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Distinct glutathione-dependent enzyme activities and a verapamil-sensitive binding of xenobiotics in a fresh-water mussel Anodonta cygnea
Vol. 164, No. 2, 1989 October
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
31, 1989
934-940
DISTINCT GLUTATHIONE-DEPENDENT ENZYME ACTIVITIES AND A VERAPAMIL-SENSITIVE BINDING OF XENOBIOTICS IN A FRESH-WATER MUSSEL ANODONTA CYGNEA
Branko Kurelec and Branka PivEeviC Ruder BoSkovid Institute, Center for Marine Research Zagreb, 41001 Zagreb, Croatia, Yugoslavia Received
September
19,
1989
SUMMARY: A fresh-water mussel Anodonra cygnet, an aquatic invertebrate resistant to pollution, possessesan inherent high potential to bind 2-acetylaminofluorene onto membrane vesicles. This binding is saturable and trypsin- and verapamil- sensitive. Simultaneously, this mussel reveals a relatively high inherent activity of glutathionedependent enzyme activities with a distinct spectrum of substrate affinities. Both these activities are similar to the elements of the molecular mechanism involved in the acquired multi-drug resistance phenomenon described in tumour cell-lines. The recognition that in organisms exposed to polluted waters a multi-xenobiotic resistance mechanism may be involved is essential for understanding both the biological impact of pollution and the development of methods for rational risk assessment in regulatory policy. 0 1989Academic Pn?SS,Inc.
There are aquatic invertebrates that can survive and reproduce in heavily polluted waters. Similar resistance to pollution was found in some marine bottom invertebrates where the concentration of pollutants in tissues and body fluids was lower than in surrounding recent sediments (1). Abscence of accumulation, caused by decreased transport of a xenobiotic to the site of action and/or by enhanced metabolic attack on it within the organism, has long been recognized as a common mechanism in the development of resistance to xenobiotics (2). Aquatic organisms seem to be simultaneously resistant to multiple toxic xenobiotics, since aquatic pollution is typically caused by a mixture of chemicals. This “multi-xenobiotic resistance” and lack of accumulation are characteristics that are analogous to the phenomenon of “multi-drug resistance” found in tumour cells that become refractory to treatment with a variety of chemotherapeutic agents (3). There, a decrease in drug accumulation is associated with the increased production of a membrane glycoprotein termed P-170 (4) which binds a drug and facilitates its efflux by an energy-dependent process (5). mdr-1, the gene coding for P-170, has been cloned (6) and its overexpression was found to be proportional to the degree of resistance in resistant cell lines (7). However, MDR is also associated with the decreased expression of the Phase I metabolizing enzyme cytochrome Abbreviations: GSH, glutathione; GST, glutathione transferase; CDNB, l-chloro-2,4dinitro-benzene; AAF, 2-acetylaminofluorene; MDR, multi-drug resistance. 0006-291x/89 Copyright All rights
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1989 by Academic Press, of reproduction in any form
Inc. reserved.
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P-450 monooxygenase (8), and increased expression of several Phase II conjugating enzymes, including both a many-fold increase in the activity of a distinct, resistancespecific isoenzyme of anionic glutathione transferase with a high level of intrinsic peroxidase and a high affinity to ethacrinic acid (9), and the alterations glutathione redox cycle (10).
in the
The focus of the present study was to determine whether fresh-water mussel Anodontcl cygnea. an organism that can survive and reproduce in heavily polluted waters, possesseselements of the molecular mechanism similar to that found to be involved in the drug-resistant tumour cell-lines. In order to obtain preliminary insight into the possible presence of glycoprotein P-170-mediated mechanism, we measured it? vitro the potential of isolated mussel membrane vesicles to bind radiolabelled 2acetylaminofluorene. a carcinogen that also induces an increase in mdr-I gene expression (11. 12), in the presence or absence of verapamil, a molecule that was found to bind specifically to the active sites of P-170. thereby inhibiting binding of other xenobiotics (5). The presence of resistance-specific glutathione-dependent enzymes in mussel organs was studied by substrate affinity characteristics of GSH-transferases with 1-chloro-2,4-dinitrobenzene
and ethacrynic acid, and of GSH-peroxidases with cumene
peroxide and hydroperoxide. Our results show that the mussel Atzodotztn cypzect possessesan inherent verapamil-sensitive potential to bind 2-acetylaminofluorene.
