The Journal of Experimental Biology 203, 37173725 (2000) Printed in Great Britain The Company of Biologists Limited 2000

0 JEB3216

3717

REDUCED ENZYMATIC ANTIOXIDATIVE DEFENSE IN DEEP-SEA FISH

BERNADETTE J. JANSSENS1,*, JAMES J. CHILDRESS2, FERNAND BAGUET1 AND JEAN-FRANCOIS REES1 1Animal Biology Unit, Universit Catholique de Louvain, Croix du Sud, 4, B-1348 Louvain-la-Neuve, Belgium and 2Department of Ecology, Evolution and Marine Biology and Marine Science Institute, University of California, Santa Barbara, CA 93106, USA
*e-mail: Janssens@bani.ucl.ac.be

Accepted 6 October; published on WWW 14 November 2000 Summary were detected in all tissues from all species, the levels of Oxygen, while being an obligate fuel for aerobic life, has SOD and GPX decreased in parallel with the exponential been shown to be toxic through its deleterious reactive reduction in the metabolic activity as estimated by citrate species, which can cause oxidative stress and lead synthase activity. In contrast, CAT was affected neither by ultimately to cell and organism death. In marine the metabolic activity nor by the depth of occurrence of the organisms, reactive oxygen species (ROS), such as the shes. High levels of metabolic and antioxidative enzymes superoxide anion and hydrogen peroxide, are generated were detected in the light organs of bioluminescent species. within respiring cells and tissues and also by photochemical The adjustment of the activity of SOD and GPX to the processes in sea water. Considering both the reduced decreased metabolic activity associated with deep-sea living metabolic rate of nektonic organisms thriving in the suggests that these antioxidative defense mechanisms deep sea and the physico-chemical conditions of this are used primarily against metabolically produced ROS, dark, poorly oxygenated environment, the meso- and whereas the maintenance of CAT activity throughout all bathypelagic waters of the oceans might be considered as depths could be indicative of another role. The possible refuges against oxidative dangers. This hypothesis reasons for the occurrence of such a reduced antioxidative prompted us to investigate the activities of the three arsenal in deep-sea species are discussed. essential enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX) constitutive of the antioxidative arsenal of cells in the tissues of 16 species of Key words: oxidative stress, deep-sea sh, reactive oxygen species, superoxide dismutase, catalase, glutathione peroxidase, oxygen, meso- and bathypelagic shes occurring between the hydrogen peroxide. surface and a depth of 1300 m. While enzymatic activities

Introduction It has long been known that oxygen is not distributed uniformly in the oceans (Sewell and Fage, 1948). Most regions of the worlds oceans are characterized by high oxygen concentrations in the euphotic zone and by a decrease to lower oxygen levels in the deep sea. In some areas, such as the Pacic Ocean, oxygen levels reach a minimum value (the oxygen minimum layer, OML) corresponding to only a few per cent of that near the surface, followed by an increase in oxygen levels at greater depths (Wyrtki, 1962). Since oxygen is essential for aerobic life, problems associated with the existence of such a gradient focused on the adaptations of organisms able to survive at the low oxygen concentrations occurring near the OML (Childress and Siebel, 1998). However, in addition to its importance as an electron acceptor in cellular energetic processes, oxygen is also a problem for living organisms because it can be toxic. Indeed, its electronic structure favors univalent reductions that lead to the formation of highly reactive intermediates, such as the superoxide anion (O2), hydrogen peroxide (H2O2) and the hydroxyl radical (OH). These intermediates, called reactive oxygen species (ROS), can oxidize most cellular constituents, such as DNA, proteins and lipids, and markedly affect the physiology of the cell, leading to the initiation of cancer or cellular death (Cadenas, 1995). Most major pathologies in humans and animals are known to involve ROS at an early stage of the disease (Chen et al., 1995), and the involvement of ROS in the aging process is well documented in many species (Pacici and Davies, 1991; Harman, 1994). Since oxygen was released into the atmosphere by photosynthetic processes, its toxicity has posed a major threat to life (Hassan and Schiavone, 1991), and all extant organisms are endowed with a complex arsenal of antioxidative defense mechanisms. Besides some small diffusible molecules, such as vitamins C and E, which are able to quench ROS in cells and tissues, specialized enzymes are aimed at neutralizing ROS. The three most important enzymes are the superoxide dismutases (SODs) that converts O2 into H2O2, which can in turn be detoxied into water and oxygen by either catalase (CAT) or peroxidases. A major peroxidase

