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Aortic arylhydrocarbon hydroxylase activity in atherosclerosis-susceptible and atherosclerosis-resistant species
Comp. Biochem. Physiol. Vol. 73B, No. 3, pp. 669 to 671, 1982 Printed in Great Britain.
0305-0491/82/110669-03503.00/0 Pergamon Press Ltd
AORTIC ARYLHYDROCARBON HYDROXYLASE ACTIVITY IN ATHEROSCLEROSIS-SUSCEPTIBLE AND ATHEROSCLEROSIS-RESISTANT SPECIES STUART I. HOGG* and ANTHONY fRYER Department of Biochemistry, University College, P.O. Box 78, Cardiff, CF1 1XL, Wales, U.K.
(Received 9 February 1982) Abstract--1. Arylhydrocarbon hydroxylase activity has been measured in the aortae of two strains of pigeon, the atherosclerosis-susceptible White Carneau and the atherosclerosis-resistant Show Racer. The activity in aortae taken from the rabbit, an atherosclerosis-susceptible species and the rat, an atherosclerosis-resistant species was also studied. 2. Aortic arylhydrocarbon hydroxylase activity was found to be present in both of the atherosclerosissusceptible species, but absent from both of the atherosclerosis-resistant species. 3. Aortic arylhydrocarbon hydroxylase activity was not induced by 3-methylcholanthrene in any of the species studied.
INTRODUCTION In industrialised societies, the polycycli6 aromatic hydrocarbons are an environmentally ubiquitous group of compounds. Of these compounds the most extensively studied is benzo(a)pyrene, a constituent of smokes and other environmental pollutants. In common with the majority of carcinogenic organic xenobiotics, benzo(a) pyrene must first undergo bioactivation for its oncogenic potential to be realised (for review, see Juchau, 1981). Benzo(a)pyrene is metabolised by the microsomal mixed functional oxidase arylhydrocarbon hydroxylase (EC 1.14.14.2) to reactive metabolites capable of covalently binding to nucleic acids and other macromolecules, so potentially giving rise to tumour formation (Miller & Miller, 1974). The metabolism of benzo(a)pyrene, together with the susceptibility of arylhydrocarbon hydroxylase to induction by a variety of agents has recently been reviewed (Gelboin, 1980). Arylhydrocarbon hydroxylase activity has been demonstrated in a number of tissues from a variety of species. The liver has consistently been found to be the tissue richest in the enzyme but lower levels of activity have been found in lung, intestine, adrenals and other extrahepatic tissues (Bast et al., 1980). In recent years, it has also been shown that arylhydrocarbon hydroxylase activity is present in homogenates prepared from the aortae of rabbits, chickens, monkeys and humans, and that the enzyme can be induced in this tissue by 3-methylcholanthrene or polychlorinated biphenyls (Juchau et al., 1976; Bond et al., 1979). Arylhydrocarbon hydroxylase activity and its induction have both been demonstrated in cultured human aortic smooth muscle cells (Juchau et al., 1979). On the basis of these observations, the latter authors suggested that the presence of this enzyme activity in the aorta may represent supportive evidence for the monoclonal theory of atherosclerosis,
* To whom all correspondence should be addressed. 669
first proposed by Benditt & Benditt (1973), and that the activated polycyclic hydrocarbon could act as an initiator of vascular smooth muscle proliferation. On the basis of these suggestions, the present paper compares the aortic activity of arylhydrocarbon hydroxylase in two strains of pigeon (Columba livia), the atherosclerosis-susceptible White Carneau (Clarkson et al., 1959; Prichard, 1965; Santerre et al., 1972) and the atherosclerosis-resistant Show Racer, together with the inducibility of the enzyme by 3-methycholanthrene. The study was extended to include another atherosclerosis-susceptible species, the rabbit (Oryctolagus cuniculus dora.) and another atherosclerosis-resistant species, the rat (Rattus nort;egicus). MATERIALS AND METHODS The sources of the animals (Columba livia) used in the experiments were as follows. Pigeons of the White Carneau strain were from a colony obtained originally from the Palmetto Pigeon Corporation, Sumpter, SC, U.S.A. and maintained in this Department (Ward & Cryer, 1981). Pigeons of the Show Racer strain were obtained from Abbot Bros, Thuxton, Norfolk, U.K. Rats of both the MRC hooded and Wistar strains, and rabbits of both the New Zealand White and the Dutch strains, were taken from colonies maintained in this Department. In all cases, mature animals of both sexes were used. Immediately following sacrifice, aortae were dissected from the animals and stripped of all extraneous material; they were then chopped finely with a scalpel and homogenised in 0.1 M sodium phosphate buffer pH 7.35, using a Potter Elvejhem, glass-teflon homogeniser. The homogenates were introduced into the incubations to give a total protein content of between 0.7 and 2.0 mg protein. Liver samples were also taken and 10% (w/v) homogenates in the same buffer were prepared give between 0.9 and 2.1 mg of protein per incubation. Arylhydrocarbon hydroxylase activity was determined using a modification of the method described by Bond et al. (1979). Homogenates of aorta or liver were incubated, with shaking, for 30 min at 37°C in a medium containing the following: NADPH (l.15mM, final concentration), MgCl2 (2.3raM), bovine serum albumin (0.5mg/ml), benzo(a)pyrene(80/IM),and sufficient Na2HPO4/NaH 2PO4
