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species variation in the activation of 2-acetylaminofluorene to mutagens, the rat being a very poor activator (Ioannides et al., 1981). In this paper we report the.
Mutation Research, 124 (1983) 325-336 Elsevier

325

MTR 00832

Activation of aromatic amines to mutagens by various animal species including man Caroline E. Phillipson and Costas Ioannides Biochemistry Department, University of Surrey, Guildford, Surrey, GUZ 5XH (Great Britain) (Received 26 May 1983) (Revision received 3 August 1983) (Accepted 16 August 1983)

Summary

2-Acetylaminofluorene, 2-aminofluorene, 4-aminobiphenyl, 2-naphthylamine, 2aminoanthracene and benzidine were assayed for mutagenicity in the Ames test in the presence of hepatic microsomal preparations derived from mouse, hamster, rat, pig and man. Prior to each mutagenicity assay all activation systems were fully characterized with respect to mono-oxygenase and mixed-function amine oxidase activities. All compounds were metabolically activated to mutagens by all activation systems, but with markedly different efficiencies, hamster being the only species which readily activated all amines.. The hamster also exhibited the highest ethoxyresorufin 0-deethylase and dimethylaniline N-oxidase activities.

The use of the Ames Salmonella/microsome test (Ames et al., 1975) is not confined to the screening of chemicals for mutagenicity but also serves as a tool to further our understanding of the pathways of activation of chemical carcinogens. Metabolic activation of chemicals which are not direct-acting mutagens is usually achieved by incorporating a hepatic microsomal preparation (S9 mix) derived from rats pretreated with Aroclor 1254, a mixture of polychlorinated biphenyls which induces a wide range of mixed-function oxidases (Alvares et al., 1973). During the last 5 years a number of workers have reported marked species differences in the activation of some carcinogens. For example preparations from hamster liver were more effective than preparations from rat liver in the activation of nitrosamines (Prival et al., 1979), 2-acetylaminofluorene and 3-methylcholanthrene (Raineri et al., 1981). Furthermore, pretreatment of animals with various monooxygenase inducers often resulted in decreased mutagenicity of carcinogens such as Address for all correspondence: Dr. C. Ioannides, Guildford, Surrey, GU2 SXH (Great Britain). 0165-1218/83/$03.00

0 1983 Elsevier Science Publishers

Biochemistry

B.V.

Department,

University

of Surrey,

326 dimethylnitrosamine (Hutton et al., 1979), whose activation does not appear to involve this enzyme system (Rowland et al., 1980; Phillipson et al., 1982; Masson et al., 1983). We therefore embarked on a study to evaluate the ability of liver preparations from various animals to activate groups of chemical carcinogens and compare it to that of human hepatic preparations. The aromatic amines comprise an important group of bladder carcinogens in animals and man which are used as dye intermediates in the rubber and plastics industries (Radomski, 1979; Kriek, 1974). We have previously reported a marked species variation in the activation of 2-acetylaminofluorene to mutagens, the rat being a very poor activator (Ioannides et al., 1981). In this paper we report the efficiency of various laboratory animals to activate six aromatic amines to mutagens and compare it to that of man. The pig has also been included in this study as this animal possesses high mixed-function amine oxidase activity (Ziegler and Mitchell, 1972), another microsomal enzyme system involved in the activation of aromatic amines (Pelroy and Gandolfi, 1980). Materials and methods Benzidine, 2-acetylaminofluorene, 2-aminoanthracene, 2-naphthylamine, dimethylaniline and all cofactors (Sigma Co., Poole, Dorset), 2-aminofluorene (Aldrich Chemical Co., Gillingham, Dorset), 4-aminobiphenyl (Phase Separations Ltd., Queensferry, Flintshire) and ethoxyresorufin and resorufin (Pierce Chemicals, Rockford, Illinois, U.S.A.) were purchased. Benzphetamine was a generous gift of UpJohn Co. Ltd., Kalamazoo, Michigan, U.S.A. Male Wistar albino rats (150-200 g) and male albino CD1 mice (35-40 g) were obtained from the Animal Breeding Unit, University of Surrey, and male golden Syrian hamsters (loo-120 g) from Wrights of Essex, Essex, Great Britain. Fresh pig liver was obtained from the local abattoir and was kept frozen at -80°C. The human liver sample was from a male killed in a traffic accident. The sample was also kept at -80°C. Hepatic post-mitochondrial supernatant (9000 X g supernatant, S9 fraction) and microsomal suspension (105 000 X g pellet resuspended) were prepared as previously described (Ioannides and Parke, 1975). The following determinations were carried out on the post-mitochondrial supernatant: benzphetamine N-demethylase (Lu et al., 1972) and mixed-function amine oxidase using dimethylaniline as substrate (Ziegler and Pettit, 1964); and on the microsomal suspension: NADPH-cytochrome c reductase (Williams and Kamin, 1962), ethoxyresorufin 0-deethylase (Burke and Mayer, 1974), cytochrome P-450 (Omura and Sato, 1964); protein was determined in both fractions (Lowry et al., 1951). Activation of the aromatic amines to mutagens in the Salmonella typhimurium strains TA1538 and TAlOO was carried out as described by Ames (Ames et al., 1975), but employing double the amount of the S9 fraction in the S9 mix, i.e. 100 pl per plate. All carcinogens were dissolved in DMSO so that less than 100 ~1 was added to each plate. Spontaneous reversion rates were in the range 14-20 and 65-120 per plate for TA1538 and TAlOO respectively.

