Artículos

 

Presence of cytochrome P₄₅₀ in the Caribbean corals Siderastrea siderea and Montastraea faveolata

 

Presencia del citocromo P₄₅₀ en las especies de coral Siderastrea siderea y Montastraea faveolata del Caribe

 

E. García*, R. Ramos and C. Bastidas

 

^* Departamento de Biología de Organismos Universidad Simón Bolívar Apartado 89000, Caracas 1080-A, Venezuela. * E-mail: emgarcia@usb.ve

 

Recibido en junio de 2004;
aceptado en julio de 2004.

 

Abstract

Cytochrome P₄₅₀ was detected in the animal tissue of the scleractinian corals Siderastrea siderea and Montastraea faveolata collected from a reef site on the west coast of Venezuela. The concentration of P₄₅₀ in these species (0.4-2.1 nmol mg^-1 of microsomal protein) was similar to the levels found in other marine invertebrates, but higher than previously reported for other organisms of the phylum Cnidaria. Maximum P₄₅₀ content was detected in samples collected during the reproductive season of S. siderea. These results confirm the presence of a functional cytochrome P₄₅₀ in two coral species. Its usefulness as a biomarker for environmental monitoring has to be further investigated.

Key words: Cytochrome P₄₅₀, coral reefs, cnidarians, scleractinians, biomarkers.

 

Resumen

Se detectó citocromo P₄₅₀ en el tejido de colonias de los corales Siderastrea siderea y Montastraea faveolata de un arrecife de la costa al oeste de Venezuela. La concentración de P₄₅₀ en estas especies (0.4-2.1 nmol mg^-1 de proteína microsomal) es similar a los niveles detectados previamente en otros invertebrados marinos, pero superior a los valores reportados para otros organismos del phylum Cnidaria. El mayor contenido de P₄₅₀ fue encontrado en muestras extraídas durante el periodo reproductivo de S. siderea. Estos resultados confirman la presencia de un citocromo P₄₅₀ funcional en dos especies de coral. La utilidad de este complejo enzimático como biomarcador en los monitoreos ambientales deberá ser investigada con mayor profundidad.

Palabras clave: Citocromo P₄₅₀, corales escleractínidos, cnidarios, biomarcadores.

 

Introduction

Techniques using molecular and cellular biomarkers have been proposed as sensitive tools for assessing the effects of environmental distress in biological systems. The cytochrome P₄₅₀ monooxygenases are among the markers currently used in field studies (Snyder, 2000). Although these enzymes metabolize a wide variety of substrates, including endogenous molecules (e.g., steroids, fatty acids, prostaglandins) (Livingston, 1991; Porte et al., 1991), their enzymatic activity is considered an indicator of exposure to pollutants, particularly to polycyclic aromatic hydrocarbons and polychlorinated biphenyls (PCBs) (Cajaraville et al., 2000). It is difficult, however, to obtain a linear relationship between the concentration of contaminants and the activity of P₄₅₀ in samples from natural environments, where endogenous compounds and xenobiotics could be acting simultaneously, or even synergistically, as inducers of such activity (Livingstone, 1993).

Many aquatic invertebrates respond to xenobiotic exposure by increasing the activity of cytochrome P₄₅₀ and of the mixed function oxidases (MFO) (Snyder, 2000). Little is known, however, about such enzymatic activities in the phylum Cnidaria. Successful detection of cytochrome P₄₅₀ and other enzymes from the MFO complex in cnidarians is limited to three anemone species (Heffernan and Winston, 1998) and the scleractinian coral Favia fragum (Gassman and Kennedy, 1992) . In contrast, Montastraea faveolata did not exhibit cytochrome P₄₅₀ activity after exposure to chlordane in a laboratory assay (Firman, 1995).

