Influence of environmental variables on the hepatic mixed-function oxidase system in bluegill sunfish, Lepomis macrochirus

Pergamon Press plc

Corn/~.Biochem.Phys~l Vol. 93C, No. I, pp. 11-21, 1989 Printed in Great Britarn

INFLUENCE OF ENVIRONMENTAL VARIABLES ON THE HEPATIC MIXED-FUNCTION OXIDASE SYSTEM IN BLUEGILL SUNFISH, LEPOMIS MACROCHIRUS BRAULIO D. JIMENEZ*and LISA S. BURT@ *Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 378316036; TGraduate Program in Ecology, The University of Tennessee, Knoxville, TN 37996, USA. Telephone: (615) 574-7321

(Received 14 June 1988) Abstract-l. MFO activity measured as 7-ethoxyresorufin 0-deethylase (EROD) was assayed in hepatic microsomes of unfed bluegills acclimated to 4, 13, 18, or 26°C and injected once intraperitoneally_with benzo(a)pyrene (BaP) at a dose of 1, 10 or 20 pg/g body weight. 2. Fish acclimated to the higher temperature exhibited elevated EROD activity after the fifth day of injection; however, fish held at 4°C did not exhibit induction of this enzyme until 18 days following the initial injection and then only at the highest dose. 3. Although the concentrations of BaP and metabolites in the liver 5 days after injection were similar for fish acclimated to three temperatures, EROD activities were greater for the fish acclimated to higher temperatures. 4. Comparison of fed and unfed fish acclimated to 26°C revealed that at all doses hrgher concentrations of cytochromes P-450 and b, accumulated in hepatic tissue of fed fish; however, no difference was observed in EROD activities at any particular dose. 5. The results of this work indicate that EROD activity increases as acclimation temperature and BaP dose increase.

the hypothesis that levels of induced MFOs in aquatic organisms are indicative of environmental contamination (Dewaide and Henderson, 1970; Burns, 1976; Stegeman and Binder, 1979) the wide variability observed in wild fish from polluted and “unpolluted” sites has made it difficult to use these enzymes in biomonitoring programs. Factors such as food and sex, as well as season and other environmental conditions, could be responsible for this great variability (Jimenez et al., 1988). Further information on the effect of environmental variables is needed so that the change in activity of these enzymes due to exposure to xenobiotics can be evaluated for biomonitoring purposes. Very little research has been conducted on the detoxification system of bluegill sunfish, Lepomis mucrochirus, especially with respect to the effects of environmental variables on the induction of the MFO system. This study expands on previous work performed on the MFO system in bluegills (Jimenez et al., 1988). Benzo(a)pyrene (BaP), a ubiquitous environmental pollutant and carcinogen, was used to determine the effects of dose, acclimation temperature, feeding status and induction time on the accumulation and metabolism of contaminants in this fish.

INTRODUCTION

Environmental organism’s

variables play an important role in an ability to respond to toxic chemicals in the

aquatic environment. Uptake of contaminants has been reported to be temperature dependent in many species of fish, with accumulation enhanced at higher temperatures (MacLeod and Pessah, 1973; Cember et al., 1978; Powell and Fielder, 1982; Jimenez et al., 1987). Conversely, lower temperatures increase the retention of organic compounds in the tissues of fish (Collier et al., 1978). Because uptake and elimination rates of organic pollutants are influenced by environmental factors, it is important to know how detoxification systems in aquatic organisms respond to these factors. Metabolism of organic xenobiotics in many aquatic organisms is accomplished by the mixedfunction oxidase (MFO) system, a multienzyme complex that metabolizes both foreign and endogenous compounds (steroids and fatty acids) through a series of oxidative reactions. These enzymes are also responsible for the metabolic activation of some xenobiotic compounds to highly reactive intermediates which are thought to be ultimate carcinogens (Cummings and Prough, 1983; Jefcoate, 1983; Wolf, 1986). Compounds such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), as well as an array of drugs, have been shown to induce the hepatic MFO system in fish (Burns, 1976; Gerhart and Carlson, 1978; Elcombe and Lech, 1979; Stegeman, 1979; Stegeman et al., 1981; Anderson and Koivusaari, 1985). Consequently, these enzyme activities have been suggested as a means of environmental monitoring (Payne et al., 1984). Although a substantial body of research has supported

MATERIALSANDMETHODS Chemicals

Stock solutions of benzo(a)pyrene (BaP) were prepared by dissolving BaP (Aldrich; gold label) in Mazola corn oil at concentrations of 1, 10 and 20mg/ml. Tritiated 1,3,6 rH-BaP(78.9 Ci/mmol, Amersham Searle), purified by high performance liquid chromatography to >95% radiochemical purity, was added to label the stock solutions 11

12

BRAULIOD. JIMENEZand LISA S. BURTIS

to a specific activity of 0.025 mCi/mmol. Radioactivity was determined by using Aqueous Counting Scintillant (ACS, Amersham) on a Packard Tri-Carb 4640 scintillation counter.