and a relatively high
inherent glutathione-dependent enzyme activities with a distinct. resistance-specific spectrum of substrate affinities. MATERIALS
AND METHODS
Specimens of Anodonta cygnea, 14-18 cm, 150-2 10 g, were collected during May-July 1989 from a brook suplying waters for carp-farm Draganidi. near Zagreb. A group of mussels was exposed to a flow of polluted (defined in 13) Sava River waters or to a flow of tap-water saturated with a Diesel-2 oil as described (14). Membrane vesicles from digestive gland-, gills- and mantle- tissues were prepared according to the nethod of Riqrdan and Liy4 (15). _ Total and nonspecific binding of 2-acetylamino(9C)ammofluorene ( C-AAF; 55.3 mCi/mmol, Amersham, England) was measured as described by Cornwell et al. (5), except that aliquots of ves@ preparation containing 1OOpg protein were incubated in the presence of 50 nM of C-AAF and that filtration was done on glass fiber filters (Whatman GFK) pretreated with 10% human serum. GSH peroxidase activity was assayed in postmitochonrial fraction of 20% tissue homogenates using hydrogen- or cumene- peroxides as substrates (16). GST was assayed using 1 mM I-chloro-2,4-dinitro-benzene and 1 mM glutathione, or, 0.2 mM ethacrynic acid and 0.25 mM glutathione. respectively. according to the procedure of Habig and Jacoby (17). Benzo(a)pyrene monooxygenase activity was determined according Nebert and Gelboin (18) and proteins were determined by the method of Lowry et al. (19). RESULTS AND DISCUSSION Measurements of the association of 14 C-AAF with digestive gland vesicles prepared from mussels. collected at a pristine environment, revealed that vesicles accumulate a considerable amount of 14C AAF (Fig. 1A). The effect of premcubatlon of vesicles with verapamil at 10yglml was profound: it reduced the AAF binding by 80% 935
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gland
FIGURE 1. The effect of verapamil treatment oo 14C-AAF binding in mussel membrane vesicles.A, the binding in digestive gland- (gland), gills-, and mantlevesicles derived from mussels collected at pristine site. B, the binding in digestive gland
vesiclesfrom musselsexposedfor 14 days to a flow of SavaRiver wateftj (Sava),or for 4 days to a water saturated with Diesel-2 oil (Diesel). Total binding of C-AAF (SO nM) on vesicles (1OOpg protein/assay) was measured after 20-min incubation at 23’C in the absence(-) or presence(+) of IO&ml verapamil (VP). Nonspecificbinding (NB) was assessed by including 1OOpM unlabelled AAF in the reaction mixture. Data are expressed as the means f SD from determinations in 4 specimens.
compared to untreated vesicles. Similar dramatic effect of verapamil was observed with binding of vinblastine on vesicles derived from a multi-drug resistant KB-C4 cell line (5). There, association of vinblastine with vesicles was reduced by verapamil to the level bound by vesicles derived from a drug-sensitive KB-3-1 cells. In the absence of a corresponding “xenobiotic-sensitive”
reference mussel, we adopted a level of binding
from a verapamil-treated mussel as a “normal” level of binding in a hypothetical “sensitive” mussel. Similarly to the behaviour of the vinblastine binding in drugresistant cells, binding of l4 C-AAF to mussel vesicles was reduced by 48% after 20 min incubation of vesicles with 10 ng of trypsin/mg of vesicle protein, or by 90% after 20 min incubation in the presence of 100&M concentration of unlabelled AAF (not shown), suggesting that binding in mussel vesicles involves a protein component and a saturable process, respectively. The saturation process may well explain the 57 and 77% reduction of 14C-AAF binding to vesicles derived from mussels exposed to Sava River waters, or to waters polluted with Diesel-2 oil, respectively (Fig 1B). The apparent inhibition of binding by some unknown chemicals out of the myriad of compounds present in the polluted Sava River waters, or in water polluted with Diesel-2 oil, suggests that the protein component in mussel vesicles possessesthe affinity of binding compounds that are chemically unrelated to AAF or verapamil, substances that obviously were not present in waters to which our mussels were exposed. This cross-affinity in binding, together with a considerable capacity of trypsin- and verapamil- sensitive AAF binding in a saturable manner, strongly suggests that binding in mussels involves a molecular mechanism similar to that found in membrane vesicles from MDR cells and identified as 936
