Hepatic microsomal 4,5,6-trichloroguaiacol glucuronidation in five species of fish

Comp. Biochem. Physiol. Vol. 93B, No. 3, pp. 653-656, 1989

0305-0491/89 $3.00+ 0.00 © 1989 Maxwell Pergamon Macmillanpie

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HEPATIC MICROSOMAL 4,5,6-TRICHLOROGUAIACOL G L U C U R O N I D A T I O N IN FIVE SPECIES OF FISH LARS FORLIN,*t TOMMY ANDERSSON* and CARL AXEL WACHTMEISTER~ *Department of Zoophysiology, University of G6teborg, Box 25059, 400 31 G6teborg, Sweden, and ,Wallenberglaboratoriet, University of Stockholm, S-I04 05 Stockholm, Sweden (Tel: 46 031 85 3000) (Received 14 November 1988)

Abstract--1. Hepatic microsomal UDP glucuronyl transferase activity towards 4,5,6-trichloroguaiacol was characterized in five species of fish. The simple extraction technique used to determine glucuronidation rates was sufficiently accurate, as judged by high pressure liquid chromatography. 2. UDP glucuronyl transferase activities were found to vary considerably between fish species. The level of UDP glucuronyl transferase activity in rainbow trout is among the highest in vitro conjugating activities reported in fish. 3. Intraperitoneal injection of fl-naphthoflavone resulted in an almost doubling of the UDP glucuronyl transferase activity in rainbow trout.

INTRODUCTION In the late 50s and early 60s evidence was found that fish have the capacity to form glucuronide conjugates in vitro (Dutton and Montgomery, 1958; Stevenson and Dutton, 1962). Although relatively little attention has been paid to conjugating reactions in fish, it has been demonstrated since then that fish are also capable of forming sulfate, glutathione and amino acid conjugates in vitro (Chambers and Yarbrough, 1976; Balk et al., 1980; Gregus et al., 1983; Castrrn et al., 1984). Conjugation is an important part of the detoxification process in fish since this biotransformation dramatically increases the polarity of the toxic substance, thereby facilitating excretion through the bile and urine. In 1976 it was proposed that the levels of biotransformation products of certain pollutants in fish could be quantified by monitoring their concentrations in bile (Statham et al., 1976). In bleached kraft mill effluents large amounts of chlorinated derivatives of phenolic compounds (chlorophenols, -catechols and -guaiacols) have been found. Some of these compounds are released in very large quantities from the mills but only accumulate to a moderate degree in fish tissues (Landner et al., 1977; Renberg et al., 1980). In fish exposed to pulp mill effluents, however, high amounts of chlorinated compounds, mainly conjugates, have been identified in the bile (Oikari and ,~n/is, 1985; Oikari, 1986; Oikari and Kunnamo-Ojala, 1987). 4,5,6-Trichloroguaiacol, a compound found in pulp mill effluents, was recently used as a model compound for studies in which the chemical analysis of bile was evaluated as an environmental monitoring method (F6rlin and Wachtmeister, 1989). In rainbow trout glucuronidation appears to be the major route by which this compound is conjugated. This reaction is catalyzed by microsomal UDP glucuronyl trans-

tAuthor to whom correspondence should be addressed. 653

ferase, which is found in high concentrations in the liver. Recently, unusually high levels of hepatic microsomal UDP glucuronyl transferase activity toward 4,5,6-trichloroguaiacol were detected in rainbow trout (Frrlin et al., 1985). In Sweden certain species of fish, including perch, flounder, fourhorn sculpin and blenny have been used or proposed for use in various environmental monitoring programs. It was therefore considered of interest to characterize the activity of hepatic microsomal UDP glucuronyl transferase with the aglycon substrate 4,5,6-trichloroguaiacol in these species of fish. MATERIALS AND METHODS Chemicals

4,5,6-Trichloroguaiacol and 4,5,6-trichloro-[~4C-CH3]guaiacol were synthesized according to the method recently described by Bergman and Wachtmeister (1987). UDP glucuronic acid was purchased from Sigma Chemicals. All other chemicals used were of analytical grade. Fish

