Effects of complex waste mixtures on hepatic monooxygenase activities in brown bullheads (Ictalurus nebulosus)

Environmental Pollution 62 (1989) 113 128

Effects of Complex Waste Mixtures on Hepatic Monooxygenase Activities in Brown Bullheads (lctalurus

nebulosus)

Evan P. Gallagher* & Richard T. Di Giulio Ecotoxicology Laboratory, School of Forestry and Environmental Studies, Duke University, Durham, NC 27706, USA (Received 12 October 1988; accepted 10 July 1989) ABSTRACT Hepatic M F O components (cytochrome P-450, cytochrome b s, and ethoxyresorufin O-deethylase, EROD ) were measured in brown bullheads (Ictalurus nebulosus) inhabiting a creek receiving a complex mixture of organics and trace metals. The activities of these same enzymes were also measured in bullheads from an uncontaminated reference site to assess the relative ability of M F O parameters to serve as a biomarker of aquatic pollution. Bullheads analyzed from the polluted site had lower hepatic microsomal P-450 (p < 0"01) concentrations and similar EROD activities per mg protein as compared to bullheads from the reference site. However, analysis o f enzyme turnover ratios revealed greater E R O D activity per mg cytochrome P-450 (p < 0"05) in the fish from the polluted site. No differences in cytochrome b 5 activities were observed between the two groups. As compared to the reference site, bullheads collected from the polluted creek had an increased occurrence of l i p and lower jaw lesions and liver damage, including elevated liver~body weight ratios. Accordingly, the monooxygenase activities measured in this study were not reliable indicators of chemical pollution or contaminant stress in bullheads in the polluted creek. Further research is needed concerning contaminant interactions, particularly among organic pollutants and metals and their effects on monooxygenase activities.

INTRODUCTION In the last decade, a major research priority in the field of aquatic toxicology has focused on the potential for utilizing various biochemical and * To whom correspondence should be addressed. 113 Environ. Pollut. 0269-7491/89/$03'50 © 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain

114

Evan P. Gallagher, Richard T. Di Giulio

physiological responses of aquatic organisms to serve as early indicators of chemical exposure or xenobiotic related stress. These indices, or biomarkers, would theoretically provide sensitive tools for assessing both the presence and impacts of various contaminants in the aquatic environment. Obviously, the sensitivity and reliability of any biological indicator necessitates a thorough understanding of its mechanism of action. Also, knowledge of either a quantitative change in biochemistry, physiology or morphology carries little meaning unless the relationship of the perturbation to the overall physiological status of the organism is understood. Finally, field validation is essential for the application of any biochemical or physiological index as a biomonitoring tool. Much of the research in this area has concerned the ability of many environmentally relevant types of organic pollutants to induce MFO activity in fish (Payne & Penrose, 1975; Brown, 1976; Stegeman, 1980; Melancon et al., 1987). Exposure to polycyclic aromatic hydrocarbons (PAHs), for example, has resulted in MFO induction in fish sampled from areas contaminated by crude oil (Burns, 1976; Stegeman, 1978; Baumann et al., 1982; Payne et al., 1987). Exposure to polychlorinated and polyhalogenated biphenyls has also been shown to induce certain MFO activities in fish (Hill et al., 1976; Dent, 1978; Melancon et al., 1981). Many of these studies involve comparisons of hepatic MFO activities in fish sampled from polluted locations to fish from clean sites. Although these studies typically reveal higher MFO activities in fish from polluted locations, this is not always the case (Ahokas et al., 1976; Fabacher & Baumann, 1985). Unfortunately, a serious obstacle to the application of monooxygenases and other biotransformation enzymes as pollutant biomarkers lies in the fact that the overwhelming majority of real world pollution scenarios involve complex mixtures, as opposed to pure compounds. Interactive effects among environmental chemicals may substantially alter the toxicokinetics, metabolism, and toxicity of isolated compounds. For example, many industrial solvents are hepatotoxic and through either direct or indirect means may destroy or inhibit certain drug metabolizing enzymes (Plaa, 1986). Another important class of aquatic pollutants are trace metals. However, the effects of these compounds on fish monooxygenases have remained virtually unknown. Relatedly, reports concerning interactions of metals and organic compounds on biotransformation enzymes are also scarce. One such study (George & Young, 1986) examined the effects of single and co-exposure to cadmium and 3-methylcholanthrene (3-MC) in the plaice (Pleuronectes platessa). The authors found that 3-MC increased EROD, glucuronyl transferase and glutathione S-transferase activities while cadmium, though not affecting glucuronyl transferase activity, did inhibit EROD and GSH transferase activities. Cotreatment with both

