- Email: [email protected]
Differential Inhibition of Xenobiotic-Metabolizing Carboxylesterases by Organotins in Marine Fish
Ecotoxicology and Environmental Safety 46, 258}264 (2000) Environmental Research, Section B doi:10.1006/eesa.2000.1928, available online at http://www.idealibrary.com on
Differential Inhibition of Xenobiotic-Metabolizing Carboxylesterases by Organotins in Marine Fish Saif M. Al-Ghais, Shakeel Ahmad, and Basheer Ali Marine Environment Research Center, Environmental Research and Wildlife Development Agency, P. O. Box 45553, Abu Dhabi, United Arab Emirates Received July 9, 1999
The hydrolytic metabolism of xenobiotics in the liver of two tropical marine 5sh, Siganus canaliculatus and Acanthopagrus latus, was found to be catalyzed by both microsomal and cytosolic carboxylesterases; the latter forms were more active than the former. Remarkably greater e7ciency of S. canaliculatus for p-nitrophenylacetate hydrolysis was attributed to manyfold higher Vmax and lower Km values of hepatic microsomal and cytosolic carboxylesterases of S. canaliculatus as compared with those of A. latus. Comparative characterization of the in vitro responses of hepatic microsomal and cytosolic carboxylesterases to the organotin group of marine pollutants=tributyltin (TBT), triphenyltin (TPT), and dibutyltin (DBT), a relatively persistent metabolite of TBT=revealed species-, isozymic form, and organotin structure-related di4erences in the hydrolytic detoxication. In general, carboxylesterases of S. canaliculatus exhibited severalfold greater susceptibility to organotin inhibition and DBT was the most potent inhibitor (IC50 in micromolar range). Notably, the IC50 of SnCl2 , a metal present in all the compounds studied, was higher than 2 mM. Cytosolic forms in both species were more sensitive to organotin inhibition than microsomal counterparts. In line with these di4erences the nature of inhibition of cytosolic and microsomal carboxylesterases by organotins was competitive and noncompetitive, respectively. These results suggest that organotins may aggravate the toxicity of other environmental contaminants in 5sh and other aquatic organisms. Moreover, highly sensitive cytosolic carboxylesterases of S. canaliculatus liver may serve as molecular biomarkers of organotin pollution. 2000 Academic Press
INTRODUCTION
There has been an increasing awareness in the recent past that alkyl and aryl tin compounds are of environmental concern owing to their ecotoxicological impact, bioaccumulation potential, prolonged persistence in deep-sea sediment, and direct introduction into the aquatic environment, particularly coastal areas, mainly by leaching from antifouling paints applied on ships, boats, aquaculture nets, and other marine installations and as a result of their
employment in agriculture as biocides and in industry as plastic stabilizers and catalysts and preservatives for wood, textile and paper (WHO, 1990; Morcillo et al., 1997; Kannan et al., 1997; Takahashi et al., 1997). Hydrolytic metabolism of xenobiotics by nonspeci"c carboxylesterases plays a major role in the detoxication of organophosphate pesticides (Wallace and Dargan, 1987; Anderson et al., 1988; Straus and Chambers, 1995), phathalate ester plasticizers (Wo!ord et al., 1981; Barron et al., 1989), oil spill dispersants (Payne, 1982; Swall and Tjeerdema, 1991), and other environmental chemical pollutants in "sh and other aquatic animals. The vast literature available on mammalian carboxylesterases has revealed that these hydrolases are localized in the microsomal and cytoplasmic fractions of hepatic and extrahepatic tissues as multiple isoenzymes (Ali and Kaur, 1983; Hosokawa et al., 1990; Khanna et al., 1992; Heymann et al., 1993; Cashman et al., 1996) and display broad substrate speci"city toward chemicals containing a carboxylester, amide, or thioester bond. The microsomal forms, which account for the bulk of mammalian liver carboxylesterase/amidase activity, are selectively induced by foreign chemicals such as polycyclic aromatic hydrocarbons and DDT (Kaur and Ali, 1983a; Hosokawa et al., 1988), drugs such as methaqualone (Kaur and Ali, 1983b), hormones (Kaur et al., 1991), and mancozeb, a fungicide (Siddiqui et al., 1990), and are inhibited by organophosphate and carbamate pesticides (Cashman et al., 1996). Characterization of xenobiotic-metabolizing esterases and their responses environmental chemicals in "sh and aquatic invertebrates has generally been con"ned to electrophoretic separation of their multiple molecular forms present in the postmitochondrial or cytosolic fractions of liver (Varma and Frankel, 1980; Salamastrakis and Haritos, 1988; Ozretic and Krajnovic-Ozretic, 1992; Soldano et al., 1992) and inhibition of in vitro hydrolytic activity of isolated isoenzymes (Ozretic and Krajnovic-Ozretic, 1992) and tissue homogenates (Straus and Chambers, 1995) by orgnophosphate and carbamate pesticides. Lately, "sh esterases have
258 0147-6513/00 $35.00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved.
INHIBITION OF FISH CARBOXYLESTERASES BY ORGANOTINS
been evaluated as biomarker of exposure to multiple pollutants present in the aquatic environment (Huang et al., 1997). Previous studies on the e!ects of organotin compounds on xenobiotic-metabolizing enzymes in "sh and their toxicological implications have found that tributyltin (TBT) and triphenyltin (TPT) inhibit cytochrome P450-dependent monooxygenases in liver (Fent and Bucheli, 1994; Fent and Stegeman, 1993) and glutathione S-transferase activity in liver and kidney of marine "sh (Al-Ghais and Ali, 1999). The purpose of this study was to make comparative assessment of the in vitro e!ects of TBT, TPT and DBT, which is also a major and relatively persistent metabolite of TBT in "sh (Morcillo et al., 1997; Takahashi et al., 1997), on hydrolytic detoxication of xenobiotics by carboxylesterases in the liver of Siganus canaliculatus and Acanthopagrus latus, two widely occurring commercially important "sh inhabiting the southern Arabian Gulf waters along the Abu Dhabi coast of United Arab Emirates. MATERIALS AND METHODS
Chemicals Bovine serum albumin, p-nitrophenyl acetate, potassium chloride, and tromethamine (Tris) were purchased from Sigma Chemical Company (St. Louis, MO). Tributyltin chloride (96%), triphenyltin chloride (95%), dibutyltin chloride (96%), and tin chloride were obtained from Aldrich Chemical Company (Milwaukee, USA. MI). Fish Samples (60}120 g) of S. canaliculatus (rabbit"sh) and A. latus (seabream) were captured with the help of a trap from the southern Arabian Gulf waters along the Abu Dhabi coast of United Arab Emirates. Tissue Preparation Liver homogenate was prepared in ice-cold 1.15% KCl bu!ered with 0.01 M Tris}HCl, pH 7.4, with the help of a Potter}Elvehjem homogenizer "tted with a Te#on pestle. Subcellular fractions of liver (nuclei, mitochondria, microsomes, and cytosol) were isolated by di!erential centrifugation of liver homogenate in an IEC-Centra-MP4R (USA) centrifuge and Beckman Optima L-70 (USA) ultracentrifuge by the procedure described earlier (Khanna et al., 1992). Analysis Procedure Carboxylesterase activity in tissue preparations was determined by monitoring the hydrolysis of p-nitrophenyl acetate, a nonspeci"c model substrate, at 412 nm spectrophotometrically (Kaur et al., 1991; Khanna et al., 1992).
