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Hydrocarbon metabolism and cortisol balance in oil-exposed ringed seals, Phoca hispida
03064492~82~03013344603.0010 Q 1982 Pcrgamon Press Ltd
Camp. Biochem. Physiol. Vol. 72C. No. 1. pp. 133-136. 1982 Printed in Great Britain
HYDROCARBON METABOLISM AND CORTISOL BALANCE IN OIL-EXPOSED RINGED SEALS, PHOCA HISPZDA F. RAINER ENGELHARDT Department
of Indian Affairs and Northern Development. Northern Environmental Ottawa. Ontario KlA OH4. Canada
Protection.
(Receioed 19 August 1981)
Abstract-l.
Ringed seals were exposed experimentally to oil contamination. by feeding of a [‘4C]naphthalene marked crude oil in fish for up to 4 days at a rate of 5 ml/day. 2. Mixed function oxygenase (MFO) activity. measured as aryl hydrocarbon hydroxylase in liver and kidney, was found to be induced, in particular in kidney tissue where the activity increased 3-fold. 3. MFO induction correlated with a high degree of conversion of crude oil hydrocarbons to watersoluble metabolites. Most of the radioactivity was found in the polar fraction of plasma and urine. 4. Plasma cortisol levels were somewhat elevated by captive holding. and increased markedly after oil-exposure. Cortisol half-life decreased after oil exposure from 13 to 1 hr.
lNTRODUCTlON Increased activity in the development of marine, and particularly arctic, hydrocarbon reserves has given additional relevance to a continued assessment of the effects of petroleum contamination on marine mammals. Only few data exist for this group of animals which would allow accurate generalizations or predictions of oil effects. Sea otters (Enhydra lutris), for instance, have been shown to experience extensive thermoregulatory distress involving increased metabolism to compensate for heat loss (Costa & Kooyman, 1980). Polar bears (Ursus maritimus) as well experience thermal problems when coated with oil (0ritsland et al., 1981). Oil ingestion and uptake in polar bears led to serious clinical disturbances, in particular anemia and renal insufficiency (Engelhardt, 1981). Clinical and thermoregulatory responses have been measured by Geraci & Smith (1976) in ringed seals (Phoca hispida) and in harp seal (Phoca groenlandim) pups, following acute exposure to crude oil by immersion and ingestion. Clinical effects were minimal, but eye lesions resulted from 24 hr of exposure by the seals to a simulated oil slick. No thermoregulatory distress was noted. Petroleum hydrocarbons were absorbed, however, and deposited in an array of tissues, whether exposure was by immersion or ingestion (Engelhardt et al., 1977; Engelhardt. 1978). A relatively rapid clearance of stored hydrocarbons occurred, probably involving the metabolic conversion of hydrocarbons for excretion in urine and bile. While such studies demonstrate that there can be an effect of oil exposure on marine mammals, either serious in the case of sea otters and polar bears or less so in the two species of seals examined, these experiments have only been acute exposures assessing overt responses. For various reasons, including logistical problems of working with large mammals or in the field in the north, these experiments have not been able to assess chronic exposures, nor have they identified physiological mechanisms and functions which may be involved in chronic responses.
One of the consequences of exposures to a variety of lipophihc contaminants is an induction of the activity of a number of tissue enzymes of the mixed function oxygenase(MFO)system, especially in hepatic tissue. These enzymes are mainly microsomal oxygenases involving the cytochrome P450 system, reviewed by Sato & Omura (1978). Polychlorinated biphenyls, various pesticides, and polyaromatic hydrocarbons have been variously suggested to have an inducing effect on the MFO system (De Bruin, 1976; Hodgson, 1974; Payne, 1977). It is the effects of polyaromatic hydrocarbons as found in petroleum which will be implicated in this study. A possible chronic response is that the induction of the MFO system may result in imbalances in steroid hormones, compromising reproduction, stress responses and other steroid-regulated functions. The microsomal system, particularly as linked to cytochrome P450 in the liver, is known also to metabolize steroid hormones (Brenner, 1977; Conney & Klutch, 1963; Omura, 1978; Sato, 1978). It can be proposed that an induction of the enzyme system by contaminants can cause alterations in steroid metabolism by an increased breakdown rate. Although no quantitative dose-biochemical response relationships are defined, it is recognized that steroid hormone attributed abnormalities frequently accompany pollution by lipophilic organics in fish and marine mammals (Britton, 1975; Davidson & Sell, 1974; Helle et al., 1976; Nava, 1980; Peakall, 1967). The study described here does not test MFO or steroid responses as a consequence of chronic hydrocarbon contamination, but rather seeks to identify initially a mechanism which may be induced by oil exposure in seals, where such an induction results in an increased metabolism of contaminant hydrocarbons and steroids, and a possible steroid imbalance. METHODS AND MATERIALS Ringed seals were captured by net at Cape Parry, N.W.T. Four juvenile seals, l-2yr old and in good con133
F.