Hepatic monooxygenase activities in brown bullheads

115

compounds decreased all enzymes activities measured. The aforementioned study was particularly significant in that it demonstrated a synergistic inhibition of certain drug metabolizing enzymes in an aquatic species exposed to PAH/metal mixture. Accordingly, in field situations dominated by mixtures of inducing and non-inducing compounds it may not be possible to relate monooxygenase activities, alone, to the health of the affected environment. To our knowledge, there are not reports of field trials involving monooxygenase induction and cytochrome P-450 levels in fish inhabiting areas contaminated by organics (inducers and non-inducers) and trace metals. In such scenarios, interpretation of piscine hepatic monooxygenase data would necessitate qualitative and quantitative characterization of pollutants present at multiple biological levels. Unfortunately, if the ultimate goal is to use monooxygenase induction as an early warning indicator in the context of a biomonitoring program, it may not be feasible for a federal, state or local regulatory agency to employ such expensive testing on a routine basis. Therefore, for this study we looked for a field site affected by complex mixtures of contaminants for which an extensive analytical data base could be obtained. The objective being to test environmental induction of monooxygenase activity as biomarkers of mixture contamination. In this report, hepatic monooxygenase activity in brown bullheads inhabiting a creek containing a complex mixture of organic and metal wastes was compared to bullheads from a nearby nonpolluted site sharing otherwise similar chemical and physical conditions.

MATERIALS A N D METHODS Chemicals 7-ethoxyresorufin was obtained from Molecular Probes Inc. (Junction City, OR). NADPH, crystalline bovine albumin, glycerol, sodium dithionite, 5,6benzoflavone (BNF) and potassium chloride and tris buffers were obtained from Sigma Chemical Co. (St Louis, MO). Liquid nitrogen was obtained from the cryogenics laboratory at Duke University. Other reagents used in this study were of the highest quality available from commercial sources. Field sites Brown bullheads (lctalurus nebulosus) were collected from Hancock and Slocum creeks during October/November 1985 and July/August 1986 using both fyke nets and hook and line. Both creeks are tributaries of the Neuse

116

Evan P. Gallagher, Richard T. Di Giulio

River and are located in Pamlico County, NC. The sources of pollutant input to Slocum Creek included both runoff from landfills on Cherry Point Marine Air Station (MAS), located on the eastern perimeter of the creek near Havelock, NC, and three upstream wastewater treatment plants. The landfills were used from the early 1970s until 1984 to receive waste from a marine aircraft rework facility and sludge from the base's industrial treatment plant. Waste products stored in landfills included paints, organic solvents, waste oil and filings and plating sludges containing trace metals (NC Office of Hazardous Waste, unpublished closure plan for Cherry Point MAS landfills, 1984). Many of these pollutants entered the creek as a result of direct leaching from waste lagoons, overland runoff during storms, and from groundwater contamination by percolation of surface water through contaminated fill. Groundwater beneath the landfills takes about 250 days to discharge into the creek (NC Office of Hazardous Waste, unpublished file data, 1984). Although the landfills were closed in 1984, leaching and runoffto Slocum Creek continued during the course of the study. Hancock Creek is an isolated drainage area located approximately 3.5 km east of Slocum Creek. Hancock Creek is physically isolated from the landfills and does not receive groundwater discharges from the base (NC Office of Hazardous Waste, unpublished file data, 1984). There are no treatment plants located along Hancock Creek and, according to state and Marine Corps environmental management personnel, the creek does not receive any significant source of contamination (pers. comm.). Chemical analyses of Slocum Creek sediments, water, and biota were conducted by independent laboratories under contact to the United States Marine Corps (USMC, 1984). All analyses were conducted using EPA standard methods for priority pollutant analyses (USMC, 1984).

General procedures Animals All fish used in this work were selected for length and weight uniformity and were outside of their normal spawning season. The approximate age of the fish was determined by length-frequency method based upon data collected for brown bullheads in the southeastern United States (Carlander, 1969). Prior to sacrifice, the animals were examined for evidence of gross external lesions (non-specific). Bullheads collected via fyke nets were transported to a holding pen in a nearby catfish pond until sacrifice. All fish were killed within 6h of capture. Bullheads captured with hook and line were killed immediately by a blow to the head with a blunt instrument followed by severing of the spinal cord. After weighing (1986 samples only), livers were excised and the presence/absence of gross liver abnormalities was recorded.

Hepatic monooxygenase activities in brown bullheads

117

Liver tissues were then washed in ice-cold 1.15 % w/v KC1 to remove excess hemoglobin, blotted dry, placed in plastic vials and submerged in liquid nitrogen overnight until tissue preparation. Tissue preparation

All procedures were carried out on ice (0-4°C). Livers were rapidly thawed, weighed and rinsed with ice-cold KCI buffer. Microsomes were prepared by the method of Eriksson et al. (1978). Each tissue was weighed and subsequently homogenized in 0.15M KC1 buffer (pH 7"4) at a total tissue volume of 20%. Four passes of the tissue through a Brinkman (Westbury, NY) polytron motorized tissue processer were used to homogenize the tissues. The microsomal fraction was isolated from a 10 000g supernatant by centrifuging at 105 000g for 1-5 h. The pellets were resuspended in a 1:4 w/v of 0.15 M KC1 and 0"1M tris (pH 7.4) with 20% glycerol per g wet tissue weight (Forlin & Andersson, 1985). Glycogen pellets which sedimented as a separate layer were discarded. The homogenates were then aliquoted into 2 ml tubes and stored at - 7 0 ° C until use. Protein concentrations were assayed by the method of Lowry et al. (1951) within 72h of tissue preparation using crystalline bovine serum albumin as standard. Monooxygenase assays

