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Influence of the antioxidant PBN and teratogen 5-azacytidine on rat embryo development ex vivo

The influence of the antioxidant PBN (N-tert-butyl-a-phenylnitrone) and the DNA demethylating agent 5-azacytidine (5-azaC) on the most critical stage of mammalian development (gastrulation) was investigated ex vivo. Microsurgically isolated 9.5-day-old Fisher rat embryos were cultivated for two weeks at the air-liquid surface in Eagle's MEM with 5-azacytidine (5 μM) and/or PBN (22.6 µM) and controls in MEM or in MEM with 50% rat serum. Explant diameters were measured by an ocular micrometer at the beginning of culture and then every other day. Growth areas were determined in arbitrary units and data normalized to those obtained in MEM. 5-azaC impaired growth in comparison to MEM by approximately 40%. PBN applied with 5-azaC ameliorated growth of 5-azaC treated explants by approximately 25%, and in comparison to control grown in MEM by 25%, although it was less than in the medium with serum. Ex vivo in a chemically defined proteinless medium, it was possible to discover an ameliorating influence of PBN alone upon the embryo development, which was not possible before in the complicated in vivo system during gestation. These results are of importance for new therapeutic strategies in human medicine using antioxidants and epigenetic drugs.

PBN; 5-azacytidine; rat; in vitro culture; growth

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