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Cold adaptation and oxidative metabolism of Antarctic fish

Cold adaptation and oxidative metabolism of Antarctic fish Ital. J. Zool., SUPPLEMENT 1: 33-36 (2000) INTRODUCTION Cold adaptation and oxidative metabolism of Antarctic fish Studies of organisms living under the selective pres- sure of extreme climatic enviroments may provide in- sight into the basic mechanisms of thermal adaptation. ANNALISA COLELLA In this perspective, the Antarctic enviroment may be considered a sort of 'natural laboratory' where the biol- Istituto di Chimica e Chimica Clinica, Università Cattolica del Sacro Cuore "Agostino Gemelli", ogy and molecular basis of cold adaptation can be stud- largo F. Vito 1, I-00168 Roma (Italy) ied in endemic marine species. The temperature of the Antarctic Ocean, in fact, is MARIA PATAMIA constantly at -1.86° C, the equilibrium temperature of Centro del CNR per la Chimica dei Recettori sea water and ice. This is a condition which would be e delle Molecole biologicamente attive, lethal for temperate and tropical fish. However, Antarc- Università Cattolica del Sacro Cuore "Agostino Gemelli", Facoltà di Medicina, tic fishes have been forced to become highly special- largo F. Vito 1, I-00168 Roma (Italy) ized in facing cold conditions (Somero & De Vries, 1967), developing a number of physiological and bio- ANTONIO GALTIERI chemical adaptation mechanisms. One of the main char- Dipartimento di Chimica Organica e Biologica, Università di Messina, acteristics of their biochemistry is the synthesis of freez- Salita Sperone 31, I-98166 Messina (Italy) ing-point depressing molecules (antifreeze molecules), which protect the organism from ice crystal formation in BRUNO GIARDINA a non-colligative way (De Vries, 1988). A further aspect Istituto di Chimica e Chimica Clinica, is the modification of some hematological characteristics Università Cattolica del Sacro Cuore "Agostino Gemelli", and such as a markedly reduced level of hematocrit, with Centro del CNR per la Chimica dei Recettori the extreme case represented by the family of Chan- e delle Molecole biologicamente attive, nichthyidae which completely lack erythrocytes and he- Università Cattolica del Sacro Cuore "Agostino Gemelli", moglobin (Hemmingsen & Douglas, 1970; Hureau et al., Facoltà di Medicina, largo F. Vito 1, I-00168 Roma (Italy) 1977, Wells et al., 1980). This finding has been ex- plained considering that the very low temperature of sea water would involve a great increase of blood vis- cosity which, in order to facilitate the cardiac work, is counteracted by a significant decrease in the number of erythrocytes and therefore of hemoglobin. Other peculiar and important characteristics of these fishes are their preferential utilization of the lipid meta- ABSTRACT bolic pathway (Sidell, 199D and the increase of mito- A well known characteristic of the oxidative metabolism of chondrial oxygen consumption (Van den Thillart & Antarctic fishes is their preferential utilization of the lipid metabol- Modderkolk, 1978; Van den Thillart & De Bruin, 1981; ic pathway. Since oxidative metabolism may lead to a significant Dahlhoff et al, 199D. production of oxygen-derived free radicals, which may have sev- eral harmful effects on biological structures, it seemed worthwhile All these biological characteristics appear to be an ex- to investigate the presence, in various tissues from different treme case of cellular and molecular adaptation to low Antarctic fishes, of those ions and molecules which are known to temperature since Antarctic fishes die when the water tem- be at the basis of the antioxidant defense mechanisms. Hence, the perature rises by only a few degrees centigrade. Hence, we Coenzyme Q and trace elements (selenium, copper, and zinc) may say that the fitness of Antarctic fishes to their enviro- content of tissues from several Antarctic fishes was determined and compared with that of non-Antarctic species. Unlike temper- ment is paid for by an extreme degree of stenothermality, ate fishes, in which the Coenzyme Q is Q , all the Antarctic 10 and therefore by a significant loss of biological flexibility. species examined displayed the homolog Q form. This particular Given the observed preferential utilization of the lipid finding was related to the difference in the crystallisation temper- metabolic pathway, we focused our attention on the ature existing between Coenzyme Q and Coenzyme Q . Taken 10 9 together, the results indicate a higher level of antioxidant defenses presence of copper, zinc, and selenium (with respect to for Antarctic species, with respect to temperate fishes. This may Superoxide dismutase, glutathione peroxidase, and cata- be considered a stimulating basis for further studies on the adap- lase) and on the concentration of coenzyme Q (CoQ) in tation mechanisms centered on the oxidative metabolism of or- various tissues of some Antarctic fishes. Moreover, we