www.elsevier.com/locate/marpolbul

Marine Pollution Bulletin 48 (2004) 784–789

The occurrence of NADPH-ferrihemoprotein reductasein Corbula caribea, from a natural oil seep at La Brea, Trinidad

Azad Mohammed *, John Agard

Department of Life Sciences, The University of the West Indies, St. Augustine, Trinidad, Trinidad and Tobago

Abstract

Corbula caribea is the most common non-polychaete macrofaunal organism identified at a large natural oil seep at La Brea in

south Trinidad. It is hypothesized that these animals may possess (NADPH-ferrihemoprotein reductase) a component of the Mixed

Function Oxygenase system (MFO), which may allow them to ameliorate the potentially deleterious effects resulting from exposure

to the high levels of petroleum hydrocarbons within this environment. This study was designed to determine whether organisms

from the seep site showed greater enzyme activity when compared to organisms from a non-seep reference site.

NADPH-ferrihemoprotein reductase activity was determined by incubating 10 lm cryostat sections with nitro-blue tetrazolium.

The reaction product was determined by visual assessment and quantified by measuring the relative mean stain intensity. The intense

staining, indicative of enzyme activity was evident in the digestive epithelia of seep animals. Observations indicated that organisms

from the seep showed more intense staining, indicating greater enzyme activity, when compared to animals from a non-seep ref-

erence site. The relative stain intensity of NADPH-ferrihemoprotein reductase determined for organisms from the seep was 61.30.

This was significantly higher than the stain intensity determined for organisms from the non-seep reference site (7.11). This sup-

ported visual assessments, which suggested that the seep organisms showed higher enzyme activity than organisms from the non-

seep site.

The results suggest that NADPH-ferrihemoprotein reductase may be present in Corbula caribea from the seep site and not in

those from the non-seep site. It is possible that this enzyme may contribute to these animals ability to tolerate chronic exposure to

petroleum hydrocarbons and offer then a selective advantage for survival the seep environment.

� 2003 Elsevier Ltd. All rights reserved.

Keywords: NADPH-ferrihemoprotein reductase; Natural oil seep; Corbula caribea; Mixed function oxidase; Trinidad

1. Introduction

Petroleum hydrocarbons pose a pervasive treat to the

survival of organisms. However, many organisms pos-

sess an innate ability to tolerate or reduce its toxic ef-fects. This ability is reportedly linked to the presence of

the Mixed Function Oxygenase (MFO) enzyme system

(Bayne et al., 1985). The MFO system consists of at least

two protein components:

1. Cytochrome P-450––an iron containing haem protein

and

2. NADPH-ferrihemoprotein reductase (NADPHCytochrome P-450 or neotetrazolium reductase).

* Corresponding author. Tel.: +1-868-662-2002x2047; fax: +1-868-

663-5241.

E-mail address: mohc0134@trini.com (A. Mohammed).

0025-326X/$ - see front matter � 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/j.marpolbul.2003.11.009

Various studies have identified MFO activity in dif-

ferent marine invertebrates, particularly crustaceans and

polychaetes. Such studies include: MFO activity in the

stomach of the blue crab, Callinecties sapidus (Singer

et al., 1980); pollution effects on MFO activity in themarine polychaete, Nereis virens (Fries and Lee, 1984);

Payne (1977) identified MFO activity in teleost, some

crustaceans, an elasmobranch, annelids and an echino-

derm but was generally absent from gastropods and

pelecypod molluscs, coelenterates and macrophytic

algae. Lee (1981) reported MFO activity in 18 marine

invertebrates belonging to four phyla (Annelida,

Arthropoda, Echinodermata and Mollusca) and Leeand Singer (1980) detected activity in the lower portions

of the intestine of Capitella capitata after exposure to

either petroleum or its components.

Cytochrome P-450 activity has been identified in a

variety of organisms, including fishes, mammals, birds

Fig. 1. Map showing the proximity of the seep and non-seep reference

sites, in relation to petroleum hydrocarbon contamination in the Gulf-

of-Paria, Trinidad and Tobago (adapted from Agard et al., 1988).