SIt;ARI I. Ho{;(; and ANI'HONY CRYER
670
butler 10.1 MI pH 7.35, to bring the total volume to 1.0 ml. Incubations were carried out in sealed tubes which had been previously gassed with oxygen. For each separate incubation, a control in which enzyme activity was prevented by the prior addition of 1.0 ml acetone, was also run. The benzola)pyrene, which was dissolved in methanol. was added immediately before the commencement of the incubation. Enzyme activity in the test incubations was terminated by the addition, after 30 min, of 1.0 ml acetone. 3.25 ml of hexane was then added to each tube and the mixture was incubated with shaking at 37'C for a further 10 min. A 1-ml sample of the resulting organic phase was removed, and hydroxylated benzo(a)pyrene was extracted using 3 ml of 1 M-NaOH. The concentration of the hydroxylated products present in the extract was then determined using a Perkin Elmer Fluorescence Spectrophotometer (MPF-2A) with an excitation wavelength of 396 nm and with fluorescence detection at 522 nm. Known concentrations of quinine sulphate in 0.05 M-H2SO~t were used Io calibrate the instrument. Protein was determined by the method of Lowry et al. (1951) using dry bovine serum albumin as a standard. In experiments designed to determine the level of indt, ction of enzyme activity, animals were injected intraperitoneally with 3-methylcholanthrene in corn oil (40 mg/kg body wt) 48 hr prior to sacrifice. Control animals were injected with corn oil only.
RESU LTS A r y l h y d r o c a r b o n hydroxylase activity was determined as described in the Materials and M e t h o d s section and the activity expressed according to the definition of Nebert & Gelboin (1968). Thus, one unit of activity is defined as the a m o u n t of enzyme that catalyzes the formation, in a 30-min incubation at 3 7 C , of hydroxylated benzo(a)pyrene product causing a fluorescence equivalent to 1 pmol of 3-hydroxybenzo(a)pyrene. Activities were calculated on the basis that 36 pmol/ml of authentic 3-hydroxybenzo(a)pyrene emits a fluorescence equal to that given by 0.3/~g/ml of quinine sulphate in 0.05 M-H2SO,~ at the appropriate wavelengths (Rickert & Fouts, 1970). Figure 1 shows the a r y l h y d r o c a r b o n hydroxylase activity determined in h o m o g e n a t e s of liver and aorta taken from the four animal species under study. In all four species, arylhydrocarbon hydroxylase activity was detectable in h o m o g e n a t e s of liver. The activities for the four species were found to cover a wide range of values: in addition to this interspecies variation the individual values a m o n g a single group also showed a wide distribution, as observed previously with mouse (Gelboin et al., 1972). In view of this, relatively large n u m b e r s of animals (compared to some previous studies on the activity of the enzyme) were used. A statistical analysis of the activities was not found to be useful however because of the wide variations a n d the data in Fig. 1 illustrate the activities recorded for each individual animal. The widest range of hepatic activities was recorded for the White Carneau pigeon with the rat and the Show Racer pigeon showing the lowest values and the least variation. Although all four species showed considerable levels of hepatic enzyme activity, only the rabbit and the White Carneau pigeons had any detectable activity present in the aorta (Fig. 1). Relatively large n u m b e r s of rats (two strains) and Show Racer pigeons were studied without any individual being found with
measurable arylhydrocarbon hydroxylase activity. The enzyme activity detected in the aortae of rabbil and White Carneau pigeons, although well within the limits of detection of the assay system, were low compared to the hepatic values of all four species. The range of activities in the aortae of White Carneau pigeons was wider than that of the rabbits. W h e n groups of animals were pretreated by the intraperitoneal injection of 3-methylcholanthrene, no induction of aortic arylhydroxylase activity was observed in any of the animals lTable 11. The activit) present in the liver was induced to varying degrees b ) this pretreatmenk the effect being particularly pronounced in the case of the rat (Table 1/. DISCUSSION The reactions involved in the metabolism of environmental xenobiotics are generally classified according to a two-phase system (Williams, 1959). The first phase for c o m p o u n d s such as polycyclic hyd r o c a r b o n s is an oxidative process catalyzed by the so-called mixed function oxidase group of enzymes (Mason. 1957). The result of one such oxidation is the hydroxylated derivative of benzo{a)pyrene, formed by the action of a r y l h y d r o c a r b o n hydroxylase. Although the subsequent conjugation reaction ("phase lI"l involves reactions that are well characterised in a n u m b e r of tissues (Hirom & Millburn~ 1981), no studies have as yet indicated whether such reactions occur in vascular tissues. However, the presence of
2oo;. •
i/.