259
Unless otherwise stated, the enzyme assay mixture (3 ml) consisting of 0.1 M Tris}HCl bu!er, pH 7.4, 2.5 mM pnitrophenyl acetate, and an appropriate amount of liver preparation as the enzyme source was incubated at 283C for 10 min. To investigate in vitro e!ects of organotin compounds and SnCl on carboxylesterase activity varying con centrations of the compounds dissolved in ethanol or an equal amount of ethanol alone in the control were preincubated with the enzyme prior to addition of substrate. Protein was determined by the procedure of Lowry et al. (1951) using bovine serum albumin as standard. RESULTS
Subcellular localization of carboxylesterase activity toward p-nitrophenyl acetate, a nonspeci"c model substrate, in the liver of S. canaliculatus and A. latus revealed recovery of more than 75% of the total hydrolytic activity of homogenate in the microsomal and cytosolic fractions, the latter being endowed with two-to fourfold higher activity than the former (Fig. 1). The constitutive level of hepatic carboxylesterases in S. canaliculatus as indicated by their speci"c activities in homogenate, microsomal, and cytosolic fractions was 10}20 times higher than that of A. latus (Table 1). Further characterization of carboxylesterase activity was carried out by studying the relationship between reaction velocity and substrate concentration. The maximum velocity (< ) of
substrate hydrolysis and Michaelis}Menten constant (K ) K were estimated by the double-reciprocal plot of Lineweaver and Burk (1934) and are presented in Table 2. The < of
microsomal and cytosolic carboxylesterases in S. canaliculatus, an indicator of total enzyme present, were 5.9and 7.8-fold higher, respectively, than the values for A. latus. However, the apparent K of microsomal and cytosolic K forms of enzyme, which is inversely proportional to the enzyme a$nity for substrate, in S. canaliculatus were 2.0 and 12.2 times lower than those of A. latus. The carboxylesterase activity of liver homogenate, which is an index of overall e$ciency of hydrolytic metabolism of xenobiotics, was inhibited by organotin compounds, TBT, TPT, and DBT, in vitro in a concentration-dependent manner in both species (Fig. 2). A comparison of the enzyme inhibitory potency of organotins in terms of IC values (the inhibitor concentration causing 50% loss of enzyme activity) revealed species- and inhibitor-related variations. In general, carboxylesterases of S. canaliculatus were many times more susceptible to organotin inhibition and DBT was found to be the most potent inhibitor in both species. Interestingly, the IC values of SnCl , a metal present in all the compounds studied, were higher than 2 mM, the highest concentration examined. The responses of hepatic microsomal and cytosolic carboxylesterases to organotins in S. canaliculatus and A. latus
260
AL-GHAIS, AHMAD, AND ALI
FIG. 1. Subcellular distribution of hepatic carboxylesterase activity in nuclei, mitochondria, microsomes, and cytosol from Siganus canaliculatus and Acanthopagrus latus. Values are averages of three separate determinations.
are expressed as IC in Figs. 3 and 4, respectively. The cytosolic carboxylesterases exhibited markedly greater sensitivity to organotin inhibition in both species, and TPT, in particular, failed to a!ect the microsomal hydrolytic activity appreciably even at very high concentrations. In line with the inhibitory pattern of total hydrolytic activity of liver homogenate, DBT was the most e!ective inhibitor of microsomal and cytosolic carboxylesterases examined separately in both species. Preincubation and kinetic studies were performed to understand the nature of inhibition of "sh liver carboxylesterases by organotin compounds. No signi"cant change in the inhibition of enzyme activity was found when the compounds were preincubated with microsomal and cytosolic fractions for 1, 5, 10, 15, and 20 min prior to addition of the substrate, indicating the reversible nature of enzyme inhibition. Determination of the inhibition kinetics of S. canaliculatus liver carboxylesterases with the double-reciprocal plot of Lineweaver and Burk (1934) revealed the noncompetitive and competitive nature of inhibition of
TABLE 1 Carboxylesterase Activity in Liver of Marine Fish?
microsomal (Fig. 5) and cytosolic (Fig. 6) carboxylesterases, respectively by organotin compounds. DISCUSSION
The present study demonstrates that Siganus canaliculatus is remarkably more e$cient than Acanthopagrus latus in the hydrolytic detoxication of xenobiotics by hepatic microsomal and cytosolic carboxylesterases. This may be attributed to considerably greater < and lower
K values of hepatic microsomal and cytosolic carboxylesK terases of S. canaliculatus as compared with those of A. latus. Marked species- and substrate-related di!erences in the hydrolysis of chemicals by microsomal and cytosolic hydrolases of liver have been demonstrated in freshwater "sh, guppy, carp, goldenorfe, zebra"sh, and rainbow trout (Soldano et al., 1992). It was further observed that the cytosolic esterases selectively hydrolyze arylester (phenylacetate) and phosphomonoester (p-nitrophenylphosphate) linkages but are devoid of any detectable activity toward an amide
TABLE 2 Kinetic Constants for Carboxylesterase Activity in Fish Liver Fractions?
Enzyme activity (lmol/min/mg protein) Microsomes
Liver fraction
Siganus canaliculatus
Acanthopagrus latus
Homogenate Microsomes Cytosol
2.61$0.10 1.95$0.12 4.76$0.19
0.172$0.014 0.201$0.015 0.222$0.008
Fish species
?Values represent means$SE of seven Siganus canaliculatus and six Acanthopagrus latus investigated separately.
K K
Siganus canaliculatus 1.18$0.05 Acanthopagrus latus 2.43$0.29
Cytosol
<
K K
<
2.83$0.27 0.48$0.04
0.32$0.03 3.90$0.23
5.22$0.27 0.67$0.06
?Values represent means$SE of K (mM) and < (lmol/min/mg protein) K
constants obtained from four separate "sh preparations.
