Effects of PCB on xenobiotic biotransformation enzyme activities in the liver and 21-hydroxylation in the head kidney of juvenile rainbow trout

AOUATIC TllXICOUlCY Aquatic Toxicology 39 (1997) 215-230

Effects of PCB on xenobiotic biotransformation enzyme activities in the liver and 21-hydroxylation in the head kidney of juvenile rainbow trout Sonja University

of Gtitehorg.

Blom*,

Depurtrnent

Lars Fiirlin

ofZoophysiolog_v, S-413 90 Giitehorg, Swckn

Received 6 March 1997; accepted 20 April 1997

Abstract We examined the effects of handling induced-stress combined with tetrachlorobiphenyl (TCB) exposure and the effects of long-term exposure to PCB on selected detoxification enzymes in the liver and kidney, and on the head kidney 21-hydroxylation of a 17ct-hydroxyprogesterone, an enzymatic step in cortisol biosynthesis in rainbow trout (Oncorhynchus mykiss). Our findings suggest that experimental conditions, such as stress, play an important role in mediating detoxification responses in rainbow trout. TCB together with stress caused significantly elevated liver etoxyresorufin-0-deethylase (EROD) and etoxycoumarine-0-dethylase (ECOD) activities, whereas 14 days TCB treatment alone did not alter the enzyme activities significantly. The UDP-glucuronosyltransferase (UGT) activity increased significantly in fish recovering from stress. The 21-hydroxylation of a 17a-hydroxyprogesterone was not affected by TCB, stress or by PCB. The induced ECOD activity could be inhibited by aNF to control levels, indicating the existence of a non-inducible form of CYP exhibiting ECOD activity. Long-term exposure to PCBs induced UGT, glutathione reductase (GR) and glutathione tansferase (GT) activities in the liver. The rapid induction of UGT by TCB compared with PCB suggests that TCB is a potent UGT-inducing congener in the PCB mixture. The different induction patterns of CYP-dependent activities and the GR, GT and UGT activities suggest differential regulation of these enzymatic activities, 0 1997 Elsevier Science B.V. Keywotds:

PCB;

UGT;

GT; GR;

21-Hydroxylation:

Stress

_

1. Introduction The detoxification (monooxygenation) are

mainly

catalysed

*Correspondence

of organic lipophilic compounds is mediated and phase II (conjugation) reactions. The phase by cytochrome

P450

(CYP)-dependent

author.

0166-445X/97/$17.00 U 1997 Elsevier Science B.V. All rights PII SO 166-445X(97)00035-0

reserved.

reactions.