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glycoprotein PI70 (4). If this mechanism, demonstrated in mussel in vitro, is accompanied with an in vivo decrease in xenobiotic accumulation, similarly as in drugresistant cells, then it is likely that such a decrease in xenobiotic accumulation in mussel may confer at least a part of their resistance to pollution. Interestingly, the binding capacity is highest in membrane vesicles derived from mantle tissue. folowed by digestive gland- and gills- tissues (Fig. 1A). This distribution may reflect a strategic positioning of this mechanism in the first-contact and/or most-exposed organ. Study of the GSH-dependent enzyme activities in the mussel digestive gland postmitochondrial fraction showed that high CDNB-GST activity is accompanied by a high level of organic GSH-peroxidase activity. using cumene hydroperoxide as a substrate (Fig. 2A). Simultaneous finding of a high GST activity using ethacrynic acid as a substrate renders a distinct spectrum of GSH-dependent enzyme activities
that is
similar to the spectrum of anionic GST from adriamycin-resistant MCF-7 cells described by Batist et al. (9). Interestingly. gills and mantle tissues did not show such a distinct spectrum, since their CDNB-GST activity was much lower compared to the CDNB-GST activity in the digestive gland tissue (Fig. 2B). While the activity of GST in mussel digestive gland using CDNB as a substrate was in the same range as in noninduced rat liver tissue (20), fish liver tissue (21). or crustacean hepatopancreas tissue (22), the specific activities of some regulatory enzymes which represent important metabolic pathways through glycolysis, glucogenolysis, and glucose utilisation. were in moluscs an order of magnitude less than in vertebrates (23). Thus, it seems that the activities of
gills 0 FIGURE
mantle
CDNB
2. Glutathione-dependent
SAVA
DIESEL
gland
gland
gland
Eil Ethacr.
&
Cu-0,
H H202
enzyme activities in mussel Anodonta
cygneu.
A, activitiesin postmitochondrial fractions of digestive gland- (gland), gills-, and mantle- homogenatesfrom musselscollected at pristine site. B, activitiesin postmitochondrial fractions of digestivegland homogenatesfrom musselsexposedfor 14 days to a flow of SavaRiver waters (Sava)or for 4 days to a flow of water saturatedwith Diesel-2 oil (Diesel). GST activity with CDNB and ethacrynicacid (Ethacr) was assayed with 0.5 mg protein, and GSH-peroxidaseactivity with cumene peroxide (Cu-02) and hydrogen peroxide (H202) with 1 mg protein. Data are expressedasthe means f SD from determinations in 4 specimens. 937
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Phase II detoxifying enzymes, found in mussel Anodonm cygnen are indeed relatively high compared to their own metabolism, and may be involved as a molecular mechanism responsive for their resistance to pollution. Interestingly, exposure of mussels to Sava River waters and to waters polluted with Diesel-2 oil increased CDNB-GST activity by 75 and 81%, respectively, whereas ethacrynic-GST, and GSH-peroxidases with both cumene- and hydrogen- peroxide substrates did not change their characteristic, MDRlike profile (Fig 2B). Similar induction of GST with simultaneous non-induction, or a decrese in GSH-peroxidase activity, was found in mice treated with Arcclor 1254 (24). We were not able to detect the activity of benzo(a)pyrene monooxygenase activity in postmitochondrial fractions of organs either from mussels collected at pristine sites, or from mussels exposed for 3 weeks to Sava River waters, or from mussels exposed for 8 days to waters polluted with Diesel-2 oil. Thus, relatively high activity of Phase II enzymes in mussel Anodonm cygnea was accompanied by the undetectable and uninducible activity of a representative Phase I enzyme. In contrast to the MDR phenomenon in tumonr cell lines, where elements of a dual mechanism involved in the resistance were acquired after a treatment of cells by a single drug, the presence of both the verapamil-sensitive binding capacity of membrane vesicles and of GHS-dependent enzymes with distinct, resistance-specific substrate affinity characteristics, were found in our untreated mussels and seems to be inherent in this species. It has been shown that similar de novo expression of the dual mechanism of resistance phenotype may occur in medically untreated human colorectal and breast cancer, or rat liver hyperplastic nodules, where they were probably induced by carcinogen treatment (I 1, 12, 25). The tie ~ZOVO MDR-like resistance found in a population of our mussel was at species-, or at least at particular population- level, and its etiology may well be explained as a predictable response to selection pressure by a population characterized by natural variation (2). It is obvious that development of resistance in mussels occured early in their evolutionary history, probably as the response of the population to the selection pressure posed in the form of Nature*s pesticides, the major “toxic chemicals” (26) ingested by an phytoplancton filter-feeder. Although etiologies in these resistances may differ, it may well be that they represent biochemically similar events. Thus, several important features that must be induced before the development of MDR in tumour-cell lines, or before the development of resistance to carcinogens and hepatotoxins in rat hyperplastic liver nodules, are already preexisting in a natural population of mussel Anodontn cygnet from a pristine environment. Our preliminary tests have shown that similar basic elements of the resistance mechanism are taxonomically broadly distributed also among marine invertebrate Phylla (to be published). Better characterisation of the molecular mechanism involved in multixenobiotic resistance in aquatic invertebrates, as well as capitalisation on the present 938
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knowledge in the chemical combating resistance to xenobiotics in pests (2) may help us to reinterpret and understand past uncertainties in the toxicity tests, bioaccumulation phenomena (14), dose-effect relationships, realistic exposure assessments (13), and the use of organisms as “bioindicators” (27). These new insights may improve our present ranking of environmental hazards by, for example. introduction of a new class of compounds that act as inhibitors of the resistance mechanism, like verapamil and buthionine sulfoximine, referred as “chemosensitizers” (28). Thus, the recognition of the presence of multixenobiotic resistance phenomenon in organisms exposed to polluted waters is essential for understanding both the biological impact of pollution and the development of methods for rational risk assessment in regulatory policy. ACKNOWLEDGMENTS. This work was supported by the Authority for Scientific Research of Croatia and the US Environmental Protection Agency through the USYugoslav Joint Board on Scientific and Technological Cooperation, the UNEP/WHO Mediterranean Action Plan, Athens, and the Commission for Molecular Biology of the Academy of Science and Literature, Mainz, FRG. We thank Drs. E. Jackim. D. Nacci. and V. PravdiC for critically reviewing this manuscript. REFERENCES 1.
2. 3. k!: 6. 7. 8. 9. 10. 11. 12. 13.
17. 18. 19.
Malins, D. C., Hodgins, H. O., Varanasi. U.. Macleod. W. D.. McCain. B. B. and Chan, S. L. (1984) In Concepts in Marine Pollution Measurements (H. V. White, Ed.), pp. 405-426. A Maryland Sea Grant Publication, University of Maryland, College Park, MD. Graham-Bryce, I. J. (1987) In Combating Resistance to Xenobiotics (M. G. Ford, D. W. Holloman, B. P. S. Khambay. and R. M. Sawicki, Eds.), pp. 11 -25, Ellis Horwood Ltd, Chichester, England. Juliano, R. L., and Ling, V. (1976) Biochim. Biophys. Acta 455. 152-162. Kartner, N., Riordan, J. R., and Ling, V. (1983) Science 221, 1285-1288. Cornwell, M. M., Gottesman, M. M., and Pastan, I. H. (1986) J. Biol. Chem. 261, 792 l-7928. Ueda, K., Cornwell, M. M., Gottesman, M. M., Pastan I., Roninson, I. B.. Ling, V., and Riordan, J. R. (1986) Biochem. Biophys. Res. Commun. 141. 956-962. Shen, D.-W., Fojo, A., Chin, J. E. Roninson, I. B., Richert, N., Pastana. 1.. and Gottesman, M. M. (1986) Science 232, 643-645. Roomi, M. W., Ho, R. K., Sarma, D. S. R.. and Farber, E (1985) Cancer Res. 45, 564-571. Batist, G., Tulpule, A. Sinha, B. K., Katki, A. G., Myers, C. E., and Cowan. K. H. (1986) J. Biol. Chem. 261, 15544-15549. Kramer, R. a., Z&her, J., and Kim, G. (1988) Science 241. 694-697. Rinaudo, S. J. A., and Farber, E (1986) Carcinogenesis 7,523-528. Fairchild, C. R., Ivy, S. P.. Rushmore, T., Lee, G., Koo, P., Goldsmith, M. E., Myers, C. E., Farber, E., and Cowan, K. H. (1987) Proc. Natl. Acad. Sci. USA 84, 7701-7705. Kurelec, B., Garg, A., Krca, S.? Chacko. M.. and Gupta, R. C. (1989) Carcinogenesis 10, 1337-1339. Kurelec, B., and Krca, S. (1989) Comp. Biochem. Physiol. 92C, 371-376. Riordan, J. R., and Ling, V. (1979) J. Biol. Chem. 254. 12701-12705. Reddy, C. C., Tu, C.-P. D., Burgess J. R., Ho, C.-Y.. Scholz, R. W., and Massaro, E. J. (1981) Biochem. Biophys. Res. Commun. 101, 970-978. Habig, W. H., and Jacoby, W. B. (1981) Methods Enzymol. 77. 218-231. Nebert, D. W., and Gelboin, H. V. (1968) J. Biol. Chem. 243, 6242-6249. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (195 1) J. Biol. Chem. 193. 265-275. 939
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20. 21.
