Xenobiotica the fate of foreign compounds in biological systems
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Hepatic mixed-function oxidases of ferret J. Shavila, C. Ioannides, L. J. King & D. V. Parke To cite this article: J. Shavila, C. Ioannides, L. J. King & D. V. Parke (1992) Hepatic mixed-function oxidases of ferret, Xenobiotica, 22:8, 1003-1014, DOI: 10.3109/00498259209049906 To link to this article: http://dx.doi.org/10.3109/00498259209049906
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Date: 04 May 2016, At: 03:48
XENOBIOTICA,
1992, VOL. 22,
NO.
8, 1003-1014
Hepatic mixed-function oxidases of ferret
J. SHAVILA, C. IOANNIDES, L. J. K I N G and D. V. PARKET Molecular Toxicology Group, Division of Toxicology, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 SXH, U K
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Received 1 November 1991 ;accepted 1 April 1992
1 . Ferret liver mixed-function oxidase enzymes have been quantified using a variety of substrates and the activities have been compared with those found in rat liver. 2. Ferret liver total cytochrome P-450 is only 30% of that of rat liver and exhibits higher 7-ethoxyresorufin 0-deethylase (EROD) activity, and lower lauric acid hydroxylase activity than rat liver; other mixed-function oxidases are at similar levels of activity in both species. 3. Induction with 3-methylcholanthrene (MC), similar to MC-induction in rat, increases the total P-450 of ferret liver by 140%, but does not increase P-450 reductase or microsomal protein. EROD specific activity (pmol/min per mg protein) is increased 20fold by MC treatment.
4. Turnover number of EROD for control liver microsomes of ferret, hamster, mouse, guinea pig and rat were 460, 69, 44, 36 and 35pmol/min per nmol P-450, respectively, indicating the much higher value for ferret than for any of the rodent species studied. 5. Ferret liver EROD activity is inhibited by the P4501A1 inhibitor, anaphthoflavone. Use of monospecific antibodies in ELISA, Western blot and enzymeinhibition techniques has shown that EROD activity in ferret liver is attributable to two enzyme proteins orthologous with rat liver cytochromes P4501A1 and 1A2, with the former predominating. M C induces both P4501A enzyme proteins in ferret liver, as in rat liver, with P4501A1 activity predominating.
Introduction In recent years, the ferret (Mustella putoriusfuro) has been proposed as a nonrodent species for use in the safety evaluation of drugs and chemicals, as a surrogate for human and a less emotive substitute for the dog (Thornton et al. 1979). T h e high susceptibility of the ferret to organophosphate-induced delayed neuropathy, in contrast to rodents and most other laboratory animals, has led to this species being proposed as a suitable model for assessing the potential delayed neurotoxic effects of pesticides and other chemicals (Tanaka et al. 1991). On the other hand, the low susceptibility of the ferret to spontaneous neoplasia (Cotchin 1980) makes this animal an appropriate non-rodent species for carcinogenicity testing. However, only few studies have been published concerning the metabolism of drugs and toxic chemicals in this species (Ioannides et al. 1977, Pope et al. 1980). For these reasons it is important to evaluate more fully hepatic drug ?To whom correspondence should be addressed. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
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metabolism in the ferret, and to compare this, both qualitatively and quantitatively, with studies in rodents and man. Preliminary chronic toxicity studies carried out with ferrets revealed that this species was particularly susceptible to the development of fatty liver, normally an indicator of chemical toxicity. However, control untreated ferrets similarly developed fatty liver and it was considered that this condition was non-pathological and was probably diet-related. As administration of medium-chain length triglycerides to rats has been associated with changes in the cytochrome P-450 isoenzyme profiles in rat (Barnett et al. 1988, 1990a and b), it was considered that diet, and the development of fatty liver, in the ferret might well change the hepatic cytochrome P-450 isoenzyme pattern. Since the only drug metabolism studies in ferrets were conducted before the complex nature of the cytochrome P-450 superfamily was established (Ioannides et al. 1977), and only very limited isoenzyme studies were undertaken, it was considered highly desirable to undertake more detailed studies on the hepatic mixed-function oxidases and cytochromes P450 of ferret liver, and then to ascertain the effects of diet on these enzymic parameters. The present study was concerned with determining the constitutive drug-metabolizing cytochromes P-450 of ferret liver, and comparing their specific activities with those of rat liver; furthermore, as cytochrome P4501 activity appeared to be very much higher in ferret than in rat, the effects of pretreatment with 3-methylcholanthrene (MC), a specific inducer of cytochrome P4501, were also studied. Effects of diet on ferret liver mixed-function oxidases will be described in a subsequent publication.
Experimental Materials Cytochrome c, NADP, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, dimethyl sulphoxide, 3-methylcholanthrene, a-naphthoflavone, and dimethylnitrosamine (Sigma Chemical Co. Ltd., Poole, Dorset, UK); paracetamol (BDH, Poole, Dorset, UK); Soluene-350 and Dimilume (Packard Instrument Co., Reading, UK); 7-methoxy-, 7-ethoxy-, and 7-pentoxy-resorufin, and resorufin (Molecular Probes, Eugene, OR., USA); 7-benzyloxyresorufin (Boehringer, East Sussex, UK); ethylmorphine (Rhbne-Poulenc, Dagenham, Essex, UK); Glu-P-1 (Wako Fine Chemicals, Neuss 1, Germany); and 3H-paracetamol (Amersham International, Bucks., UK) were purchased. Benzphetamine (Upjohn Co., Kalamazoo, MI., USA) was a generous gift. Disposable plastic tubes and agar (London Analytical and Bacteriological Media Ltd., Salford, Lancs., UK), Oxoid nutrient broth No. 2 (Oxoid Ltd., Basingstoke, Hants., UK), and Salmonella typhzmurium, strain TA98 (a gift from Dr B. N. Ames, Berkeley, CA., USA) were used in the Ames test for Glu-P-1 activation. Animals Mature albino male ferrets (1.1-1.4 kg body weight) purchased from Grayston Breeders Ltd., Ringwood, Hants., UK, were housed individually under uniform laboratory conditions of 50% humidity at 22°C and 12 h light/dark cycle in the University of Surrey Experimental Biology Unit, and fed a diet of cat food (Pedigree Pet Foods, 40g/day) and dog biscuits (SDS Laboratory diets, 40g/day), with water available ad libitum, a diet which has been shown not to produce fatty liver in the ferret (Shavila 1990). Ferrets were acclimatized for 2 weeks before commencement of studies. Male Wistar albino rats (1 50-2008 body weight) and male mice (20-25 g body weight) University of Surrey, Experimental Biology Unit, and adult male Dunkin Hartley guinea pigs and golden Syrian hamsters, purchased from Grayston Breeders Ltd., Ringwood, Hants., were housed singly in cages with sawdust bedding and maintained under the same uniform laboratory conditions as the ferrets. Rats, mice, hamsters and guinea pigs were fed Spratts animal diet No. 1 and given water, ad libitum. All animal species were allocated randomly to the various experimental groups. For treatment with 3-methylcholanthrene (MC), animals were dosed i.p. with M C in corn oil (25rng/kg) once daily for 3 consecutive days after the 2-week acclimatization period. All animals were killed 24h after the last injection. Control animals received corn oil only. Ferrets were killed by exsanguination immediately following anaesthesia with ketamine (25 mg/kg) plus xylazine (2 mg/kg) administered intramuscularly (Moreland and Glaser 1985); all other animals
Ferret liver mixed-function oxidases
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were killed by cervical dislocation. Livers were removed, the gallbladders were removed from the livers of ferrets, mice and guinea pigs, and livers from each species were scissor-minced and then homogenized in 0 . 2 5 ~sucrose in a Potter-Elvehjem glass-tenon homogenizer to a liver concentration of 25% w/v. The homogenate was centrifuged at IOOOOg,, at 4°C for 30min, and the supernatant further centrifuged at 100 OOOg,, at 4°C for 60 min; the microsomal pellet was resuspended in 50 mM potassium phosphate buffer, p H 7.25, containing 20% v/v glycerol, further centrifuged at 100000 for 60min, and the washed microsomal pellet reconstituted in the phosphate-glycerol buffer for enzyme determinations.