134
RAINER E~GELHARDT
dition at 27-33 kg in weight, were used in the oil exposure experiment. Dosing was carried out for 4 days ar a rate of 5 ml of Norman Wells crude oil’dav, fed in 100 II of arctic char. The oil was radiolabelled with a naphthalene marker. at a sp. act. of 3.7 pCi!pmol [‘JC]naphthalene per 3.7 ml oil. Blood samples were taken both immediately before the onset of oil dosing and the day after 4 daily doses had been given. A cortisol clearance study was carried out just before treatment, as well as on the Sth day of the experiment. An additional 4 animals were shot and represented comparative wild control values. Blood samples for hydrocarbon and cortisol determinations were taken from plantar vessels into ammonium heparin. Sampling was restricted to the forenoon, with exception of the cortisol clearance study, in order to minimize the effect of diurnal variations. Tissues were taken immediately post mortem and then frozen until analysis. Mixed function oxidase activity was determined in liver and kidney tissues of 4 wild ringed seals and in the 4 oil-exposed seals. The analysis was for aryl hydrocarbon hydroxylase (AHH). based on a Huorometric measurement of the rate of formation of 3-OH-benzo[u]pyrene from a benzo[a]pyrene substrate by tissue microsomes in accordance with the method of Nebert & Gelboin (1968). Hydrocarbon determinations were carried out for the naphthalene marker using hexane extraction of a tissue homogenate, carried out for I2 hr at 4’C under agitation. The method was standardized and had an over 95”, extraction efficiency for unmodified labelled crude oil. The hexane fraction represented non-polar hydrocarbons, while the water-based extracted remainder was defined to retain mainly polar metabolized hydrocarbons. Radioactivity was determined in each fraction by liquid scintillation counting (LSC). Total hydrocarbon residues were assessed in liver and kidney tissues by a steam distillation-spectrotluorometry method described by Engelhardt et al. (1977). -
Cortisol levels were determined in plasma by radioimmunoassay (RIA), using Corning Immo-Phase methods and reagents as described in Engelhardt & Ferguson (1980). A cortisol clearance test was carried out before and on the day following 4 days of oil ingestion. Each seal was i.p. injected with 4OpCi of radiolabelled cortisol (sp. act. 1 mCij4.78 pg [‘Hlcortisol) in 2 ml ethanol and saline, 1: 10. The rate of disappearance of cortisol from circulation was assessed by blood sampling every hour for 6 hr. RESULTS
Ingestion of the crude oil doses in fish evidenced no unusual behavioral disturbances. There was some evidence of diarrhea and a copious production of darkly colored urine in all seals. Aryl hydrocarbon hydroxylase activity was present in both liver and kidney tissues, and was found to be greater especially in kidney tissues after oil exposure. as compared to levels found in wild ringed seals (Table 1). The great majority of radioactivity of the naphthalene label in oil was present as polar residues when measured in plasma and urine (Table 2). Although the polar fractions of tissues were not assayed, it was found that hexane extracts of liver and kidney and muscle tissues contained only trace amounts of radioactivity, whereas a low-metabolic tissue such as blubber showed high activity in hexane extracts, at 300-800 dpmig. Total hydrocarbon levels were found to be correspondingly low in liver and kidney, at
averaging
at less than
14 ng;ml
(Table
3).
