Corine J. Houtman

Toxicological profiling of sediments using in vitro bioassays, with emphasis on endocrine disruption

In vitro bioassays are valuable tools for screening environmental samples for the presence of bioactive (e.g., endocrine-disrupting) compounds. They can be used to direct chemical analysis of active compounds in toxicity identification and evaluation (TIE) approaches. In the present study, five in vitro bioassays were used to profile toxic potencies in sediments, with emphasis on endocrine disruption. Nonpolar total and acid-treated stable extracts of sediments from 15 locations in the Rhine Meuse estuary area in The Netherlands were assessed. Dioxin-like and estrogenic activities (using dioxin-responsive chemical-activated luciferase gene expression [DR-CALUX] and estrogen-responsive chemical-activated luciferase gene expression [ER-CALUX] assays) as well as genotoxicity (UMU test) and nonspecific toxic potency (Vibrio fischeri assay) were observed in sediment extracts. For the first time, to our knowledge, in vitro displacement of thyroid hormone thyroxine (T4) from the thyroid hormone transport protein thransthyretin by sediment extracts was observed, indicating the presence of compounds potentially able to disrupt T4 plasma transport processes. Antiestrogenic activity was also observed in sediment. The present study showed the occurrence of endocrine-disrupting potencies in sediments from the Dutch delta and the suitability of the ER- and DR-CALUX bioassays to direct endocrine-disruption TIE studies.

Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays

Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical-analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC-MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical-analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical-analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes.

Human health risk assessment of the mixture of pharmaceuticals in Dutch drinking water and its sources based on frequent monitoring data.

The presence of pharmaceuticals in drinking water is a topic of concern. Previous risk assessments indicate that their low concentrations are very unlikely to pose risks to human health, however often conclusions had to be based on small datasets and mixture effects were not included. The objectives of this study were to a) investigate if pharmaceuticals in surface and polder water penetrate in drinking water, b) assess the lifelong exposure of consumers to pharmaceuticals via drinking water and c) assess the possible individual and mixture health risks associated with this exposure. To fulfill these aims, a 2-year set of 4-weekly monitoring data of pharmaceuticals was used from three drinking water production plants. The 42 pharmaceuticals that were monitored were selected according to their consumption volume, earlier detection, toxicity and representation of the most relevant therapeutic classes. Lifelong exposures were calculated from concentrations and compared with therapeutic doses. Health risks were assessed by benchmarking concentrations with provisional guideline values. Combined risks of mixtures of pharmaceuticals were estimated using the concept of Concentration Addition. The lifelong exposure to pharmaceuticals via drinking water was calculated to be extremely low, i.e. a few mg, in total corresponding to <10% of the dose a patient is administered on one day. The risk of adverse health effects appeared to be negligibly low. Application of Concentration Addition confirmed this for the mixture of pharmaceuticals simultaneously present. The investigated treatment plants appeared to reduce the (already negligible) risk up to 80%. The large available monitoring dataset enabled the performance of a realistic risk assessment. It showed that working with maximum instead of average concentrations may overestimate the risk considerably.

Biomonitoring of estrogenic exposure and identification of responsible compounds in bream from Dutch surface waters.

The exposure to and effects of estrogenic compounds in male breams from Dutch freshwater locations were investigated. Ovotestis was observed infrequently (maximum frequency 16%). However, plasma vitellogenin (VTG) concentration was elevated highly at some locations. Estrogenic activities in male bream plasma, liver, and in gastrointestinal content were measured in the estrogen-responsive chemical-activated luciferase gene expression (ER-CALUX) assay. Plasma concentrations of vitellogenin correlated very well with the estrogenic activities in gastrointestinal content. The ER-CALUX activity in gastrointestinal content thus could provide a biomarker for recent exposure to estrogenic compounds, and the gastrointestinal content was chosen as investigative matrix for the toxicity identification and evaluation ([TIE]; bioassay-directed fractionation) of estrogenic compounds in bream. The approach consisted of a reversed-phase high-performance liquid chromatography fractionation of gastrointestinal content extract, directed by ER-CALUX and followed by gas chromatography analysis. The estrogenic hormones 17beta-estradiol and its metabolite estrone were identified as major contributors to the activity at all locations (except the reference location), independent of the presence or absence of a known source of estrogenic activity, such as a sewage treatment plant. Chemical screening showed the presence of other pollutants, such as a lower chlorinated dioxin and the disinfectants clorophene and triclosan. However, these compounds did not have high estrogenic potencies and their concentrations were not high enough to contribute significantly to the observed estrogenic activity.