Juvenile rainbow trout, Salmo gairdneri (approx. weight 100 g), were obtained from a local hatchery, Antens Laxodling AB. The fish were kept in aquaria provided with recirculating, filtered and aerated freshwater at a temperature of 10°C. Flounder, Platichthusflesus, were caught in gill nets outside Stenungsund (west coast of Sweden), and perch, Perca fluviatilis, were caught outside Norrsundet and Forsmark (east coast of Sweden). Fourhorn sculpin, Myoxocephalus quadricornis, were supplied by Dr. B. E. Bengtsson, Swedish Environment Protection Board, Studsvik. Blenny, Zoarces zoarces, were caught outside Kungsbacka (west coast of Sweden) by Alvar Jakobsson, Swedish Environment Protection Board, Kungsbacka. Experimental treatments

The rainbow trout treated with fl-naphthoflavone (BNF) were first acclimated for two weeks. They then received a single (i.p.) injection of either BNF (50 mg/kg body weight) dissolved in peanut oil or peanut oil only. Thereafter, they were kept in small aquaria (501), each aquarium was

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provided with a continuous flow (4 l/hr) of filtered fleshwater (temperature lff'C). The fish were starved throughout the experiment.

Sampling and preparation of enzymes Liver tissue for determining UDP glucuronyl transferase activities were frozen in liquid nitrogen according to F6rlin and Andersson (1985). The rainbow trout treated with BNF were sampled 7 and 14 days postinjection. Microsomes were prepared using a previously described procedure (F6rlin, 1980). The microsomes were suspended in Tris-HC1 buffer, pH 7.4, containing 20% glycerol to get microsomal preparations corresponding to 0.5 g wet weight liver/ml (8 12 mg protein/ml). The microsomes were stored at - 80°C prior to the analysis, which was performed within a month. UDP glucuronyltransferase The incubation conditions were designed to give linear conversion of the substrate over time as well as pH and enzyme (microsomes) concentration. The standard incubation mixture for UDP glucuronyl transferase with the aglycon substrate 4,5,6-trichloroguaiacol contained 2-4 mg protein/ml, 1.5 mM 14C-labelled 4,5,6-trichloroguaiacol (specific activity: 0.22 mCi/mmol), added in 10/tl methanol, 9 mM UDPGA, 0.2% (w/v) digitonin and 0.1 M phosphate buffer (pH 7.5), giving a total volume of 1351zl. The incubations were run at 20c:C. They were initiated by the addition of UDPGA and terminated, after 5 min with rainbow trout microsomes and 10 min with flounder, perch, fourhorn sculpin and blenny microsomes, by adding 0.9 ml 3% trichloroacetic acid. The pH of the mixture was adjusted to 8 with 5 M KOH, and 0.5 ml was transferred to tubes containing 5 ml dichloromethane. The tubes were shaken vigorously to remove unmetabolized 4,5,6-trichloroguaiacol. The radioactivity in 200/~1 of the aqueous phase and 10ml of Instagel (Packard Downers Grove) were measured in a Rackbeta 1217 liquid scintillation counter. UDP glucuronyltransferase activity towards p-nitrophenol was determined according to Anderson et al. (1985). The standard incubations with rainbow trout, flounder, blenny and fourhorn sculpin microsomes contained 445 mg microsomal protein/ml, 0.35 mM p-nitrophenol, 4.5mM UDPGA, 0.2% digitonin, 0.5 M phosphate buffer (pH 7.0), giving a total volume of 150 ld. In the incubations with the perch microsomes a concentration of 0.05% digitonin was used. The reactions were determined at 20°C, started by the addition of the microsomes, and terminated after 20 rain by adding 0.9 ml 3% trichloroacetic acid. High pressure liquid chromatography (HPLC) HPLC was performed using a Shimadzu LC4A instrument equipped with a RP-18 column ( 5 # m × 150ram, Lichrosorb~ E. Merck, Darmstadt FRG) and an injection loop allowing the injection of no more than 50/H. Elution was performed using a gradient from 10% v/v solution A and 90% solution B to 100% A in 25 min, following by 15 min with 100% A (A = methanol, d = 0.79, B = 200kd H3PO 4 d = 1.71 dissolved in methanol (100ml)+water (900 ml)). The solvents were continuously degassed under a slow stream of helium. Fractions were collected (0.4 ml, 0.5 min) in vials and the level of radioactivity determined by liquid scintillation after addition of a Packard 299 TM solution (10 ml per vial). The counting efficiencies at various levels of eluate water content were determined by adding appropriate internal standards. Characterization of labelled polar component formed in the in vitro glucuronidation experiment Isolation through adsorption on octadecyl-modified silica gel. A SEP PAC a C18-cartridge (RP-18, 9 × 9 m m ID) (Waters Assoc., Milford, MA, USA) was activated before use by passing methanol (10 ml) through it, followed by 0.6 M acetic acid (pH 2.5, 10 ml). A flow rate not exceeding