Table I. Effect of oil ingestion at 5 ml,‘day on mixed function oxygenase induction, measured as aryl hydrocarbon hydroxylase activity in ringed seal (Phoccl hispida) liver and kidney Condition 2 day oil-exposed 4 day oil-exposed
Non-exposed
Enzyme activity Liver Kidney
Seal no. 1
3.5
2 3 4 Average 5 6 7 8 Average
4.7 4.1 21.3 8.4 3.5 8.3 6.5 6.1
13.0 5.9 10.6 2.4 6.7 2.4 2.9 1.2 I.2 1.9
Activity is expressed in pmolimg microsomal protein of 3-OH benzo(a)pyrene formed.
Captivity
and the experimental
handling
caused
an
approximately 4-fold increase in plasma cortisol, which was elevated further by exposure to crude oil. Oil treatment caused increases of 59-408?; in cortisol as compared to the pre-exposure captive levels. An additional cortisol change was a shortened ha!flife in circulation after oil exposure. The pre-oil halflife of an injected label was calculated to be an average of 1; hr, decreasing after 4 days of oil ingestion to an average of I hr. Each of the three seals tested showed a decrease. DlSCUSSION
The effectiveness of hydrocarbon metabolism in ringed seals was evidenced previously by a rapid clearance of oil fractions after exposure by ingestion (Engelhardt et (II., 1977; Engelhardt, 1978). A dose of 5 ml oil,‘day was given for several days in those studies as well, resulting in tissue accumulation during the dosing period. Clearance mechanisms effected an apparently exponential decrease so that only trace amounts of oil hydrocarbons remained in tissues by 1 month after exposure. Metabolic activation of the MFO system can be assumed to be responsible, shown by increased AHH leve!s in kidney tissue in the study reported. Seals are somewhat unusual in that it was especially kidney tissue rather than liver
Table 2. Activity (dpmiml) in rmged seal (Phocn hispido) plasma and urine following ingestion of a labelled crude oil, 5 PCi [14C]naphthalene;5 ml oil per day Sample Non-polar fracrion Plasma Urine Polar fraction Plasma Urine *T--trace
quantities.
2 days Seal 5
Seal 6
4 days Seal 7
T*
T
T
Seal 8 T
66
100
2050 1360 17.100 63.-100 59.200
1270 -
109
300
Hydrocarbons
and cortisol in ringed seals
135
Table 3. Effect of oil ingestion at 5 ml/day on plasma cortisol balance in the ringed seal (Phoca hispida) Plasma concentration Condition Wild, non-exposed
Captive. non-exposed
Captive. oil-exposed
Seal no.
1
ng/ml
X change
Half-life (hr)
2 3 4 Average 5 6 7 8 Average 5
14 < 12.5 < 12.5 16 <14 60 66 105 13 61 248
+313
I& 2 lf 24 13 -
6 7 8 Average
105 180 66 150
+59 +71 +408 +213
1t lt t 1
which was induced. Other enzymes of the MFO system may have shown a different tissue specificity in response. A high MFO detoxification capacity was
shown by the great differential between polar or metabolized and non-polar fractions. The several hundredfold greater proportion of polar fractions in urine correlates with a high renal MFO activity. It is of interest to compare the clearance responses of polar bears to these findings. A massive oil exposure in these animals resulted in hydrocarbon uptake and retention, extensive even 1 month after the oiling event (Engelhardt, 1981). It may be speculated that the bears showed a less effective MFO induction, although evidence in support of this hypothesis is not available. There are a number of considerations to these finding which need elaboration. As a consequence of the seal study being carried out in the field, there was undoubtedly some loss of AHH activity due to handling. Although the tissues were frozen immediately, 2 weeks elapsed before they could be stored at -70°C. Further, the assay was specific for the formation of phenols, and thus may have underestimated metabolism potential since other products may also have been formed. The integrity of neither hepatic nor renal tissues had likely been affected, since Geraci & Smith (1976) showed no significant pathology resulting from either oil immersion or ingestion. The polar bear study as well showed a lack of liver involvement as a toxic response to oil (Engelhardt, 1981; F. A. Juck in Britsland et al., 1981). This does not, however, preclude a biochemical lesion in these tissues. It has been shown, although not in marine mammals, that polyaromatic hydrocarbons can inhibit enzyme activities, such as ATPases (Ikemoto et al., 1969; Kaminski et al., 1970). Inhibition of ion-dependent ATPases resulting from the in ho and in oitro presence of aromatic hydrocarbons has been demonstrated in fish (Wong & Engelhardt, 1981). Four days of exposure to oil resulted in elevations of cortisol levels in circulation. This relatively nonspecific stress response was not likely a factor in the induction of AHH. The enzyme was not inducible by