The reduced carbon monoxide binding spectra of cytochrome P-450 was assayed by the method of Matsubara et al. (1976) using an extinction coefficient of 106 c m - 1 mM- 1. This method proved to be more useful than the widely used Omura & Sato (1964) procedure due to hemoglobin contamination of microsomal fractions. Microsomal preparations containing 1 mg protein were placed in 1 ml 0.1M phosphate buffer (pH 7.4) in sample and reference cells of a Shimadzu UV-260 (Kyoto, Japan) split-beam spectrophotometer. After recording the baseline, CO was bubbled through both cuvettes at a rate of 1 bubble per second for 20-30 s. The sample cuvette was then reduced with a few mg of sodium dithionite. The difference spectra of the CO-treated samples were then measured and recorded. Cytochrome b5 was assayed via the method of Guengerich (1982) utilizing N A D H difference spectra assuming an extinction coefficient of 171 c m - 1 mM- 1 Ethoxyresorufin O-deethylase (EROD) activity was measured by a modification of the method of Burke & Mayer (1974). The reaction mixture, prepared in a fluorometer cuvette at room temperature, contained 0" 1M trisHC1 buffer, pH 7.8, 0.5-1-0mg microsomal protein, 2/~M ethoxyresorufin, and 50 #M N A D P H in a final volume of 2.5 ml. Maximum rates of E R O D activity were obtained with 1-2 #M substrate concentrations with a decrease in the deethylation rate occurring at higher concentrations (data not shown). The minimal amount of methanol used as a vehicle for ethoxyresorufin did

118

Evan P. Gallagher, Richard T. Di Giulio

not seem to interfere with the kinetics of the assay. These findings are similar to those of other researchers (Burke & Mayer, 1974; Stegeman, 1981). For the purpose of this study, the first 2 min of the reaction were used to calculate reaction velocities to avoid substrate depletion. The reaction rate was found to be linear using protein concentrations from 0" 1 to 3 mg (data not shown). The deethylation of ethoxyresorufin did not take place in the absence of liver microsomes or after heat denaturation, indicating that the reaction was catalyzed by the MFO system.

Statistical analysis The Student's t-test was used to analyze data collected for MFO comparisons and liver/body weight ratios among bullheads sampled from the two field sites (Buhyoff& Hull, 1982). Unless otherwise indicated, results were considered significant at p < 0-05.

RESULTS

Gross external observations Often fish sampled from Slocum Creek during October 1985, two (20%) had multiple non-specific lesions on the lip or lower jaw, and three (30%) had pale and/or fatty livers. In addition, one of four (25%) fish sampled from the same site in 1986 exhibited multiple non-specific lesions on the lip and a fatty liver. None of the fish sample from the reference site (Hancock Creek) during either period revealed any gross skin or liver lesions. Liver weight as a percentage of body weight was calculated during the 1986 sampling period and was significantly greater (p < 0-05) in bullheads from Slocum Creek (Table 1).

Monooxygenase activities Hepatic monooxygenase activities of brown bullheads collected during 1985 and 1986 are also presented in Table 1. Bullheads sampled from Hancock Creek during the summer of 1986 exhibited significantly higher levels of cytochrome P-450 concentrations than those collected in Slocum Creek (p < 0.01). However, no statistically significant differences were observed in either cytochrome b 5 or EROD activities in the same fish based on sampling location. EROD activity normalized per mg cytochrome P-450 (enzyme turnover) was significantly higher (p < 0"05) in Slocum Creek bullheads. The same trends in these parameters were observed in fish collected during the fall of 1985, although differences in cytochrome b 5 activity between the two

Hepatic monooxygenase activities in brown bullheads

119

TABLE 1 Hepatic Microsomal Monooxygenase Activities in Brown Bullheads Collected from Slocum Creek and Hancock Creek During 1985-1986 (mean + S E M )

Location

Hancock Creek 1985 b Slocum Creek 1985 c Hancock C r e e k 1986

Slocum Creek 1986

n

Liver (% body wt.)~

Cytochrome Cytochrome

EROD (nmol/min/mg)

EROD/P-450 (nmol EROD/ min/mg P-450)

0.141

0-100

0.381

0.181

0"083

0-081

0.446

(0.25)

0.266 (0'006)*

0"119 (0'010)

0-100 (0"009)

0.376 (0-010)

2.91 (0-22)**

0"218 (0"007)

0-092 (0"010)

0-105 (0"013)

(0.054)**

P-450 (nmol/mg)

b5 (nm/mg)

--

0"262

-4

6

1.89

0"480

" (g liver weight/g body weight) x 100. b Livers from l0 individuals were pooled for analyses. c Livers from 4 individuals were pooled for analyses. * Significantly greater than corresponding values obtained for the SIocum Creek sample p < 0.001. ** Significantly greater than corresponding values obtained for the Hancock Creek sample p < 0.05.

groups appeared more pronounced. As in the 1986 sampling period, higher b 5 activity was seen in fish taken from the clean site (Hancock Creek). Statistical analyses of 1985 data or of groups based on sampling period (i.e. 1985 vs. 1986 samples) could not be performed due to pooling of livers in the fall 1985 analysis. All field sampled brown bullheads exhibited a reduced CO-ligated cytochrome P-450 soret maximum at 450 nm.

