determine changes in estrogen response element (ERE)-mediated target gene expression or rapid action-mediated effects.Results: The three EDCs showed strong estrogenic activity as agonists for ERα in a dose-dependent manner. At lower concentrations, BPA acted as an antagonist for ERα in Ishikawa cells and BPAF acted as an antagonist for ERβ in HeLa cells, whereas Zea was only a partial antagonist for ERα. ERE-mediated activation by BPA and BPAF was via the AF-2 function of ERα, but Zea activated via both the AF-1 and AF-2 functions. Endogenous ERα target genes and rapid signaling via the p44/42 MAPK pathway were activated by BPA, BPAF, and Zea.Conclusion: BPA and BPAF can function as EDCs by acting as cell type–specific agonists (≥ 10 nM) or antagonists (≤ 10 nM) for ERα and ERβ. Zea had strong estrogenic activity and activated both the AF-1 and AF-2 functions of ERα. In addition, all three compounds induced the rapid action-mediated response for ERα.     

Observations on the Estrogenic Activity and Concentration of 17β-Estradiol in the Discharges of 12 Wastewater Treatment Plants in Southern Australia

There is very little information on the overall level of estrogenic activity, or concentrations of specific hormonal compounds in wastewater treatment plant (WWTP) discharges in Australia, compared with Europe, Japan, and North America. To partly address this, in 2004, water samples were collected as “grab” or “spot” samples from 12 WWTP facilities across southern Victoria at the point at which effluent enters the environment, either as recycled water or direct discharge to the receiving water. The WWTPs were of a variety of treatment types and served a diverse range of rural and regional municipalities. For instance, of the 12 WWTPs, 3 served municipalities with populations greater than 100,000, 4 with populations between 20,000 and 100,000, and 5 with populations less than 5,000. The principal treatment process in six was an activated sludge system, and three were trickle-filter-based systems. The remaining plants fall into a “miscellaneous” category, each plant having a mixture of treatment processes within the overall systems. The estrogenic activity and 17β-estradiol concentrations of the samples were assessed using a yeast-based, in vitro reporter gene assay and enzyme-linked immunosorbant assays, respectively. Most of the effluents showed estrogenic activity in the assays (hER, no response: 7.9 ng/L EEQ; mER, no response: 44.5 ng/L EEQ). There was no correlation between estrogenic response and the results of a concurrent toxicity assay, suggesting that a lack of bioassay response was related to lack of estrogenic compounds, rather than the direct toxic effect of the sample. Estradiol concentrations were for the most part in the range 2–5 ng/L, with one sample at 18 ng/L. Despite the assurance our results might provide (of minimal impact in most cases if there is significant dilution), there is still a need for further extensive on-ground reassurance research to provide data for higher-level risk assessment by industry and government agencies. In particular, more research is warranted to verify the estrogenic activity and to expand the range of specific hormone/metabolites reported in these studies. Moreover, studies are required to determine if the estrogenic activity reported in this and other recent Australian studies is sufficient to induce a physiological effect in exposed aquatic organisms, especially Australian native fish.     

Occurrence and Partitioning of Phenolic Endocrine-Disrupting Chemicals (EDCs) Between Surface Water and Suspended Particulate Matter in the North Tai Lake Basin, Eastern China

Concentrations and distribution of octylphenol (OP), nonylphenol (NP), and bisphenol A (BPA) in surface water and suspended particulate matter (SPM) from the north Tai Lake basin, China were studied. Aqueous and particulate (dry weight) concentrations for OP, NP, and BPA varied from 10.5–1,175 ng/L to <1.52–5,365 ng/g, respectively. The spatial distribution of endocrine-disrupting chemicals (EDCs) in dissolved and particulate phases showed that the amount of EDCs in water that were adsorbed to SPM gradually increased from upstream to downstream. There were good correlations between particulate EDCs and particulate organic carbon, with correlation coefficients of 0.46–0.57. Regression analysis of in situ SPM–water partition coefficients (log K’ _oc) and log K _ow for EDCs indicated that the hydrophobicity of chemicals greatly contributed to their SPM–water partitioning. Strong positive correlations (r = 0.68–0.82) among in situ log K’ _oc of OP, NP, and BPA and flow velocity of water were observed, indicating the critical importance of riverine hydrodynamics on the sorption of these compounds.     

