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83 Screening for the Activity of Histone Deacetylation Inhibitors in the Gastrulating Embryo-Derived Teratoma Biological System
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  1. Milvija Plazibat1,2,3,
  2. Ana Katušić Bojanac2,4,
  3. Marta Himerleich Perić2,4,
  4. Gordana Jurić-Lekić5,6,
  5. Katarina Raković4,7,
  6. Ozren Gamulin5,8,
  7. Škrabić Marko5,8,
  8. Maria Krajačić5,
  9. Jure Krasić2,4,
  10. Nino Sinčić2,4,
  11. Gordana Jurić-Lekić4,6,
  12. Floriana Bulic-Jakus2,4
  1. 1Hospital Zabok, Department of Pediatrics, Zabok, Croatia
  2. 2Centre of Excellence for Reproductive and Regenerative Medicine, Unit for Biomedical Investigation of Reproduction and Development, School of Medicine, University of Zagreb, Zagreb, Croatia
  3. 3University of Osijek, Faculty of Medicine, Dental Medicine and Health, Osijek, Croatia
  4. 4University of Zagreb, School of Medicine, Department of Medical Biology, Croatia
  5. 5University of Zagreb, School of Medicine, Department of Physics,Zagreb, Croatia
  6. 6University of Zagreb, School of Medicine, Department of Histology and Embryology
  7. 7Karolinska Institute, Department of Microbiology, Tumor and Cell Biology, Stockholm, Sweden
  8. 8Center of Excellence for Advanced Materials and Sensing Devices, Research Unit New Functional Materials, Zagreb, Croatia

Abstract

In our in vitro natural 3D biological system, rodent embryos explanted at the time of formation of the three germ-layers (gastrulation), develop a teratoma-like structure. We present the results of screening for the activity of Histone Deacetylation Inhibitors (HDIs) in this biological system.

9.5-days-old Fisher rat embryos-proper were cultivated for 14 days in Eagle’s MEM and 50% rat serum with 2mM or 1mM valproate or trichostatin A

(66 nM) at the air-liquid interface. Histone acetylation was assessed by western blotting and apoptosis and cell proliferation by immunohistochemistry and stereology. Spent media metabolomes were analyzed by IR-spectroscopy (FTIR).

Valproate and TSA both negatively influenced growth of explants and differentiation of the neural tissue. Valproate 2mM significanty increased histone acetylation and apoptosis in explants. In spent media metabolomes the amide I α-helices and the elevated ratio of A(CH3)/A(CH2, as biomarkers for histone acetylation, and a higher intensity of the 1625 cm-1 absorption band for the parallel β-strand structure and CH2 vibrations of lipids at

2853 cm-1, as biomarkers of apoptosis, were assessed.

Our in vitro biological screening system detected embryotoxic/anti-tumor impact of both HDIs. FTIR spectroscopy was able to discern biomarkers of histone acetylation and apoptosis in spent media metabolomes, thus upgrading our biological in vitro system for faster screening of embryotoxic/antitumor agents.

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