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Hepatic microsomal monooxygenases of sea birds
Marine Environmental Research 14 (1984) 416-419
Hepatic Microsomal Monooxygenases of Sea Birds
C. H. Walker, G. C. Knight, J. K. Chipman & M. J. J. Ronis Department of Physiology and Biochemistry, University of Reading, Whiteknights, Reading, Berkshire RG6 2A J, Great Britain
The present study was part of an investigation into the biochemical basis for the strong bioaccumulation of persistent organochlorine pollutants by many fish-eating birds. Hepatic microsomal monooxygenase was assayed in six species of sea birds from British and Irish coastal waters using as substrates the organochlorine compounds aldrin (1,2,3,4,10,10hexachloro- 1,4,4a,5,8,8a-hexahydro-exo- 1,4-endo-5,8-dimethanonaphthalene) and HCE (1,2,3,4,9,9-hexachloro-l,4,4a,5,6,7,8,8a,octahydroexo-7,8-epoxy-l,4-methanonaphthalene). The razorbill (Alca torda), guillemot (Uria aalge), shag (Phalacrocorax aristotelis), cormorant (Phalacrocorax carbo) and Manx shearwater (Puffinus puffinus) had much lower activities than the puffin (Fratercula arctica). When these values were compared with other data from the literature on a scale of relative activity, the high activities of the puffin were similar to those of mammals and certain omnivorous or herbivorous birds, whereas the low activities of the other five species were similar to those of fish (Walker & Knight, 1981). In the three auks, enzyme activities were highly variable and resolved into two or more peaks when frequency diagrams were plotted. This was not so with the shag. In the razorbill, individuals of low activity (Group I) were compared with individuals of higher activity (Group II) (Knight & Walker, 1982). Individuals from Group II had, on average, 5.6-8.5-fold higher monooxygenase activity at saturating substrate concentrations than did Group I, although there was only a 1.6-fold difference in microsomal cytochrome P-450 between the groups. Lineweaver-Burke plots for 416 Marine Environ. Res. 0141-1136/84/$03.00 © Elsevier Applied Science Publishers Ltd,
England, 1984. Printed in Great Britain.
Hepatic microsomal monooxygenases o]sea birds
417
aldrin epoxidation gave apparent K m values of 25-56 and 3-17 nmol/mg protein for Groups I and II respectively. This suggests that Group II possesses a form (or forms) of monooxygenase of relatively high affinity for aldrin which is either at low concentration or absent from Group I. The difference in activity between the groups increased as the concentration of aldrin fell towards levels which are realistic for organochlorine pollutants. A plot for trout liver microsomes fell between those for the two groups of razorbills, whilst a plot for male rat liver microsomes showed that this species maintains a much higher activity than the others down to environmentally realistic concentrations. Thus, at relatively low substrate concentration, the rat and the razorbills of Group II showed higher hepatic microsomal monooxygenase activities than razorbills of Group I or trout. This separation of monooxygenase activities into two or more peaks in razorbills and other auks was not related to sex, time of season, geographical location or tissue levels of organochlorine pollutants. In view of this, and the fact that no such distribution was found in the sedentary shag which was feeding in the same areas, it seemed unlikely that the more active individuals had been induced by pollutants. Had induction occurred during the breeding season, a similar effect might have been anticipated in the shag; also substantial differences in the extent of induction might have been expected between areas as different as the Isle of May and the Saltee Islands. Had induction occurred in the auks during migration it should have declined or disappeared during the breeding season, and might have been expected to bear some relationship to organochlorine residues, the class of sea-borne pollutants most likely to cause a long-lasting induction of microsomal enzymes. More probably, these variations are an expression of genetic differences. The highly active individuals may be representatives of resistant strains which have arisen as a consequence of the selective pressure of PCBs and other persistent pollutants over many years. PCBs were implicated in the large kill of guillemots and razorbills in the Irish Sea in 1969, which was followed by substantial reductions in numbers of breeding birds at Welsh and Irish coastal sites in 1970 (Knight & Walker, 1982). To test the rate of elimination of a persistent organochlorine compound by a species with low monooxygenase activity, cormorants were given dieldrin by i.p. injection. Dieldrin concentrations were determined in blood samples, and in whole carcases at death. Rates of loss of dieldrin were determined as a percentage of original dose (carcases) or
418
c. H. Walker, G. C. Knight, J. K. Chipman, M. J. J. Ronis
of initial concentration in blood. The rate of loss of dieldrin from whole bodies after dosing at 1.5 mg/kg was similar to that from blood after a dose of 5mg/kg, but somewhat slower than that from blood after 1.5 mg/kg. The loss of half the original dose from the body was estimated to occur in 20-30 days. This value may be compared with half-lives estimated for male rats which contained similar tissue levels of dieldrin (Robinson et al., 1969; Moriarty, 1975). After feeding rats 10ppm dieldrin in their diet for 8 weeks, 0.23 ppm of the chemical was found in brain. Cormorants given a single i.p. injection of dieldrin were estimated to contain 0.1-0.2 ppm of dieldrin in brain. Thus the rats had, evidently, somewhat higher tissue concentrations than the cormorants at the start of experimentation. The loss of chemical from adipose tissue and brain of rats was biphasic: in the case of adipose tissue, which accounts for much of the body burden of dieldrin, half the original concentration was lost within 4 days. In another experiment, rats given single i.v. doses of 0.25 mg/kg excreted 50 ~ of the dose in bile within 2'5 days (Bedford & Hutson, 1976). In these experiments male rats lost half of their body load of dieldrin within 4 days, a rate which was at least 5 times more rapid than that found in cormorants in the present study. This adds to the growing body of evidence that the rate of elimination of liposoluble organochlorine pollutants can be limited by low rates of metabolism (Walker, 1981). The very low monooxygenase activities found in five species of sea birds should result in slow rates of metabolism of persistent pollutants, and correspondingly efficient bioaccumulation. This work was supported by a grant from the NERC. The authors are grateful to Mr S. Hallam for skilled technical assistance and to Dr D. C. Cabot and Dr M. P. Harris and their colleagues for arranging the collection of sea birds under licence.