1!1~
.AORTA
LIVER
Fig. 1. Arylhydrocarbon hydroxylase activities of homogenates of liver and aorta of four animal species. Activities, expressed as U per mg of protein, were determined as described in the Materials and Methods section and each symbol represents the value for an individual animal.
Aortic arylhydrocarbon hydroxylase activity Table I. The effect of pretreatment with 3-methylcholanthrene on the arylhydrocarbon hydroxylase activities of hepatic and aortic homogenates Degree of induction Liver Mean value White Carneau pigeon 3.42 Show Racer pigeon 1.37 Rat 37.07
Aorta
Range O-13.39 0-4.1 7.38-90.88
ND ND ND
n= 8 n= 3 n= 5
ND = None detected. ~'Degree of Induction" is obtained by comparing values for pretreated animals with the mean value for untreated animals.
arylhydrocarbon hydroxylase activity in the aortae of atherosclerotic species could provide the potential for the local formation of carcinogenic derivatives that, as suggested in studies on h u m a n aortae, could stimulate smooth muscle proliferation during the initiation of a t h e r o m a t o u s plaque formation (see Benditt & Gown, 1980). The presence of arylhydrocarbon hydroxylase activity in the aortae of atherosclerosis-susceptible species and its absence in those of atherosclerosis-resistant species may further suggest a predisposition in the aortic tissue of atherosclerosis-susceptible species to the effect of environmental carcinogens, and represents a potentially i m p o r t a n t difference between the two groups of animals. Acknowledgements--We wish to thank Mr G. E. Ward for his technical assistance with the pigeon colony. This work was made possible by the financial assistance provided by the British Heart Foundation (Grant No. 784).
REFERENCES BAST R. C. JR, BASTB. S. & GELaOIN H. V. (1980) Extrahepatic Metabolism of Drugs and Other Foreign Compounds (Edited by GRAM T. E.), pp. 27%294. S. P. Medical and Scientific Books. BENDITT E. P. & BENDITT J. M. (1973) Evidence for a monoclonal origin of human atherosclerotic plaques. Proc. HatH. Acad. Sci. U.S.A. 70, 1753-1756. BENDITT E. P. & GOWN A. M. (1980) Atheroma: the artery wall and the environment. Int. Rev. exp. Pathol. 21, 55 118.