3718 B. J. JANSSENS AND OTHERS

is glutathione peroxidase (GPX), which utilizes glutathione (GSH) as an electron donor (Ahmad, 1995). Accordingly, in addition to aerobic life in a hypoxic environment such as the OML being a real physiological challenge, it can also be seen as benecial because it relieves organisms from a major danger. From that point of view, the deep sea appears to be a somewhat protected place to live. Marine organisms face two threatening sources of ROS: one is exogenous and is linked to the presence of ROS in sea water; the other is endogenous and is associated with metabolic processes in the organisms. The mitochondrial respiratory chain, which continuously generates O2 in every cell at a rate of 15 % of the total oxygen consumed, is the main producer of ROS (Boveris and Cadenas, 1982). Work by Petasne and Zika (1987) and Van Baalen and Marler (1966) has revealed that superoxide anions and hydrogen peroxide are formed in sea water exposed to sunlight. Measurements in the Gulf of Mexico indicated maximal levels of 2108 mol l1 and 107 to 2107 mol l1 for O2 and H2O2, respectively (Zika et al., 1985). Because these ROS are produced by photochemical reactions, they are restricted to near the euphotic zone and their concentration decreases rapidly with increasing depth. As a consequence, levels of ROS were undetectable at depths below 100 m. From these data, one could expect deep-sea organisms to face a reduced threat from ROS. First, the absence of exogenously produced ROS eliminates dangers associated with them. Second, the general decrease in the metabolic rate with increasing depth of occurrence of shes, crustaceans and cephalopods (Childress, 1995; Childress and Siebel, 1998) should be accompanied by a decrease in the amounts of ROS produced in their tissues. Thus, it is possible that these reduced dangers linked to oxygen toxicity could have led deep-sea organisms to reduce their biochemical defenses against oxidative damage. To test this hypothesis, we investigated the activity of the three most important antioxidative enzymes in a variety of tissues of meso- and bathypelagic shes occurring between a depth of 1300 m and the surface. Materials and methods Meso- and bathypelagic shes were collected in the San Clemente Basin (32N, 119W; 2000 m maximal depth), northeastern Pacic, during a 15-day cruise aboard the R/V Point Sur. Animals were captured with an opening/closing Mother Tucker trawl (10 m2 mouth), equipped with a 30 l thermally insulated cod end for reducing mechanical damage and heat shock to the animals during ascent (Childress et al., 1978). Once on board, the animals were immediately transferred to seawater tanks (5 C), where they were identied. Selected individuals were immediately frozen in liquid nitrogen and then stored in a freezer at 20 C. Others were rapidly dissected on ice, and the resulting tissue samples were also frozen in liquid nitrogen before storage at 20 C. After the cruise, samples were stored at 80 C until processed. Taking into account the number of individuals captured by Table 1. Minimum depth of occurrence (MDO) and number of individuals of sh species used in this study
Symbol a b c d e f g h i j k l m n o p Species Symbolophorus californiensis Tritophorus mexicanus Bathylagus wesethi Argyropelecus affinis Idiacanthus antrostomus Servivomer sector Stomias atriventer Scopelogadus vaillant Poromitra crassiceps Borostomias panamensis Cyclothone acclinidens Anoplogaster cornuta Scopelengys tristis Parvilux ingens Bajacalifornia burragei Eurypharynx pelicanoides MDO (m) 10 25 25 200 250 300 300 350 400 500 500 550 650 700 1000 1300 N 15 15 5 12 4 2 7 4 2 2 8 3 3 2 1 2

The symbols are those used in the gures.