10ml/min was maintained by using a disposable syringe. The combined aqueous phases (1.8 ml, containing 4500 dpm 14-C) from several incubation experiments with nonconjugated trichloroguajacol removed by extraction with dichloromethane (cf above}--were acidified to pH ca 2.5 with 1.5 M acetic acid and forced through the SEP PAC cartridge. Thereafter 0.6 M acetic acid (5 ml) was added and, finally, the cartridge was eluted with acetone (2 × 5 ml, in the opposite flow direction). The combined eluates were evaporated to dryness under a stream of nitrogen, and the residue was dissolved in methanol-water (1:1 v/v, 100/d). HPLC-analysis. The methanol water solution described above was injected (20/~1) and analysed by HPLC using the general procedure given above. A single radioactive peak (elution time approx. 19 min: 620 dpm above background), corresponding to ca 70% recovery of the amount originally applied on SEP-PAC, was obtained. Assuming a sate detection limit of 1.5 × background (40dpm/fraction/, wc concluded that the sample was devoid of activity at the elution time of free 4,5,6-trichloroguaiacol (2L5min). Hence, the content of free phenol after extraction with dichloromethane (cf above) should have been less than 3%. RESULTS AND DISCUSSION Hepatic and extra-hepatic microsomal U D P glucuronyltransferase activities have been measured in several species o f fish. p - N i t r o p h e n o l is tYequently used as an aglycon substrate in fish, but other exogenous (e.g. 1-naphthol, T F M ) and e n d o g e n o u s (e. bilirubin, testosterone, estradiol-17fl) have also been used (Lindstr6m-Sepp~i et al., 1981; Gregus et at., 1983; A n d e r s s o n et al., 1985). In the present study 4,5,6-trichloroguaiacol was conjugated to its glucuronide in the presence o f U D P G A and hepatic microsomes from rainbow trout, perch, flounder, f o u r h o r n sculpin or blenny (Table 1). The level o f UDP glucuronyl transferase activity towards 4,5,6-trichloroguaiacol was c o m p a r a b l e to that towards p - n i t r o p h e n o l in four o f the fish species tested (perch, flounder, f o u r h o r n sculpin and blenny). In rainbow trout, however, a 5-fold higher level o f activity was observed (Table 1). It is notable that the level o f U D P glucuronyl transferase activity towards 4,5,6-trichloroguaiacol in rainbow trout is a m o n g the highest in vitro conjugating activities reported in fish. Optimal conditions for the enzyme incubations were f o u n d to be fairly similar in all five fish liver microsomes. W h e n using the microsomes from rainbow trout, however, a shorter incubation time was necessary to obtain a linear time-activity relationship. A surfactant (digitonin) increased the enzyme activity in the fish microsomes. In the present study the activity o f U D P glucuronyl transferase was in-

Table 1. Hepatic microsomal UDP glucuronyhransferaseactivities with the aglycon substrates p-nitrophenol (PNP/ and 4,5,6-trichloroguaiacol (TCG) in rainbow trout, flounder, perch. fopurhorn sculpin and blenny UDPGT UDPGT (PNP) (nmol/mg (TCG)(nmol/mg protein/min) protein/mini Rainbow trout 0.82 _+0.08 (6) 4.2 + 0.9 (6) Flounder 0.57 ± 0.17 (6) 0.64 + 0.12 (6) Perch 0.21 ± 0.06 (6) 0.15 + 0.08 (6) Fourhorn sculpin 0.16 ± 0.04 (6) 0.22 ± 0.07 (6) Blenny 0.55 ± 0.09 (6) 0.61 _+0.12 (6) Mean + S.D. (n).