A. Mohammed, J. Agard / Marine Pollution Bulletin 48 (2004) 784–789 785

and invertebrates. Quattrochi and Lee (1984) reported

cytochrome P-450 activity in several marine crabs,

Libinia emarginata, Callinectes sapidus, Menippe merce-

narie and Uca minax. Lee et al. (1982) identified in-creased activity of Cytochrome P-450 in the crabs,

Callinectes sapidus, Serarina cinerum and Uca pugilator,

and the polychaete (Neries virens) following exposure to

oil. Lee et al. (1982) also identified cytochrome P-450

activity in Uca minax collected from a heavily oiled area,

Uca pugilator collected from an oil spill site, Callinectes

sapidus from a heavy industrial area and in Sesarma

cinerum maintained in marsh sediment. Livingstone(1988) has also reported elevated levels of cytochrome

P-450 and cytochrome b5 in the digestive glands of

mussels exposed to diesel oil. Cytochrome P-450 activity

has also been reported in fish (Platichthys flesus), mol-

luscs (Mytilus edulis and Littorina littorea) and in crabs

(Carcinus maenas), (IOC/UNESCO, 1986). Kurelec

(1985) and Kurelec et al. (1986) suggested that in mol-

lusc, in which levels of P-450 are low, flavin-containingmonooxygenases may be the predominant phase 1

enzyme.

NADPH-ferrihemoprotein reductase is involved in

the electron transfer system of the endoplasmic reticu-

lum. According to Bayne et al. (1985), Moore (1988)

reported that there was no definitive evidence to sug-

gest induction of MFO activity in bivalves, but evidence

of induction of components of the system (NADPH-ferrihemoprotein reductase) has been observed.

However, studies have shown increased activity of

microsomal NADPH-ferrihemoprotein reductase in

Mytilus edulis blood cells following exposure to pheno-

barbital (Moore, 1979). Moore et al. (1987) also re-

ported elevated levels of NADPH-ferrihemoprotein

reductase activity in L. littorea from contaminated sites,

when compared to animals for a clean reference site, andincreased activity following exposure to 400 lg/l of

phenanthrene. Moore (1988) also showed that elevated

levels of NADPH-ferrihemoprotein in the digestive cells

ofMytilus edulis and L. littorea, correlated directly with

total PAH concentration.

The La Brea seep in Trinidad was identified as one of

the three largest natural oil seeps known (Wilson et al.,

1974). Persistent oil intrusion saturates this area withpetroleum hydrocarbons (Agard et al., 1988), making

the environment harsh to the survival of animals.

However, the occurrence of thriving benthic communi-

ties with this environment despite the persistent oil

intrusion, may suggest that organisms here have innate

homeostatic mechanisms to help reduce potentially toxic

effects. In a study conducted in parallel with this work,

Agard et al. suggested that Corbula caribea was the mostabundant non-polychaete organism (46 individuals per

sq. meter) identified at the seep. Surveys conducted

during this research on the western Gulf coast of Trin-

idad, failed to identify any areas with such high densities

of organisms. This present study focused on determining

if NADPH-ferrihemoprotein reductase can be identified

in organisms from the seep site.

2. Methodology

Corbula caribea was collected from a natural oil seep

at La Brea and a non-seep reference site at Carenage,

about 50 km (Fig. 1) north of the seep site. Animals were

collected using as a petit ponar grab and transported to

the laboratory alive.

2.1. Tissue preparation for cytochemistry

Organisms were immediately removed from their

shells and rinsed twice in molluscan saline [0.4 M NaCl,

0.05 M CaCl2 and 0.08 M MgCl2], embedded in a small

amount of Tissue Tek (O.C.T 4583, Sukura Finetechni-

cal Co.) and frozen at )70 �C. Tissue blocks were sub-sequently sectioned at 10 lm using a Re�ıchert Jung

Cryocut 1800 (Eine Gesellsenaft Der Cambridge

instrument) at a cabinet temperature of )15 �C. Sectionswere then flash dried on to clean slides and stored at

)70 �C. Histochemical analysis was done within 24 h

(Pearse, 1980; Kiernan, 1981; Moore, 1988).

Fig. 2. Sections of the stomach of Corbula caribea, following treatment

with the control media: (1) sections of animals from the seep site (·100)and (2) sections of animals from the reference site (·100).

