Wim De Coen

Recent advances in recognition elements of food and environmental biosensors: A review

A sensitive monitoring of contaminants in food and environment, such as chemical compounds, toxins and pathogens, is essential to assess and avoid risks for both, human and environmental health. To accomplish this, there is a high need for sensitive, robust and cost-effective biosensors that make real time and in situ monitoring possible. Due to their high sensitivity, selectivity and versatility, affinity-based biosensors are interesting for monitoring contaminants in food and environment. Antibodies have long been the most popular affinity-based recognition elements, however recently a lot of research effort has been dedicated to the development of novel recognition elements with improved characteristics, like specificity, stability and cost-efficiency. This review discusses three of these innovative affinity-based recognition elements, namely, phages, nucleic acids and molecular imprinted polymers and gives an overview of biosensors for food and environmental applications where these novel affinity-based recognition elements are applied.

Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo

We developed and optimized a screening procedure, in which zebrafish embryos were explored as a model for the evaluation of the specific embryotoxic and teratogenic potential of chemicals. A selection of known positive (retinoic acid, valproic acid, caffeine, lithium chloride) and negative (glucose, saccharin) compounds for developmental toxicity were used to evaluate this method. We exposed embryos and evaluated embryotoxicity and morphological characteristics of the embryos at 24, 48, 72 and 144 h post fertilization. After evaluation of the induced effects, concentration-response curves were created for both embryotoxicity and teratogenic effects. Values for teratogenic indices (TI) were calculated as the ratio LC(50)/EC(50). The results obtained were compared to existing data from studies with laboratory animals and humans. We demonstrated that our classification of the compounds, based on TI values, allows to distinguish teratogens from non-teratogens and supports the application of zebrafish embryos as an alternative method for developmental toxicity studies to predict effects in mammals.

Perfluorinated Substances in Human Food and Other Sources of Human Exposure

The widespread distribution and degradation of PFCs in the environment results in a very complex exposure pattern, which makes it difficult to define the relative contribution to human exposure from different exposure pathways. The present review is designed to provide an overview of the existing data on levels of PFCs measured in the human diet and in drinking water. Data on levels of PFCs in the human diet are rather scarce, but the level in the fish appear to be well documented. Among PFCs, PFOS and PFOA are the best studied compounds in fish from both experimental and monitoring studies. Recently, the number of publications that address other PFCs has increased, but the total number available is still limited. In general, we discovered that care should be exercised when using the reviewed data, because, in the majority of publications, quality control and/or details on analysis are, at least partly, lacking. It has been well documented that PFOA and PFOS have the potential to accumulate in fish and concentrations up to 7 and 170 ng/g wwt, respectively in edible fish species have been found. PFOS is the most crucial and prominent compound identified, followed by the PFOA. Also, in aquatic invertebrate such as shrimps, mussels, clams, and oysters, high PFOS levels have been reported (up to 387 ng/g wwt). However in most publications PFC level reported in molluscs were less than 1 ng/g wwt. Positive correlations were found between PFC body burden and self reported fish consumption. In recognition of the potential for human exposure to PFCs via fish consumption, the Minnesota Department of Health has recently issued fish consumption advisories for contaminated sections of the Mississippi River. It is interesting to note that 79% of the reviewed publications on PFCs in the whole fish homogenates exceed the that threshold. Moreover, five of the PFC concentration reported in muscles tissue exceeded the advisory level of 38 ng/g wwt. Even though several authors concluded that consumption of contaminated food and drinking water constitutes the major exposure pathway for humans, only a few reports on PFCs in composite food exist. Food can be contaminated in an indirect way, because PFCs are widely used in food-packaging coatings and cooking materials. On the other hand, PFCs can also enter food organisms via environmental routes such as inhalation or adsorption from air. In a few studies, composite samples, duplicate diet samples, or other food items were analyzed for several PFCs, PFOS and PFOA, PFHpA, PFHxA, and PFHxS were meAsured and displayed concentrations ranging from-detected up to 15 ng/g wwt. In one study, a very high PFOA concentration of 118 ng/g were reported, but overall, PFC levels are below 10 ng/g wwt. It is important to note that, among all studies reviewed, PFCs were found in a maximum of 50% of the analyzed samples and generally only in 10% or less of samples analyzed. In contrast to what is observed in fish and other food items PFOA levels in drinking water (ND - 50 ng/L) and other PFCs (1-3 ng/L). In one study, extremely high values (519 ng/L) were measured in drinking water of a contaminated area in the Ruhr region. In Spain, bottled water was analyzed and four PFCs (PFOA, PFNA,PFDA and PFHpA) were found at low levels (<1 ng/L). Because of higher levels found in drinking water at several locations, some provisional drinking water guideline values for PFOS and PFOA have already been established, e.g., in the UK, Bavaria, and Minnesota. Since PFCs are present both in food and drinking water, Tolerable Daily Intake values for PFOS and PFOA have also been proposed by several institutes in Europe and in the USA. The ingestion of dust through hand-to-mouth transfer from indoor house dust can also be a potential source of PFC exposure, especially for toddlers and children. In publications on PFCs in indoor dust, the mean PFOS and PFOA levels varied between 39 and 1,200 ng/g and between 11 and 220 ng/g, respectively. Overall, it is clear that there is still lack of PFC exposure data for food and beverages, which renders the assessment of the contribution of the diet to total human PFC exposure uncertain. It is, therefore, appropriate that several scientific projects have recently been launched that addresses the assessment of human exposure to PFCs and related compounds from dietary sources.