species and their minimum depth of occurrence (MDO), 16 species were selected to cover as far as possible the range between the surface and a depth of 1300 m (Table 1). Minimum depth of occurrence is dened as the minimum depth below which 90 % of the population of the species concerned can be found (Childress and Nygaard, 1973). Sample preparation Eleven tissues and organs were selected: eyes, brain, gills, heart, liver, stomach, kidney, white muscle, red muscle, skin and skin with photophores (Aksnes and Njaa, 1981; Filho et al., 1993). After the dissection, which was carried out on ice, similar tissues of each species were pooled, weighed and homogenized for 20 s at 9500 revs min1 with an electronic homogenizer (Polytron) in 5 volumes of phosphate-buffered saline (PBS) containing 1 % Triton X-100. Supernatants were obtained by a 30 min centrifugation at 4000 revs min1 (2880 g) at 4 C (Minifuge GL, Heraeus). Part of the supernatant was stored at 80 C, while the rest was dialysed (Medicell International Ltd; 1214 kDa) overnight at 4 C against the PBS-Triton buffer. Some samples were assayed directly for CAT while others were stored at 80 C. Assay for catalase (EC 1.11.1.6) The chemiluminescent method of Maral et al. (1977) was adapted for 96-well microplates to process numerous samples and replicates simultaneously. The assay for catalase (CAT) was performed on fresh samples immediately after dialysis. The measurements were carried out at 25 C on a PCcontrolled microplate luminometer (Berthold) equipped with two 50 l injectors. A rst injection of 106 mol l1 H2O2 solution initiated its reduction by the CAT contained in the diluted sample (50 l), which was added to 100 l of

Enzymatic antioxidative defence in deep-sea sh 3719

100 mmol l1 phosphate buffer (pH 7.8) containing 0.6 mmol l1 EDTA. After a 30 min incubation, the injection of 50 l of a solution containing 20 mmol l1 luminol and 11.6 units ml1 horseradish peroxidase produces an emission of light whose intensity was proportional to the H2O2 remaining in the mixture. CAT activity in the sample was estimated with puried bovine liver CAT solubilized in the homogenization buffer as standard and is expressed in international units. Assay for superoxide dismutase (EC 1.15.1.1) A new microplate assay method was developed to detect the very low activities in many samples. The method is based on the chemiluminescence of an imidazolopyrazinone molecule (CLZm) when this reacts with O2 (Nakano, 1990). Exploiting this chemiluminescent property, total superoxide dismutase (SOD) activity was estimated by the inhibition of light production in conditions where the enzymatically generated O2 is the rate-limiting factor of the luminescence. In these circumstances, SOD competes with CLZm for O2, and the luminescence will become weaker as the activity of SOD in the sample increases. The injection of 50 l of hypoxanthine solution (34 mg ml1) into a mixture of 25 l of diluted sample, 165 l of 100 mmol l1 phosphate buffer (pH 7.8) containing 0.6 mmol l1 EDTA and 0.01 % bovine serum albumin (BSA), 5 l of 0.8 mol l1 CLZm and 5 l of xanthine oxidase (27.56 munits ml1) initiates the emission of light. The assay was carried out at 25 C, and SOD activity was calculated from a standard curve established with Cu,Zn-SOD puried from bovine erythrocytes in PBS-Triton buffer. The activity of SOD is expressed in international units. Assay for glutathione peroxidase (EC 1.11.1.9) Glutathione peroxidase (GPX) activity was measured according to the spectrophotometric method of Paglia and Valentine (1967). Diluted samples (65 l) were added to 1 ml of 50 mmol l1 Tris-HCl buffer (pH 7.6) containing 0.1 mmol l1 EDTA, 0.14 mmol l1 NADPH, 1 mmol l1 GSH and 1 unit