Effects of fifteen PBDE metabolites, DE71, DE79 and TBBPA on steroidogenesis in the H295R cell line

Polybrominated diphenyl ethers (PBDEs) and tetrabromobisphenol A (TBBPA) are brominated flame retardants that are produced in large quantities and are commonly used in construction materials, textiles, and as polymers in electronic equipment. Environmental and human levels of PBDEs have been increasing in the past 30 years, but the toxicity of PBDEs is not fully understood. Studies on their effects are relatively limited, and show that PBDEs are neurotoxins and potential endocrine disrupters. Hydroxylated (OH) and methoxylated (MeO) PBDEs have also been reported in the adipose tissue, blood and milk of wild animals and humans. In the present study, 15 PBDE metabolites, two BDE mixtures (DE71 and DE79), and TBBPA were studied individually to determine their effects on ten steroidogenic genes, aromatase activity, and concentrations of two steroid hormones (testosterone and 17beta-estradiol) in the H295R human adrenocortical carcinoma cell line. Exposure to 0.05 microM 2'-OH-BDE-68 significantly induced the expression of CYP11A, CYP11B2, CYP17, CYP21, 3betaHSD2, 17betaHSD1, and 17betaHSD4, and the expression of StAR was induced by 6-OH-BDE-90 at the three exposure concentrations. Exposure to DE71 and DE79 resulted in dose-dependent trend towards induction, but these effects were not significant. Exposure to 0.5 microM 2-OH-BDE-123 and 2-MeO-BDE-123 resulted in significantly greater aromatase activity. However, none of the compounds affected sex hormone production at the concentrations tested. Generally, OH-BDEs had a much stronger ability to affect steroidogenic gene expression than MeO-BDEs.     

The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

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 17??-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.

Methods

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.

Results

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.

Discussion and conclusions

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).

Perspectives

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.     

Modulation of steroidogenesis by coastal waters and sewage effluents of Hong Kong, China, using the H295R assay

BACKGROUND, AIM, AND SCOPE: 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. MATERIALS AND METHODS: 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). RESULTS: 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. DISCUSSION: 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. CONCLUSIONS: 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. RECOMMENDATIONS AND PERSPECTIVES: 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.     

Individual PCBs as predictors for concentrations of non and mono-ortho PCBs in human milk

32 Dutch human milk samples were analyzed for PCBs with either HRGC-ECD or HRGC-LRMS in the NCI mode. Samples were collected from three different locations in The Netherlands: Amsterdam, Rotterdam and Groningen. Quantitatively, no differences could be observed between the three localities, while in addition the congener specific pattern showed a striking similarity for all individual samples. Only principal component analysis revealed slight individual differences. Based on similarities in the PCB profiles, linear relationships were calculated between 2,3′4,4′,5-PnCB (#118) or 2,2′4,4′5,5′HxCB (#153) and the most relevantnon andmonoortho PCBs exhibiting dioxinlike activity. These PCBs included 2,3,3′,4,4′-PnCB (#105), 3,3′,4,4′5-PnCB (#126) 2,3,3′,4,4′,5-HxCB (#156), 2,3,3′,4,4′,5′-HxCB (#157), 2,3′,4,4′,5,5′-HxCB (#167) and 3,3′,4,4′,5′5-HxCB (#169). Good linear relationships were observed between individual PCBs. Based on the results of this study, PCB #118 can be used to predict concentrations of the PCBs #105 and #126. PCB #153 can be used as a predictor for the PCBs #156, #157, #167 and #169, but also for the total toxic equivalencies (TEQs) ofnon andmonoortho PCBs present in human milk. This method using certain PCBs as predictors for other toxicological relevant congeners, can be useful and cost effective, e.g. for epidemiological studies. However, before applied a number of conditions should be met. These are:

1. A stable composition of the PCB matrix should be established.
2. A possible time dependent change in composition of the matrix should first be excluded when used over different time periods.

     

