Development of a new test for developmental toxicology with the aim to substantially decrease the number of living mammals which would be used for the same purpose. To this aim a multisystem biological approach was developed by testing at least 5 drugs at different concentrations within the range of human serum concentration in therpy.
Further experiments were carried out about gene expression under the effect of several drugs, essentially the same as described before and about the effect on the offspring.
These effects were found in all the systems which constitute our 'multisystem developmental toxicology test'.
a) sea urchin embryos
Prof. Giudice found by in situ hybridization that after treatment with valproate, which induces exogastrulation, the location in the zygote of the product of one maternal gene relevant the establishment of the animal-axis, like bep 1, is changed
b) Dictyostelium discoideum. It was established that the cellular slime mold Dictyostelium discoideum can be used as a model system to evaluate acute and developmental toxicity of chemicals and drugs. Cell growth, cell volume impaired chemotaxis delayed develompent and impaired expression of developmentally regulated genes were selected as endpoints. The system has been tested with valproic acid and teratogenic and non-teratogenic derivatives. It responds exactly as the well documented embryonic mouse model.
1) Prof. Polito has extended to methotrexate and diazepam the observation that intrabdominal injection of the above drugs into adult Drosophila transgenic phemales produces a change in the pattern of expression of the beta gal reporter gene in the ovaries.
NOC niclei oocytes; NNCEV nuclei nurse cells early vitellogenesis; NNCLV Nuclei nurse cells late vitellogenesis; Nuclei germarium cells; OOP ooplasm; NFC nuclei follicular cells. Minus and plus signs show the levels of beta-galactosidase.
The drug is given microinjecting the product to test into adult females of Drosophila. A volume of 400 nl a solution of different concentration was injected in adult fly abdomen for any drug to be tested and in the pictures is shown the amount of drug injected.
2) Prof. Morata found that intrabdominal injection of normal adult Drosophila females with Phenytoin produces aberration in 5-8% of larvae and of valproate in 10% of these. Valproate also induces increase of apoptosis expecially in the cephalic regions.
d) Yeast: Prof. Planta calculated the IC50 values for Doxorubicin, Dactinomycin, Valproate (VPA), 4-yn VPA and 2en VPA under standardized growth conditions, respiratory or fermentatives taking as end point the increase in number of cells. Clear effects were found for all the drugs tested and much lower effect for analogs known to be non toxic in humans, therefore stressing the sensitivity and specificity of the system for hydrosoluble drugs.
Attempts were made to make the system suitable also for hydrofobic drugs (like phenytoin), by adding to the culture medium 1% DMSO and 0,2% oleate. Under these conditions most hydrofobic drugs remain in solution in the medium.
e) Human keratinocytes: Dr. Pellegrini has tried the effect of thalidomide and phenotoin on these cultures and found no effect. Dosage of internal metabolites suggests an alternative pathway of these drugs in epidermis than in other tissues.
f) Drug dosage. Prof. Caccia has measured the concentration of the tested drugs and metabolites in ovaries and carcasses of Drosophila after injection in the fly abdomen. It was found that Drosophila metabolizes these drugs through metabolic pathways similar to those of mammals.
MAJOR SCIENTIFIC BREAKTHROUGHS:
It was confirmed that our 'multisystem approach' is suitable, as previously suggested, as an alternative to living mammals, for studies of developmental toxicology, for hydrosoluble drugs, and may be, with some modifications suggested by the last experiments, also for hydrofobic ones.
Funding SchemeCSC - Cost-sharing contracts
60325 Frankfurt Am Main
1081 HV Amsterdam