Assays

Microsomal protein concentrations were determined ,by the Coomassie Blue dye-binding method (Bradford, 1976). A Bio-Rad standard assay kit was employed with serum albumin used as standard, and absorbance measurements General animals treatment were made using a Gilford 2600 spectrophotometer. The activity of 7-ethoxyresorufin 0-deethylase (EROD) Bluegill sunfish were obtained from Melton Hill Reservoir in Anderson County, Tennessee. Fish, averaging 2l.&3Og was determined fluorometrically at 30°C according to the methods of Egan et al. (1983). Activity was normalized in each, were collected during a period from June 1985 through terms of the level of cytochrome P-450 present in the June 1986. The fish were nlaced in separate flow-throueh “living stream” tanks, and gradually acclimated to one if microsomal fraction. The activity is expressed as picomoles four final temperatures (4, 13, 18 or 26°C). bv channinn the of product per minute per nanomole of cytochrome P-450. ambient water temperature 1°C per day.‘Ali fish were fed Concentrations of cytochrome b, and cytochrome P-450 during the acclimation period, which lasted for at least a were determined spectrophotometrically by a modification of the methods of Omura and Sato (1964). Microsomes were month. diluted to concentrations of -‘l mg protein per ml of resuspension buffer (0.2 M Tris, 30% glycerol, 1 mM dithTime course experiments iothreitol, 1mM EDTA). and difference spectra were obTime course experiments were conducted at 4 and 18°C. tained. Cytochrome b, was quantified by obtaining a Fish were denied food for a period of 2 weeks before being NADH difference spectrum (424-4 13 nm) and assuming an injected intraperitoneally (i.p.) with ‘H-BaP at a dose of extinction coefficient of 185 cm/mM, and the concentration 20 pg BaP per gram of body weight (b.w.), and were not fed is expressed as nanomoles of cytochrome b, per mg of throughout the remaining-experiment. The control group protein. The concentration of cytochrome P-450 was deterreceived only corn oil injections, and were kept upstream in mined from the sodium dithionite (Na,S,O,) difference the same flow-through tank, while the other groups were spectrum (450-490 nm) of carbon monoxide-treated microkept downstream in increasing order of injected dose. Fish somes by using an extinction coefficient of 91 cm/mM. were sacrificed 5 days after i.p. injection, and livers from Cytochrome P-450 concentration is expressed as nmol of four animals were pooled to obtain sufficient material for cytochrome P-450 per mg of protein. microsomal isolation. Fish acclimated to 18°C received an extra injection of BaP which was administered 5 days after Liuer BaP accumulation the first dose. Fish were sacrificed at 0,2, 5 and 10 days, and The fraction of BaP absorbed by the liver was estimated hepatic microsomes were isolated. The second injection at 5 by determining the total radioactivity in the liver and days was to insure maximum induction at the later sampling gallbladder and expressed as g of BaP equivalents per gram time. of liver. It was assumed that the radioactivity (BaP and metabolites) in the gallbladder had been processed by the Temperature and dose liver and stored in the gallbladder because-the fish were not Fish acclimated to 4, 13 or 26°C were denied food for two fed. Tissue was digested by using Soluene-100 tissue soluweeks before i.p. injection with BaP at 1, 10 or 20 pg/g b.w. bilizer, and radioactivity determined. Fish were sacrificed 5 days after injection, and livers pooled for microsomal isolation. The control group received only corn oil injections, and were kept upstream in the same Sodium dodecyl sulfate-polyacrylamide gel electroflow-through tank, while the other groups were kept downphoresis (SDS-PAGE) was performed according to the stream in increasing order of injected dose. methods of Laemmli (1970). Slab gels were prepared by using 4% stacking gel and either 8 or 10% acrylamide Feeding experiments separating gel. Microsomal samples were prepared by dilutOne group of fish acclimated to 26°C were fed Purina ing the thawed suspensions 1: 1 with a buffer solution trout chow ad libitum, and another group was denied food (0.125 M TrisCl with a pH of 6.8, 4% SDS, 20% glycerol throughout the experiment. Injection with BaP at different and 10% 2-mercaptoethanol). High mol. wt protein standoses was conducted as previously described, and fish were dards ranging from 29,000 to 205,000 obtained from Sigma sacrificed after 5 days. Chemical Companv were likewise diluted with buffer. APproximately 9dpgVof microsomal protein was loaded inio each sample well, and a constant current of 6OmA was Microsomal isolation applied. Gels were stained with a solution of 0.125% Fish hepatic microsomes were prepared by differential Coomassie Blue R-250 (Sigma), 50% methanol, and 10% centrifugation (Mckee et al., 1983) with several modiacetic acid. fications. The fish were sacrificed by severing their spinal Densitometric analysis involved scanning the gels with a cords, and their livers were immediately removed, and LKB-Bromma Ultrascan XL Laser Densitometer in conplaced in ice-cold phosphate buffer (0.05 M Sodium phosjunction with an IBM AT computer. All data were collected phate, 0.15 M KCI, pH 7.5). Livers were sequentially hothrough use of the LKB-Bromma Gel Scan software, which mogenized using a Tekmar tissue homogenizer, and a determines the area of each electrophoretic peak by means motor-driven Potter-Elvehjem glass with a Teflon piston. of integration using Gaussian distribution. The area of each The homogenates were centrifuged at 10,OOOgfor 30 min in peak in relation to the total area of the profile is reported. a Beckman J-21B centrifuge, and the supernatants were During the evaluation of the induction of a particular band, placed on ice while the pellet was resuspended in phosphate its relative area and mol. wt are compared to those of its buffer and again centrifuged at 10.000~ for 20 min. The controls. Each electrophoretic profile illustrated in our supernatantswere then pooled and centrifuged at 105,OOOg figures represents the average of two replicates from sepafor 60 min in a Beckman L3-50 Ultracentrifuge. Microsomal rate microsomal samples. pellets were resuspended in 0.3-l .Oml of Tris buffer (0. I M Tris, 1.0 mmol EDTA, 10% glycerol, pH 7.4) by using a Statistical analyses Braun Sonic 1510 at 50 watts for 20sec. Microsome susFor each of the responses of interest (EROD activity, pensions were frozen in liquid nitrogen and stored at -90°C cytochromes b, and P-450 concentrations), analysis of variuntil assayed.