Enzyme determinations Total cytochrome P-450 and cytochrome b, (Omura and Sat0 1964), NADPH-cytochrome c (P-450) reductase (Williams and Kamin 1962), and microsomal protein (Lowry et al. 1951) were determined, using the liver microsomal preparations. Lauric acid hydroxylase, quantifying the sum of 11- and 12-hydroxylauric acids (Parker and Orton 1980), ethylmorphine N-demethylase, benzphetamine N-demethylase and dimethylnitrosamine N-demethylase (60 mM) (Holtzman et al. 1968), and the 7-alkoxyresorufin 0-deethylases, 7-methoxy- (MROD), 7-ethoxy- (EROD), 7-pentoxy- (PROD), and 7-benzyloxy- (BROD) (Burke et al. 1989, were all determined using liver microsomal preparations. Inhibition of 7-ethoxyresorufin 0-deethylase (EROD) by the specific inhibitors of P4501, namely, a-naphthoflavone was studied using ferret and rat liver microsomal preparations and various concentrations of the inhibitor in DMSO. Inhibition of several alkoxyresorufin 0-dealkylase activities (EROD, PROD aiid BROD) by addition of monospecific polycional antibodies (2-25 mg protein/nmol total P-450) was studied by incubating liver microsomes from MC-treated ferrets (0.63 nmol P-450/mg protein) with anti-P4501Al and anti-P4501A2 (gifts from Dr C. R. Wolf, ICRF Unit, Edinburgh, Scotland), anti-P4502BI (a gift from Dr R. J. Makowski, University of Surrey) or pre-immune sera (control) in 0.1 ml phosphate-buffered saline, pH 7.4, for 35 min at room temperature, then further incubating with the alkoxyresorufin substrates and determining the production of resorufin in the normal manner. E L f S A anaIysis for P4501Al Enzyme-linked immunosorbent assay analysis (ELISA) of cytochrome P4501A1 present in ferret liver microsomes constitutively, or after pretreatment with MC, was carried out by the method of Voller et al. (1978). Microsomes were solubilized with sodium cholate (1 mg/mg protein) and Emulgen 911 (0.2 mg/mg protein). T h e primary antibody, anti-P4501Al, was diluted 1:lOOOO and applied to the wells (0.2 ml/well) which had previously been coated with microsomal protein (antigen); the wells initially incubated with pre-immune serum served as blank. Microsomal protein plus primary antibody was then incubated with the secondary antibody of donkey/anti-sheep labelled with horseradish peroxidase. The substrate solution (H,O, plus o-phenylenediamine) was added, and the plates incubated for 30min to develop the colour which was measured with a Dynatech plate reader at 492 nm.
Western blot analysis for cytochromes P-450 Immunoblot (Western blot) analysis was carried out as described by Towbin et al. (1979). Liver microsomes from ferrets and rats were subjected to SDS-polyacrylamide gel electrophoresis (Laemmli 1970) and transferred to nitrocellulose paper. After blocking with 10 mM phosphate-buffered saline, pH 7.4, containing 1yo w/w bovine serum albumin, the nitrocellulose was incubated with the polyclonal monospecific antibodies P4501A1 and A2 (raised in rabbit) at dilutions of 1:lOOOO. Detecting antibody (donkey-anti-rabbit) conjugated to horseradish peroxidase, was used at a dilution of 1:2000 in phosphate-buffered saline, p H 7.4, containing 1yo bovine serum albumin. Immunopositive bands were detected by treatment with diaminobenzidine chloride (1 mg/ml) and H,O, (30vol. diluted 1:SOOO) for 30s then rinsing the nitrocellulose paper with water. Enzymic reactions f o r cytochrome P4501A2 Metabolic activation of the precarcinogen Glu-P-1 (2 pg/plate) to mutagenic intermediates, an enzymic reaction catalyzed by P4501A2, was determined using the Ames test (Maron and Ames 1983) and employing a fresh, overnight culture of S. typhimurium strain TA98, in the presence of 10% v/v ferret liver microsomal preparation from control and MC-treated ferrets supplemented with glucose 6phosphate dehydrogenase (1 unitlplate). The mixed precarcinogen, bacteria and activation system were pre-incubated at 37" for 1 h. Determination of the metabolic activation of paracetamol to covalently bound intermediates, a further enzymic reaction catalyzed by P4501A2, was carried out according to Ioannides et al. (1983). Statistical analysis was performed, using Student's t-test, on an IBM PC X T computer.
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Results Basal enzyme activities Quantification of total cytochrome P-450 in ferret liver preparations was originally made by the method of Bend et al. (1972) from the dithionite-difference spectra of CO-treated liver microsomes, because of the high contamination with haemoglobin. However, it was subsequently shown that there were no significant differences in the specific contents of cytochrome P-450 when measured by the original method of Omura and Sat0 (1964) from the CO-difference spectrum of dithionite-reduced microsomes, and the method of Bend et al. (1972) (results not shown), so that the method of Omura and Sat0 (1964) was used for all subsequent determinations. Ferret basal total cytochrome P-450 content of liver was 0.24 f0.05 nmol/mg microsomal protein, which is only some 30% of that present in adult rat liver; similarly, the basal cytochrome b, content was 0.1 1fO.05 nmol/mg microsomal protein, which is only some 20% of that in rat liver (table 1). NADPHcytochrome c reductase activity and microsomal protein content were similar in both species. T h e mixed-function oxidases, benzphetamine N-demethylase, ethylmorphine N-demethylase, and the 7-alkoxyresorufin 0-dealkylases (tables 1 and 2) all had higher basal activities in ferret liver than in rat liver when activities are expressed per nmol P-450. Part of this species difference is due to the higher cytochrome
Table 1 .
Comparison of the hepatic microsomal monoxygenase system of ferret and rat Ferret
Liver parameter
Rat
Control
MC-treated
Control
MC-treated
Cytochrome P-450 (nmol/mg protein)
0.24f0.05
0.57&0.14$
0.87fO.OSt
2.15 kO.11'
Cytochrome b, (nmol/mg protein)
0.11 kO.05
0.29 0.023
NADPH-Cytochrome c reductase (nmol/min per mg protein) Microsomal protein (mg/g wet wt. liver)
34f9
26f3
55f6
49+7
31.5 f 5.1
37.2k3.5
27.4f2.2
16.1 3.5$
Benzphetamine N-demethylase (nmol/min per nmol P-450) (nmol/min per mg protein)
19.7k6.5 4.7k0.1
12.4f5.2 7.1 *0.7*
6.1 f 1.27 5.3 f 0 . 1
7.5f1.7 16&0.2*
Ethylmorphine N-demethylase (nmol/min per nmol P-450) (nmol/min per mg protein)
15.2 k 3.2 3.6 k 0 . 2
19.5 k 4 . 8 1 1 . 1 &0.7*
9.8 f1.97 8.5+0.lt
7.6 f 2.8 16f0.3'
Dimethylnitrosamine Ndemethylase (nmol/min per nmol P-450) (nmol/min per mg protein)
2657 6k0.4
26+3 14+0.4*
12f2t 10 f 0.1t
13f4 28 k 0.4*
Lauric acid hydroxylase (nmol/min per nmol P-450) (nmol/min per mg protein
2.4k0.7 0.6f0.1
1.9 kO.5 1 . 1 kO.1
3.3 k 1.8 2.9 f 0.1t
5.5 2.3 12+0.3*
Values are means f S D of groups of three male ferrets and six male rats. $Statistically different from control at P i 0.05; tstatistically different from control ferret at PiO.01. 'Statistically different from control ferret at P 4 times that of rat, but the MC-induced level was only 50% greater in ferret. In contrast, ferret hepatic basal lauric acid hydroxylase was only 20-70% that of rat, and MC-induced activity was only 10-35% that of rat, as expressed in terms of microsomal protein and total cytochrome P-450 respectively.
Species differences in hepatic EROD activity To examine further this high level of EROD activity in ferret liver, the hepatic EROD activities of a number of rodent species were determined. As can be seen from table 3, EROD activity in ferret liver is 30-300% greater than in hamster, mouse, guinea pig and rat, when expressed per mg protein and, because of the much lower total cytochrome P-450 activity in ferret than in rodent liver, is 6- to 12-fold greater than in rodent liver when expressed per nmol P-450. Nature of ferret EROD activity Ferret liver EROD activity was inhibited by a-naphthoflavone; this was greater with MC-induced EROD activity in ferret, and was much greater with ferret than with rat liver (table 4). Effects of addition of monospecific polyclonal antibodies against rat cytochromes P4501A1, P4501 A2, and P4502B on the alkoxyresorufin 0-dealkylase activities of ferret liver microsomal preparations (EROD, PROD and BROD) are shown in table 5. From the inhibition of these enzyme activities by the specific antisera it would appear that MC-induced ferret liver EROD activity is associated with an orthologue or closely-related form of rat liver cytochrome P4501A1 and, to a lesser extent, with an orthologue or closely-related form of rat liver P4501A2.