DISCUSSION It has been suggested that measurement of certain piscine hepatic microsomal enzyme activities (e.g. P-450, EROD, AHH) may be useful in monitoring aquatic pollution (Payne & Penrose, 1975; Burns, 1976; Fingerman et al., 1983; Payne et aL, 1987). Spectrally determined microsomal cytochrome P-450 content, while not an indicator of catalytic function, reflects the collective presence of constitutive and inducible cytochromes P-450 (Omura & Sato, 1964). Enzyme activities such as EROD, AHH and ECOD are routinely employed to assess inducible cytochromes P-450 in fish (Stegeman, 1981; Williams & Buhler, 1983, 1984; Payne et al., 1987). However, immunochemical studies have shown that the major constitutive isozyme (LM2) as well as the major BNF-inducible P-448 isozyme (LM4B) of rainbow trout show a greater than 10-fold specificity

120

Evan P. Gallagher, Richard T. Di Giulio

towards ethoxyresorufin, as opposed to ethoxycoumarin or other commonly employed substrates (Williams & Buhler, 1983, 1984). Other reports have showed that polyclonal antibodies raised against rainbow trout LM4 inhibit EROD activity not only in trout, but in other freshwater and marine species as well (Williams & Buhler, 1984; Varanasi et al., 1986). Collectively, these and other studies suggest that fish species contain similar inducible cytochromes P-450. Since fish do not respond to phenobarbital type inducers (Buhler & Rasmusson, 1968), EROD activity is often used in conjunction with cytochrome P-450 content to indicate exposure of fish to PAHs and certain organochlorines (Melancon et al., 1981, 1987; Payne et al., 1987). The results of this study indicate that discretion should be employed when drawing conclusions about environmental contamination based solely upon monooxygenase activities. The bullheads analyzed from our polluted site (Slocum Creek) generally exhibited less P-450 content per mg microsomal protein, similar EROD activity per mg protein, and greater EROD activity per mg cytochrome P-450 as compared to fish sampled from the nonpolluted site (Hancock Creek). On interpreting the data one must consider the types of pollutants the fish were exposed to and the effects of these compounds on both constitutive and inducible forms of P-450. Although our reference site (Hancock Creek) was not analyzed for pollutants there is strong evidence to support that it is clean, or at the least, minimally contaminated (see materials and methods). Furthermore, a comparison of EROD activity in bullheads sampled from both creeks to bullheads held in either dechlorinated city water or polluted river water (Melancon et al., 1987) suggests that neither of our groups were MFO-induced. In Melancon's study, EROD activity in bullheads held in dechlorinated city water for 31 days (0.175 +_0-044 nmol/ min/mg protein) was significantly lower than in those held in Kinnikinnic River water (0-624 _+0"087 nmol/min/mg protein) for the same period. EROD activity in our groups ranged from 0.081nmol/min/mg protein (Slocum Creek sample, November 1985) to 0-105 ___0.013 nmol/min/mg protein (Slocum Creek sample, August 1986). In addition, all field sampled bullhead in this study exhibited enzyme turnover values less than 0.5 nmol EROD/min/mg cytochrome P-450 (Table 1), consistent with values for similar species sampled from nonpolluted sites (Fabacher & Baumann, 1985; Melancon et al., 1987). Chemical analysis of Slocum Creek water, sediments, and biota was carried out by independent analytical laboratories under contract to Cherry Point MAS. The results of inorganics analyses (Table 2) reveal elevated levels of copper, chromium, mercury and nickel in the sediments or water column as compared to other sites examined in North Carolina by the Division of Environmental Management (NCDEM, 1983). Bioaccumu-

Hepatic monooxygenase activities in brown bullheads

121

TABLE 2 R a n g e a n d M e d i a n C o n c e n t r a t i o n s o f I n o r g a n i c C o n t a m i n a n t s in S l o c u m C r e e k S e d i m e n t s a n d Biota ~

Ecosystem component

Number of individuals analyzed

Metal concentrations (range) Ag

Cr

Cu

11

<0.25

< 1-30

Bluegill Sunfish

10

<0.25

Blue Crab

3

0-37 3.60 (0"7)~ 1.0y 1.30-1.90 (1.1) 1.0 <0.025

Wedge Clam

4

BIOTA ~ Largemouth Bass

SEDIMENT h Bullhead sampling site Leachate Site"