Scleractinian corals inhabit nearshore environments where they constitute an important asset both in terms of their aesthetic value and by providing refuge for fish and other marine biota. These environments are often heavily contaminated with hydrocarbons and their derivates (e.g., Guzmán and Jarvis, 1996). Hence, it is important to study the potential involvement of detoxification enzymes such as P₄₅₀ in order to understand the adaptive response of cnidarians to the environmental changes induced by anthropogenic activity.

Samples were collected from the Morrocoy National Park, which is located on the west coast of Venezuela. During several decades this park has been subjected to the impact of industrial activity, tourism, coastal development and sedimentation of terrestrial inputs, such as those derived from runoff (Bastidas and García, 1999). This situation has resulted in an overall deterioration of the coral reef conditions (Bone et al., 2001; Laboy-Nieves et al., 2001), similar to that of other Caribbean localities (e.g., Hughes, 1994). Sediments collected concurrently in different localities within the park contain hydrocarbons, but this contamination varies geographically and seasonally (Jaffé et al., 1998; García and Farina, 2000). Despite these conditions, coral species such as Siderastrea siderea and Montastraea faveolata are still present in these reefs, suggesting that they have mechanisms for tolerating such levels of environmental stress.

The purpose of this paper is to demonstrate the existence of cytochrome P₄₅₀ in the tissue of S. siderea and M. faveolata. Our results contribute to elucidate the presence of this enzyme in cnidarians, particularly in corals, for which studies are scarce.

 

Materials and methods

Study site and sampling procedures

Fourteen colonies of S. siderea and ten colonies of M. faveolata were collected from the Playa Caimán reef, in the Morrocoy National Park. These colonies were collected during the dry and rainy seasons (table 1), corresponding to the non-reproductive and reproductive seasons, respectively, of both species (Szmant, 1986; Soong, 1991; Guzmán and Holst, 1993) . Corals were frozen in liquid nitrogen, transported to the laboratory and stored at -80°C until tissue extraction.

Enzyme preparation

The coral tissue was removed with a water-pick (Johannes and Wiebe, 1970) from the coral skeleton using a homogenization buffer, following Gassman and Kennedy (1992), as modified by Heffernan and Winston (1998). The homogenization buffer also included protease inhibitors and antioxidants: 0.15 M KCl, 0.02 M Hepes, N-2-hydroxyethyl piperazine-N'-(2-ethane sulfonic acid) pH 7.4, 2 mM dithiothreitol (DTT), 1 mM ethylenediamine tetraacetic acid (EDTA), and 1 mM Phenylmethylsulfonylfluoride (PMSF).

The tissue was homogenized on ice with an Ultra-Turrax homogenizer, which disrupts the animal tissue but preserves the integrity of the zooxanthellae. The homogenate was serially centrifuged at 2000 g for 5 min at 4°C using a Sorvall RC-26 Plus Super Speed centrifuge to remove the zooxanthellae and the remaining skeletal elements. The supernant was centrifuged at 110,000 g for 60 min in a Beckman L8-55 ultra-centrifuge to sediment the microsomes. The microsomal fraction was resuspended in homogenization buffer with 20% glycerol (v/v) and stored at -80°C for no longer than 15 days prior to analysis. The protein concentration was measured using Bradford's (1976) method for all fractions. All enzymatic tests were run in duplicate.

Enzyme assay

Cytochrome P₄₅₀ was measured according to Omura and Sato (1964a). The carbon monoxide (CO) difference spectrum was recorded by adding sodium dithionite (DTN) and performing a background correction prior to the addition of CO. The P₄₅₀ content was determined in a 1-cm cuvette containing approximately 100-400 µg of microsomal protein solubilized with 10 mM potassium phosphate pH 7.4, which contained 1 mM EDTA, 1 mM DTT and 150 mM NaCl. The spectra were scanned (400-500 nm) at room temperature (22°C) on a dual beam Perkin Elmer Lambda 35 spectrophotometer. The P₄₅₀ concentration was calculated using the extinction coefficient (e = 450-490 = 91 mM^-1 cm^-1). If a peak was observed at 420 nm, the concentration of P₄₂₀ was calculated in the same way but using an extinction coefficient of 110 mM^-1 cm^-1 (Omura and Sato, 1964b; Schenkman and Jansson, 1998).

