Environ. Sci. Technol. 2006, 40, 2851-2858
Humic Substances and Crude Oil Induce Cytochrome P450 1A Expression in the Amazonian Fish Species Colossoma macropomum (Tambaqui) A L I N E Y . O . M A T S U O , * ,† BRUCE R. WOODIN,‡ CHRISTOPHER M. REDDY,§ ADALBERTO L. VAL,† AND JOHN J. STEGEMAN‡ Laboratory of Ecophysiology and Molecular Evolution, National Institute for Research in the Amazon (INPA), Av. Andre´ Arau ´ jo, 2936, Aleixo, Manaus, Amazonas 69083-000, Brazil, and Biology Department and Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
Cytochrome P450 1A (CYP1A) induction is used widely as a biomarker of exposure to pollutants, such as petroleum hydrocarbons, yet CYP1A inducibility has been characterized in few tropical fish. Using Western blot analysis, catalytic assay, and immunohistochemistry, we evaluated CYP1A induction in an Amazonian fish (tambaqui; Colossoma macropomum) acclimated to humic substances (HS) and acutely exposed to crude oil. HS are ubiquitous in Amazonian waters, and they are known to affect the bioavailability of pollutants. CYP1A activity was also measured in fish exposed for 10 days to a range of concentrations of HS from both natural and commercial sources. Crude oil induced CYP1A expression in tambaqui, as expected. Exposure to both HS and crude oil resulted in greater levels of CYP1A expression relative to that in fish exposed to petroleum alone. Interestingly, CYP1A induction was also observed in fish exposed to HS alone. Induction by HS was concentrationdependent, and activity was higher in fish exposed to HS from the commercial source than in fish exposed to the HS from the natural source. The use of CYP1A as a biomarker of exposure to pollutants such as petroleum hydrocarbons in fish living in environments rich in humic substances should be considered with caution given that HS themselves induce CYP1A expression. Our results suggest that there may be as yet unknown CYP1A inducing components (aryl hydrocarbon receptor agonists) in humic substances.
Introduction Members of the cytochrome P450 1 family (CYP1), particularly CYP1A, are catalysts for the oxidative biotransformation * Corresponding author phone: (574) 631-2665; fax: (574) 6317413; e-mail:
[email protected]. Present address: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, 46556. † National Institute for Research in the Amazon (INPA). ‡ Biology Department, Woods Hole Oceanographic Institution. § Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution. 10.1021/es052437i CCC: $33.50 Published on Web 03/14/2006
2006 American Chemical Society
(phase I reactions) of planar substrates including polycyclic aromatic hydrocarbons (PAHs) and some polychlorinated biphenyls (PCBs). Induction of CYP1A by PAH-type pollutants occurs through activation of the aryl hydrocarbon receptor (AHR) transcription factor that controls the expression of several genes, most prominently CYP1A. CYP1A induction has been used widely as a biomarker of environmental exposure of vertebrates to AHR agonists (1). Prominent sources of PAHs in the environment include petroleum and the incomplete combustion of organic matter (2). Although the metabolism of chemicals by CYP1 enzymes to more polar and hence more readily excretable products is generally favorable to the organism, the oxidation of some PAHs results in more toxic intermediates including highly carcinogenic derivatives (3-4). CYP1A induction is therefore related to the susceptibility of fish to the toxic effects of crude oil components. In 1988, petroleum extraction began at Coari along the Urucu River in the Amazon basin of Brazil. Estimates have suggested that this reserve may contain over 5.5 billion gallons of crude oil; the average extraction of petroleum in 2002 was 2.4 million gallons per day (5). Following extraction, the crude oil is transported by ship from the Urucu River to the refinery located in Manaus (approximately 600 km). The risk of oil contamination or spills during transport is of particular concern in this region, which harbors an abundant and uniquely diverse population of fish and other wildlife. One factor that may affect the toxicity of petroleum in Amazonian waters is the presence of high concentrations of humic compounds originating from the decay of vegetation and animals. Humic substances (HS) constitute the major fraction of dissolved organic carbon (DOC) in aquatic ecosystems (6) and are characterized by molecular weights from 500 to 5000 Da, a moderately hydrophobic nature, and a variety of functional groups (carboxylic, phenolic hydroxyl, hydroxyl, carbonyl) (6-8). The concentration of HS is low in most freshwaters around the world (0.50-4.0 mg C/L) (6), but can exceed 50 mg C/L in streams along the Rio