Environ. Sci. Technol. 1997, 31, 1198-1205
Induction of Cytochrome P4501A in the Intertidal Fish Anoplarchus purpurescens by Prudhoe Bay Crude Oil and Environmental Induction in Fish from Prince William Sound BRUCE R. WOODIN,* ROXANNA M. SMOLOWITZ, AND JOHN J. STEGEMAN* Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
Cytochrome P4501A (CYP1A) induction is a sensitive and specific adaptive response in fish exposed to xenobiotics including petroleum hydrocarbons. CYP1A expression was examined in the intertidal fish Anoplarchus purpurescens collected from or caged at reference sites and sites oiled by the Exxon Valdez spill in Prince William Sound. Immunoblotting of hepatic microsomes showed that the content of CYP1A in fish at oiled sites was up to 6-fold greater than that in reference site fish. Fish injected with the CYP1A inducer β-naphthoflavone showed a 70-fold induction of CYP1A, to levels six times the highest level seen in the field. To model the field exposure, fish were maintained over oiled sediments and/or fed amphipods collected from an oiled site. Hepatic microsomal CYP1A was induced 49-fold in fish exposed to oiled sediments but rapidly returned to control levels after fish were removed from oil exposure. Immunohistochemistry showed CYP1A induction in multiple organs. CYP1A staining in hepatic and some extrahepatic cells was highly correlated (r 2 g 0.95) with the hepatic CYP1A content detected by immunoblot. Oiled food induced CYP1A most strongly in intestinal mucosal epithelial and endothelial cells. Relatively low levels of CYP1A were observed in liver, gill, and gonad of fish exposed to oil through the diet, consistent with the metabolism of dietary hydrocarbons by intestinal CYP1A. Exposure to oiled sediment alone strongly induced CYP1A in endothelial cells in all organs examined. Thus, oil present in Prince William Sound sediments more than 1 year after the spill was able to induce CYP1A in intertidal fish. The caging and laboratory experiments indicate that the induction of CYP1A observed in field specimens of A. purpurescens from oiled sites was due primarily to persistent spill-derived hydrocarbons.
Introduction On March 24, 1989, the Exxon Valdez ran aground on Bligh’s Reef in Prince William Sound, AK, spilling 11 million gal of Prudhoe Bay crude oil in North America’s worst oil spill to date. In the months following, thousands of miles of shoreline were contaminated to varying degrees by oil from the spill * Address reprint requests to B.R.W. All other correspondence should be sent to J.J.S.
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(1). Weathering and physical cleanup and bioremediation programs removed much of the oil visible on surface sediments, but substantial amounts remained for months in the subsurface sediments at many sites (2). Determining the degree to which oil in sediments after such spills can elicit biological effects is important in evaluating the continuing risk to biota and the success of remediation attempts. Biochemical alterations that can be linked to hydrocarbon exposure in marine animals may indicate the amount and geographical and temporal extent of the remaining oil. One of the most sensitive and specific biochemical parameters for assessing sublethal exposure to certain classes of environmental pollutants is the induction of cytochromes P450 (CYP) in the CYP1A gene subfamily. CYP1A mRNA, protein, and associated catalytic activities are induced in fish exposed to polychlorinated biphenyls (PCB), chlorinated dioxins, chlorinated dibenzofurans, and polycyclic aromatic hydrocarbons (PAHs) (3-7). Some chemicals in these classes induce CYP1A through the action of the aryl hydrocarbon receptor (AhR) (8). CYP1A proteins catalyze the oxidation of many of these same pollutant chemicals, the first step in biotransformation pathways that render these lipophilic chemicals more polar and hence more readily eliminated. Prudhoe Bay crude oil contains PAH (9) that could induce and be metabolized by CYP1A enzymes. The oxidation of some PAH components of Prudhoe Bay crude oil by CYP1A, including benzo[a]pyrene, benzanthracene, and chrysene yields highly reactive intermediates that can bind to proteins and nucleic acids (10) resulting in toxicity or mutagenicity (11). As demonstrated in studies of temperate or boreal fishes, CYP1A is expressed at very low levels in liver of untreated fish from clean environments (12). Elevated levels of CYP1A in fish from the environment are thus indicative of exposure to xenobiotic chemical inducers. The CYP1A content in liver of fish living in polluted environments is highly correlated with the amounts of specific hydrocarbon pollutants measured at the sites of collection or within the tissues of the fish (13, 14). In the present studies, the induction of CYP1A was evaluated in an intertidal fish collected at sites that were impacted by the Exxon Valdez oil spill and from reference sites that were not impacted. The species selected was the high cockscomb prickleback (Anoplarchus purpurescens), an abundant and readily obtainable fish in many intertidal systems. We also examined the ability of oil in Prince William Sound sediments more than 1 year after the initial spill to induce CYP1A by caging fish at reference and oiled sites. Fish from a reference site were also exposed in the laboratory to heavily-oiled sediments collected from an impacted field site and fed amphipods collected in the oiled zone. PAH can bioaccumulate from sediments to amphipods (15), a primary food source for many intertidal fish, including the intertidal fish used in this study. Our objectives were to evaluate the degree of CYP1A induction by environmental levels of oil relative to the animal’s capacity to respond and to determine the role of the route of exposure on CYP1A expression in various tissues. The rapidity of the induction response and its decline after the removal of oil were examined in the fish maintained over oiled sediments. Fish were also injected with β-naphthoflavone (BNF), a known inducer of CYP1A, to produce a strong response against which to compare the levels of CYP1A measured in the field and experimentally oiled fish. Tissue and cell type specific induction of CYP1A in various organs from fish exposed to oiled sediments and food in the laboratory was determined using immunohistochemical techniques. The data presented here enhance our understanding of the effects of oil spills and the role of exposure routes in determining the sites of action of PAH.