by phase 1 I reactions Although

the liver is the major organ of detoxification of lipophilic xenobiotics in fish, the kidney also plays an important role in detoxification (Pesonen et al., 1987). Measurement of CYPIA-dependent etoxyresorufin-0-deethylase (EROD) and etoxycoumarine-0-deethylase (ECOD) activities is an established method for assessing exposure to xenobiotics like PCB (Di Giulio et al., 1995). We previously noted inhibition of induced ECOD activity. when liver microsomes were prepared from _juvenile rainbow trout injected i.p. with isosafrol or P-naphthoflavone (PNF) (unpublished results). Therefore we also studied the inhibition of ECOD activity after TCB induction. CYP is also involved in the production of endogenous compounds, including steroid hormones such as cortisol. Steroidogenesis includes several enzymatic steps involving different forms of CYP (Miller, 1988). The head kidney is the major glucocorticoid-producing organ in teleost fish (Nandi and Bern, 1965). We have studied the head kidney 2 I -hydroxylation of I7a-hydroxyprogesterone, leading to the formation of glucucorticoids. This enzymatic reaction is a branch step in steroidogenesis because the substrate 17a-hydroxyprogesterone can be converted into by 17a-hydroxylase glucocorticoids by 2 I -hydroxylase or into androstenedione (CYPI 7). Glutathione (GSH) provides a diverse number of critical cellular defensive functions. For example, it is involved in detoxification of electrophilic xenobiotics in phase II reactions initiated by glutathione-S-transferase (GT) (Otto and Moon, 1995). GT catalyses the conjugation between the sulfuryl group of GSH and the xenobiotic. GT activity varies depending on the level of reduced GSH in the cell. The reduction of GSH in the cell is catalysed by glutathione reductase (GR). UDPglucuronosyltransferases (UGT). a multigene family of membrane-bound enzymes (Senati et al., 1994). catalyse the conjugation and excretion of endogenous compounds, such as steroids, as well as a multitude of xenobiotics, including environmental pollutants. In mammals, the UGT isoform. which preferentially conjugates planar phenols, is induced by an Ah-receptor-mediated mechanism (Nebert et al., 1990). It has been suggested that phase I and phase II enzyme activities after toxicant exposure could be used to predict the fate of a toxicant in an organism (Kleinow et al., 1987). Polychlorinated biphenyls (PCBs) are ubiquitous pollutants in the aquatic environment. Although PCBs are no longer produced or used, they are resistant to biodegradation (Safe. 1994) and therefore accumulate along food chains, reaching highest concentrations in the tissues of top predators like fish (Livingstone et al., 1994). Previous studies have shown that exposure of fish to PCB impairs steroid production (Freeman et al.. 1980: Miranda et al.. 1992; Sivarajah et al., 1978). In fish, organic lipophilic compounds riced to be detoxificated and biotransformed to more water-soluble forms before they can be excreted. Many studies have examined the effects of PCB on CYP-monooxygenase and conjugating enzyme systems and in fish (Andersson and Fbrlin, 1992; Andersson et al.. 1985; Brumley et al., 1995: 1978; Celander and Forlin. 1995; Eggens and Boon. 1996; Forlin and Lidman, Lindstriim-Seppa ct al.. 1994; Otto and Moon, 1995). PCBs comprise a theoretical maximum of 209 variously chlorinated congeners of which the coplanar ones, like

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117

3,4,3’,4’-tetrachlorobiphenyl (TCB), induce CYPlA forms and EROD activity in fish (Gooch et al., 1989; Lindstrom-Seppa et al., 1994). This induction is mediated by an intracellular Ah-receptor (Stegeman et al., 1992). Many endogenous and environmental factors, e.g., sex, age, temperature and type of food, can affect the activity of xenobiotic biotransformation enzymes (Koivusaari et al., 1983; Lindstrdm-Seppl, 1985) and it has been shown that CYPlA induction can be enhanced by cortisol in rainbow trout liver cells in culture (Devaux et al., 1992). The induction of CYPlA has been successfully used as a tool in detecting exposure to polyaromatic hydrocarbons (PAH) or PCBs in many biomonitoring programmes (Goksoyr and Fiirlin, 1992; Eggens and Boon, 1996; Forlin et al., 1994). The aim of the present study was to investigate the effects of PCB/TCB exposure and handling stress on the induction of biotransformation enzymes and on head kidney 21-hydroxylation of 17a-hydroxyprogesterone.

2. Materials

and methods

2.1. Chemiculs TCB was a generous gift from professor Ake Bergman, Wallenberg Laboratory, University of Stockholm. Clophen A50 (PCB) was obtained from Bayer Chemicals. All other chemicals were of the highest purity available from Aldrich, Amersham, BioRad, Boehringer-Mannheim, Merck or Sigma. 2.2. Long-term