<0.25~).61 ( < 0.25) 0-33 1.9(~7.10 (6.0) 1.0 < 8.0

33.3

< 1.30

0.47-1.10 (0.82) 1.0

1.90-3.30 (2.6) 1.0 2.30~3.30 (2,6) 1'0

51.8-266 (121) 1.0 440

< 8.0-38.0 (8,0) 0.67 154

ttg

Ni

<0.5(~1-0 (0-5) 0-64 <0.50-0.5 (<0.5) 0.10 <0-5

2-(~75-0 (12.0) 1.0 1-(~30.0 (3.4) 1.0 < 1.0

<0-5

1.10-1.70 (1.30) 1.0

0.63-1.84 (0.77) 1-0 2-16

< 50.0

<50-0

a mg/kg dry weight. Data reflect whole body concentration of metals. b /~g/g dry weight. Sediment samples were taken from three transects approximately fifteen yards apart at bullhead sampling site. ' One transect taken from area of Slocum Creek receiving maximum runoff from the landfills. This area lies about 0.8 km east of bullhead sampling site. These data were extracted from USMC (1984). Values in parentheses represent median concentrations. I Indicates occurrence ( × 100%) of compound in samples.

lation of mercury, chromium, and nickel by largemouth bass (Micropterus salmoides) and bluegill sunfish (Lepomis machrochirus), silver by blue crabs (Callinectes sapidus), and chromium, silver and nickel by wedge clams (Rangea cuneata) is reported in Table 2. The extent of silver and nickel contamination of Slocum Creek sediments cannot be assessed due to high detection limits of these chemicals during the chemical analyses (8"0 and 50"0 mg/kg, respectively). However, since these compounds were bioaccumulated by clams and blue crabs (Table 2), they were probably present in either sediment or sediment associated food particulates. Relative to other sites in North Carolina, elevated levels of nickel, zinc, chromium, copper and cadmium were also detected in sediments, striped bass (Ro¢cus linaetus), bluegill sunfish and spot (Leiostomos xanthrus) from Slocum Creek during studies conducted by the NC Department of Environmental Management during 1981-1983 (NCDEM, 1983).

Evan P. Gallagher, Richard T. Di Giulio

122

TABLE 3 R a n g e a n d M e d i a n C o n c e n t r a t i o n s o f O r g a n i c C o m p o u n d s [ # g / k g ] F o u n d in S l o c u m C r e e k Biota c Compound

Species Largemouth bass n=ll

VOLA TILE ORGANICS" Chloroform <25 24000 (6000) a 0.64 e Methylene chloride <25-18000 (37) 0.36 Toluene < 25-20 000 (<25) 0.27 2-butanone <25-20000 (1 100) 0.64 o-xylene <25-21 000 (37) 0.55 2-hexanone < 25-180 (<25) 0.10 PESTIC1DES/PCBs b Dieldrin <0-3-36.0 (5'0) 0-82 4,4'-DDE <0.3

4,4'-DDD

PCB-1254

<0.7-8.0 ( < 0.7) 0"45 <0.3-110 (40) 0.73

Bluegill sunfish n=12

Wedge clam n=4

Blue crab n=3

Channel Brown catfish bullhead n=l n=l

<25-21000 (31) 0.50

<25 18000 (8 500) 0.75

13000-20000 (14000) 1.0

NM

NM

<25-20000 (150) 0.67 < 25-20 000 (31) 0-75 <25-24000 (2000) 0.75 <25-19000 (< 25) 0.33 < 25-300 (<25) 0.17

<25-23000 (14000) 0-75 < 25

16000-18000 (18 000) 1.0 < 25

NM

NM

NM

NM

<25

NM

NM

<25

<25-540 (295) 0.67 <25

NM

NM

< 25

< 25

NM

NM

< 1.0-16.0 (4"0) 0-75 < 1-0-7.0 ( < 1"0) 0.17 <3.0-6.0

NM

NM

<0.4

<0.3

NM

NM

100

7.0

NM

NM

<0-9

<0.7

NM

NM

110

570

(3.0) 0.58 <10-150 (70) 0.67

Volatile organic concentrations reflect whole body analysis. b Pesticide/PCB concentrations reflect tissue fillet analysis. c Data extracted from USMC (1984). a Values in parentheses represent median concentrations. e Indicates occurrence ( x 100%) of compound in samples.