786 A. Mohammed, J. Agard / Marine Pollution Bulletin 48 (2004) 784–789

2.2. Determination of NADPH-ferrihemoprotein reduc-

tase

NADPH-ferrihemoprotein reductase activity wasdemonstrated using 10 lm cryostat sections. The incu-

bating medium was prepared by dissolving 6 mM

NADPH and 5 mM nitro-blue tetrazolium in an aliquot

of stock solution purged with nitrogen for several min-

utes. The stock solution contained 0.1 M HEPES buffer

(pH 8.0), 20 M MgCl2, 20% (w/v) polyvinyl alcohol and

15 mg sodium azide. Sections were incubated with

NADPH-media for 30 minutes in the dark at 37 �C, in aclosed atmospheric chamber purged with nitrogen. The

chamber was kept moist by lining the base with damped

tissue paper. After incubation, the slides were washed in

running distilled water and mounted using glycerol jelly.

Sections were also incubated with a control medium

containing no NADPH (Pearse, 1980; Kiernan, 1981;

Moore, 1988). A visual assessment was done to deter-

mine the formation of the insoluble reaction products(purple, blue or black formazans) in sections of organ-

isms from both sites. NADPH-ferrihemoprotein reduc-

tase activity between sites was ranked based on the

presence (rank 1) or absence (rank 0) of reaction prod-

ucts and the data analysed using the Mann–Whitney

U -statistic.

Quantification of the reaction product was attempted

using the Sigma Scan Pro Version 5.0 software to esti-mate the relative staining intensity. Stained cryostat

sections were scanned directly into the software and a

grid (10 · 10) was superimposed onto the image. A

minimum of 10 readings of the uncalibrated pixel

intensity was made per section of tissue. The relative

staining intensity was calculated as follows:

Relative stain intensity

¼ ð100� relative mean pixel intensityÞ

where relative mean pixel intensity ¼ mean pixel inten-sity of stained sections=mean pixel intensity of control

sections� 100.

3. Results

The presence of the reaction products following his-

tochemical analysis may suggest the presence ofNADPH-ferrihemoprotein reductase in Corbula caribea.

Histochemical methods showed intense staining in the

digestive epithelia of animals from the seep site.

Sections of the stomach and the intestinal region

(Fig. 2) showed no staining characteristic of NADPH-

ferrihemoprotein reductase activity, following treat-

ment with control media. However, sections incubated

with NADPH media, showed intense staining indica-tive of formazan deposition and consistent with

NADPH-ferrihemoprotein reductase activity. Forma-

zan deposition was most evident in the intestinal and

the stomach epithelia (Figs. 3 and 4). Closer exami-

nation of the stomach epithelium showed differential

deposition of reaction products within the epithelia

with more intense staining in the stomach epitheliathan in the intestine.

Investigations of organisms from the reference site

(Fig. 1) showed relatively little staining when compared

to organisms from the seep site. Treatment with the

NADPH media showed very low staining intensity in

both the intestine and stomach (Figs. 3 and 4) epithelia,

indicative of low formazan deposition and consistent

with low NADPH-ferrihemoprotein reductase activity.The stain intensity was significantly less (P < 0:001)than that observed in sections from animals collected at

the seep site (Figs. 3 and 4). Statistical analysis of the

ranked responses, bases on the presence or absence of

NADPH-ferrihemoprotein reductase activity, using the

Mann–Whitney U -statistic showed that responses for

animals from the seep were significantly higher than that

for animals from reference site ð �U ¼ 21Þ.Statistically significant differences were also observed

for the relative mean staining intensity for organisms

from the two sites. The relative mean staining intensity

Fig. 3. Sections of the intestine showing the varying degrees of staining following treatment with the NADPH media: (1) sections of animals from

reference site (·100) and (2) sections of animals from the seep (·100).

Fig. 4. Sections of the stomach showing the varying degrees of staining following treatment with the NADPH media: (1) sections of the animals from

reference site (·100) and (2) sections of animals from the seep (·100).

Table 1

Relative mean staining intensity of NADPH-ferrihemoprotein reductase in Corbula caribea from three sites in Trinidad

Sites Relative mean stain intensity of NADPH-ferrihemoprotein reductase in Corbula caribea, as a

percent of the controls

Intensity of the controls Relative mean stain intensity

Seep site 231.85± 16.46 61.30± 3.46

Non-seep contaminated 200.04± 15.66 7.11± 6.21

A. Mohammed, J. Agard / Marine Pollution Bulletin 48 (2004) 784–789 787

for organisms from the seep was 61.30 ± 3.46 as com-

pared to 7.11 ± 6.21 (Table 1) for the non-seep uncon-

taminated site (ANOVA; Fcal ¼ 57:63, Ftab ¼ 9:55, P <0:001Þ.