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Alin, P., Jensson, H., Guthenberg, C., Danielson, U. H., Tahir, M. K., and Mannervik, B. (1985) Anal. B&hem. 146, 313-320. James, M. O., Bowen, E. R., Dansatte. P. M.. and Bend, J. R. (1979) Chem. Biol. Interact. 25, 321-344. Keeran, W. S., and Lee, R. F. (1987) Arch. Biochem. Biophys. 255, 233-243. Ebberink, R. H. M., and de Zwaan, A. (1980) J. Comp. Physiol. 137, 165-171. Makary, M., Kim, H. L., Safe, S., Womack, J.: and Ivie, G. W. (1988) Comp. B&hem. Physiol. 91C, 425-429. Cowan, K. H., Batist, G., Tulpule, A., Sinha, B. K., and Myers, C. E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9328-9332. Ames, B. N. (1989) Environ. Mol. Mutagenesis 14, 66-77. Kurelec, B (1985) Biochem. Biphys. Res. Commun. 127, 773-778. Kartner, N., and Ling, V. (1989) Scientific American, March 1989, 44-51.
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DISTINCT GLUTATHIONE-DEPENDENT ENZYME ACTIVITIES AND A VERAPAMIL-SENSITIVE BINDING OF XENOBIOTICS IN A FRESH-WATER MUSSEL ANODONTA CYGNEA
Branko Kurelec and Branka PivEeviC Ruder BoSkovid Institute, Center for Marine Research Zagreb, 41001 Zagreb, Croatia, Yugoslavia Received
September
19,
1989
SUMMARY: A fresh-water mussel Anodonra cygnet, an aquatic invertebrate resistant to pollution, possessesan inherent high potential to bind 2-acetylaminofluorene onto membrane vesicles. This binding is saturable and trypsin- and verapamil- sensitive. Simultaneously, this mussel reveals a relatively high inherent activity of glutathionedependent enzyme activities with a distinct spectrum of substrate affinities. Both these activities are similar to the elements of the molecular mechanism involved in the acquired multi-drug resistance phenomenon described in tumour cell-lines. The recognition that in organisms exposed to polluted waters a multi-xenobiotic resistance mechanism may be involved is essential for understanding both the biological impact of pollution and the development of methods for rational risk assessment in regulatory policy. 0 1989Academic Pn?SS,Inc.
There are aquatic invertebrates that can survive and reproduce in heavily polluted waters. Similar resistance to pollution was found in some marine bottom invertebrates where the concentration of pollutants in tissues and body fluids was lower than in surrounding recent sediments (1). Abscence of accumulation, caused by decreased transport of a xenobiotic to the site of action and/or by enhanced metabolic attack on it within the organism, has long been recognized as a common mechanism in the development of resistance to xenobiotics (2). Aquatic organisms seem to be simultaneously resistant to multiple toxic xenobiotics, since aquatic pollution is typically caused by a mixture of chemicals. This “multi-xenobiotic resistance” and lack of accumulation are characteristics that are analogous to the phenomenon of “multi-drug resistance” found in tumour cells that become refractory to treatment with a variety of chemotherapeutic agents (3). There, a decrease in drug accumulation is associated with the increased production of a membrane glycoprotein termed P-170 (4) which binds a drug and facilitates its efflux by an energy-dependent process (5). mdr-1, the gene coding for P-170, has been cloned (6) and its overexpression was found to be proportional to the degree of resistance in resistant cell lines (7). However, MDR is also associated with the decreased expression of the Phase I metabolizing enzyme cytochrome Abbreviations: GSH, glutathione; GST, glutathione transferase; CDNB, l-chloro-2,4dinitro-benzene; AAF, 2-acetylaminofluorene; MDR, multi-drug resistance. 0006-291x/89 Copyright All rights
0
$1.50
1989 by Academic Press, of reproduction in any form
Inc. reserved.