INHIBITION OF FISH CARBOXYLESTERASES BY ORGANOTINS
261
FIG. 2. Inhibition of carboxylesterase activity in "sh liver homogenate by organotin compounds. Values are averages of "ve or six individual measurements. Values indicated as 2000 represent IC '2000 lM.
(acetanilide) or aliphatic ester (ethyl butyrate), whereas microsomal hydrolases are capable of metabolizing all the substrates. The speci"c activity of microsomal carboxylesterases of S. canaliculatus liver is comparable to that of hepatic microsomal hydrolases catalyzing phenyl acetate metabolism in freshwater "shes (0.5}4.5) lmol/min/mg protein) (Soldano et al., 1992) and rat (1.5 lmol/min/mg protein) reported earlier (Kaur et al., 1991). However, at variance with the 5}10 times lower levels of hepatic cytosolic carboxylesterases compared with microsomal forms reported in freshwater "shes (Soldano et al., 1992) and rat (Khanna et al., 1992), cytosolic enzymes seem to contribute considerably to the hydrolytic detoxication of xenobiotics in marine "sh liver. Species-related di!erences
in glutathione S-transferase-catalyzed xenobiotic detoxication in hepatic and extrahepatic tissues of S. canaliculatus and other marine "sh inhabiting the Arabian Gulf have been reported earlier (Al-Ghais and Ali, 1995, 1999; AlGhais, 1997). The e!ectiveness of organotins as inhibitors of hepatic carboxylesterase activity was found to be dependent on the inhibitor structure, isozymic forms, and "sh species investigated in the present study. The hydrolytic metabolism of xenobiotics by cytosolic carboxylesterases in S. canaliculatus was most susceptible to inhibition by organotins, TBT, TPT, and DBT, and, therefore, has potential to serve as molecular biomarker of orgnotin pollution. Interestingly, the di!erence in the inhibitory responses of microsomal and
FIG. 3. Di!erential inhibition of microsomal and cytosolic carboxylesterases by organotins in Siganus canaliculatus liver. Values are averages of "ve or six individual determinations. Values indicated as 2000 represent IC '2000 lM.
262
AL-GHAIS, AHMAD, AND ALI
FIG. 4. Di!erential inhibition of microsomal and cytosolic carboxylesterases by organotins in Acanthopagrus latus liver. Values are averages of "ve or six individual determinations. Values indicated as 2000 represent IC '2000 lM.
cytosolic carboxylesterases was further re#ected in their nature of inhibiton, i.e., noncompetitive and competitive, respectively. In line with these observations a recent study has reported strong inhibition of glutathione S-transferasemediated xenobiotic detoxication in liver and kidney of S. canaliculatus and Sparus sarba by TBT, TPT, and DBT at micromolar concentrations in vitro and markedly greater sensitivity of the former species to organotin inhibition. Among the organotins tested, DBT was the most potent enzyme inhibitor in both species. Greater inhibitory e!ectiveness of DBT compared with TBT and TPT is contrary to the general structure}activity and physical property relationships reported for inhibition of glutathione S-transfer-
ase activity in "sh (Al-Ghais and Ali, 1999) and rat (Henry and Byington, 1976), induction of mitochondrial swelling and hemolysis (Henry and Byington, 1976), and toxicity caused by organotin compounds (WHO, 1990; Morcillo et al., 1997). These studies on the biochemical mechanism of action and tissue distribution of organotins have revealed high a$nity of these compounds toward amino acids, peptides, and proteins having }SH and "NH groups rather than lipids (Henry and Byington, 1976; Morcillo et al., 1997; Kannan et al., 1997). Though the explanation for distinct patterns of "sh liver carboxylesterase inhibition by dialkyl and trialkyl tins is not known at present, it may be attributed to the possible additional role of the carbohydrate
FIG. 5. Lineweaver}Burk plot indicating noncompetitive inhibition of hepatic microsomal carboxylesterases by organotins in Siganus canaliculatus. Values are averages of three separate determinations.
INHIBITION OF FISH CARBOXYLESTERASES BY ORGANOTINS
263
FIG. 6. Lineweaver}Burk plot indicating competitive inhibition of hepatic cytosolic carboxylesterases by organotin compounds in Siganus canaliculatus. Values are averages of three separate determinations.
moiety of the glycoprotein molecule of carboxylesterases and/or serine or other amino acids constituting the active site in organotin}enzyme interaction (Hosokawa et al., 1990).
Mohammed Al-Bowardi, Managing Director, for advice and encouragement and Dr. Walter H. Pearson for helpful discussions and kind cooperation.
REFERENCES CONCLUSION
It may be concluded from the current study that both microsomal and cytosolic carboxylesterases are responsible for the hydrolytic detoxication of aquatic chemical pollutants in the liver of S. canaliculatus and A. latus. A markedly greater rate of xenobiotic detoxication in S. canaliculatus indicates that this species may be at an advantage when exposed to deleterious environmental chemicals. Organotin compounds, TBT, TPT, and DBT, were found to inhibit hydrolytic xenobiotic metabolism in both species; however, there were marked species-, isozymic form-, and orgnotin structure-related di!erences in the magnitude of inhibition. Cytosolic carboxylesterases of S. canaliculatus liver were most susceptible to organotin inhibition and, therefore, may serve as potential molecular biomarkers of organotin pollution. These results further suggest that organotins, when present in a mixture of chemical pollutants, may enhance the toxicity of environmental chemicals undergoing hydrolytic detoxication. ACKNOWLEDGMENTS The authors are grateful to H. H. Sheikh Khalifa bin Zayed Al Nahyan, the Crown Prince of Abu Dhabi and President of ERWDA, and H. H. Sheikh Hamdan bin Zayed Al Nahyan, Chairman of the Board, for their generous support of this research program. Thanks are due Mr.