671
BOND J. A., OMIECINSKI C. J. 8z JUCHAU M. R. (1979) Kinetics, activation and induction of aortic mono-oxygenases biotransformation of benzo(a)pyrene. Biochem. Pharmac. 28, 305-311. CLARKSON T. B., PRICHARD R. W., NETSKY M. G. 8z, LOPLAND H. B. (1959) Atherosclerosis in pigeons. Archs Path. 68, 143-147. GELBOIN H. V. (1980) Benzo(a)pyrene metabolism, activation and carcinogenesis: role and regulation of mixedfunction oxidases and related enzymes. Physiol. Rev. 60, 1107-1166. GELBOIN H. V., KINOSHITAN. & WIEBEL F. J. (1972) Microsomal hydroxylases: induction and role in polycyclic hydrocarbon carcinogenesis and toxicity. Fedn Proc. Fedn Am. Socs exp. Biol. 31, 1298-1309. HIROM P. C. & MILLBURN P. (1981) Foreign Compound Metabolism in Mammals. Vol. 6, p.. 111-132. The Royal Society of Chemistry, London. JUCHAU M. R. (1981) The Biochemical Basis of Chemical Teratogenesis (Edited by JUCHAU M. R.), pp. 63-94. Elsevier/N. Holland, New York & Amsterdam. JUCHAU M. R., BOND J. A. & BENDITT E. P. (1976) Aryl 4-monooxygenase and cytochrome P-450 in the aorta: possible role in atherosclerosis. Proc. HatH. Acad. Sci. U.S.A. 73, 3723-3725. JUCHAU M. R., BOND J. A., KOCAN R. A. & BENDITT E. P. (1979) Proc. Third Int. Symp. Polynuclear Aromatic Hydrocarbons (Edited by JONES P. W. & LEBER P.), p. 639. Ann Arbor Science, Ann Arbor, Michigan. LOWRY O. H., ROSEBROUGHN. J., PARR A. U & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MASON H. S. (1957) Mechanisms of oxygen metabolism. Adv. Enzymol. 19, 79-223. MILLER E. C. & MILLER J. A. (1974) Molecular Biology o[ Cancer (Edited by BUSCH H., pp. 377-401. Academic Press, New York. NEBERT D. W. & GELaOIN H. V. (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J. biol. Chem. 243, 6242-6249. PRIC8ARD, R. W. (1965) Comparative atherosclerosis (Edited by ROBERTS J. C. & STRAPS R.), pp. 45-49. Harper & Row, Publishers, New York. RICKERT D. E. & POUTS J. R. (1970) Benzpyrene pretreatmerit changes the kinetics and pH optimum for aniline hydroxylation in vitro but not those for benzphetamine demethylation in vitro by rat liver microsomes. Biochem. Pharmac. 19, 381-390. SANTERRE R. F., WIGHT T. N., SMITH S. C. t~ BRANNIGAN D. (1972) Spontaneous atherosclerosis in pigeons. Am. J. Path. 67, 1 22. WARD G. E. & CRYER A. (1981) The establishment and maintenance of a colony of White Carneau Pigeons. J. Inst. Anita. Tech. 32, 71 76. WILLIAMS R. T. (1959) Detoxication Mechanisms, 2nd edn. Chapman & Hall, London.
0305-0491/82/110669-03503.00/0 Pergamon Press Ltd
AORTIC ARYLHYDROCARBON HYDROXYLASE ACTIVITY IN ATHEROSCLEROSIS-SUSCEPTIBLE AND ATHEROSCLEROSIS-RESISTANT SPECIES STUART I. HOGG* and ANTHONY fRYER Department of Biochemistry, University College, P.O. Box 78, Cardiff, CF1 1XL, Wales, U.K.
(Received 9 February 1982) Abstract--1. Arylhydrocarbon hydroxylase activity has been measured in the aortae of two strains of pigeon, the atherosclerosis-susceptible White Carneau and the atherosclerosis-resistant Show Racer. The activity in aortae taken from the rabbit, an atherosclerosis-susceptible species and the rat, an atherosclerosis-resistant species was also studied. 2. Aortic arylhydrocarbon hydroxylase activity was found to be present in both of the atherosclerosissusceptible species, but absent from both of the atherosclerosis-resistant species. 3. Aortic arylhydrocarbon hydroxylase activity was not induced by 3-methylcholanthrene in any of the species studied.