Estrogen-Like Properties of Fluorotelomer Alcohols as Revealed by MCF-7 Breast Cancer Cell Proliferation

We investigated estrogen-like properties of five perfluorinated compounds using a combination of three in vitro assays. By means of an E-screen assay, we detected the proliferation-promoting capacity of the fluorotelomer alcohols 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH) and 1H,1H,2H,2H-perfluoro-decan-1-ol (8:2 FTOH). The more widely environmentally distributed compounds perfluoro-1-octane sulfonate, perfluorooctanoic acid, and perfluorononanoic acid did not seem to possess this hormone-dependent proliferation capacity. We investigated cell cycle dynamics using flow cytometric analyses of the DNA content of the nuclei of MCF-7 breast cancer cells. Exposure to both fluorotelomer alcohols stimulated resting MCF-7 cells to reenter the synthesis phase (S-phase) of the cell cycle. After only 24 hr of treatment, we observed significant increases in the percentage of cells in the S-phase. In order to further investigate the resemblance of the newly detected xenoestrogens to the reference compound 17β-estradiol (E2), gene expression of a number of estrogen-responsive genes was analyzed by real-time polymerase chain reaction. With E2, as well as 4-nonylphenol and the fluorotelomer alcohols, we observed up-regulation of trefoil factor 1, progesterone receptor, and PDZK1 and down-regulation of ERBB2 gene expression. We observed small but relevant up-regulation of the estrogen receptor as a consequence of exposures to 6:2 FTOH or 8:2 FTOH. The latter finding suggests an alternative mode of action of the fluorotelomer alcohols compared with that of E2. This study clearly underlines the need for future in vivo testing for specific endocrine-related end points.

Locomotor activity in zebrafish embryos: a new method to assess developmental neurotoxicity.

