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Metabolism of a model environmental carcinogen by bivalve molluscs
Marine Environmental Research 17 (1985) 137-140
Metabolism of a Model Environmental Carcinogen by Bivalve Molluscs
Robert S. Anderson* Sloan-Kettering Institute for Cancer Research, 145 Boston Post Road, Rye, New York 10580, USA
Bivalve molluscs have been used as monitors of marine pollution because they concentrate xenobiotics of many kinds in their tissues. Marine pollutants often produce stress responses such as reduced scope f o r growth, lysosomal destabilization, altered immune responsiveness and other sequelae. The ability of bivalves to metabolize organic compounds via mixed-function oxygenases ( MFO) was demonstrated by the epoxidation of aldrin t and benzo[a]pyrene ( BP); 2 this work has been corroborated and extended in many subsequent studies. Biotransformation oJ" BP and other environmental procarcinogens can generate either activated or detoxified metabolites. In this paper the BP metabolites produced by digestive gland homogenates of the clam ( M e r c e n a r i a m e r c e n a r i a ) and the oyster ( C r a s s o s t r e a virginica) are described. Control bivalves produced the proximate carcinogen ( BP 7,8-dihydrodiol), as well as various minimally reactive BP diols and monohydroxylated metabolites. Seven days after injection oj'the potent inducer o f mammalian M FO, Aroclor 1254, BP 7,8diol production was augmented in M e r c e n a r i a , and atypical quinones and phenols were seen in both species. However, in the Ames bacterial mutagenicity assay, homogenates from A roclor-treated clams failed to activate benzo[a]pyrene. The biological significance oJBP metabolism in bivalves is unknown; however, it may prove to be a useful index of pollution. * Present addl:ess: Chemical Research and Development Center, ATTN: SMCCR-RSB, Aberdeen Proving Ground, Maryland 21010, USA. 137 Marine Environ. Res. 0141-1136/85/$03-30 ~ Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain
138
Robert S. Anderson
The inducibility of aryl hydrocarbon hydroxylase ( A H H ) by certain environmental contaminants has been used as a measure of pollutant exposure in marine animals such as fish. The enzyme appears to be present in bivalves, but at comparatively low specific activity. Furthermore, attempts to detect elevated A H H levels in bivalves collected from polluted sites have met with little success. 3"'t In this study, Aroclor 1254 injected into the hemolymph sinus of the anterior adductor muscle, 7 days prior to sampling, caused little A H H induction in clams and only slight ( < 2-fold) induction in the 250 mg group of oysters (Table 1). The specific activity of A H H ranged from about 2-5 pmoles BP.min/mg protein, as measured by the extraction procedures of Abramson and Hutton 5 using t4BP as substrate. Generally, the majority of the label occurred in the aqueous phase, possibly representing B P conjugates or macromolecular adducts. In order to ascertain the identity and potential biological activity of the BP metabolites produced, analysis by high-performance liquid chromatography ( H P L C ) was carried out (see Refs 6 and 7 for methods). The identities of the metabolites reported in Table 2 were established by coelution with authentic standards. Only 9,10-diol and 7,8-diol were recovered after incubation with control M e r c e n a r i a preparations. Control Crassostrea enzyme preparations made these diols and smaller amounts of 3-OH, 6-OH, 9 - O H and 12-OH BP. Based on total TABLE 1
t'~C-Benzo[a]pyrene Metabolism by Digestive Gland Homogenates in the Aryl Hydrocarbon Hydroxylase Assay Sample NaOH-soluble size metabolites M. mercenaria
C. virginica
HzO-sohtble metabolites
Total"
Vehiclecontrol Aroclor 1254 (250 mg) (1 000mg)
4
0-065+ 0-089 0.134_-20-012 0-199+ 0.088
5 6
0-041 + 0.044 0-037+0.051
0-037+_0.006 0.144+0-037
Vehicle control Aroclor 1254 (250 mg) (1 000 mg)
6
0.024+ 0.049
0.058+_0-020 0.082+ 0.046
6 6
0.015+0.007 0.040+0-039
0.127+_0-015 0-143___0-010b 0-051+0.011 0-091+0.043
a nmoles BP metabolized/mg protein/45 min. b Induction significant at 0-05 level (t-test).