Lepomis MFO activity

13 RESULTS

ante (ANOVA) techniques were used to test for sigmficant effects of treatment factor (e.g. main effects due to dose or temperature), as well as interaction between treatments. After an effect (main or interaction effect) was found to be significant, contrasts were defined to help interpret the main effects and interactions (Snedecor and Cochran, 1980). Prior to the ANOVA, each response was examined for homogeneity of variance across the different treatment groups in order to determine if there was a need for transforming the observations. For all responses (EROD activity, cytochromes P-450 and b, concentrations) from each experiment, a log-transformation of the observations was used to improve the homogeneity of assumed variance. The primary treatment factors of interest in the temperature experiment were (1) dose of BaP, (2) water temperature and (3) interactions of factors (1) and (2). However, for the feeding experiments, the water temperature was held constant, and feeding status (fed or unfed) was introduced as an additional factor. Therefore, for the feeding experiments, the experimental factors of interest were the main effects of (1) BaP dose, (2) feeding status and (3) their interaction. For the time course experiment, the number of days after the initial injected dose was the only factor of interest, because both water temperature and BaP dose were held constant. Hence, the results of this experiment were analyzed as a one-way ANOVA. When the time effect was significant, Tukey’s multiple comparison procedure was used to determine which days after injection differed significantly.

Time course of induction The time of maximum induction for EROD activity after a single i.p. injection with 20 pg BaP/g b.w.

was determined in fish acclimated to 18°C (Fig. la). The highest activity was observed at day five. The activity, at both day five and day ten was significantly (P < 0.01) higher (19-fold) than that at day two. There was no significant difference in activity at 5 and 10 days after injection, despite the administration of a second dose on day five. Even so, cytochromes P-450 and b, (Fig. lb) from these fish showed no significant changes after BaP injection. Levels of cytochromes b, and P-450 did appear to follow the general trend of the EROD activities, with higher levels 5 and 10 days after injection. Because fish acclimated to 4°C did not induce EROD activity 5 days after injection of BaP at a dose of 20 pg/g b.w. (Fig. 2), a time course experiment at this temperature was performed. Induction of EROD activity took more than 11 days. Effects of temperature and dose

Hepatic microsomal EROD activity was measured in bluegills acclimated to 4, 13 or 26°C and injected

4800 ^o 2 A 4000

a

E E E E f E

3200

2400

,P

/ (3)

FIRST DOSE

SECOND DOSE I

5 TIME (days)

Fig. 1. Time course of (A) EROD activity and (B) hepatic microsome concentrations of cytochromes P-450 (dashed line and solid squares) and b, (solid line and open circles) in unfed bluegills acclimated to 18°C and injected with 20 pg BaP/g body weight of day zero and again at day five. Each point represents the mean f SE; the number of observations is indicated in parentheses. Each observation represents the data obtained from a fish liver.

BRAULIOD. JMENEZand

LISA S. BURTIS

I

I

I

260

-

260 C f

8 p” I 220 P = E ,E 160 E E 5 g 140 5 z 5 F Y 8

100

60

ii

1

5

11

16

TIME (days)

Fig. 2. Time course of the incorporation of 3H-BaP radioactivity in fish livers (solid line and open circles) and hepatic EROD activity (dashed line and solid squares) in bluegills acclimated to 4°C and injected with 20 pg BaP/g body weight. The mean & SE and the number of observations are indicated. Each observation represent the pool of four hvers.

