Ron L.A.P. Hoogenboom

Rapid yeast estrogen bioassays stably expressing human estrogen receptors α and β, and green fluorescent protein: a comparison of different compounds with both receptor types

Previously, we described the construction of a rapid yeast bioassay stably expressing human estrogen receptor α (hERα) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, the properties of this assay were further studied by testing a series of estrogenic compounds. Furthermore, a similar assay was developed based on the stable expression of human estrogen receptor β (hERβ). When exposed to 17β-estradiol, the maximum transcriptional activity of the ERβ cytosensor was only about 40% of the activity observed with ERα, but the concentration where half-maximal activation is reached (EC50), was about five times lower. The relative estrogenic potencies (REP), defined as the ratio between the EC50 of 17β-estradiol and the EC50 of the compound, of the synthetic hormones dienestrol, hexestrol and especially mestranol were higher with ERα, while DES was slightly more potent with ERβ. The gestagens progesterone and medroxyprogesterone-acetate showed no response, whereas the androgen testosterone showed a very weak response. The anabolic agent, 19-nortestosterone showed a clear dose-related response with estrogen receptor α but not β. The phytoestrogens coumestrol, genistein, genistin, daidzein, daidzin and naringenin were relatively more potent with ERβ. Ranking of the estrogenic potency with ERα was: 17β-estradiol ⪢ 8-prenylnaringenin > coumestrol > zearalenone ⪢ genistein ⪢ genistin > naringenin. The ranking with the ERβ was: 17β-estradiol ⪢ coumestrol > genistein > zearalenone > 8-prenylnaringen ⪢ daidzein > naringenin > genistin ⪢ daidzin. The hop estrogen 8-prenylnaringenin is relatively more potent with ERα. These data show that the newly developed bioassays are valuable tools for the rapid and high-throughput screening for estrogenic activity.

Dietary exposure to dioxins and dioxin-like PCBs in The Netherlands anno 2004

In this study, representative occurrence data for PCDD/Fs and dioxin-like PCBs in food were obtained and used to estimate dietary exposure of the Dutch population. Food composite samples were analyzed as well as single fish and vegetables samples. Total dioxin concentrations in animal products ranged from 0.05 pg TEQ/g product in poultry to 2.5 pg TEQ/g product (using TEF(2006)) in fish (shrimp), with 0.12pg TEQ/g product being the lowest concentrations measured in fish (tuna). In vegetable products, concentrations ranged from 0.00002 pg TEQ/g product (white kale) to 0.19 pg TEQ/g (oils and fats). A long-term dietary exposure distribution was calculated using Monte Carlo Risk Assessment software. The lower bound median exposure of the Dutch population to PCDD/Fs and dioxin-like PCBs was estimated at 0.8 pg WHO-TEQ/kgbw/d, half of which were dioxin-like PCBs. Dairy was the main source (38%) due to its high consumption. Time-trend analysis shows that the exposure to dioxins has further decreased by 35% over the past five years. This is due to lower levels of dioxin-like compounds in most of the foods, mainly influenced by lower levels in meat and milk. The use of the new TEFs gives an exposure reduction of 10% with respect to TEF(1998). Still, 4% of the Dutch population exceeds the exposure limit of 14 pg/kgbw/week as set by the EU.

A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists

Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17β-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound, of 5α-dihydrotestosterone, methyltrienolone, and 17β-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action.

Subacute effects of hexabromocyclododecane (HBCD) on hepatic gene expression profiles in rats.

Hexabromoyclododecane (HBCD), used as flame retardant (FR) mainly in textile industry and in polystyrene foam manufacture, has been identified as a contaminant at levels comparable to other brominated FRs (BFRs). HBCD levels in biota are increasing slowly and seem to reflect the local market demand. The toxicological database of HBCD is too limited to perform at present a solid risk assessment, combining data from exposure and effect studies. In order to fill in some gaps, a 28-day HBCD repeated dose study (OECD407) was done in Wistar rats. In the present work liver tissues from these animals were used for gene expression profile analysis. Results show clear gender specificity with females having a higher number of regulated genes and therefore being more sensitive to HBCD than males. Several specific pathways were found to be affected by HBCD exposure, like PPAR-mediated regulation of lipid metabolism, triacylglycerol metabolism, cholesterol biosynthesis, and phase I and II pathways. These results were corroborated with quantitative RT-PCR analysis. Cholesterol biosynthesis and lipid metabolism were especially down-regulated in females. Genes involved in phase I and II metabolism were up-regulated predominantly in males, which could explain the observed lower HBCD hepatic disposition in male rats in this 28-day study. These sex-specific differences in gene expression profiles could also underlie sex-specific differences in toxicity (e.g. decreased thyroid hormone or increased serum cholesterol levels). To our knowledge, this is the fist study that describes the changes in rat hepatic gene profiles caused by this commonly used flame retardant.

