Effect of TRIS and bicarbonate as buffers on anesthetic efficacy of tricaine methane sulfonate in zebrafish (Danio rerio)

Tricaine methane sulfonate (TMS), often called MS-222, is the most common anesthetic used with fishes. Because it is very acidic (pKa about 3) it must be neutralized especially when used in soft fresh water. Much of the literature on fish anesthetics recommends neutralizing with bicarbonate. However, much of the zebrafish literature uses the protocol in “The Zebrafish Book” that recommends neutralizing with TRIS. Three considerations when comparing these buffers are: first, TRIS has the advantage that the pH tends to remain constant, whereas the pH of solutions containing bicarbonate tends to increase as CO2 diffuses from the water to air; second, the CO2 produced by bicarbonate may have some sedative effects in and of itself; and third, there is some evidence that the efficacy of TMS changes with pH. In the present study, we compared the efficacy of TMS using these two buffers and show that there is no substantial difference in anesthetic properties in zebrafish

Modulation of cadmium-induced mitochondrial dysfunction and volume changes by temperature in rainbow trout (Oncorhynchus mykiss)

We investigated how temperature modulates cadmium (Cd)-induced mitochondrial bioenergetic disturbances, metal accumulation and volume changes in rainbow trout (Oncorhynchus mykiss). In the first set of experiments, rainbow trout liver mitochondrial function and Cd content were measured in the presence of complex I substrates, malate and glutamate, following exposure to Cd (0-100 μM) at three (5, 13 and 25 °C) temperatures. The second set of experiments assessed the effect of temperature on Cd-induced mitochondrial volume changes, including the underlying mechanisms, at 15 and 25 °C. Although temperature stimulated both state 3 and 4 rates of respiration, the coupling efficiency was reduced at temperature extremes due to greater inhibition of state 3 at low temperature and greater stimulation of state 4 at the high temperature. Cadmium exposure reduced the stimulatory effect of temperature on state 3 respiration but increased that on state 4, consequently exacerbating mitochondrial uncoupling. The interaction of Cd and temperature yielded different responses on thermal sensitivity of state 3 and 4 respiration; the Q10 values for state 3 respiration increased at low temperature (5-13 °C) while those for state 4 increased at high temperature (13-25 °C). Importantly, the mitochondria accumulated more Cd at high temperature suggesting that the observed greater impairment of oxidative phosphorylation with temperature was due, at least in part, to a higher metal burden. Cadmium-induced mitochondrial volume changes were characterized by an early phase of contraction followed by swelling, with temperature changing the kinetics and intensifying the effects. Lastly, using specific modulators of mitochondrial ion channels, we demonstrated that the mitochondrial volume changes were associated with Cd uptake via the mitochondrial calcium uniporter (MCU) without significant contribution of the permeability transition pore and/or potassium channels. Overall, it appears that high temperature exacerbates Cd-induced mitochondrial dysfunction and volume changes in part by increasing metal uptake through the MCU

Interactions of copper and thermal stress on mitochondrial bioenergetics in rainbow trout, Oncorhynchus mykiss

Thermal stress may influence how organisms respond to concurrent or subsequent chemical, physical and biotic stressors. To unveil the potential mechanisms via which thermal stress modulates metals-induced bioenergetic disturbances, the interacting effects of temperature and copper (Cu) were investigated in vitro. Mitochondria isolated from rainbow trout livers were exposed to a range of Cu concentrations at three temperatures (5, 15 and 25 °C) with measurement of mitochondrial complex I (mtCI)-driven respiratory flux indices and uncoupler-stimulated respiration. Additional studies assessed effects of temperature and Cu on mtCI enzyme activity, induction of mitochondrial permeability transition pore (MPTP), swelling kinetics and mitochondrial membrane potential (MMP). Maximal and basal respiration rates, as well as the proton leak, increased with temperature with the Q10 effects being higher at lower temperatures. The effect of Cu depended on the mitochondrial functional state in that the maximal respiration was monotonically inhibited by Cu exposure while low and high Cu concentrations stimulated and inhibited the basal respiration/proton leak, respectively. Importantly, temperature exacerbated the effects of Cu by lowering the concentration of the metal required for toxicity and causing loss of thermal dependence of mitochondrial respiration. Mitochondrial complex I activity was inhibited by Cu but was not affected by incubation temperature. Compared with the Ca positive control, Cu-imposed mitochondrial swelling exhibited variable kinetics depending on the inducing conditions, and was highly temperature-sensitive. A partial reversal of the Cu-induced swelling by cyclosporine A was observed suggesting that it is in part mediated by MPTP. Interestingly, the combination of high Cu and high temperature not only completely inhibited mitochondrial swelling but also greatly increased the RCR relative to the controls. Copper exposure also caused marked MMP dissipation which was reversed by N-acetyl cysteine and vitamin E suggesting a role of ROS in this response. Taken together, Cu impairs oxidative phosphorylation in part by inhibiting the ETC, stimulating proton leak, inducing MPTP and dissipating MMP, with high temperature exacerbating these effects. Thus environmental temperature rise due to natural phenomenon or global climate change may sensitize fish to Cu toxicity by exacerbating mitochondrial dysfunction