PCB exposure

of rainbow

trout

About 250 cultured juvenile rainbow trout (0. my&s), of both sexes and with an average weight of approximately 9.5 g, were obtained from Antens Fiskodling, a local Fish Hatchery close to Goteborg. The fish were kept in concrete basins provided with aerated, dechlorinated, filtered, recirculating fresh water at a temperature of 7-9°C. A 12 h light/l2 h dark photoperiod was used. The fish were acclimated to these conditions for two weeks prior to the experiment. They were then itrjected intraperitoneally (i.p.) with either 5 ml/kg body weight (b.w.) of peanut oil (carrier vehicle) or 100 mg PCB (Clophen A5O)/kg b.w.) and transferred to 250-L PCV tanks supplied with aerated, dechlorinated fresh water at a temperature of 779°C. Commercial trout pellets 1.25% (food weight/fish weight) were supplied every 3 or 4 days. Six or ten individuals from each treatment group were sampled 2. 5, 15, 35. 105 and 140 days post-injection. _?.3. TCB dose-response Fifty-four rainbow trout (average weight approx. 150 g) were divided into nine groups of six fish, with each group kept in a separate 50-I glass aquarium provided with aerated, dechlorinated and filtered fresh water at a temperature of 7--~9”C. A 12 h light/l2 h dark photoperiod was used. The fish were injected intraperitoneally

with 5 ml/b.w. peanut oil (carrier vehicle), 0.3 mg TCB/kg b.w. or 0.3 mg TCB/kg b.w. Six individuals from each treatment group were sampled 2, 14 and 28 days post-injection. 2.4.

TCB-stress

Rainbow trout (average weight approx. 150 g) were divided into four experimental groups and acclimated in the 250-I PVC tanks supplied with aerated and dechlorinated fresh water at a temperature of 9910°C. The fish were injected intraperitoneally with either 5 ml/kg body weight (b.w.) of peanut oil (carrier vehicle) or 0.1 mg TCB/kg b.w. One control group and one TCB group were subjected to stress by restraining them in a hand net for 5 min during each of 14 consecutive days. Before each sampling, the fish from all groups were chased for about 2 min. Six individuals from each group were sampled 14 and 28 days post-injection. 2.5. Sutnpling The fish were caught with a hand net and killed by a blow to the head. The head kidneys. trunk kidneys and livers were then excised, weighed and immediately placed on ice-cold buffer. The head kidney was rinsed in 1.0 M K-phosphate buffer containing 1.O M EDTA, 0.1 mM dithiothreitol and 20 pM butylated hydroxytoluene. Head kidney microsomes were prepared essentially as described by Pesonen and Andersson (1987) and stored in liquid nitrogen until use. The trunk kidney and liver were rinsed in 0.1 M Na/K-phosphate buffer pH 7.4, containing 0.15 M KCI. Trunk kidney microsomes were prepared essentially as described by Pesonen and Andersson (1987). The microsomal fractions from the liver were isolated as described by Forlin (1980) and stored in small aliquots at -80°C before use.

Microsomal protein contents were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. Total CYP levels were measured using the method described by Matsubara et al. (1974) (e=91 mM_‘cm-‘) with an Aminco DW 2a UV/VIS spectrophotometer. EROD activity was measured fluorometrically as described by Forlin et al. (1994) using a Perkin-Elmer LS-5 spectrofluorometer and resorufin as internal standard in each series. ECOD activity was determined as described by Goksoyr et al. (1987). UGT activity was measured as described by Andersson et al. (1985). We measured GT activity as described by Habig et al. (1974). and GR activity as described by Carlberg and Mannervik (1985). The 21 -OH activity was measured as previously described by Blom et al. (manuscript). To study the in vitro effects of PCB. Clophen A50 was dissolved in acetonitrile. and 15 pl was mixed with the microsomal suspensions prior to addition of other reaction components. The control reaction mixture received an equivalent volume of acetonitrile.

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39 11997) 215-230

2.7. St&sties

All data were expressed as means + SD. The data were tested day-wise by oneway analysis of variance (ANOVA), and, whenever a significant treatment effect was indicated, the Duncan multiple comparison test was carried out.