The German bakery waste incident; use of a combined approach of screening and confirmation for dioxins in feed and food

During the last six years several incidents have occurred with dioxins in feed, stressing the need for rapid screening methods for these compounds. The most recent incident was the contamination of bakery waste used for animal feed due to the use of waste wood for drying of the material. In addition to Germany, the material was also shipped to the Netherlands. Levels up to 12ng TEQ/kg have been detected, being about 15 times over the current limit of 0.75ng TEQ/kg. In the Netherlands a combined strategy of screening with the CALUX-bioassay and the HRGC/HRMS confirmatory method was used to rapidly control the incident. Pigs were contaminated by the incident but only to a very limited extent. Despite the rather low limits for pig meat, the CALUX bioassay showed excellent performance, once again confirming the value of this assay.

Screening for modulatory effects on steroidogenesis using the human H295R adrenocortical cell line: a metabolomics approach.

The recently OECD validated H295R steroidogenesis assay provides an in vitro alternative to evaluate the potential interference of exogenous compounds with endogenous steroid hormone synthesis. Currently, this assay is used for a simple negative-positive screening of compounds using testosterone and estradiol levels as end points, measured with specific enzyme immunoassays (EIAs) or targeted liquid chromatography (LC) and gas chromatography (GC)-mass spectrometry (MS) methods. However, recent developments in LC-MS and bioinformatics allow for more comprehensive approaches to evaluate changes in steroid profiles. In the current work, the H295R cell model was combined with a metabolomics approach to monitor changes in metabolite profiles in both a targeted and untargeted way. H295R cells were exposed for 48 h to model compounds, i.e., forskolin, abiraterone, prochloraz, ketoconazole, trilostane, formestane, aminoglutethimide, fadrozole, etomidate, and metyrapone, known to affect steroidogenesis. After exposure, the levels of 9 natural steroids were determined by a quantitative targeted GC-MS/MS method and compared to a metabolomics method using Ultra Performance Liquid Chromatography-Time-of-Flight-Mass Spectrometry (UPLC-ToF-MS). Like the EIAs, both methods were suited for negative-positive screening, but the MS methods also generated specific fingerprints, allowing chemical class prediction of the compound under investigation. Although the targeted GC-MS/MS was more sensitive, which was an advantage regarding analysis of the estrogens 17β-estradiol and estrone, the untargeted UPLC-ToF-MS was able to evaluate effects on the synthesis of the corticosteroids. Moreover, untargeted comparison of the aligned chemical profiles allowed identification of all m/z-values that are differential between exposed and nonexposed H295R cells. In conclusion, application of a comprehensive metabolite profiling methodology not only provides a tool to screen compounds for steroidogenic modulating properties, but also allows chemical class prediction. As such, steroid profiling methodologies in conjunction with the H295R assay can contribute to the prioritization of chemicals for additional safety testing.

Kaolinic clay derived PCDD/Fs in the feed chain from a sorting process for potatoes

At the end of 2004, during a routine monitoring project, high levels of PCDDs in milk from two farms were found. Using a bioassay and the congener patterns obtained by HRGC/HRMS, the source was traced back to the use of kaolinic clay for sorting potatoes in a production process of French fries. Rest products, especially peelings after scrubbing, were used as feed for dairy cows. Levels of PCCD/Fs in this product amounted to 44 ng WHO(1998)-TEQ kg(-1) (88% dw). The maximum level observed in milk was 20 pg WHO(1998)-TEQ g(-1) fat. A Physiologically Based PharmacoKinetic (PB-PK) model was used to model three data obtained before eliminating the source in order to estimate the starting time of the contamination of the cows, the steady-state level after prolonged contamination and the kinetics of the decrease in the levels after removal of the source. Samples of milk were continuously collected for several months showing a decrease to levels below the product limit of 3 pg WHO(1998)-TEQ g(-1) fat within 2 months, in excellent agreement with the decrease predicted by the PB-PK model. Different batches of clay were sampled and analysed, showing varying levels of especially PCDDs. All clays were confirmed to be kaolinic clay using X-ray analysis. Other by-products used for animal feed were also contaminated and led to precautionary measures at a few hundred farms, especially pig farms. However, levels in other animal derived products like pig meat did not exceed the product limits.