 

Results

Colonies of S. siderea and M. faveolata consistently exhibited a typical 448-450-nm peak corresponding to the CO-reduced cytochrome P₄₅₀ complex (figs. 1 y 2). The microsomal cytochrome P₄₅₀ spectra were well resolved when DTN was added prior to the CO. In the extracts from both coral species the maximum peak at 450 nm was attained about 2-5 min after the addition of CO. Often, the 450-nm peak was accompanied with a positive reading at 420 nm. This value, however, was never higher than the absorbance obtained at 450 nm for the same sample.

The concentration of cytochrome P₄₅₀ in S. siderea showed seasonal fluctuations (table 1). The concentrations in February were significantly lower than the concentrations found in September, when this species reproduces, i.e., broadcasts its gametes (Soong, 1991; Guzmán and Holst, 1993). In each species, the P₄₅₀ concentration was very similar amongst colonies, and highly consistent between years for S. siderea (February 2002 and March 2003, table 1).

Montastraea faveolata also showed the presence of cytochrome P₄₅₀ in March. Furthermore, the mean concentration of cytochrome P₄₅₀ was significantly higher in M. faveolata than in S. siderea during the dry season studied (Student-t P < 0.01, table 1). The microsomal cytochrome P₄₅₀ spectra for M. faveolata differed from the spectra obtained for S. siderea in the reproductive season. Three colonies of M. faveolata collected in July 2003 only showed a peak of absorbance at 420 nm. Since no further activity was detected at 450 nm, it is difficult to compare these results with the values obtained for individuals collected in other seasons of the year, when the main activity was found exclusively at 450 nm. The cytochrome P₄₂₀ concentration for these colonies varied between 2.05 and 36.10 nmol mg^-1 microsomal protein.

 

Discussion

The P₄₅₀ concentrations in S. siderea and in M. faveolata were within the range reported for other marine invertebrates (0.43-2.77 nmol mg^-1 microsomal protein) and were substantially higher than those reported for other species of the phylum Cnidaria. In the sea anemone Bunodosoma cavernata, the P₄₅₀ content observed within a pool of 30 organisms was 52 pmol mg^-1 microsomal protein (Heffernan and Winston, 1998), whereas in the coral Favia fragum, Gassman and Kennedy (1992) detected a mean value of 0.09 nmol mg^-1 microsomal protein when working with individual colonies using a method similar to the one used in this study.

The detection of P₄₅₀ and some other components of the MFO system in marine invertebrates has proved difficult due to technical problems during the preparation of the microsomal fraction (Snyder, 2000). It is likely that this is why detection of such activity has been elusive for most studies in cnidarians.

Despite these difficulties, strong evidence points towards the existence of P₄₅₀ and MFO activity in these organisms. Heffernan and Winston (1998) reported the activity of the NAD(P)H-dependent cytochrome c (P₄₅₀) reductase in the microsomal fraction of the sea anemones Anthopleura elegantissima, A. xanthogrammica and B. cavernata. Nevertheless, P₄₅₀ was only found in the last species. Gassman and Kennedy (1992) detected the activity of glutathione-S-transferase (GST) and the presence of P₄₅₀ in the microsomal fraction of the coral F. fragum. In this study, we showed the presence of P₄₅₀ in the microsomal fraction of S. siderea and M. faveolata. Furthermore, these two coral species exhibited GST and NAD(P)H cytochrome c reductase activity (Ramos and García, 2003). These results strongly suggest the existence of a functional P₄₅₀-dependent MFO system in cnidarians, similar to that found in other marine invertebrates.