Effect of nonylphenol on steroidogenesis of rat Leydig cells

Nonylphenol is the primary final biodegradation product of nonylphenol polyethoxylate (NPE), a non-ionic surfactant that is frequently incorporated into pesticide and detergent formulation. Recent researchers have hypothesized that environmental/ occupational exposure to nonylphenol poses adverse effects on reproductive system of humans and wildlife species. During our study, in vivo and in vitro experiments were performed to examine the effect of nonylphenol on testosterone biosynthesis of rat Leydig cells. In experiment in vivo, serum testosterone (T) as well as luteinizing hormone (LH) levels were detected after animals had been treated with different doses (0 mg/kg/day, 125 mg/kg/day, and 250 mg/kg/day) of nonylphenol for 50 days by gavage, and the final result revealed that testosterone level dramatically declined at the dose of 250 mg/kg/day, while LH level ascended at the dose of 125 mg/kg/day and 250 mg/kg/day. In experiment in vitro, primary cultured Leydig cells were exposed to nonylphenol for 48 h, including low concentrations (0 mg/L, 0.0011 mg/L, 0.0033 mg/L, 0.0055 mg/L, 0.011 mg/L, 0.022 mg/L) and higher concentrations (0.11 mg/L, 0.55 mg/L, 1.1 mg/L, 1.65 mg/L, 2.2 mg/L, 2.75 mg/L, 3.3 mg/L, 5.5 mg/L). Increase of testosterone levels was observed at low concentrations of nonylphenol while reduction was detected at higher concentrations.     

Dioxin-like and non-dioxin-like PCBs in human serum of Slovak population

In the present study, non-ortho, mono-ortho and other ortho-substituted PCB congeners were analysed in individual blood serum samples taken from healthy adults (196 males and 119 females) in the polluted area of the Michalovce district and in the background area of the Stropkov/Svidnik districts in Eastern Slovakia by high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). The samples from general population living in villages and towns of two regions were taken between August 2001 and February 2002 within the project of the EC's 5th Framework Programme (PCBRISK, ). The medians of dioxin-like PCBs (dl-PCBs)--expressed as TEQs on lipid basis--of all males (24.7 pg g(-1) lipid) and females (21.4 pg g(-1) lipid) were comparable, but there was a significant difference between both of the areas studied. In the district of Michalovce, the medians of males (47.3 pg g(-1) lipid) and females (41.1 pg g(-1) lipid) were 2.3 times higher than in the area of the Stropkov/Svidnik districts. The medians of total non-dioxin-like PCB concentration were 1,835 and 1,033 ng g(-1) lipid in males and females, respectively. The data show a trend toward higher concentrations of both dl- and non-dioxin-like PCBs in the older age groups. The substantial increase was observed for the 60+ age group. PCB-126 was the most abundant non-ortho congener. PCBs-118 and 156 were the predominant mono-ortho congeners. They were quantitated in all samples analysed. Congeners 153, 138(+163), 180 and 170(+190) were the main contributors to total non-dioxin-like PCB concentrations. Mean mutual ratio HexaCBs:HeptaCBs:OctaCBs in the non-dioxin-like group was 37:50:1. The results of this study represent the overall information about the congener distribution of dioxin-like and non-dioxin-like PCBs in human serum blood of adult Slovaks.     

Effects of dietary wheat bran on absorption and accumulation of PCBs in rats

Polychlorinated biphenyls (PCBs) are toxic environmental contaminants, which tend to accumulate in the food chain. Since dietary intake is the most important exposure route, PCB body burden may be affected by taking proper dietary measures. In the present study, diets were supplemented with either wheat bran or its cellulose based placebo in order to study the effect of bran consumption on the absorption of dietary PCBs and the excretion of initially stored PCBs. During the period of PCB intake, faecal PCB excretion was elevated by consumption of wheat bran as compared to the placebo. Hence, apparent faecal PCB digestibilities as well as PCB retentions in the whole body were lower in the wheat bran consuming rats. After ending PCB consumption, dietary wheat bran had only a minor effect on faecal PCB output while accumulation in the body was not affected. When PCBs were consumed for a longer time, a small but significant reduction of apparent faecal PCB digestibility was found. However, PCB content in the body kept increasing while PCB retention as percent of intake remained almost constant. Furthermore, differences among individual PCB congeners in metabolic susceptibility and hydrophobic characteristics had an impact on their accumulation in the body.     

Levels and trends of PCDD/Fs and PCBs in human milk in Finland

Word Health Organization, WHO/EURO, has coordinated two rounds of follow-up studies on levels of PCDDs, PCDFs, and PCBs in human milk which were analyzed as two pooled samples from each participating country, one from urban and the other one from rural area. Finland has taken part to both of those studies and we are now reporting results of all the second round randomly sampled human milk samples (84 samples) from Southern (20) and Eastern (64) Finland. The levels of PCDD/Fs and PCBs in human milk in Southern Finland were considerably higher than in Eastern Finland. The level of PCDD/Fs in human milk in Southern Finland was the same as in the Central Europe but the level in Eastern Finland was similar to levels in Norway and eastern parts of Europe. The concentrations of PCDD/Fs and PCBs showed a significant decrease from 1987 to 1994. Declining of PCDD/Fs and PCBs was 36 and 49% in primiparae mothers' milk, respectively. This decrease in concentrations of PCDD/F and PCB was slightly greater in Eastern than in Southern Finland.     