ganisms living in extreme environments. investigated in some detail the activity of NADH oxi- dase and NADH coenzyme Q reductase, enzymes in- KEY WORDS: Cold adaptation - Oxidative metabolism - Antiox- volved in mitochondrial respiration. idant defenses. MATERIALS AND METHODS ACKNOWLEDGEMENTS This research is part of the Italian National Programme for Specimens were collected in the vicinity of Terra Nova Bay Antarctic Research (PNRA). (Ross Sea) and of Palmer Station (Antarctica Peninsula). All the 34 A. COLELLA, M. PATAMIA, A. GALTIERI, B. GIARDINA samples were stored at -80° C until analysis. Mitochondria from fish liver (-15 g) were prepared, after homogenization of tissue 4.0- with a Potter-Hevelheim homogenizer, by gradient centrifugation in H-medium (0.7 M sucrose, 0.21 M D-mannitol, 0.002 M Hepes, Bis(trimethylsilyl)acetamide (BSA) 0.05% w/v BSA), as reported by Pedersen et al. (1978). Aliquote of homogenate were used to determine coenzyme Q, NADH oxidase, NADH CoQ reductase, > 3.5- vitamin E, selenium, zinc, and copper . To determine coenzyme Q, the homogenat e was treated with 0.25 M sodium dodecylsulphate (SDS). Proteins were then precip- itated with 2 ml of a mixture of ethanol-isopropanol (95:5), and 3.0- coenzyme Q was extracted with 5 ml of exan. The extract was then evaporated to dryness under a stream of nitrogen, redis- solved in 100 ml of ethanol and chromatographed on a HPLC ap- paratus (Beckmann). Vitamin Ε wa s measured with the method of Ericson & Soerensen (1977). 2.5· For trace elements determination, an aliquot of homogenate 0.0033 0.0034 0.0035 0.0036 was digested in acid medium with a microwave system (MLS-1200 Mega Milestone s.r.l. Bergamo, Italy). Zinc and coppe r were de- 1/T (Κ"1) termined with Computerized Stripping Potentiometry using the "Tracelab™" (Radiometer A/S Copenhagen-Denmark) as de - Fig. 1 - Arrhenius plot of NADH oxidase activity for liver mito- scribed by Gozzo et al. (1993, 1995); selenium was measured with chondria of Chaenocephalus aceratus (filled circle) and Onco- the same apparatus but in Constant Current Stripping Analysis as rynchus mykiss (filled triangle) expressed in terms of V described by Chi Hu a et al. (1987). Proteins were determined max (nmol/min/mg of proteins). with the method of Lowry et al. (1951). The overall activity of the mitochondrial respiratory chain was tested as NADH oxidase ac- tivity, as reported by Mackler (1961). ture increases. In fact, comparison with the data ob- Furthermore, the activity of the first and third respiratory chain tained in the case of trout is astonishing. In addition, at complexes was evaluted since they are mor e substrate specific and species-representative. The first complex was measured by 2° C, the V values of the enzymatic systems investi- ma x testing the activity of NADH CoQ reductase with the metho d of gated appear significantly higher in icefish with respect Youssef (1967), and th e third complex by testing that of NADH to those obtained for trout (V NADH oxidase / cytochrome c reductase according to Youssef & Stiggall (1967). ma x icefis h V NADH oxidase £ 2, and V NADH CoQre- ma x trou t ma x ductase / V NADH CoQreductase = 3.5). icefis h ma x trou t On the whole, these results indicate that the biological RESULTS activity of mitochondria is one of interesting functional and structural adaptations which still remains a fruitful The effect of temperature changes on mitochondrial area of investigation. oxygen consumption exhibits a common pattern in all Another effect of cold adaptation is shown by the de- species: the rate of respiration increases, as a function termination of antioxidant agents, which have to inacti- of temterature, up to a certain value whereupon any vate the reactive oxygen species produced by oxidative further increase leads to a sharp reduction in respiration metabolism. Our study was carried out on various tis- rate. This pattern is shown most clearly when data are presented in Arrhenius plots. The temperature at which the sharp change in slope is observed is termed the Ar- Η.Ό· rhenius break temperature (ABT) and appears to be linked to the habitat temperature of species. Although the biochemical mechanisms underlying the ABT phe- y 4.0- y nomenon per se are not fully understood, disruption of lipid-protein interactions may be partially responsible for ABTs. S 3.5- \ The strong stenothermic character of Antarctic fishes has been well brought out by recent experiments on mitochondria from the icefish Chaenocephalus aceratus. 