Impact of valproate on postimplantation embryos in an ex vivo model of rat development

Valproat (VPA), antiepileptički lijek/teratogen je također epigenetički inhibitor histonske deacetilaze (HDACi). Cilj disertacije bio je istražiti aktivnost VPA na gastrulaciju sisavaca u modelu ex vivo lišenog složenog majčinog utjecaja. Štakorski Fisher zameci stari 9,5 dana (tri zametna listića) uzgajali su se 14 dana, a ektoplacentalni konusi (podrijetlo buduće posteljice) 3 dana. Korišten je Eagle-ov MEM i serum štakora (1:1) s 1 mM ili 2 mM VPA. Pronađen je negativni utjecaj 2 mM valproata na preživljenje, rast, indeks proliferacije stanica i povećanu volumensku gustoću apoptototičnih stanica, zajedno sa značajno nižom učestalošću svih diferenciranih derivata triju zametnih listića (epidermisa, živčanog tkiva, hrskavice, mišića, crijevnog epitela) u teratomima koji se razvijaju tijekom kulture. 1 mM VPA manje je negativno utjecao na rast i na diferencijaciju živčanog tkiva. VPA je povećao acetilaciju histona H3AcK9, a da nije promijenio globalnu metilaciju DNA procijenjenu pirosekvenciranjem. Valproat je negativno utjecao na preživljenje i rast ektoplacentalnih konusa. U našem prirodnom 3D in vitro biološkom sustavu dokazana je embriotoksičnost ovisna o dozi, uzrokovana VPA-induciranom acetilacijom histona već pri gastrulaciji. Budući da je VPA istodobno negativno utjecao na razvoj embrionalnog teratoma, to bi bilo relevantno za novi terapijski pristup sličnim tumorima u ljudi.
Valproate (VPA), an antiepileptic drug/teratogen is also an epigenetic histone deacetylase inhibitor (HDACi). The aim of dissertation was to investigate VPA activity on mammalian gastrulation in the ex vivo model devoid of complex maternal influence. 9.5 day-old rat Fisher embryos (three germ layers) were cultivated for 14 days and ectoplacental cones (origin of future placenta) for 3 days. Eagle's MEM and rat serum (1:1) with 1 mM or 2 mM VPA was used. The negative influence of 2 mM valproate on survival, growth, cell proliferation index and increased volume density of apoptotic cells was assessed, together with a significantly lower incidence of all differentiated three germ layer derivatives (epidermis, neural tissue, cartilage, muscle, gut epithelium) in teratoma that develops during the culture. 1 mM VPA less negatively influenced growth and differentiation of nervous tissue alone. VPA increased H3AcK9 histone acetylation without altering global DNA methylation assessed by pyrosequencing. Valproate negatively affected survival and growth of ectoplacental cones. In our natural 3D in vitro biological system, a dose dependent embryotoxicity, caused by VPA-induced histone acetylation already at gastrulation, was demonstrated. Since VPA simultaneously negatively affected the development of embryonic teratoma, this would be relevant for a new therapeutic approach of similar tumors in humans.