REFERENCES Bedford, C. T. & Hutson, D. H. (1976). The comparative metabolism in rodents of the isomeric insecticides dieldrin and endrin. Chem. Ind. 15 May, 440 -7. Knight, G. C. & Walker, C. H. (1982). A study of the hepatic microsomal monooxygenase of sea birds and its relationship to liposoluble pollutants. Comp. Biochem. Physiol. 73C, 211-21. Moriarty, F. (1975). Exposure and residues. In: Organochlorine insecticides. persistent organic pollutants (Moriarty, F. (Ed.)). Academic Press, London, 29 72.
Hepatic microsomal monooxygenases oJ sea birds
419
Robinson, J., Roberts, M., Baldwin, M. & Walker, A. I. T. (1969). The pharmacokinetics of HEOD (Dieldrin) in the rat. Fd Cosmet. Toxicol. 7, 317-22. Walker, C. H. (1981). The correlation between in vivo and in vitro metabolism of pesticides in vertebrates. Progr. Pestic. Biochem. 1,247-85. Walker, C. H. & Knight, G. C. (1981). The hepatic microsomal enzymes of sea birds and their interaction with liposoluble pollutants. Aquat. Toxicol. 1, 343 -54.
Hepatic Microsomal Monooxygenases of Sea Birds
C. H. Walker, G. C. Knight, J. K. Chipman & M. J. J. Ronis Department of Physiology and Biochemistry, University of Reading, Whiteknights, Reading, Berkshire RG6 2A J, Great Britain
The present study was part of an investigation into the biochemical basis for the strong bioaccumulation of persistent organochlorine pollutants by many fish-eating birds. Hepatic microsomal monooxygenase was assayed in six species of sea birds from British and Irish coastal waters using as substrates the organochlorine compounds aldrin (1,2,3,4,10,10hexachloro- 1,4,4a,5,8,8a-hexahydro-exo- 1,4-endo-5,8-dimethanonaphthalene) and HCE (1,2,3,4,9,9-hexachloro-l,4,4a,5,6,7,8,8a,octahydroexo-7,8-epoxy-l,4-methanonaphthalene). The razorbill (Alca torda), guillemot (Uria aalge), shag (Phalacrocorax aristotelis), cormorant (Phalacrocorax carbo) and Manx shearwater (Puffinus puffinus) had much lower activities than the puffin (Fratercula arctica). When these values were compared with other data from the literature on a scale of relative activity, the high activities of the puffin were similar to those of mammals and certain omnivorous or herbivorous birds, whereas the low activities of the other five species were similar to those of fish (Walker & Knight, 1981). In the three auks, enzyme activities were highly variable and resolved into two or more peaks when frequency diagrams were plotted. This was not so with the shag. In the razorbill, individuals of low activity (Group I) were compared with individuals of higher activity (Group II) (Knight & Walker, 1982). Individuals from Group II had, on average, 5.6-8.5-fold higher monooxygenase activity at saturating substrate concentrations than did Group I, although there was only a 1.6-fold difference in microsomal cytochrome P-450 between the groups. Lineweaver-Burke plots for 416 Marine Environ. Res. 0141-1136/84/$03.00 © Elsevier Applied Science Publishers Ltd,
England, 1984. Printed in Great Britain.
