ENVIRONMENTAL

RESEARCH

17,284 -295 f 1978)

Hepatic Mixed-Function Oxidase Activity in Rainbow Trout Exposed to Several Polycyclic Aromatic Compounds ELLEN HEATH

GERHART

AND ROBERT

M. CARLSON'

Department of Chemistry, University of Minnesota, Duluth, Minnesota 55812 Received January 4, 1978 The effects of polycyclic aromatic hydrocarbons (PAHs) on microsomal liver enzymes were examined in rainbow trout. Various PAJ-Js (naphrhalene through benxo(a)pyrene [B(a)P]) were injected ip to screen for mixed-function oxidase (MFO) activity. Chrysene, B(a)P, and Aroclor 1254 caused MFO induction. When fish were also exposedto solubilized pyrene, fluoranthene, and B(a)P in water, bioaccumulation of B(a)P resulted in MFO induction, whereas bioaccumulation of pyrene and fluoranthene did not. Based on water and injection exposure to B(a)P, it was predicted that tissue concentrations in excess of 300 &kg B(a)P would be accompanied by MFO induction in rainbow trout.

Mixed-function oxidases (MFO) have been studied extensively in mammals (Gillette et al., 1972; Lu, 1976) and have been found to catalyze a wide variety of reactions involved in xenobiotic metabolism (Brown, 1976). A characteristic of MFO enzymes is inducibility by certain compounds such as drugs, pesticides, and polycyclic aromatic hydrocarbons (PAHs). More recently, MFO activity has been demonstrated in fishes (Stanton and Khan, 1975; Chambers and Yarbrough, 1976). Of particular interest from an environmental standpoint has been the finding that fishes exposed to petroleum under laboratory and field conditions show induced MFO activity (Burns, 1976; Gruger et al., 1977; Payne and Penrose, 1975; Payne, 1976, 1977; Kurelec et al., 1977; Yarbrough and Chambers, 1977). One MFO enzyme system, aryl hydrocarbon hydroxylase (AHH), has been suggested as an environmental monitor (Payne and Penrose, 1975) since not only does it respond to the presence of hydrocarbons, but it also activates certain PAHs to carcinogenic metabolites. MFO induction in fishes has also been demonstrated by exposure to polychlorinated biphenyls (PCBs) in both food (Gruger et al., 1976) and water (Hill et al., 1976) which resulted in induction of hepatic MFO activity in coho salmon and channel catfish, respectively. Burns (1976) showed that Fundulus heteroclitus exposed to 10 and 100 /.&kg phenylbutazone in seawater had increased aldrin epoxidation rates and cytochrome P-450 levels after 10 days. Recently, Statham et al. (1978) examined the effects of several PAHs in rainbow trout after injection. In that study, benzanthracene, 3-methylcholanthrene (3-MC), and p-naphthoflavone all caused severalfold increases in AHH activity at 10 to 100 mg/kg. In a 1Zday time-course study using the sheepshead (a teleost), two 20 mg/kg injections of 3-MC caused elevated AHH activity for the duration of the experiment (Philpot et r Author to whom reprint requests should be addressed. 284 0013-9351/78/0172-0284$02.00/O Copyright All rights

Q 1978 by Academic Press, Inc. of reproduction in any form reserved.

MFO

ACTIVITY

IN

TROUT

EXPOSED

al., 1976). Pederson et al. (1974) caused a fourfold

TO

285

PAHs

increase in AHH

activity

in

rainbow trout by injecting 3-MC. The current investigation was designed to examine several PAHs to determine whether MFO induction in rainbow trout is a response to particular (especially carcinogenic) PAHs or to general PAH exposure and to evaluate the relationship between the concentration of a recognized carcinogen, benzo(a)pyrene [B(a)P], in tissue and hepatic MFO [AHH, aniline hydroxylase (AH) and P-4501 induction. Sources