Al-Ghais, S. M. (1997). Species variation and some properties of renal glutathione S-trnsferase of "sh from Arabian Gulf. Bull. Environ. Contam. ¹oxicol. 59, 976}983. Al-Ghais, S. M., and Ali, B. (1995). Xenobiotic metabolism by glutathione S-transferase in gill of "sh from Arabian Gulf. Bull. Environ. Contam. ¹oxicol. 55, 201}208. Al-Ghais, S. M., and Ali, B. (1999). Inhibition of glutathione S-transferase catalyzed xenobiotic detoxication by organotin compounds in tropical marine "sh tissues. Bull. Environ. Contam. ¹oxicol. 62, 207}213. Ali, B., and Kaur, S. (1983). Mammalian tissue acetylsalicylic acid esterase(s): Identi"cation, distribution and discrimination from other esterases. J. Pharmacol. Exp. ¹her. 226, 589}594. Anderson, R. S., Durst, H. D., and Landis, W. G. (1988). Orgno#uorophosphate-hydrolyzing activity in an estuarine clam, Rangia cuneata. Comp. Biochem. Physiol. C 91, 575}578. Barron, M. G., Schultz, I. R., and Hayton, W. L. (1989). Presystemic branchial metabolism limits di-2-ethylhexyl phthalate accumulation in "sh. ¹oxicol. Appl. Pharmacol. 98, 49}57. Cashman, J. R., Perotti, B. Y. T., Berkman, C. E., and Lin, J. (1996). Pharmacokinetics and molecular detoxication. Environ. Health Perspect. 104, 23}40. Fent, K., and Bucheli, T. D. (1994). Inhibition of hepatic microsomal monooxygenase system by organotins in vitro in freshwater "sh. Aquat. ¹oxicol. 28, 107}126. Fent, K., and Stegeman, J. J. (1993). E!ects of tributyltin in vivo on hepatic cytochrome P450 forms in marine "sh. Aquat. ¹oxicol. 24, 219}240. Henry, R. A., and Byington, K. H. (1976). Inhibition of glutathione Saryltransferase from rat liver by organogermanium, lead, and tin compounds. Biochem. Pharmacol. 25, 2291}2295.
264
AL-GHAIS, AHMAD, AND ALI
Heymann, E., Hoppe, W., Krusselmann, A., and Tschoetschel, C. (1993). Organophosphate sensitive and insensitive carboxylesterases in human skin. Chem. Biol. Interact. 87, 217}226. Hosokawa, M., Maki, T., and Satoh, T. (1988). Di!erences in the induction of carboxylesterase isozymes in rat liver microsomes by xenobiotics. Biochem. Pharmacol. 37, 2708}2711. Hosokawa, M., Maki, T., and Satoh, T. (1990). Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans. Arch. Biochem. Biophys. 277, 219}227. Huang, T. L., Obih, P. O., Jaiswal, R., Hartley, W. R., and Thiyagarajah, A. (1997). Evaluation of liver and brain esterases in the spotted gar "sh (¸episosteus oculatus) as biomarkers of e!ect in the lower Mississippi River basin. Bull. Environ. Contam. ¹oxicol. 58, 688}695. Kannan, K., Senthilkumar, K., Loganathan, B. G., Takahashi, S., Odell, D. K., and Tanabe, S. (1997). Elevated accumulation of tributyltin and its breakdown products in bottlenose dolphins (¹ursiops truncatus) found stranded along the U.S. Atlantic and Gulf coasts. Environ. Sci. ¹echnol. 31, 296}301. Kaur, S., and Ali, B. (1983a). The e!ects of phenobarbital, 3-methylcholanthrene and benzo(a)pyrene on the hydrolysis of xenobiotics in the rat. Biochem. Pharmacol. 32, 3479}3480. Kaur, S., and Ali, B. (1983b). Selective induction of xenobiotic metabolizing esterases/amidases of liver by methaqualone consumption. ¹oxicol. Appl. Pharmacol. 70, 156}159. Kaur, S., Khanna, P., Parmar, S. S., and Ali, B. (1991). Selective stimulation of carboxylesterases metabolizing charged steroid esters by hydrocortisone. Biochem. Pharmacol. 41, 476}478. Khanna, P., Kaur, S., Sanwal, G. G., and Ali, B. (1992). Characteristics of a cytosolic arylacylamidase metabolizing thiacetazone. J. Pharmacol. Exp. ¹her. 262, 1225}1231. Lineweaver, H., and Burk, D. (1934). The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658}666. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265}275. Morcillo, Y., Borghi, V., and Porte, C. (1997). Survey of organotin compounds in the western Mediterranean using molluscs and "sh as sentinel organisms. Arch. Environ. Contam. ¹oxicol. 32, 198}203.
Ozretic, B., and Krajnovic-Ozretic, M. (1992). Esterase heterogeneity in mussel Mytilus galloprovincialis: E!ects of organophosphate and carbamate pesticides in vitro. Comp. Biochem. Physiol. C 103, 221}225. Payne, J. F. (1982). Metabolism of complex mixtures of oil spill surfactant compounds by a representative teleost (Salmo gairdneri), crustacean (Cancer irroratus), and mollusc (Chlamys islandicus). Bull. Environ. Contam. ¹oxicol. 28, 277}280. Salamastrakis, S. S., and Haritos, A. A. (1988). Physicochemical characterization and tissue distribution of multiple molecular forms of "sh (¹rachurus trachurus) esterases. Comp. Biochem. Physiol. B 91, 741}750. Siddiqui, A., Srivastava, S. P., and Ali, B. (1990). E!ect of mancozeb on hydrolytic metabolism of xenobiotics. Res. Commun. Chem. Pathol. Pharmacol. 70, 249}252. Soldano, S., Gramenzi, F., Cirianni, M., and Vittozzi, L. (1992). Xenobioticmetabolizing enzyme systems in test "sh. IV. Comparative studies of liver microsomal and cytosolic hydrolases. Comp. Biochem. Physiol. C 101, 117}123. Straus, D. L., and Chambers, J. E. (1995). Inhibition of acetylcholinesterase and aliesterases of "ngerling channel cat"sh by chlorpyrifos, parathion, and S, S, S-tributylphosphorotrithioate (DEF). Aquat. ¹oxicol. 33, 311}324. Swall, L. M., and Tjeerdema, R. S. (1991). Tissue distribution and temperature dependence of xenobiotic hydrolysis in the dungeness crab (Cancer magister). Aquat. ¹oxicol. 20, 1}12. Takahashi, S., Tanabe, S., and Kubodera, T. (1997). Butyltin residues in deep-sea organisms collected from Suruga Bay, Japan. Environ. Sci. ¹echnol. 31, 3103}3109. Varma, A. K., and Frankel, J. S. (1980). A comparison of tissue esterases in the genus Barbus by vertical gel electrophoresis. Comp. Biochem. Physiol. B 65, 267}273. Wallace, K. B., and Dargan, J. E. (1987). Intrinsic metabolic clearance of parathion and paraoxon by livers from "sh and rodents. ¹oxicol. Appl. Pharmacol. 90, 235}242. WHO (World Health Organization) (1990). ¹ributyltin Compounds. Environmental Health Criteria 116, p. 273. WHO, Geneva. Wo!ord, H. W., Wilsey, C. D., Ne!, G. S., Giam, C. S., and Ne!, J. M. (1981). Bioaccumulation and metabolism of phthalate esters by oysters, brown shrimp and sheepshead minnows. Ecotoxicol. Environ. Saf. 5, 202}210.
