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Effect-directed analysis (EDA) can be useful in identifying and evaluating potential toxic chemicals in matrixes. Previous investigations of extracts of sediments from the upper Danube River in Germany revealed acute nonspecific and mechanism-specific toxicity as determined by several bioassays. In the present study, EDA was used to further characterize these sediments and identify groups of potentially toxic chemicals. Four extracts of sediments were subjected to a novel fractionation scheme coupled with identification of chemicals to characterize their ability to disrupt steroidogenesis or cause mutagenic and/or teratogenic effects. All four whole extracts of sediment caused significant alteration of steroidogenesis and were mutagenic as well as teratogenic. The whole extracts of sediments were separated into 18 fractions and these fractions were then subjected to the same bioassays as the whole extracts. Fractions 7 to 15 of all four extracts were consistently more potent in both the Ames fluctuation and H295R assays. Much of this toxicity could be attributed to polycyclic aromatic hydrocarbons, sterols, and in fraction 7-naphthoic acids. Because the fraction containing polychlorinated biphenyls, polychlorodibenzodioxin/furan, dichlorodiphenyltrichloroethane, and several organophosphates did not cause any observable effects on hormone production or a mutagenic response, or were not detected in any of the samples, these compounds could be eliminated as causative agents for the observed effects. These results demonstrate the value of using EDA, which uses multiple bioassays and new fractionation techniques to assess toxicity. Furthermore, to our knowledge this is the first study using the recently developed H295R assay within EDA strategies. Environ. Toxicol. Chem. 2012; 31: 1053–1062. © 2012 SETAC

BACKGROUND, GOALS, AND SCOPE: In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17β-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and "negative" chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.With a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.With one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.

The present study was part of a comprehensive weight-of-evidence approach with the goal of identifying potential causes for the declines in fish populations, which have been observed during the past decades in the Upper Danube River.The specific goal was the investigation of the endocrine disrupting potential of sediment extracts from different sites along the Danube River. Parallel to the identification and quantification of target estrogens, two in vitro bioassays were employed to assess the estrogenic potential (yeast estrogen screen, YES) of the sediment samples and to evaluate their effects on the production of testosterone (T) and E2 (H295R Steroidogenesis Assay). Using a potency balance approach, the contribution of the measured compounds (Chem-EEQs) to the total endocrine activity measured by the YES (YES-EEQs) was calculated.Of the nine sediment extracts tested five extracts exhibited significant estrogenic activities in the YES, which suggested the presence of ER agonists in these samples. The xenoestrogens nonylphenol (NP) and bisphenol A (BPA) and the natural estrogen estrone (E1) were detected while concentrations of 17β-estradiol (E2) and ethinylestradiol (EE2) were less than their respective limits of quantification in all sediment extracts. A comparison of the measured YES-EEQs and the calculated Chem-EEQs revealed that as much as 6% of estrogenic activity in extracts of most sediments could be explained by two xeno- and one natural estrogen. Exposure of H295R cells to sediment extracts from four different locations in the Danube River resulted in significantly increased concentrations of E2, but only slight inhibition of T synthesis. Furthermore, application of the H295R Steroidogenesis Assay provided evidence for endocrine disrupting potencies in sediment samples from the Upper Danube River, some of which were not detectable with the YES. In conclusion, differential endocrine activities were associated with several sediments from the Upper Danube River. Further investigations will have to show whether the observed activities are of biological relevance with regard to declines in fish populations in the Upper Danube River.