ml1 glutathione reductase (EC 1.6.4.2). The consumption of NADPH was monitored at 340 nm (25 C) in a spectrophotometer (Beckman DU-8) after the addition of 0.2 mmol l1 t-butyl hydroperoxide. GPX activity was estimated from a standard curve constructed with GPX puried from bovine erythrocytes solubilized in PBS-Triton buffer and is expressed in international units. Metabolic activity Metabolic activity was estimated from the activity of citrate synthase (CS; EC 4.1.3.7), a mitochondrial enzyme that is a good indicator of the metabolic activity of the tissues (Childress and Somero, 1979). Samples (10 l) were added to 995 l of 100 mmol l1 Tris-HCl (pH 8.0) buffer containing 1 mmol l1 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) and 0.5 mmol l1 acetyl-CoA. The consumption of acetyl-CoA was measured at 25 C from the increase in absorbance at 420 nm after the addition of 5 l of 5 mmol l1 oxaloacetate (Shepherd and Garland, 1969). The activity of CS was estimated from a standard curve established with CS from chicken heart and is expressed in international units. Protein measurements were performed according to the method of Lowry et al. (1951) with bovine serum albumin as standard. Oxygen partial pressures in the sea water at different depths of the area studied were obtained from Childress (1995). Reagents All reagents were purchased from Sigma (St Louis, USA), except for Triton X-100, GSH and glutathione reductase, which were obtained from Boehringer (Mannheim, Germany). Methyl-coelenterazine [CLZm; 3,7-dihydro-2-methyl-6-(phydroxyphenyl)-8-benzylimidazo(1,2-a]pyrazin-3-one)] was synthesized as described by Inoue et al. (1975). Statistical analyses The enzymatic activities are presented as means of quadruplicate measurements with the standard error of the mean and are expressed relative to both the protein content and to the wet mass of the tissue. To compare metabolic activity levels among different shes and tissues (Childress and Somero, 1979) with various protein and water contents (Childress and Nygaard, 1973), the data were presented as a function of wet mass because this standardization procedure seems to be the most appropriate to analyze these results as a function of depth. This standardization procedure indicates the metabolic activity per unit of living tissue. However, for the activities of antioxidative enzymes, standardization to protein content seems to be more appropriate because the antioxidative status of the cell is linked to cellular targets (lipids, proteins, DNA) and not to the size, mass or water content of the animal. This standardization procedure indicates priorities for the organism in the synthesis of antioxidative proteins within the context of their global protein synthesis. Accordingly, expressing activity with respect to protein content appears to be most appropriate for comparing enzyme activities. Simple linear regressions were carried out on logtransformed data, as for previous studies on these shes (Childress and Siebel, 1998). The gures are, however, semilogarithmic plots. Condence intervals on the regression coefficients are at the 95 % level; P values are from F-tests. Results Protein content and metabolic activity of sh tissues Our measurements conrmed the previously reported exponential decrease in the protein content (Table 2) of the white muscle with increasing minimum depth of occurrence (Childress and Nygaard, 1973). Interestingly, similar relationships were found in the eyes, liver, stomach and skin (Table 2). The previously reported (Childress and Somero, 1979) exponential decrease in the metabolic activity in the white muscle, as revealed by CS activity, with increasing minimum depth of occurrence was also conrmed (Table 2)