Table 3.
Species differences in basal hepatic microsomal cytochrome P4501 as measured by 7ethoxyresorufin 0-deethylase (EROD) activity EROD activity
Species Mouse, CDI (6) Rat Wistar (5) Guinea pig, Dunkin Hartley (3) Hamster, golden Syrian (4) Ferret, albino (3)
Specific activity (pmol/min per mg protein)
Turnover number (pmol/min per nmol P-450)
55+9 32+8 47k11 97 f22 125k19
44+12 35+6 36k7 69k14 460 75
The number of each species are given in parentheses. Values are means fS D .
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Ferret liver mixed-function oxidases Table 4.
Inhibition of ferret and rat liver 7-ethoxyresorufin 0-deethylase (EROD) activity by u-naphthoflavone Ferret
(yocontrol)
Rat ("/" control)
a-Naphthoflavone (M)
Basal
MC-Induced
Basal
MC-Induced
1 x 10-'0 1 x 10-8 2.5 x 10-7
100 62 32
98 54 6
110 114 60
105 104 31
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Values are typical of three determinations with pooled liver microsomes from three ferrets or six rats, and are expressed as percentage of basal activities whicll were: 0.91 and 4.6 nmol/min per nmol P-450 for untreated and MC-induced ferrets, and 0.03 and 0.51 nmol/min per nmol P-450 for untreated and MC-induced rats, respectively.
Ferret liver P4501AI Western blot analysis of ferret liver microsomes with a monospecific anti-rat cytochrome P4501A1 showed no detectable band of immune precipitation, but liver microsomes from MC-pretreated ferrets did show a weak band which migrated to a region of molecular weight (57Kd) similar to that of rat liver P4501A1 proteins (57 Kd) and precipitated with anti-cytochrorne P4501A1 (not shown). This indicates that MC-treatment of ferrets results in the induction of a protein orthologous or closely related to rat cytochrome P4501A1. As liver microsomes from untreated ferrets showed no cytochrome interacting with rat anti-P4501Al, indicating that P4501A1 if present constitutively in ferret liver was below the limit of detection, ELISA analysis was undertaken. As it can be seen from figure 1, liver microsomes from MC-treated ferrets produced a much stronger interaction with rat anti-P4501Al than did control rnicrosomes from untreated ferrets. Ferret liver P4S01 A2 Western blot analysis of ferret liver microsomes with a monospecific polyclonal antibody to rat cytochrome P4501 A2 showed no detectable protein precipitation band corresponding to rat liver cytochrome P4501A2 in liver microsomes from
Table 5.
Inhibition of ferret liver alkoxyresorufin 0-dealkylase activities by antibodies to rat liver cytochromes P450 1A1, 1A2 and 2B1 Alkoxyresorufin 0-dealkylase activity (Yo control)
Antibody addition
EROD
Pre-immune sera Anti-P450 1Al Anti-P450 1A2 Anti-P450 2B1
100 12 47 85
PROD
BROD
89
105 92 81 93
81
89 73
7-Alkoxyresorufin 0-dealkylase activities were determined using pooled liver microsomal preparations from three ferrets pretreated with MC. Monospecific antibodies to three forms of rat liver cytochrome P-450 (P450 1A1, 1A2, and 2B1) were pre-incubated with ferret liver microsomes (20mg protein/nmol P-450) then added to the incubation mixture. Control enzyme activities (lOOo/,) were for 7-ethoxyresorufin 0-deethylase (EROD), 4.1 nmolimin per nmol P-450; for 7-pentoxyresorufin 0dealkylase (PROD), 0.07 nmol/min per nmol P-450; and for 7-benzyloxyresorufin 0-dealkylase (BROD), 0.33 nmolimin per nmol P-450.
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Hicrosomal protein (IJ g / w e l l )
Figure 1 . ELISA immunoquantification of a ferret liver microsomal protein orthologous to rat liver cytochrome P4501A1. Liver microsomes from groups of three ferrets untreated (0) or pretreated with 3-methylcholanthrene (0) were subjected to ELISA analysis with rat anticytochrome P4501A1 as primary antibody (see Experimental section for details).
untreated ferrets. However, with liver microsomes from MC-pretreated ferrets the appearance of a protein orthologous to rat liver cytochrome P4501A2 became apparent (not shown). Activation of the food pyrolysis product, Glu-P-1, to mutagenic intermediates in the Ames test and of the drug, paracetamol, to intermediates covalently bound to protein, which have been attributed largely to rat cytochromes P4501A2 and P4502E1 have also been used as an indication of P4501A2 activity in ferret liver microsomes. In the activation of Glu-P-l , liver microsomes from untreated ferrets gave 1100 200 histidine revertants/mg protein, while microsomes from MCtreated ferrets gave 12 200 & 700 revertants/mg, an 11-fold increase. In the activation of paracetamol to covalently-bound intermediates, liver microsomes from untreated ferrets gave 0.4_+0.1 nmol bound/min per mg protein, while microsomes from MC-treated ferrets gave 4.0 _+ 0.9 nmol bound/min per mg protein, a %fold increase. These results support the Western blot analysis with anti-cytochrome P4501A2, and the inhibitory effect of this antibody on the EROD activity of ferret liver microsomes, that an orthologue of rat liver cytochrome P4501A2 may be present constitutively at low concentration in ferret liver, but is readily induced some 10-fold by pretreatment with MC.
Discussion Basal mixed-function oxidases T h e cytochromes P-450 are a superfamily of enzymes abundant in rodent liver microsomes, ubiquitous to most tissues and species examined, which are concerned with the oxygenation of endogeneous and exogeneous substrates in the metabolism of lipids, steroids, drugs and environmental chemicals. It was not surprising, therefore, that the present studies have shown that ferret liver contains an active microsomal mixed-function oxidase system comprising several cytochromes P-450, as has been repeatedly demonstrated in rodent and other mammalian livers. The individual components of this system, namely, cytochromes
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P-450, cytochrome b, and NADPH-cytochrome c reductase were present at lower concentrations than found in rat liver (table l), but this was to be expected for a mammal of greater body mass (Walker 1978). T h e mixed-function oxidase specific activities (nmol product/min per m g microsomal protein) of ferret liver microsomes were of a similar order to those for rat liver only in the case of benzphetamine demethylase. T h e alkoxyresorufin dealkases (MROD, EROD, PROD and BROD) were all 2- to 4-fold more active in ferret than in rat, when expressed per mg microsomal protein, and ethylmorphine N-demethylase, dimethylnitrosamine demethylase and lauric acid hydroxylase were all lower in the ferret than in rat (tables 1 and 2). Lauric acid hydroxylase is associated with cytochrome P4504 and the a-hydroxylation of long-chain fatty acids, and is induced by the antihyperlipidaemic, peroxisomal-proliferating drugs, clofibrate, ciprofibrate (Tamburini et al. 1984) and by the anti-inflammatory, hepatotoxic drug benoxaprofen (Ayrton et al. 1991). It is possible that the lower level of activity of this cytochrome P-450 may be associated with the tendency of the ferret to lipid accumulation in the liver. Alkoxyresorufin 0-dealkylase actiwities T h e individual alkoxyresorufin 0-dealkylase activities have been associated with specific cytochromes P-450 (Winston et al. 1990, Burke et al. 1985). 7Methoxyresorufin 0-demethylase (MROD) and the 7-ethoxy (EROD) are relatively specific for cytochrome P4501 (Burke et al. 1985), the 7-pentoxy (PROD) is largely indicative of P4502B, but 7-benzyloxy (BROD) is relatively non-specific and responds to several different inducing agents (Winston et al. 1990). Activities of the different alkoxyresorufin 0-dealkylases of ferret liver indicate a 3-fold higher specific activity and a 15-fold higher turnover number (nmol product/min per nmol total P-450) for cytochrome P4501 (EROD) than is seen in rat liver; rodents generally appear to have a much lower turnover number for P4501 than is seen in the ferret (table 3). Hence, with the substrates used in the present study, it is possible to conclude that the specific activity of P4501 is markedly greater than in rat (MROD 100% increase, EROD 350% increase); P4502B is similarly greater in ferret than in rat (PROD 300% increase); P4502E is slightly lower in the ferret (dimethylnitrosamine demethylase is 60% of that in rat); P4503 is lower in ferret (ethylmorphine N-demethylase is 40% that of rat); and P4504 is also lower in ferret (20% of that in rat). However, benzphetamine N-demethylase, which is also a P4502B reaction, is the same in both animal species, for which a possible explanation is that benzphetamine is known to be metabolized by other basal forms of P-450 (Ryan and Levin 1990). Moreover, it should be noted that P4504 catalyses only the 12-hydroxylation of lauric acid, whereas the assay used in the present study does not discriminate between the 11- and 12-hydroxylations. It must be emphasized that although orthologous proteins to rat P-450s may be present in ferret, these may not necessarily have the same substrate specificity. Enzyme induction by MC Induction of ferrret liver mixed-function oxidases by MC is similar to that seen in rat, namely, increases in total cytochromes P-450 with a shift in the wavelength of the cytochrome P-450 CO-ligand difference spectrum from 450 to 448nm, and no increases in the NADPH-cytochrome c reductase or microsomal protein; indeed there is a significant decrease in hepatic microsomal protein
