Interactive effects of temperature, cadmium, and hypoxia on rainbow trout (Oncorhynchus mykiss) liver mitochondrial bioenergetics

Abstract

Fish in their natural environments possess elaborate mechanisms that regulate physiological function to mitigate the adverse effects of multiple environmental stressors such as temperature, metals, and hypoxia. We investigated how warm acclimation affects mitochondrial responses to Cd, hypoxia, and acute temperature shifts (heat shock and cold snap) in rainbow trout. We observed that state 3 respiration driven by complex I (CI) was resistant to the stressors while warm acclimation and Cd reduced complex I +II (CI + II) driven state 3 respiration. In contrast, state 4 (leak) respirations for both CI and CI + II were consistently stimulated by warm acclimation resulting in reduced mitochondrial coupling efficiency (respiratory control ratio [RCR]). Warm acclimation and Cd exacerbated their individual effect on leak respiration to further reduce the RCR. Moreover, the effect of warm acclimation on mitochondrial bioenergetics aligned with its inhibitory effect on activities of citrate synthase and both CI and CII. Unlike the Cd and warm acclimation combined exposure, hypoxia alone and in combination with warm acclimation and/or Cd abolished the stimulation of CI and CI + II powered leak respirations resulting in partial recovery of RCR. The response to acute temperature shifts indicated that while state 3 respiration returned to pre-acclimation level, the leak respiration did not. Overall, our findings suggest a complex in vivo interaction of multiple stressors on mitochondrial function that are not adequately predicted by their individual effects.

Keywords: Acclimation; Cd; Hypoxia; Mitochondrial bioenergetics; Plasticity; Rainbow trout.

Fig. 1.

Individual and combined effects of acclimation temperature, Cd, and hypoxia on mitochondrial complex I powered respiration. (A) state 3, (B) state 4, and (C) RCR. Rainbow trout were acclimated to 10 °C (control) or 20 °C, (warm-acclimated) for 50 days and exposed to (i) 10 μg/l Cd for 24 h, (ii) hypoxia (30 % air saturation) for 2 h, or (iii) 10 μg/l Cd for 24 h combined with hypoxia (30 % air saturation) for 2 h. Liver mitochondria were isolated and the respiration fueled by glutamate-malate was measured at the respective acclimation temperature. Data are means ± SEM, N = 5 independent fish. Bars with different letters represent statistically significant means (p < 0.05), three-way ANOVA, Tukey’s HSD test.

Fig. 2.

Individual and combined effects of acclimation temperature, Cd, and hypoxia on mitochondrial complex I + II powered respiration. (A) state 3, (B) state 4, and (C) RCR. Rainbow trout were acclimated to 10 °C (control) or 20 °C (warm-acclimated) for 50 days and exposed to (i) 10 μg/l Cd for 24 h, (ii) hypoxia (30 % air saturation) for 2 h, or (iii) 10 μg/l Cd for 24 h combined with hypoxia (30 % air saturation) for 2 h. Liver mitochondria were isolated and the respiration fueled by glutamate-malate-succinate was measured at the respective acclimation temperature. Data are means ± SEM, N = 5 independent fish. Bars with different letters represent statistically significant means (p < 0.05), three-way ANOVA, Tukey’s HSD test.

Fig. 3.

Individual and combined effects of acclimation temperature, Cd, and hypoxia on activities of mitochondrial enzymes. (A) citrate synthase, (B) complex I, and (C) complex II. Rainbow trout were acclimated to 10 °C (control) or 20 °C (warm-acclimated) for 50 days and exposed to (i) 10 μg/l Cd for 24 h, (ii) hypoxia (30 % air saturation) for 2 h, or (iii) 10 μg/l Cd for 24 h combined with hypoxia (30 % air saturation) for 2 h. Liver mitochondria were isolated, and activities of the enzymes were measured. Data are means ± SEM, N = 5 independent fish. Bars with different letters represent statistically significant means (p < 0.05), three-way ANOVA, Tukey’s HSD test.

Fig. 4.

Plasticity of mitochondrial complex I powered respiration following acute temperature rise. (A) complex I state 3, (B) complex I state 4, and (C) complex I RCR. Rainbow trout acclimated to 10 ° C for 50 days were exposed to (i) 10 μg/l Cd for 24 h, (ii) hypoxia (30 % air saturation) for 2 h, or (iii) 10 μg/l Cd for 24 h combined with hypoxia (30 % air saturation) for 2 h. Liver mitochondria were isolated and the respiration fueled by glutamate-malate was measured at 20 ° C. Data for the 10 ° C- and 20 ° C-acclimated fish measured at the respective acclimation temperatures were imbedded with the acute temperature rise measurements for statistical analysis. Data are means ± SEM, N = 5 independent fish. Bars with different letters represent statistically significant means (p < 0.05), three-way ANOVA, Tukey’s HSD test.

Fig. 5.

Plasticity of mitochondrial complex I powered respiration following acute temperature drop. (A) complex I state 3, (B) complex I state 4, and (C) complex I RCR. Rainbow trout acclimated to 20 ° C for 50 days were exposed to (i) 10 μg/l Cd for 24 h, (ii) hypoxia (30 % air saturation) for 2 h, or (iii) 10 μg/l Cd for 24 h combined with hypoxia (30 % air saturation) for 2 h. Liver mitochondria were isolated and the respiration fueled by glutamate-malate was measured at 10 ° C. Data for the 10 °C- and 20 ° C-acclimated fish measured at the respective acclimation temperatures were imbedded with the acute temperature drop measurements for statistical analysis. Data are means ± SEM, N = 5 independent fish. Bars with different letters represent statistically significant means (p < 0.05), three-way ANOVA, Tukey’s HSD test.

Fig. 6.

Plasticity of mitochondrial complex I + II powered respiration following acute temperature rise. (A) state 3, (B) state 4, and (C) RCR. Rainbow trout acclimated to 10 ° C for 50 days were exposed to (i) 10 μ;g/l Cd for 24 h, (ii) hypoxia (30 % air saturation) for 2 h, or (iii) 10 μg/l Cd for 24 h combined with hypoxia (30 % air saturation) for 2 h. Liver mitochondria were isolated and the respiration fueled by glutamate-malate-succinate was measured at 20 ° C. Data for the 10 ° C- and 20 ° C-acclimated fish measured at the respective acclimation temperatures were imbedded with the acute temperature rise measurements for statistical analysis. Data are means ± SEM, N = 5 independent fish. Bars with different letters represent statistically significant means (p < 0.05), three-way ANOVA, Tukey’s HSD test.

Fig. 7.

Plasticity of mitochondrial complex I + II powered respiration following acute temperature drop. (A) state 3, (B) state 4, and (C) RCR. Rainbow trout acclimated to 20 ° C for 50 days were exposed to (i) 10 μg/l Cd for 24 h, (ii) hypoxia (30 % air saturation) for 2 h, or (iii) 10 μg/l Cd for 24 h combined with hypoxia (30 % air saturation) for 2 h. Liver mitochondria were isolated and the respiration fueled by glutamate-malate-succinate was measured at 10 ° C. Data for the 10 ° C- and 20 ° C-acclimated fish measured at the respective acclimation temperatures were imbedded with the acute temperature rise measurements for statistical analysis. Data are means ± SEM, N = 5 independent fish. Bars with different letters represent statistically significant means (p < 0.05), three-way ANOVA, Tukey’s HSD test.