934
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P-450 monooxygenase (8), and increased expression of several Phase II conjugating enzymes, including both a many-fold increase in the activity of a distinct, resistancespecific isoenzyme of anionic glutathione transferase with a high level of intrinsic peroxidase and a high affinity to ethacrinic acid (9), and the alterations glutathione redox cycle (10).
in the
The focus of the present study was to determine whether fresh-water mussel Anodontcl cygnea. an organism that can survive and reproduce in heavily polluted waters, possesseselements of the molecular mechanism similar to that found to be involved in the drug-resistant tumour cell-lines. In order to obtain preliminary insight into the possible presence of glycoprotein P-170-mediated mechanism, we measured it? vitro the potential of isolated mussel membrane vesicles to bind radiolabelled 2acetylaminofluorene. a carcinogen that also induces an increase in mdr-I gene expression (11. 12), in the presence or absence of verapamil, a molecule that was found to bind specifically to the active sites of P-170. thereby inhibiting binding of other xenobiotics (5). The presence of resistance-specific glutathione-dependent enzymes in mussel organs was studied by substrate affinity characteristics of GSH-transferases with 1-chloro-2,4-dinitrobenzene
and ethacrynic acid, and of GSH-peroxidases with cumene
peroxide and hydroperoxide. Our results show that the mussel Atzodotztn cypzect possessesan inherent verapamil-sensitive potential to bind 2-acetylaminofluorene.
and a relatively high
inherent glutathione-dependent enzyme activities with a distinct. resistance-specific spectrum of substrate affinities. MATERIALS
AND METHODS
Specimens of Anodonta cygnea, 14-18 cm, 150-2 10 g, were collected during May-July 1989 from a brook suplying waters for carp-farm Draganidi. near Zagreb. A group of mussels was exposed to a flow of polluted (defined in 13) Sava River waters or to a flow of tap-water saturated with a Diesel-2 oil as described (14). Membrane vesicles from digestive gland-, gills- and mantle- tissues were prepared according to the nethod of Riqrdan and Liy4 (15). _ Total and nonspecific binding of 2-acetylamino(9C)ammofluorene ( C-AAF; 55.3 mCi/mmol, Amersham, England) was measured as described by Cornwell et al. (5), except that aliquots of ves@ preparation containing 1OOpg protein were incubated in the presence of 50 nM of C-AAF and that filtration was done on glass fiber filters (Whatman GFK) pretreated with 10% human serum. GSH peroxidase activity was assayed in postmitochonrial fraction of 20% tissue homogenates using hydrogen- or cumene- peroxides as substrates (16). GST was assayed using 1 mM I-chloro-2,4-dinitro-benzene and 1 mM glutathione, or, 0.2 mM ethacrynic acid and 0.25 mM glutathione. respectively. according to the procedure of Habig and Jacoby (17). Benzo(a)pyrene monooxygenase activity was determined according Nebert and Gelboin (18) and proteins were determined by the method of Lowry et al. (19). RESULTS AND DISCUSSION Measurements of the association of 14 C-AAF with digestive gland vesicles prepared from mussels. collected at a pristine environment, revealed that vesicles accumulate a considerable amount of 14C AAF (Fig. 1A). The effect of premcubatlon of vesicles with verapamil at 10yglml was profound: it reduced the AAF binding by 80% 935
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DIESEL
T
I
I I I I L
: mantle
gland
gland
gland
FIGURE 1. The effect of verapamil treatment oo 14C-AAF binding in mussel membrane vesicles.A, the binding in digestive gland- (gland), gills-, and mantlevesicles derived from mussels collected at pristine site. B, the binding in digestive gland
vesiclesfrom musselsexposedfor 14 days to a flow of SavaRiver wateftj (Sava),or for 4 days to a water saturated with Diesel-2 oil (Diesel). Total binding of C-AAF (SO nM) on vesicles (1OOpg protein/assay) was measured after 20-min incubation at 23’C in the absence(-) or presence(+) of IO&ml verapamil (VP). Nonspecificbinding (NB) was assessed by including 1OOpM unlabelled AAF in the reaction mixture. Data are expressed as the means f SD from determinations in 4 specimens.
