Hepatic phenylalanine hydroxylase of. Clethrionomys glareolus Schreber as a bioindicator of pollution. JOHN DONLON, BRIGID FALLON, PATRICK BARRETT,.
S64 Biochemical Society Transactions (1998) 26 76 Hepatic phenylalanine hydroxylase of Clethrionomys glareolus Schreber as a bioindicator of pollution
JOHN DONLON, BRIGID FALLON, PATRICK BARRETT, OLIVER CARROLL, PHILIP HENDERSON, and JAMES S. FAIRLEY* Departments of Biochemistry and *Zoology, University College, Galway, Ireland Recent studies have yielded some successes in terms of finding a wild rodent suitable as a terrestrial bioindicator of pollution (1-2). Other developments in the search for animal and piscine biomarkers of pollution have been reviewed (3-4). We set out to identify hepatic enzymes (other than the members of the cytochromeP-450 family) in f e d rodents that might serve as useful biomarkers of pollution. This has potential advantages as they are mammals living permanently in the field which are both common and easy to live-trap. Thus, we choose to study hepatic phenylalanine hydroxylase in two species of wild rodents:- the bank vole Clethrionomys glureolus Schreber and field mouse Apodernus sylvuticus (L). In this study, the specific activities of hepatic phenylalanine hydroxylase of bank voles and field mice were determined for groups of animals trapped at a site of suspected industrial pollution (mainly sulphur dioxide) and two reference sites. Rodents were caught in Longworth live traps, set overnight baited with oats. Each animal was killed by crushing the cranium, sexed, weighed and the liver removed, weighed and frozen in liquid nitrogen and stored at -7OOC. The control areas were two wildlife reserves administered by the National Parks and Wildlife Service. The study area was in the Askeaton district on the southern banks of Shannon Estuary where there are known to be emissions of sulphur dioxide and nitrogen oxides and where there have been a number of unexplained deaths of cattle. Sulphur dioxide is by far the most significant emissions to air in the region (up to 100,OOO tonnes annually); emanating from three major sources this constitutes 55% of national annual S @ emissions (Interim Report issued by EPA, Dublin, September 1995). The frozen livers were carefully thawed, cut into small pieces and homogenised in 3 volumes of cold 0.15 M KCI and this homogenate was centrifuged at 35,000 g for 45 min at 4OC; the supernatants were collected and unused amounts were stored at -2BC. Protein was determined by the biuret method using bovine serum albumin as standard. Phenylalanine hydroxylase activity was measured by colorimetricdetermination (5)of tyrosine formed in the presence of 5 mM dithiothreitol (6), 1 mM phenylalanine and 20 p M tetrahydrobiopterin ( B a ) or 5 mM phenylalanine and 0.5 mM dimethyltetrahydropterin (DMPHq). SDS polyacrylamide gel electrophoresis and western blotting experiments were carried out according to accepted procedures (7). Pol yclonal antisera raised against purified rat hepatic phenylalanine hydroxylase were used as source of primary antibody. The specific activities of hepatic phenylalanine hydroxylase using the two cofactors B a and D M P a for field mice are shown in Table 1 and for bank voles in Table 2. In both control and contaminated sites these were lower in voles than in mice. Another difference between the two species is that the enzyme from the field mice is relatively moreactive with BHq than with the synthetic cofactors, as determined from the ratio of activity with DMPHq to that with BHq, which reflects a more activated state of the enzyme (8-9). Whereas there was no significantdifference in specific activities between mice from the contaminated and reference sites, the specific activities of hepatic phenylalanine hydroxylase of voles were significantly diminished at the contaminated site. In prtcular male voles had only 27% of control Bfi-dependent activity (p < 0.01) and 43% of control DMPHq-dependent activity (p < 0.001). Similarly, female bank voles also showed serious losses of DMPfi-dependent activities (p < 0.01 in both instances). When the levels of immunoreactive protein were compared (western blotting) it was observed that the affected male bank voles had a normal amount of phenylalanine hydroxylase protein. Thus we conclude that the loss of catalytic activity in bank voles from the study site is due to a loss of catalwc activity by normal levels of enzyme protein.
Table 1. Scecific activitiesof hepatic Dhenvlalanine hvdroxvlase from field mice. Values are means (nmol tyrosine formed / min / mg protein) & standard error. Site
n
Sex BH4
Reference sites Askeaton
16 32 19 18
Female Male Female Male
Cofactor DMPl-4
0.77 f 0.15 0.97 f 0.08 0.93 f 0.10 1.15 f 0.21
5.00 f 0.49 6.07 f 0.38 5.97 f 0.50 6.17 f 0.67
Table 2. Specific activitiesof hepatic phenvlalanine hvdroxvlasefrom bank voles. Values are means (nmol tyrosine formed / min / mg protein) standard error. Site
n
Sex BH4
Reference sites Askeaton
21 22 13 10
Female Male Female Male
Cofactor DWfi
0.23 f 0.02 0.33 f 0.05 0.19 f 0.02 0.09 f 0.02a
2.73 f 0.20 2.47 f 0.25 1.40 f 0.28a 1.05 f 0.20b
P values verxus respective controls: a, < 0.01; b, < 0.001. The distinctly lower amount of phenylalanine hydroxylase in the livers of the bank voles than in those of field mice from the control sites may be related to their diet which, being biased to green plant cells and fruit flesh, rather than the predominantly endosperm and animal food taken by the mice, is likely to contain higher amounts of fructose. In the case of laboratory rats, we have observed (Walsh, Guerin and Donlon, see this volume) that feeding a diet containing high amounts of fructose leads to a significant diminution in the amount of hepatic phenylalanine hydroxylase. Our observations show a serious loss of hepatic phenylalanine hydroxylase among bank voles collected at a site close to a sources of atmospheric contamination, with potentially deleterious effects on the tetrahydrobiopterin-dependentactivity of this enzyme among males of the species. Similar studies on bank voles may be of value in monitoring the environmental / ecological effects of atmospheric pollUtants.
Acknowledgements We wish to thank b i n MacLoughlin for help in fieldwork; Thomas Guerin for his assistance with western blotting experiments and the National Parks and Wildlife service for permission to trap on its properties. References 1. Bhatia, A., Tobil, F., Lepschy, G., Werk, X. and Mazzucco, K. (1994) Chemosphere 28, 1525-1537 2. Schrenk, D., Lipp, H.-P., Brunner, H., Wiesmiiller, T., Hagenmaier, H.and Bock, K.W. (1991) Chemosphere 2 2 , 10111018 3. Peakall, D.B. (1991) Animal Biomarkers as Pollution Indicators. Chapman and Hall, London 4. Payne, J.F., Fancey, L.L., Rahimtula, A.D.and P0rter.E.L. (1987) Comp. Biochem. Physiol. 8 6 C , 233-245 5. Kaufman, S and Fisher, D.B. (1970) J. Biol. Chem. 245, 47454750 6.Bublitz, C. (1%9) Biochim. Biophys. Acta 19 1, 249-256 7. De Maio, A.( 1994) Protein Blotting: A Practical Approach, pp. 11-32, I R L Press at Oxford University Press Inc., NY 8. Donlon, J. and Beirne E. (1992) Biochem. Biophys. Res. Commun. 108,746-751 9. Kaufman, S. (1986) Adv. in Enzyme Regulation 2 5,37-64
