Jef Hooyberghs

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 cell-based in vitro alternative to identify skin sensitizers by gene expression

The ethical and economic burden associated with animal testing for assessment of skin sensitization has triggered intensive research effort towards development and validation of alternative methods. In addition, new legislation on the registration and use of cosmetics and chemicals promote the use of suitable alternatives for hazard assessment. Our previous studies demonstrated that human CD34(+) progenitor-derived dendritic cells from cord blood express specific gene profiles upon exposure to low molecular weight sensitizing chemicals. This paper presents a classification model based on this cell type which is successful in discriminating sensitizing chemicals from non-sensitizing chemicals based on transcriptome analysis of 13 genes. Expression profiles of a set of 10 sensitizers and 11 non-sensitizers were analyzed by RT-PCR using 9 different exposure conditions and a total of 73 donor samples. Based on these data a predictive dichotomous classifier for skin sensitizers has been constructed, which is referred to as VITOSENS. In a first step the dimensionality of the input data was reduced by selectively rejecting a number of exposure conditions and genes. Next, the generalization of a linear classifier was evaluated by a cross-validation which resulted in a prediction performance with a concordance of 89%, a specificity of 97% and a sensitivity of 82%. These results show that the present model may be a useful human in vitro alternative for further use in a test strategy towards the reduction of animal use for skin sensitization.

A new approach to study exhaled proteins as potential biomarkers for asthma

Cite this as: K. Bloemen, R. Van Den Heuvel, E. Govarts, J. Hooyberghs, V. Nelen, E. Witters, K. Desager and G. Schoeters, Clinical & Experimental Allergy, 2011 (41) 346–356.

Assessment of the developmental neurotoxicity of compounds by measuring locomotor activity in zebrafish embryos and larvae.

The developmental neurotoxic potential of the majority of environmental chemicals and drugs is currently undetermined. Specific in vivo studies provide useful data for hazard assessment but are not amenable to screen thousands of untested compounds. In this study, methods which use zebrafish embryos, eleutheroembryos and larvae as model organisms, were proposed as alternatives for developmental neurotoxicity (DNT) testing. The evaluation of spontaneous tail coilings in zebrafish embryos aged 24-26 hours post fertilization (hpf) and the swimming activity of eleutheroembryos at 120 and larvae at 144 hpf, i.e. parameters for locomotor activity, were investigated as potential endpoints for DNT testing, according to available standard protocols. The overall performance and predictive value of these methods was then examined by testing a training set of 10 compounds, including known developmental neurotoxicants and compounds not considered to be neurotoxic. The classification of the selected compounds as either neurotoxic or non-neurotoxic, based on the effects observed in zebrafish embryos and larvae, was compared to available mammalian data and an overall concordance of 90% was achieved. Furthermore, the specificity of the selected endpoints for DNT was evaluated as well as the potential similarities between zebrafish and mammals with regard to mechanisms of action for the selected compounds. Although further studies, including the screening of a large testing set of compounds are required, we suggest that the proposed methods with zebrafish embryos and larvae might be valuable alternatives for animal testing for the screening and prioritization of compounds for DNT.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters

Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching (PM) complementary sequence and other spots with one or two mismatches (MM) : in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays.

Strong disorder fixed point in absorbing-state phase transitions.

The effect of quenched disorder on nonequilibrium phase transitions in the directed percolation universality class is studied by a strong disorder renormalization group approach and by density matrix renormalization group calculations. We show that for sufficiently strong disorder the critical behavior is controlled by a strong disorder fixed point and in one dimension the critical exponents are conjectured to be exact: beta=(3-sqrt[5])/2 and nu( perpendicular )=2. For disorder strengths outside the attractive region of this fixed point, disorder dependent critical exponents are detected. Existing numerical results in two dimensions can be interpreted within a similar scenario.

Spatial interpolation of ambient ozone concentrations from sparse monitoring points in Belgium

Due to scientific interest on the one hand and political and regulatory obligations on the other hand the monitoring of ozone in the troposphere is an important issue. To this end, in Belgium as in many other countries, a fixed network of monitoring stations is operated. In order to estimate the ozone concentrations over the whole territory, a model is needed to spatially complement the sparse measurements. This paper describes the development of an interpolation scheme which is aimed at fast operational use. The model uses the population density as auxiliary data to remove a spatial trend due to titration by nitric oxide. The residuals are interpolated by kriging. As a benchmark the inverse distance weighting interpolation method is used with and without the detrending. The proposed model systematically improves the interpolation and makes a significant difference when estimating human exposure to ozone. It is generic in design, easy to implement and flexible to changes in the monitoring network.

Assessment of Chemical Skin-Sensitizing Potency by an In Vitro Assay Based on Human Dendritic Cells

The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34(+) progenitor-derived dendritic cells (CD34-DC), which allows to classify chemicals as skin (non-)sensitizing. However, skin sensitization is not an all-or-none phenomenon, and up to now, the assessment of relative potency can only be derived using the in vivo local lymph node assay (LLNA). In our study, we analyzed the feasibility to predict the sensitizing potency, i.e., the LLNA EC3 values, of 15 skin sensitizers using in vitro data from the CD34-DC-based assay. Hereto, we extended the in vitro-generated gene expression data set by an additional source of information, the concentration of the compound that causes 20% cell damage (IC20) in CD34-DC. We statistically confirmed that this IC20 is linearly independent from the gene expression changes but that it does correlate with LLNA EC3 values. In a further analysis, we applied a robust linear regression with both IC20 and expression changes of CREM and CCR2 as explanatory variables. For 13 out of 15 compounds, a high linear correlation was established between the in vitro model and the LLNA EC3 values over a range of four orders of magnitude, i.e., from weak to extreme sensitizers.

Gene profiles of a human alveolar epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis.

There are currently no accepted biological prediction models for assessing the potential of a substance to cause respiratory sensitization. New tests should be based on mechanistic understanding and should be preferentially restricted to in vitro assays. The major goal of this study was to investigate the alterations in gene expression of human alveolar epithelial (A549) cells after exposure to respiratory sensitizing and non-respiratory sensitizing chemicals, and to identify genes that are able to discriminate between both groups of chemicals. A549 cells were exposed during 6, 10, and 24 h to the respiratory sensitizers ammonium hexachloroplatinate IV, hexamethylene diisocyanate, and trimellitic anhydride, the irritants acrolein and methyl salicylate, and the skin sensitizer 1-chloro-2,4-dinitrobenzene. Overall changes in gene expression were evaluated using Agilent Whole Human Genome 4x44K oligonucleotide arrays. A Fisher linear discriminant analysis was used to obtain a ranking of genes that reflects their potential to discriminate between respiratory sensitizing and respiratory non-sensitizing chemicals. Among the 20 most discriminating genes, which were categorized into molecular and biological gene ontology (GO) terms, CTLA4 could be associated with asthma and/or respiratory sensitization. When categorizing the top-1000 genes into biological GO terms, 22 genes were associated with immune function. Using a pathway analysis tool to identify possible underlying mechanisms of respiratory sensitization, no known canonical signaling pathway was observed to be activated in the A549 cell line.