Effect-Directed Analysis of Municipal Landfill Soil Reveals Novel Developmental Toxicants in the Zebrafish Danio rerio

Effect-directed analysis (EDA) is an approach used to identify (unknown) contaminants in complex samples which cause toxicity, using a combination of biology and chemistry. The goal of this work was to apply EDA to identify developmental toxicants in soil samples collected from a former municipal landfill site. Soil samples were extracted, fractionated, and tested for developmental effects with an embryotoxicity assay in the zebrafish Danio rerio. Gas chromatograph mass selective detection (GC-MSD) chemical screening was used to reveal candidate developmental toxicants in fractions showing effects. In a parallel study, liquid chromatography-hybrid linear ion trap Orbitrap mass spectrometry was also applied to one polar subfraction (Hoogenboom et al. J. Chromatogr. A2009, 1216, 510-519). EDA resulted in the identification of a number of previously unknown developmental toxicants, which were confirmed to be present in soil by GC-MS. These included 11H-benzo[b]fluorene, 9-methylacridine, 4-azapyrene, and 2-phenylquinoline, as well as one known developmental toxicant (retene). This work revealed the presence of novel contaminants in the environment that may affect vertebrate development, which are not subject to monitoring or regulation under current soil quality assessment guidelines.

Rapid activity-directed screening of estrogens by parallel coupling of liquid chromatography with a functional gene reporter assay and mass spectrometry

In this study we developed a new LC nanofractionation platform that combines a human cell (BG1.Luc) gene reporter assay with a high resolution mass spectrometer for the detection and identification of estrogenic and anti-estrogenic compounds in environmental waters. The selection of this assay was based on its high sensitivity and selectivity, which is required for environmental trace level detection. We modified an autosampler and controlled it with in-house developed software to collect fractions in the low second range in microtiter plates. This ensured that chromatographic separation was maintained and allowed straightforward hyphenation with the bioassay. After bioassay testing, bioassay chromatograms were reconstructed and directly correlated with MS chromatograms that were obtained in parallel. This enabled to pinpoint bioactives in the MS chromatogram within a single fractionation cycle and results in a significant increase in throughput compared to traditional EDA studies. The sensitivity of the platform was low enough for environmental waters (80nM for bisphenol A and 320pM and 3.2nM for estradiol and estriol, respectively). In addition, the ability of the platform to detect anti-estrogens was successfully demonstrated as well. Finally, real samples were analysed.

A multicomponent snapshot of pharmaceuticals and pesticides in the river Meuse basin

The river Meuse serves as a drinking-water source for more than 6 million people in France, Belgium, and The Netherlands. Pharmaceuticals and pesticides, both designed to be biologically active, are important classes of contaminants present in this river. The variation in the presence of pharmaceuticals in time and space in the Dutch part of the Meuse was studied using a multicomponent analytical method for pharmaceuticals combined with univariate and multivariate statistical analyses of the results. Trends and variation in time in the presence of pharmaceuticals were investigated in a dead-end side stream of the Meuse that serves as an intake point for the production of drinking water, and 93% of the selected compounds were detected. Highest concentrations were found for the antidiabetic metformin. Furthermore, a spatial snapshot of the presence of pharmaceuticals and pesticides was made along the river Meuse. Principal component analysis was successfully applied to reveal that wastewater-treatment plant effluent and water composition at the Belgian border were the main factors determining which compounds are found at different locations. The Dutch part of the river basin appeared responsible for approximately one-half of the loads of pharmaceuticals and pesticides discharged by the Meuse into the North Sea. The present study showed that multicomponent monitoring in combination with principal component analysis is a powerful tool to provide insight into contamination patterns in surface waters.