3.0- As is evident from the data reported in Figures 1 and 2, ^ \ in the case of C. aceratus the Arrhenius plot obtained for the activity of NADH oxidase, as well as that of 2.5- NADH coenzyme Q reductase, is reversed up to 2° C 0.0033 0.0034 0.0035 0.0036 indicating that, within the range of temperature exam- ined, we are to the left of the ABT of the systems under (Κ"1) 1/T investigation. Even if on the basis of the present data Fig. 2 - Arrhenius plot of NADH Coenzyme Q reductase activity we do not know the precise temperature of the break for liver mitochondria of Chaenocephalus aceratus (filled circle) point, it seems in the proximity of 2° C, which points to and Oncorynchus mykiss (filled triangle) expressed in terms of the fragility of these systems with respect to tempera- V (nmol/min · mg of proteins"1). ma x COLD ADAPTATION IN ANTARCTIC FISH sues of six Antarctic fishes, three nototheniids (Pagothe- higher in all the others. Coenzyme Q content reaches its nia bernacchii, Notothenia coriiceps, Notothenia gibber- maximum value in Notothenia coriiceps, the most active ifrons) and three channichthyids (Chionodraco hama- Antarctic species: here, in fact, its value is about five tus, Chaenocepbalus aceratus, Champspcephalus gun- times higher than that of Mugil cephalus and about nari); the same types of experiments were performed three times higher than that of the other Antarctic fishes. on tissues of two non-Antarctic fishes {Mugil cephalus, As far as the other parameters are concerned, the lev- Oncocbynchus mykiss). els of selenium and zinc seem to be of particular signif- icance since they show values from four to five times The obtained results show an overall increase of the higher with respect to Mugil cephalus. antioxidant defenses, as reported in Tables I and II. The main feature immediately apparent is the different form of coenzyme Q which characterizes the Antarctic fishes; in fact, while temperate fishes such as Mugil cephalus DISCUSSION are generally characterized by the presence of CoQ , this cofactor is substituited by C0Q9 in both nototheni- Previous studies (Eastman & De Vries, 1982) have ids and channichthyids. pointed out that the substantial corporeal accumulation Table I shows the plasma levels of Coenzyme Q and of lipid in Antarctic fishes is not only linked to buoyan- vitamin Ε observed in the selected Antarctic species in cy regulation but also to specific requirements of ener- comparison with those of Oncochynchus mykiss. The gy metabolism. Moreover, besides these fundamental content of both Coenzyme Q and vitamin Ε is higher in roles, other aspects, and not minor ones, have to be all Antarctic fishes, the level of the latter in plasma from considered. These are based upon the observation that Notothenia coriiceps, about 12 times higher than in tem- some temperate-zone fishes display a substantial accu- perate fish, being particularly impressive. mulation of intracellular lipid during cold acclimation (Egginton & Sidell, 1989). Hence, on the basis of this In Table II, the levels of Coenzyme Q, vitamin E, sele- observation and of the combined solubility and diffu- nium, zinc and copper in the muscle tissues of Antarctic siveness characteristics of oxygen in lipids, two other species are reported in comparison with those of the additional roles of intracellular lipid inclusions in the temperate marine fish Mugil cephalus. The level of oxygen economy of oxidative tissues of Antarctic fish Coenzyme Q is comparable to that of Mugil cephalus have been hypothesized. These may be summarized as only in the case of Pagothenia bernacchii, being always TABLE I - Coenzyme Q and Vitamin Ε content of plasma, expressed as ng/mg of proteins, in Antarctic fishes and in a temperate fresh- water fish (the results are means of three determinations on three different specimens for each species) Oncohynchus Notothenia Notothenia Champsocephalus coriiceps gibberifrons mykiss gunnari CoQ (ng/mg prot.) 0.7 ± 0.4 1 0 — 0.4 CoQ (ng/mg prot.) 1.9 + 1.6 ± 0.4 3.5 ±0. 5 VitaminE (ng/mg prot.) 11.0 ± 2.1 142.0 40.0 ±6.1 17.9 ±8.3 ±3. 3 TABLE II - Coenzyme Q and trace elements content of muscle tissues, expressed as ng/mg of proteins, of Antarctic fishes and one Mediter- ranean fish (the results are means of three determinations on three different specimens for each species). Mugil Chionodraco Pagothenia Notothenia Chaenocephalus cephalus hamatus coriiceps aceratus bernacchii io (ng/mg prot.) 36.0 ± 8.1 CoQ (ng/mg prot.) 152 .0 54.4 ±8. 3 27.5 ±6. 1 ±8.6 53.6 ±7.1 VitaminE (ng/mg prot.) 32.2 86.0 27.6 ±5.4 2 34.6 ±6. 3 ±9. 1 73. ±9.3 ±6.3 Copper (ng/mg prot.) 3.0 ±0. 5 ± 2.4 5.2 ± 1.4 5.9 Selenium (ng/mg prot.) 9.0 ±3.7 48.0 ±9.7 35.0 ±9.3 Zinc (ng/mg prot.) 2.2 ±0. 