INTRODUCTION In industrialised societies, the polycycli6 aromatic hydrocarbons are an environmentally ubiquitous group of compounds. Of these compounds the most extensively studied is benzo(a)pyrene, a constituent of smokes and other environmental pollutants. In common with the majority of carcinogenic organic xenobiotics, benzo(a) pyrene must first undergo bioactivation for its oncogenic potential to be realised (for review, see Juchau, 1981). Benzo(a)pyrene is metabolised by the microsomal mixed functional oxidase arylhydrocarbon hydroxylase (EC 1.14.14.2) to reactive metabolites capable of covalently binding to nucleic acids and other macromolecules, so potentially giving rise to tumour formation (Miller & Miller, 1974). The metabolism of benzo(a)pyrene, together with the susceptibility of arylhydrocarbon hydroxylase to induction by a variety of agents has recently been reviewed (Gelboin, 1980). Arylhydrocarbon hydroxylase activity has been demonstrated in a number of tissues from a variety of species. The liver has consistently been found to be the tissue richest in the enzyme but lower levels of activity have been found in lung, intestine, adrenals and other extrahepatic tissues (Bast et al., 1980). In recent years, it has also been shown that arylhydrocarbon hydroxylase activity is present in homogenates prepared from the aortae of rabbits, chickens, monkeys and humans, and that the enzyme can be induced in this tissue by 3-methylcholanthrene or polychlorinated biphenyls (Juchau et al., 1976; Bond et al., 1979). Arylhydrocarbon hydroxylase activity and its induction have both been demonstrated in cultured human aortic smooth muscle cells (Juchau et al., 1979). On the basis of these observations, the latter authors suggested that the presence of this enzyme activity in the aorta may represent supportive evidence for the monoclonal theory of atherosclerosis,
* To whom all correspondence should be addressed. 669
first proposed by Benditt & Benditt (1973), and that the activated polycyclic hydrocarbon could act as an initiator of vascular smooth muscle proliferation. On the basis of these suggestions, the present paper compares the aortic activity of arylhydrocarbon hydroxylase in two strains of pigeon (Columba livia), the atherosclerosis-susceptible White Carneau (Clarkson et al., 1959; Prichard, 1965; Santerre et al., 1972) and the atherosclerosis-resistant Show Racer, together with the inducibility of the enzyme by 3-methycholanthrene. The study was extended to include another atherosclerosis-susceptible species, the rabbit (Oryctolagus cuniculus dora.) and another atherosclerosis-resistant species, the rat (Rattus nort;egicus). MATERIALS AND METHODS The sources of the animals (Columba livia) used in the experiments were as follows. Pigeons of the White Carneau strain were from a colony obtained originally from the Palmetto Pigeon Corporation, Sumpter, SC, U.S.A. and maintained in this Department (Ward & Cryer, 1981). Pigeons of the Show Racer strain were obtained from Abbot Bros, Thuxton, Norfolk, U.K. Rats of both the MRC hooded and Wistar strains, and rabbits of both the New Zealand White and the Dutch strains, were taken from colonies maintained in this Department. In all cases, mature animals of both sexes were used. Immediately following sacrifice, aortae were dissected from the animals and stripped of all extraneous material; they were then chopped finely with a scalpel and homogenised in 0.1 M sodium phosphate buffer pH 7.35, using a Potter Elvejhem, glass-teflon homogeniser. The homogenates were introduced into the incubations to give a total protein content of between 0.7 and 2.0 mg protein. Liver samples were also taken and 10% (w/v) homogenates in the same buffer were prepared give between 0.9 and 2.1 mg of protein per incubation. Arylhydrocarbon hydroxylase activity was determined using a modification of the method described by Bond et al. (1979). Homogenates of aorta or liver were incubated, with shaking, for 30 min at 37°C in a medium containing the following: NADPH (l.15mM, final concentration), MgCl2 (2.3raM), bovine serum albumin (0.5mg/ml), benzo(a)pyrene(80/IM),and sufficient Na2HPO4/NaH 2PO4
SIt;ARI I. Ho{;(; and ANI'HONY CRYER
670
butler 10.1 MI pH 7.35, to bring the total volume to 1.0 ml. Incubations were carried out in sealed tubes which had been previously gassed with oxygen. For each separate incubation, a control in which enzyme activity was prevented by the prior addition of 1.0 ml acetone, was also run. The benzola)pyrene, which was dissolved in methanol. was added immediately before the commencement of the incubation. Enzyme activity in the test incubations was terminated by the addition, after 30 min, of 1.0 ml acetone. 3.25 ml of hexane was then added to each tube and the mixture was incubated with shaking at 37'C for a further 10 min. A 1-ml sample of the resulting organic phase was removed, and hydroxylated benzo(a)pyrene was extracted using 3 ml of 1 M-NaOH. The concentration of the hydroxylated products present in the extract was then determined using a Perkin Elmer Fluorescence Spectrophotometer (MPF-2A) with an excitation wavelength of 396 nm and with fluorescence detection at 522 nm. Known concentrations of quinine sulphate in 0.05 M-H2SO~t were used Io calibrate the instrument. Protein was determined by the method of Lowry et al. (1951) using dry bovine serum albumin as a standard. In experiments designed to determine the level of indt, ction of enzyme activity, animals were injected intraperitoneally with 3-methylcholanthrene in corn oil (40 mg/kg body wt) 48 hr prior to sacrifice. Control animals were injected with corn oil only.