In response to concerns about chemical substances that can alter the function of endocrine systems and may result in adverse effects on human and ecosystem health, a number of in vitro tests have been developed to identify and assess the endocrine disrupting potential of chemicals and environmental samples. One endpoint that is frequently used in in vitro models for the assessment of chemical effects on the endocrine system is the alteration of aromatase activity (AA). Aromatase is the enzyme responsible for converting androgens to estrogens. Some commonly used aromatase assays, including the human microsomal assay that is a mandatory test in US-EPA’s endocrine disruptor screening program (EDSP), detect only direct effects of chemicals on aromatase activity and not indirect effects, including changes in gene expression or transcription factors. This can be a problem for chemical screening initiatives such as the EDSP because chemicals can affect aromatase both indirectly and directly. Here we compare direct, indirect, and combined measurements of AA using the H295R cell line after exposure to seven model chemicals. Furthermore, we compare the predictability of the different types of AA measurements for 17β-estradiol (E2) and testosterone (T) production in vitro. H295R cells were exposed to forskolin, atrazine, letrozole, prochloraz, ketoconazole, aminoglutethimide, and prometon for 48 h. Direct, indirect, and combined effects on aromatase activity were measured using a tritiated water-release assay. Direct effects on aromatase activity were assessed by exposing cells only during the conduct of the tritium-release assay. Indirect effects were measured after exposing cells for 48 h to test chemicals, and then measuring AA without further chemical addition. Combined AA was measured by exposing cells prior and during the conduction of the tritium-release assay. Estradiol and testosterone were measured by ELISA. Exposure to the aromatase inhibitors letrozole, prochloraz, ketoconazole, and aminoglutethimide resulted in greater indirect aromatase activity after a 48-h exposure due to presumed compensatory mechanisms involved in aromatase activity regulation. Forskolin and atrazine caused similar changes in hormone production and enzyme profiles, and both chemicals resulted in a dose-dependent increase in E2, T, and indirect AA. Neither of these two chemicals directly affected AA. For most of the chemicals, direct and combined AA and E2 were good predictors of the mechanism of action of the chemical, with regard to AA. Indirect aromatase activity was a less precise predictor of effects at the hormone level because of presumed feedback loops that made it difficult to predict the chemicals’ true effects, mostly seen with the aromatase inhibitors. Further, it was found that direct and indirect AA measurements were not reliable predictors of effects on E2 for general inducers and inhibitors, respectively. Differential modulation of AA and hormone production was observed in H295R cells after exposure to seven model chemicals, illustrating the importance of measuring multiple endpoints when describing mechanisms of action in vitro. For future work with the H295R, it is recommended that a combination of direct and indirect aromatase measurements is used because it was best in predicting the effects of a chemical on E2 production and its mechanism of action. Further, it was shown that direct AA measurements, which are a common way to measure AA, must be used with caution in vitro.

