Stress response gene activation protects sea urchin embryos exposed to X-rays

Abstract

We used Paracentrotus lividus sea urchin embryos, a well-established model in developmental biology and ecotoxicology, for investigation on stress/anti-apoptotic protein expression elicited in response to harmful ionizing radiation, such as X-rays. We evaluated the acute effects of a high-dose exposure (5 Gy) on P. lividus analyzing by Western blotting the accumulation levels of HSP60, HSP70, BAG3 and a putative p63 at 24 and 48 h after irradiation. We found an increase in the HSP70, BAG3, and p63 protein levels only 48 h after irradiation, whereas no HSP60 increase was detected either at 24 or 48 h. Levels of the mRNA coding for HSP70 and p63 were also investigated by relative RT-PCR and were found to increase 24 h after irradiation, returning to their initial levels at 48 h. Results demonstrate the presence of an adaptive regulatory mechanism operating at the transcriptional level at 24 h, followed by a translational activation at 48 h post-irradiation. In conclusion, our findings confirm the sea urchin embryo as a sensible bioindicator of cell damage and we propose this model for studies on the protective pathways activated in response to X-rays. The novel result of the involvement of BAG3 and p63 in the response to X-rays, never tested so far in any other embryonic system, opens the way for their use as biomarkers of X-ray hazards.

Fig. 1

Effects of X-rays on protein levels 24 and 48 h after irradiation. Western blot tests of total lysates from unexposed (0 Gy) and exposed (5 Gy) embryos were reacted with anti-hsp70, anti-hsp60, anti-Bag-3, anti-p53, respectively, shown in panels a, b, c, d. Each histogram reports the volumetric analysis data obtained from Western blot shown on the upper part (representative WB images). Each bar represents the mean of two/three independent WB experiments ± SE. Asterisks (*) indicate significant difference between control and exposed embryos by the ANOVA test (P < 0.05) followed by the Tukey test

Fig. 2

Semiquantitative analysis of hsp70 and p53-like mRNA levels in embryos exposed to X-rays. Each bar represents the mean of the two independent experiments ± SE with six replicates in samples analyzed 24 h after irradiation and three replicates in samples analyzed 48 h after irradiation. Values obtained from measurements of band intensities for each amplification product were normalized to the Pl-S24 values. Asterisks (*) indicate significant difference between control and exposed embryos by the ANOVA test (P < 0.05) followed by the Tukey test