Negro, a major tributary of the Amazon River (9, 10). In sedimentrich waters, such as those of the Rio Solimo˜es (or Upper Amazon), a significant portion of the HS is adsorbed to silt or clay particles, so the concentration varies from 5 to 15 mg C/L (11). The concentration of HS in the water also varies seasonally with changes in depth of the water. Water levels at the confluence of the Rio Negro and the Amazon River vary by as much as 8-11 m seasonally. Humic substances affect the bioavailability of organic contaminants through different mechanisms of binding and adsorption (12). A number of studies have indicated decreased bioavailability of lipophilic pollutants to aquatic organisms in the presence of HS (12-18). In a few cases, however, HS have been reported to increase the solubility of petroleum hydrocarbons, possibly resulting in higher bioavailability to aquatic organisms (19-21). HS can also affect the physiology of living organisms directly (22-25), a factor that is largely overlooked in ecotoxicological studies. Although some studies have addressed CYP genes and CYP1A inducibility in tropical fish (27-29), CYP1A regulation has not been addressed in fish in the Amazon basin. We therefore examined expression of CYP1A in a native Amazonian fish species (tambaqui; Colossoma macropomum) acclimated to HS and exposed to petroleum hydrocarbons. In addition, CYP1A catalytic activity was analyzed in fish acclimated to natural and commercial sources of HS alone at different concentrations. VOL. 40, NO. 8, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Experimental Section Laboratory Acclimation of Tambaqui. Juvenile tambaqui were obtained from a fish farm and acclimated for 60 days in 100 L fiberglass tanks at the Laboratory of Ecophysiology and Molecular Evolution (INPA). Amazonian waters are typically soft, so acclimation was in water with the following composition: Na+ ) 34, Ca2+ ) 11, K+ ) 15, Mg2+ ) 0.8, all in µmol/L; humic content ,1 mg C/L; pH 6.0. Water temperature was 28 ( 2 °C for all experimental procedures. All fish were fed Purina fish chow, at 2% of body weight per day. Exposure of Tambaqui to Crude Oil and HS. Tambaqui (32 ( 2 g) were exposed to HS alone, crude oil alone, or HS + crude oil. For the exposures involving HS, we used Aldrich humic acid [AHA] (Sigma) as a surrogate for HS, because isolation and concentration of HS from Amazonian waters was not logistically possible. Stock solutions of AHA were prepared by dissolving AHA in deionized water; the solutions were stirred for 6 h and kept refrigerated until use. For the experiments involving HS in combination with oil, we used HS concentrations of 22 mg C/L, equivalent to midrange concentrations seen in the Rio Negro. A subset of fish was acclimated for 10 days to water containing AHA at this concentration, in fiberglass tanks that had not been used for any oil or other chemical exposures. Feeding was suspended 24 h before the exposures. Fish acclimated to either soft water or to HS were transferred to glass aquaria with one fish per aquarium with 15 L of water. Exposure to crude oil was accomplished by adding it directly to the soft water or humic-enriched water at 2.8% (v/v) in aquaria holding the fish. Aquaria were aerated and covered with plastic lids during the experiments. The crude oil was from the Urucu Reserve (Amazonas, Brazil) and is a light type crude oil with 0.06% sulfur, 7% naphtha, 5 mg/L nickel, 5 mg/L vanadium, and a high-boiling fraction of 86.8 (30). Further properties of this crude oil, including the PAH content, are not available at this time. The concentration of Urucu crude oil used in these experiments represents half the 96-hour LC50 value for this oil in tambaqui (A. Val, unpublished data). Following exposure, fish were deeply anesthetized in less than 60 s with a high dose (1 g/L) of the anesthetic MS-222 (Sigma), after which livers were excised within 15 s and frozen immediately in liquid nitrogen, timing that should obviate an effect of anesthetic on liver P450. Tissues were held in liquid nitrogen for preparation of microsomal fractions and analysis of ethoxyresorufin-O-deethylase (EROD) activity and CYP1A protein levels. Portions of gill and liver were fixed in 10% neutral buffered formalin for immunohistochemistry. Exposure to Varying Concentrations and Sources of HS. In a separate experiment, the effects of HS on CYP1A were tested further by exposing soft water acclimated tambaqui (12 ( 3 g) to different concentrations of HS from both natural and commercial sources. For this study, we used smaller fish because the supply of natural HS was limited. The natural HS was natural organic matter (NOM) isolated through reverse osmosis from Luther Marsh (Ontario, Canada), provided by Richard Playle (Wilfrid Laurier University). The commercial HS was AHA. The DOC content of concentrated stock solutions of NOM (1858 mg C/L) was previously determined in filtered samples in a total organic carbon analyzer 5050A (Shimadzu). The DOC content of AHA has been estimated at about 40% (31). Fish were exposed to 0, 20, 40, or 80 mg C/L of AHA, and 40 or 80 mg C/L of NOM for 10 days. The water plus HS was renewed every other day. Exposures to each dose and type of HS were done in one opaque polyethylene container per concentration, each containing 12 fish in 15 L of water. These 2852