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following M C treatment of the ferret. However, no overt pathology was histologically apparent. EROD specific activity, indicative of P4501, was increased 20-fold in ferret liver and 60-fold in rat liver after MC-induction; and the corresponding turnover numbers were increased 7- and 25-fold for ferret and rat liver, respectively (table 2). EROD activity is attributable to the enzymic actions of both cytochrome P4501A1 and P4501A2, although the turnover number of the former is an order of magnitude greater than that of the latter isozyme (Astrom and De Pierre 1985). As can be seen from table 5, an antibody to rat liver P4501A1 inhibits ferret liver EROD activity by 88%, whereas an antibody to P4501A2 inhibits only by 53%. I n both rat and ferret liver, M C treatment induces the synthesis of both P4501 proteins, and in rat liver the synthesis of P4501A1 is greater than that of P4501A2. Inhibition by a-naphthoflavone (an inhibitor of P4501) of rat liver EROD activity after MC-induction (69%) is greater than before M C treatment (40%) (table 4). Similarly, in the ferret, a-naphthoflavone inhibits 94% of the EROD activity of MC-induced liver microsomes, but inhibits only 68% of the EROD activity of the basal enzymes (table 4). Inhibition of P4501 activity by a-naphthoflavone is more marked with ferret liver microsomal preparations than with rat, indicating that differences may exist in the nature of the active sites of the rat and ferret P4501 proteins. Moreover, M C treatment of ferrets resulted in a much greater induction of EROD (20-fold, representing largely P4501A1 as shown immunologically; table 5) than of Glu-P-1 or paracetamol bioactivation (10-fold, representing P4501A2 but not 1Al). T h e more specific immunoquantitative determination of P4501A1 indicates that MC-induction increases the concentration of this ferret liver enzyme protein to at least five times the basal level (figure 1). Comparison of ferret and rat T h e relatively small group size (three ferrets and guinea pigs, and six rats and mice), and the exclusive use of male animals, does impose certain limitations on the conclusions that may be drawn from these preliminary studies, but earlier work (Ioannides et al. 1977, Pope et al. 1980) showed that, unlike rat, no major sex differences in drug metabolism were seen in the ferret. A comparison of drug metabolism in male ferret and male rat was therefore considered tenable. T h e four-fold greater concentration of basal P4501 in male ferret liver than in male rat liver, and the similar concentration of these two enzyme proteins after MC induction, indicates that the ferret may be as sensitive an experimental animal for carcinogenicity and chemical toxicity studies as the rat. For several workers have shown that the two cytochrome P4501 proteins are the major enzymes catalyzing the oxidative activation of toxic chemicals to mutagens, carcinogens, neoantigens and other reactive intermediates; Guengerich (1988) and Ioannides and Parke (1990) reported that some 90% of carcinogens are activated by these cytochrome P4501 enzyme proteins. One other isoform of cytochrome P-450 that is known to activate carcinogens, and to cause chemical toxicity and carcinogenicity by oxygen radical production, is P4502E1, activity of which has been indicated in ferret liver by the presence of dimethylnitrosamine N-demethylase activity, although Western blots with rat liver P4502E1 polyclonal antibody did not exhibit any immuno-reactive band in the P-450 region. T h u s ferret liver contains comparable or greater levels of at least two of the three activating microsomal cytochromes P-450, namely 1Al and 1A2. T h e ferret would therefore
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appear to be-at least in respect of hepatic drug metabolism-a suitable nonrodent species in the safety evaluation of new drugs and other chemicals since, like the dog (McKillop 1985) it exhibits a high level of P4501 activity in liver. Furthermore, the ferret, like man, is a periodic feeder with a gallbladder, whereas rodents are continuous feeders and the rat has no gallbladder, which should lead to the ferret exhibiting similar pharmacokinetics to man for drugs and chemicals that are orally administered and/or enterohepatically circulated. However, the ferret is a hibernating species which undergoes seasonal changes in body weight, basal metabolism and hepatic lipid accumulation, that might well affect the metabolism of xenobiotics.
Acknowledgements Downloaded by [RMIT University Library] at 03:48 04 May 2016
We are grateful to Glaxo Laboratories for their financial support of this study.
References ASTROM, A,, and DEPIERRE,J. W., 1985, Metabolism of 2-acetylaminofluorene by eight different forms of cytochrome P-450 isolated from rat liver. Carcinogenesis, 6, 113-120. AYRTON,A. D., IOANNIDES, C. and PARKE,D. V., 1991, Induction of P450 I and IV families and peroxisomal proliferation in the liver of rats treated with benoxaprofen. Biochemical Pharmacology, 42, 109-115. BARNETT, C . R., FLATT, P. R., and IOANNIDES, C., 1988, Role of ketone bodies in the diabetes-induced changes in hepatic mixed-function oxidase activities. Biochimica et Biophysica Acta, 967, 250-254. BARNETT, C. R., FLATT, P. R., and IOANNIDES, C., 1990a, Induction of hepatic microsomal P450 I and IIB proteins by hyperketonaemia. Biochemical Pharmacology, 40, 393-397. BARNETT, C. R., FLATT,P. R., and IOANNIDES, C., 1990b, Hyperketonaemia markedly modulates the metabolic activation of chemical carcinogens. Chemico-Biological Interactions, 74, 281-289. BEND,J. R., HOOK,G . E. R., EASTERLING, R. F., GRAM,T . E., and FOUTS,J. R. 1971, A comparative study of hepatic and pulmonary microsomal mixed-function oxidase systems in rabbits. Journal of Pharmacology and Experimental Therapeutics, 183, 206-217. BURKE,M . D., THOMPSON, S., ELCOMBE,C. R., HALPERT,J. HAAPARANTA, T., and MAYER,R. T., 1985, Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochemical Pharmacology, 34, 3337-3345. COTCHIN, E., 1980, Smooth muscle hyperplasia and neoplasia in the ovaries of domestic ferrets (mustela putorius furo). Journal of Pathology, 130, 169-171. GUENCERICH, F . P., 1988, Roles of cytochrome P-450 enzymes in chemical carcinogenesis and cancer chemotherapy. Cancer Research, 48, 2946-2954. HOLTZMANN, J. L., GRAM,T . E., GIGON, P. L., and GILLETTE, J. R., 1968, The distribution of the components of mixed-function oxidase between rough and smooth endoplasmic reticulum of liver cells. Biochemical Journal, 110, 407-41 2. IOANNIDES, C., SWEATMAN, B., RICHARDS, R., and PARKE,D . V., 1977, Drug metabolism in the ferret: effect of age, sex and strain. General Pharmacology, 8, 243-249. IOANNIDES, C., STEELE,C. M., and PARKE, D. V., 1983, Species variation in the metabolic activation of paracetamol to toxic intermediates: role of cytochrome P-450 and P-448. Toxicology Letters, 16, 55-61. IOANNIDES, C., and PARKE, D. V., 1990, The cytochrome P450 I gene family of microsomal hemoproteins and their role in the metabolic activation of chemicals. Drug Metabolism Reviews, 22, 1-85. L A E M M L t , V. K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T 4 . Nature, 227, 680-684. LOWRY,0. H., ROSEBOROUGH, N. J., FARR,A. L., and RANDALL, R. J., 1951, Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. MARON,D . M., and AMES, B. N., 1983, Revised methods for the salmonella mutagenicity test. Mutation Research, 113, 173-215. MCKILLOP,D., 1985, Effects of phenobarbitone and 8-naphthoflavone on hepatic microsomal drugmetabolising enzymes of the beagle dog. Biochemical Pharmacology, 34, 3137-3142. MORELAND, A. F., and GLASER,C., 1985, Evaluation of ketamine, ketamine-xylazine and ketaminediazepam anesthesia in the ferret. Laboratory Animal Science, 35, 287-290. OMURA, T . , and SATO,R., 1964, The carbon monoxide binding pigment of liver microsomes. Journal of Biological Chemistry, 239, 2379-2385.