Fish UDP glucuronyl transferase

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phenol are apparently similar, we suggest that in fish these substrates are conjugated by the same UDP glucuronyl transferase(s) or closely related ones. In fish exposed to radioactively labelled 4,5,6trichloroguaiacol most of 4,5,6-trichloroguaiacolderived radioactivity in the bile was identified as 200 glucuronides (F6rlin and Wachtmeister, 1989). Recently it was proposed that, in fish, conjugation is an important step limiting the rate of release of a glucuronide into the gall bladder (F6rlin and "I Haux, 1985). The great differences in hepatic UDP 0 . . . . . . . . . . . . . . . . 15 20 25 glucuronyl transferase activities among the teleost TIME, rain species used in this study imply that corresponding Fig. 1. High pressure liquid chromatography chro- interspecific differences exist in the efficiency with matogram showing radioactive components formed in the in which 4,5,6-trichloroguaiacol and possibly other vitro glucuronidation of [14C-CH31-4,5,6-trichloroguaiacol. chlorinated phenolic compounds is eliminated. For further details see Materials and Methods.

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REFERENCES

creased 2-fold by digitonin. This degree of latency is Andersson T., Pesonen M. and Johansson C. (1985) similar to the 2- to 3-fold increase in UTP glueDifferential induction of cytochrome P-450-dependent uronyl transferase activity previously reported in fish monooxygenase, epoxide hydrolase, glutathione transferase and UDP glucuronosyltransferase activities in the (H~inninen et al., 1984; Andersson et al., 1985). To liver of rainbow trout by fl-naphthoflavone or Clophen reduce problems with activation caused by various A50. Biochem. Pharm. 34, 3309-3314. treatments and the enzyme preparation, the activity of U D P glucuronyl transferase should be measured in Balk L., Meijer J., Seideg~rd J., Morgenstern R. and DePierre J. (1980) Initial characterizartion of drugits activated state (Andersson et al., 1985). metabolizing systems in the liver of the Northern-Pike, The results clearly indicate that 4,5,6-trichloroEsox lucius. Drug Metab. Dispos. 8, 98-103. guaiacol is a good aglycon substrate for hepatic Bergman A. and Wachtmeister C. A. (1987) Phase transfer microsomal U D P glucuronyltransferase in fish. The mediated synthesis of radiolabelled alkylarylethers and developed radiometric method included a dichlorosulfides. J. labelled comp. Radiopharm. 24, 925-930. methane extraction step used to separate the Castr6n M. and Oikari A. (1984) Optimal assay conditions for liver UDP-glucuronosyltransferase from the rainbow 4,5,6-trichloroguaiacol from its glucuronide. The trout, Salmo gairdneri. Comp. Biochem. Physiol. 7612, simple extraction technique used to determine gluc365-369. uronidation rates appeared to be sufficiently accurate Chambers J. E. and Yarbrough J. D. (1976) Xenobiotic for the present purposes, as judged by HPLC biotransformation systems in fish. Comp, Biochem. measurements (Fig. 1). PhysioL 55C, 77-84. Treatment of rainbow trout with BNF, a strong Dutton G. J. and Montgomery J. P. (1958) Glucuronide cytochrome P-450 inducer, resulted in the near doubsynthesis in fish and the influence of temperature. Biochem. J. 70, 17. ling of UDP glucuronyl transferase activities (Table 2). This effect appeared 14 days after the F6rlin L. (1980) Effect of Clophen A50, 3-methylcholanthrene, pregnenolone-16ct-earbonitrile and phenointraperitoneal injection of BNF. A similar pattern of barbital on hepatic microsomal cytochrome P-450increase was also observed when p-nitrophenoi was dependent monooxygenase system in rainbow trout, used as a substrate for the hepatic microsomal UDP Salmo gairdneri, of different age and sex. Toxicol. Appl. glucuronyl transferase in rainbow trout (Andersson PharmacoL 54, 420-430. et al., 1985; F6rlin and Haux, 1985). Andersson et al. F6rlin L. and Andersson T. (1985) Storage conditions of (1985) further presented evidence for the presence of rainbow trout liver cytochrome P-450 and conjugating different forms of UDP glucuronyl transferases in enzymes. Comp. Biochem. Physiol. 80B, 569-572. rainbow trout, based on the nonparallel pattern by F6rlin L., Ahlman M., Bengtsson B.-E., Svanberg O. and which three different UDP glucuronyl transferase Wachtmeister C. A. (1985) Studies on the metabolism of 4,5,6-trichloro-[t4C--CH3]-guaiacol in fish. In Nordic activities were induced by BNF or polychlorinated Symposium on Bleaching Effluents, p. 91. Abo Akedemi, biphenyls. In light of the suggested presence of UDP Finland. glucuronyl transferase isoenzymes in fish and the fact F6rlin L. and Haux C. (1985) Increased excretion in the bile that induction patterns of UDP glucuronyl transof 17fl-[3H]estradiol-derived radioactivity in rainbow ferase towards 4,5,6-trichloroguaiacol and p-nitrotrout treated with fl-naphthoflavone. Aquatic Toxicol. 6, 197-208. F6rlin L. and Wachtmeister C. A. (1989) Fishbile analysis Table 2. Hepatic microsomal 4,5,6for monitoring of low concentrations of polar xenobiotics trichloroguaiacol glucuronidation in in water. In Advanced Hazard Assessment of Chemicals in fl-naphthoflavone (BNF) treated rainthe Aquatic Environment (Edited by Landen L.). Springer bow trout (in press). 7 days 14 days Gregus Z., Watkins J. B., Thompson T. N., Harvey M. J., Control 5.2 + 0.7 (5)* 4.9 + 0.9 (5) Rozman K. and Klassen C. D. (1983) Hepatic phase I and BNF 7.9 + 1.4 (5) 9.8 + 1.5 (5)? phase II biotransformation in quail and trout: com* M e a n s + S.D. (n) (nmol/mg protein/ parison to other species commonly used in toxicity testmin). ing. Toxicol. Appl. Pharrnacol. 67, 430--441. tP < 0.05. H/inninen O., Lindstr6m-Sepp/i P., Koivusaari U., Vaisanen