doses of BaP (Fig. 3a). Significantly (P < 0.0001) higher EROD levels were observed at higher acclimation temperatures and at higher doses. At doses of 10 and 2Opg BaP/g b.w., activity levels in fish acclimated to 26°C were approximately three times higher than those in fish acclimated to 13°C. Fish held at 4°C however, showed little or no induction of EROD activity at any of the doses tested 5 days after injection. It should be noted that, even 18days after injection of 2Opg BaP/g b.w. in fish acclimated to 4°C EROD activity was only 140 pmol/min/nmol P-450 in contrast to 600 pmol in fish acclimated to 13°C. Because initially there was no induction at any dose in 4°C acclimated fish, we explored the possibility that there was greater absorption of BaP from the i.p. cavity at higher acclimation temperatures. Five days after i.p. injection, we measured the amount of radioactivity (both parent BaP compound and metabolites) in livers of fish acclimated to one of three different temperatures (Fig. 3b). Very little radioactivity could be detected in the fish livers at a dose of 1 pg BaP/g b.w., regardless of the acclimation temperature. However, at a dose of 10 pg BaP/g b.w., it appears that the incorporation of ‘H-BaP in the liver is temperature dependent, with highest accumulation at 26°C. At the highest dose (2Opg BaP/g b.w.), the concentrations of total radioactivity accumulated by the livers were similar at all three temperatures (P > 0.05), although the concentration seems to be slightly lower in fish acclimated to 4°C. The incorporation of 3H-BaP into the livers of fish from the 4°C time-course experiment did not show any significant (P > 0.05) change from day 11 to day 18 (Fig. 2). The amounts of total BaP found in the liver of these fish were similar to the levels found in fish injected with the same dose at either 13 or 26°C (Fig. 3b). The similarities in the accumulations of with different

total radioactivity in the livers of fish acclimated to different temperatures suggest that the differences in EROD induction at these three temperatures is not solely the result of temperature-dependent differences in the concentration of BaP received by the liver. Analyses of cytochromes P-450 and b, (Fig. 4a, b) revealed a significant dose effect (P < 0.0001) for all temperatures. The concentration of cytochrome P450 at a dose of 2Opg BaP/g b.w. is significantly higher (P < 0.001) than concentrations at doses of 1 or 10 pg BaP/g b.w. for the same temperature; these results suggest that cytochrome P-450 is being produced after the injection of high concentrations of contaminant into unfed fish. Effects of feeding

The fed and the unfed fish injected with different doses of BaP were not different in their EROD activity (Fig. 5a). As dosages increased, significantly higher (P < 0.01) EROD activity was observed in both the fed and the unfed fish than in the corn oil injected fish (without BaP). The levels of cytochromes P-450 and b, varied significantly (P < 0.016) (1) in the fed and the unfed fish injected with BaP and (2) at different doses (Fig. 5b, c). Higher levels of cytochromes P-450 and bS were found in the fed fish than in the unfed fish. Both the fed and the unfed control fish, however, exhibited similar levels of cytochromes P-450 and b5 regardless of their nutritional status, but they contained significantly less (P < 0.001) cytochrome P-450 than fish receiving BaP. Fed fish showed a dramatic increase in levels of both cytochromes P-450 and b, in contrast to control fish, with the greatest increase occurring in the fish administered 1 pg BaP/g b.w. and with lower rates of increase at progressively higher doses. Unfed fish, did not exhibit major differences in the concentrations of cytochrome br after doses of 1

Lepomis MFO activity

and 10 pg BaP/g b.w. It was only at a dose of 20 pg BaP/g b.w. that an effect was observed (Fig. 5~). Electrophoresis

Microsomal cytochrome P-450 with mol. wts ranging between 45,000-60,000 have been reported for various organisms (Lu and West, 1980; Haugen et al., 1975; Alvares and Siekevits, 1973; Stegeman, 1981). In our studies with bluegills, a series of polypeptide bands (59,000, 58,000, 57,000 and 54,000) characteristic of the cvtochrome P-450 region, as well as other bands of higher mol. wts (l-lO,OOO, 98,000 and

15

94,000), were observed to be induced in bepatic microsomes of BaP-treated animals (Figs 6 and 7). SDS-PAGE of hepatic microsomes from BaPinjected (2Opg BaP/g b.w.) fish which were acclimated to 13, 18 or 26°C exhibited induction of three major protein bands (59,000, 58,000 and 57,000; Figs 6b and 7a, b). There was an approximately two-fold increase in these bands when compared to the controls, with the 58,000 and 57,000 bands having the greatest induction. Fish injected with BaP at 26°C also showed induction of a 54-kDa band. Induction of high mol. wt bands (110,000, 98,000 and 94,000)

1600

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220 _

g 0 Z .z 3 z

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100 -

9

a

DOSE (ug BaPlg body weight)

Fig. 3. The effectsof temperature [4”C (solid line with open circles), 13°C (dashed line with solid squares), 26°C (dotted line closed circles)] and dose (1, 10 or 20 pg BaP/g body weight) on (A) EROD activity and (B) accumulation of ‘H-BaP by fish livers in unfed bluegills 5 days after injection. The mean f SE and the number of observations are indicated. Each observation represent the pool of four livers.

BRAIJLIOD. JIMENEZand LISA S. BURTIS

16

I 10

DOSE (pg BaPlg

body

welght)

Fig. 4. Effects of temperature [4”C (solid line with open circles), 13’C (dashed line with solid squares), 26°C (dotted line closed circles)] and dose (1, 10 or 20 pg BaP/g body weight) on (A) hepatic microsomal cytochrome P-450 and (B) on cytochrome b, in unfed bluegills 5 days after injection. The mean f SE and the number of observations are indicated. Each observation represents the pool of four livers.

which are outside the cytochrome P-450 region was observed in injected fish acclimated to higher temperatures. Fish which were injected fish acclimated to higher temperatures. Fish which were injected with BaP at 4°C exhibited a 1.5-fold increase of the 59,000 band and a three-fold increase of the 57,000 band. No induction of the 58,000 band or high mol. wt bands was observed in the 4”C-acclimated fish. The electrophoretic profiles obtained from 5 and 10 day BaP post-injected fish acclimated to 18°C were very similar. DISCUSSION