6 ±2.4 8.5 6.6 ±4.3 A. COLELLA, M. PATAMIA, A. GALTIERI, B. GIARDINA De Vries A. L., 1988 - The role of antifreeze glycopeptides and follows: a) the presence of lipid droplets may accelerate peptides in the freezing avoidance of Antartic fishes. Comp. the rate of oxygen diffusion between capillaries and mi- Biochem. Physiol., 90B: 539-545. tochondria; b) intracellular lipid may function as a ma- Eastman J. T., De Vries A. L., 1982 - Buoyancy studies of notothe- jor oxygen store, damping out otherwise large varia- nioid fishes in McMurdo Sound, Antarctica, Copeia, 1982: 385- tions in oxygen supply to mitochondria during changes 393. Egginton S., Sidell B. D., 1989 - Thermal acclimation induces in muscular activity. adaptive changes in subcellular structure of fish skeletal mus- These peculiar characteristics, together with the cle. Am. J. Physiol., 256: R1-R9. greater activation of oxidative metabolism could be at Ericson T., Soerensen B., 1977 - High performance liquid chro- the basis of an increased production of lipoperoxides matography of vitamine Ε. Acta Pharmac. Suec , 14: 478-483. Gozzo M. L., Lippa S., Barbaresi G., Morosi R., 1993 - Computer- and oxygen-derived free radicals. On the whole, Antarc- ized stripping potentiometry applied to copper determination tic fishes, in comparison with temperate ones, have to in plasma and urine samples. In: Trace Elements and Free Rad- cope with a higher oxidative stress which could be very icals in Oxidative Diseases. Chamonix, April 5th-9th, p. 92. harmful for some of their biological structures. This hy- Gozzo M. L., Lippa S., Colacicco L., Callà C., Barbaresi G., Giardi- pothesis seems to be supported by data presented here na B., 1995 - Computerized stripping potentiometry applied to zinc determination in plasma and urine samples. In: Fourth In- showing an overall increase of antioxidant defences in ternational Congress of International Society for Trace Elements Antarctic fishes. Research in Humans. Taormina (Italy), Sept. 25th-28th, p. 132. Another important feature of Antarctic fishes is the Hemmingsen Ε. Α., Douglas E. L., 1970 - Respiratory and circula- tory adaptations to the absence of hemoglobin in Chan- presence of the Q form of coenzyme Q, instead of the nichthyid fishes. In: G. A. Llano (ed.), Adaptations within Q form normally found in cells from temperate fishes 1 0 Antarctic ecosystems. Smithsonian Inst., Washington DC, pp . as well as in those of most vertebrates. It has been hy- 479-487. pothesized that this could be due to the different crys- Hureau J. C., Petit D., Fine J. M., Marneux M., 1977 - New cyto- tallization temperature of Coenzyme Q homologs (+ logical, biochemical and physiological data on the colqrless blood of the Channichthyidae. In: Adaptation within Antartic 0.5° C for Q and + 9.7° C for Q ). Hence, in the mi- 9 lo ecosystems. Gulf, Houston, Texas, pp. 459-477. croenvironments, in vivo, where crystallization of the Lowry O. M., Rosenbrough N. J., Farr L., Randall R. J., 1951 - Pro- compound cannot be excluded, this could impair the tein measurement with the Folin phenol reagent. J. Biol. Chem., correct functioning of the respiratory chain with great 193: 265-275. trouble resulting for the whole organism. On the basis Mackler B., 1961 - Electron transport particle of yeast. Biochem. Biophys. Acta, 50: 141-145. of this consideration, the presence of Q^ seems the re- Pedersen P. L., Greenwalt J. W., Reynafarje B., Hullihen J., Decker sult of an adaptive strategy by which the efficiency of G. L., Soper J. W., Bustamente E., 1978 - Preparation and char- one of the main metabolic pathways could be main- acterization of mitochondria and submitochondrial particles of tained at a reasonable level. rat liver and liver-derived tissues. Methods Cell. Biol., 20: 411- Moreover, in parallel with these biochemical adapta- Sidell B. D., 1991 - Physiological roles of high lipid content in tis- tions, we have to consider the very narrow temperature sues of Antarctic fish species. In: G. Di Prisco, B. Maresca & Β. range in which the various complexes of the respiratory Tota (eds), Biology of Antarctic Fish. Springer-Verlag, Berlin, chain maintain their biological activity. The stringent Heidelberg, New York, pp. 220-231. Somero G. N., De Vries A. L., 1967 - Temperature tollerance of stenothermality which characterizes the biological activ- some Antartic fishes. Sciences, 156: 257-258. ity of mitochondria of Antarctic fish, and in particular Van den Thillart G., De Bruin G., 1981 - Influence of environmen- the icefish, appears to be the cost that has to be paid tal temperature on mitochondrial membranes. Biochem. Bio- for the impressive fitness of these organisms to their ex- phys. Acta, 640: 439-447. treme environment. Van den Thillart G., Modderkolk J., 1978 - The effect of acclima- tion temperature on the activation energy of state III respiration and on the unsaturation of membrane lipids of goldfish mito- chondria. Biochem. Biophys. Acta, 510: 38-51. REFERENCES Wells R. M. G., Ashby M. D., Duncan S. J., Macdonald J. A., 1980 - Comparative studies of the erythrocytes and haemoglobins in Chi Hua C., Jagner D., Renman L., 1987 - Determination of seleni- nototheniid fishes from Antarctica. J. Fish Biol., 17: 517-527. um by means computerized flow constant current stripping at Youssef H., 1967 - Preparation and properties of NADH: carbon fibre electrodes. Anal. Chim. Acta, 197: 257-264. ubiquinone oxidoreductase (complex I) E.C.I.6.5.3. Methods Dahlhoff E., O'Brien J., Somero G. Ν., Vetter R. D., 1991 - Tem- Enzymol., 10: 13-16. perature effects on mitochondria from hydrothermal vent inver- Youssef H., Stiggall D. L., 1967 - Preparation and properties of tebrates: evidence for adaptation to elevated and variable habi- NADH cytochrome c oxidoreductase (complex I-III). Methods tat temperature. Physiol. Zool., 64: 1490-1508. Enzymol., 10: 9-12. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Italian Journal of Zoology Taylor & Francis

