The identification of cellular targets of 17ß estradiol using a lytic (T7) cDNA phage display approach.

To unravel the mechanism of action of chemical compounds, it is crucial to know their cellular targets. A novel in vitro tool that can be used as a fast, simple and cost effective alternative is cDNA phage display. This tool is used in our study to select cellular targets of 17ß estradiol (E2). It was possible to select two potential cellular targets of E2 out of the T7 Select™ Human Breast cDNA phage library. The selected cellular targets, autophagy/beclin-1 regulator 1 (beclin 1) and ATP synthase F(0) subunit 6 (ATP6) have so far been unknown as binding proteins of E2. To confirm the E2 binding properties of these selected proteins, surface plasmon resonance (SPR) was used. With SPR the K(d) values were determined to be 0.178±0.031 and 0.401±0.142 nM for the ATP6 phage and beclin 1 phage, respectively. These K(d) values in the low nM range verify that the selected cellular proteins are indeed binding proteins for E2. The selection and identification of these two potential cellular targets of E2, can enhance our current understanding of its mechanism of action. This illustrates the potential of lytic (T7) cDNA phage display in toxicology, to provide important information about cellular targets of chemical compounds.

cDNA phage display as a novel tool to screen for cellular targets of chemical compounds.

cDNA phage display is frequently used in drug development to screen for cellular target of drugs. However, in toxicology, cDNA phage display remains unexplored, although it has large potential in this field. In this study, cDNA phage display is demonstrated as a novel tool to screen for interactions between chemical compounds and cellular targets. The knowledge of these target interactions is valuable to have a more complete understanding of the mechanisms of action of chemical compounds. Bisphenol A (BPA) was selected as a model compound for this study. By selection of the cellular proteins that bind BPA with cDNA phage display, it was possible to identify a known cellular target of BPA, tubulin alpha and a possible novel cellular target of BPA, transforming acidic coiled-coil containing protein 3. Both these cellular proteins are involved in the mechanism of cell division. The disruption of cell division is a known non-genomic effect of BPA. Non-genomic effects are not mediated by differences in gene expression and therefore important mechanistic information might be missed with the widely used differential gene expression techniques for mode of action research. This cDNA phage display technique can provide important additional information about the interaction of chemical compounds with cellular targets that mediates these non-genomic actions and therefore gives complementary information to toxicogenomic studies to obtain a more complete understanding of the mechanism of action of chemical compounds.

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.

Alterations in the energy budget of Arctic benthic species exposed to oil-related compounds.

We studied cellular energy allocation (CEA) in three Arctic benthic species (Gammarus setosus (Amphipoda), Onisimus litoralis (Amphipoda), and Liocyma fluctuosa (Bivalvia)) exposed to oil-related compounds. The CEA biomarker measures the energy budget of organisms by biochemically assessing changes in energy available (carbohydrates, protein and lipid content) and the integrated energy consumption (electron transport system activity (ETS) as the cellular aspect of respiration). Energy budget was measured in organisms subjected to water-accommodated fraction (WAF) of crude oil and drill cuttings (DC) to evaluate whether these compounds affect the energy metabolism of the test species. We observed significantly lower CEA values and higher ETS activity in G. setosus subjected to WAF treatment compared to controls (por=0.19). Different responses to oil-related compounds between the three test species are likely the result of differences in feeding and burrowing behavior and species-specific sensitivity to petroleum-related compounds.