Effects of polluted seawater on the proliferation and HSP70 CDNA transcripts in Protoreaster nodosus linn.

Abstract

Sea stars are potential indicators of both environmental and ecological conditions of their marine habitat. This study determined the effects of polluted seawater on the proliferation of primary cells and level of heat shock protein 70 (Hsp70) cDNA transcripts in primary cells and arm tissue of chocolate chip sea star (Protoreaster nodosus). Polluted water collected from Manila Bay (14º32'19.8"N, 120º58'30.1"E) was analyzed for heavy metals and inorganic nutrients concentrations. Water concentrations at 0%, 10%, 30%, and 50% were used to treat the primary cells, where MTT assay was performed. Live sea stars were also treated with the same concentrations of polluted seawater. Arm tips measuring 1cm were amputated before and after treatment, RNA was extracted and underwent RT-PCR for the Hsp70 analysis. Cells were treated with 0%, 5%, 10%, and 15% polluted seawater concentrations, total RNA was extracted and subjected to RT-PCR. Manila Bay water tested positive for SO4-, Cl- and PO43- at 983, 2448 and 2.7 mg/L, respectively. Linear regression analysis of percent cytotoxicity in cells indicated 50% lethal concentration (LC50) at 15%. No significant differences in the cytotoxicity of the Manila Bay water and the positive controls (p>0.05) were determined. A strong, positive correlation between the concentrations and cytotoxicity of polluted seawater and positive controls (ZnSO4 • 7H2O, 3CdSO4 • 8H2O, and PbCl2) were established. Comparison of mean integrated density values between the Hsp70 cDNA profiles of both P. nodosus primary cells and arm tissues suggested heightened stress response at increased concentration of polluted seawater (p>0.05). Increasing concentration of polluted seawater caused increased cytotoxicity in cells and stress response in both cells and tissues of P. nodosus.