RESU LTS A r y l h y d r o c a r b o n hydroxylase activity was determined as described in the Materials and M e t h o d s section and the activity expressed according to the definition of Nebert & Gelboin (1968). Thus, one unit of activity is defined as the a m o u n t of enzyme that catalyzes the formation, in a 30-min incubation at 3 7 C , of hydroxylated benzo(a)pyrene product causing a fluorescence equivalent to 1 pmol of 3-hydroxybenzo(a)pyrene. Activities were calculated on the basis that 36 pmol/ml of authentic 3-hydroxybenzo(a)pyrene emits a fluorescence equal to that given by 0.3/~g/ml of quinine sulphate in 0.05 M-H2SO,~ at the appropriate wavelengths (Rickert & Fouts, 1970). Figure 1 shows the a r y l h y d r o c a r b o n hydroxylase activity determined in h o m o g e n a t e s of liver and aorta taken from the four animal species under study. In all four species, arylhydrocarbon hydroxylase activity was detectable in h o m o g e n a t e s of liver. The activities for the four species were found to cover a wide range of values: in addition to this interspecies variation the individual values a m o n g a single group also showed a wide distribution, as observed previously with mouse (Gelboin et al., 1972). In view of this, relatively large n u m b e r s of animals (compared to some previous studies on the activity of the enzyme) were used. A statistical analysis of the activities was not found to be useful however because of the wide variations a n d the data in Fig. 1 illustrate the activities recorded for each individual animal. The widest range of hepatic activities was recorded for the White Carneau pigeon with the rat and the Show Racer pigeon showing the lowest values and the least variation. Although all four species showed considerable levels of hepatic enzyme activity, only the rabbit and the White Carneau pigeons had any detectable activity present in the aorta (Fig. 1). Relatively large n u m b e r s of rats (two strains) and Show Racer pigeons were studied without any individual being found with
measurable arylhydrocarbon hydroxylase activity. The enzyme activity detected in the aortae of rabbil and White Carneau pigeons, although well within the limits of detection of the assay system, were low compared to the hepatic values of all four species. The range of activities in the aortae of White Carneau pigeons was wider than that of the rabbits. W h e n groups of animals were pretreated by the intraperitoneal injection of 3-methylcholanthrene, no induction of aortic arylhydroxylase activity was observed in any of the animals lTable 11. The activit) present in the liver was induced to varying degrees b ) this pretreatmenk the effect being particularly pronounced in the case of the rat (Table 1/. DISCUSSION The reactions involved in the metabolism of environmental xenobiotics are generally classified according to a two-phase system (Williams, 1959). The first phase for c o m p o u n d s such as polycyclic hyd r o c a r b o n s is an oxidative process catalyzed by the so-called mixed function oxidase group of enzymes (Mason. 1957). The result of one such oxidation is the hydroxylated derivative of benzo{a)pyrene, formed by the action of a r y l h y d r o c a r b o n hydroxylase. Although the subsequent conjugation reaction ("phase lI"l involves reactions that are well characterised in a n u m b e r of tissues (Hirom & Millburn~ 1981), no studies have as yet indicated whether such reactions occur in vascular tissues. However, the presence of
2oo;. •
i/.
1!1~
.AORTA
LIVER
Fig. 1. Arylhydrocarbon hydroxylase activities of homogenates of liver and aorta of four animal species. Activities, expressed as U per mg of protein, were determined as described in the Materials and Methods section and each symbol represents the value for an individual animal.
Aortic arylhydrocarbon hydroxylase activity Table I. The effect of pretreatment with 3-methylcholanthrene on the arylhydrocarbon hydroxylase activities of hepatic and aortic homogenates Degree of induction Liver Mean value White Carneau pigeon 3.42 Show Racer pigeon 1.37 Rat 37.07
Aorta
Range O-13.39 0-4.1 7.38-90.88
ND ND ND
n= 8 n= 3 n= 5
ND = None detected. ~'Degree of Induction" is obtained by comparing values for pretreated animals with the mean value for untreated animals.
arylhydrocarbon hydroxylase activity in the aortae of atherosclerotic species could provide the potential for the local formation of carcinogenic derivatives that, as suggested in studies on h u m a n aortae, could stimulate smooth muscle proliferation during the initiation of a t h e r o m a t o u s plaque formation (see Benditt & Gown, 1980). The presence of arylhydrocarbon hydroxylase activity in the aortae of atherosclerosis-susceptible species and its absence in those of atherosclerosis-resistant species may further suggest a predisposition in the aortic tissue of atherosclerosis-susceptible species to the effect of environmental carcinogens, and represents a potentially i m p o r t a n t difference between the two groups of animals. Acknowledgements--We wish to thank Mr G. E. Ward for his technical assistance with the pigeon colony. This work was made possible by the financial assistance provided by the British Heart Foundation (Grant No. 784).