3. Results Mortality was negligible during the experimental periods. No signs of external abnormalities were apparent in the PCB or TCB experiments. The LSI did not differ between treatment groups on any occasion in the longterm exposure to PCB. No-significant differences were seen in the LSI (liver somatic index calculated as (liver weight) X (body weight)-’ x 100)) during the dose-response study. The LSI was significantly lower in the stressed TCB group 14 days post-TCB injection compared with the controls (Table 1). No significant differences in LSI were observed between the groups 28 days post-injection (Table 2). Effects of the long-term exposure to PCB on EROD activity, CYPlAl protein levels and CYPl AlmRNA levels have previously been reported by Celander and Forlin (1995). Injection of rainbow trout with 0.3 mg TCB/kg b.w. resulted in a significant induction of liver microsomal EROD activities compared with controls (Fig. 1). Treatment with 0.03 mg TCBlkg b.w. did not result in any significant effects on EROD activity. Injection of rainbow trout with 0.1 mg TCB/kg b.w. together with 14 days of handling-induced stress resulted in significantly elevated liver microsomal EROD activities, whereas TCB treatment alone did not have any significant effect (Table 1). Table I LSI and enzyme activities measured groups were chased daily with a hand -___________

14 days net

post-TCB

injection.

During

these

14 days

two of the

14 days

Control

Stress ______

TCB

TCB-stress

LSI P450 (nmollmg protein) Liver EROD activity (pmollmg protein X mitt) Liver ECOD activity (pmollmg protein X min) Kidney EROD activity (pmollmg protein X min) GS activity (nmol/mg protein X min) Head kidney 21.OH* activity (pmol/mg protein X min)

1.17~0.16” 0.21 f 0.032.“’

I .09 f 0.2@ 0.16~0.01”

1.10~0.101‘ 0.21 rt 0.05”’

0.88 + 0.06” 0.24 + 0.07”

75.Ok45.1,’

44.2 * 21.3“

426.8 f 485.8””

1203+132@

2.90?

1.23,’

2.47 ?

I .29”

6.86 2 4.37;“’

12.7 f I I .O”

1.85f

1.13;“’

I .90 *

I .74.“’

7.13 + 8.95~’

31.8f22.5”

500 f 7@’

580 f 180”

570 + 120;1

550 t 7W’

66.2 + 19.7,’

92.7 ?49.1>’

78.8 t 32.4;’

16.2 i 23.1,’

All data are expressed as mean f SD. I”’ Rows followed by one or more of the same letters are not significantly *21-hydroxylase.

different

(pcO.05).

Fig.

I. Total

mg TCB/kg 2 SE:

n=

EROD b.w.

6. ("/'<

activity (pmoi/(mg

protclnXmm))

or (m) 0.3 mg TCB/kg

b.w.

m liver mxrosomes

treated

rainbow

trout.

from (_I) control.

Values

are presented

(ci) 0.03 as means

0.0.5).

However, 28 days post-injection, fish in the TCB group showed significantly elevated EROD activities. Handling stress only did not have any significant effect on the indLkction level (Table 1). The pattern of effects on ECOD activity was the same as that observed for EROD activity (Tables 1 and 2). Kidney microsomal EROD

Table LSI

2 and enzyme

activities

fish \scre left to recover

measured from

1X days post-TCB

the handling

induction

After

being chased

~~_. _~ Control

28 days

for

13 days the

strew> Stress rrco\ery

_~

~_

_~~ TC‘B

TCB

stress recovery _~ LSI P450 (ngmol/mg Liver EROD (pmolhig (pmolhng

Ih 0.21i 0.0s

0.x52 0.06 0.25k o.ow”

I. IOk 0.I I 0.34+ 0.IJ.“’

0.95IL0.23 0.M 2 0.09”

49.2 f 35.4’

61 .o t 55.2,’

I657 t 1306”

2018?