Differential Inhibition of Xenobiotic-Metabolizing Carboxylesterases by Organotins in Marine Fish Saif M. Al-Ghais, Shakeel Ahmad, and Basheer Ali Marine Environment Research Center, Environmental Research and Wildlife Development Agency, P. O. Box 45553, Abu Dhabi, United Arab Emirates Received July 9, 1999
The hydrolytic metabolism of xenobiotics in the liver of two tropical marine 5sh, Siganus canaliculatus and Acanthopagrus latus, was found to be catalyzed by both microsomal and cytosolic carboxylesterases; the latter forms were more active than the former. Remarkably greater e7ciency of S. canaliculatus for p-nitrophenylacetate hydrolysis was attributed to manyfold higher Vmax and lower Km values of hepatic microsomal and cytosolic carboxylesterases of S. canaliculatus as compared with those of A. latus. Comparative characterization of the in vitro responses of hepatic microsomal and cytosolic carboxylesterases to the organotin group of marine pollutants=tributyltin (TBT), triphenyltin (TPT), and dibutyltin (DBT), a relatively persistent metabolite of TBT=revealed species-, isozymic form, and organotin structure-related di4erences in the hydrolytic detoxication. In general, carboxylesterases of S. canaliculatus exhibited severalfold greater susceptibility to organotin inhibition and DBT was the most potent inhibitor (IC50 in micromolar range). Notably, the IC50 of SnCl2 , a metal present in all the compounds studied, was higher than 2 mM. Cytosolic forms in both species were more sensitive to organotin inhibition than microsomal counterparts. In line with these di4erences the nature of inhibition of cytosolic and microsomal carboxylesterases by organotins was competitive and noncompetitive, respectively. These results suggest that organotins may aggravate the toxicity of other environmental contaminants in 5sh and other aquatic organisms. Moreover, highly sensitive cytosolic carboxylesterases of S. canaliculatus liver may serve as molecular biomarkers of organotin pollution. 2000 Academic Press
INTRODUCTION
There has been an increasing awareness in the recent past that alkyl and aryl tin compounds are of environmental concern owing to their ecotoxicological impact, bioaccumulation potential, prolonged persistence in deep-sea sediment, and direct introduction into the aquatic environment, particularly coastal areas, mainly by leaching from antifouling paints applied on ships, boats, aquaculture nets, and other marine installations and as a result of their
employment in agriculture as biocides and in industry as plastic stabilizers and catalysts and preservatives for wood, textile and paper (WHO, 1990; Morcillo et al., 1997; Kannan et al., 1997; Takahashi et al., 1997). Hydrolytic metabolism of xenobiotics by nonspeci"c carboxylesterases plays a major role in the detoxication of organophosphate pesticides (Wallace and Dargan, 1987; Anderson et al., 1988; Straus and Chambers, 1995), phathalate ester plasticizers (Wo!ord et al., 1981; Barron et al., 1989), oil spill dispersants (Payne, 1982; Swall and Tjeerdema, 1991), and other environmental chemical pollutants in "sh and other aquatic animals. The vast literature available on mammalian carboxylesterases has revealed that these hydrolases are localized in the microsomal and cytoplasmic fractions of hepatic and extrahepatic tissues as multiple isoenzymes (Ali and Kaur, 1983; Hosokawa et al., 1990; Khanna et al., 1992; Heymann et al., 1993; Cashman et al., 1996) and display broad substrate speci"city toward chemicals containing a carboxylester, amide, or thioester bond. The microsomal forms, which account for the bulk of mammalian liver carboxylesterase/amidase activity, are selectively induced by foreign chemicals such as polycyclic aromatic hydrocarbons and DDT (Kaur and Ali, 1983a; Hosokawa et al., 1988), drugs such as methaqualone (Kaur and Ali, 1983b), hormones (Kaur et al., 1991), and mancozeb, a fungicide (Siddiqui et al., 1990), and are inhibited by organophosphate and carbamate pesticides (Cashman et al., 1996). Characterization of xenobiotic-metabolizing esterases and their responses environmental chemicals in "sh and aquatic invertebrates has generally been con"ned to electrophoretic separation of their multiple molecular forms present in the postmitochondrial or cytosolic fractions of liver (Varma and Frankel, 1980; Salamastrakis and Haritos, 1988; Ozretic and Krajnovic-Ozretic, 1992; Soldano et al., 1992) and inhibition of in vitro hydrolytic activity of isolated isoenzymes (Ozretic and Krajnovic-Ozretic, 1992) and tissue homogenates (Straus and Chambers, 1995) by orgnophosphate and carbamate pesticides. Lately, "sh esterases have
258 0147-6513/00 $35.00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved.
INHIBITION OF FISH CARBOXYLESTERASES BY ORGANOTINS
been evaluated as biomarker of exposure to multiple pollutants present in the aquatic environment (Huang et al., 1997). Previous studies on the e!ects of organotin compounds on xenobiotic-metabolizing enzymes in "sh and their toxicological implications have found that tributyltin (TBT) and triphenyltin (TPT) inhibit cytochrome P450-dependent monooxygenases in liver (Fent and Bucheli, 1994; Fent and Stegeman, 1993) and glutathione S-transferase activity in liver and kidney of marine "sh (Al-Ghais and Ali, 1999). The purpose of this study was to make comparative assessment of the in vitro e!ects of TBT, TPT and DBT, which is also a major and relatively persistent metabolite of TBT in "sh (Morcillo et al., 1997; Takahashi et al., 1997), on hydrolytic detoxication of xenobiotics by carboxylesterases in the liver of Siganus canaliculatus and Acanthopagrus latus, two widely occurring commercially important "sh inhabiting the southern Arabian Gulf waters along the Abu Dhabi coast of United Arab Emirates. MATERIALS AND METHODS
Chemicals Bovine serum albumin, p-nitrophenyl acetate, potassium chloride, and tromethamine (Tris) were purchased from Sigma Chemical Company (St. Louis, MO). Tributyltin chloride (96%), triphenyltin chloride (95%), dibutyltin chloride (96%), and tin chloride were obtained from Aldrich Chemical Company (Milwaukee, USA. MI). Fish Samples (60}120 g) of S. canaliculatus (rabbit"sh) and A. latus (seabream) were captured with the help of a trap from the southern Arabian Gulf waters along the Abu Dhabi coast of United Arab Emirates. Tissue Preparation Liver homogenate was prepared in ice-cold 1.15% KCl bu!ered with 0.01 M Tris}HCl, pH 7.4, with the help of a Potter}Elvehjem homogenizer "tted with a Te#on pestle. Subcellular fractions of liver (nuclei, mitochondria, microsomes, and cytosol) were isolated by di!erential centrifugation of liver homogenate in an IEC-Centra-MP4R (USA) centrifuge and Beckman Optima L-70 (USA) ultracentrifuge by the procedure described earlier (Khanna et al., 1992). Analysis Procedure Carboxylesterase activity in tissue preparations was determined by monitoring the hydrolysis of p-nitrophenyl acetate, a nonspeci"c model substrate, at 412 nm spectrophotometrically (Kaur et al., 1991; Khanna et al., 1992).