The OECD Validation Program of the H295R Steroidogenesis Assay for the Identification of In Vitro Inhibitors and Inducers of Testosterone and Estradiol Production. Phase 2: Inter-Laboratory Pre-Validation Studies (8 pp)

Background, Goals and Scope In response to concerns that have been raised about chemical substances that may alter the function of endocrine systems and result in adverse effects on human health, an OECD initiative was undertaken to develop and validate in vitro and in vivo assays to identify chemicals that may interfere with endocrine systems of vertebrates. Here we report on studies that were conducted to develop and standardize a cell-based screening assay using the H295R cell line to prioritize chemicals that may act on steroidogenic processes in humans and wildlife. These studies are currently ongoing as part of the ‘Special Activity on the Testing and Assessment of Endocrine Disruptors’ within the OECD Test Guidelines Program to review, develop, standardize, and validate a number of in vitro and in vivo toxicological assays for testing and assessment of chemicals concerning their potential to interact with the endocrine system of vertebrates. Study Design Six laboratories from five countries participated in the pre-validation studies. Each laboratory tested the effects of three model chemicals on the production of testosterone (T) and estradiol (E2) using the H295R Steroidogenesis Assay. Chemicals tested were well described inducers or inhibitors of steroidogenic pathways (forskolin, prochloraz and fadrozole). All experiments were conducted in 24 well plates following standard protocols. Six different doses per compound were analyzed in triplicate per plate. A quality control (QC) plate was run in conjunction with the chemical exposure plate to account for inter-assay variation. Each chemical exposure was conducted two or three times. Results All laboratories successfully detected increases and/or decreases in hormone production by H295R cells after exposure to the different model compounds and there was good agreement in the pattern of response for all groups. Forskolin increased both T and E2 while fadrozole and prochloraz decreased production of both hormones. All chemicals affected hormone production in a dose-dependent manner with the exception of fadrozole which caused maximum inhibition of E2 at the two least concentrations tested. Some inter-laboratory differences were noted in the alteration of hormone production measured in chemically exposed cells. However, with the exception of the production of T measured at one laboratory in cells exposed to forskolin, the EC₅₀s calculated were comparable (coefficients of variation 34–49%) for all hormones. Discussion and Perspectives The results indicated that the H295R Steroidogenesis Assay protocol was robust, transferable and reproducible among all laboratories. However, in several instances that were primarily related to one laboratory there were unexplained minor uncertainties related to the inter-laboratory hormone production variation. Based on the findings from this Phase 2 prevalidation study, the H295R Steroidogenesis Assay protocol is currently being refined. The next phase of the OECD validation program will test the refined protocol among the same group of laboratories using an extended set of chemicals (∼30) that will include positive and negative chemical controls as well as a broad spectrum of different potential inducers and inhibitors of steroidogenic pathways. Submission Editor: Dr. Carsten Brühl (bruehl@uni-landau.de)     

Methods for the analysis of PCBs in human food, faeces and serum.

A method was developed to determine trace concentrations of a range of individual PCB congeners in biological samples (serum, food and faeces) using GC-MS, to prepare a mass balance of PCBs in humans. A simple method for the analysis of PCBs in human serum, which excluded an extraction step, was first employed. Results indicated that the recoveries of 13C12 PCB spikes were variable. A soxhlet extraction step was added and was found to be efficient and reproducible. A quality control routine and method validation results are presented. In batch tests of the methods presented it was found that the serum analysis method gave within batch mean 13C12 spike recoveries of 98-120% and standard deviations between 6 and 20%. The food/faeces analysis method gave within-batch mean 13C12 spike recoveries of 88-100%, and within batch standard deviations between 4 and 12%. The batch to batch mean recovery for serum analysis was 100%, with an RSD of 9% for high spikes and 10% for low spikes. For food/faeces analysis the batch to batch average recovery was 110%, with an RSD of 5% for high spikes and 9% for low spikes.     

溶液中阴离子和腐殖酸对UV/H2O2降解2,4-二氯酚的影响

研究了UV/H2O2工艺对2,4-二氯酚(2,4-DCP)的去除效果和水中阴离子、腐殖酸对该工艺降解2,4-DCP的影响.结果表明:UV/H2O2工艺可以有效地去除水中2,4-DCP,光降解过程符合一级反应动力学模型;在H2O2投加量为8 mg/L、1个30 W低压汞灯照射下,2,4-DCP在蒸馏水和自来水中反应速率常数分别为0.023 2、0.016 2 min-1;NO-3、Cl-、HCO-3对2,4-DCP光降解有抑制作用,当3种阴离子摩尔浓度为0.5、10.0、20.0 mmol/L时,对2,4-DCP光降解的抑制程度为HCO-3＞NO-3＞Cl-;腐殖酸在低浓度时,促进光降解反应进行,在高浓度时,2,4-DCP的光降解受到抑制.自来水中的反应速率常数低于蒸馏水中的反应速率常数是由于水中多种阴离子和腐殖酸影响的结果.     