Cold adaptation and oxidative metabolism of Antarctic fish

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Abstract

Ital. J. Zool., SUPPLEMENT 1: 33-36 (2000) INTRODUCTION Cold adaptation and oxidative metabolism of Antarctic fish Studies of organisms living under the selective pres- sure of extreme climatic enviroments may provide in- sight into the basic mechanisms of thermal adaptation. ANNALISA COLELLA In this perspective, the Antarctic enviroment may be considered a sort of 'natural laboratory' where the biol- Istituto di Chimica e Chimica Clinica, Università Cattolica del Sacro Cuore "Agostino Gemelli", ogy and molecular basis of cold adaptation can be stud- largo F. Vito 1, I-00168 Roma (Italy) ied in endemic marine species. The temperature of the Antarctic Ocean, in fact, is MARIA PATAMIA constantly at -1.86° C, the equilibrium temperature of Centro del CNR per la Chimica dei Recettori sea water and ice. This is a condition which would be e delle Molecole biologicamente attive, lethal for temperate and tropical fish. However, Antarc- Università Cattolica del Sacro Cuore "Agostino Gemelli", Facoltà di Medicina, tic fishes have been forced to become highly special- largo F. Vito 1, I-00168 Roma (Italy) ized in facing cold conditions (Somero & De Vries, 1967), developing a number of physiological and bio- ANTONIO GALTIERI chemical adaptation mechanisms. One of the main char- Dipartimento di Chimica Organica e Biologica, Università di Messina, acteristics of their biochemistry is the synthesis of freez- Salita Sperone 31, I-98166 Messina (Italy) ing-point depressing molecules (antifreeze molecules), which protect the organism from ice crystal formation in BRUNO GIARDINA a non-colligative way (De Vries, 1988). A further aspect Istituto di Chimica e Chimica Clinica, is the modification of some hematological characteristics Università Cattolica del Sacro Cuore "Agostino Gemelli", and such as a markedly reduced level of hematocrit, with Centro del CNR per la Chimica dei Recettori the extreme case represented by the family of Chan- e delle Molecole biologicamente attive, nichthyidae which completely lack erythrocytes and he- Università Cattolica del Sacro Cuore "Agostino Gemelli", moglobin (Hemmingsen & Douglas, 1970; Hureau et al., Facoltà di Medicina, largo F. Vito 1, I-00168 Roma (Italy) 1977, Wells et al., 1980). This finding has been ex- plained considering that the very low temperature of sea water would involve a great increase of blood vis- cosity which, in order to facilitate the cardiac work, is counteracted by a significant decrease in the number of erythrocytes and therefore of hemoglobin. Other peculiar and important characteristics of these fishes are their preferential utilization of the lipid meta- ABSTRACT bolic pathway (Sidell, 199D and the increase of mito- A well known characteristic of the oxidative metabolism of chondrial oxygen consumption (Van den Thillart & Antarctic fishes is their preferential utilization of the lipid metabol- Modderkolk, 1978; Van den Thillart & De Bruin, 1981; ic pathway. Since oxidative metabolism may lead to a significant Dahlhoff et al, 199D. production of oxygen-derived free radicals, which may have sev- eral harmful effects on biological structures, it seemed worthwhile All these biological characteristics appear to be an ex- to investigate the presence, in various tissues from different treme case of cellular and molecular adaptation to low Antarctic fishes, of those ions and molecules which are known to temperature since Antarctic fishes die when the water tem- be at the basis of the antioxidant defense mechanisms. Hence, the perature rises by only a few degrees centigrade. Hence, we Coenzyme Q and trace elements (selenium, copper, and zinc) may say that the fitness of Antarctic fishes to their enviro- content of tissues from several Antarctic fishes was determined and compared with that of non-Antarctic species. Unlike temper- ment is paid for by an extreme degree of stenothermality, ate fishes, in which the Coenzyme Q is Q , all the Antarctic 10 and therefore by a significant loss of biological flexibility. species examined displayed the homolog Q form. This particular Given the observed preferential utilization of the lipid finding was related to the difference in the crystallisation temper- metabolic pathway, we focused our attention on the ature existing between Coenzyme Q and Coenzyme Q . Taken 10 9 together, the results indicate a higher level of antioxidant defenses presence of copper, zinc, and selenium (with respect to