259
Unless otherwise stated, the enzyme assay mixture (3 ml) consisting of 0.1 M Tris}HCl bu!er, pH 7.4, 2.5 mM pnitrophenyl acetate, and an appropriate amount of liver preparation as the enzyme source was incubated at 283C for 10 min. To investigate in vitro e!ects of organotin compounds and SnCl on carboxylesterase activity varying con centrations of the compounds dissolved in ethanol or an equal amount of ethanol alone in the control were preincubated with the enzyme prior to addition of substrate. Protein was determined by the procedure of Lowry et al. (1951) using bovine serum albumin as standard. RESULTS
Subcellular localization of carboxylesterase activity toward p-nitrophenyl acetate, a nonspeci"c model substrate, in the liver of S. canaliculatus and A. latus revealed recovery of more than 75% of the total hydrolytic activity of homogenate in the microsomal and cytosolic fractions, the latter being endowed with two-to fourfold higher activity than the former (Fig. 1). The constitutive level of hepatic carboxylesterases in S. canaliculatus as indicated by their speci"c activities in homogenate, microsomal, and cytosolic fractions was 10}20 times higher than that of A. latus (Table 1). Further characterization of carboxylesterase activity was carried out by studying the relationship between reaction velocity and substrate concentration. The maximum velocity (< ) of
substrate hydrolysis and Michaelis}Menten constant (K ) K were estimated by the double-reciprocal plot of Lineweaver and Burk (1934) and are presented in Table 2. The < of
microsomal and cytosolic carboxylesterases in S. canaliculatus, an indicator of total enzyme present, were 5.9and 7.8-fold higher, respectively, than the values for A. latus. However, the apparent K of microsomal and cytosolic K forms of enzyme, which is inversely proportional to the enzyme a$nity for substrate, in S. canaliculatus were 2.0 and 12.2 times lower than those of A. latus. The carboxylesterase activity of liver homogenate, which is an index of overall e$ciency of hydrolytic metabolism of xenobiotics, was inhibited by organotin compounds, TBT, TPT, and DBT, in vitro in a concentration-dependent manner in both species (Fig. 2). A comparison of the enzyme inhibitory potency of organotins in terms of IC values (the inhibitor concentration causing 50% loss of enzyme activity) revealed species- and inhibitor-related variations. In general, carboxylesterases of S. canaliculatus were many times more susceptible to organotin inhibition and DBT was found to be the most potent inhibitor in both species. Interestingly, the IC values of SnCl , a metal present in all the compounds studied, were higher than 2 mM, the highest concentration examined. The responses of hepatic microsomal and cytosolic carboxylesterases to organotins in S. canaliculatus and A. latus
260
AL-GHAIS, AHMAD, AND ALI
FIG. 1. Subcellular distribution of hepatic carboxylesterase activity in nuclei, mitochondria, microsomes, and cytosol from Siganus canaliculatus and Acanthopagrus latus. Values are averages of three separate determinations.
are expressed as IC in Figs. 3 and 4, respectively. The cytosolic carboxylesterases exhibited markedly greater sensitivity to organotin inhibition in both species, and TPT, in particular, failed to a!ect the microsomal hydrolytic activity appreciably even at very high concentrations. In line with the inhibitory pattern of total hydrolytic activity of liver homogenate, DBT was the most e!ective inhibitor of microsomal and cytosolic carboxylesterases examined separately in both species. Preincubation and kinetic studies were performed to understand the nature of inhibition of "sh liver carboxylesterases by organotin compounds. No signi"cant change in the inhibition of enzyme activity was found when the compounds were preincubated with microsomal and cytosolic fractions for 1, 5, 10, 15, and 20 min prior to addition of the substrate, indicating the reversible nature of enzyme inhibition. Determination of the inhibition kinetics of S. canaliculatus liver carboxylesterases with the double-reciprocal plot of Lineweaver and Burk (1934) revealed the noncompetitive and competitive nature of inhibition of
TABLE 1 Carboxylesterase Activity in Liver of Marine Fish?
microsomal (Fig. 5) and cytosolic (Fig. 6) carboxylesterases, respectively by organotin compounds. DISCUSSION
The present study demonstrates that Siganus canaliculatus is remarkably more e$cient than Acanthopagrus latus in the hydrolytic detoxication of xenobiotics by hepatic microsomal and cytosolic carboxylesterases. This may be attributed to considerably greater < and lower
K values of hepatic microsomal and cytosolic carboxylesK terases of S. canaliculatus as compared with those of A. latus. Marked species- and substrate-related di!erences in the hydrolysis of chemicals by microsomal and cytosolic hydrolases of liver have been demonstrated in freshwater "sh, guppy, carp, goldenorfe, zebra"sh, and rainbow trout (Soldano et al., 1992). It was further observed that the cytosolic esterases selectively hydrolyze arylester (phenylacetate) and phosphomonoester (p-nitrophenylphosphate) linkages but are devoid of any detectable activity toward an amide
TABLE 2 Kinetic Constants for Carboxylesterase Activity in Fish Liver Fractions?
Enzyme activity (lmol/min/mg protein) Microsomes
Liver fraction
Siganus canaliculatus
Acanthopagrus latus
Homogenate Microsomes Cytosol
2.61$0.10 1.95$0.12 4.76$0.19
0.172$0.014 0.201$0.015 0.222$0.008
Fish species
?Values represent means$SE of seven Siganus canaliculatus and six Acanthopagrus latus investigated separately.
K K
Siganus canaliculatus 1.18$0.05 Acanthopagrus latus 2.43$0.29
Cytosol
<
K K
<
2.83$0.27 0.48$0.04
0.32$0.03 3.90$0.23
5.22$0.27 0.67$0.06
?Values represent means$SE of K (mM) and < (lmol/min/mg protein) K
constants obtained from four separate "sh preparations.