Aryl Hydrocarbon Receptor and Estrogen Receptor Ligand Activity of Organic Extracts from Road Dust and Diesel Exhaust Particulates

A wide variety of contaminants derived from diesel and gasoline engines, tire, asphalt, and natural organic compounds is found in road dust. Polycyclic aromatic compounds (PACs) are the important toxic targets among various contents in road dust and diesel exhaust particulates (DEPs), and endocrine-disrupting activity of PACs was suggested. In the present study, aryl hydrocarbon receptor (AhR) ligand activity was confirmed in the extract of both road dust and DEPs. In the separation of the extracts for both road dust and DEPs with reversed-phase HPLC, it was found that polar fractions contributed to significant AhR ligand activity in both a mouse hepatoma (H1L1) cell system and a yeast system. Furthermore, the contribution of these polar fractions was higher in DEPs than in road dust, probably because of the greater concentration of oxy-PAHs in DEPs than in road dust. The contribution of contaminants associated with the polar region to AhR ligand activity was also evident following the separation of road dust with normal-phase HPLC. Additionally, remarkable estrogen receptor (ER) ligand activity was detected in the highly polar region separated with normal-phase HPLC. It is suggested that many unknown AhR or ER ligand active compounds are contained in the polar region.     

Specific in vitro toxicity of crude and refined petroleum products: 3. Estrogenic responses in mammalian assays

Current petroleum risk assessment considers only narcosis as the mode of action, but several studies have demonstrated that oils contain compounds with dioxin-like, estrogenic or antiestrogenic, and androgenic or antiandrogenic activities. The present study is the third in a series investigating the specific toxic effects of 11 crude oils and refined products. By employing recombinant mammalian cells stably transfected with the human estrogen receptor alpha (ERα) or beta (ERβ), and expressing the luciferase protein (ERα-U2OS-Luc and ERβ-U2OS-Luc assay), the estrogenicity or antiestrogenicity of oils was studied. All oils, except for two refined oils and one crude oil, induced estrogenic responses. The calculated estrogenic potencies of the oils were six to nine orders of magnitude lower than the potency of 17β-estradiol (E2). Upon coexposure to a fixed concentration of E2 and increasing concentrations of oils, additive, antagonistic, and synergistic effects were revealed. One nautical fuel oil was tested in the human breast carcinoma cell line MCF-7, in which it induced cell proliferation up to 70% relative to the maximal induction by E2. At its minimum effect concentration of 25?mg/L, the oil was also capable of inducing mRNA expression of the estrogen-dependent protein pS2 by a factor of two. The present results indicate that oils naturally contain potentially endocrine-disrupting compounds that are able to influence the estrogenicity of other compounds and may cause biological responses beyond receptor binding.     

Bioavailability and biodegradation of nonylphenol in sediment determined with chemical and bioanalysis

The surfactant nonylphenol (NP) is an endocrine-disrupting compound that is widely spread throughout the environment. Although environmental risk assessments are based on total NP concentrations, only the bioavailable fraction possess an environmental risk. The present study describes the bioavailability and biodegradability of NP over time in contaminated river sediment of a tributary of the Ebro River in Spain. The bioavailable fraction was collected with Tenax TA(R) beads, and biodegradation was determined in aerobic batch experiments. The presence of NP was analyzed chemically using gas chromatography-mass spectrometry and indirectly as estrogenic potency using an in vitro reporter gene assay (ER(alpha)-luc assay). Of the total extractable NP in the sediment, 95%+/-1.5% (mean +/- standard error) desorbed quickly into the water phase. By aerobic biodegradation, the total extractable NP concentration and the estrogenic activity were reduced by 97%+/-0.5% and 94%+/-2%, respectively. The easily biodegradable fraction equals the potential bioavailable fraction. Only 43 to 86% of the estrogenic activity in the total extractable fraction, as detected in the ER(alpha)-luc assay, could be explained by the present NP concentration. This indicates that other estrogenic compounds were present and that their bioavailability and aerobic degradation were similar to that of NP. Therefore, we propose to use NP as an indicator compound to monitor estrogenicity of this Ebro River sediment. To what extent this conclusion holds for other river sediments depends on the composition of the contaminants and/or the nature of these sediments and requires further testing.     