Siderastrea siderea and Montastraea faveolata expressed cytochrome P₄₅₀ in colonies from their natural environment. The major peak occurred at 450 nm and a second component at 420 nm. This spectral pattern differed from that reported for other marine invertebrates, such as anemones and echinoderms (den Besten, 1998; Heffernan and Winston, 1998), for which the highest peak occurred at 418 and 420 nm, respectively. Asteria rubens and Echinus esculentus only showed a 420-nm peak in June and July, during the reproductive season (den Besten et al., 1990; den Besten 1998). In this study, M. faveolata also showed this pattern with only one peak at 420 nm in July, before the onset of the reproductive season. It is important to emphasize that, at the present time, our findings regarding the shifting pattern of P₄₂₀ activity concomitant with the onset of the reproductive season can only be interpreted as a coincidence. Any further relationship of P₄₂₀ and its role in the M. faveolata reproductive activity remain to be proved. High P₄₂₀ activity in other organisms has been related to the size and reproductive status of the individuals, and to environmental pollution gradients (Livingston, 1991). In the literature, the absorbance peak at 420 nm has been interpreted in different ways. Omura and Sato (1964b) referred to P₄₂₀ as the solubilized form of cytochrome P₄₅₀. Livingstone et al. (1989) and Livingston (1991) suggested that P₄₂₀ is a degraded form of P₄₅₀, due to high proteolytic activity within the microsomes of marine invertebrates. On the other hand, Heffernan and Winston (1998) consider that P₄₁₈ is unlikely to be a degraded form of P₄₅₀.

Interspecific and intraspecific variability in the concentration of P₄₅₀ has been commonly found in marine invertebrates (Snyder, 2000) such as anemones, polychaetes, equinoderms, mollusks and crustaceans (Livingstone, 1991; den Besten, 1998; Heffernan and Winston, 1998; James and Boyle, 1998; Lee, 1998). Overall, the content of cytochrome P₄₅₀ in S. siderea and M. faveolata was similar to that in other marine invertebrates. These results contrasted with the very low concentration of P₄₅₀ reported in the coral F. fragum (Gassman and Kennedy, 1992) and its absence in M. faveolata (Firman, 1995). Our results show that the content of P₄₅₀ in M. faveolata was 1.5 times higher than in S. siderea, both collected in March 2003.

In the Caribbean, the reproductive seasons of S. siderea and M. faveolata spans from July to September (Szmant, 1986; Soong, 1991; Guzmán and Holst, 1993; Bastidas et al., in press). The seasonal fluctuations of P₄₅₀ in S. siderea seemed to be coincident with its reproductive cycle. Lower levels of P₄₅₀ were detected in February, during the non-reproductive period of the year, whereas higher levels of P₄₅₀ were found in samples collected when this species attains its reproductive peak (September). Also, a difference in the absorbance spectrum of M. faveolata was observed during the reproductive season, when this shifted from a dominance at 450 nm to a unique peak at 420 nm. For the month of July, during the reproductive season, the activity ratio of cytochrome P₄₂₀/P₄₅₀ is about 5. In other marine crustaceans and echinoderms, increased cytochrome activity has been related to reproductive activity, by playing a role in the regulation of steroid synthesis and in the production of gametes (Lee et al., 1981; Lee, 1982; den Besten, 1998; Hall, 1988). Assuming that P₄₅₀ and P₄₂₀ are the oxidized/reduced forms of the same cytochrome, it could be speculated that the increase in activity observed may be involved in the reproduction of this coral species.

In conclusion, our results show: (1) a detectable P₄₅₀ enzymatic system in S. siderea and M. faveolata; (2) levels of P₄₅₀ in S. siderea and M. faveolata similar to those previously reported for other marine invertebrates and the largest ever reported for cnidarians; and (3) a seasonal variation of the content of cytochrome P₄₅₀ in S. siderea, this being highest during the reproductive period. Studies to elucidate the level of enzymatic activity in different seasons and localities, as well as the potential role of P₄₅₀ as a detoxifying mechanism in S. siderea and M. faveolata are currently underway in our laboratory.