Currently, neurotoxicity testing defined by OECD and FDA is based solely on in vivo experiments, using large numbers of animals, being expensive, time-consuming and unsuitable for screening numerous chemicals. The great demand for thousands of chemicals yet to be evaluated, urges the development of alternative test methods which are cheaper, faster and highly predictive for developmental neurotoxicity. In this study, we developed a new method to assess locomotor activity in early life stage of zebrafish at 24 h post fertilization (hpf), in comparison to locomotor activity of zebrafish larvae at 96 to 192 hpf. We hypothesized that this endpoint at early life stages could be used to predict the developmental neurotoxic potential of chemicals and performed exposure studies with chlorpyrifos to demonstrate this. Furthermore, the case study with chlorpyrifos was used to critically evaluate behavioral data analysis and improve method sensitivity. The approach for data analysis using distribution plots for parameters on locomotor activity, next to mean values allowed to obtain more accurate information from the same set of behavioral data, both for embryos and larvae. Embryos exposed to chlorpyrifos, within the range 0.039 to 10 mg/l, exhibited a significant concentration-dependent increase in the frequency and total duration of their spontaneous tail coilings at 24-26 hpf. Larvae exhibited altered swimming activity, as evidenced by a significant decrease in the total duration of movement and an increase in mean turn angle in the range 0.18 to 0.75 mg/l chlorpyrifos. Methodological evaluation showed that locomotor effects in larvae were most pronounced and reproducible at 96 hpf, compared to older individuals (120, 144, 168 and 192 hpf). These new methods based on locomotor activity at early life stages of zebrafish allowed to classify chlorpyrifos as a developmental neurotoxicant. Further research to judge the validity of these alternative methods is currently performed with an extended set of expected positive or negative chemicals for developmental neurotoxicity.

A biology-based approach for mixture toxicity of multiple endpoints over the life cycle.

Typical approaches for analyzing mixture ecotoxicity data only provide a description of the data; they cannot explain observed interactions, nor explain why mixture effects can change in time and differ between endpoints. To improve our understanding of mixture toxicity we need to explore biology-based models. In this paper, we present an integrated approach to deal with the toxic effects of mixtures on growth, reproduction and survival, over the life cycle. Toxicokinetics is addressed with a one-compartment model, accounting for effects of growth. Each component of the mixture has its own toxicokinetics model, but all compounds share the effect of body size on uptake kinetics. The toxicodynamic component of the method is formed by an implementation of dynamic energy budget theory; a set of simple rules for metabolic organization that ensures conservation of mass and energy. Toxicant effects are treated as a disruption of regular metabolic processes such as an increase in maintenance costs. The various metabolic processes interact, which means that mixtures of compounds with certain mechanisms of action have to produce a response surface that deviates from standard models (such as ‘concentration addition’). Only by separating these physiological interactions from the chemical interactions between mixture components can we hope to achieve generality and a better understanding of mixture effects. For example, a biology-based approach allows for educated extrapolations to other mixtures, other species, and other exposure situations. We illustrate our method with the interpretation of partial life-cycle data for two polycyclic aromatic hydrocarbons in Daphnia magna.

Molecular targets of TBBPA in zebrafish analysed through integration of genomic and proteomic approaches

Tetrabromobisphenol-A (TBBPA) is nowadays one of the most frequently used brominated flame retardants (BFRs) and can be considered as a high production volume chemical. Over the last decade, numerous reports of increasing concentrations of BFRs in the environment and humans have been published. However, the toxicological knowledge on TBBPA, and more specifically its molecular mode of action, is rather fragmentary. In this study two populations of adult zebrafish (Danio rerio) were exposed for 14 days to 0.75 microM and 1.5 microM TBBPA. Subsequently, we employed a combined transcriptomic and proteomic approach to evaluate the molecular effects of TBBPA in zebrafish liver. Oligonucleotide microarrays were used to study the effects on gene expression levels. These results were validated through real-time PCR. The proteome of the liver was analysed by means of differential in-gel electrophoresis (DiGE), an innovative application of traditional 2D-PAGE. Combination of the extracted datasets allowed reassembling of individual molecular responses into a comprehensive overview of affected molecular pathways. Interpretation of the results depicted an interference of thyroid and Vitamin A homeostasis in the exposed zebrafish, TBBPA also elicited responses indicating onset of oxidative stress and general stress responses. Additionally, numerous differentially expressed transcripts could be associated with defence mechanisms or corresponded to metabolizing enzymes. Furthermore, cellular metabolism was clearly affected, illustrated as disturbance of e.g. lipid, carbohydrate, and organic acid metabolic processes. Summarizing, these results enabled us to hypothesize several working mechanisms of TBBPA and demonstrated the potential of a combined genome and proteome approach to generate detailed mechanistic toxicological information.