Clustering of sex hormone disruptors in Singapore's marine environment

Abnormal sexual differentiation and other reproductive abnormalities in marine animals indicate the presence in seawater of endocrine-disrupting compounds (EDCs) that perturb the function of the sex hormone signaling pathways. However, most studies to date have reported on EDC effects in freshwater and sewage samples, and there is a paucity of bioassay data on the effects of EDCs in marine waters. Our aims in this study were to devise robust methodologies suitable for extracting potential EDCs and to measure their summated effects on activities of androgen receptors (ARs) and estrogen receptors (ER-alpha and ER-beta) in marine samples from Singapore's coastal waters. In this study, we examined the ability of C18, hydrophilic and lipophilic balance, and diol cartridges to extract potential EDCs from seawater samples. Extracts from C18 cartridges exhibited the highest sex hormone bioactivities in reporter gene assays based on a human cell line expressing AR, ER-alpha, and ER-beta. Examination of extracts from 20 coastal locations showed high androgenic and estrogenic agonist activities in confined clusters closest to the main island of Singapore. Sex hormone activity declined rapidly in clusters farther from the main coastline and in more open waters. Unexpectedly, surface and mid-depth samples from the confined high-activity clusters, in the presence of hormone, exhibited AR and ER-alpha activities that were 200-900% higher than those observed for the cognate hormone alone. This enhanced sex hormone activity suggests that analyses of complex seawater mixtures may uncover unusual bioactivities that may not be obvious by studying individual compounds. Our data present a "snapshot" of the sex hormone disruptor activity in Singapore's marine environment and indicate that C18 extraction for EDCs used in conjunction with reporter gene bioassays represents a robust and sensitive methodology for measuring summated androgenic and estrogenic activities in seawater.     

Evaluation of the estrogenic potential of river and treated waters in the Paris area (France) using in vivo and in vitro assays

For many years, surface waters have been shown to be contaminated by endocrine-disrupting compounds (EDCs), which can cause adverse effects on human and wildlife growth, development, and reproduction. It is therefore of primary importance to determine if drinking water could be contaminated by EDCs when produced from polluted surface waters. It is also essential to determine if disinfection by-products can account for estrogenic activity in treated waters. The estrogenic potential of river and treated waters was investigated using an in vivo assay. Adult male zebrafish were placed in three drinking water treatment plants (DWTPs) in the Paris area and exposed for 1 month to the two types of waters. After exposure, vitellogenin (VTG) was measured in the plasma of fish using a competitive ELISA. In addition, an in vitro assay (MELN cells) was used to assess the estrogenic potential of 10 major chlorination by-products. No significant induction of VTG was observed in fish exposed to river or treated waters. Among the 10 chlorination by-products tested, only 2-chlorophenol was found to be weakly estrogenic at concentrations up to 1mg/L. Therefore, the risk for the three DWTPs studied to produce drinking water with significant level of estrogenic substances appears to be low.     