Development of a luminescent mutagenicity test for high-throughput screening of aquatic samples

The Salmonella reversion based Ames test is the most widely used method for mutagenicity testing. For rapid toxicity assessment of e.g. water samples and for effect-directed analysis, however, the Ames test suffers from lack of throughput and is regarded as a laborious, time consuming method. To achieve faster analysis, with increased throughput, a (downscaled) luminescent derivative of the Ames Salmonella/microsome fluctuation test has been developed through expression of the Photorhabdus luminescens luciferase in the Salmonella TA98 and TA100 strains. The applicability of this test is demonstrated by analysis of environmentally relevant compounds, a suspended particulate matter extract and an industrial effluent sample. Use of the luminescent reporter reduced the required detection time from 48 to 28h with a specificity of 84% for responses reported in the literature to a set of 14 mutagens as compared to 72% in the unmodified fluctuation test. Testing of the same compounds in a downscaled luminescent format resulted in an 88% similarity with the response found in the regular luminescent format. The increase in throughput, faster analysis and potential for real-time bacterial quantification that luminescence provides, allows future application in the high-throughput screening of large numbers of samples or sample fractions, as required in effect-directed analysis in order to accelerate the identification of (novel) mutagens.

Steroid hormonal bioactivities, culprit natural and synthetic hormones and other emerging contaminants in waste water measured using bioassays and UPLC-tQ-MS

Emission of compounds with biological activities from waste water treatment plant (WWTP) effluents into surface waters is a topic of concern for ecology and drinking water quality. We investigated the occurrence of hormone-like activities in waste water sample extracts from four Dutch WWTPs and pursued to identify compounds responsible for them. To this aim, in vitro reporter gene bioassays for androgenic, anti-androgenic, estrogenic, glucocorticoid and progestogenic activity and a UPLC-tQ-MS target analysis method for 25 steroid hormones used in high volumes in pharmacy were applied. Principal component analysis of the data was performed to further characterize the detected activities and compounds. All five types of activities tested were observed in the WWTP samples. Androgenic and estrogenic activities were almost completely removed during WW treatment, anti-androgenic activity was only found in treated WW. Glucocorticoid and progestogenic activities persisted throughout the treatment. The androgenic activity in both influent could predominantly be attributed to the presence of androstenedione and testosterone. Anti-androgenic activity was explained by the presence of cyproterone acetate. The glucocorticoid activity in influent was fully explained by prednicarbate, triamcinolone acetonide, dexamethasone and amcinonide. In effluent however, detected hormones could only explain 10-32% of the activity, indicating the presence of unknown glucocorticoids or their metabolites in effluent. Progesterone and levonorgestrel could explain the observed progestogenic activity. The principle component analysis confirmed the way in which hormones fit in the spectrum of other emerging contaminants concerning occurrence and fate in WWTPs.

Effect-Directed Analysis of Endocrine Disruptors in Aquatic Ecosystems

The topic of endocrine disruption in the aquatic environment is a clear example of a problem-driven research area. Field observations of endocrine abnormalities in wild life have prompted the growth of scientific attention and concern about the topic. Multiple studies have reported the presence of endocrine disrupting activities in various compartments of the aquatic environment, without, at the time, knowing the cause of the observations. The application of effect-directed analysis (EDA) has shown to be a valuable approach in investigating the nature of the compounds responsible for endocrine disrupting activities in environmental samples. Various research groups have applied EDA approaches and thereby successfully identified compounds responsible for endocrine disrupting effects. The research field of endocrine disruption is thus one of the research areas that has extensively experienced the benefits of EDA. This chapter describes the issue of endocrine disruption in the aquatic environment and discusses examples of the application of EDA for the identification of responsible compounds.