of the Aromatic

METHODS Test Compounds

Aroclor 1254, Monsanto Chemical Company (Lot No. KB-02 606), 1,2,4trimethylnaphthalene (unpublished results); phenanthrene (recrystallized, sublimed), pyrene (sublimed), and fluoranthene (sublimed), Aldrich Chemical Company; and benzo(a)pyrene, Sigma Chemical Company. GC analysis indicated greater than 99.9% purity. Bioassay a. Injection

experiments. Rainbow trout were obtained from the Genoa National Fish Hatchery, Genoa, Wisconsin, and held in flowing Lake Superior water for 6 months prior to use. The fish used in the experiments weighed from 30 to 50 g and had a total length range of 14 to 17 cm. All fishes sampled were sexually immature and were 1 year old at the start of the 6-month experimental period. Several PAHs were administered in sterile peanut oil by intraperitoneal injection. After anesthetizing in 100 mg/liter MS-222 (ethyl m-aminobenzoate methanesulfonic acid salt), each fish received a 0.2~ml injection with a 22-gauge needle. The standard dose of B(a)P was approximately 30 mg/kg. B(a)P was given in doses from 3 pg/kg to 300 mg/kg in dose-response experiments. Controls received 0.2-ml injections of peanut oil. Experiments were carried out in 55-liter stainless-steel tanks supplied with 10 + 1°C Lake Superior water by a Mount - Brungs proportional dilutor, at a rate to give a tank turnover time of 2.5 hr. The photoperiod was 16:8 (1ight:dark). Fish were fed 3% of their body weight per day (Zeigler Bros., 3/16-in. pellets) during the experiments except on the day they were to be sacrificed. The tanks were siphoned daily. The water chemistry data during the experiments were: alkalinity, 40.8 5 0.6 mg/liter CaCO,; hardness, 45.1 t 0.3 mg/liter CaCO,; conductivity, 90 ? 3 pmhos/ cm, pH 7.05 to 7.79; and dissolved oxygen, 92 + 5% saturation. b. PAH flowthrough exposures. Conditions were similar to those described above. Rainbow trout from the same cohort were exposed to both pyrene (pyr) and fluoranthene (fl) in one experiment and to B(a)P in the other. During each experiment aliquots of a stock solution containing pydfl or B(a)P in acetone were diluted 1: 1000 with lake water and added daily to a 20-liter reservoir. A control reservoir was filled with 0.1% acetone in lake water. Nominal acetone levels for both control and experimental tanks were 10 @liter (ppm). FM1 (Fluid Metering, Inc.) lab pumps with stainless-steel fittings and tubing were used to meter reservoir solutions into the tanks. Three water samples were

286

GERHART

AND

CARLSON

taken during each experiment. Lake Superior water at 10 t 1°C for the pyr/fl experiment and 13 * 1°C for the B(a)P experiment was delivered at a rate of 25 literlhr (2.2-hr tank turnover time) and 16.7 liter/hr (3.3-hr tank turnover time), respectively. Fish for enzyme and tissue analyses were taken on Days 3,7, 10, and 21 for the pyr/fl experiment and on Day 10 for the B(a)P experiment. Preparation

of Microsomes

The trout were killed by cervical dislocation at 0930 + 30 min, and the livers were quickly removed and placed in cold 1.15% KCl. Individual livers were blotted and weighed before homogenizing. Microsomes were then prepared by grinding two to three pooled livers in 4 vol of 1.15% KC1 using a Potter-Elvehjem homogenizer with a Teflon pestle. Homogenates were centrifuged at 15,OOOg for 20 min and the supernatant was spun at 100,000g for 1 hr on a Beckman LS-50 ultracentrifuge (OOC). The microsomal pellet was rinsed three times with 1.15% KC1 and resuspended in 1.15% KC1 by sonicating (Branson Instruments) 3 set after gentle homogenizing. Enzyme Assays

