Polychlorinated biphenyls (PCBs) are ubiquitous industrial compounds found in almost every component of the terrestrial and marine ecosystem. Most of the PCB congeners bind to the aryl hydrocarbon receptor and in turn cause expression of stress response genes. Here we report for the first time that PCB 118 acts in the marine sponge Geodia cydoniurn as an inducer of 2 chaperones, the 14-3-3 protein(s) (a protein targeting molecule) and the heat shock protein HSP70 (a chaperone, primarily involved in folding of proteins). While the cDNA encoding the latter protein has been cloned previously, the 14-3-3 cDNA from sponges is reported in this study. The full-length cDNA clone of G. cydoniurn, GC14-3-3, has a size of 912 nucleotides (nt) and contains a 744 nt long potential open reading frame; the relative molecular weight (M,) of the deduced aa sequence is 28 378 Da. The sponge polypeptide is closely related to the deduced polypeptides of the 14-3-3 sequences belonging to isoforms q and y. Using the cDNAs, coding for the 14-3-3 and the HSP70 protelns as well as antibodies raised against these 2 proteins, it was demonstrated that neither chaperone can be detected in the absence of PCB. However, after incubation of sponge tlssue w t h PCB 118 the transcripts of the 2 chaperones are detectable after 12 h, while the corresponding proteins appear after 1 d . Subsequently, the levels of the transcripts and of the proteins increase steably. From these data we conclude that the 2 chaperones, 14-3-3 and HSP70, are useful biomarkers in sponges. Due to the broad cross-reactivity of their antibodies throughout the Metazoa, these chaperones may be useful biomarkers for monitoring environmental contaminants, as shown here for PCB 118, in all organisms.
The marine sponge Suberites domuncula was used as a bioindicator to study the effects of cadmium on the occurrence of DNA strand breakage and on the induction of the expression of the stress biomarkers, heat shock protein 70 (HSP70) and glucose-regulated protein 78 (GRP78) homolog. The cDNA encoding GRP78 homolog from S. domuncula was isolated and characterized. The GRP78 cDNA has a length of 2.1 kb and displays characteristic features of the HSP70 family; it encodes an aa sequence of Mr 72,000. Exposure of S. domuncula to 1 mg/L of cadmium chloride for 24 h caused a strong (16. 6-fold) increase in cadmium content to 7.7 microg/g wet weight of sponge tissue; after an incubation period of 6 days, the accumulation was 20.4-fold. The increase in cadmium content was paralleled by a transient decrease in zinc content at days 1 and 3. Exposure of S. domuncula to cadmium chloride also resulted in a marked increase in the number of DNA single strand breaks, as assessed by a recently developed fast and sensitive microplate assay. The maximum increase in DNA damage was observed after an incubation of 12 h in the presence of 1 mg/L of cadmium chloride; after longer incubation, the number of damaged sites decreased, most likely due to DNA repair. Quantitative analysis of the expression of HSP70 (Mr 73 kDa) revealed that onset of maximal levels of HSP70 depends on the concentration of cadmium chloride in the ambient seawater. Maximal induction (8.9-fold increase compared to control) of HSP70 following exposure to 1 mg/L of cadmium chloride was found after 12 h, while longer incubation periods (3-6 days) were needed to reach maximum levels of HSP70 in the presence of lower concentrations of cadmium chloride (0.1 mg/L and 0.01 mg/L). Northern blot analysis revealed that the level of the 2.0 kb sponge GRP78 homolog mRNA transiently increased under cadmium stress; the maximum increase in the presence of 0.1 mg/L of cadmium chloride was observed at day 3. Our results suggest that sponges are useful indicator organisms to assess the genotoxic risks of cadmium pollution in marine environments.
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