Hepatic monooxygenase activities in brown bullheads

123

As demersal fish, bullheads spend much of their lives in contact with sediments, thus increasing interaction with contaminants. The uptake and bioaccumulation of contaminants from sediments by bottom dwelling fish is well documented (Varanasi & Gmur, 1981; Malins et al., 1985) and it is likely that the bullheads sampled from Slocum Creek bioaccumulated sediment metals to some degree. As previously discussed, the effects of trace metals on fish metabolizing systems remains relatively unknown. In mammalian systems, additions of 0"01-10mu solutions of copper, iron, zinc, tin and cadmium completely inhibited aryl hydroxylase activity in the microsomal fraction of hamster fetal cells (Nebert & Gelboin, 1968). Yoshida & coworkers (1976) observed a decreased synthesis in cytochrome P-450 in mice exposed to cadmium. Similarly, Alvares et al. (1972) documented a 40%-50% decrease in cytochrome P-450 activity as well as ethylmorphine n-demethylase and aniline hydroxylase activities in hepatic microsomes of rats after administration of 5 mg/kg PbC12 iv, or 5 mg/kg CHaHgCli p. Cadmium, an inhibitor of EROD and glutathione S-transferase (GST) activities in fish (George & Young, 1986), was found in both largemouth bass (0.104).18mg/kg) and bluegill sunfish (1.30-1.90mg/kg) collected from Slocum Creek (USMC, 1984). However, it is difficult to determine how these levels of contamination may have affected monooxygenase activities in our sample of bullheads. Analysis of pesticides and PCBs (Table 3) revealed elevated levels of PCB1254 in tissue fillets of all species analyzed. This compound has been shown to be a potent inducer of MFO activity in several fish species (Hill et al., 1976; Addison et al., 1978; Melancon et al., 1981). The pesticide DDT or its metabolites were detected in all species analyzed (Table 3); however, these compounds do not appear to affect MFO activities in fish (Buhler & Rasmusson, 1968). Assessment of contamination by PAHs in Slocum Creek is impossible to the extremely high detection limits (670-6600/~g/kg) on these and other neutral organics during chemical analyses. It is likely that some level of PAH contamination exists due to the large volume of waste oil which was stored in the landfills and subsequently leaked into the creek over a 10-20 year period. As previously discussed, organic solvents constituted the major class of compounds stored in the landfills near Slocum Creek. Analysis of fish and shellfish from Slocum Creek revealed elevated levels of methylene chloride, chloroform, toluene, and other volatile organics (Table 3). This finding is relatively uncommon in the light of the short residence time of these compounds in aquatic ecosystems and probably reflects their steady leaching from the waste site. While most organic solvents are not considered classic mammalian MFO inducers, pentane, 2-heptanone, chloroform and toluene, as well as many other solvents, cause spectral changes in

124

Evan P. Gallagher, Richard T. Di Giulio

microsomal P-450 (Mclean, 1967; Yoshida & Kumaoka, 1975). The maximal spectral changes produced vary with the compound, with some substrates producing greater spectral change than others. Of greater relevance to this project is the potential for these compounds to induce hepatotoxicity in Slocum Creek bullheads. Thirty per cent (3 of 10) of the bullheads collected from the polluted site during 1985, and twenty five per cent (1 of 4) collected during 1986 had discolored or fatty livers. In addition, bullheads sampled from Slocum Creek had a significantly higher liver/somatic index when compared to fish from the clean site (Table 1). Since the liver has primary responsibility for biotransformation, any compound that interferes with its normal functioning may potentially affect xenobiotic metabolism. Solvents such as carbon tetrachloride, chloroform, and ethanol are potent mammalian hepatotoxins (Zimmerman, 1978). Acute injury by these compounds usually consists of liver necrosis and fat accumulation, and chronic exposure to these agents may result in marked alteration in liver structure and cirrhosis (Plaa, 1986). Fish dosed with high levels of carbon tetrachloride exhibit a disruption of biochemical liver functions as exemplified by changes in lipids, plasma protein, glycogen and cholesterol (Gingerich et al., 1978; Statham et al., 1978). Relatedly, the oxidation of chloroform by mammalian liver is enhanced by phenobarbital pretreatment which also results in a potentiation of hepatotoxicity (Docks & Kirshna, 1976). Slocum Creek bullheads were exposed to a myriad of compounds, including MFO inducers, non-inducers and hepatotoxicants. It is highly possible that exposure to non-inducing compounds (metals, solvents, etc.) or hepatotoxicants (carbon tetrachloride) may have either reduced the synthesis of, or increased destruction of, cytochromes P-450 in general, while the inducing effect of appropriate compounds may have stimulated the production of inducible isozymes of P-450. As a result, although total cytochrome P-450 content (constitutive and inducible isozymes) are reduced, this reduction may have been localized more in constitutive isozymes of P-450, while the inducible forms have been increased slightly. These events would account for the increased turnover number in the presence of decreased total P-450 content. Since monooxygenase activity is only one, fairly labile, form of cytochrome(s) P-450, use of more sophisticated immunochemical techniques would be necessary to completely evaluate P-450 isozyme content. In addition, one cannot discount the effects of temperature, age, sex and seasonal variation in any aquatic field study related to the use of monooxygenase components as indicators of biological stress (Stegeman, 1979; Stegeman & Chevion, 1980). In our experiment, only gonadally mature animals from the two sites were collected, and these animals were taken during similar time periods outside of spawning. In addition, water

Hepatic monooxygenase activities in brown bullheads

125

temperature measurements taken at the two sites indicated differences of less than 2°C. However, we did not attempt to sex the animals, and cannot dismiss possible bias due to this factor. In conclusion, measurement of certain M F O related activities in wild bullheads were not reliable as indicators of contamination of Slocum Creek. Our observations do not necessarily conflict with those who have observed induction of these responses in field sites contaminated by petroleum hydrocarbons or polychlorinated biphenyls. In such circumstances, M F O measurements may be able to identify potential water quality problems or serve as an early warning system for biological stress. Unfortunately, many pollutant-impacted natural waters contain compounds or mixtures of chemicals which may either have no effect or act to suppress M F O activity. Such situations may render measurements of M F O activity meaningless in the context of decision making regarding biological degradation of these sites. The environmental contamination scenario that exists at Slocum Creek is not an altogether unique one, i.e. an aquatic system containing an elevated concentration of a potential myriad of trace metals and organic compounds. Further research concerning the effects of trace metals and their interactions with organic compounds on the M F O system is needed if we are to understand and make use of measurements of these enzymes in systems containing contaminant mixtures.