2.0 I + I .I)5

I .4; i 0.45~~

I I .Y 2

17.9 F

13.3+ 7.20

12.5 5 4.W

167% 159”

I33 f

29.8”

501

I104’~

activity

protein X mm)

EROD

(pmol/mg

0.X’) ? 0. activity

protein X min)

Liver ECOD Kidney

protem)

9.7x”

I 1.o”

acti\it)

1

protein X mm

GS actiuty (nmol/mg CR

protein

X mln)

570 2 1704

760 + 30

620 * 190’

6 IOk

protcm

X niin)

40.0 I? x.0’

360 & 5.0

50.0 + 14.0

86.0 2

77.3 + 30.5,’

54.5 -t

63.6 2 18.2,’

42.4 2 24.7,’

activit)

(miiol/mg

Head kidney 2 I -OH* (pmohig All

protein X min)

data arc expressed

“’ Rous

followed

*1 I -hvdrouylasc.

I3.W

actlvit)

IS.9

_.

.~

as mcnn F SD.

b) one or more of the same letters NC not sigmficantly

diKerent

(p < 0.05).

S. Blow, L. FtirlinlAquatic

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221

3Y (1997) 215-230

0.025 M ANF Fig. 2. Inhibition of ECOD activity with aNF. ECOD activity in microsome preparations to which only I5 VI of ethanol had been added was referred to as 100% for the individual fish. (~)=microsomes from control fish; (m) = activity in microsomes from fish treated for 28 days with 0.1 mg TCB/ kg b.w.

*

I

0

50

I

100

I

150 days

Fig. 3. Total UGT activity (pmol/(mg proteinxmin)) in liver microsomes from (0) control, or (m) PCB-treated rainbow trout. Values are presented as means+SE: II= IO; except for the PCB I40 days where 17= 6. (*, p < 0.05).

activities were 5- to IO-fold lower than in the liver but showed an induction pattern similar to that of EROD activity in the liver (Tables 1 and 2). The total content of liver CYP was significantly elevated in the stressed fish injected with TCB 14 days post-injection (Table 1) and in both TCB-treated groups 28 days post-injection (Table 2). In vitro effects of a-naphthoflavone (aNF) on liver microsomal ECOD activities are shown in Fig. 2. ECOD activity was inhibited in a dose-dependent manner by CL-NF in liver microsomes from the TCB-treated fish. Addition of 0.025 yM CXNF to the reaction mixture resulted in a reduction of ECOD activity to levels reported for control fish (Fig. 2). In the study of long-term exposure to PCB, the liver microsomal UGT activity of exposed rainbow trout was significantly elevated on days 105 and 140 post-injection, but not on post-injection days 2, 5. 15 or 35 (Fig. 3). Injection of rainbow trout with TCB only resulted in significantly elevated UGT activities on day 28 post-injection (Fig. 4). The 14 days of daily handling stress had no effect on liver microsomal UGT activity, but after an additional 14 days, during which no stress was applied, UGT activities were significantly elevated compared with the control (Fig. 4). In the long-term exposure of rainbow trout to PCB, the cytosolic GT and GR activities were signilicantly elevated 105 and 140 days post-injection (Fig. 5). However. no ctrects on these activities were observed in the experiment where trout were TCB (Tables 1 and 2).

14 days

28 days

Fig. 4. Total CJGT activity (pmol/(mg protein X min)) an liver n~~crosotmcs from (open square) control, (horizontally hatched square) stressed, (hatched square) TCB or (filled square) stressed TCB-treated rainbow trout. The shaded area represents the period of handling-stress. Values are presented as means * SD: n = 6. (*/J < 0.05).

S. Blom. L. FtirlinlAquatic

is

Toxicology 39 (1997) 215-230

223

1 I

I 5 i

Fig. 5. Total glutathione reductase content (nmol/(mg proteinxmin)) in liver cytosol from (0) control, or (w) PCB-treated rainbow trout. Values are presented as means 2 SE; n= 10, except for the PCB 140 days where n = 6. (*p < 0.05).

Head kidney microsomal 21-hydroxylase activity towards 17a-hydroxyprogesterone was not significantly affected by the PCB or TCB treatments (Tables 1 and 2).