for Antarctic species, with respect to temperate fishes. This may Superoxide dismutase, glutathione peroxidase, and cata- be considered a stimulating basis for further studies on the adap- lase) and on the concentration of coenzyme Q (CoQ) in tation mechanisms centered on the oxidative metabolism of or- various tissues of some Antarctic fishes. Moreover, we ganisms living in extreme environments. investigated in some detail the activity of NADH oxi- dase and NADH coenzyme Q reductase, enzymes in- KEY WORDS: Cold adaptation - Oxidative metabolism - Antiox- volved in mitochondrial respiration. idant defenses. MATERIALS AND METHODS ACKNOWLEDGEMENTS This research is part of the Italian National Programme for Specimens were collected in the vicinity of Terra Nova Bay Antarctic Research (PNRA). (Ross Sea) and of Palmer Station (Antarctica Peninsula). All the 34 A. COLELLA, M. PATAMIA, A. GALTIERI, B. GIARDINA samples were stored at -80° C until analysis. Mitochondria from fish liver (-15 g) were prepared, after homogenization of tissue 4.0- with a Potter-Hevelheim homogenizer, by gradient centrifugation in H-medium (0.7 M sucrose, 0.21 M D-mannitol, 0.002 M Hepes, Bis(trimethylsilyl)acetamide (BSA) 0.05% w/v BSA), as reported by Pedersen et al. (1978). Aliquote of homogenate were used to determine coenzyme Q, NADH oxidase, NADH CoQ reductase, > 3.5- vitamin E, selenium, zinc, and copper . To determine coenzyme Q, the homogenat e was treated with 0.25 M sodium dodecylsulphate (SDS). Proteins were then precip- itated with 2 ml of a mixture of ethanol-isopropanol (95:5), and 3.0- coenzyme Q was extracted with 5 ml of exan. The extract was then evaporated to dryness under a stream of nitrogen, redis- solved in 100 ml of ethanol and chromatographed on a HPLC ap- paratus (Beckmann). Vitamin Ε wa s measured with the method of Ericson & Soerensen (1977). 2.5· For trace elements determination, an aliquot of homogenate 0.0033 0.0034 0.0035 0.0036 was digested in acid medium with a microwave system (MLS-1200 Mega Milestone s.r.l. Bergamo, Italy). Zinc and coppe r were de- 1/T (Κ"1) termined with Computerized Stripping Potentiometry using the "Tracelab™" (Radiometer A/S Copenhagen-Denmark) as de - Fig. 1 - Arrhenius plot of NADH oxidase activity for liver mito- scribed by Gozzo et al. (1993, 1995); selenium was measured with chondria of Chaenocephalus aceratus (filled circle) and Onco- the same apparatus but in Constant Current Stripping Analysis as rynchus mykiss (filled triangle) expressed in terms of V described by Chi Hu a et al. (1987). Proteins were determined max (nmol/min/mg of proteins). with the method of Lowry et al. (1951). The overall activity of the mitochondrial respiratory chain was tested as NADH oxidase ac- tivity, as reported by Mackler (1961). ture increases. In fact, comparison with the data ob- Furthermore, the activity of the first and third respiratory chain tained in the case of trout is astonishing. In addition, at complexes was evaluted since they are mor e substrate specific and species-representative. The first complex was measured by 2° C, the V values of the enzymatic systems investi- ma x testing the activity of NADH CoQ reductase with the metho d of gated appear significantly higher in icefish with respect Youssef (1967), and th e third complex by testing that of NADH to those obtained for trout (V NADH oxidase / cytochrome c reductase according to Youssef & Stiggall (1967). ma x icefis h V NADH oxidase £ 2, and V NADH CoQre- ma x trou t ma x ductase / V NADH CoQreductase = 3.5). icefis h ma x trou t On the whole, these results indicate that the biological RESULTS activity of mitochondria is one of interesting functional and structural adaptations which still remains a fruitful The effect of temperature changes on mitochondrial area of investigation. oxygen consumption exhibits a common pattern in all Another effect of cold adaptation is shown by the de- species: the rate of respiration increases, as a function termination of antioxidant agents, which have to inacti- of temterature, up to a certain value whereupon any vate the reactive oxygen species produced by oxidative further increase leads to a sharp reduction in respiration metabolism. Our study was carried out on various tis- rate. This pattern is shown most clearly when data are presented in Arrhenius plots. The temperature at which the sharp change in slope is observed is termed the Ar- Η.Ό· rhenius break temperature (ABT) and appears to be linked to the habitat temperature of species. Although the biochemical mechanisms underlying the ABT phe- y 4.0- y nomenon per se are not fully understood, disruption of lipid-protein interactions may be partially responsible for ABTs. S 3.5- \ The strong stenothermic character of Antarctic fishes has been well brought out by recent experiments on mitochondria from the icefish Chaenocephalus aceratus. 