Changes of cellular stress response related hsp70 and abcb1 transcript and Hsp70 protein levels in Siberian freshwater amphipods upon exposure to cadmium chloride in the lethal concentration range

Abstract

The induction of cellular stress response systems, heat shock protein hsp70/Hsp70 and multixenobiotic transporter abcb1, by cadmium chloride (CdCl₂) was explored in amphipod species with different stress adaptation strategies from the Lake Baikal area. Based on the lethal concentrations (LC) of CdCl₂, the sensitivities of the different species to CdCl₂ were ranked (24 hr LC50 in mg/L CdCl₂ (mean/95% confidence interval)): Gammarus lacustris (1.7/1.3-2.4) < Eulimnogammarus cyaneus (2.9/2.1-4.0) < Eulimnogammarus verrucosus (5.7/3.8-8.7) < Eulimnogammarus vittatus (18.1/12.4-26.6). Conjugated dienes, indicating lipid peroxidation, were significantly increased after 24 hr exposures to 5 mg/L CdCl₂ only in the more CdCl₂-sensitive species G. lacustris and E. cyaneus. Upon treatment with 0.54 to 5.8 mg/L CdCl₂ for 1, 6 and 24 hrs, hsp70 transcript levels were generally more increased after the longer exposure times and in the more CdCl₂-sensitive species. Relating the CdCl₂ exposure concentrations to LCx values revealed that across the species the increases of hsp70 transcript levels were comparatively low (up to 2.6-fold) at CdCl₂ concentrations ≤LC50. Relative hsp70 transcript levels were maximally increased in E. cyaneus by 5 mg/L CdCl₂ ( $\widehat{=}$ LC70) at 24 hrs (9.1-fold increase above the respective control). When G. lacustris was exposed to 5 mg/L CdCl₂ ( $\widehat{=}$ LC90) for 24 hrs, the increase in hsp70 was in comparison to E. cyaneus considerably less pronounced (3.0-fold increase in hsp70 levels relative to control). Upon exposure of amphipods to 5 mg/L CdCl₂, increases in Hsp70 protein levels compared to untreated controls were highest in E. cyaneus at 1 and 6 hrs (5 mg/L CdCl₂ $\widehat{=}$ LC70) and in E. verrucosus at 24 hrs (5 mg/L CdCl₂ $\widehat{=}$ LC45). Thus, when the fold increases in Hsp70 protein levels in the different amphipod species were related to the respective species-specific LCx values a similar bell-shaped trend as for hsp70 transcript levels was seen across the species. Transcript levels of abcb1 in CdCl₂exposed individuals of the different amphipod species varied up to 4.7-fold in relation to the respective controls. In contrast to hsp70/Hsp70, abcb1 transcripts in CdCl₂ exposed individuals of the different amphipod species did not indicate similar levels of induction of abcb1 at equal LCx levels across the species. Induction of hsp70 and abcb1 genes and Hsp70 proteins by CdCl₂ in the lethal concentration range shows that these cellular responses are rather insensitive to CdCl₂ stress in the examined amphipod species. Furthermore, the increase of expression of these cellular defense systems at such high stress levels suggests that induction of these genes is not related to the maintenance of normal metabolism but to mitigation of the effects of severe toxic stress.

Keywords: Amphipods; Cadmium; Cellular stress response; Conjugated dienes; Heavy metal; Lake Baikal; P-glycoprotein; Toxicity; Transcript levels; hsp70.