INHIBITION OF FISH CARBOXYLESTERASES BY ORGANOTINS
261
FIG. 2. Inhibition of carboxylesterase activity in "sh liver homogenate by organotin compounds. Values are averages of "ve or six individual measurements. Values indicated as 2000 represent IC '2000 lM.
(acetanilide) or aliphatic ester (ethyl butyrate), whereas microsomal hydrolases are capable of metabolizing all the substrates. The speci"c activity of microsomal carboxylesterases of S. canaliculatus liver is comparable to that of hepatic microsomal hydrolases catalyzing phenyl acetate metabolism in freshwater "shes (0.5}4.5) lmol/min/mg protein) (Soldano et al., 1992) and rat (1.5 lmol/min/mg protein) reported earlier (Kaur et al., 1991). However, at variance with the 5}10 times lower levels of hepatic cytosolic carboxylesterases compared with microsomal forms reported in freshwater "shes (Soldano et al., 1992) and rat (Khanna et al., 1992), cytosolic enzymes seem to contribute considerably to the hydrolytic detoxication of xenobiotics in marine "sh liver. Species-related di!erences
in glutathione S-transferase-catalyzed xenobiotic detoxication in hepatic and extrahepatic tissues of S. canaliculatus and other marine "sh inhabiting the Arabian Gulf have been reported earlier (Al-Ghais and Ali, 1995, 1999; AlGhais, 1997). The e!ectiveness of organotins as inhibitors of hepatic carboxylesterase activity was found to be dependent on the inhibitor structure, isozymic forms, and "sh species investigated in the present study. The hydrolytic metabolism of xenobiotics by cytosolic carboxylesterases in S. canaliculatus was most susceptible to inhibition by organotins, TBT, TPT, and DBT, and, therefore, has potential to serve as molecular biomarker of orgnotin pollution. Interestingly, the di!erence in the inhibitory responses of microsomal and
FIG. 3. Di!erential inhibition of microsomal and cytosolic carboxylesterases by organotins in Siganus canaliculatus liver. Values are averages of "ve or six individual determinations. Values indicated as 2000 represent IC '2000 lM.
262
AL-GHAIS, AHMAD, AND ALI
FIG. 4. Di!erential inhibition of microsomal and cytosolic carboxylesterases by organotins in Acanthopagrus latus liver. Values are averages of "ve or six individual determinations. Values indicated as 2000 represent IC '2000 lM.
cytosolic carboxylesterases was further re#ected in their nature of inhibiton, i.e., noncompetitive and competitive, respectively. In line with these observations a recent study has reported strong inhibition of glutathione S-transferasemediated xenobiotic detoxication in liver and kidney of S. canaliculatus and Sparus sarba by TBT, TPT, and DBT at micromolar concentrations in vitro and markedly greater sensitivity of the former species to organotin inhibition. Among the organotins tested, DBT was the most potent enzyme inhibitor in both species. Greater inhibitory e!ectiveness of DBT compared with TBT and TPT is contrary to the general structure}activity and physical property relationships reported for inhibition of glutathione S-transfer-
ase activity in "sh (Al-Ghais and Ali, 1999) and rat (Henry and Byington, 1976), induction of mitochondrial swelling and hemolysis (Henry and Byington, 1976), and toxicity caused by organotin compounds (WHO, 1990; Morcillo et al., 1997). These studies on the biochemical mechanism of action and tissue distribution of organotins have revealed high a$nity of these compounds toward amino acids, peptides, and proteins having }SH and "NH groups rather than lipids (Henry and Byington, 1976; Morcillo et al., 1997; Kannan et al., 1997). Though the explanation for distinct patterns of "sh liver carboxylesterase inhibition by dialkyl and trialkyl tins is not known at present, it may be attributed to the possible additional role of the carbohydrate
FIG. 5. Lineweaver}Burk plot indicating noncompetitive inhibition of hepatic microsomal carboxylesterases by organotins in Siganus canaliculatus. Values are averages of three separate determinations.
INHIBITION OF FISH CARBOXYLESTERASES BY ORGANOTINS
263
FIG. 6. Lineweaver}Burk plot indicating competitive inhibition of hepatic cytosolic carboxylesterases by organotin compounds in Siganus canaliculatus. Values are averages of three separate determinations.
moiety of the glycoprotein molecule of carboxylesterases and/or serine or other amino acids constituting the active site in organotin}enzyme interaction (Hosokawa et al., 1990).
Mohammed Al-Bowardi, Managing Director, for advice and encouragement and Dr. Walter H. Pearson for helpful discussions and kind cooperation.
REFERENCES CONCLUSION
It may be concluded from the current study that both microsomal and cytosolic carboxylesterases are responsible for the hydrolytic detoxication of aquatic chemical pollutants in the liver of S. canaliculatus and A. latus. A markedly greater rate of xenobiotic detoxication in S. canaliculatus indicates that this species may be at an advantage when exposed to deleterious environmental chemicals. Organotin compounds, TBT, TPT, and DBT, were found to inhibit hydrolytic xenobiotic metabolism in both species; however, there were marked species-, isozymic form-, and orgnotin structure-related di!erences in the magnitude of inhibition. Cytosolic carboxylesterases of S. canaliculatus liver were most susceptible to organotin inhibition and, therefore, may serve as potential molecular biomarkers of organotin pollution. These results further suggest that organotins, when present in a mixture of chemical pollutants, may enhance the toxicity of environmental chemicals undergoing hydrolytic detoxication. ACKNOWLEDGMENTS The authors are grateful to H. H. Sheikh Khalifa bin Zayed Al Nahyan, the Crown Prince of Abu Dhabi and President of ERWDA, and H. H. Sheikh Hamdan bin Zayed Al Nahyan, Chairman of the Board, for their generous support of this research program. Thanks are due Mr.