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M., Julkununen A. and Juvonen R. (1984) Glucuronidation and glucosidation reactions in aquatic species in boreal regions. Biochem. Soc. Trans. 12, 12-21. Landner L., Lindstr6m M., Karlsson J., Nordon J. and S6rensen L. (1977) Bioaccumulation in fish of chlorinated phenols from kraft pulp mill bleachery effluents. Bull. environ. Contam. Toxicol. lg, 663~573. Lindstr6m-Sepp/i P., Koivusaari U. and H/inninen O. (1981) Extrahepatic xenobiotic metabolism in northEuropean freshwater fish. Comp. Biochem. Physiol. 69C, 259-263. Oikari A. and An/is E. (1985) Chlorinated phenolics and their conjugates in the bile of trout (Salmo gairdneri) exposed to contaminated waters. Bull. environ. Contain. Toxicol. 35, 802-809. Oikari A. (1986) Metabolites of xenobiotics in the bile of fish in waterways polluted by pulpmill effluents. Bull. environ. Contam. Toxicol. 36, 429-436.

Oikari A. and Kunnamo-Ojala T. (1987) Tracing of xenobiotic contamination in water with the aid of fish bile metabolites: A field study with caged rainbow trout (Salmo gairdneri). Aquatic. Toxicol. 9, 327-341. Renberg L., Svanberg O., Bengtsson B.-E. and Sundstr6m G. (1980) Chlorinated guaiacols and catechols bioaccumulation potential in bleaks (Alburnus alburnus, Pisces) and reproductive and toxic effects on the harpacticoid Nitrocra spinipes (Crustacea). Chemosphere 9, 143-150. Statham C. N., Melancon M. J. Jr. and Lech J. J. (1976) Bioconcentration of xenobiotics in trout bile: A proposed monitoring aid for some waterborne chemicals. Science 193, 680-681. Stevenson I. H. and Dutton G. J. (1962) Glucuronide synthesis in kidney and gastrointestinal tract. Biochem. J. 82, 330-340.