A C K N O W L E D G E M ENTS Many thanks to Bill Rogers of the Cherry Point MAS Office of Environmental Affairs, and to Kent Nelson and A1 Little of the North Carolina Wildlife Commission for their gracious assistance during the field sampling. This work was supported in part by the North Carolina Department of Natural Resources and Community Development-Duke Fellows program. REFERENCES Addison, R. F., Zinck, M. E. & Willis, D. E. (1978). Induction of hepatic mixedfunction oxidase (MFO) enzymes in trout (Savelinus fontinalis) by feeding aroclor 1254 or 3-methylcholanthrene. Comp. Biochem. Physiol., 61C, 323-5. Ahokas, J. T., Pelkonen, O. & Kari, N. T. (1976). Cytochrome P-450 and drug induced spectral interactions in the hepatic microsomes of trout Salmo trutta lacustris. ,4cta PharmacoL Toxicol., 38, 440-9. Alvares, A. P., Leigh, S., Cohn, J. & Kappas, A. (1972). Lead and methyl mercury: Effects of acute exposure on cytochrome P-450 and the mixed function oxidase system in the liver. J. Exp. Med., 135, 1406-20.

126

Evan P. Gallagher, Richard T. Di Giulio

Baumann, P. C., Smith, W. D. & Ribick, M. (1982). Hepatic tumor rates and polynuclear aromatic hydrocarbon levels in two populations of brown bullhead (Ictalurus nebulosus). In Polynuclear Aromatic Hydrocarbons: Sixth International Symposium on Physical and Biological Chemistry, ed. M. W. Cooke, A. J. Dennis & G. L. Fisher, Columbus, Battelle Press, pp. 93-102. Brown, G. W. (1976). Effects of polluting substances on enzymes of aquatic organisms. J. Fish. Res. Board Can., 33, 2018-22. Buhler, D. R. & Rasmusson, R. E. (1968). The oxidation of drugs by fishes. Comp. Biochem. Physiol., 25, 223 39. Buhyoff, G. J. & Hull, R. B. (1982). Statistical Processing System Version 4.2 For Apple II. Apple Computer Inc. Cupertino, CA. Burke, D. M. & Mayer, R. T. (1974). Ethoxyresorufin: Direct fluorometric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Disp., 2, 583-8. Burns, K. (1976). Microsomal mixed function oxidases in an estuarine fish, Fundulus heteroclitus, and their induction as a result of environmental contamination. Comp. Biochem. Physiol., 53B, 443-6. Carlander, K. D. (1969). Handbook of Freshwater Fisher)' Biology, Vol. 1. Iowa State University Press, Iowa, pp. 534-7. Dent, J. G. (1978). Characteristics of cytochrome P-450 and mixed function oxidase enzymes following treatment with PBBs. Environ. Health Perspect., 23, 301-8. Docks, E. L. & Kirshna, G. (1976). The role of glutathione in chloroform induced hepatotoxicity. Exp. Mol. Pathol., 24, 13-22. Eriksson, L. C., DePierre, J. W. & Pallner, G. (1978). Preparation and properties of microsomal fractions. Pharmacol. Ther. A., 2, 281-317. Fabacher, D. L. & Baumann, P. C. (1985). Enlarged livers and hepatic microsomal mixed function oxidase components in tumor bearing brown bullheads from a chemically contaminated river. Environ. Toxicol. Chem., 4, 703-10. Fingerman, S. W., Brown, L. H., Lynn, M. & Short Jr, E. C. (1983). Responses of channel catfish to xenobiotics: Induction and partial characterization of a mixed function oxygenase. Arch. Environ. Contam. ToxicoL, 12, 195-201. Forlin, L. & Andersson, T. (1985). Storage conditions of rainbow trout liver cytochrome P-450 and conjugating enzymes. Comp. Biochem. Physiol., 80B, 569-72. George, S. G. & Young, P. (1986). The time course of effects of cadmium and 3methylcholanthrene on activities of enzymes of xenobiotic metabolism and metallothionein levels in the plaice, Pleuronetes platessa. Comp. Biochem. Physiol., 83C, 37~,4. Gingerich, W. H., Weber, L. J. & Larson, R. E. (1978). Carbon tetrachloride-induced retention of sulfobromophthalein in the plasma of rainbow trout. Toxicol. Appl. Pharmacol., 43, 147-58. Guengerich, F. P. (1982). Microsomal enzymes involved in toxicology--Analysis and separation. In Principles andMethods of Toxicology, ed. A. W. Hayes, New York, Raven Press, pp. 609-34. Hill, D. W., Hejtmancik, E. & Camp, B. S. (1976). Induction of hepatic microsomal enzymes by aroclor 1254 in Ictalurus punctatus (channel catfish). Bull. Environ. Contam. ToxicoL, 16, 495-502. Lowry, O. H., Rosebrough, N. J., Farr, A. C. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-75. Malins, D. C., Krahn, M. M., Brown, D. W., Myers, M. S., McLain, B. B. & Chan,