TABLE 1 ENZYME ACTIVITIES OF THE PREPARATIONS USED IN THE MUTAGENICITY ASSAYS Mixed function oxidases and mixed function amine oxidase were determined in all activation systems prior to each mutagenicity assay. This table represents only a typical set of activities. Animal species

N-Oxidation of dimethylaniline (nmoles/min per mg pt)

N-Demethylation of benzphetamine (nmoles H C H O / rain per mg pt)

O-Decthylation of ethoxyresorufin (pmoles/min per mg pt)

NADPH-cytochrome c reductase (nmoles/min per mg pt)

Cytochrome P-450 (nmoles/mg pt)

Mouse a Hamster Rat Pig Human

8.2-21.4 17.1 12.3 16.7 4.5

1.52 2.16 4.80 0.21 0.84

26-87 269 46 44 68

51 90 71 84 50

1.03 1.43 0.86 0.33 0.37

a Marked variations were observed in the N-oxidation of dimethylaniline and O-deethylation of ethoxyresorufin among mice. 6000

5000

4000 L

o

3000

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I

~ 1

I 20

| 30

I 40

2-Ace ty 1amino f l uorene ( u g / p l a t e )

Fig. 1. Metabolic activation of 2-aeetylaminofluorene to mutagens in Salmonella typhimurium strain TA1538. Liver microsomal preparations were isolated from the mouse (D), hamster (,x), rat (O), pig (I) and man (o). Each determination was carried out in triplicate.

328 Results Prior to each mutagenicity assay all activation systems were fully characterized with respect to mono-oxygenases and mixed-function amine oxidase. A typical set of activities is shown in Table 1. The human sample exhibited the lowest mixed-function amine oxidase while the highest levels were observed in the pig and hamster. The latter species also had the highest content of cytochromes P-450 while low levels were always observed in the human and pig hepatic microsomes. The cytochrome P-450-dependent N-demethylation of benzphetamine was low in the human and pig liver while the rat exhibited the highest activity. The cytochrome P-448-mediated O-deethylation of ethoxyresorufin was markedly higher in the hamster (5-fold) than in any other animal species. The rate of NADPH-dependent reduction of cytochrome c was similar in all animal species. Hepatic preparations from all animals activated all aromatic amines but at markedly different rates (Figs. 1-6). The rat, in contrast to the other species, had a very limited capacity to convert 2-acetylaminofluorene to mutagens (Fig. 1). 2Aminofluorene and 2-aminoanthracene were activated by all hepatic preparations with similar efficiency (Figs. 2 and 3). The activation of 2-naphthylamine was catalysed markedly more efficiently by the hamster than any of the other species

8000

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"---o SO00 = 5000

> 4000

3000 z

2000

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I 20 2-Aminofluorene

I 30

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(~g/pl ate)

Fig. 2. Metabolic activation of 2-aminofluoreneto mutagens in Salmonella typhimurium strain TA1538. Symbols as in Fig. 1.