Effects

oftemperature

Acclimation temperature plays an important role in the activity of the hepatic mixed-function oxidase system of bluegill sunfish. Because body temperatures of poikilotherms correspond closely to habitat temperature, organisms require mechanisms (e.g. temperature compensation) for maintaining their physiological and metabolic activities despite fluctuating environmental temperatures. Two basic enzyme mechanisms that seem possible for achieving rate compensation are (1) alterations in the concentration of enzymes in tissues and (2) changes in the catalytic

efficiencies of enzymes (Hochachka and Somero, 1984). Previous research has demonstrated this temperature compensation phenomenon for MFO enzyme activity in some fish species (Egaas and Varanasi, 1982; Stegeman, 1979; Ankley et al., 1985). In addition, metabolism of contaminants such as BaP has been found to be greater in cold-acclimated (4°C) than in warm-acclimated (17°C) liver cells from rainbow trout (Andersson and Koivusaari, 1986a). Conversely, higher levels of hepatic EROD activity have also been reported both in laboratory bluegills acclimated to higher temperatures and in wild fish collected during the summer (Jimenez et al., 1988). Similarly, experiments using BaP have indicated that hydroxylase activity increases at higher acclimation temperatures, as evidenced in fed bluegills (Karr et al., 1985), channel catfish 48 hr after BaP injection (Fingerman et al., 1983) and sheepshead (James and Bend, 1980). Furthermore, Shugart et al. (1987) reported higher levels of liver DNA and hemoglobin BaP adducts in bluegill sunfish acclimated to higher temperatures. This research supports the findings that EROD activity increases with higher temperatures after the fish are injected with BaP (Fig. 3a). The injection of radioisotopes at different acclimation temperatures

Lepomis

MFO activity

2000

17

I A

,000

-

1600 -

5: ,400

-

LT i g ,200

-

E E

I 0.16

I

I C

T

---.____ -.--.____ 26.C

FED

I 10

DOSE (Ug SIP/g body wright)

Fig. 5. Effects of dose (BaP) on (A) EROD activity, (B) cytochrome P-450 and (C) cytochrome b, in fed (dashed line and solid squares) and unfed bluegills (solid line and open circles). The mean f SE and the number of observations are indicated. Each observation represent the pool of four livers.

18

BRAULIO

JIMENEZand LISA S. BURTIS

D.

0.16 2oug

BaP/g

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body weight

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MIGRATIONAL DISTANCE (MOLECULAR WEIGHT IN kDA)

47

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MIGRATIONAL DISTANCE (MOLECULAR WEIGHT IN kDa)

Fig. 6. Densitometric profiles (SDS-PAGE) indicating mol. wts (kDa) of microsomal proteins from control (shaded area) fish and fish injected with BaP at a dose of 2Opg/g body weight (solid line) and acclimated to (A) 4°C and (B) 13°C. Each profile represents the average of two different replicate samples (from different fish) run in the same gel.

Fig. 7. Densitometric profiles (SDS-PAGE) indicating mol. wts (kDa) of microsomal proteins from control (shaded area) fish and injected with BaP at a dose of 20 fig/g body weight fish (solid line) and acclimated to (A) 18°C or (B) 26°C. Each profile IS the average of two dtfferent replicate samples (from different fish) run in the same gel.

provided a means of assessing changes in EROD activity due to the effects of temperature and dose. The amounts of radioactivity accumulated in the livers of fish dosed with 20 pg BaP/g b.w. (measured

responsive to inducers and require more time for induction to occur (Egaas and Varanasi, 1982; Stegeman, 1979; Andersson and Koivusaari, 1985). There could be many possible reasons for this delay or suppression. Andersson and Kiouvusaari (1985) suggested that this phenomenon could be due to low rates of protein synthesis of the MFO enzymes and/or to an influence of temperature on the pharmacokinetics of the inducing agent. Another explanation, however, is that there could be a decrease in the synthesis, activation or transport of cytosolic factors necessary for induction of the MFO enzymes. Furthermore, these factors may be receptors for Polyaromatic hydrocarbons and/or secondary messengers which mediate the induction event. Specific binding experiments injecting mice with 3H-tetrachlorodibenzo-p-dioxin (TCDD), a very potent inducer of cytochrome P-450, support this hypothesis. The binding of the TCDD receptors to nuclear sites is a temperature-sensitive step (Greenlee, 1979); Greenlee reports that the specific nuclear binding was maximal after incubation for 1 hr at 25°C and was more than three times greater than the specific binding measured at 0°C.

