HP acclimatization of yellow eels improves oxidative phosphorylation together with supposed concomitant changes in electron leak and ROS production.
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UHM 2006, Vol. 33, No. 3 - Reactive oxygen species and hydrostatic pressure
Does hydrostatic pressure have an effect on reactive oxygen species in the eel? A. AMÉRAND, A. VETTIER, P. SÉBERT and C. MOISAN UHPM-UPCI- EA3879 - Faculty of Medicine - CS 93837 29238 Brest Cedex3 (France)
Amerand A, Vettier A, Sebert, Moisan C. Does hydrostatic pressure have an effect on reactive oxygen species in the eel? Undersea Hyperb Med 2006; 33(3):157-160.Eels are submitted to hydrostatic pressure (HP) during their spawning migration (about 6000 Km). Before migration, they change from the yellow to the silver stage (silvering process). The effects of HP in relation to the silvering process have been studied on aerobic metabolism and more precisely on reactive oxygen species (ROS) metabolism. HP acclimatization of yellow eels improves oxidative phosphorylation together with supposed concomitant changes in electron leak and ROS production. Therefore hydroxyl radical (OH•) production, superoxyde dismutase and catalase activities, malondialdehyde content and in parallel oxygen consumption were measured in the red muscle of long-term pressure exposed and control group yellow and silver eels. At atmospheric pressure, yellow eels exhibited significantly higher oxygen consumption and OH• production than silver eels; and significantly lower malondialdehyde content. This could be due to the increase in membrane fluidity induced by the silvering process. Long-term HP exposure decreases yellow eel oxygen consumption which becomes similar to that of the silver stage. In parallel there is a decrease in OH• production and concomitantly antioxidant enzyme activities follow the same tendency. Thus the respiratory chain improvement in pressure acclimatized yellow eels is accompanied by a ROS production decrease which could mean an electron leak decrease.
INTRODUCTION In yellow European eels (non-migrating stage), high pressure induces physiological and morphological events which could prepare them for the spawning migration (1). It seems that pressure acclimatization mimics the silvering process, in terms of pressure resistance of muscle aerobic metabolism (2). The physiological state of pressure acclimatized yellow eels can be compared to those of silver eels at atmospheric pressure. The acclimatization of yellow eels increases the efficiency of oxidative phosphorylation, through mitochondrial enzymatic complex readjustment (3) thus allowing optimization of electron transport. Under normal physiological conditions, there is an electron leak at the semi-ubiquinone level (4). Consequently, we hypothesize that the acclimatization of yellow eels could change the electron leak and reactive oxygen species (ROS) production (already Copyright © 2006 Undersea and Hyperbaric Medical Society, Inc
suspected (5). In fact, the electron leak is known to be the starting point of ROS production (like the highly reactive: hydroxyl radical OH•) (6). This hypothesis was tested by conducting a hydrostatic pressure acclimatization comparative study between yellow eels and silver eels (migrating stage) in terms of ROS metabolism using OH• quantification, superoxide dismutase (SOD) and catalase (CAT) activity measurements (antioxidant enzymes) and malondialdehyde (MDA) quantification (index of membrane ROS impact) in red muscle and in parallel muscle oxygen consumption measurement. METHODS Animals Twenty-eight yellow and silver eels (Anguilla anguilla) respectively averaging 100 g and 350 g in weight were used for the experiment. They were stored before the 157
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UHM 2006, Vol. 33, No. 3 - Reactive oxygen species and hydrostatic pressure
Antioxidant enzyme activity measurements were performed on frozen sections of eels, and more precisely on superficial red muscle. The red muscle was homogenised (in 75 mM TRIS and 5 mM EDTA buffer pH 7.4) and centrifuged at 5000 g for 10 min and the supernatant centrifuged at 12000 g for 15 min at 4 °C. The last supernatant was used for SOD and CAT activity measurements. SOD activity was determined spectrophotometrically at 480 nm by the method involving the inhibition of the adrenalineadrenochrome reaction (10). CAT activity involved quantifying the tissue catalase H2O2 breakdown rate using a spectrophotometer (240 nm). This technique was adapted according to Beers et al. (11), and the assay concentration of H2O2 was 10 mM in 75 mM TRIS and 5 mM buffer pH 7,4. A thiobarbituric acid (TBA) was used to determine MDA concentration in red muscle from frozen sections of eels using HPLC procedures. The MDA/TBA complex was detected at 532nm. The technique has been previously described by Sébert et al.(12) and applied to fish red muscle.
experiment in 40 L polyethylene tanks where tap water was continuously renewed and aerated. Protocol Seven yellow eels and seven silver eels were put in an experimental tank placed in a hyperbaric chamber connected to a high-pressure water circulation system (7). During the experiment, oxygen content and temperature were controlled. After 5 days at atmospheric pressure, the hyperbaric chamber was compressed at a rate of 0.2 MPa.min1 to 10.1 MPa. After 21 days at this pressure, decompression was performed at 0.2 MPa.min-1. On reaching atmospheric pressure, the eels were killed and sampling performed. During the experimental period, control groups (7 yellow eels and 7 silver eels) were maintained at atmospheric pressure in a similar experimental tank. All measurements were performed at 15 °C. Oxygen consumption and OH• quantification were performed on permeabilised red muscle fibre preparations. This technique was adapted by Theron et al (3) according to Veksler et al and Letellier et al (8, 9). Red muscle fibres were treated by a saponin solution which perforates cell membrane but it does not affect the mitochondria. The rate of oxygen consumption was measured by a confinement method on freshly permeabilised red muscle fibres using pyruvate plus malate and ADP (state 3) at saturating concentrations (3). Oxygen consumption was expressed in nmolO2.min-1.mg-1 of tissues. An indirect method was used to quantify OH• production with salicylic acid as the OH• trapper. The hydroxylation of salicylic acid gave two stable metabolites which were quantified by HPLC coupled with electrochemical detection as reported earlier (5). OH• production was expressed in ngOH•.mg-1 of muscle for 30 minutes of salicylate incubation.
Data analysis The results shown in this study are expressed as mean ± SEM. The statistical significance has been evaluated using the Student’s t test. RESULTS In control groups, the results (Fig. 1) show a significantly higher oxygen consumption in yellow eels (+30 %, p