788 A. Mohammed, J. Agard / Marine Pollution Bulletin 48 (2004) 784–789

4. Discussion

Cytochemical analyses suggest that NADPH-ferrihe-

moprotein may be present in the digestive epithelia ofthe marine bivalve Corbula caribea found at the seep

site. According to Bayne et al. (1985), Moore (1988)

suggested that there was no definitive evidence of induc-

tion of MFO activity in bivalves, though evidence of

NADPH-neotetrazolium reductase and NADPH pro-

ducing enzymes was present. Studies conducted in par-

allel with this work, Agard et al. suggested that Corbula

caribeawas the most abundant non-polychaete organism(46 individuals per sq. meter) identified at the seep.

Additional surveys conducted during the duration of this

project along the Gulf coast of Trinidad, failed to iden-

tify such densities at any other location even at the ref-

erence site. This enzyme is involved in the detoxification

pathway for petroleum hydrocarbons, in particular

PAHs. It is probable that this enzyme may contribute to

the ability of Corbula caribea to adapt survive within theseep environment. Agard et al. (1988) reported varying

concentration of petroleum hydrocarbons (3.0–1824.4

lg/g dry wt. Crysene equivalent) along the West Coast of

Trinidad. The area, which they identified as showing the

highest contamination (Fig. 1), coincided with the seep

site under investigation. The reference site showed only

low levels of petroleum hydrocarbons.

Analysis of Corbula caribea, obtained from two sitesshowed significant differences in stain intensity, which

suggest that there was a difference in enzyme activity

between the seep and non-seep organisms. Animals from

the seep showed higher staining intensity when com-

pared to animals from an uncontaminated reference site.

It is therefore possible that the presence of NADPH-

ferrihemoprotein reductase may be an adaptive response

to exposure to high levels of petroleum hydrocarbons.The differential NADPH-ferrihemoprotein reductase

activity observed in animals from both sites may not be

due to geographical separation of populations, but may

be more closely related to the levels of hydrocarbons

within the environment. It is therefore probable that the

presence of elevated levels of NADPH-ferrihemoprotein

reductase in Corbula caribea at the seep may be an

adaptive response to the high levels of hydrocarbonsin the environment. Further, it is evident that there

is a higher concentration of NADPH-ferrihemoprotein

reductase in the stomach than in the intestine. Moore

(1988), also observed higher NADPH-ferrihemoprotein

reductase activity in Mytilus edulis and L. littorea col-

lected from contaminated sites, than in animals collected

from an uncontaminated site. Similar results were also

reported for L. littorea from contaminated sites (Mooreet al., 1987). The stomach would represent the region

where the most direct exposure would take place. Food

material and sediment particles that would be ingested

may undoubtedly contain high levels of hydrocarbons,

which would have to be detoxified before toxic effect

can manifest. This detoxification can be facilitated by

the NADPH-ferrohemoprotein reductase. It may also

be possible that these organisms associated miofaunalcommunities in areas such as gills which can also serve

to reduce the toxic effect of the hydrocarbons through

absorption and biotransformation, however, this has yet

to be investigated.

The hydrocarbons within the area may also serve as a

carbon source for organisms within the seep ecosystem.

Dhanraj (1999) was able to identify significantly high

levels of oil degrading bacteria at the seep site. Thesebacteria may be able to succeed in transforming the

carbon from the petroleum hydrocarbons and incorpo-

rating it into the food chain. This may be one of the

contributing factors for the high numbers of organisms

identified at the seep.

References

Agard, J.B.R., Gobin, J., Boodosingh, M., 1988. Petroleum residues in

surficial sediments from the Gulf-of-Paria, Trinidad. Mar. Pollut.

Bull. 19, 231–233.

Bayne, B.L., Brown, D.A., Burns, K., Dixon, D.R., Ivanovici, A.,

Livingstone, D.R., Lowe, D.M., Moore, M.N., Stebbing, A.R.D.,

Widdows, J., 1985. The Effects of Stress and Pollution on Marine

Animals. Praeger Publishers, New York.