The Need and Potential of Biosensors to Detect Dioxins and Dioxin-Like Polychlorinated Biphenyls along the Milk, Eggs and Meat Food Chain

Dioxins and dioxin-like polychlorinated biphenyls (DL-PCBs) are hazardous toxic, ubiquitous and persistent chemical compounds, which can enter the food chain and accumulate up to higher trophic levels. Their determination requires sophisticated methods, expensive facilities and instruments, well-trained personnel and expensive chemical reagents. Ideally, real-time monitoring using rapid detection methods should be applied to detect possible contamination along the food chain in order to prevent human exposure. Sensor technology may be promising in this respect. This review gives the state of the art for detecting possible contamination with dioxins and DL-PCBs along the food chain of animal-source foods. The main detection methods applied (i.e., high resolution gas-chromatography combined with high resolution mass-spectrometry (HRGC/HRMS) and the chemical activated luciferase gene expression method (CALUX bioassay)), each have their limitations. Biosensors for detecting dioxins and related compounds, although still under development, show potential to overcome these limitations. Immunosensors and biomimetic-based biosensors potentially offer increased selectivity and sensitivity for dioxin and DL-PCB detection, while whole cell-based biosensors present interpretable biological results. The main shortcoming of current biosensors, however, is their detection level: this may be insufficient as limits for dioxins and DL-PCBs for food and feedstuffs are in pg per gram level. In addition, these contaminants are normally present in fat, a difficult matrix for biosensor detection. Therefore, simple and efficient extraction and clean-up procedures are required which may enable biosensors to detect dioxins and DL-PCBs contamination along the food chain.

Levels of polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs) and dioxin-like PCBs in free range eggs from Vietnam, including potential health risks

Chicken and duck eggs collected from three different areas in Vietnam were examined for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs). These regions included a background area, an area sprayed with Agent Orange and the Bien Hoa airbase area where Agent Orange was handled by the US Army. The latter area now is inhabited and people keep their own laying hens. Egg samples were first screened with an in vitro reporter gene bioassay and a selection was analyzed by GC/HRMS. Samples from Bien Hoa airbase showed very high PCDD/F levels, up to 249 pg dioxin-equivalents (TEQ)/g fat, mainly due to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the sprayed areas, levels (3.2-8.2 pg TEQ g(-1)) were comparable to those observed in background areas (3.2-8.2 pg TEQ g(-1) fat). The estimated average consumption of 22 g d(-1) of the highly contaminated eggs will result in a 2-fold exceedance of the current exposure limits for adults and 5-fold for children, even without considering other contaminated food sources. This indicates a potential health risk from consumption of these highly contaminated eggs, which were not yet considered as a source for exposure to PCDD/Fs of people living in the highly contaminated areas.

Endocrine-Disrupting Effects of Thioxanthone Photoinitiators

Photoinitiators used in food packaging ink, such as 2-isopropylthioxanthone (2-ITX), have been shown to migrate into food and beverages. Recently, several studies indicated that 2-ITX might be an endocrine-disrupting chemical. In this work, the effects of 2-ITX, 4-isopropylthioxanthone (4-ITX), 2,4-diethylthio xanthone (2,4-diethyl-TX), 2-chlorothioxanthone (2-chloro-TX), and 1-chloro-4-propoxythioxanthone (1-chloro-4-propoxy-TX) on steroidogenesis and androgen and estrogen receptor-mediated transcription activation have been studied using human H295R adrenocarcinoma cells and yeast hormone bioassays, respectively. None of the compounds showed androgenic or estrogenic activities, but clear antiandrogenic and antiestrogenic activities were observed for 2-ITX, 4-ITX, and 2,4-diethyl-TX, whereas 2-chloro-TX showed only antiandrogenic activity. In an adapted version of the H295R steroidogenesis assay, using gas chromatography-tandem mass spectrometry analysis of H295R media, all five compounds increased levels of 17ß-estradiol and estrone. H295R cells incubated with 2-ITX also showed significantly reduced androgen and increased pregnenolone and progesterone levels. Expression of particular steroidogenic genes, including the one encoding for aromatase (CYP19A1), was significantly upregulated after incubation of H295R cells with 2-ITX, 4-ITX, and 2,4-diethyl-TX. In line with the increased CYP19A1 mRNA expression, 2-ITX increased catalytic activity of aromatase in H295R cells as measured by cognate aromatase assays. The results indicate that thioxanthone derivatives can act as potential endocrine disruptors both at the level of nuclear receptor signaling and steroid hormone production.