Ionizing radiation-induced gene modulations, cytokine content changes and telomere shortening in mouse fetuses exhibiting forelimb defects

Dev Biol. 2008 Oct 15;322(2):302-13. doi: 10.1016/j.ydbio.2008.07.032. Epub 2008 Aug 7.

Abstract

Several lines of evidence have linked limb teratogenesis to radiation-induced apoptosis and to the p53 status in murine fetuses. In previous reports, we studied the occurrence of various malformations after intrauterine irradiation and showed that these malformations were modulated by p53-deficiency as well as by the developmental stage at which embryos were irradiated. In this new study, we focused onto one particular phenotype namely forelimb defects to further unravel the cellular and molecular mechanisms underlying this malformation. We measured various parameters expected to be directly or indirectly influenced by irradiation damage. The mouse fetuses were irradiated at day 12 p.c. (post conception) and examined for forelimb defects on gestational days 15, 16, 17 and 19 of development. The release of inflammatory cytokines was determined in the amniotic fluid on day 16 p.c. and the mean telomere lengths assessed at days 12, 13 and 19 p.c. Differential gene expression within the forelimb bud tissues was determined using Real Time quantitative PCR (RTqPCR) 24 h following irradiation. Apoptosis was investigated in the normal and malformed fetuses using the TUNEL assay and RTqPCR. First, we found that irradiated fetuses with forelimb defects displayed excessive apoptosis in the predigital regions. Besides, overexpression of the pro-apoptotic Bax gene indicates a mitochondrial-mediated cell death. Secondly, our results showed overexpression of MKK3 and MKK7 (members of the stress-activated MAP kinase family) within the malformed fetuses. The latter could be involved in radiation-induced apoptosis through activation of the p38 and JNK pathways. Thirdly, we found that irradiated fetuses exhibiting forelimb defects showed a marked telomere shortening. Interestingly, telomere shortening was observed as the malformations became apparent. Fourthly, we measured cytokine levels in the amniotic fluid and detected a considerable inflammatory reaction among the irradiated fetuses as evidenced by the increase in pro-inflammatory cytokine levels. Altogether, our data suggest that transcriptional modulations of apoptotic, inflammation, stress, and DNA damage players are early events in radiation-induced forelimb defects. These changes resulted in harsh developmental conditions as indicated by a marked increase in cytokine levels in the amniotic fluid and telomere shortening, two features concomitant with the onset of the forelimb defect phenotype in our study.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Abnormalities, Radiation-Induced / etiology
  • Abnormalities, Radiation-Induced / metabolism*
  • Abnormalities, Radiation-Induced / pathology
  • Amniotic Fluid / metabolism
  • Animals
  • Apoptosis
  • Cytokines / metabolism*
  • DNA Damage
  • Female
  • Fetus / abnormalities
  • Fetus / metabolism*
  • Forelimb / abnormalities
  • Forelimb / metabolism*
  • Limb Buds / cytology
  • Limb Buds / radiation effects
  • MAP Kinase Signaling System / radiation effects
  • Male
  • Maternal Exposure / adverse effects*
  • Mice
  • Mice, Inbred C57BL
  • Telomere / metabolism
  • Telomere / ultrastructure*
  • Tumor Suppressor Protein p53

Substances

  • Cytokines
  • Tumor Suppressor Protein p53