3720 B. J. JANSSENS AND OTHERS

Citrate synthase activity (munits g1 wet mass)

Table 2. Stastically signicant relationships between biochemical variables and the minimum depth of occurrence in tissues of meso- and bathypelagic shes collected off California
P a bS.D. Protein content (mg g1 wet mass) versus minimum depth of occurrence (m) Eyes 0.0228 196.37 0.320.12 Liver 0.0090 411.58 0.410.14 Stomach 0.0062 259.82 0.490.15 White muscle 0.0580 85.63 0.360.18 Skin 0.0277 533.79 0.680.27 Citrate synthase activity (munits g1 wet mass) versus minimum depth of occurrence (m) Eyes 0.0037 16 481 0.640.18 Stomach 0.0349 34 544 0.620.27 White muscle 0.0406 6 701 0.430.19 Superoxide dismutase activity synthase activity (munits mg1 protein) Brain 0.0004 0.57 Gills 0.0126 0.17 Liver 0.0021 2.29 Stomach 0.0100 2.20 Kidney 0.0126 0.90 Red muscle 0.0466 0.10 White muscle 0.0001 0.19 Glutathione peroxidase activity synthase activity (munits mg1 protein) Brain 0.0076 2.43 Gills 0.0140 4.01 Liver 0.0003 6.62 Stomach 0.0041 8.58 Kidney 0.0073 9.39 White muscle 0.0055 0.82 (units mg1 protein) versus citrate 0.910.18 1.140.04 0.700.18 0.510.17 0.830.25 1.110.24 1.100.20 versus citrate 0.970.30 0.780.27 0.920.18 0.570.16 0.840.22 0.970.29

104

a d b

103

c g f e

k j

n m o l p

102

h i

200 400 600 800 1000 1200 1400 Minimum depth of occurrence (m)

Fig. 1. Exponential relationship between the citrate synthase (CS) activity (munits g1 wet mass) of the eyes and the minimum depth of occurrence (MDO) (m) of meso- and bathypelagic shes: CS activity=16 841MDO0.64. Each letter represents one species (see Table 1). Values are means S.E.M. (N=4).