The presence of a variety of pollutants in the aquatic environment that can potentially interfere with the production of sex steroid hormones in wildlife and humans has been of increasing concern. The aim of the present study was to investigate the effects of extracts from Hong Kong marine waters, and influents and effluents from wastewater treatment plants on steroidogenesis using the H295R cell bioassay. After exposing H295R cells to extracts of water, the expression of four steroidogenic genes and the production of three steroid hormones were measured.Water samples were collected during the summer of 2005 from 24 coastal marine areas and from the influents and effluents of two major waste water treatment plants (WWTPs) in Hong Kong, China. Samples were extracted by solid phase extraction (SPE). H295R cells were exposed for 48 h to dilutions of these extracts. Modulations of the expression of the steroidogenic genes CYP19, CYP17, 3betaHSD2, and CYP11beta2 were determined by measuring mRNA concentrations by real-time polymerase chain reaction (Q-RT-PCR). Production of the hormones progesterone (P), estradiol (E2), and testosterone (T) was quantified using enzyme linked immunosorbent assays (ELISA).Extracts from samples collected in two fish culture areas inhibited growth and proliferation of H295R cells at concentrations greater or equal to 10(5) L equivalents. The cells were exposed to the equivalent concentration of active substances in 10,000 L of water. Thus, to observe the same level of effect as observed in vitro on aquatic organisms would require a bioaccumulation factor of this same magnitude. None of the other 22 marine samples affected growth of the cells at any dilution tested. Twelve of the marine water samples completely inhibited the expression of CYP19 without affecting E2 production; inhibition of CYP17 expression was observed only in one of the samples while expression of CYP11beta2 was induced as much as five- and ninefold after exposure of cells to extracts from two locations. The expression of the progesterone gene 3betaHSD2 was not affected by any of the samples; only one sample induced approximately fourfold the production of E2. Although more than twofold inductions were observed for P and T production, none of these values were statistically significant to conclude effects on the production of these two hormones. While influents from WWTPs did not affect gene expression, an approximately 30% inhibition in the production of E2 and a 40% increase in P occurred for the exposure with influents from the Sha Tin and Stonecutters WWTPs, respectively. Effluents from WWTPs did not affect the production of any of the studied hormones, but a decrement in the expression of the aldosterone gene CYP11beta2 was observed for the Sha Tin WWTP exposure. No direct correlation could be established between gene expression and hormone production.Observed cytotoxicity in the two samples from fish culture areas suggest the presence of toxic compounds; chemical analysis is required for their full identification. Although effluents from WWTPs did not affect hormone production, other types of endocrine activity such as receptor-mediated effects cannot be ruled out. Interactions due to the complexity of the samples and alternative steroidogenic pathways might explain the lack of correlation between gene expression and hormone production results.Changes observed in gene expression and hormone production suggest the presence in Hong Kong coastal waters of pollutants with endocrine disruption potential and others of significant toxic effects. The aromatase and aldosterone genes seem to be the most affected by the exposures, while E2 and P are the hormones with more significant changes observed. Results also suggest effectiveness in the removing of compounds with endocrine activity by the WWTPs studied, as effluent samples did not significantly affect hormone production. The H295R cell showed to be a valuable toll in the battery required for the analysis of endocrine disrupting activities of complex environmental samples.Due to the intrinsic complexity of environmental samples, a combination of analytical tools is required to realistically assess environmental conditions, especially in aquatic systems. In the evaluation of endocrine disrupting activities, the H295R cell bioassay should be used in combination with other genomic, biological, chemical, and hydrological tests to establish viable modes for endocrine disruption and identify compounds responsible for the observed effects.

The H295R cell bioassay was used to evaluate the potential endocrine disrupting effects of 18 of the most commonly used pharmaceuticals in the United States. Exposures for 48 h with single pharmaceuticals and binary mixtures were conducted; the expression of five steroidogenic genes, 3betaHSD2, CYP11beta1, CYP11beta2, CYP17 and CYP19, was quantified by Q-RT-PCR. Production of the steroid hormones estradiol (E2), testosterone (T) and progesterone (P) was also evaluated. Antibiotics were shown to modulate gene expression and hormone production. Amoxicillin up-regulated the expression of CYP11beta2 and CYP19 by more than 2-fold and induced estradiol production up to almost 3-fold. Erythromycin significantly increased CYP11beta2 expression and the production of P and E2 by 3.5- and 2.4-fold, respectively, while production of T was significantly decreased. The beta-blocker salbutamol caused the greatest induction of CYP17, more than 13-fold, and significantly decreased E2 production. The binary mixture of cyproterone and salbutamol significantly down-regulated expression of CYP19, while a mixture of ethynylestradiol and trenbolone, increased E2 production 3.7-fold. Estradiol production was significantly affected by changes in concentrations of trenbolone, cyproterone, and ethynylestradiol. Exposures with individual pharmaceuticals showed the possible secondary effects that drugs may exert on steroid production. Results from binary mixture exposures suggested the possible type of interactions that may occur between drugs and the joint effects product of such interactions. Dose-response results indicated that although two chemicals may share a common mechanism of action the concentration effects observed may be significantly different.

The present studies were undertaken to evaluate the utility of the H295R system as an in vitro assay to assess the potential of chemicals to modulate steroidogenesis. The effects of four model chemicals on the expression of ten steroidogenic genes and on the production of three steroid hormones were examined. Exposures with individual model chemicals as well as binary mixtures were conducted. Although the responses reflect the known mode of action of the various compounds, the results show that designating a chemical as "specific inducer or inhibitor" is unwise. Not all changes in the mixture exposures could be predicted based on results from individual chemical exposures. Hormone production was not always directly related to gene expression. The H295R system integrates the effects of direct-acting hormone agonists and antagonists as well as chemicals affecting signal transduction pathways for steroid production and provides data on both gene expression and hormone secretion which makes this cell line a valuable tool to examine effects of chemicals on steroidogenesis.