even high prolonged
cortisol levels in other studies (Nava, 1980). It is not suggested that it is the AHH induction which was instrumental in an increased cortisol breakdown and shortened half-life. Aryl hydrocarbon hydroxylase was used only as a marker for the induction of the MFO system, including enzymes specific for adrenocorticosteroids (Sato and Omura, 1978). It would now be of great interest to determine to what degree the increased cortisol breakdown is balanced by greater synthesis. Cortisol has been indicated to have a functional role in harp seals, probably involved in thermogenic and fasting lipid metabolism, as well as water balance (Engelhardt & Ferguson, 1980). The annual molt cycle involves cortisol regulation in phocid seals such as the harbor seal (Phoca uituha) and the harp seal (Engelhardt & Ferguson 1980; Riviere et al., 1977). A disturbance in cortisol cycle and balance, or its response capacity, may be expected to be deleterious to seals. The ultimate question arising is whether an eventual adreno-cortical exhaustion for cortisol and other steroids would result from chronic polyaromatic hydrocarbon exposure. This question is continued to be investigated in marine mammals, assessing not only polyaromatic hydrocarbons but also other relevant contaminants found to be associated with reproductive and other physiological problems in seals and cetaceans. Acknowledgements-1 would like to express my appreciation to Drs T. G. Smith and J. R. Geraci for continued access to and support at the Cape Parry field laboratory. This study was supported by grants to the author from the Natural Sciences and Engineering Research Council and from the Department of Indian Affairs and Northern Development through the Northern Research Fund at the University of Ottawa.
REFERENCES BRENNERR. R. (1977) Metabolism of endogenous substrates by microsomes. Drug Metub. Rev. 6, 155-212. BRITTONW. M. (1975) Influence of high levels of DDT in
136
F. RAINERENGELIURDT
the diet on liver microsomal estrogen metabolism in the laying hen. Bull. enuir. Contam. Toxic. 13. 698-702. CONNEYA. H. & KLUTCH A. (1963) Increased activity of androgen hydroxylases in liver microsomes of rats pretreated with phenobarbital and other drugs. .I. biol. Chem. 238, 161l-1617. COSTAD. P. & K~~YMANG. L. (1980) Effects of oil contamination in the sea otter, Enhydra lurris. Rep. Outer Continental Shelf Environmental Assessment Program, N.O.A.A., Alaska. DAVISONK. & SELLJ. (1974) Dieldrin and DDT effects on reproduction and some hepatic mixed function oxidases in the mallard duck. Archs encir. Conram. Toxic. 2, 302-314. DE BRUN A. (1976) Biochemical Toxicology of Enkonmental Agents. Elsevier-North Holland, New York. ENGELHARDT F. R. (1978) Petroleum hydrocarbons in arctic ringed seals. Phoca hispida, following experimental oil exposure. Proc. Assessment Ecol. Impacts Oil Spills, pp. 614-428. Keystone, CO, June 1978. American Institute of Biological Sciences. ENGELHARDTF. R. (1981) Oil pollution in polar bears: exposure and clinical effects. Proc. 4rh Arctic Marine Oil Spill Tech. Seminnr, pp. 139-179. Environmental Protection Service, Canada. Edmonton, June 1981. ENGELHARDTF. R. & FERGU~CJN J. M. (1980) Adaptive hormone changes in harp seals, Phoca groenlandica. and grey seals, Halichoerus grypus, during the postnatal period. Gen. romp. Endocr. 40, 43-5. ENGELHARDTF. R., GERACI J. R. & SMITH T. G. (1977) Uptake and clearance of petroleum hydrocarbons in the rinned seal. Phoca hisoida. J. Fish. Res. Bd Can. 34. 11~3-1147.’ GERACI J. R. & SMITH T. G. (1976) Direct and indirect effects of oil on ringed seals (Phoca hispida) of the Beaofort Sea. J. Fish. Res. Bd Can. 33, 19761984. HELLEE., OLSSONM. & JENSENS. (1976) DDT and PCB levels and reproduction in ringed seals from the Bothnian Bay. Ambio 5. 261-263. HODG~ONE. (1974) Comparative studies of cytochrome P-450 and its interaction with pesticides. In Surcival in
Toxic Enoironmenrs (Edited by KHAN M. A. 0. & BEDERKAJ. P., Jr.), pp. 213-260. Academic Press, New York. IKEMOT~F., IWATAS. & NARI~AN. (1969) Studies on the enucleated lens 3. Cation content in naphthalene cataractous lens. J. pharmac. Sot. Jap. 89,745-749. KAMINSKI M., KARBOWSKI A. & JONEKJ. (1970) Some histochemical renal changes in mice during acute poisoning with benzene. Folia Hisrochem. Cvtochem. 8. 63-75. NAVA M. E. L. (1980) Petroleum effects on mixed function oxidases and cortisol balance in the North American eel, Anguilla rostrata M.Sc. thesis, University of Ottawa. NEBERTD. W. & GELB~XNH. V. (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. J. biol. Chem. 243, 6242-6249.