Al-Ghais, S. M. (1997). Species variation and some properties of renal glutathione S-trnsferase of "sh from Arabian Gulf. Bull. Environ. Contam. ¹oxicol. 59, 976}983. Al-Ghais, S. M., and Ali, B. (1995). Xenobiotic metabolism by glutathione S-transferase in gill of "sh from Arabian Gulf. Bull. Environ. Contam. ¹oxicol. 55, 201}208. Al-Ghais, S. M., and Ali, B. (1999). Inhibition of glutathione S-transferase catalyzed xenobiotic detoxication by organotin compounds in tropical marine "sh tissues. Bull. Environ. Contam. ¹oxicol. 62, 207}213. Ali, B., and Kaur, S. (1983). Mammalian tissue acetylsalicylic acid esterase(s): Identi"cation, distribution and discrimination from other esterases. J. Pharmacol. Exp. ¹her. 226, 589}594. Anderson, R. S., Durst, H. D., and Landis, W. G. (1988). Orgno#uorophosphate-hydrolyzing activity in an estuarine clam, Rangia cuneata. Comp. Biochem. Physiol. C 91, 575}578. Barron, M. G., Schultz, I. R., and Hayton, W. L. (1989). Presystemic branchial metabolism limits di-2-ethylhexyl phthalate accumulation in "sh. ¹oxicol. Appl. Pharmacol. 98, 49}57. Cashman, J. R., Perotti, B. Y. T., Berkman, C. E., and Lin, J. (1996). Pharmacokinetics and molecular detoxication. Environ. Health Perspect. 104, 23}40. Fent, K., and Bucheli, T. D. (1994). Inhibition of hepatic microsomal monooxygenase system by organotins in vitro in freshwater "sh. Aquat. ¹oxicol. 28, 107}126. Fent, K., and Stegeman, J. J. (1993). E!ects of tributyltin in vivo on hepatic cytochrome P450 forms in marine "sh. Aquat. ¹oxicol. 24, 219}240. Henry, R. A., and Byington, K. H. (1976). Inhibition of glutathione Saryltransferase from rat liver by organogermanium, lead, and tin compounds. Biochem. Pharmacol. 25, 2291}2295.
264
AL-GHAIS, AHMAD, AND ALI
Heymann, E., Hoppe, W., Krusselmann, A., and Tschoetschel, C. (1993). Organophosphate sensitive and insensitive carboxylesterases in human skin. Chem. Biol. Interact. 87, 217}226. Hosokawa, M., Maki, T., and Satoh, T. (1988). Di!erences in the induction of carboxylesterase isozymes in rat liver microsomes by xenobiotics. Biochem. Pharmacol. 37, 2708}2711. Hosokawa, M., Maki, T., and Satoh, T. (1990). Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans. Arch. Biochem. Biophys. 277, 219}227. Huang, T. L., Obih, P. O., Jaiswal, R., Hartley, W. R., and Thiyagarajah, A. (1997). Evaluation of liver and brain esterases in the spotted gar "sh (¸episosteus oculatus) as biomarkers of e!ect in the lower Mississippi River basin. Bull. Environ. Contam. ¹oxicol. 58, 688}695. Kannan, K., Senthilkumar, K., Loganathan, B. G., Takahashi, S., Odell, D. K., and Tanabe, S. (1997). Elevated accumulation of tributyltin and its breakdown products in bottlenose dolphins (¹ursiops truncatus) found stranded along the U.S. Atlantic and Gulf coasts. Environ. Sci. ¹echnol. 31, 296}301. Kaur, S., and Ali, B. (1983a). The e!ects of phenobarbital, 3-methylcholanthrene and benzo(a)pyrene on the hydrolysis of xenobiotics in the rat. Biochem. Pharmacol. 32, 3479}3480. Kaur, S., and Ali, B. (1983b). Selective induction of xenobiotic metabolizing esterases/amidases of liver by methaqualone consumption. ¹oxicol. Appl. Pharmacol. 70, 156}159. Kaur, S., Khanna, P., Parmar, S. S., and Ali, B. (1991). Selective stimulation of carboxylesterases metabolizing charged steroid esters by hydrocortisone. Biochem. Pharmacol. 41, 476}478. Khanna, P., Kaur, S., Sanwal, G. G., and Ali, B. (1992). Characteristics of a cytosolic arylacylamidase metabolizing thiacetazone. J. Pharmacol. Exp. ¹her. 262, 1225}1231. Lineweaver, H., and Burk, D. (1934). The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658}666. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265}275. Morcillo, Y., Borghi, V., and Porte, C. (1997). Survey of organotin compounds in the western Mediterranean using molluscs and "sh as sentinel organisms. Arch. Environ. Contam. ¹oxicol. 32, 198}203.
Ozretic, B., and Krajnovic-Ozretic, M. (1992). Esterase heterogeneity in mussel Mytilus galloprovincialis: E!ects of organophosphate and carbamate pesticides in vitro. Comp. Biochem. Physiol. C 103, 221}225. Payne, J. F. (1982). Metabolism of complex mixtures of oil spill surfactant compounds by a representative teleost (Salmo gairdneri), crustacean (Cancer irroratus), and mollusc (Chlamys islandicus). Bull. Environ. Contam. ¹oxicol. 28, 277}280. Salamastrakis, S. S., and Haritos, A. A. (1988). Physicochemical characterization and tissue distribution of multiple molecular forms of "sh (¹rachurus trachurus) esterases. Comp. Biochem. Physiol. B 91, 741}750. Siddiqui, A., Srivastava, S. P., and Ali, B. (1990). E!ect of mancozeb on hydrolytic metabolism of xenobiotics. Res. Commun. Chem. Pathol. Pharmacol. 70, 249}252. Soldano, S., Gramenzi, F., Cirianni, M., and Vittozzi, L. (1992). Xenobioticmetabolizing enzyme systems in test "sh. IV. Comparative studies of liver microsomal and cytosolic hydrolases. Comp. Biochem. Physiol. C 101, 117}123. Straus, D. L., and Chambers, J. E. (1995). Inhibition of acetylcholinesterase and aliesterases of "ngerling channel cat"sh by chlorpyrifos, parathion, and S, S, S-tributylphosphorotrithioate (DEF). Aquat. ¹oxicol. 33, 311}324. Swall, L. M., and Tjeerdema, R. S. (1991). Tissue distribution and temperature dependence of xenobiotic hydrolysis in the dungeness crab (Cancer magister). Aquat. ¹oxicol. 20, 1}12. Takahashi, S., Tanabe, S., and Kubodera, T. (1997). Butyltin residues in deep-sea organisms collected from Suruga Bay, Japan. Environ. Sci. ¹echnol. 31, 3103}3109. Varma, A. K., and Frankel, J. S. (1980). A comparison of tissue esterases in the genus Barbus by vertical gel electrophoresis. Comp. Biochem. Physiol. B 65, 267}273. Wallace, K. B., and Dargan, J. E. (1987). Intrinsic metabolic clearance of parathion and paraoxon by livers from "sh and rodents. ¹oxicol. Appl. Pharmacol. 90, 235}242. WHO (World Health Organization) (1990). ¹ributyltin Compounds. Environmental Health Criteria 116, p. 273. WHO, Geneva. Wo!ord, H. W., Wilsey, C. D., Ne!, G. S., Giam, C. S., and Ne!, J. M. (1981). Bioaccumulation and metabolism of phthalate esters by oysters, brown shrimp and sheepshead minnows. Ecotoxicol. Environ. Saf. 5, 202}210.