Tp0300

Tissue samples were removed from -80°C and maintained on ice. A 10 mg subsample was dissected for biochemical analysis. Total protein and enzyme activities were determined by absorbance using a microplate reader (FLUOstar OPTIMA, BMG Labtech). Total protein was assessed at 584 nm absorbance using an assay kit employing the Lowry method with Peterson's modification (Sigma-Aldrich TP0300). Superoxide dismutase (SOD) activity was assessed at 450 nm absorbance using an assay kit employing inhibition of xanthine oxidase activity (Sigma-Aldrich 19160) . Glutathione peroxidase (GPx) activity was assessed at 340 nm absorbance using an assay kit employing NADPH oxidation in the presence of excess glutathione reductase, reduced glutathione, and corresponding peroxide (Abcam ab102530).

Tissue from individuals used to assess respiratory response to sublethal toxicity was homogenised at 4°C in Tris-HCl pH 7.6 buffer. Following 10 mins incubation at 4°C, the homogenates were centrifuged at 1000g for 10 min at 4°C and supernatant was retained for analysis. Total protein and enzyme activities were determined using a microplate reader (FLUOstar OPTIMA, BMG Labtech). Total protein was assessed at 584 nm absorbance employing the Lowry method with Peterson's modification using an assay kit (Sigma-Aldrich TP0300). Superoxide dismutase (SOD) activity was assessed at 450 nm absorbance employing inhibition of xanthine oxidase activity using an assay kit (Sigma-Aldrich 19160).
Glutathione peroxidase (GPx) activity was assessed at 340 nm absorbance employing NADPH oxidation in the presence of excess glutathione reductase, reduced glutathione, and corresponding peroxide using an assay kit (Abcam ab102530).
The effects of temperature and hydrostatic pressure on biochemical responses to copper and cadmium were analysed using two-way ANOVAs: data were normal (Shapiro-Wilk test) and homoscedastic (Levene's test) (p > 0.05). The post-hoc multiple comparison Holm-Sidak test was used to determine which treatments produced the differences.

Membrane protein levels were measured by a commercially available assay kit (Sigma, SL, USA, Cat no; TP0300). The membrane pellet was re-suspended in an isotonic buffer with 2% SDS and were divided into aliquots (one for each assay) so the concentration of proteins in samples are the same.