REFERENCES BAST R. C. JR, BASTB. S. & GELaOIN H. V. (1980) Extrahepatic Metabolism of Drugs and Other Foreign Compounds (Edited by GRAM T. E.), pp. 27%294. S. P. Medical and Scientific Books. BENDITT E. P. & BENDITT J. M. (1973) Evidence for a monoclonal origin of human atherosclerotic plaques. Proc. HatH. Acad. Sci. U.S.A. 70, 1753-1756. BENDITT E. P. & GOWN A. M. (1980) Atheroma: the artery wall and the environment. Int. Rev. exp. Pathol. 21, 55 118.
671
BOND J. A., OMIECINSKI C. J. 8z JUCHAU M. R. (1979) Kinetics, activation and induction of aortic mono-oxygenases biotransformation of benzo(a)pyrene. Biochem. Pharmac. 28, 305-311. CLARKSON T. B., PRICHARD R. W., NETSKY M. G. 8z, LOPLAND H. B. (1959) Atherosclerosis in pigeons. Archs Path. 68, 143-147. GELBOIN H. V. (1980) Benzo(a)pyrene metabolism, activation and carcinogenesis: role and regulation of mixedfunction oxidases and related enzymes. Physiol. Rev. 60, 1107-1166. GELBOIN H. V., KINOSHITAN. & WIEBEL F. J. (1972) Microsomal hydroxylases: induction and role in polycyclic hydrocarbon carcinogenesis and toxicity. Fedn Proc. Fedn Am. Socs exp. Biol. 31, 1298-1309. HIROM P. C. & MILLBURN P. (1981) Foreign Compound Metabolism in Mammals. Vol. 6, p.. 111-132. The Royal Society of Chemistry, London. JUCHAU M. R. (1981) The Biochemical Basis of Chemical Teratogenesis (Edited by JUCHAU M. R.), pp. 63-94. Elsevier/N. Holland, New York & Amsterdam. JUCHAU M. R., BOND J. A. & BENDITT E. P. (1976) Aryl 4-monooxygenase and cytochrome P-450 in the aorta: possible role in atherosclerosis. Proc. HatH. Acad. Sci. U.S.A. 73, 3723-3725. JUCHAU M. R., BOND J. A., KOCAN R. A. & BENDITT E. P. (1979) Proc. Third Int. Symp. Polynuclear Aromatic Hydrocarbons (Edited by JONES P. W. & LEBER P.), p. 639. Ann Arbor Science, Ann Arbor, Michigan. LOWRY O. H., ROSEBROUGHN. J., PARR A. U & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MASON H. S. (1957) Mechanisms of oxygen metabolism. Adv. Enzymol. 19, 79-223. MILLER E. C. & MILLER J. A. (1974) Molecular Biology o[ Cancer (Edited by BUSCH H., pp. 377-401. Academic Press, New York. NEBERT D. W. & GELaOIN H. V. (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J. biol. Chem. 243, 6242-6249. PRIC8ARD, R. W. (1965) Comparative atherosclerosis (Edited by ROBERTS J. C. & STRAPS R.), pp. 45-49. Harper & Row, Publishers, New York. RICKERT D. E. & POUTS J. R. (1970) Benzpyrene pretreatmerit changes the kinetics and pH optimum for aniline hydroxylation in vitro but not those for benzphetamine demethylation in vitro by rat liver microsomes. Biochem. Pharmac. 19, 381-390. SANTERRE R. F., WIGHT T. N., SMITH S. C. t~ BRANNIGAN D. (1972) Spontaneous atherosclerosis in pigeons. Am. J. Path. 67, 1 22. WARD G. E. & CRYER A. (1981) The establishment and maintenance of a colony of White Carneau Pigeons. J. Inst. Anita. Tech. 32, 71 76. WILLIAMS R. T. (1959) Detoxication Mechanisms, 2nd edn. Chapman & Hall, London.