compared to untreated vesicles. Similar dramatic effect of verapamil was observed with binding of vinblastine on vesicles derived from a multi-drug resistant KB-C4 cell line (5). There, association of vinblastine with vesicles was reduced by verapamil to the level bound by vesicles derived from a drug-sensitive KB-3-1 cells. In the absence of a corresponding “xenobiotic-sensitive”
reference mussel, we adopted a level of binding
from a verapamil-treated mussel as a “normal” level of binding in a hypothetical “sensitive” mussel. Similarly to the behaviour of the vinblastine binding in drugresistant cells, binding of l4 C-AAF to mussel vesicles was reduced by 48% after 20 min incubation of vesicles with 10 ng of trypsin/mg of vesicle protein, or by 90% after 20 min incubation in the presence of 100&M concentration of unlabelled AAF (not shown), suggesting that binding in mussel vesicles involves a protein component and a saturable process, respectively. The saturation process may well explain the 57 and 77% reduction of 14C-AAF binding to vesicles derived from mussels exposed to Sava River waters, or to waters polluted with Diesel-2 oil, respectively (Fig 1B). The apparent inhibition of binding by some unknown chemicals out of the myriad of compounds present in the polluted Sava River waters, or in water polluted with Diesel-2 oil, suggests that the protein component in mussel vesicles possessesthe affinity of binding compounds that are chemically unrelated to AAF or verapamil, substances that obviously were not present in waters to which our mussels were exposed. This cross-affinity in binding, together with a considerable capacity of trypsin- and verapamil- sensitive AAF binding in a saturable manner, strongly suggests that binding in mussels involves a molecular mechanism similar to that found in membrane vesicles from MDR cells and identified as 936
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glycoprotein PI70 (4). If this mechanism, demonstrated in mussel in vitro, is accompanied with an in vivo decrease in xenobiotic accumulation, similarly as in drugresistant cells, then it is likely that such a decrease in xenobiotic accumulation in mussel may confer at least a part of their resistance to pollution. Interestingly, the binding capacity is highest in membrane vesicles derived from mantle tissue. folowed by digestive gland- and gills- tissues (Fig. 1A). This distribution may reflect a strategic positioning of this mechanism in the first-contact and/or most-exposed organ. Study of the GSH-dependent enzyme activities in the mussel digestive gland postmitochondrial fraction showed that high CDNB-GST activity is accompanied by a high level of organic GSH-peroxidase activity. using cumene hydroperoxide as a substrate (Fig. 2A). Simultaneous finding of a high GST activity using ethacrynic acid as a substrate renders a distinct spectrum of GSH-dependent enzyme activities
that is
similar to the spectrum of anionic GST from adriamycin-resistant MCF-7 cells described by Batist et al. (9). Interestingly. gills and mantle tissues did not show such a distinct spectrum, since their CDNB-GST activity was much lower compared to the CDNB-GST activity in the digestive gland tissue (Fig. 2B). While the activity of GST in mussel digestive gland using CDNB as a substrate was in the same range as in noninduced rat liver tissue (20), fish liver tissue (21). or crustacean hepatopancreas tissue (22), the specific activities of some regulatory enzymes which represent important metabolic pathways through glycolysis, glucogenolysis, and glucose utilisation. were in moluscs an order of magnitude less than in vertebrates (23). Thus, it seems that the activities of
gills 0 FIGURE
mantle
CDNB
2. Glutathione-dependent
SAVA
DIESEL
gland
gland
gland
Eil Ethacr.
&
Cu-0,
H H202
enzyme activities in mussel Anodonta
cygneu.