0-078+ 0.047 0-181+0-125
Metabolism o]a carcinogen by bivah'e molluscs
139
TABLE 2
High-Performance Liquid Chromatography of ttC-B P Metabolites Produced by Bivalve Digestive Gland Homogenates (cpm 45 min) Mercenaria mercenaria
Control
9, t0-diol 4,5-diol 7,8-diol
Aroclor Aroctor (250mg) (lO00mg)
48 297
1128
1,6 quinone 3,6 quinone 6,12 quinone
113 108 113
12-OH 6-OH 9-OH 10-OH 2-OH 3-OH
175 210 627 1064
772 106 428
Crassostrea virginica
Control
Aroclor Aroclor (250mg) (lO00mg)
485
160 148 589
IL34
269
74
281 172 73 217
125 120
469 551 573
63
metabolite cpms recovered by HPLC, both Aroclor concentration treatments induced BP 7,8-diol formation by clams, and high dose groups in both species produced 9,10-, 4,5- and 7,8-diol. The induction regimen was also associated with change in the phenol and quinone metabolite profiles. In terms of potential biological activity the presence of the proximate BP carcinogenic form in both control and Aroclor-treated bivalves is of primary interest. However, AHH preparations from clams showed limited ability to activate BP when used as enzyme sources of the Salmonella mutagenesis assay. For example, reacting 10 #g BP with clam digestive gland homogenate ( ~ 2 mg protein) resulted in the production of 1 5 + 7 ( N = 8 , TA 98) his + revertants/plate, after correcting for spontaneous mutation and other forms of background. A comparable preparation reacting BP with rat liver homogenate produced 774 + 232 (N = 4, TA98) his + revertants. Apparently, Mercenaria enzymes can activate aromatic amine procarcinogens in the Ames assay, s For example, 626 + 238 (N = 8) TA 98 his + revertants were counted after incubation of clam digestive gland homogenate with 10#g 2-aminoanthracene. Clearly, additional systematic studies of chemical carcinogenesis using marine bivalves are in order. Aromatic amines might
140
Robert S. Anderson
possibly prove useful as model oncogens, based on their solubility and activation by molluscan enzymes.
REFERENCES 1. Khan, M. A. Q., Kamal, A., Wolin, R. J. & Runnels, J. Bull. Environ. Contain. Toxicol., 8, 219-28 (1972). 2. Anderson, R. S. Benzo[a]pyrene metabolism in the American oyster, Crassostrea virginica, EPA Ecol. Res. Ser. Monogr., EPA-600,'3-78-009, 1978. 3. Payne, J. F. Mar. Environ. Res., 8, 112-16 (1977). 4. Stegeman, J. J. In 1980-81 Annual Sea Grant Report 15, Woods Hole Oceanographic Institution, 1981. 5. Abramson, R. K. & Hutton, J. J. Cancer Res., 35, 23-9 (1975). 6. Anderson, L. M., Deschner, E. E., Angel, M. & Herrmann, S. L. Oncology, 39, 369-77 (1982). 7. Elnenaey, E. A. & Schoor, W. P. Anal. Biochem., 111, 393-400 (1981). 8. Anderson, R. S. & D66s, J. E. Mutation Res., 116, 247-56 (1983).
Metabolism of a Model Environmental Carcinogen by Bivalve Molluscs
Robert S. Anderson* Sloan-Kettering Institute for Cancer Research, 145 Boston Post Road, Rye, New York 10580, USA
Bivalve molluscs have been used as monitors of marine pollution because they concentrate xenobiotics of many kinds in their tissues. Marine pollutants often produce stress responses such as reduced scope f o r growth, lysosomal destabilization, altered immune responsiveness and other sequelae. The ability of bivalves to metabolize organic compounds via mixed-function oxygenases ( MFO) was demonstrated by the epoxidation of aldrin t and benzo[a]pyrene ( BP); 2 this work has been corroborated and extended in many subsequent studies. Biotransformation oJ" BP and other environmental procarcinogens can generate either activated or detoxified metabolites. In this paper the BP metabolites produced by digestive gland homogenates of the clam ( M e r c e n a r i a m e r c e n a r i a ) and the oyster ( C r a s s o s t r e a virginica) are described. Control bivalves produced the proximate carcinogen ( BP 7,8-dihydrodiol), as well as various minimally reactive BP diols and monohydroxylated metabolites. Seven days after injection oj'the potent inducer o f mammalian M FO, Aroclor 1254, BP 7,8diol production was augmented in M e r c e n a r i a , and atypical quinones and phenols were seen in both species. However, in the Ames bacterial mutagenicity assay, homogenates from A roclor-treated clams failed to activate benzo[a]pyrene. The biological significance oJBP metabolism in bivalves is unknown; however, it may prove to be a useful index of pollution. * Present addl:ess: Chemical Research and Development Center, ATTN: SMCCR-RSB, Aberdeen Proving Ground, Maryland 21010, USA. 137 Marine Environ. Res. 0141-1136/85/$03-30 ~ Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain
138
Robert S. Anderson
