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Ciencias marinas

versión impresa ISSN 0185-3880

Cienc. mar vol.31 no.1a Ensenada mar. 2005




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:


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



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.



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.



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).



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.



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.



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.



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