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Introduction: The aim of the BMBF-project “Development of a biotransformating system for the metabolic activation of validated in-vitro-systems to assess embryotoxicity” is to further develop already established alternative methods for animal testing.
In this dissertation, embryotoxic substances and their metabolites (cyclophophamide/acrolein, retinol/all-transretinoic acid, valpromide/valproic acid, albendazole/albendazole sulfoxide, acetylaminofluorene/ N-hydroxy-acetylaminofluorene) have been tested in the WEC with rat embryos to simulate the maternal metabolism and to establish precise concentration-response-curves, followed by examination of substance combinations (in 80/20 and 50/50 ratios) on the embryo.
Substances and methods: First, the substances were tested at increasing concentrations und then combined with their associated metabolite as part of a 80/20 and 50/50 ratio, in the WEC with rat embryos (9,5 days old).
The embryos were incubated under defined conditions in a rotating system for 48 hours and the results were evaluated on the basis of a morphologic scoring system.
Results: The five examined substances (cyclophosphamide, retinol, valpromide, albendazole,
acetylaminofluorene) originate from different substance groups, however, they displayed similarities in the evoked malformations of rat embryos. With increasing concentrations, all substances led to a reduction of size, protein content, somites, and yolk sac vasculogenesis and a decrease in scoring parameters.
More specifically, substance-specific alterations were detected.
Mostly, the development of the telencephalon was affected. From a concentration of 200 μg/ml (716,5 μM) upward, cyclophosphamide led to the missing of the eye anlage and a stunted telencephalon. Retinol caused also alterations of the telencephalon (at 2,5 μg/ml / 8,73 μM): 66,6 % of the embryos had an open cranial neuroporus. At a concentration of 5,0 μg/ml (17,5 μM), 100 % of the embryos were abnorm, the telencephalon displayed an uneven surface and several hamorrhagies; the eye- and ear anlagen were completely missing. Albendazole caused an impared development of the telencephalon, but with a closed neural tube. Acetylaminofluorene (50 μg/ml bzw. 223,9 μM) led to the development
of placodes of the nose and open neural tubes. At a concentration of 200 μg/ml (895,7 μM) or higher, the telencephalon did not exist anymore, but placodes of the nose and hamorrhagies could be found in all rat embryos. The cranial neural tube was open.
Valpromide did not cause any microscopically visible alterations of the telencephalon. Characteristic for cyclophosphamide, valpromide and acetylaminofluorene was the incomplete rotation of the trunk, causing a “squirrel-like” position. Retinol and albendazole showed an
effect on the heart. At 2,5 μg/ml, retinol caused an incomplete amnion development, displaying microscopic malfunctions of the cardiac loop, and albendazole caused a malformation of the pericard. Characteristically, retinol also caused a complete adhesion of amnion and trunk at a concentration of 5 μg/ml (17,5 μM). At 2,5 μg/ml (8,7 μM) the composition of the hind limbs and tail was retarded. Valpromide did not induce specific or characteristic malformations.
At higher concentrations, the rat embryos did not completely rotate, and at the highest tested concentration, most embryos were malformed. The 80/20 and 50/50 combinations of most of the substances caused effects and were in agreement with the results observed with compounds when tested alone.
Discussion: Comparing the test compounds with their metabolites, alterations of the rat embryos were found to be mostly in the same body parts and similar to effects described in earlier publications. Most notably was the higher toxicity found among the retinol/all-transretinoic acid-combination in comparison to the testings of retinol and its metabolite alone. At the second concentration (0,83 μM) of 80/20 combination, open cranial neuropori and an incomplete rotation of the trunk was visible, while by testing retinol and its metabolite alone, statistically significant alterations occurred only at concentrations of 1,66 μM and higher. This effect was even more pronounced with the 50/50 combination: The first alterations (incomplete rotation and open neuropori) were already observed at levels of 0,33 μM (0,1 μg/ml).
Converse results, however, were found with albendazole/albendazole sulfoxide combination.
The metabolite was less toxic than the parent compound. As a result, both combinations displayed less pronounced effects than the parent substance alone.
Conclusion: The WEC with rat embryos is appropriate for analysing various substances in combination, thus simulating the situation induced by maternal metabolic activity.
The results will be an essential prerequisite for the development and integration of a metabolizing system
into the WEC.