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PARKER, G . L., and ORTON,T . C., 1980, In Biochemistry, Biophysics and Regulation of Cytochrome P-4.50, Edited by J . A. Gustafsson, J. Duke, A. Mode and J. Rafter, (Elsevier: Amsterdam) pp. 373-377. POPE, D. J., GILBERT,A. P., EASTER,D. J., CHAN,R. P., TURNER, J. C., GOTTFRIED, S., and PARKE, D. V., 1980, T h e metabolism of bifluranol by rat, dog and ferret. Journal of Pharmacy and Pharmacology, 33, 302-308. RYAN,D. E., and LEVIN,W., 1990, Purification and characterization of hepatic microsomal cytochrome P-450. Pharmacology and Therapeutics, 45, 153-239. SHAVILLA, J., 1990, The toxicological significance of fatty liver in ferrets. PhD Thesis. University of Surrey, UK. TAMBURINI, P., MASSON, H. A,, BAINS, S. K., MAKOWSKI, R. J., MORRIS,B., and GIBSON,G. G., 1984, Multiple forms of cytochrome P-450. Purification characterisation and comparison of a novel clofibrate-induced isoenzyme with other major forms of cytochrome P-450. European Journal of Biochemistry, 139, 235-246. S. J., LEHNING, E. J., and AULERICH, R. J., 1991, Delayed neurotoxic effects TANAKA, D . J R . , BURSIAN, of bis( 1-methylethyl)phosphorofluoridate (DFP) in the European ferret: a possible mammalian model for organophosphorus-induced delayed neurotoxicity. Neurotoxicology, 12, 209-224. THORNTON, P. C., WRIGHT,P. A., SACRA, P. J., and GOODIER, T . E. W., 1979, T h e ferret, Mustela putorius furo, as a new species in toxicology. Laboratory Animals, 13, 1 19- 124. TOWBIN, H., STAEHELIN, T . , and GORDON,J., 1979, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Science, 76, 4350-4355. D. E., 1978, Enzyme immunoassays with special reference to VOLLER, A., BARTLETT, A., and BIDWELL, ELISA techniques. Journal of Clinical Pathology, 31, 507-520. WALKER, C. H., 1978, Species differences in microsomal monooxygenase activity and the relationship of biological half-lives. Drug Metabolism Reoiews, 7, 295-323. C. H., and KAMIN,H., 1962, Microsomal triphosphopyridine adenine dinucleotide cytoWILLIAMS, chrome c reductase of liver. Journal of Biological Chemistry, 237, 587-595. WINSTON, G. W., NARAYAN, S., and BOUNDS, P. L., 1990, Profiles of ethanol-induced microsomal alkoxyresorufin (alkoxyphenoxazone) 0-dealkylation: comparison with phenobarbital- and Aroclor 1254-induced systems. Alcohol and Alcoholism, 2 5 , 667-672.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Hepatic mixed-function oxidases of ferret J. Shavila, C. Ioannides, L. J. King & D. V. Parke To cite this article: J. Shavila, C. Ioannides, L. J. King & D. V. Parke (1992) Hepatic mixed-function oxidases of ferret, Xenobiotica, 22:8, 1003-1014, DOI: 10.3109/00498259209049906 To link to this article: http://dx.doi.org/10.3109/00498259209049906
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Date: 04 May 2016, At: 03:48
XENOBIOTICA,
1992, VOL. 22,
NO.
8, 1003-1014
Hepatic mixed-function oxidases of ferret
J. SHAVILA, C. IOANNIDES, L. J. K I N G and D. V. PARKET Molecular Toxicology Group, Division of Toxicology, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 SXH, U K
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Received 1 November 1991 ;accepted 1 April 1992
1 . Ferret liver mixed-function oxidase enzymes have been quantified using a variety of substrates and the activities have been compared with those found in rat liver. 2. Ferret liver total cytochrome P-450 is only 30% of that of rat liver and exhibits higher 7-ethoxyresorufin 0-deethylase (EROD) activity, and lower lauric acid hydroxylase activity than rat liver; other mixed-function oxidases are at similar levels of activity in both species. 3. Induction with 3-methylcholanthrene (MC), similar to MC-induction in rat, increases the total P-450 of ferret liver by 140%, but does not increase P-450 reductase or microsomal protein. EROD specific activity (pmol/min per mg protein) is increased 20fold by MC treatment.
4. Turnover number of EROD for control liver microsomes of ferret, hamster, mouse, guinea pig and rat were 460, 69, 44, 36 and 35pmol/min per nmol P-450, respectively, indicating the much higher value for ferret than for any of the rodent species studied. 5. Ferret liver EROD activity is inhibited by the P4501A1 inhibitor, anaphthoflavone. Use of monospecific antibodies in ELISA, Western blot and enzymeinhibition techniques has shown that EROD activity in ferret liver is attributable to two enzyme proteins orthologous with rat liver cytochromes P4501A1 and 1A2, with the former predominating. M C induces both P4501A enzyme proteins in ferret liver, as in rat liver, with P4501A1 activity predominating.
Introduction In recent years, the ferret (Mustella putoriusfuro) has been proposed as a nonrodent species for use in the safety evaluation of drugs and chemicals, as a surrogate for human and a less emotive substitute for the dog (Thornton et al. 1979). T h e high susceptibility of the ferret to organophosphate-induced delayed neuropathy, in contrast to rodents and most other laboratory animals, has led to this species being proposed as a suitable model for assessing the potential delayed neurotoxic effects of pesticides and other chemicals (Tanaka et al. 1991). On the other hand, the low susceptibility of the ferret to spontaneous neoplasia (Cotchin 1980) makes this animal an appropriate non-rodent species for carcinogenicity testing. However, only few studies have been published concerning the metabolism of drugs and toxic chemicals in this species (Ioannides et al. 1977, Pope et al. 1980). For these reasons it is important to evaluate more fully hepatic drug ?To whom correspondence should be addressed. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
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metabolism in the ferret, and to compare this, both qualitatively and quantitatively, with studies in rodents and man. Preliminary chronic toxicity studies carried out with ferrets revealed that this species was particularly susceptible to the development of fatty liver, normally an indicator of chemical toxicity. However, control untreated ferrets similarly developed fatty liver and it was considered that this condition was non-pathological and was probably diet-related. As administration of medium-chain length triglycerides to rats has been associated with changes in the cytochrome P-450 isoenzyme profiles in rat (Barnett et al. 1988, 1990a and b), it was considered that diet, and the development of fatty liver, in the ferret might well change the hepatic cytochrome P-450 isoenzyme pattern. Since the only drug metabolism studies in ferrets were conducted before the complex nature of the cytochrome P-450 superfamily was established (Ioannides et al. 1977), and only very limited isoenzyme studies were undertaken, it was considered highly desirable to undertake more detailed studies on the hepatic mixed-function oxidases and cytochromes P450 of ferret liver, and then to ascertain the effects of diet on these enzymic parameters. The present study was concerned with determining the constitutive drug-metabolizing cytochromes P-450 of ferret liver, and comparing their specific activities with those of rat liver; furthermore, as cytochrome P4501 activity appeared to be very much higher in ferret than in rat, the effects of pretreatment with 3-methylcholanthrene (MC), a specific inducer of cytochrome P4501, were also studied. Effects of diet on ferret liver mixed-function oxidases will be described in a subsequent publication.
