Stress to cadmium monitored by metallothionein gene induction in Paracentrotus lividus embryos.

We used Paracentrotus lividus sea urchin embryos as a bio-indicator to study the effects of exposure to subacute/sublethal cadmium concentrations on the expression of the stress marker metallothionein (MT) gene. To this purpose a full length cDNA was obtained by RT-PCR from total RNA extracted from prism stage embryos. On the basis of cDNA sequence analyses and comparison of the deduced amino acidic sequence with those reported for other sea urchin species we concluded that protein product of this cDNA constitutes a new member of the metallothionein gene family which has been referred to as Pl-MT3. Northern blot analysis showed that basal levels of Pl-MT3 mRNA, having an apparent size of 700b, are expressed in all the developmental stages analyzed from early cleavage to pluteus. When embryos were continuously cultured in the presence of subacute/sublethal doses of CdCl2, and harvested at cleavage, swimming blastula, late gastrula and pluteus stages (6, 12, 24 and 48 hrs after fertilization, respectively) a time and dose-dependent increase in the transcription levels of the Pl-MT3 gene were observed. This finding was confirmed by RT-PCR experiments and compared to the statistical analysis of morphological effects on sea urchin embryo development. While microscopical inspection revealed the occurrence of abnormalities only after 24 hrs, Northern blotting studies indicated an over-expression of metallothionein gene already after 12 hrs; the latter effect could be observed by RT-PCR even after 6 hrs. Therefore, we correlate the over-expression of Pl-MT3 to the stress induced by CdCl2 treatment and propose the use of the sea urchin embryo as a very fast and convenient bio-indicator in the detection of environmental pollution by heavy metals.