Aryl hydrocarbon hydroxylase (AHH) and aniline hydroxylase (AH) were assayed by the methods of Nebert and Gelboin (1968) and Imai et al. (1966), respectively. Aliquots of the microsomal preparation containing 0.4 to 2.0 mg of protein were added to incubation vials (without substrate) and frozen for 24 to 48 hr until analysis. The incubation mixture for both assays (total volume, 0.9 ml) contained 50 PM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] buffer, pH 7.50; 0.6 mg of NADPH (Sigma, Type I); 3 PM MgC&; and 200 ~1 of microsomal suspension. The assays were initiated by adding 100 ,ul of substrate: 100 nM B(a)P in acetone and 10 PM aniline-HCl. Incubations were carried out in a shaking water bath under dimmed lights. The water bath temperature was 28.5”C (Pederson et al., 1974) and the incubation times were 30 min (AHH) and 20 min (AH). Each sample was measured in duplicate and blanks were run with each set of assays. Hydroxylated B(a)P was measured at 396-nm excitation and 522-nm emission on an Aminco-Bowman spectrophotofluorometer calibrated against 3-hydroxy B(a)P (received through the courtesy of the National Institutes of Health). For AH determinations the p-aminophenol content was measured at 630 nm on a Beckman DB-G spectrophotometer equipped with a recorder scale expander. Sublimed p-aminophenol was used as the standard. With the remaining microsomal preparation, cytochrome P-450 concentration was determined after diluting with 25% glycerol in 0.1 M TES buffer, pH 7.40. The CO difference spectrum was measured on a Beckman DB-G spectrophotometer according to Omura and Sato (1964). Protein was determined using a microbiuret method with the reagent formulation of Honn and Chavin, (1975). Crystalline bovine serum albumen was used as the standard. For most samples, only one measurement was made of P-450 concentration on each sample day with replicates generally within 15%.

MFO

ACTIVITY

IN

TROUT

EXPOSED

TO

PAHs

287

Tissue Analyses

After the livers were removed, the fish were gutted (kidney remaining), and rinsed with water, methanol, and finally water. Fish from each exposure tank were combined and ground thoroughly in a blender. The homogenized fish were stored in 8-02 glass jars with foil-lined lids at -20°C. At the time of extraction, 10 g (wet wt) was mixed with 40 g of Na,SO, and subjected to Soxhlet extraction with 170 ml of methylene chloride for 5 hr. After concentration in a Kuderna-Danish evaporator, interfering lipids were removed from samples containing B(a)P using high-pressure liquid chromatography (HPLC). The HPLC apparatus (Waters Associates) consisted of the following items: one M-6000 pump, a Model U6K injector, and a 254~nm uv detector. Methylene chloride was used as the carrier solvent at a flow rate of 4.0 ml/min. A 2-ml concentrated fish extract was quantitatively injected onto two stainless-steel Styragel columns (each 37-75 pm; pore size, 60 A; 122 cm x 7.8 mm i.d.) connected in series. Lipids from the pyr/fl samples were separated with a gel permeation chromatograph by a method similar to Veith et al. (1975). All solvents (Burdick and Jackson) were redistilled. After air concentrating to 300 ~1, PAHs were quantitated using a Varian 1700 gas chromatograph fitted with a flame ionization detector (detector, 300°C; injector, 250°C; column, 225°C). The 2 m x 2 mm i.d. glass column was packed with 3% OV-101 on 80-100 mesh Gas Chrom Q. PAH concentrations were calculated by computer program using the regression estimate and error treatment in Peters et al. (1974). Extraction recoveries for pyr/fl(69 ? 6%, 72 + 10%) and for B(a)P (74 t 11%) were used to adjust the tissue concentration data. Water Analyses