OMURAT. (1978) Cytochrome P-450 containing oxygenase systems. In Cytochrome P-450 (Edited by SATO R. & OUURAT.), pp. 138-163. Academic Press, New York. GRITSLANDN. A., ENGELHARDT F. R., JUCK F. A., HURST R. & WATTSP. W. (1981) Effect of crude oil on polar bears. Dep. Indian @airs and Northern Development Canada, Publ. No. QS-8283-020-EE-Al Environmental Studies Report, No. 24. PAYNE J. F. (1977) Mixed function oxidases in marine organisms in relation to petroleum hydrocarbon metabolism and detection. Mar. Pollur. Bull. 8, 112-116. PEAKALLD. B. (1967) Pesticide induced enzyme breakdown of steroids in birds. Nature, Land. 216. 505-506. RIVIEREJ. E., ENGELHARDT F. R. & SOLOMON J. (1977) The relationship of thyroxine and cortisol to the moult of the harbour seal Phoca citulina. Gen. camp. Endocr. 31, 398-401.
SATOR. (1978) Distribution and physiological functions. In C_vrochrome P-450 (edited by SATOR. & OMURAT.), pp. 23-25. Academic Press, New York. SATOR. & OMURAT. (1978) Cyrochrome P-450. Academic Press, New York. WONG M. P. & ENGELHARDTF. R. (1981) The effects of petroleum hydrocarbons on ionic balance and branchial ATPases in rainbow trout. Proc. Symp. int. Sot. Petroleum Industry Biologisrs, Denver, September 1981.
Camp. Biochem. Physiol. Vol. 72C. No. 1. pp. 133-136. 1982 Printed in Great Britain
HYDROCARBON METABOLISM AND CORTISOL BALANCE IN OIL-EXPOSED RINGED SEALS, PHOCA HISPZDA F. RAINER ENGELHARDT Department
of Indian Affairs and Northern Development. Northern Environmental Ottawa. Ontario KlA OH4. Canada
Protection.
(Receioed 19 August 1981)
Abstract-l.
Ringed seals were exposed experimentally to oil contamination. by feeding of a [‘4C]naphthalene marked crude oil in fish for up to 4 days at a rate of 5 ml/day. 2. Mixed function oxygenase (MFO) activity. measured as aryl hydrocarbon hydroxylase in liver and kidney, was found to be induced, in particular in kidney tissue where the activity increased 3-fold. 3. MFO induction correlated with a high degree of conversion of crude oil hydrocarbons to watersoluble metabolites. Most of the radioactivity was found in the polar fraction of plasma and urine. 4. Plasma cortisol levels were somewhat elevated by captive holding. and increased markedly after oil-exposure. Cortisol half-life decreased after oil exposure from 13 to 1 hr.