The novel brominated flame retardants (NBFRs), 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), Bis(2-ethylhexyl)-2,3,4,5-tetrabromophtalate (TBPH), and 1,2,5,6-tetrabromocyclooctane (TBCO) are components of flame retardant mixtures including Firemaster 550 and Saytex BC-48. Despite the detection of these NBFRs in environmental and biotic matrices, studies regarding their toxicological effects are poorly represented in the literature. The present study examined endocrine disruption by these three NBFRs using the yeast YES/YAS reporter assay and the mammalian H295R steroidogenesis assay. Activation of the aryl hydrocarbon receptor (AhR) was also assessed using the H4IIE reporter assay. The NBFRs produced no TCDD-like effects in the H4IIE assay or agonistic effects in the YES/YAS assays. TBB produced a maximal antiestrogenic effect of 62% at 0.5mgL(-1) in the YES assay while TBPH and TBCO produced maximal antiandrogenic effects of 74% and 59% at 300mgL(-1) and 1500mgL(-1), respectively, in the YAS assay. Significant effects were also observed in the H295R assay. At 0.05mgL(-1), 15mgL(-1), and 15mgL(-1) TBB, TBPH, and TBCO exposures, respectively resulted in a 2.8-fold, 5.4-fold, and 3.3-fold increase in concentrations of E2. This is one of the first studies to demonstrate the in vitro endocrine disrupting potentials of TBB, TBPH, and TBCO.

PLoS one 8(10), e75596 (2013). doi:10.1371/journal.pone.0075596, Published by PLoS [u.a.], Lawrence, Kan.

There is increasing concern over the risk of environmentally relevant doses of bisphenol A (BPA) on human endocrine systems. Effects of BPA on steroidogenesis and the related molecular mechanisms were investigated in H295R human adenocarcinoma cells. This immortal cell line is unique in expressing all the enzymes of the steroidogenic pathways. The effects of BPA on steroidogenesis, 17β-estradiol (E2) metabolism, and aromatase activity were examined in H295R cells exposed to BPA from 3.0 × 10(-1) to 3.0 × 10(3) ng/ml. Concentrations of BPA in basic cell culture materials were verified. Stable CYP17A-knockdown H295R cells were developed to verify the mechanism of inhibited steroidogenesis by BPA. Background concentrations of BPA in control cell culture media ranged from 0.03 to 0.38 ng/ml. Significantly lesser concentrations of androstenedione, testosterone, cortisol, and cortisone were caused by exposure to 30-3000 ng BPA/ml. In contrast, sconcentrations of estrone (E1) and E2 were significantly greater in BPA-exposed H295R cells. Lesser production of androstenedione and testosterone by H295R cells exposed to BPA was the most sensitive endpoint (no observable effect concentrations < 30 ng BPA/ml). CYP17A knockdown in H295R cells resulted in less production of both 17α hydroxyprogesterone and androstenedione. The results are consistent with the hypothesis that in H295R cells, BPA selectively inhibits 17,20-lyase but not 17α-hydroxylase. The primary mechanism causing increased E2 in the medium was inhibition of E2 metabolism rather than greater aromatase (CYP19) activity. These results suggest that BPA has the potential to interfere with cellular steroidogenesis in humans through multiple molecular mechanisms.