Decolorization of RBBR by plant cells and correlation with the transformation of PCBs

An extracellular H2O2-requiring Remazol Brilliant Blue R (RBBR) decolorizing enzyme activity was detected after cultivation of cells of various plant species both in liquid medium and when growing on agar plates containing RBBR. Level of the enzyme activity was compared with the ability to metabolize polychlorinated biphenyls (PCBs). The ability to decolorize RBBR was tested in the presence and absence of PCBs. The cultures with high PCB-transforming activity proved to exhibit RBBR oxidase much more resistant towards the influence of PCBs. In addition low activities of lignin peroxidase (LiP) and manganese dependent peroxidase (MnP) were detected in medium and in plant cells. No correlation of MnP and LiP activities with PCB degradation could be found. The RBBR decolorization could be used as a rough screening method for plant cultures able to metabolize PCBs.     

Effects of H2O2 on growth,metabolic activity and membrane integrity in three strains of Microcystis aeruginosa

Environmental Science and Pollution Research - The application of hydrogen peroxide (H2O2) as a management tool to control Microcystis blooms has become increasingly popular due to its short...     

Levels of dioxins, furans and PCBs in human serum and milk of people living in Greece

In an attempt to evaluate the background levels of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), non-ortho, mono-ortho and indicator polychlorinated biphenyls (PCBs) in people living in an urban (Athens) and a rural (Kozani) area of Greece, blood serum and mother milk samples were analyzed. Analytical results are reported in this study. Statistically significantly differences were observed between regions, sexes and ages. Contamination levels in blood and human milk from Greece reported here are low compared to the previously reported dioxin data from other European countries and give no indication of particular health risk.     

PCBs and other organochlorines in human tissue samples from the Welsh population: II--Milk

Polychlorinated biphenyl (PCB) and SigmaDDT (i.e. p,p'-DDT + p,p'-DDE = p,p'-DDD) concentrations were determined from the analysis of 115 Welsh breast milk samples collected in 1990 and 1991. Fifty PCB congeners were screened, of which 24 were identified in most samples. The PCB congener pattern was consistent between individual milk samples, with IUPAC congeners 28, 138, 153 and 180 being the most abundant and accounting for an average of 50% of the SigmaPCB concentrations determined. PCB concentrations varied between 2 and 70 ng g(-1) whole milk, were positively correlated with age, and negatively correlated with the total lactation period and with the percent lipid content of the milk. PCB pattern distributions differed between milk and adipose tissue samples. Human milk had a higher proportion of tri- (18 and 28), tetra- (44, 52 and 66) and pentachlorinated biphenyls (101) compared to human adipose tissue. SigmaDDT concentrations ranged from 0.3 to 71 ng g(-1) of whole milk, with p,p'-DDE contributing towards an average of 92% of the SigmaDDT concentrations. SigmaDDT levels were also positively correlated with age and negatively associated with the lactation period, though these correlations were rather weak. No significant differences in the SigmaPCB and SigmaDDT concentrations were noted between milk samples from donors living in rural and urban locations, or between the subjects' body weight, smoking habits or diet.     

溶液中阴离子对UV/H2O2降解十二烷基苯磺酸钠的影响及其机理

研究了UV/H2O2工艺对十二烷基苯磺酸钠(LAS)的去除效果、溶液中阴离子对LAS降解的影响及机理.结果表明:UV/H2O2工艺可以有效地去除水中的LAS;在H2O2投加量为8 mg/L,14 W低压汞灯照射下,LAS在蒸馏水和自来水中的反应速率常数分别为0.018 0 、0.012 2 min-1;NO-3、Cl-、SO2-4和HCO-3对LAS光降解有抑制作用,当该4种离子摩尔浓度均分别为5、10、15 mmol/L时,对LAS光降解的抑制程度为HCO-3》NO-3》Cl-》SO2-4,且随着离子摩尔浓度的增大,抑制作用增强;LAS在自来水中的反应速率常数低于在蒸馏水中的反应速率常数是由于水中多种离子影响的结果.     

Ruthana date extract inhibited proliferation of human hepatocellular carcinoma (HepG2) cells by modulation of BAX gene

Environmental Science and Pollution Research - Cancer response to chemotherapeutic agents and its side effects remain a challenge for the development of new anticancer compounds. Dates are consumed...