Dhanraj, D.K., 1999. The ecology of hydrocarbon degrading bacteria

at a natural oil seep. M.Phil Thesis. Department of Life Sciences,

University of the West Indies, St Augustine, Trinidad and

Tobago.

Fries, C.R., Lee, R.F., 1984. Pollutant effects on the mixed function

oxygenase (MFO) and reproductive system of marine polychaete

(Nieris virens). Mar. Biol. 79, 187–193.

Intergovernmental Oceanographic Commission (IOC), 1986. IOC

workshop on the biological effects of pollutants. No. 53, UNESCO,

48p.

Kiernan, J.A., 1981. Histological and Histochemical Methods, Theory

and Practices. Pergamon Press, Oxford.

Kurelec, B., 1985. Exclusion activation of aromatic amines in the

marine mussel (Mytilus edulis) by FAD containing monooxygena-

ses. Biochem. Biophys. Res. Commun. 127, 773–788.

Kurelec, B., Brituik, S., Krea, S., Zahn, R.K., 1986. Metabolic fate of

aromatic amines in the mussel (Mytilus galoprovincialis). Mar. Biol.

91, 523–527.

Lee, R.F., 1981. Mixed Function Oxygenase (MFO) in marine

invertebrates. Mar. Biol. Lett. 2, 87–105.

Lee, R.F., Singer, S.C., 1980. Detoxifying enzyme systems in marine

polychaetes: increases in activity after exposure to aromatic

hydrocarbons. Rap. P.-V. R�eun. Cons. Int. Explor. Mer. 179,

29–32.

Lee, R.F., Conner, J.W., Page, D., Ray, L.E., Giam, C.S., 1982.

Cytochrome P-450 dependent Mixed Function Oxygenase system

in marsh crabs. Physiol. Mech. Mar. Pollut. Toxic.

Livingstone, D.R., 1988. Responses of microsomal NADPH-cyto-

chrome c reductase activity and cytochrome P-450 in the digestive

glands inMytilus edulis and Littorina littorea to environmental and

experimental exposure to pollutants. Mar. Ecol. Prog. Ser. 46 (1–

3), 37–43 (MEPS special. Biological Effects of Pollutants: Results

of a Practical Workshop).

Moore, M.N., 1988. Cytochemical responses of the lysosomal

system and NADPH-ferrihemoprotein reductase in the molluscan

A. Mohammed, J. Agard / Marine Pollution Bulletin 48 (2004) 784–789 789

digestive cells to environmental and experimental exposure to

xenobiotics. Mar. Ecol. Prog. Ser. 46, 81–89.

Moore, M.N., 1979. Cellular responses to polyaromatic hydrocarbons

and phenobarbital inMytilus edulis. Mar. Environ. Res. 2, 255–263.

Moore, M.N., Pipe, R.K., Farrar, S.V., Thomson, S., Donkin, P.,

1987. Lysosomal and microsomal responses to oil-derived hydro-

carbons in Littorina littorea. In: Capuzzo, J.M., Kester, D.R.

(Eds.), Oceanic Processes in Marine Pollution; Biological Processes

and Waste in the Oceans, 1. Robert E. Kriege Publishing

Company, Florida, pp. 89–97.

Pearse, A.G.E., 1980. Histochemistry: Theoretical and Applied, Vol. 1:

Preparation and optical technique. In: Preparation and Optical

Technique, vol. 1. J&A Churchill Publications, London.

Payne, J.F., 1977. Mixed Function Oxygenase in marine organisms in

relation to petroleum hydrocarbon metabolism and detection. Mar.

Pollut. Bull. 8 (5), 112–116.

Quattrochi, L.C., Lee, R.F., 1984. Microsomal cytochrome P-450

from marine crabs. Comp. Biochem. Physiol. 79C (1), 171–

176.

Singer, S.C., Paul Jr., E.M., Gonsoulin, F., Lee, R.F., 1980. Mixed

Function Oxygenase activity in the Blue crab, Callinectes sapidus,

characterization of enzyme activity from stomach tissue. Comp.

Biochem. Physiol. 65C, 129–134.

Wilson, R.D., Monaghan, P.H., Osanik, A., Price, L.C., Rogers,

M.A., 1974. Natural marine oil seeps. Science 184, 857–

865.