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containers had not been previously used for oil or other chemical exposures. After 10 days, 4-6 fish from each group of 12 were randomly sampled and sacrificed to collect liver for analysis of both EROD and benzyloxyresorufin-Odebenzylase (BROD). Preparation of Microsomal Fractions. Frozen and fixed tissue samples were transported to Woods Hole, where the hepatic microsomal fractions were prepared and the assays were performed. Liver was defrosted on ice, homogenized with buffer, and subjected to differential centrifugation (32). Microsomal fractions were immediately frozen and stored in liquid nitrogen until use. Protein content was measured with bicinchoninic acid (33) (Pierce Kit-Perbio). Enzyme Activity. Microsomal EROD and BROD rates were determined using modifications of the microplate-based fluorometric assay described elsewhere (34). 7-Ethoxy- or 7-benzyloxyresorufin (Molecular Probes) was dissolved in methanol. Prior to assays, these substrates were diluted in assay buffer to a final assay concentration of 2 µmol/L. Enzyme activity was measured in duplicate in 48-well plates (CytoFluor Series 4000). Immunoblotting. Microsomal proteins (60 µg from control and HS-acclimated fish and 2-5 µg from oil-exposed fish) were electrophoretically resolved on 5-20% gradient gels (Precast Snap-A-Gel, Jule Inc.), along with molecular weight and CYP1A standards. Transfer, staining, and detection of CYP1A with monoclonal antibody Mab 1-12-3 were conducted as before (35). Staining was visualized by enhanced chemiluminescence, and images were scanned and analyzed for molecular weight and CYP1A content (nmol of scup CYP1A equivalents/mg microsomal protein), using NIH Image Software (Macintosh). Immunohistochemistry. Formalin-fixed gill and liver samples from tambaqui exposed to crude oil, with or without HS, were embedded in paraffin and examined for CYP1A expression by immunohistochemistry (36). Gills were decalcified prior to embedding, using serial formic acid-sodium citrate solutions. Sections (5 µm) were mounted on slides (Superfrost/Plus; Fisher Scientific), and dried at room temperature for 2 days prior to staining. The primary antibody was MAb 1-12-3, and the secondary antibody was goat antimouse IgG. CYP1A staining in the cells was detected by optical microscopy, and the signal was scored as described previously (36-37). Analysis of HS for Contaminants. The AHA and NOM used in these experiments were analyzed for the presence of known AHR agonists, in particular PAHs and PCBs. Concentrated aqueous solutions of NOM, AHA, and a blank (distilled water) were extracted three times with methylene chloride. The extracts were reduced in volume and injected onto a gas chromatograph (Agilent 6890N series) connected to a 5973 network mass selective detector (70 eV ionization energy) operating in the full scan mode. The resulting chromatograms were analyzed for PAHs, PCBs, and other noteworthy compounds. The approximate detection limits were 0.20-0.25 ng of an individual PAH or PCB per mg of organic C, in either HS. Statistical Analysis. To minimize differences in variance between treated and control groups, CYP1A, BROD, and EROD values (X) were transformed prior to statistical analysis using a log (X + 1) transform. Differences between all pairs of treatment means were determined using the TukeyKramer multiple-comparisons test PRISM (GraphPad Software, Inc.). Because CYP1A levels were below the limit of detection (LOD) for all control samples, the LOD for CYP1A was estimated as described in the results section below, and it was compared to the lower 99% confidence interval of exposure means for this parameter. Log-transformed dose response EROD and BROD data for AHA exposures were
FIGURE 1. Western blot analysis of CYP1A in liver microsomes of tambaqui (N ) 6) following exposure to different treatments. Gel loading in control and AHA groups was 60 µg/well. Loading in treatments petro and the AHA + petro groups was 2-5 µg/well. CYP1A protein concentration was below the limit of detection by this technique in the control group (