 

Acknowledgements

We would like to thank G. Farache for his advice during the study and helpful comments during the preparation of the manuscript. We also thank L. Márquez, H. Guzmán, R. Cipriani and F. Osborn for critical reviews of this work, and are grateful to three anonymous reviewers whose comments improved the manuscript. This research was supported by FONACIT, under project S1-2001000368.

 

References

Bastidas, C. and García, E., (1999). Metal content on the reef coral Porites astreoides: An evaluation of river influence and 35 years of chronology. Mar. Pollut. Bull., 38: 899-907.         [ Links ]

Bastidas, C., Ramos, R., Croquer, A., Leon, A., Weinberger, C., Kortnik, V., and Marquez L. (in press). Coral mass- and split-spawning at a coastal and an offshore Venezuelan reefs, southern Caribbean. Hydrobiologia, XX: 1-6.         [ Links ]

Bone, D., Cróquer, A., Klein, E., Pérez, D., Losada, F., Martín, A., Bastidas, C., Rada, M., Galindo, L. y Penchaszadeh, P. (2001). Programa CARICOMP: Monitoreo a largo plazo de los ecosistemas marinos del Parque Nacional Morrocoy, Venezuela. Interciencia, 26: 457-162.         [ Links ]

Bradford, M.M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-Dye binding. Anal. Biochem., 72: 248-254.         [ Links ]

Cajaraville, M.P., Bebianno, M.J., Blasco, J., Porte, C., Sarasquete, C. and Viarengo, A. (2000). The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: A practical approach. Sci. Total Environ., 247: 295-311.         [ Links ]

den Besten, P.J. (1998). Cytochrome P₄₅₀ monooxygenase system in echinoderms. Comp. Biochem. Physiol., 121C: 139-146.         [ Links ]

den Besten, P.J., Herwig, H.J., Donselaar, E.G. and van Livingstone, D.R. (1990). Cytochrome P₄₅₀ monooxygenase system and benzo(a)pyrene metabolism in equinoderms. Mar. Biol., 107: 171-177.         [ Links ]

Firman, J.C. (1995). Chronic toxicity of pesticides to reef-building corals: Physiology, biochemical, cellular and developmental effects (Montastraea faveolata, Porites divaricata, Aiptasia pallida). Ph.D. dissertation, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 125 pp.         [ Links ]

García, E. y Farina, O. (2000). Evaluación de la calidad del agua y de los indicadores de la contaminación del Parque Nacional Morrocoy y sus áreas de influencia. En: Estudio Integral del Sistema Parque Nacional Morrocoy con Vías al Desarrollo de Planes de Uso y Gestión para su Conservación. Primer Informe Técnico CONICIT, Caracas, Venezuela, 33 pp.         [ Links ]

Gassman, N.J. and Kennedy, C.J. (1992). Cytochrome P₄₅₀ content and xenobiotic metabolizing enzyme activities in the scleractinian coral, Faviafragum (ESPER). Bull. Mar. Sci., 50: 320-330.         [ Links ]

Guzmán, H.M. and Holst, I. (1993). Effects of chronic oil-sediment pollution on the reproduction of the Caribbean reef coral Siderastrea siderea. Mar. Pollut. Bull., 26: 276-282.         [ Links ]

Guzmán, H.M. and Jarvis, K.E. (1996). Vanadium century record from Caribbean reef corals: A tracer of oil pollution in Panama. Ambio, 25: 523-526.         [ Links ]

Hall, P.F. (1988). Cytochromes P-450 and the regulation of steroid synthesis. Steroid, 48: 131-136.         [ Links ]

Heffernan, L.M. and Winston, G.W. (1998). Spectral analysis and catalytic activities of the microsomal mixed-function oxidase system of the sea anemone (phylum: Cnidaria). Comp. Biochem. Physiol., 121C: 371-383.         [ Links ]