lNTRODUCTlON Increased activity in the development of marine, and particularly arctic, hydrocarbon reserves has given additional relevance to a continued assessment of the effects of petroleum contamination on marine mammals. Only few data exist for this group of animals which would allow accurate generalizations or predictions of oil effects. Sea otters (Enhydra lutris), for instance, have been shown to experience extensive thermoregulatory distress involving increased metabolism to compensate for heat loss (Costa & Kooyman, 1980). Polar bears (Ursus maritimus) as well experience thermal problems when coated with oil (0ritsland et al., 1981). Oil ingestion and uptake in polar bears led to serious clinical disturbances, in particular anemia and renal insufficiency (Engelhardt, 1981). Clinical and thermoregulatory responses have been measured by Geraci & Smith (1976) in ringed seals (Phoca hispida) and in harp seal (Phoca groenlandim) pups, following acute exposure to crude oil by immersion and ingestion. Clinical effects were minimal, but eye lesions resulted from 24 hr of exposure by the seals to a simulated oil slick. No thermoregulatory distress was noted. Petroleum hydrocarbons were absorbed, however, and deposited in an array of tissues, whether exposure was by immersion or ingestion (Engelhardt et al., 1977; Engelhardt. 1978). A relatively rapid clearance of stored hydrocarbons occurred, probably involving the metabolic conversion of hydrocarbons for excretion in urine and bile. While such studies demonstrate that there can be an effect of oil exposure on marine mammals, either serious in the case of sea otters and polar bears or less so in the two species of seals examined, these experiments have only been acute exposures assessing overt responses. For various reasons, including logistical problems of working with large mammals or in the field in the north, these experiments have not been able to assess chronic exposures, nor have they identified physiological mechanisms and functions which may be involved in chronic responses.
One of the consequences of exposures to a variety of lipophihc contaminants is an induction of the activity of a number of tissue enzymes of the mixed function oxygenase(MFO)system, especially in hepatic tissue. These enzymes are mainly microsomal oxygenases involving the cytochrome P450 system, reviewed by Sato & Omura (1978). Polychlorinated biphenyls, various pesticides, and polyaromatic hydrocarbons have been variously suggested to have an inducing effect on the MFO system (De Bruin, 1976; Hodgson, 1974; Payne, 1977). It is the effects of polyaromatic hydrocarbons as found in petroleum which will be implicated in this study. A possible chronic response is that the induction of the MFO system may result in imbalances in steroid hormones, compromising reproduction, stress responses and other steroid-regulated functions. The microsomal system, particularly as linked to cytochrome P450 in the liver, is known also to metabolize steroid hormones (Brenner, 1977; Conney & Klutch, 1963; Omura, 1978; Sato, 1978). It can be proposed that an induction of the enzyme system by contaminants can cause alterations in steroid metabolism by an increased breakdown rate. Although no quantitative dose-biochemical response relationships are defined, it is recognized that steroid hormone attributed abnormalities frequently accompany pollution by lipophilic organics in fish and marine mammals (Britton, 1975; Davidson & Sell, 1974; Helle et al., 1976; Nava, 1980; Peakall, 1967). The study described here does not test MFO or steroid responses as a consequence of chronic hydrocarbon contamination, but rather seeks to identify initially a mechanism which may be induced by oil exposure in seals, where such an induction results in an increased metabolism of contaminant hydrocarbons and steroids, and a possible steroid imbalance. METHODS AND MATERIALS Ringed seals were captured by net at Cape Parry, N.W.T. Four juvenile seals, l-2yr old and in good con133
F.