(munits mg1 protein)

associated with light organs, when present, showed a signicantly higher CS activity (P<0.05). The size of the sh could be correlated neither with the minimum depth of occurrence nor with CS activity. Superoxide dismutase Although SOD activity was detected in all tissues from all species, large differences were observed among tissues and species. The highest mean SOD activity was detected in the photophores (416.40 units mg1 protein), while the lowest levels were found in the eyes (16.46 units mg1 protein). Variations of up to three orders of magnitude were found in a single organ. For example, the SOD level in the liver of the deepest-living species, Eurypharynx pelicanoides (minimum depth of occurrence 1300 m), was 1.02 units mg1 protein, but it was 175.72 units mg1 protein in Argyropelecus affinis (minimum depth of occurrence 200 m). Although there appeared to be a trend for SOD activity (units g1 wet mass) to decrease with increasing depth of occurrence, attempts to link SOD activity to either the minimum depth of occurrence or the ambient oxygen concentration did not yield signicant relationships. In contrast, the activity of SOD was tightly correlated to CS activity in most tissues and organs (Fig. 2). The relationship is best described as an exponential increase in SOD activity (units mg1 protein) with increasing CS activity (munits mg1 protein) following the equation SOD activity =a(CS activity)b, with b ranging from a minimum 0.51 in the stomach to more than 1 in the muscles and gills (Table 2). Although high CS activity was always associated with high levels of SOD in the tissues, large variations in SOD activity were observed at low CS levels (<25 munits mg1 protein), suggesting that other factors could be important in determining

Glutathione peroxidase activity (munits mg1 protein) versus superoxide dismutase activity (units mg1 protein) Eyes 0.0027 3.23 1.050.28 Brain 0.0001 3.97 1.090.18 Gills 0.0122 22.65 0.430.15 Liver 0.0003 7.69 0.950.20 Stomach 0.0001 5.42 0.970.09 Kidney 0.0278 26.31 0.700.25 White muscle 0.0001 3.56 0.870.16 Skin 0.0021 9.12 0.800.20 All relationships are described by y=axb. P, probability.

and was also demonstrated in the eyes (b=0.64; Fig. 1) and the stomach (b=0.62). Although CS activity was detected in all the tissue of all species, some activities tended to be very low when expressed as a function of protein content or wet mass. Among species, the level of CS in the liver of the deepest-living species, Eurypharynx pelicanoides (minimum depth of occurrence 1300 m), was only 2.5 % of that assayed in the myctophid Symbolophorus californiensis, a species reaching the surface. Compared with all other tissues, the skin

Enzymatic antioxidative defence in deep-sea sh 3721

n d Superoxide dismutase activity (units mg1 protein) 100 m o i 10 k f c h b e n j g l Glutathione peroxidase activity (munits mg1 protein) a 1000 o g m 100 i 10 c k p h e f l j d a

p 0 50 100 150 200 250 300 350 Citrate synthase activity (munits mg1 protein)

50

100

150

200

250

300

350

Citrate synthase activity

(munits mg1 protein)

Fig. 2. Exponential relationship between superoxide dismutase (SOD) (units mg1 protein) and citrate synthase (CS) activities (munits mg1 protein) in the liver of meso- and bathypelagic shes: SOD activity=2.29(CS activity)0.70. Each letter represents one species (see Table 1). Values are means S.E.M. (N=4).

Fig. 3. Exponential relationship between glutathione peroxidase (GPX) (munits mg1 protein) and citrate synthase (CS) activities (munits mg1 protein) in the liver of meso- and bathypelagic shes: GPX activity=6.62(CS activity)0.92. Each letter represents one species (see Table 1). Values are means S.E.M. (N=4).

SOD activity when the metabolic rate is low. As an example, it can be seen that SOD levels in the liver of Scopelengys tristis and Parvilux ingens are higher than would be expected from their CS activity. Glutathione peroxidase The activity of GPX shows a very similar pattern to that of SOD, i.e. large variations among tissues and species and a dependence on the metabolic activity as measured by CS activity. The highest activities were again associated with the photophores (P<0.05 compared with all other tissues), with levels (9308.87 munits mg1 protein) far higher than in the liver, which ranked second (541.62 munits mg1 protein). Very low levels were measured in the eyes (68.18 munits mg1 protein) and the red muscle (71.45 munits mg1 protein). Variations in activity of three orders of magnitude were observed in most tissues. In the liver, the GPX activity measured in Eurypharynx pelicanoides (minimum depth of occurrence 1300 m) reached 39.3 munits mg1 protein, while the level in Symbolophorus californiensis (minimum depth of occurrence 10 m) was 1522.76 munits mg1 protein. The relationship between GPX activity (munits mg1 protein) and CS activity (munits mg1 protein) was very similar to that described between SOD and CS, with GPX activity=a(CS activity)b and with b ranging from a minimum of 0.57 in the stomach to 0.97 in the white muscle and brain (Fig. 3; Table 2). Here again, high CS activity was associated with high levels of the antioxidative enzyme while large variations in GPX activity were found at low CS levels (<25 munits mg1 protein), suggesting that other factors also affect GPX activity when metabolic rate is low.