Association between particulate matter and emergency room visits, hospital admissions and mortality in Spokane, Washington

There is conflicting evidence regarding the association between different size fractions of particulate matter (PM) and cardiac and respiratory morbidity and mortality. We investigated the short-term associations of four size fractions of particulate matter (PM(1), PM(2.5), PM(10), and PM(10-2.5)) and carbon monoxide with hospital admissions and emergency room (ER) visits for respiratory and cardiac conditions and mortality in Spokane, Washington. We used a log-linear generalized linear model to compare daily averages of PM and carbon monoxide with daily counts of the morbidity and mortality outcomes from January 1995 to June 2001. We examined pollution lags ranging from 0 to 3 days and compared our results to a similar log-linear generalized additive model. Effect estimates tended to be smaller and have larger standard errors for the generalized linear model. Overall, we saw no association with respiratory ER visits and any size fraction of PM. However, there was a suggestion of greater respiratory effect from fine PM when compared to coarse fraction. Carbon monoxide was associated with both all respiratory ER visits and visits for asthma at the 3-day lag. We feel that carbon monoxide may be serving as a marker for combustion-derived pollutants, which is one large component of the diverse air pollutant mixture. We also found no association with any size fraction of PM or CO with cardiac hospital admissions or mortality at the 0- to 3-day lag. We found no consistent associations between any size fraction of PM and cardiac or respiratory ER visits or hospital admissions.     

Characterization of estrogenic activity of riverine sediments from the Czech Republic

Extracts of sediments from rivers in an industrialized area in the Czech Republic were used to evaluate suitability of a simple in vitro bioassay system to detect estrogen receptor (ER)-mediated activity in the complex mixture. Total estrogenic activity was detected by measuring luciferase activity in a stably transfected cell line containing an estrogen-responsive element linked to a luciferase reporter gene. For appropriate interpretation of ER-mediated activity, the effect of sediment extracts on the cell cytotoxicity was assessed at the same time. All sediment samples elicited considerable estrogenic activity. Fractionation of the extracts along with bioassay testing and subsequent instrumental analysis allowed the estrogenic fractions to be identified. The Florisil fraction, which was intermediate in polarity, was the most estrogenic. Instrumental analysis documented that the concentration of the degradation products of alkylphenol ethoxylates did not occur at sufficient concentrations to account for the estrogenic activity. Mass-balance calculations and testing of fractions confirmed that certain polycyclic aromatic hydrocarbons (PAHs) or their metabolites were the most likely compounds contributing to estrogenicity. Some other compounds, such as PCNs and PAH derivatives, that were present in the first and second fraction were tested for their potential estrogenic activity. Their ER-mediated activity and contribution to the overall responses of the complex extracts were very low. The concentrations of 17β-estradiol present in the bioassay media was an important factor for the evaluation of (anti)estrogenicity of single compound(s) or complex mixtures. Received: 15 June 2001/Accepted: 27 February 2002     

Relationship between sex steroid and vitellogenin concentrations in flounder (Platichthys flesus) sampled from an estuary contaminated with estrogenic endocrine-disrupting compounds

High concentrations of vitellogenin (VTG; egg yolk protein) have previously been found in male flounder (Platichthys flesus) from several UK estuaries; these levels have been ascribed to the presence of estrogenic endocrine-disrupting compounds (EDCs). Gonadal abnormalities, including intersex, have also been recorded in these estuaries. However, there is no firm evidence to date that these two findings are causally linked or that the presence of estrogenic EDCs has any adverse population effects. In the present study, we examined the relationship between concentrations of VTG and sex steroids (11-oxo-testosterone in males and 17beta-estradiol in females) in specimens of flounder captured from the estuary of the River Mersey. We first questioned whether the high concentrations of VTG in male and immature female flounder were indeed caused by a direct effect of exogenous EDCs and not indirectly via the endogenous secretion of 17beta-estradiol. The data favored the direct involvement of estrogenic EDCs. We then questioned whether the presence of estrogenic EDCs not only stimulated inappropriate VTG synthesis but whether it might also have had a negative effect on endogenous steroid secretion. It should be noted that the predicted consequences of a drop in steroid secretion include smaller gonads, smaller oocytes, fewer numbers of sperm, and depressed spawning behavior. This question was more difficult to answer because of the strong effect of the seasonal reproductive cycle and stage of maturation on steroid concentrations. However, matched by month of capture and stage of maturation, both 17beta-estradiol in females and 11-keto-testosterone in males were in most cases significantly lower in those years when VTG concentrations were higher.