5 days after injection into fish held at 13 or 26°C and measured 18 days after injection into fish held at 4°C) were similar regardless of the acclimation temperature (Figs 2 and 3b); however, EROD activities were quite different (Fig. 3a). These results suggest that the differences in hepatic EROD activity are due to effects of temperature rather than dose. The effects of temperature on enzyme activity were distinct in fish acclimated to low temperatures (4°C). No induction was observed in EROD activity in these fish 5 days after injection with high doses of contaminant. Even 18 days after injection, when liver accumulation was greater in 4°C fish than in 26 or 13°C fish, EROD activity in 4°C fish was three to ten times less than that observed at the higher temperatures. These results indicate that the time delay does not appear to be due to less incorporation of the contaminant by the liver at lower temperatures; indeed, the amount of total BaP accumulated by this organ is similar regardless of the acclimation temperature tested. Therefore, we conclude that low temperatures delay and suppress the induction of EROD activity in bluegills. Previous research using Fundulus heteroclitus and Salmo gairdneri supports our findings that fish at colder temperature are less

Efects

of feeding

The extent of EROD induction was similar in fed and unfed fish injected with BaP at 26°C (Fig. 5a).

Lqomis

MFO activity

Cytochrome P-450 concentrations did not differ between fed and unfed fish. However, higher levels of cytochromes P-450 and b, were found in the livers of injected fed fish than in the liver of unfed fish injected with the same dose of BaP (Fig. Sb, c). This suggests that in fed bluegills, BaP induces cytochrome P-450s which are not associated with EROD activity. In unfed fish, however, the induction of these cytochromes P-450 is greatly reduced. Previous research utilizing fed and unfed bluegills demonstrated that the unfed fish possess lower EROD activity than the fed fish and that this activity is temperature dependent (Jimenez et al., 1988). These findings are consistent with those of Andersson (1986b), who reported lower MFO activity (7-ethoxycoumarin) in isolated liver cells from starved rainbow trout than in cells from fed trout, but similar cytochrome P-450 concentration. In addition, he found induction of cytochrome P-450 and MFO activity in B-naphthoflavone-treated liver cells from the fed and the starved rainbow trout. Effects of dose Higher doses of BaP resulted in an increase in EROD activity in both unfed and fed fish (Fig. 5a) this effect was observed in fish acclimated to temperatures of 13°C or higher (Fig. 3a). Likewise, Gerhart and Carlson (1978) found that, in fed rainbow trout at lO”C, aryl hydrocarbon hydroxylase (AHH) activity increased as the dose of BaP increased. At lower acclimation temperatures (4°C) in our experiments, however, no dose effect was evident. This lack of response could not be attributed entirely to the fact that EROD induction was delayed in the cold-acclimated fish (Fig. 2). Even after 18 days, when induction appeared maximal in the 4°C fish, EROD activity was very much lower than that observed in fish at the higher acclimation-temperatures (Fig. 3a). Previous research using Fundulus heteroclitus and Salmo gairdneri supports our finding that fish at colder temperatures are less responsive to inducers and require more time for induction to occur (Stegeman, 1979; Egaas and Varanasi, 1982; Andersson and Koivusaari, 1985). We have demonstrated that the time delay is not due to less incorporation of the pollutant by the liver at colder temperatures; since the concentrations of total BaP assimilated by this organ are similar irrespective of the acclimation temperature tested. Cytochromes P450 and b, were induced at low concentrations (1 kg BaP/g, Fig. 5b, c) in both the fed and the unfed injected fish; it is unusual to see this type of response in fish. Electrophoresis The different responses of the MFO system to organic compounds have been reported to involve the synthesis of numerous isozymes, each with its own individual specificity (Nebert, 1979; Lu and West, 1980). The response of this system to temperature acclimation has resulted in the appearance of different isozymic forms in many fish systems (Hochachka and Somero, 1984; Baldwin and Hochachka, 1970; Shaklee et al., 1977). We have found both quantitative and qualitative changes in hepatic microsomal enzyme profile after injection with BaP.

19

Microsomes from injected fish acclimated to 4 (18 days after injection), 13, 18 and 26°C exhibited highest induction of 57,000 and/or a 58,000 polypeptide band (Figs 6 and 7). This suggests that EROD activity may be associated to either of these bands. Conversely, microsomes isolated from 4°C injected (10 and 20 pg BaP/g b.w.) fish did not show induction of the 57,000 band. Elcombe and Lech (1979) found that /?-naphthoflavone and Aroclor 1242 induced the synthesis of a new protein band at 57,000 in rainbow trout microsomes which was only faint in the controls. Peroxidase activity was found to be associated with this band and in the bands at 59,500, 51,000, 48,000 and 45,000, indicating that they were all hemoproteins. In their report they suggest that 57,000 protein may therefore be associated with high monooxygenase activity in response to BaP, ethoxyresorufin and ethoxycoumarin. Stegeman et al. (1981) found similar results which additionally indicated a species difference. Rainbow trout treated with 3-methylcholanthrene (3-MC) produced a microsomal protein band at 57,000 that was only a shoulder in controls fish. In experiments using scup, however, it was a prominent band at 54,000 that was higher in the treated fish. Peroxidase activity also indicated the presence of cytochrome P-450 in this band. Although the results of these experiments support our findings, further research involving the isolation and characterization of these two bands is required before EROD activity can be definitely attributed to either of these bands in bluegill sunfish. Summary and conclusions We have shown that EROD activity in bluegill sunfish injected with BaP is dependent on temperature and on the dose of inducer. By affecting the level of MFO activity, habitat temperature and contaminant dose levels can vary the metabolism and detoxication of xenobiotic compounds. Of the parameters studied, EROD activity appeared to be the most reliable and consistent indicator of the presence of BaP exposure, but the level of activity induced by a given exposure was highly dependent upon temperature. Levels of cytochromes b, and P-450 are useful and sensitive biological indicators, but the response at 4°C was inconsistent. These findings are in agreement with other reports on the effects of pollutants on cytochromes P-450 and bS (Payne et al., 1984; Gerhart and Carlson, 1978; Burns, 1976; Karr et al., 1985).

