Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro

Mužić, Vedrana and Katušić Bojanac, Ana and Jurić-Lekić, Gordana and Himelreich, Marta and Tupek, Katarina and Šerman, Ljiljana and Marn, Nina and Sinčić, Nino and Vlahović, Maja and Bulić-Jakuš, Floriana (2013) Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro. Croatian Medical Journal, 54 (5). pp. 489-495. ISSN 0353-9504

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AIM: To establish an organotypic in vitro model of limb bud development to verify whether epigenetic drug and teratogen 5-azacytidine (5azaC) has an effect on limb buds independent of its effects on the placenta. ----- METHODS: Fischer strain rat fore- and hindlimb buds were microsurgically isolated from 13 days old embryos and cultivated in vitro for two weeks at the air-liquid interface in Eagle's minimum essential medium (MEM) with 50% rat serum. 30 μmol of 5azaC was added to the fresh medium. Overall growth was measured by an ocular micrometer. Routine histology, immunohistochemical detection of the proliferating cell nuclear antigen (PCNA), and stereological quantification of PCNA expression were performed. ----- RESULTS: At four time points, significantly lower overall growth was detected for fore- and hindlimb bud explants cultivated with 5azaC in comparison to controls. After the culture period, numerical density of the PCNA signal for both types of limb buds was lower than for controls (P<0.001). Limb buds were initially covered by immature epithelium and contained mesenchyme, myotubes, single hemangioblasts, hemangioblast aggregates, blood islands, and capillaries. Regardless of the treatment, cartilage and epidermis differentiated, but cells and structures typical for vasculogenesis disappeared. ----- CONCLUSION: Our findings, obtained outside of the maternal organism, stress the importance of compromised cell proliferation for 5azaC impact on limb buds. This investigation points to the necessity to establish alternatives to in vivo research on animals using teratogenic agents.

Item Type: Article
MeSH: Animals ; Azacitidine/pharmacology ; Cell Differentiation/drug effects ; Cell Proliferation/drug effects ; Epigenomics ; Female ; Humans ; Limb Buds/cytology ; Limb Buds/drug effects ; Limb Buds/growth & development ; Organ Culture Techniques ; Pregnancy ; Proliferating Cell Nuclear Antigen/analysis ; Rats ; Rats, Inbred F344 ; Teratogens/pharmacology
Departments: Katedra za histologiju i embriologiju
Katedra za medicinsku biologiju
Depositing User: Marijan Šember
Status: Published
Katušić Bojanac, AnaUNSPECIFIED
Jurić-Lekić, GordanaUNSPECIFIED
Himelreich, MartaUNSPECIFIED
Tupek, KatarinaUNSPECIFIED
Šerman, LjiljanaUNSPECIFIED
Bulić-Jakuš, FlorianaUNSPECIFIED
Date: 28 October 2013
Date Deposited: 19 Sep 2014 09:42
Last Modified: 13 Jul 2020 11:31
Subjects: /
Related URLs:
URI: http://medlib.mef.hr/id/eprint/2136

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