Experimental Materials Cytochrome c, NADP, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, dimethyl sulphoxide, 3-methylcholanthrene, a-naphthoflavone, and dimethylnitrosamine (Sigma Chemical Co. Ltd., Poole, Dorset, UK); paracetamol (BDH, Poole, Dorset, UK); Soluene-350 and Dimilume (Packard Instrument Co., Reading, UK); 7-methoxy-, 7-ethoxy-, and 7-pentoxy-resorufin, and resorufin (Molecular Probes, Eugene, OR., USA); 7-benzyloxyresorufin (Boehringer, East Sussex, UK); ethylmorphine (Rhbne-Poulenc, Dagenham, Essex, UK); Glu-P-1 (Wako Fine Chemicals, Neuss 1, Germany); and 3H-paracetamol (Amersham International, Bucks., UK) were purchased. Benzphetamine (Upjohn Co., Kalamazoo, MI., USA) was a generous gift. Disposable plastic tubes and agar (London Analytical and Bacteriological Media Ltd., Salford, Lancs., UK), Oxoid nutrient broth No. 2 (Oxoid Ltd., Basingstoke, Hants., UK), and Salmonella typhzmurium, strain TA98 (a gift from Dr B. N. Ames, Berkeley, CA., USA) were used in the Ames test for Glu-P-1 activation. Animals Mature albino male ferrets (1.1-1.4 kg body weight) purchased from Grayston Breeders Ltd., Ringwood, Hants., UK, were housed individually under uniform laboratory conditions of 50% humidity at 22°C and 12 h light/dark cycle in the University of Surrey Experimental Biology Unit, and fed a diet of cat food (Pedigree Pet Foods, 40g/day) and dog biscuits (SDS Laboratory diets, 40g/day), with water available ad libitum, a diet which has been shown not to produce fatty liver in the ferret (Shavila 1990). Ferrets were acclimatized for 2 weeks before commencement of studies. Male Wistar albino rats (1 50-2008 body weight) and male mice (20-25 g body weight) University of Surrey, Experimental Biology Unit, and adult male Dunkin Hartley guinea pigs and golden Syrian hamsters, purchased from Grayston Breeders Ltd., Ringwood, Hants., were housed singly in cages with sawdust bedding and maintained under the same uniform laboratory conditions as the ferrets. Rats, mice, hamsters and guinea pigs were fed Spratts animal diet No. 1 and given water, ad libitum. All animal species were allocated randomly to the various experimental groups. For treatment with 3-methylcholanthrene (MC), animals were dosed i.p. with M C in corn oil (25rng/kg) once daily for 3 consecutive days after the 2-week acclimatization period. All animals were killed 24h after the last injection. Control animals received corn oil only. Ferrets were killed by exsanguination immediately following anaesthesia with ketamine (25 mg/kg) plus xylazine (2 mg/kg) administered intramuscularly (Moreland and Glaser 1985); all other animals
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were killed by cervical dislocation. Livers were removed, the gallbladders were removed from the livers of ferrets, mice and guinea pigs, and livers from each species were scissor-minced and then homogenized in 0 . 2 5 ~sucrose in a Potter-Elvehjem glass-tenon homogenizer to a liver concentration of 25% w/v. The homogenate was centrifuged at IOOOOg,, at 4°C for 30min, and the supernatant further centrifuged at 100 OOOg,, at 4°C for 60 min; the microsomal pellet was resuspended in 50 mM potassium phosphate buffer, p H 7.25, containing 20% v/v glycerol, further centrifuged at 100000 for 60min, and the washed microsomal pellet reconstituted in the phosphate-glycerol buffer for enzyme determinations.
Enzyme determinations Total cytochrome P-450 and cytochrome b, (Omura and Sat0 1964), NADPH-cytochrome c (P-450) reductase (Williams and Kamin 1962), and microsomal protein (Lowry et al. 1951) were determined, using the liver microsomal preparations. Lauric acid hydroxylase, quantifying the sum of 11- and 12-hydroxylauric acids (Parker and Orton 1980), ethylmorphine N-demethylase, benzphetamine N-demethylase and dimethylnitrosamine N-demethylase (60 mM) (Holtzman et al. 1968), and the 7-alkoxyresorufin 0-deethylases, 7-methoxy- (MROD), 7-ethoxy- (EROD), 7-pentoxy- (PROD), and 7-benzyloxy- (BROD) (Burke et al. 1989, were all determined using liver microsomal preparations. Inhibition of 7-ethoxyresorufin 0-deethylase (EROD) by the specific inhibitors of P4501, namely, a-naphthoflavone was studied using ferret and rat liver microsomal preparations and various concentrations of the inhibitor in DMSO. Inhibition of several alkoxyresorufin 0-dealkylase activities (EROD, PROD aiid BROD) by addition of monospecific polycional antibodies (2-25 mg protein/nmol total P-450) was studied by incubating liver microsomes from MC-treated ferrets (0.63 nmol P-450/mg protein) with anti-P4501Al and anti-P4501A2 (gifts from Dr C. R. Wolf, ICRF Unit, Edinburgh, Scotland), anti-P4502BI (a gift from Dr R. J. Makowski, University of Surrey) or pre-immune sera (control) in 0.1 ml phosphate-buffered saline, pH 7.4, for 35 min at room temperature, then further incubating with the alkoxyresorufin substrates and determining the production of resorufin in the normal manner. E L f S A anaIysis for P4501Al Enzyme-linked immunosorbent assay analysis (ELISA) of cytochrome P4501A1 present in ferret liver microsomes constitutively, or after pretreatment with MC, was carried out by the method of Voller et al. (1978). Microsomes were solubilized with sodium cholate (1 mg/mg protein) and Emulgen 911 (0.2 mg/mg protein). T h e primary antibody, anti-P4501Al, was diluted 1:lOOOO and applied to the wells (0.2 ml/well) which had previously been coated with microsomal protein (antigen); the wells initially incubated with pre-immune serum served as blank. Microsomal protein plus primary antibody was then incubated with the secondary antibody of donkey/anti-sheep labelled with horseradish peroxidase. The substrate solution (H,O, plus o-phenylenediamine) was added, and the plates incubated for 30min to develop the colour which was measured with a Dynatech plate reader at 492 nm.
Western blot analysis for cytochromes P-450 Immunoblot (Western blot) analysis was carried out as described by Towbin et al. (1979). Liver microsomes from ferrets and rats were subjected to SDS-polyacrylamide gel electrophoresis (Laemmli 1970) and transferred to nitrocellulose paper. After blocking with 10 mM phosphate-buffered saline, pH 7.4, containing 1yo w/w bovine serum albumin, the nitrocellulose was incubated with the polyclonal monospecific antibodies P4501A1 and A2 (raised in rabbit) at dilutions of 1:lOOOO. Detecting antibody (donkey-anti-rabbit) conjugated to horseradish peroxidase, was used at a dilution of 1:2000 in phosphate-buffered saline, p H 7.4, containing 1yo bovine serum albumin. Immunopositive bands were detected by treatment with diaminobenzidine chloride (1 mg/ml) and H,O, (30vol. diluted 1:SOOO) for 30s then rinsing the nitrocellulose paper with water. Enzymic reactions f o r cytochrome P4501A2 Metabolic activation of the precarcinogen Glu-P-1 (2 pg/plate) to mutagenic intermediates, an enzymic reaction catalyzed by P4501A2, was determined using the Ames test (Maron and Ames 1983) and employing a fresh, overnight culture of S. typhimurium strain TA98, in the presence of 10% v/v ferret liver microsomal preparation from control and MC-treated ferrets supplemented with glucose 6phosphate dehydrogenase (1 unitlplate). The mixed precarcinogen, bacteria and activation system were pre-incubated at 37" for 1 h. Determination of the metabolic activation of paracetamol to covalently bound intermediates, a further enzymic reaction catalyzed by P4501A2, was carried out according to Ioannides et al. (1983). Statistical analysis was performed, using Student's t-test, on an IBM PC X T computer.
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Results Basal enzyme activities Quantification of total cytochrome P-450 in ferret liver preparations was originally made by the method of Bend et al. (1972) from the dithionite-difference spectra of CO-treated liver microsomes, because of the high contamination with haemoglobin. However, it was subsequently shown that there were no significant differences in the specific contents of cytochrome P-450 when measured by the original method of Omura and Sat0 (1964) from the CO-difference spectrum of dithionite-reduced microsomes, and the method of Bend et al. (1972) (results not shown), so that the method of Omura and Sat0 (1964) was used for all subsequent determinations. Ferret basal total cytochrome P-450 content of liver was 0.24 f0.05 nmol/mg microsomal protein, which is only some 30% of that present in adult rat liver; similarly, the basal cytochrome b, content was 0.1 1fO.05 nmol/mg microsomal protein, which is only some 20% of that in rat liver (table 1). NADPHcytochrome c reductase activity and microsomal protein content were similar in both species. T h e mixed-function oxidases, benzphetamine N-demethylase, ethylmorphine N-demethylase, and the 7-alkoxyresorufin 0-dealkylases (tables 1 and 2) all had higher basal activities in ferret liver than in rat liver when activities are expressed per nmol P-450. Part of this species difference is due to the higher cytochrome
Table 1 .