Acknowledgements-The authors would like to express their appreciation to Carmen Cadilla, G. R. Southworth, S. W. Christensen, J. F. McCarthy and J. J. Beauchamp for their critical review of this manuscript and to Dr Carl Burtis, Norman Lee and Z. Egan for their support with the analysis on the centrifugal fast analyzer. This research was supported by the Oak Ridge Y12 Plant, Department of Environmental Management, Health, Safety, Environment and Accountability Division and by the Gak Ridge National Laboratory Director’s Exploratory Studies Program. The Oak Ridae Y-12 Plant is operated by Martin M&etta Energy Systems Inc., for the US Department of Energy under Contract No. DE-AC0584OR21400. This is Publication No. 3169 of the Environmental Sciences Division, Oak Ridge National Laboratory.

BRAULIOD. JIMENEZand LISA S. BURTIS

20 REFERENCES

Alvares A. P. and Siekevitz P. (1973) Gel electrophoresis of partially purified cytochromes P-450 from liver microsomes of variously-treated rats. Biochem. biophys. Res. Commun. 54, 923-929.

tetrachlorodibenzo-p-dioxin in C57BL/6J and DBA/U mice. J. biol. Chem. 254, 98149821. Haugen D. A., van der Hoeven J. A. and Coon M. J. (1975) Purified liver cytochrome P-450. J. biol. Chem. 250, 3567-3570.

Hochachka P. W. and Somero G. N. (1984) In Biochemical Adaptation, p. 537. Princeton University Press, New Jersey. James M. 0. and Bend J. R. (1980) Polycyclic aromatic gairdneri. Toxic. appl. Pharmac. 80, 43-50. hydrocarbon induction of cytochrome P-450 dependent Andersson T. and Koivusaari U. (1986a) Oxidative and mixed function oxidases in marine fish. Toxic. appl. conjugative metabolism of xenobiotics in isolated liver Pharmac. 54, 117-133. cells from thermally acclimated rainbow trout. Aquat. Jefcoate C. R. (1983) Integration of xenobiotic metabolism Toxic. 8, 85-92. in carcinogen activation and detoxication. In Biological Andersson T. (1986b) Cytochrome P-450-dependent metabBasis of Detoxication (Edited by John Caldwell and olism in isolated liver cells from fed and starved rainbow William B. Jakoby), pp. 31-76. Academic Press, New trout, Salmo gairdneri. Fish Physiol. Biochem. 1, 105-l 11. York. Ankley G. T., Reinert R. E., Wade A. E. and White R. A. (1985) Temperature compensation in the hepatic mixed Jimenez B. D.. Burtis L. S., Ezell G. H., Egan B. Z., Lee N. E., Beauchamp J. J. and McCarthy J. F. (1988) The mixed function oxidase system of bluegill. Comp. Biochem. function oxidase system of bluegill sunfish Lepomis macPhysiol. 18, 1255129. rochirus: correlation of activities found in experimental Baldwin J. and Hochachka P. W. (1970) Function and wild fish. Envir. Toxic. Chem. 7, (in press). significance of the isozymes in thermal acclimatization acetylcholinesterase from trout brain. Biochem. J. 116, Jimenez B. D., Cirmo C. P. and McCarthy J. F. (1987) 8831887. Effects of feeding and temperature on uptake, elimiBradford M. M. (1976) A rapid and sensitive method for the nation, and metabolism of benzo(a)pyrene in the bluegill quantitation of protein utilizing the principle of proteinsunfish (Lepomis macrochirus). Aquat. Toxic. 10, 41-57. dye binding. Analyt. Biochem. 72, 248-254. _ Karr S. W., Reinert R. E. and Wade A. E. (1985) The effects Buhler D. R. and Rasmusson M. E. (1968) The oxidation of temperature on the cytochrome P-450 system of therof drugs by fishes. Comp. Biochem. Physiol. 25, 223-239. mally acclimated bluegill. Comp. Biochem. Physiol. 8OC, 135-139. Burns K. A. (1976) Microsomal mixed function oxidases in an estuarine fish, Fundulus heteroclitus, and their in- Koivusaari U., Harri M. and Hanninen 0. (1981) Seasonal duction as a result of environmental contamination. variation of hepatic biotransformation in female and male Comp. Biochem. Physiol. 53B, 443446. rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. Campbell C. M. and Davies P. S. (1978) Temperature 7oc, 149-157. acclimation in the teleost, Blennius pholis: changes in Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. enzyme activity and cell structure. Comp. Biochem. Physiol. 61B, 165-167. Nature, Lond. 227, 68CM85. Lu A. Y. and West S. B. (1980) Multiplicity of mammalian Cember H., Curtis E. H. and Blaylock B. G. (1978) Mercury microsomal cytochromes P-450. Pharmac. Rev. 31, bioconcentration in fish: temperature and concentration effects. Envir. Poll&. 17, 31 l-319. 277-295. Collier T. K., Thomas L. C. and Malins D. C. (1978) MacLeod J. C. and Pessah E. (1973) Temperature effects on mercury accumulation, toxicity and metabolic rate in Influence of environmental temperature on disposition of rainbow trout (Salmo gairdeneri). J. Fish. Res. Bd Can. dietary naphthalene in coho salmon (Oncorhynchus kit30, 485492. sutch): isolation and identification of individual metaboMcKee M. J., Hendricks A. C. and Ebel R. E. (1983) Effects lites. Comp. Biochem. Physiol. 61, 23-28. of naphthalene on benzo[a]pyrene hydroxylase and cytoCummings S. W. and Prough R. A. (1983) Metabolic chrome P-450 in Fundulus heteroclitus. Aquat. Toxic. 3, formation of toxic metabolites. In Biological Basis of 103-l 14. Detoxicarion (Edited by Caldwell J. and Jakoby W. B.), Nebert D. W. (1979) Multiple forms of inducible drugpp. l-30. Academic Press, New York. metabolizing enzymes: a reasonable mechanism by which Dewaide J. H. and Henderson P. T. (1970) Seasonal vari. any organism can cope with adversity. Molec. Cell. ation of hepatic drug metabolism in the roach, Leuciscus Andersson T. and Koivusaari U. (1985) Influence of environmental temperature on the induction of xenobiotic metabolism by B-naphthoflavone in rainbow trout, Sulmo

