engleski

Valproate enhances apoptosis of gastrulating mammalian embryo cultivated in vitro

Valproate is a known antiepileptic responsible for the valproate syndrome (FVS) in children whose mothers have used it during pregnancy (1). In 2014 The European Medicines Agency warned against the use of valproate in treating ep- ilepsy but also some other illnesses such as e.g. the migraine (2). A recent study has described 29 cases of FVS diagnosed in Ireland from 1995-2016. Among malformations were those of the neural tube, heart, limbs, craniofacial etc. (2). However, all effects are still not completely explained at the molecular level (3, 4). Today it is clear that anomalies of development of specific organs are in- duced at the specific time-windows or critical phases that correlate with the phases of organ development (5). On the basis of his experiments with various teratogenic agents, the founder of the Zagreb School of Mammalian Embryology academician N. Škreb (5) has proposed an “all or none” hypothesis regarding the early postimplantation development at the Pallanza conference already in 1960 (6). Before the formation of the mesoderm, the young mammalian embryo is either destroyed by the teratogen or recovers completely, and therefore these early stages are not susceptible to teratogens (5). The formation of the mesoderm and the three definitive germ-layers takes place at gastrulation. Therefore, gas- trulation is the first and the most critical phase of mammalian development where the early embryo loses its plasticity. At that stage the „all or none rule” is lost and the influence of a teratogenic agent may be visible after birth (5, 6, 7). In this study we aimed to investigate the direct effect of valproate on the development of the gastrulating rat embryo (embryo proper) devoid of its extra- embryonic parts and outside of the maternal environment. 198 Rad 533. Medical Sciences, 45 (2018) : 179-200 M. Belicza: Abstracts Fischer rat embryos were microsurgically isolated and grown in the original organ-culture model with the Eagle’s Minimum Essential Medium and 50% rat serum (controls). Valproate (2mM or 1mM) was added to the culture medium and cultures were grown for 3 days or 2 weeks with 5% CO2 in ahumidified incubator. We used immunohistochemistry to detect cleaved caspase 3 as the marker of apoptosis and the Proliferating Cell Nuclear Antigen as the marker of proliferation. Stereology was used to calculate the volume density (Vv ) of the apoptotic marker and proliferation index was calculated. Growth was measured by an ocular micrometer. Acetylated histone expression was detected by the Western blot. By measuring overall growth, from the third day onwards, a significantly lower growth was discovered in cultures with valproate that was also lower in embryos treated with the higher dose. After three days of culture, in embryos treated with the higher dose of valproate, a significantly higher apoptotic activ- ity (Vv of cleaved caspase-3) was discovered than in controls and in embryos treated with the lower dose of valproate (Figure 1). After two weeks in culture, the proliferation index (PCNA index) was significantly lower in embryos treated with a higher dose of valproate. Higher dose also inhibited differentiation, espe- cially of the nervous tissue. Higher expression of the acetylated histone H3 was discovered in embryos treated with valproate. It can be concluded that valproate negatively affects developmental process- es already at the gastrulation stage, and its impact is dose-dependent. Apoptosis enhanced by the higher dose (2mM) may be connected to the enhanced histone acetylation caused by valproate that is an epigenetic drug and inhibitor of the histone deacetylase (3).

apoptosis ; valproate ; embryo ; gastrulation ; in vitro

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