Comparison of the hepatic microsomal monoxygenase system of ferret and rat Ferret
Liver parameter
Rat
Control
MC-treated
Control
MC-treated
Cytochrome P-450 (nmol/mg protein)
0.24f0.05
0.57&0.14$
0.87fO.OSt
2.15 kO.11'
Cytochrome b, (nmol/mg protein)
0.11 kO.05
0.29 0.023
NADPH-Cytochrome c reductase (nmol/min per mg protein) Microsomal protein (mg/g wet wt. liver)
34f9
26f3
55f6
49+7
31.5 f 5.1
37.2k3.5
27.4f2.2
16.1 3.5$
Benzphetamine N-demethylase (nmol/min per nmol P-450) (nmol/min per mg protein)
19.7k6.5 4.7k0.1
12.4f5.2 7.1 *0.7*
6.1 f 1.27 5.3 f 0 . 1
7.5f1.7 16&0.2*
Ethylmorphine N-demethylase (nmol/min per nmol P-450) (nmol/min per mg protein)
15.2 k 3.2 3.6 k 0 . 2
19.5 k 4 . 8 1 1 . 1 &0.7*
9.8 f1.97 8.5+0.lt
7.6 f 2.8 16f0.3'
Dimethylnitrosamine Ndemethylase (nmol/min per nmol P-450) (nmol/min per mg protein)
2657 6k0.4
26+3 14+0.4*
12f2t 10 f 0.1t
13f4 28 k 0.4*
Lauric acid hydroxylase (nmol/min per nmol P-450) (nmol/min per mg protein
2.4k0.7 0.6f0.1
1.9 kO.5 1 . 1 kO.1
3.3 k 1.8 2.9 f 0.1t
5.5 2.3 12+0.3*
Values are means f S D of groups of three male ferrets and six male rats. $Statistically different from control at P i 0.05; tstatistically different from control ferret at PiO.01. 'Statistically different from control ferret at P 4 times that of rat, but the MC-induced level was only 50% greater in ferret. In contrast, ferret hepatic basal lauric acid hydroxylase was only 20-70% that of rat, and MC-induced activity was only 10-35% that of rat, as expressed in terms of microsomal protein and total cytochrome P-450 respectively.
Species differences in hepatic EROD activity To examine further this high level of EROD activity in ferret liver, the hepatic EROD activities of a number of rodent species were determined. As can be seen from table 3, EROD activity in ferret liver is 30-300% greater than in hamster, mouse, guinea pig and rat, when expressed per mg protein and, because of the much lower total cytochrome P-450 activity in ferret than in rodent liver, is 6- to 12-fold greater than in rodent liver when expressed per nmol P-450. Nature of ferret EROD activity Ferret liver EROD activity was inhibited by a-naphthoflavone; this was greater with MC-induced EROD activity in ferret, and was much greater with ferret than with rat liver (table 4). Effects of addition of monospecific polyclonal antibodies against rat cytochromes P4501A1, P4501 A2, and P4502B on the alkoxyresorufin 0-dealkylase activities of ferret liver microsomal preparations (EROD, PROD and BROD) are shown in table 5. From the inhibition of these enzyme activities by the specific antisera it would appear that MC-induced ferret liver EROD activity is associated with an orthologue or closely-related form of rat liver cytochrome P4501A1 and, to a lesser extent, with an orthologue or closely-related form of rat liver P4501A2.
Table 3.
Species differences in basal hepatic microsomal cytochrome P4501 as measured by 7ethoxyresorufin 0-deethylase (EROD) activity EROD activity
Species Mouse, CDI (6) Rat Wistar (5) Guinea pig, Dunkin Hartley (3) Hamster, golden Syrian (4) Ferret, albino (3)
Specific activity (pmol/min per mg protein)
Turnover number (pmol/min per nmol P-450)
55+9 32+8 47k11 97 f22 125k19
44+12 35+6 36k7 69k14 460 75
The number of each species are given in parentheses. Values are means fS D .
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Ferret liver mixed-function oxidases Table 4.
Inhibition of ferret and rat liver 7-ethoxyresorufin 0-deethylase (EROD) activity by u-naphthoflavone Ferret
(yocontrol)
Rat ("/" control)
a-Naphthoflavone (M)
Basal
MC-Induced
Basal
MC-Induced
1 x 10-'0 1 x 10-8 2.5 x 10-7
100 62 32
98 54 6
110 114 60
105 104 31
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Values are typical of three determinations with pooled liver microsomes from three ferrets or six rats, and are expressed as percentage of basal activities whicll were: 0.91 and 4.6 nmol/min per nmol P-450 for untreated and MC-induced ferrets, and 0.03 and 0.51 nmol/min per nmol P-450 for untreated and MC-induced rats, respectively.
Ferret liver P4501AI Western blot analysis of ferret liver microsomes with a monospecific anti-rat cytochrome P4501A1 showed no detectable band of immune precipitation, but liver microsomes from MC-pretreated ferrets did show a weak band which migrated to a region of molecular weight (57Kd) similar to that of rat liver P4501A1 proteins (57 Kd) and precipitated with anti-cytochrorne P4501A1 (not shown). This indicates that MC-treatment of ferrets results in the induction of a protein orthologous or closely related to rat cytochrome P4501A1. As liver microsomes from untreated ferrets showed no cytochrome interacting with rat anti-P4501Al, indicating that P4501A1 if present constitutively in ferret liver was below the limit of detection, ELISA analysis was undertaken. As it can be seen from figure 1, liver microsomes from MC-treated ferrets produced a much stronger interaction with rat anti-P4501Al than did control rnicrosomes from untreated ferrets. Ferret liver P4S01 A2 Western blot analysis of ferret liver microsomes with a monospecific polyclonal antibody to rat cytochrome P4501 A2 showed no detectable protein precipitation band corresponding to rat liver cytochrome P4501A2 in liver microsomes from
Table 5.
Inhibition of ferret liver alkoxyresorufin 0-dealkylase activities by antibodies to rat liver cytochromes P450 1A1, 1A2 and 2B1 Alkoxyresorufin 0-dealkylase activity (Yo control)
Antibody addition
EROD
Pre-immune sera Anti-P450 1Al Anti-P450 1A2 Anti-P450 2B1
100 12 47 85
PROD
BROD
89
105 92 81 93
81
89 73
7-Alkoxyresorufin 0-dealkylase activities were determined using pooled liver microsomal preparations from three ferrets pretreated with MC. Monospecific antibodies to three forms of rat liver cytochrome P-450 (P450 1A1, 1A2, and 2B1) were pre-incubated with ferret liver microsomes (20mg protein/nmol P-450) then added to the incubation mixture. Control enzyme activities (lOOo/,) were for 7-ethoxyresorufin 0-deethylase (EROD), 4.1 nmolimin per nmol P-450; for 7-pentoxyresorufin 0dealkylase (PROD), 0.07 nmol/min per nmol P-450; and for 7-benzyloxyresorufin 0-dealkylase (BROD), 0.33 nmolimin per nmol P-450.
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Hicrosomal protein (IJ g / w e l l )
Figure 1 . ELISA immunoquantification of a ferret liver microsomal protein orthologous to rat liver cytochrome P4501A1. Liver microsomes from groups of three ferrets untreated (0) or pretreated with 3-methylcholanthrene (0) were subjected to ELISA analysis with rat anticytochrome P4501A1 as primary antibody (see Experimental section for details).
untreated ferrets. However, with liver microsomes from MC-pretreated ferrets the appearance of a protein orthologous to rat liver cytochrome P4501A2 became apparent (not shown). Activation of the food pyrolysis product, Glu-P-1, to mutagenic intermediates in the Ames test and of the drug, paracetamol, to intermediates covalently bound to protein, which have been attributed largely to rat cytochromes P4501A2 and P4502E1 have also been used as an indication of P4501A2 activity in ferret liver microsomes. In the activation of Glu-P-l , liver microsomes from untreated ferrets gave 1100 200 histidine revertants/mg protein, while microsomes from MCtreated ferrets gave 12 200 & 700 revertants/mg, an 11-fold increase. In the activation of paracetamol to covalently-bound intermediates, liver microsomes from untreated ferrets gave 0.4_+0.1 nmol bound/min per mg protein, while microsomes from MC-treated ferrets gave 4.0 _+ 0.9 nmol bound/min per mg protein, a %fold increase. These results support the Western blot analysis with anti-cytochrome P4501A2, and the inhibitory effect of this antibody on the EROD activity of ferret liver microsomes, that an orthologue of rat liver cytochrome P4501A2 may be present constitutively at low concentration in ferret liver, but is readily induced some 10-fold by pretreatment with MC.