134
RAINER E~GELHARDT
dition at 27-33 kg in weight, were used in the oil exposure experiment. Dosing was carried out for 4 days ar a rate of 5 ml of Norman Wells crude oil’dav, fed in 100 II of arctic char. The oil was radiolabelled with a naphthalene marker. at a sp. act. of 3.7 pCi!pmol [‘JC]naphthalene per 3.7 ml oil. Blood samples were taken both immediately before the onset of oil dosing and the day after 4 daily doses had been given. A cortisol clearance study was carried out just before treatment, as well as on the Sth day of the experiment. An additional 4 animals were shot and represented comparative wild control values. Blood samples for hydrocarbon and cortisol determinations were taken from plantar vessels into ammonium heparin. Sampling was restricted to the forenoon, with exception of the cortisol clearance study, in order to minimize the effect of diurnal variations. Tissues were taken immediately post mortem and then frozen until analysis. Mixed function oxidase activity was determined in liver and kidney tissues of 4 wild ringed seals and in the 4 oil-exposed seals. The analysis was for aryl hydrocarbon hydroxylase (AHH). based on a Huorometric measurement of the rate of formation of 3-OH-benzo[u]pyrene from a benzo[a]pyrene substrate by tissue microsomes in accordance with the method of Nebert & Gelboin (1968). Hydrocarbon determinations were carried out for the naphthalene marker using hexane extraction of a tissue homogenate, carried out for I2 hr at 4’C under agitation. The method was standardized and had an over 95”, extraction efficiency for unmodified labelled crude oil. The hexane fraction represented non-polar hydrocarbons, while the water-based extracted remainder was defined to retain mainly polar metabolized hydrocarbons. Radioactivity was determined in each fraction by liquid scintillation counting (LSC). Total hydrocarbon residues were assessed in liver and kidney tissues by a steam distillation-spectrotluorometry method described by Engelhardt et al. (1977). -
Cortisol levels were determined in plasma by radioimmunoassay (RIA), using Corning Immo-Phase methods and reagents as described in Engelhardt & Ferguson (1980). A cortisol clearance test was carried out before and on the day following 4 days of oil ingestion. Each seal was i.p. injected with 4OpCi of radiolabelled cortisol (sp. act. 1 mCij4.78 pg [‘Hlcortisol) in 2 ml ethanol and saline, 1: 10. The rate of disappearance of cortisol from circulation was assessed by blood sampling every hour for 6 hr. RESULTS
Ingestion of the crude oil doses in fish evidenced no unusual behavioral disturbances. There was some evidence of diarrhea and a copious production of darkly colored urine in all seals. Aryl hydrocarbon hydroxylase activity was present in both liver and kidney tissues, and was found to be greater especially in kidney tissues after oil exposure. as compared to levels found in wild ringed seals (Table 1). The great majority of radioactivity of the naphthalene label in oil was present as polar residues when measured in plasma and urine (Table 2). Although the polar fractions of tissues were not assayed, it was found that hexane extracts of liver and kidney and muscle tissues contained only trace amounts of radioactivity, whereas a low-metabolic tissue such as blubber showed high activity in hexane extracts, at 300-800 dpmig. Total hydrocarbon levels were found to be correspondingly low in liver and kidney, at
averaging
at less than
14 ng;ml
(Table
3).
Table I. Effect of oil ingestion at 5 ml,‘day on mixed function oxygenase induction, measured as aryl hydrocarbon hydroxylase activity in ringed seal (Phoccl hispida) liver and kidney Condition 2 day oil-exposed 4 day oil-exposed
Non-exposed
Enzyme activity Liver Kidney
Seal no. 1
3.5
2 3 4 Average 5 6 7 8 Average
4.7 4.1 21.3 8.4 3.5 8.3 6.5 6.1
13.0 5.9 10.6 2.4 6.7 2.4 2.9 1.2 I.2 1.9
Activity is expressed in pmolimg microsomal protein of 3-OH benzo(a)pyrene formed.
Captivity
and the experimental
handling
caused
an
approximately 4-fold increase in plasma cortisol, which was elevated further by exposure to crude oil. Oil treatment caused increases of 59-408?; in cortisol as compared to the pre-exposure captive levels. An additional cortisol change was a shortened ha!flife in circulation after oil exposure. The pre-oil halflife of an injected label was calculated to be an average of 1; hr, decreasing after 4 days of oil ingestion to an average of I hr. Each of the three seals tested showed a decrease. DlSCUSSION
The effectiveness of hydrocarbon metabolism in ringed seals was evidenced previously by a rapid clearance of oil fractions after exposure by ingestion (Engelhardt et (II., 1977; Engelhardt, 1978). A dose of 5 ml oil,‘day was given for several days in those studies as well, resulting in tissue accumulation during the dosing period. Clearance mechanisms effected an apparently exponential decrease so that only trace amounts of oil hydrocarbons remained in tissues by 1 month after exposure. Metabolic activation of the MFO system can be assumed to be responsible, shown by increased AHH leve!s in kidney tissue in the study reported. Seals are somewhat unusual in that it was especially kidney tissue rather than liver
Table 2. Activity (dpmiml) in rmged seal (Phocn hispido) plasma and urine following ingestion of a labelled crude oil, 5 PCi [14C]naphthalene;5 ml oil per day Sample Non-polar fracrion Plasma Urine Polar fraction Plasma Urine *T--trace
quantities.