4. Discussion Earlier studies have shown that the LSI in juvenile rainbow trout can increase after treatment with PCB (Flirlin and Lidman, 1978; Lidman et al., 1976). In the present study no increased LSI levels were observed in fish treated with PCB or TCB. However, decreased LSI levels were observed in fish subjected to both TCB treatment and handling stress. Neither TCB treatment nor handling stress alone resulted in liver weight changes in the TCB-stress and dose-response studies. It is not known why the combination of handling stress and TCB treatment resulted in reduced liver sizes in the fish; thus additional studies with combined treatments are called for. Changes in liver weight could have been caused by increases or decreases in energy reserves in the form of glycogen and fat. Effects of long-term exposure to PCB on liver EROD activity, CYPlAl protein and mRNA levels in rainbow trout have been reported elsewhere (Celander and Fiirlin, 1995). In brief, the induction of CYPlAl mRNA was reported to be accompanied by the enhancement of CYPlAl protein levels and EROD activity. CYPlAl protein levels and EROD activities continued to increase throughout the experimental period of 140 days (Celander and Forlin, 1995). Treatment of rainbow trout with TCB affected liver EROD activity in a

dose-dependent manner, i.e. 0.03 mg/kg b.w. did not cause significant effects, whereas 0.3 mg TCB/kg b.w. caused a strong enhancement of EROD activity that lasted for 28 days. It has previously been shown that high concentrations ( > 0.1 FM) of TCB can inhibit the EROD activity in a fish hepatoma cell line (Hahn et al.. 1993). The significant increases in rainbow trout liver EROD and ECOD activities, as well as the increase in trunk kidney EROD activity by TCB (0.1 mg/kg b.w.) were in agreement with previous studies (Andersson et al.. 1985; Brumley et al., 1995; Celander and Fiirlin. 1995; Eggens and Boon, 1996; Fijrlin and Lidman, 1981; Otto and Moon, 1995). Inductions of the CYP-dependent EROD and ECOD activities in the liver were pronounced when the TCB treatment was combined with handling stress. It has previously been noted that TCB elicits toxic responses similar to those reported for TCDD (Safe, 1994). Using whole-body autography on rainbow trout injected with radiolabelled TCDD. Hektoen et al. (1994) showed that the radioactivity was largely confined to abdominal and subcutaneous adipose tissues. If the fate of TCB in rainbow trout is similar to that of TCDD, the pronounced increases in EROD and ECOD activities induced by handling stress could reflect a higher metabolic rate in these fish. resulting in larger doses of the TCB reaching the circulation and, eventually, the liver. Synergistic effects on rainbow trout hepatocyte EROD activity by PNF and cortisol were demonstrated by Devaux et al. (1992). and glucocorticoids have been used to maintain CYP levels in hepatocytes in culture (Dubois et al.. 1996). The quicker induction of EROD activity when TCB treatment was combined with handling stress could have been a result of synergistic effects of cortisol and TCB, but no statistically significant differences between the TCB group subjected to handling stress and the TCB group kept under normal conditions were detected on any of the sampling occasions. In the present study, cxNF inhibited the in vitro ECOD activity in the liver in a dose-dependent manner in all microsomes from TCB-injected fish. We previously noted the same kind of inhibition of the induced ECOD activity when liver microsomes were prepared from juvenile rainbow trout injected i.p. with isosafrol or PNF (unpublished results). This clearly indicates the existence of a non-inducible form of CYP exhibiting ECOD activity (Senegarof, 1982) that is not inhibited by aNF at concentrations used in the present study (5 0.025 FM). aNF at low concentrations (5 I yM) has previously been regarded as a specific inhibitor of CYPlAl and CYPlA2 (Tassaneeyakul et al.. 1993): however. Takahashi et al. (1995) stated that (xNF cannot be regarded as a specific inhibitor of CYPlA activity in fish, even at low tnicromolar concentrations. In mammalian liver microsomes, at least two different isoforms of CYPIA are present, and recently Berndtson and Chen (1994) sequenced a second form of CYPl A in trout liver. The identity of the CYP isoform catalysing the ECOD activity that was not inhibited by aNF in the present study remains unknown. EROD activity was inhibited in a similar manner in the control and induced microsomes (result not shown), in accordance with previous reports (Takahashi et al., 1995). The liver is considered to be the major target organ of PCB-induced toxicity (Safe. 1994). In the present study the patterns of time-dependent increase in