Hepatic monooxygenase activities in brown bullheads

127

S. C. (1985). Toxic chemicals in marine sediment and biota from Mukilto, Washington: Relationships with hepatic neoplasms and other hepatic lesions in English sole (Parophrys vetulus). JNCI, 74, 487-93. Matsubara, T., Koike, M., Touchi, A., Tochino, T. & Sugeno, K. (1976). Quantitative determination of cytochrome P-450 in rat liver homogenate. Anal, Biochem., 75, 596-603. Mclean, A. E. M. (1967). Effect of hexane and carbon tetrachloride on microsomal cytochrome P-450. Biochem. Pharmacol., 16, 2030-3. Melancon, M. J., Elcombe, C. R., Vodicnik, M. J. & Lech, J. J. (1981). Induction of cytochrome P-450 and mixed function oxidase activity by polychlorinated biphenyls and b-naphthoflavone in carp (Cyprinus carpis). Comp. Biochem. Physiol., 69C, 218-26. Melancon, M. J., Yeo, S. E. & Lech, J. J. (1987). Induction of hepatic microsomai monooxygenase activity in fish by exposure to river water. Environ. Toxicol. Chem., 6, 127-36. Nebert, D. W. & Gelboin, H. V. (1968). Substrate inducible microsomal aryl hydroxylase in mammalian cell culture. J. Biol. Chem., 243, 6242-9. North Carolina Division of Environmental Management (1983). Basic water monitoring program data review 1981-1982. Department of Natural Resources and Community Development Report 83-10. Raleigh, NC. Omura, T. & Sato, R. (1964). The carbon monoxide binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239, 2370-8. Payne, J. F. & Penrose, W. R. (1975). Induction ofaryl hydrocarbon benzo(a)pyrene hydroxylase in fish by petroleum. Bull. Environ. Contam. Toxicol., 14, 112-16. Payne, J. F., Fancey, L. L., Rahimtula, A. D. & Porter, E. W. (1987). Review and perspective on the use of mixed-function oxygenase enzymes in biological monitoring. Comp. Biochem. Physiol., 86C, 233~,5. Plaa, G. L. (1986). Toxic responses of the liver. In Toxicology--The Basic Science of Poisons, ed. C. D. Klaassen, M. O. Amdur & J. Doull, Macmillan Publishing, New York, pp. 286-309. Statham, C. N., Croft, W. A. & Lech, J. J. (1978). Uptake, distribution, and effects of carbon tetrachloride in rainbow trout (Salmo gairdneri). Toxicol. Appl. Pharmacol., 45, 131~,0. Stegeman, J. J. (1978). Influence of environmental contamination on cytochrome P-450 mixed-function oxidases in fish: Implications for recovery in the Wild Harbor Marsh. J. Fish. Res. Board Can., 35, 668-74. Stegeman, J. J. (1979). Temperature influence on basal activity and induction of mixed function oxidase activity in Fundulus heteroclitus. J. Fish. Res. Board Can., 36, 1400-6. Stegeman, J. J. (1980). Mixed function oxygenase studies in monitoring for effects of organic pollution. Rapp. P. -II. Re'un. Cons. Int. Explor. Mer., 179, 33-8. Stegeman, J. J. (1981). Induction of hepatic cytochrome P-450 in fish and the indication of environmental induction in scup (Stenotomas chrysops). Aquat. Toxicol., 1, 197-212. Stegeman, J. J. & Chevion, M. (1980). Sex differences in cytochrome P-450 and mixed function oxygenase activity in gonadally mature rainbow trout. Biochem. Pharmacol., 29, 553-8. USMC (1984). Division of Environmental Affairs report on landfill contamination to Slocum Creek. Havelock, NC, 421 pp. Varanasi, U. & Gmur, D. J. (1981). Hydrocarbons and metabolites in English sole

128

Evan P. Gallagher, Richard T. Di Giulio

(Parophrys vetulus) exposed simultaneously to (3H)-benzo(a)pyrene and (14C)naphthalene in oil contaminated sediment. Aquat. Toxicol., 1, 49-67. Williams, D. & Buhler, D. (1983). Comparative properties of purified cytochrome P-448 from/~-naphthoflavone treated rats and rainbow trout. Comp. Biochem. PhysioL, 75C, 25-32. Williams, D. & Buhler, D. (1984). Benzo[a]pyrene hydroxylase catalyzed by purified isozymes of cytochrome P-450 from /3-naphthoflavone fed rainbow trout. Biochem. Pharmacol., 33, 3742. Yoshida, T., Ito, Y. & Suzuki, Y. (1976). Inhibition of hepatic drug metabolizing enzymes by cadmium in mice. Bull. Environ. Toxicol., 15, 402-5. Yoshida, Y. & Kumaoka, H. (1975). Studies on the substrate-induced spectral changes of cytochrome P-450 in liver microsomes. J. Biochem., 78, 455-68. Zimmerman, H. L. (1978). Hepatotoxicity. New York, Appleton Century Crofts, 198-217.