Hepatic microsomal monooxygenases o]sea birds
417
aldrin epoxidation gave apparent K m values of 25-56 and 3-17 nmol/mg protein for Groups I and II respectively. This suggests that Group II possesses a form (or forms) of monooxygenase of relatively high affinity for aldrin which is either at low concentration or absent from Group I. The difference in activity between the groups increased as the concentration of aldrin fell towards levels which are realistic for organochlorine pollutants. A plot for trout liver microsomes fell between those for the two groups of razorbills, whilst a plot for male rat liver microsomes showed that this species maintains a much higher activity than the others down to environmentally realistic concentrations. Thus, at relatively low substrate concentration, the rat and the razorbills of Group II showed higher hepatic microsomal monooxygenase activities than razorbills of Group I or trout. This separation of monooxygenase activities into two or more peaks in razorbills and other auks was not related to sex, time of season, geographical location or tissue levels of organochlorine pollutants. In view of this, and the fact that no such distribution was found in the sedentary shag which was feeding in the same areas, it seemed unlikely that the more active individuals had been induced by pollutants. Had induction occurred during the breeding season, a similar effect might have been anticipated in the shag; also substantial differences in the extent of induction might have been expected between areas as different as the Isle of May and the Saltee Islands. Had induction occurred in the auks during migration it should have declined or disappeared during the breeding season, and might have been expected to bear some relationship to organochlorine residues, the class of sea-borne pollutants most likely to cause a long-lasting induction of microsomal enzymes. More probably, these variations are an expression of genetic differences. The highly active individuals may be representatives of resistant strains which have arisen as a consequence of the selective pressure of PCBs and other persistent pollutants over many years. PCBs were implicated in the large kill of guillemots and razorbills in the Irish Sea in 1969, which was followed by substantial reductions in numbers of breeding birds at Welsh and Irish coastal sites in 1970 (Knight & Walker, 1982). To test the rate of elimination of a persistent organochlorine compound by a species with low monooxygenase activity, cormorants were given dieldrin by i.p. injection. Dieldrin concentrations were determined in blood samples, and in whole carcases at death. Rates of loss of dieldrin were determined as a percentage of original dose (carcases) or
418
c. H. Walker, G. C. Knight, J. K. Chipman, M. J. J. Ronis
of initial concentration in blood. The rate of loss of dieldrin from whole bodies after dosing at 1.5 mg/kg was similar to that from blood after a dose of 5mg/kg, but somewhat slower than that from blood after 1.5 mg/kg. The loss of half the original dose from the body was estimated to occur in 20-30 days. This value may be compared with half-lives estimated for male rats which contained similar tissue levels of dieldrin (Robinson et al., 1969; Moriarty, 1975). After feeding rats 10ppm dieldrin in their diet for 8 weeks, 0.23 ppm of the chemical was found in brain. Cormorants given a single i.p. injection of dieldrin were estimated to contain 0.1-0.2 ppm of dieldrin in brain. Thus the rats had, evidently, somewhat higher tissue concentrations than the cormorants at the start of experimentation. The loss of chemical from adipose tissue and brain of rats was biphasic: in the case of adipose tissue, which accounts for much of the body burden of dieldrin, half the original concentration was lost within 4 days. In another experiment, rats given single i.v. doses of 0.25 mg/kg excreted 50 ~ of the dose in bile within 2'5 days (Bedford & Hutson, 1976). In these experiments male rats lost half of their body load of dieldrin within 4 days, a rate which was at least 5 times more rapid than that found in cormorants in the present study. This adds to the growing body of evidence that the rate of elimination of liposoluble organochlorine pollutants can be limited by low rates of metabolism (Walker, 1981). The very low monooxygenase activities found in five species of sea birds should result in slow rates of metabolism of persistent pollutants, and correspondingly efficient bioaccumulation. This work was supported by a grant from the NERC. The authors are grateful to Mr S. Hallam for skilled technical assistance and to Dr D. C. Cabot and Dr M. P. Harris and their colleagues for arranging the collection of sea birds under licence.
REFERENCES Bedford, C. T. & Hutson, D. H. (1976). The comparative metabolism in rodents of the isomeric insecticides dieldrin and endrin. Chem. Ind. 15 May, 440 -7. Knight, G. C. & Walker, C. H. (1982). A study of the hepatic microsomal monooxygenase of sea birds and its relationship to liposoluble pollutants. Comp. Biochem. Physiol. 73C, 211-21. Moriarty, F. (1975). Exposure and residues. In: Organochlorine insecticides. persistent organic pollutants (Moriarty, F. (Ed.)). Academic Press, London, 29 72.
Hepatic microsomal monooxygenases oJ sea birds
419
Robinson, J., Roberts, M., Baldwin, M. & Walker, A. I. T. (1969). The pharmacokinetics of HEOD (Dieldrin) in the rat. Fd Cosmet. Toxicol. 7, 317-22. Walker, C. H. (1981). The correlation between in vivo and in vitro metabolism of pesticides in vertebrates. Progr. Pestic. Biochem. 1,247-85. Walker, C. H. & Knight, G. C. (1981). The hepatic microsomal enzymes of sea birds and their interaction with liposoluble pollutants. Aquat. Toxicol. 1, 343 -54.