The similar evolution of SOD with metabolic activity (units mg1 protein) and GPX activity (munits mg1 protein) is further illustrated by the existence of a signicant relationship, GPX activity=a(SOD activity)b, linking the activities of the two antioxidative enzymes in most tissues (Table 2; Fig. 4). The value of the exponent b, which characterizes the relationships, is close to 1 in most tissues (eyes, brain, liver, stomach, kidney, white muscle and skin), indicating a linear
n 1000 Glutathione peroxidase activity (munits mg1 protein) o g m e k f i h c j a d

l 100 p 10

50 100 150 Superoxide dismutase activity (units mg1 protein)

200

Fig. 4. Exponential relationship between glutathione peroxidase (GPX) (munits mg1 protein) and superoxide dismutase (SOD) activities (units mg1 protein) in the liver of meso- and bathypelagic shes: GPX activity=7.69(SOD activity)0.95. Each letter represents one species (see Table 1). Values are means S.E.M. (N=4).

3722 B. J. JANSSENS AND OTHERS

relationship. In the gills, where b is only 0.43, it appears that the activity of SOD increases faster than that of GPX (Table 2). Catalase Contrary to what was observed for SOD and GPX, no relationship between CAT and CS activity could be found in any tissue whether data were expressed as a function of the wet mass or of the protein content. Although large variations occurred among tissues, the activity of CAT in a single tissue varied little among species. In most tissues, the highest activity of CAT was found in the liver, representing 3499 % of the total CAT activity of the sh. The lowest activity was found in the white muscle, where its content varied from 0.01 to 0.64 % of the total CAT activity. It is interesting to note that skin containing photophores has a higher CAT activity than skin lacking these luminous organs; this relationship was independent of the minimum depth of occurrence of the sh. Discussion The decline in metabolic activity of pelagic shes and crustaceans with increasing minimum depth of occurrence has been reported in many studies (for a review, see Childress, 1995), and has also recently been observed in pelagic cephalopods (Siebel et al., 1997). Many variables could be implicated in such a decline: reduced residual light, temperature, available nutrients, the low-oxygen environment or increased pressure, among others. Childress (1995) reviewed these variables and refuted their possible implications in the evolution of metabolic rates. According to Childress (1995), the lower metabolic rates of deep-sea shes, crustaceans and cephalopods are functionally adaptive for life in the deep sea and the oxygen minimum layer and are not specically evolved adaptations to the low food availability or low O2 levels in these habitats. Several lines of evidence, including the observation that no decrease in metabolic activity occurs in the brain of deep-sea shes although such a decrease can be measured in white muscles, have led to the hypothesis that a decrease in visual prey/predator interactions was the key factor in the evolution of lower metabolic rates at depth, through a reduction in the locomotor abilities needed for foraging. This view is consistent with observations on chaetognaths (Thuesen and Childress, 1993) and medusae (Thuesen and Childress, 1994), which show no decrease in metabolic activities with increasing depth of occurrence. Our results conrm previous data indicating that the metabolic activity of the brain does not decrease with increasing depth of occurrence (Childress and Somero, 1979). Interestingly, the present work indicates that the decline in metabolic activity occurs not only in the muscles of sh but also in their eyes, a result that could support the above model. However, the reduction in ocular CS activity seems to take place at a faster rate (b=0.64) than the reduction in the overall metabolic rate when data from all tissues are combined (b=0.43), a result that might be related to the rapid decline in ambient light levels with increasing depth. Such a decline would certainly decrease the operational cost of the visual system. Also, the depth-related decline in CS activity in the stomach may indicate that the metabolic needs of tissues involved in the digestion of prey could have adjusted to those of the energy-consuming locomotory system. This result suggests that selection could have favored low-energyconsuming digestion processes acting over longer periods in a context of lower densities of prey with a lower nutritional value. Whatever the exact reasons for these declines in metabolic activity in pelagic organisms could be, the present data demonstrate that these reduced metabolic requirements are associated with lower activities of two essential antioxidative enzymes, superoxide dismutase and glutathione peroxidase. The decline in the activities of both enzymes cannot be ascribed to some experimental artifact associated with an excessive assay temperature favoring enzymes from species thriving in warmer conditions, i.e. near the surface. Because of the dependency of the reaction between SOD and O2 on diffusion rate, SOD is little affected by temperature. Issels et al. (1986) demonstrated that SOD activity increased by a factor of only 1.6 when the temperature was raised from 20 to 60 C. In any case, experimental procedures limited the exposure of the enzymes to 25 C to a maximum of 15 min. Controls indicated that the exposure of these enzymes extracted from the deepest-living species to 25 C for 15 min did not affect their activity. Also, the possible interference of the assay temperature with the determination of CS activity seems very unlikely because the results are very similar to those reported in other studies in which this assay was carried out at a lower temperature (Childress and Somero, 1979; Childress, 1995). If one considers that the reduced CS activity with increasing depth could be accomplished by reduced numbers of mitochondria rather than reduced enzymatic efficiency or reduced quantities of enzymes within mitochondria, the possibility arises that the reduced activities of SOD and GPX could simply reect the attenuation of mitochondrial densities. However, although both antioxidant enzymes are partly localized within mitochondria, their reduced activities with decreasing CS activity cannot be explained by their intracellular distribution within cells. Indeed, mitochondrial Mn-based SOD activity of vertebrates accounts for only 1020 % of the total SOD activity, while the remainder represents Cu,Zn-SOD located in the cytosol (McCord and Fridovich, 1988). In the case of GPX, several forms coexist within cells, but only 10 % of the total activity is located in the mitochondrial matrix (Meister, 1995). If one assumes that the reduction in CS activity with increasing depth corresponds exclusively to a decreasing mitochondrial density within cells, then this could account only for a maximum reduction of 1020 % in both SOD and GPX activity. Our results indicate a far more drastic reduction in activity of both enzymes. To support this, differential assays of Mn-SOD and Cu,Zn-SOD were performed according to the method of Nakano (1990) on liver homogenates from the shes described in this study. These revealed that Cu,Zn-SOD represents 75.915.74 % of