3.0- As is evident from the data reported in Figures 1 and 2, ^ \ in the case of C. aceratus the Arrhenius plot obtained for the activity of NADH oxidase, as well as that of 2.5- NADH coenzyme Q reductase, is reversed up to 2° C 0.0033 0.0034 0.0035 0.0036 indicating that, within the range of temperature exam- ined, we are to the left of the ABT of the systems under (Κ"1) 1/T investigation. Even if on the basis of the present data Fig. 2 - Arrhenius plot of NADH Coenzyme Q reductase activity we do not know the precise temperature of the break for liver mitochondria of Chaenocephalus aceratus (filled circle) point, it seems in the proximity of 2° C, which points to and Oncorynchus mykiss (filled triangle) expressed in terms of the fragility of these systems with respect to tempera- V (nmol/min · mg of proteins"1). ma x COLD ADAPTATION IN ANTARCTIC FISH sues of six Antarctic fishes, three nototheniids (Pagothe- higher in all the others. Coenzyme Q content reaches its nia bernacchii, Notothenia coriiceps, Notothenia gibber- maximum value in Notothenia coriiceps, the most active ifrons) and three channichthyids (Chionodraco hama- Antarctic species: here, in fact, its value is about five tus, Chaenocepbalus aceratus, Champspcephalus gun- times higher than that of Mugil cephalus and about nari); the same types of experiments were performed three times higher than that of the other Antarctic fishes. on tissues of two non-Antarctic fishes {Mugil cephalus, As far as the other parameters are concerned, the lev- Oncocbynchus mykiss). els of selenium and zinc seem to be of particular signif- icance since they show values from four to five times The obtained results show an overall increase of the higher with respect to Mugil cephalus. antioxidant defenses, as reported in Tables I and II. The main feature immediately apparent is the different form of coenzyme Q which characterizes the Antarctic fishes; in fact, while temperate fishes such as Mugil cephalus DISCUSSION are generally characterized by the presence of CoQ , this cofactor is substituited by C0Q9 in both nototheni- Previous studies (Eastman & De Vries, 1982) have ids and channichthyids. pointed out that the substantial corporeal accumulation Table I shows the plasma levels of Coenzyme Q and of lipid in Antarctic fishes is not only linked to buoyan- vitamin Ε observed in the selected Antarctic species in cy regulation but also to specific requirements of ener- comparison with those of Oncochynchus mykiss. The gy metabolism. Moreover, besides these fundamental content of both Coenzyme Q and vitamin Ε is higher in roles, other aspects, and not minor ones, have to be all Antarctic fishes, the level of the latter in plasma from considered. These are based upon the observation that Notothenia coriiceps, about 12 times higher than in tem- some temperate-zone fishes display a substantial accu- perate fish, being particularly impressive. mulation of intracellular lipid during cold acclimation (Egginton & Sidell, 1989). Hence, on the basis of this In Table II, the levels of Coenzyme Q, vitamin E, sele- observation and of the combined solubility and diffu- nium, zinc and copper in the muscle tissues of Antarctic siveness characteristics of oxygen in lipids, two other species are reported in comparison with those of the additional roles of intracellular lipid inclusions in the temperate marine fish Mugil cephalus. The level of oxygen economy of oxidative tissues of Antarctic fish Coenzyme Q is comparable to that of Mugil cephalus have been hypothesized. These may be summarized as only in the case of Pagothenia bernacchii, being always TABLE I - Coenzyme Q and Vitamin Ε content of plasma, expressed as ng/mg of proteins, in Antarctic fishes and in a temperate fresh- water fish (the results are means of three determinations on three different specimens for each species) Oncohynchus Notothenia Notothenia Champsocephalus coriiceps gibberifrons mykiss gunnari CoQ (ng/mg prot.) 0.7 ± 0.4 1 0 — 0.4 CoQ (ng/mg prot.) 1.9 + 1.6 ± 0.4 3.5 ±0. 5 VitaminE (ng/mg prot.) 11.0 ± 2.1 142.0 40.0 ±6.1 17.9 ±8.3 ±3. 3 TABLE II - Coenzyme Q and trace elements content of muscle tissues, expressed as ng/mg of proteins, of Antarctic fishes and one Mediter- ranean fish (the results are means of three determinations on three different specimens for each species). Mugil Chionodraco Pagothenia Notothenia Chaenocephalus cephalus hamatus coriiceps aceratus bernacchii io (ng/mg prot.) 36.0 ± 8.1 CoQ (ng/mg prot.) 152 .0 54.4 ±8. 3 27.5 ±6. 1 ±8.6 53.6 ±7.1 VitaminE (ng/mg prot.) 32.2 86.0 27.6 ±5.4 2 34.6 ±6. 3 ±9. 1 73. ±9.3 ±6.3 Copper (ng/mg prot.) 3.0 ±0. 5 ± 2.4 5.2 ± 1.4 5.9 Selenium (ng/mg prot.) 9.0 ±3.7 48.0 ±9.7 35.0 ±9.3 Zinc (ng/mg prot.) 2.2 ±0. 