I

rutilus L. Comp. Biochem. Physiol. 32, 489-497.

Biochem. 27, 2746.

Egaas E. and Varanasi U. (1982) Effects of polychlorinated biphenyls and environmental temperature on in vitro formation of benzo(a)pyrene metabolites by liver of trout (Salmo gairdneri).

Biochem. Pharmac. 31, 561-566.

Egan B. 2, Lee N. E., Burtis C. A., Kao J. Y. and Holland J. M. (1983) IJse of laser-excited fluorescence to measure mixed:function oxidase activity. Clin. Chem. 29, 16161619. Elcombe C. R. and Lech J. J. (1979) Induction and characterization of hemoprotein(s)P-450 and monooxygenation in rainbow trout (Salmo gairdneri). Toxic, appl. Pharmac. 49, 437-450.

Fingerman S. W., Brown L. A., Lynn M. and Short E. C., Jr (1983) Responses of channel catfish to xenobiotics: induction and partial characterization of a mixed function oxygenase. Enoir. Contam. Toxic. 12, 195-201. Gerhart E. H. and Carlson R. M. (1978) Hepatic mixedfunction oxidase activity in rainbow trout exposed to several polycyclic aromatic hydrocarbons. Envir. Res. 17, 284-295.

Greenlee

W.

F.

(1979) Nuclear

uptake

of

2,3,7,8-

Omura T. and Sato R. (1964) The carbon monoxide-binding pigment of liver microsomes. J. biol. Chem. 239, 237G2378.

Payne J. F., Bauld C., Dey A. C., Kiceniuk J. W. and Williams U. (1984) Selectivity of mixed-function oxygenase enzyme induction in flounder (Pseudopleuronectes americanus) collected at the site of the Baie Verte, Newfoundland oil spill. Comp. Biochem. Physiol. 79C, 15-19. Powell J. H. and Fielder D. R. (1982) Temnerature and accumulation of DDT by sea Mullet (hfugil cephalus L.). Mar. Pollut. Bull. 13, 28-230.

Shugart L. McCarthy J. F., Jimenez B. D. and Daniels J. (1987) Analysis of adduct formation in the bluegill sunfish (Lepomis macrochirus) between benzo[a]pyrene and DNA of the liver and hemoglobin of the erythrocyte. Aquat. Toxic. 9, 319-325. Shaklee J. B., Christiansen J. A., Side11B. D., Prosser C. L. and Whitt G. S. (1977) Molecular aspects of temperature acclimation in fish: contributions of changes in enzyme patterns to _metabolic _ _ _ reorganization in green sunfish. J. exp. Zool. ZUl, l-20.

Lepomis MFO activity

G. W. and Cochran W. G. (1980) Statistical Mefhoa!s, 7th edn. Iowa State University Press, Ames,

&decor

Iowa. Stegeman J. J. (1979) Temperature influence on basal activity and induction of mixed function oxygenase activity in Fundulus heteroclitus. J. Fish. Res. Bd Can. 36, 14o(r1405. Stegeman J. J. and R. L. Binder (1979) High benzo(a)pyrene

21

hydroxylase activity in the marine fish Srenofomus versicolor. Biochem. Pharmac. 28, 16861688.

Stegeman J. J.. Klotz A. V.. Woodin B. R. and Paior A. M. (1981) Induction of hepatic cytochrome P-450 in fish and the indication of environmental induction in scup (Srenotomus chrysops). Aquar. Toxic. 1, 197-212. Wolf C. R. (1986) Cytochrome P-450s: polymorphic multigene families involved in carcinogen activation. Trends Genet. 2, 209-214.