Hughes, T.P. (1994). Catastrophes, phase-shift, and large-scale degradation of Caribbean coral reefs. Science, 265: 1547-1551.         [ Links ]

Jaffé, R., Leal, I., Alvarado, J., Gardinali, P. and Sericano, J. (1998). Baseline study on the levels of organic pollutants and heavy metals bivalves from the Morrocoy National Park, Venezuela. Mar. Pollut. Bull., 36: 925-929.         [ Links ]

James, M.O. and Boyle, S.M. (1998). Cytochrome P₄₅₀ in Crustacea. Comp. Biochem. Physiol., 121C: 157-172.         [ Links ]

Johannes, R.E. and Wiebe, W.J. (1970). Method for determination of coral tissue, biomass and composition. Limnol. Oceanogr., 15: 822-824.         [ Links ]

Laboy-Nieves, E.D., Klein, E., Conde J., Losada, F., Cruz, J. and Bone, D. (2001). Mass mortality of tropical marine communities in Morrocoy, Venezuela. Bull. Mar. Sci., 68: 163-179.         [ Links ]

Lee, R.F. (1982). Mixed function oxygenases (MFO) in marine invertebrates. Mar. Biol. Lett., 2: 87-107.         [ Links ]

Lee, R.F. (1998). Annelid cytochrome P-450. Comp. Biochem. Physiol., 121C: 173-179.         [ Links ]

Lee, R.F., Singer, S.C. and Page, D.S. (1981). Responses of cytochrome P-450 systems in marine crab and polychaetes to organic pollutants. Aquat. Toxicol., 1: 355-365.         [ Links ]

Livingstone, D.R. (1991). Organic xenobiotic metabolism in marine invertebrates. Adv. Comp. Environ. Physiol., 7: 45-185.         [ Links ]

Livingstone, D.R. (1993). Biotechnology and pollution monitoring: Use of molecular biomarkers in the aquatic environment. J. Chem. Tech. Biotechnol., 57: 195-211.         [ Links ]

Livingstone, D.R., Kirchin, M.A. and Wiseman, A. (1989). Cytochrome P₄₅₀ and oxidative metabolism in mollusks. Xenobiotica, 19: 1041-1062.         [ Links ]

Omura, T. and Sato, R. (1964a). The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239: 2370-2378.         [ Links ]

Omura, T. and Sato, R. (1964b). The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification and properties. J. Biol. Chem., 239: 2379-2385.         [ Links ]

Porte, C., Cole, M., Albagaiges, J. and Livingstone, D.R. (1991). Responses of mixed-function oxygenase and antioxidase enzyme system of Mytilus sp. to organic pollution. Comp. Biochem. Physiol., 100C: 183-186.         [ Links ]

Ramos, R. y García, E. (2003). Determinación de la glutation transferasa, citocromo c NADPH reductasa y citocromo P₄₅₀ en dos especies de corales escleractinidos en el Parque Nacional Morrocoy, Venezuela. Décimo Congreso Latinoamericano de Ciencias Marinas, San José, Costa Rica, 379 pp.         [ Links ]

Schenkman, J.B. and Jansson, I. (1998). Spectral analyses of cytochromes P₄₅₀. In: I.R. Phillips and E.A. Shephard, E.A. (eds.), Cytochrome P₄₅₀ Protocols. Methods in Molecular Biology, 107. Humana Press, Totowa, NJ, pp. 25-33.         [ Links ]

Snyder, M.J. (2000). Cytochrome P₄₅₀ enzymes in aquatic invertebrates: Recent advances and future directions. Aquat. Toxicol., 48: 529-547.         [ Links ]

Soong, K. (1991). Sexual reproductive patterns of shallow-water reef corals in Panama. Bull. Mar. Sci., 49: 832-846.         [ Links ]

Szmant, A.M. (1986). Reproductive ecology of Caribbean reef corals. Coral Reefs, 5: 43-54.         [ Links ]