6 ±2.4 8.5 6.6 ±4.3 A. COLELLA, M. PATAMIA, A. GALTIERI, B. GIARDINA De Vries A. L., 1988 - The role of antifreeze glycopeptides and follows: a) the presence of lipid droplets may accelerate peptides in the freezing avoidance of Antartic fishes. Comp. the rate of oxygen diffusion between capillaries and mi- Biochem. Physiol., 90B: 539-545. tochondria; b) intracellular lipid may function as a ma- Eastman J. T., De Vries A. L., 1982 - Buoyancy studies of notothe- jor oxygen store, damping out otherwise large varia- nioid fishes in McMurdo Sound, Antarctica, Copeia, 1982: 385- tions in oxygen supply to mitochondria during changes 393. Egginton S., Sidell B. D., 1989 - Thermal acclimation induces in muscular activity. adaptive changes in subcellular structure of fish skeletal mus- These peculiar characteristics, together with the cle. Am. J. Physiol., 256: R1-R9. greater activation of oxidative metabolism could be at Ericson T., Soerensen B., 1977 - High performance liquid chro- the basis of an increased production of lipoperoxides matography of vitamine Ε. Acta Pharmac. Suec , 14: 478-483. Gozzo M. L., Lippa S., Barbaresi G., Morosi R., 1993 - Computer- and oxygen-derived free radicals. On the whole, Antarc- ized stripping potentiometry applied to copper determination tic fishes, in comparison with temperate ones, have to in plasma and urine samples. 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Llano (ed.), Adaptations within Q form normally found in cells from temperate fishes 1 0 Antarctic ecosystems. Smithsonian Inst., Washington DC, pp . as well as in those of most vertebrates. It has been hy- 479-487. pothesized that this could be due to the different crys- Hureau J. C., Petit D., Fine J. M., Marneux M., 1977 - New cyto- tallization temperature of Coenzyme Q homologs (+ logical, biochemical and physiological data on the colqrless blood of the Channichthyidae. In: Adaptation within Antartic 0.5° C for Q and + 9.7° C for Q ). Hence, in the mi- 9 lo ecosystems. Gulf, Houston, Texas, pp. 459-477. croenvironments, in vivo, where crystallization of the Lowry O. M., Rosenbrough N. J., Farr L., Randall R. J., 1951 - Pro- compound cannot be excluded, this could impair the tein measurement with the Folin phenol reagent. J. Biol. Chem., correct functioning of the respiratory chain with great 193: 265-275. trouble resulting for the whole organism. On the basis Mackler B., 1961 - Electron transport particle of yeast. Biochem. Biophys. Acta, 50: 141-145. of this consideration, the presence of Q^ seems the re- Pedersen P. L., Greenwalt J. W., Reynafarje B., Hullihen J., Decker sult of an adaptive strategy by which the efficiency of G. L., Soper J. W., Bustamente E., 1978 - Preparation and char- one of the main metabolic pathways could be main- acterization of mitochondria and submitochondrial particles of tained at a reasonable level. rat liver and liver-derived tissues. Methods Cell. Biol., 20: 411- Moreover, in parallel with these biochemical adapta- Sidell B. D., 1991 - Physiological roles of high lipid content in tis- tions, we have to consider the very narrow temperature sues of Antarctic fish species. In: G. Di Prisco, B. Maresca & Β. range in which the various complexes of the respiratory Tota (eds), Biology of Antarctic Fish. Springer-Verlag, Berlin, chain maintain their biological activity. The stringent Heidelberg, New York, pp. 220-231. Somero G. N., De Vries A. L., 1967 - Temperature tollerance of stenothermality which characterizes the biological activ- some Antartic fishes. Sciences, 156: 257-258. ity of mitochondria of Antarctic fish, and in particular Van den Thillart G., De Bruin G., 1981 - Influence of environmen- the icefish, appears to be the cost that has to be paid tal temperature on mitochondrial membranes. Biochem. Bio- for the impressive fitness of these organisms to their ex- phys. Acta, 640: 439-447. treme environment. Van den Thillart G., Modderkolk J., 1978 - The effect of acclima- tion temperature on the activation energy of state III respiration and on the unsaturation of membrane lipids of goldfish mito- chondria. Biochem. Biophys. Acta, 510: 38-51. REFERENCES Wells R. M. G., Ashby M. D., Duncan S. J., Macdonald J. A., 1980 - Comparative studies of the erythrocytes and haemoglobins in Chi Hua C., Jagner D., Renman L., 1987 - Determination of seleni- nototheniid fishes from Antarctica. J. Fish Biol., 17: 517-527. um by means computerized flow constant current stripping at Youssef H., 1967 - Preparation and properties of NADH: carbon fibre electrodes. Anal. Chim. Acta, 197: 257-264. ubiquinone oxidoreductase (complex I) E.C.I.6.5.3. Methods Dahlhoff E., O'Brien J., Somero G. Ν., Vetter R. D., 1991 - Tem- Enzymol., 10: 13-16. perature effects on mitochondria from hydrothermal vent inver- Youssef H., Stiggall D. L., 1967 - Preparation and properties of tebrates: evidence for adaptation to elevated and variable habi- NADH cytochrome c oxidoreductase (complex I-III). Methods tat temperature. Physiol. Zool., 64: 1490-1508. Enzymol., 10: 9-12.

Journal

Italian Journal of Zoology – Taylor & Francis

Published: Jan 1, 2000

Keywords: Cold adaptation; Oxidative metabolism; Antioxidant defenses

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