EXPOSURE PATTERNS OF PERFLUOROOCTANE SULFONATE IN AQUATIC INVERTEBRATES FROM THE WESTERN SCHELDT ESTUARY AND THE SOUTHERN NORTH SEA

Over the past decades little research has been conducted on the environmental behavior and effects of fluorinated organochemicals (FOCs). Recently it has been reported that perfluorooctane sulfonic acid (PFOS) is occurring worldwide. Little is known about the PFOS levels in organisms originating from the southern North Sea and the Western Scheldt estuary. In this study, we determined, for the first time, the PFOS-exposure levels in Crangon crangon, Carcinus maenas, and Asterias rubens from these ecosystems. Concentrations on a wet-weight basis in soft tissues of shrimp, crab, and starfish ranged from 19 to 520 ng/g, from 24 to 877 ng/g, and from 9 to 176 ng/g, respectively. These results show the existence of a PFOS pollution gradient in organisms along the Western Scheldt estuary, with the highest concentrations near Antwerp. The range of PFOS levels in shrimp and crab are slightly higher in coastal regions compared with sampling sites in open water. This study shows widespread distribution of PFOS in the Belgian and Dutch marine and estuarine environment at rather high concentrations.

An integrated transcriptomic and proteomic approach characterizing estrogenic and metabolic effects of 17 α-ethinylestradiol in zebrafish (Danio rerio)

Nowadays there is much concern about the presence of endocrine disrupting compounds (EDCs) in the environment due to their ability to interfere with the endocrine system. In the presented study, adult zebrafish (Danio rerio) were exposed to 30 ng L(-1) 17alpha-ethinylestradiol (EE2) for 4 and 28 days. The underlying molecular mechanisms of EE2 were studied in the zebrafish liver by applying a combined transcriptomics and proteomics approach. In addition, we assessed the added value of such an integrated-omics approach. Oligo microarrays, spotted with 3479 zebrafish-specific oligos, were employed to generate differential gene expression levels. The proteomic responses were evaluated by means of differential in-gel electrophoresis (DiGE), combined with MALDI-tandem mass spectrometry. Assessment of the major biological functions of the differentially expressed transcripts and proteins illustrated that both individual platforms could profile a clear estrogenic interference, next to numerous metabolism-related effects and stress responses. Cross-comparison of both transcriptomics and proteomics datasets displayed limited concordance, though, thorough revision of the results illustrated that transcriptional effects were projected on protein level as downstream effects of affected signalling pathways. Overall, this study demonstrated that a proteomics approach can lift the biological interpretation of microarrays to a higher level, and moreover, opens a window for identification of possible new biomarkers.

An omics based assessment of cadmium toxicity in the green alga Chlamydomonas reinhardtii

The effects of cadmium were assessed in the freshwater alga Chlamydomonas reinhardtii. Algae were exposed to concentrations of 0, 8.1 or 114.8 μM of cadmium and growth rates, gene transcription and metabolite profiles were examined after 48 and 72 h of exposure. In algae exposed to 8.1 μM Cd, several genes were differentially transcribed after 48 h but no adverse growth related effects were detected. A transient effect on both gene transcription patterns and metabolite profiles could be discerned after 48 h of exposure but the majority of these changes disappeared after 72 h. In contrast, all effects were more pronounced at the 114.8 μM cadmium exposure. Here growth was clearly reduced and transcription of a large number of genes involved in oxidative stress defense mechanisms was differentially increased. Metabolites involved in the glutathione synthesis pathway (an important antioxidant defense) were also affected but the effects of cadmium were found to be more pronounced at the transcript level than in the metabolome, suggesting that the former exhibits greater sensitivity toward cadmium exposure.