Enzymatic antioxidative defence in deep-sea sh 3723

the total SOD activity in the liver of surface-living sh, 92.139.39 % in sh living between 250 and 500 m, and 84.238.96 % (means S.E.M., N=4) in deeper living species. These data are consistent with results obtained for other marine sh. For example, Cu,Zn-SOD in the liver of saithe and mackerel represents 83 % and 93 % of the total SOD content, respectively (Aksnes and Njaa, 1981). The correlation between the decline in both SOD and GPX activity and oxidative metabolism indicates that levels of both antioxidant enzymes were adjusted to the endogenous source of ROS. Since superoxide is known to be produced continuously in all aerobic cells during the transfer of electrons in mitochondria, a lower rate of oxygen consumption would be expected to reduce the rate of O2 production and, thus, lower requirements for the corresponding detoxication systems. Similarly, the decreased production of superoxide together with a reduction in the lipid content should reduce the risks of formation of H2O2 and lipid peroxides, thus diminishing the requirement to maintain high GPX activities in the tissues. The analysis of the possible role of exogenous sources of ROS and the inuence of the oxygen content of the sea water will require further studies of populations thriving in different geographical areas at different depth and oxygen concentrations. It is interesting to note that three of the deepest-living species studied, Scopelengys tristis (650 m), Parvilux ingens (700 m) and Bajacalifornia burragei (1000 m), while showing a very low CS activity, contained higher levels of SOD and GPX than could be expected from the relationships described (Figs 2, 3). This suggests that other sources of ROS might be present in these deep-living species. It is well known that many pollutants present in aquatic systems, such as organochlorines, exert toxic effects related to oxidative stress. In response to increasing levels of organic xenobiotics, contaminated animals show an induction of mono-oxygenases (Stohs, 1995), such as cytochrome-P450-associated ethoxyresorun-O-deethylase (EROD), and higher activities of antioxidative enzymes (Rodriguez-Ariza et al., 1993; Bainy et al., 1996). Kramer et al. (1984) showed that the amount of unmetabolized 4,4dichlorophenyltrichloroethane (4,4-DTT) and other pollutants in deep-sea benthic shes is far higher than that found in surface-living sh. Some deep-sea sh appear to contain approximately 50 times more polychlorinated biphenyls (PCB) than surface-living sh. In addition, very high activities of EROD and other xenobiotic-metabolizing enzymes have been detected in deep-sea sh (Frlin et al., 1995). Thus, it is possible that the high antioxidative activities found in some pelagic deep-sea species could be linked to the oxidative stress due to pollutants contaminating the deep-sea biota. Considering the above statements, it is very surprising that the activity of CAT, which catabolizes the production of oxygen and water from H2O2, remained unchanged while metabolic rates declined with increasing depth of occurrence. In epipelagic shes, close positive correlations were found between the CAT and SOD activity of most tissues (Filho et al., 1993), and CAT activity would have been expected to decrease in the same manner as does SOD activity. Several hypotheses can be proposed for this unexpected observation. First, it could indicate that the dangers associated with H2O2 do not decrease with increasing depth of occurrence and, while the endogenous sources undergo a rapid decline, the activity of CAT could be essentially devoted to the elimination of exogenous H2O2 diffusing into the sh tissue. It is remarkable that the activity of CAT does not vary with the depth, while that of GPX shows a rapid decline to very low levels, although both enzymes are involved in the detoxication of peroxides. Since GPX can decompose H2O2 and lipidic peroxides while CAT is devoted solely to the catabolism of H2O2, it is surprising that the reduced enzyme activity is that detoxifying the wider range of ROS. The possible reason for this apparent preference for CAT might be related to the limited resources available in the deep sea. Indeed, CAT requires neither cofactors nor energy for its activity, while GPX consumes glutathione, which is oxidized upon reaction with GPX and must then be recycled by a NADPH-consuming enzyme (glutathione reductase). In an environment characterized by a decline in the resources available, favoring the least energyconsuming enzyme could be an efficient strategy, in particular when the total amount of lipid targets is decreased. In a preliminary series of experiments, we assayed H2O2 in water samples taken nightly at depths of 250, 700 and 1200 m. Although the H2O2 content of the sea water increased from 250 to 700 m, levels remained much lower (31011 to 2.81012 mol l1) than those reported for surface waters (Van Baalen and Marler, 1966) and could, therefore, probably not account for the high CAT activities in deep-living species. A second reason could lie in the possible exposure of the sh to important occasional oxidative stress events. The respective shares of the two enzymes in the catabolism of H2O2 could explain the maintenance of CAT activity. It has been shown in mammalian cells that, although CAT decomposes H2O2 very efficiently, the enzyme is active only at rather high H2O2 concentrations (Chance et al., 1979). Thus, CAT normally plays a relatively minor role in the catabolism of H2O2 at low rates of H2O2 generation (Jones et al., 1981), but becomes indispensable when the rate of H2O2 production is enhanced, such as during oxidative stress. Godin and Garnett (1992) demonstrated that a low GPX activity could be compensated by a high CAT activity, the activities of these two enzymes being negatively correlated in various mammalian species. So, it is possible that GPX would maintain normal cell function and adjust to the metabolic activity of the cell, while CAT would be part of a stress response mechanism regardless of the low metabolic rate or the oxygen concentration in the surrounding water. One more interesting nding is that the light organs show signicantly higher activities of the three antioxidative enzymes and of CS, while their protein content is similar to that of skin lacking photophores. It is possible that these high levels of antioxidative defense in the light organs are devoted to catabolizing ROS generated by the high metabolic activity that also characterizes this tissue. However, the observation

3724 B. J. JANSSENS AND OTHERS

that CAT level is also higher than in skin lacking photophores suggests that this antioxidative arsenal could be related to the bioluminescent reactions taking place in these organs. These reactions may involve radical intermediates and ROS, which could be potentially harmful to the cells. Also, the luciferin in these sh, coelenterazine or some derivatives, is very sensitive to oxygen and ROS (Rees et al., 1998), and an elevated antioxidative status could therefore help preserve the dietaryacquired luciferin against spontaneous autoxidation. The high metabolic activity of the light organs may be related to the occurrence of energy-consuming inhibitory mechanisms maintaining luminous cells in a resting state, as described previously in the bioluminescent sh Porichthys notatus (Rees and Baguet, 1987, 1989). This research was supported by the UCL Scientic Development Fund and the FRIA (National Scientic Research Fund) (B.J.J.) and by NSF grant OCE 9415543 to J.J.C.. J.-F.R. is senior researcher associate of the Belgian Fonds National de la Recherche Scientique. References
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