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EROD activity were similar in the liver and the kidney after a single injection of TCB; however, kidney microsomal EROD activities were 5- to lo-fold lower (Tables 1 and 2). This is consistent with previous results reported by Huuskonen et al. (1996) who found a 4- and 2-fold induction in the liver and kidney, respectively, following 6 days i.p. exposure to O.lmg/kg TCB. Pesonen et al. (1987) showed that trunk kidney EROD activity calculated on a per-CYP basis was IO-fold higher than the corresponding hepatic activity. In rainbow trout, the CYP content of kidney is approximately 5-fold lower than that of liver (Pesonen et al., 1990). Studying a food-chain model, Connolly (1991) found that dietary uptake exceeded uptake across the gill for all four PCB congeners tested (including TCB) and was the dominant route for the higher chlorinated congeners. Administration of a xenobiotic by intraperitoneal injection resembles the uptake of compounds through the gastrointestinal tract after exposure via the diet (Klaasen, 1975). The liver may thus serve as the primary target organ, whereas only a minor fraction of the intraperitoneally injected xenobiotics may reach the kidney. The high induction in the kidney in the present study further indicates the importance of the trunk kidney in the detoxification of xenobiotics. The metabolism of PCBs yields methylsulfonyl PCB metabolites. Such metabolites bind with high affinity to the kidney (Safe, 1994). The possible role of these metabolites in PCB toxicity in the kidney has yet to be revealed. PCBs induce enzymes other than CYPs associated with drug metabolism, including GT and UGT (Otto and Moon, 1995). The induction with 100 mg Clophen ASO/kg b.w. did not occur until 105 days post-injection. Andersson et al., 1985 found that the UGT activity toward p-nitrophenol was markedly increased 4 weeks after a single injection of 500 mg Clophen ASO/kg b.w. It is clear that the PCB mixture Clophen A50 is a potent inducer of UGT activity in rainbow trout liver but, as noted before by Andersson et al., 1985, the rate of induction of UGT activity towards p-nitrophenol in rainbow trout was relatively slow compared with the CYP-dependent activities in the liver. The inducing capacity of Aroclor (PCB) mixtures stems almost exclusively from the coplanar fraction of the PCB mixture (Stegeman et al., 1992). The Clophen A50 mixture closely resembles Aroclor 1254 in congener composition (Detlef et al., 1989). The coplanar PCBs, like TCB, competitively bind with relatively high affinity to the cytosolic Ah-receptor. We found that the UGT activity toward p-nitrophenol was markedly increased 4 weeks after a single injection of 0.1 mg TCB/kg b.w.. This is consistent with previous findings by Huuskonen et al. (1996) who found a slight (non-significant) increase in UGT activity following TCB treatment (0.1-5 mg/kg during 6 days). The more rapid induction of UGT by TCB compared with Clophen A50 suggests that TCB is one of the most potent UGT-inducing congeners in the PCB mixture of Clophen A50. We also found a significant increase in UGT activity following handling stress. Glucuronide formation is catalysed by a family of UGTs which use a number of xenobiotic compounds as substrates, and many of the different isoforms have broad substrate specificities (Burchell et al., 1995). It is possible that the altered metabo-