2 days Seal 5
Seal 6
4 days Seal 7
T*
T
T
Seal 8 T
66
100
2050 1360 17.100 63.-100 59.200
1270 -
109
300
Hydrocarbons
and cortisol in ringed seals
135
Table 3. Effect of oil ingestion at 5 ml/day on plasma cortisol balance in the ringed seal (Phoca hispida) Plasma concentration Condition Wild, non-exposed
Captive. non-exposed
Captive. oil-exposed
Seal no.
1
ng/ml
X change
Half-life (hr)
2 3 4 Average 5 6 7 8 Average 5
14 < 12.5 < 12.5 16 <14 60 66 105 13 61 248
+313
I& 2 lf 24 13 -
6 7 8 Average
105 180 66 150
+59 +71 +408 +213
1t lt t 1
which was induced. Other enzymes of the MFO system may have shown a different tissue specificity in response. A high MFO detoxification capacity was
shown by the great differential between polar or metabolized and non-polar fractions. The several hundredfold greater proportion of polar fractions in urine correlates with a high renal MFO activity. It is of interest to compare the clearance responses of polar bears to these findings. A massive oil exposure in these animals resulted in hydrocarbon uptake and retention, extensive even 1 month after the oiling event (Engelhardt, 1981). It may be speculated that the bears showed a less effective MFO induction, although evidence in support of this hypothesis is not available. There are a number of considerations to these finding which need elaboration. As a consequence of the seal study being carried out in the field, there was undoubtedly some loss of AHH activity due to handling. Although the tissues were frozen immediately, 2 weeks elapsed before they could be stored at -70°C. Further, the assay was specific for the formation of phenols, and thus may have underestimated metabolism potential since other products may also have been formed. The integrity of neither hepatic nor renal tissues had likely been affected, since Geraci & Smith (1976) showed no significant pathology resulting from either oil immersion or ingestion. The polar bear study as well showed a lack of liver involvement as a toxic response to oil (Engelhardt, 1981; F. A. Juck in Britsland et al., 1981). This does not, however, preclude a biochemical lesion in these tissues. It has been shown, although not in marine mammals, that polyaromatic hydrocarbons can inhibit enzyme activities, such as ATPases (Ikemoto et al., 1969; Kaminski et al., 1970). Inhibition of ion-dependent ATPases resulting from the in ho and in oitro presence of aromatic hydrocarbons has been demonstrated in fish (Wong & Engelhardt, 1981). Four days of exposure to oil resulted in elevations of cortisol levels in circulation. This relatively nonspecific stress response was not likely a factor in the induction of AHH. The enzyme was not inducible by
even high prolonged
cortisol levels in other studies (Nava, 1980). It is not suggested that it is the AHH induction which was instrumental in an increased cortisol breakdown and shortened half-life. Aryl hydrocarbon hydroxylase was used only as a marker for the induction of the MFO system, including enzymes specific for adrenocorticosteroids (Sato and Omura, 1978). It would now be of great interest to determine to what degree the increased cortisol breakdown is balanced by greater synthesis. Cortisol has been indicated to have a functional role in harp seals, probably involved in thermogenic and fasting lipid metabolism, as well as water balance (Engelhardt & Ferguson, 1980). The annual molt cycle involves cortisol regulation in phocid seals such as the harbor seal (Phoca uituha) and the harp seal (Engelhardt & Ferguson 1980; Riviere et al., 1977). A disturbance in cortisol cycle and balance, or its response capacity, may be expected to be deleterious to seals. The ultimate question arising is whether an eventual adreno-cortical exhaustion for cortisol and other steroids would result from chronic polyaromatic hydrocarbon exposure. This question is continued to be investigated in marine mammals, assessing not only polyaromatic hydrocarbons but also other relevant contaminants found to be associated with reproductive and other physiological problems in seals and cetaceans. Acknowledgements-1 would like to express my appreciation to Drs T. G. Smith and J. R. Geraci for continued access to and support at the Cape Parry field laboratory. This study was supported by grants to the author from the Natural Sciences and Engineering Research Council and from the Department of Indian Affairs and Northern Development through the Northern Research Fund at the University of Ottawa.
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