S0013-936X(96)00719-5 CCC: $14.00
1997 American Chemical Society
FIGURE 1. Sites of collection and caging of A. purpurescens in Prince William Sound. The track of the oil spilled by the grounded Exxon Valdez is indicated by shading. Collection sites are indicated by 2.
Materials and Methods Animal Selection and Collection. Anoplarchus purpurescens were collected by hand or dip net from beneath stones in the upper to mid-intertidal zone (exposed at low tide). This species exhibits a very limited home range, approximately 15 m. Based on standard length, fish ranged in age from 1 to 4 years. Spawning occurs in late winter, and at the time of sampling (June 15-25, 1990), gonads were either undetectable or very small (gonadosomatic index ) 1.5 ( 1.7, females; 1.0 ( 0.8, males). Collection Sites. Fish were collected from five sites (Figure 1): three sites that were oiled to varying degrees as a result of the March 24, 1989, Exxon Valdez spill of Prudhoe Bay crude oil (Disk, Ingot, and Knight Islands) and two reference sites that were not directly impacted by the spill based on visual inspection and records of oil movement (Hinchinbrook Island and Port Fidalgo). Some oiled sites were chosen because bioremediation and sediment sampling studies were being conducted at the site or in the beach segment immediately adjacent to the sites. Caging Experiments. On May 27, 1990, 45 fish ranging in standard length from 6.9 to 12.5 cm were collected at the reference site on Hinchinbrook Island. Twenty-three fish were caged in the intertidal zone at the Hinchinbrook Island site in two mesh cages (11-12 fish/cage). The following day, 11 fish/cage were placed in the intertidal zone at Ingot Island (Alaska Department of Environmental Conservation; ADEC Site IN24) and Knight Island (ADEC Site KN211). The fish were examined 2 weeks later and determined to be in good health based on external appearance. It was determined at that time that amphipods, a primary component of the diet of these fish, were present in the cage. All of the caged fish had full guts containing amphipods at the time of sampling (day 26 or day 28). β-Naphthoflavone Treatment. Fish were collected from the control site at Hinchinbrook Island and shipped to the Woods Hole Oceanographic Institution in Woods Hole, MA. Fish were maintained in flowing, filtered seawater at 15 °C with aeration and were fed daily with frozen brine shrimp for
7 weeks prior to treatment. Fish were injected intraperitoneally on days 0 and 3 with β-naphthoflavone (BNF) in corn oil, at a dose of 10 mg of BNF/kg of body weight at each injection or with an equivalent volume of corn oil (10 µL/g of fish). This dosing regimen has been found to produce a strong CYP1A response in fish. Fish were killed by cervical scission on day 6, and livers were weighed and grouped in pools of 1-3 livers per pool (0.06-0.10 g total) to yield four pools per treatment type. Laboratory Exposure to Oiled Sediment and Food. Heavily oiled sediments were collected from Rua Cove on Knight Island (ADEC Site KN213, station 94) on June 27, 1990, and were shipped to Woods Hole, where they were stored sealed at 4 °C for 6 months prior to use. Sixteen fish were placed in tanks with unoiled sediment and 62 were placed in tanks with oiled sediment. The unoiled sediment was clean sand from glacial deposits on Cape Cod. Eight fish in a tank with the clean sand and 8 fish in a tank with oiled sediment were fed daily a diet of oiled amphipods collected from Knight Island (KN211) to examine the role of oil-derived hydrocarbons in the natural diet of these fish on CYP1A. All other fish were fed frozen brine shrimp. Six large fish (>6 g each) or pools of smaller fish (