One hundred milliliters of methylene chloride (pyr/fl) or hexane [B(a)P] was placed into a 2-liter volumetric flask followed by 1900 ml of tank water which had been filtered through a Gelman glass fiber filter (0.2-10 PM). A 1.5-in. Tefloncoated stirring bar was introduced and the sample was extracted by emulsifying the contents for 2 hr. The organic layer was dried over Na,SO, and concentrated for GC analysis in a Kuderna-Danish evaporator. The recovery efficiency for known samples was 95 +- 4%. Effects

of Several PAHs

RESULTS on MFO Activity

The effects of several aromatic compounds (injected dose, 30 mg/kg) on MFO parameters indicated that in general it was the higher molecular weight compounds that caused induction (Fig. 1). Cytochrome P-450 levels were elevated by injection with Aroclor 1254 (a PCB mixture), pyrene, chrysene, and B(a)P. The CO difference spectra of induced P-450 did not show the spectral shift to 448 nm characteristic of mammalian systems. This observation is consistent with other studies on piscine P-450 measurements (Philpot et al., 1976; Stegeman and Sabo, 1976; Ahokas et al., 1977). AHH activity showed a dramatic 1Zfold increase 3 days after B(a)P administration. Injected Aroclor 1254 also caused significant hydroxylase induction. This latter observation is in agreement with previous studies where PCBs in food have induced AHH activity in coho salmon (Gruger et al., 1976, 1977) and PCBs in water caused very high AH and N-demethylase activities in channel catfish (Hill et al., 1976). The response of AH to injection of

P-450

naph

1254

phen

rw

f luor

chrys

B(o)P

FIG. 1. Effects of polycyclic aromatic hydrocarbons (PAHs) on microsomal enzymes in rainbow trout. The two bars for each compound are values obtained 3 and 5 days after ip injection of 30 mg/kg PAH. For each compound tested, the three enzyme assays were done using the microsomal fraction obtained from two or three pooled livers. AHH and AH assays were done in duplicate with the error bars representing the standard deviation. The mean and standard deviation of seven control fish samples are indicated by the horizontal lines (a = P < 0.05). Abbreviations: naph, 1,2,4trimethyhtaphthalene; 1254, Aroclor 1254; phen, phenanthrene: pyr, pyrene; fluor, fluoranthene: chrys, chrysene; B(a)P. benzo(a)pyrene.

TIME

SERIES

ANALYSIS

TABLE PAH TISSUE CONCENTRATIONS PAH concentration G-&g) w

AND

Time (day)

MFO

OF

1

MEASUREMENTS

P-4506 (nM/mg protein)

289

HEALTH

DURING

WATER

AHH (pM/mg protein/min)

UPTAKE

(pM/mg

EXPERIMENTS”

AH protein/min)

fl

0.107 kO.048

2.18 kO.43 1.18 20.27 1.14 +0.02 2.50 k-o.43 1.72 kO.80

0.238* &0.018 0.115 to.028 0.175 20.032

3.80* kO.62 1.66 20.88 1.76 20.25

95 2 8 82 k 7

318 + 10 273 k 14

3 7

0.083 0.107

150 * 11

408 t 20

10

0.103

281 k 23

1,250 k 38

21

0.071

0 0 (controls, n = 6)

368 k 24

10

73.1 2 24’

10

0 (controls, n = 2)

34.52* k5.14 11.47 21.13 20.01 e3.21

o The mean water concentrations during the exposures were 3.89 + 0.08 /.@I, pyrene; 3.31 ? 0.08 &l, fluoranthene; and 0.40 ? 0.21 &l, B(a)P. Control values were obtained by taking several (n) three-fish samples from control tanks at various times during the experiments. Induced enzyme levels (P < 0.05) are indicated by asterisks. b During the pyr/fl experiment only one measurement of P-450 was made on microsomes prepared from three pooled livers; duplicates were run during the B(a)P exposure. c The exposure tank contained 50 mg/l ground coal (co.125 mm) in addition to B(a)P. Water concentrations of B(a)P determined on filtered samples were

Hepatic mixed-function oxidase activity in rainbow trout exposed to several polycyclic aromatic compounds.

ENVIRONMENTAL RESEARCH 17,284 -295 f 1978) Hepatic Mixed-Function Oxidase Activity in Rainbow Trout Exposed to Several Polycyclic Aromatic Compound...
915KB Sizes 0 Downloads 0 Views