A, activitiesin postmitochondrial fractions of digestive gland- (gland), gills-, and mantle- homogenatesfrom musselscollected at pristine site. B, activitiesin postmitochondrial fractions of digestivegland homogenatesfrom musselsexposedfor 14 days to a flow of SavaRiver waters (Sava)or for 4 days to a flow of water saturatedwith Diesel-2 oil (Diesel). GST activity with CDNB and ethacrynicacid (Ethacr) was assayed with 0.5 mg protein, and GSH-peroxidaseactivity with cumene peroxide (Cu-02) and hydrogen peroxide (H202) with 1 mg protein. Data are expressedasthe means f SD from determinations in 4 specimens. 937
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Phase II detoxifying enzymes, found in mussel Anodonm cygnen are indeed relatively high compared to their own metabolism, and may be involved as a molecular mechanism responsive for their resistance to pollution. Interestingly, exposure of mussels to Sava River waters and to waters polluted with Diesel-2 oil increased CDNB-GST activity by 75 and 81%, respectively, whereas ethacrynic-GST, and GSH-peroxidases with both cumene- and hydrogen- peroxide substrates did not change their characteristic, MDRlike profile (Fig 2B). Similar induction of GST with simultaneous non-induction, or a decrese in GSH-peroxidase activity, was found in mice treated with Arcclor 1254 (24). We were not able to detect the activity of benzo(a)pyrene monooxygenase activity in postmitochondrial fractions of organs either from mussels collected at pristine sites, or from mussels exposed for 3 weeks to Sava River waters, or from mussels exposed for 8 days to waters polluted with Diesel-2 oil. Thus, relatively high activity of Phase II enzymes in mussel Anodonm cygnea was accompanied by the undetectable and uninducible activity of a representative Phase I enzyme. In contrast to the MDR phenomenon in tumonr cell lines, where elements of a dual mechanism involved in the resistance were acquired after a treatment of cells by a single drug, the presence of both the verapamil-sensitive binding capacity of membrane vesicles and of GHS-dependent enzymes with distinct, resistance-specific substrate affinity characteristics, were found in our untreated mussels and seems to be inherent in this species. It has been shown that similar de novo expression of the dual mechanism of resistance phenotype may occur in medically untreated human colorectal and breast cancer, or rat liver hyperplastic nodules, where they were probably induced by carcinogen treatment (I 1, 12, 25). The tie ~ZOVO MDR-like resistance found in a population of our mussel was at species-, or at least at particular population- level, and its etiology may well be explained as a predictable response to selection pressure by a population characterized by natural variation (2). It is obvious that development of resistance in mussels occured early in their evolutionary history, probably as the response of the population to the selection pressure posed in the form of Nature*s pesticides, the major “toxic chemicals” (26) ingested by an phytoplancton filter-feeder. Although etiologies in these resistances may differ, it may well be that they represent biochemically similar events. Thus, several important features that must be induced before the development of MDR in tumour-cell lines, or before the development of resistance to carcinogens and hepatotoxins in rat hyperplastic liver nodules, are already preexisting in a natural population of mussel Anodontn cygnet from a pristine environment. Our preliminary tests have shown that similar basic elements of the resistance mechanism are taxonomically broadly distributed also among marine invertebrate Phylla (to be published). Better characterisation of the molecular mechanism involved in multixenobiotic resistance in aquatic invertebrates, as well as capitalisation on the present 938
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knowledge in the chemical combating resistance to xenobiotics in pests (2) may help us to reinterpret and understand past uncertainties in the toxicity tests, bioaccumulation phenomena (14), dose-effect relationships, realistic exposure assessments (13), and the use of organisms as “bioindicators” (27). These new insights may improve our present ranking of environmental hazards by, for example. introduction of a new class of compounds that act as inhibitors of the resistance mechanism, like verapamil and buthionine sulfoximine, referred as “chemosensitizers” (28). Thus, the recognition of the presence of multixenobiotic resistance phenomenon in organisms exposed to polluted waters is essential for understanding both the biological impact of pollution and the development of methods for rational risk assessment in regulatory policy. ACKNOWLEDGMENTS. This work was supported by the Authority for Scientific Research of Croatia and the US Environmental Protection Agency through the USYugoslav Joint Board on Scientific and Technological Cooperation, the UNEP/WHO Mediterranean Action Plan, Athens, and the Commission for Molecular Biology of the Academy of Science and Literature, Mainz, FRG. We thank Drs. E. Jackim. D. Nacci. and V. PravdiC for critically reviewing this manuscript. REFERENCES 1.
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