The inducibility of aryl hydrocarbon hydroxylase ( A H H ) by certain environmental contaminants has been used as a measure of pollutant exposure in marine animals such as fish. The enzyme appears to be present in bivalves, but at comparatively low specific activity. Furthermore, attempts to detect elevated A H H levels in bivalves collected from polluted sites have met with little success. 3"'t In this study, Aroclor 1254 injected into the hemolymph sinus of the anterior adductor muscle, 7 days prior to sampling, caused little A H H induction in clams and only slight ( < 2-fold) induction in the 250 mg group of oysters (Table 1). The specific activity of A H H ranged from about 2-5 pmoles BP.min/mg protein, as measured by the extraction procedures of Abramson and Hutton 5 using t4BP as substrate. Generally, the majority of the label occurred in the aqueous phase, possibly representing B P conjugates or macromolecular adducts. In order to ascertain the identity and potential biological activity of the BP metabolites produced, analysis by high-performance liquid chromatography ( H P L C ) was carried out (see Refs 6 and 7 for methods). The identities of the metabolites reported in Table 2 were established by coelution with authentic standards. Only 9,10-diol and 7,8-diol were recovered after incubation with control M e r c e n a r i a preparations. Control Crassostrea enzyme preparations made these diols and smaller amounts of 3-OH, 6-OH, 9 - O H and 12-OH BP. Based on total TABLE 1
t'~C-Benzo[a]pyrene Metabolism by Digestive Gland Homogenates in the Aryl Hydrocarbon Hydroxylase Assay Sample NaOH-soluble size metabolites M. mercenaria
C. virginica
HzO-sohtble metabolites
Total"
Vehiclecontrol Aroclor 1254 (250 mg) (1 000mg)
4
0-065+ 0-089 0.134_-20-012 0-199+ 0.088
5 6
0-041 + 0.044 0-037+0.051
0-037+_0.006 0.144+0-037
Vehicle control Aroclor 1254 (250 mg) (1 000 mg)
6
0.024+ 0.049
0.058+_0-020 0.082+ 0.046
6 6
0.015+0.007 0.040+0-039
0.127+_0-015 0-143___0-010b 0-051+0.011 0-091+0.043
a nmoles BP metabolized/mg protein/45 min. b Induction significant at 0-05 level (t-test).
0-078+ 0.047 0-181+0-125
Metabolism o]a carcinogen by bivah'e molluscs
139
TABLE 2
High-Performance Liquid Chromatography of ttC-B P Metabolites Produced by Bivalve Digestive Gland Homogenates (cpm 45 min) Mercenaria mercenaria
Control
9, t0-diol 4,5-diol 7,8-diol
Aroclor Aroctor (250mg) (lO00mg)
48 297
1128
1,6 quinone 3,6 quinone 6,12 quinone
113 108 113
12-OH 6-OH 9-OH 10-OH 2-OH 3-OH
175 210 627 1064
772 106 428
Crassostrea virginica
Control
Aroclor Aroclor (250mg) (lO00mg)
485
160 148 589
IL34
269
74
281 172 73 217
125 120
469 551 573
63
metabolite cpms recovered by HPLC, both Aroclor concentration treatments induced BP 7,8-diol formation by clams, and high dose groups in both species produced 9,10-, 4,5- and 7,8-diol. The induction regimen was also associated with change in the phenol and quinone metabolite profiles. In terms of potential biological activity the presence of the proximate BP carcinogenic form in both control and Aroclor-treated bivalves is of primary interest. However, AHH preparations from clams showed limited ability to activate BP when used as enzyme sources of the Salmonella mutagenesis assay. For example, reacting 10 #g BP with clam digestive gland homogenate ( ~ 2 mg protein) resulted in the production of 1 5 + 7 ( N = 8 , TA 98) his + revertants/plate, after correcting for spontaneous mutation and other forms of background. A comparable preparation reacting BP with rat liver homogenate produced 774 + 232 (N = 4, TA98) his + revertants. Apparently, Mercenaria enzymes can activate aromatic amine procarcinogens in the Ames assay, s For example, 626 + 238 (N = 8) TA 98 his + revertants were counted after incubation of clam digestive gland homogenate with 10#g 2-aminoanthracene. Clearly, additional systematic studies of chemical carcinogenesis using marine bivalves are in order. Aromatic amines might
140
Robert S. Anderson
possibly prove useful as model oncogens, based on their solubility and activation by molluscan enzymes.
REFERENCES 1. Khan, M. A. Q., Kamal, A., Wolin, R. J. & Runnels, J. Bull. Environ. Contain. Toxicol., 8, 219-28 (1972). 2. Anderson, R. S. Benzo[a]pyrene metabolism in the American oyster, Crassostrea virginica, EPA Ecol. Res. Ser. Monogr., EPA-600,'3-78-009, 1978. 3. Payne, J. F. Mar. Environ. Res., 8, 112-16 (1977). 4. Stegeman, J. J. In 1980-81 Annual Sea Grant Report 15, Woods Hole Oceanographic Institution, 1981. 5. Abramson, R. K. & Hutton, J. J. Cancer Res., 35, 23-9 (1975). 6. Anderson, L. M., Deschner, E. E., Angel, M. & Herrmann, S. L. Oncology, 39, 369-77 (1982). 7. Elnenaey, E. A. & Schoor, W. P. Anal. Biochem., 111, 393-400 (1981). 8. Anderson, R. S. & D66s, J. E. Mutation Res., 116, 247-56 (1983).