lism during stress leads to the production of endogenous metabolites that are glucuronidated in the liver and therefore induce UGT activity. In the present study, we noted a significant increase in GT activity after 140 days of exposure to 100 mg Clophen ASO/kg b.w. Andersson et al. (1985) reported a significant induction of GT activity after 4 weeks of treatment with 500 mg Clophen ASO/kg b.w. The GT activity in rainbow trout liver seemed to be induced by PCB in a dose-dependent manner, but further studies would be required to confirm this. We measured the activity of GT against 1-chloro-2,4_dinitrobenzene (CDNB), explaining why only references using this substrate have been cited. GT activity in the cytosolic fraction of the liver was not affected by treatment with 0.1 mg TCB/kg b.w. during the experimental period of 4 weeks. Using 5 mg TCB/kg b.w., Otto and Moon (1995) noted a 2%fold increase in GT activity 6 weeks post-injection. It therefore seems clear that the dose and duration of exposure in our TCB experiment were not sufficient to induce GT activity. The dissimilar patterns of induction of CYP-dependent activities as well as the GT and UGT activities suggest that these xenobiotic metabolising enzymes are differentially regulated in the rainbow trout liver (Andersson et al., 1985). We saw a significant induction of the GR activity in liver microsomes after longterm exposure to PCB. GR helps to maintain the ratio of reduced GSH to oxidised GSH in the cell at a proper level. GSH is not only important as a substrate for the phase II enzyme GT, but also provides various functions related to protein metabolism and oxyradical scavenging (Di Giulio et al., 1995 ; Stegeman et al., 1992). The xenobiotic metabolism sometimes yields increased amounts of oxyradicals and organoradicals in the cell. Therefore the increase in GR activity could reflect increases in the demand for GSH for both phase II detoxification reactions and radical scavenging. In the present study, GR activity proved to be just as sensitive as the GT and UGT activities to the PCB treatment. Therefore, it would be interesting to learn more about the relations between this enzyme system and the xenobiotic metabolism. GR activity was not significantly affected by the TCB treatment, although on the second sampling occasion activities tended to be increased in the TCB group stressed for the first 14 days. Since GR activity was only measured in the TCBstress experiment, it remains to be determined whether higher doses of TCB would have aflected the activity of this enzyme in the absence of stress. No effects on 21-hydroxylase activities were noted in the TCB study or in the study of long-term exposure to PCB. In mammals, it has been demonstrated that the increase in plasma cortisol seen after treatment with TCDD is coupled to a decrease in the CYP21 (P45Oc21) activity in rat adrenal gland (Mebus and Piper, 1986). PCBs also inhibit various adrenal steroid hydroxylases in the guinea pig (Safe, 1994). Handling stress resulted in a slight increase in the 21-hydroxylation rate in the head kidney, but this increase was not significant, and experiments with larger sample sizes are needed to verify that handling stress actually increases the interrenal 21-hydroxylase activity. To test the hypothesis if 21-hydroxylation in the rainbow trout head kidney is differentially regulated by PCB than the adrenal 21hydroxylation in mammals, we followed the in vitro procedure described by Gold-

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man and Yawetz (1992) who found that 13.5 uM Aroclor 1254 inhibited the rate of 21-hydroxylation in microsomes prepared from guinea pig adrenal cortex. We found that the 21-hydroxylation rate, following the addition of Clophen A50, dissolved in acetonitrile to the test tube, increased with Clophen A50 concentrations up to 15 pM. Above this concentration, the rate decreased, but remained higher than the rate in microsomes treated with acetonitrile when the Clophen A50 concentration in the reaction mixture was 300 pM (unpublished results). Our findings suggest that experimental conditions, such as stress, play an important role in mediating detoxification responses in rainbow trout. The effects of experimental conditions need to be examined further, especially with regard to the regulation of UGT and 21-hydroxylation.

Acknowledgements

We would like to thank Associate Professor Tommy B. Andersson for engaging us in valuable discussions. Mrs. Aina Stenborg and Mrs. Barbro Blomgren provided excellent technical assistance. This study was supported by grants from the Swedish Environmental Protection Agency (SNV), Helge Ax:son Johnsons Stiftelse, Stiftelsen Lars Hiertas Minne, Giiteborgs Kungliga Vetenskaps och Vitterhetssamhalle, Clas Groschinskys Minnesfond, Anna Ahrenbergs Fond and Vilhelm and Martina Lundgrens Vetenskapsfond.

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