Discussion Basal mixed-function oxidases T h e cytochromes P-450 are a superfamily of enzymes abundant in rodent liver microsomes, ubiquitous to most tissues and species examined, which are concerned with the oxygenation of endogeneous and exogeneous substrates in the metabolism of lipids, steroids, drugs and environmental chemicals. It was not surprising, therefore, that the present studies have shown that ferret liver contains an active microsomal mixed-function oxidase system comprising several cytochromes P-450, as has been repeatedly demonstrated in rodent and other mammalian livers. The individual components of this system, namely, cytochromes
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P-450, cytochrome b, and NADPH-cytochrome c reductase were present at lower concentrations than found in rat liver (table l), but this was to be expected for a mammal of greater body mass (Walker 1978). T h e mixed-function oxidase specific activities (nmol product/min per m g microsomal protein) of ferret liver microsomes were of a similar order to those for rat liver only in the case of benzphetamine demethylase. T h e alkoxyresorufin dealkases (MROD, EROD, PROD and BROD) were all 2- to 4-fold more active in ferret than in rat, when expressed per mg microsomal protein, and ethylmorphine N-demethylase, dimethylnitrosamine demethylase and lauric acid hydroxylase were all lower in the ferret than in rat (tables 1 and 2). Lauric acid hydroxylase is associated with cytochrome P4504 and the a-hydroxylation of long-chain fatty acids, and is induced by the antihyperlipidaemic, peroxisomal-proliferating drugs, clofibrate, ciprofibrate (Tamburini et al. 1984) and by the anti-inflammatory, hepatotoxic drug benoxaprofen (Ayrton et al. 1991). It is possible that the lower level of activity of this cytochrome P-450 may be associated with the tendency of the ferret to lipid accumulation in the liver. Alkoxyresorufin 0-dealkylase actiwities T h e individual alkoxyresorufin 0-dealkylase activities have been associated with specific cytochromes P-450 (Winston et al. 1990, Burke et al. 1985). 7Methoxyresorufin 0-demethylase (MROD) and the 7-ethoxy (EROD) are relatively specific for cytochrome P4501 (Burke et al. 1985), the 7-pentoxy (PROD) is largely indicative of P4502B, but 7-benzyloxy (BROD) is relatively non-specific and responds to several different inducing agents (Winston et al. 1990). Activities of the different alkoxyresorufin 0-dealkylases of ferret liver indicate a 3-fold higher specific activity and a 15-fold higher turnover number (nmol product/min per nmol total P-450) for cytochrome P4501 (EROD) than is seen in rat liver; rodents generally appear to have a much lower turnover number for P4501 than is seen in the ferret (table 3). Hence, with the substrates used in the present study, it is possible to conclude that the specific activity of P4501 is markedly greater than in rat (MROD 100% increase, EROD 350% increase); P4502B is similarly greater in ferret than in rat (PROD 300% increase); P4502E is slightly lower in the ferret (dimethylnitrosamine demethylase is 60% of that in rat); P4503 is lower in ferret (ethylmorphine N-demethylase is 40% that of rat); and P4504 is also lower in ferret (20% of that in rat). However, benzphetamine N-demethylase, which is also a P4502B reaction, is the same in both animal species, for which a possible explanation is that benzphetamine is known to be metabolized by other basal forms of P-450 (Ryan and Levin 1990). Moreover, it should be noted that P4504 catalyses only the 12-hydroxylation of lauric acid, whereas the assay used in the present study does not discriminate between the 11- and 12-hydroxylations. It must be emphasized that although orthologous proteins to rat P-450s may be present in ferret, these may not necessarily have the same substrate specificity. Enzyme induction by MC Induction of ferrret liver mixed-function oxidases by MC is similar to that seen in rat, namely, increases in total cytochromes P-450 with a shift in the wavelength of the cytochrome P-450 CO-ligand difference spectrum from 450 to 448nm, and no increases in the NADPH-cytochrome c reductase or microsomal protein; indeed there is a significant decrease in hepatic microsomal protein
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following M C treatment of the ferret. However, no overt pathology was histologically apparent. EROD specific activity, indicative of P4501, was increased 20-fold in ferret liver and 60-fold in rat liver after MC-induction; and the corresponding turnover numbers were increased 7- and 25-fold for ferret and rat liver, respectively (table 2). EROD activity is attributable to the enzymic actions of both cytochrome P4501A1 and P4501A2, although the turnover number of the former is an order of magnitude greater than that of the latter isozyme (Astrom and De Pierre 1985). As can be seen from table 5, an antibody to rat liver P4501A1 inhibits ferret liver EROD activity by 88%, whereas an antibody to P4501A2 inhibits only by 53%. I n both rat and ferret liver, M C treatment induces the synthesis of both P4501 proteins, and in rat liver the synthesis of P4501A1 is greater than that of P4501A2. Inhibition by a-naphthoflavone (an inhibitor of P4501) of rat liver EROD activity after MC-induction (69%) is greater than before M C treatment (40%) (table 4). Similarly, in the ferret, a-naphthoflavone inhibits 94% of the EROD activity of MC-induced liver microsomes, but inhibits only 68% of the EROD activity of the basal enzymes (table 4). Inhibition of P4501 activity by a-naphthoflavone is more marked with ferret liver microsomal preparations than with rat, indicating that differences may exist in the nature of the active sites of the rat and ferret P4501 proteins. Moreover, M C treatment of ferrets resulted in a much greater induction of EROD (20-fold, representing largely P4501A1 as shown immunologically; table 5) than of Glu-P-1 or paracetamol bioactivation (10-fold, representing P4501A2 but not 1Al). T h e more specific immunoquantitative determination of P4501A1 indicates that MC-induction increases the concentration of this ferret liver enzyme protein to at least five times the basal level (figure 1). Comparison of ferret and rat T h e relatively small group size (three ferrets and guinea pigs, and six rats and mice), and the exclusive use of male animals, does impose certain limitations on the conclusions that may be drawn from these preliminary studies, but earlier work (Ioannides et al. 1977, Pope et al. 1980) showed that, unlike rat, no major sex differences in drug metabolism were seen in the ferret. A comparison of drug metabolism in male ferret and male rat was therefore considered tenable. T h e four-fold greater concentration of basal P4501 in male ferret liver than in male rat liver, and the similar concentration of these two enzyme proteins after MC induction, indicates that the ferret may be as sensitive an experimental animal for carcinogenicity and chemical toxicity studies as the rat. For several workers have shown that the two cytochrome P4501 proteins are the major enzymes catalyzing the oxidative activation of toxic chemicals to mutagens, carcinogens, neoantigens and other reactive intermediates; Guengerich (1988) and Ioannides and Parke (1990) reported that some 90% of carcinogens are activated by these cytochrome P4501 enzyme proteins. One other isoform of cytochrome P-450 that is known to activate carcinogens, and to cause chemical toxicity and carcinogenicity by oxygen radical production, is P4502E1, activity of which has been indicated in ferret liver by the presence of dimethylnitrosamine N-demethylase activity, although Western blots with rat liver P4502E1 polyclonal antibody did not exhibit any immuno-reactive band in the P-450 region. T h u s ferret liver contains comparable or greater levels of at least two of the three activating microsomal cytochromes P-450, namely 1Al and 1A2. T h e ferret would therefore
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appear to be-at least in respect of hepatic drug metabolism-a suitable nonrodent species in the safety evaluation of new drugs and other chemicals since, like the dog (McKillop 1985) it exhibits a high level of P4501 activity in liver. Furthermore, the ferret, like man, is a periodic feeder with a gallbladder, whereas rodents are continuous feeders and the rat has no gallbladder, which should lead to the ferret exhibiting similar pharmacokinetics to man for drugs and chemicals that are orally administered and/or enterohepatically circulated. However, the ferret is a hibernating species which undergoes seasonal changes in body weight, basal metabolism and hepatic lipid accumulation, that might well affect the metabolism of xenobiotics.
Acknowledgements Downloaded by [RMIT University Library] at 03:48 04 May 2016
We are grateful to Glaxo Laboratories for their financial support of this study.
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