Concentration-dependent response relationships provide essential information on the characteristics of chemical-induced effects on toxicological end points, which include effect (inhibition or induction), potency, and efficacy of the chemical. Recent developments in systems biology and high throughputtechnologies have allowed simultaneous examination of many chemicals at multiple end point levels. While this increase in the quantity of information generated offers great potential, it also poses a significant challenge to environmental scientists to efficiently manage and interpret these large data sets. Here we present a novel method, ToxClust, that allows clustering of chemicals on the basis of concentration-response data derived with single or multiple end points. This method utilizes a least distance-searching algorithm (LDSA) to measure the pattern dissimilarity of concentration-response curves between chemicals and their relative toxic potency. ToxClust was tested using simulated data and chemical test data collected from the human H295R cell-based in vitro steroidogenesis assay. ToxClust effectively identified similar patterns of simulated data and responses to the exposure with the five model chemicals and separated them into different groups on the basis of their dissimilarities. These observations demonstrate that ToxClust not only provides an effective data analysis and visualization tool, but also has value in hypothesis generation and mechanism-based chemical classification.

An in vitro steroidogenesis assay using H295R human adenocarcinoma cells has been suggested as a possible alternative to gonad explant assays for use as a Tier I screening assay to detect endocrine active chemicals capable of modulating steroid hormone synthesis. This study is one of the first to investigate the utility of the H295R assay for predicting effects and/or understanding mechanisms of action across species and tissues. Six chemicals, including one selective aromatase inhibitor (fadrozole), four fungicides (fenarimol, ketoconazole, prochloraz, and vinclozolin), and one herbicide (prometon), were tested in both the H295R steroidogenesis assay, and an in vitro steroidogenesis assay using fathead minnow ovary explants. All six chemicals caused significant alterations in 17beta-estradiol (E2) and/or testosterone (T) production in vitro. Effects of ketoconazole, prochloraz, and prometon were similar in both assays. However, there were differences in the profile of responses for T for fadrozole and fenarimol, and for T and E2 for vinclozolin. In terms of sensitivity, steroid production in the H295R assay was most sensitive for detecting the effects of fadrozole, fenarimol, and prochloraz, but was less sensitive than the fathead minnow ovary explant assay to the effects of ketoconazole and vinclozolin. The H295R assay was consistently less variable (among replicates) than the fathead minnow ovary explant assay. However, the ovary explant assay was more predictive of in vivo effects of the six chemicals on fathead minnows than the H295R system. Further characterization of autoregulatory capacities, interaction of steroid-hormone receptor pathways with steroidogenesis, and metabolic capabilities of each system are needed for either system to provide clear and informative insights regarding a chemical's mechanism of action. Overall, however, results of this study suggest that both the H295R and fathead minnow ovary explant assays have utility for identifying endocrine-active chemicals in screening-type applications.

To identify and prioritize chemicals that may alter steroidogenesis, an in vitro screening assay based on measuring alterations in hormone production was developed using the H295R human adrenocortical carcinoma cell line. Previous studies indicated that this cell line was useful to screen for effects on gene expression of steroidogenic enzymes. This study extended that work to measure the integrated response on production of testosterone (T), estradiol (E2), and progesterone/pregnenolone (P) using an ELISA. Under optimized culture and experimental conditions, the basal release of P, T and E2 into the medium was 7.0 ± 1.2 ng/ml, 1.6 ± 0.4 ng/ml, and 0.51 ± 0.13 ng/ml, respectively. Model chemicals with different modes of action on steroidogenic systems were tested. Exposure to forskolin resulted in dose-dependent increases in all three hormones with the greatest relative increase being observed for E2. This differed from cells exposed to prochloraz or ketoconazole where P concentrations increased while T and E2 concentrations decreased in a dose-dependent manner. In cells exposed to fadrozole, E2 decreased in a dose-dependent manner while T and P only decreased at the greatest dose tested. Aminoglutethimide decreased P and E2 concentrations but increased T concentrations. Vinclozolin reduced both P and T but resulted in a slight increase in E2. The alteration in the patterns of hormone production in the H295R assay was consistent with the modes of action of the chemicals and was also consistent with observed effects of these chemicals in animal models. Based on these results, the H295R in vitro system has potential for high throughput screening to not only characterize the effects of chemicals on endocrine systems but also to prioritize chemicals for additional testing.