Mapping the extracellular and membrane proteome associated with the vasculature and the stroma in the embryo.

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.

An alternative test battery in detecting ocular irritancy of agrochemicals.

Using animals in toxicological screening is a controversial issue. To get knowledge about eye irritation, recently only the in vivo Draize-test is accepted, which is one of the most criticized methods because of the injuries inflicted on the test animals. In recent years various in vitro methods have been developed to replace the primary irritation test. Most of these tests are in process of validation. The Draize rabbit eye test, or some modification of this test is essentially the only method for determining ocular irritation that is accepted by authorities. Several in vitro methods have been used to investigate the toxicity of potential eye irritants with the aim of replacing in vitro eye irritation testing. This study reports the results of an alternative approach for predicting irritation potential of agrochemicals. The approach was a two-stage test battery in vitro. The first stage was a cytotoxicity test, the MTT assay. The second stage was the HET-CAM test. The chick chorioallantoic membrane (CAM), being a connective tissue sheet with a visible blood supply, has been proposed as a substrate to identify the eye irritation potential of chemicals. During the HET-CAM test the chemicals are placed directly onto the chorioallantoic membrane. The changes of the vascular injury (haemorrhage, lysis or coagulation) are indications of the potential of the chemical to damage mucous membranes in vivo. MTT assay is a simple method to determine the viability of cells in the presence of a chemical. Cells are cultured with several concentrations of a substance or product, then the ratio of cell destruction is determined. LCC50 (concentration lethal for the 50 % of the cells) is assessed. This is in correlation of the eye irrigative potential of the chemical. In our studies comparative screening was performed with 6 agrochemicals to establish parallel data on alternative test battery (HET-CAM, MTT) and in vivo (Draize) results. In most cases, this study showed a good correlation between in vitro and in vivo data. By these results the present form of an alternative test battery (HET-CAM and MTT) can be proposed as a prescreen method of eye irritation tests.

The use of HET-CAM test in detecting the ocular irritation.

Agrochemicals must undergo numerous toxicological tests before registration. One of these experiments is the examination of eye irritation potential. To get knowledge about eye irritation, recently only the in vivo Draize-test is accepted, which is one of the most criticized methods because of the injuries inflicted on the test animals. Several in vitro methods have been used to investigate the toxicity of potential eye irritants with a view to replacing in vivo eye irritation testing. In the HET-CAM test chemicals are placed in direct contact with chorioallantoic membrane of the hen's egg. The occurrence of vascular injury or coagulation in response to a compound is the basis for employing this technique as an indication of the likelihood that a chemical would damage mucous membranes (especially the eye) in vivo. In our studies comparative screening was performed with a set of agrochemicals to establish paralell data on in vitro (HET-CAM) and in vivo (Draize) results in case of 6 agrochemicals. The solutions to be tested are added to the membrane and left in contact for 5 minutes and the membrane is examined for vascular damage at set time periods. Irritancy is scored according to the severity and speed at which damage occurs providing an indication of the likely irritant effect of the compound. Our study showed good correlation between results obtained by the HET-CAM test and those of the Draize rabbit eye test most cases. The present form of the HET-CAM test can be proposed as a pre-screen method of eye irritation tests.