329 6000

5000

~4000

3000

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2

4

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2-Ami noan th racene ( u g / p l a t e )

Fig. 3. Metabolicactivationof 2-aminoanthraceneto mutagens in Salmonella typhimurium strain TA100. Symbols as in Fig. 1.

(Fig. 4). When the mutagenicity of 4-arninobiphenyl was investigated, the pig, in contrast to the other animal species, had poor capacity to activate this carcinogen (Fig. 5). Finally, only the hamster and mouse were effective activators of benzidine (Fig. 6).

Discussion

The initial metabolism of aromatic amines involves N- and ring hydroxylations and N-acetylation (Clayson and Garner, 1976; Tanaka et al., 1981; Brouns et al.,

330 1200

I000

800

aJ 600 o

400

2O0

I0

I

I

20

30

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2-Naphthylamine (ug/plate)

Fig. 4. Metabolic activation of 2-naphthylamineto mutagensin Salmonella typhimurium strain TA100. Symbols as in Fig. 1.

1982). Ring hydroxylation is considered to be a deactivation pathway while N-bydroxylation is recognized as the first step in the activation of aromatic amines to their ,reactive intermediates (Weisburger and Weisburger, 1973; Miller et al., 1961; Sakai et al., 1978; Irving, 1979). The N-hydroxy derivatives serve only as proximate carcinogens and may undergo further metabolism through conjugation with glucuronic acid and sulphate, N-deacetylation, N-acetylation and N, O-acyl transfer (Sakai et al., 1978; Schut et al., 1978; Weeks et al., 1978; Wirth and Thorgeirsson, 1981). The activation systems employed in the present mutagenicity studies lack acetylase activity (McCann and Ames, 1977) and generally have very limited phase II enzyme

331

100(

750

~ 500 z

/

250

I 25

I

I

50 75 4-Aminobiphenyl (~g/plate)

I I00

Fig. 5. M e t a b o l i c a c t i v a t i o n o f 4 - a m i n o b i p h e n y l to m u t a g e n s in Salmonella typhimurium strain TA1538. S y m b o l s as i n Fig. 1.

activity (Greim et al., 1980). Therefore under the present conditions only N- and ring-hydroxylated products are formed; since the latter are non-mutagenic (Masson et al., 1983a), mutagenicity can be assumed to be due to the N-hydroxy metabolites. Furthermore, the mutagenicity of N-hydroxy-4-aminobiphenyl was not enhanced in the presence of $9 demonstrating that it cannot be converted to the ultimate carcinogen. Recently, it has been demonstrated that in the case of 2-acetylaminofluorene activation could also be mediated by rat cytosolic preparations (Forster et al., 1981a and b). However, in the uninduced animal efficiency of activation was very poor. The N-hydroxylation of aromatic amines may be catalysed by (a) the cytochrome P-450-dependent mono-oxygenases (Gutmann and Bell, 1977; Thorgeirsson et al., 1973), (b) the mixed-function amine oxidase (Ziegler and Mitchell, 1972; Pelroy and Gandolfi, 1980) and (c) by active forms of oxygen generated during the metabolism of arachidonic and by the prostaglandin endoperoxide synthetase enzyme system

332 1250

750

500

250

;~

~0

7~

1'00

Benzidine (~g/plate)

Fig. 6. Metabolic activation of benzidine to mutagens in SalmonellatyphimuriumstrainTA1538. Symbols as in Fig. 1.

present in extrahepatic tissues (Kadlubar et al., 1982). In the present study hepatic mono-oxygenases and mixed function amine oxidase were determined prior to each mutagenicity assay to ensure the metabolic integrity of each activation system. The activities exhibited by the human activation system were always within the upper range reported in recent extensive studies (Jakobsson et al., 1982). During our initial studies we observed marked species variations in the activation of 2-acetylaminofluorene to mutagens, the rat being a very poor activator (Ioannides et al., 1981). This is confirmed in the present study and is compatible with previous reports that the rat exhibits poor N-hydroxylase activity (Razzouk and Roberfroid,

333 1982). Similarly the rat was a poor activator of 4-aminobiphenyl and 2-naphthylamine, but readily activated 2-aminofluorene and 2-aminoanthracene. In fact the last two compounds were activated to the same extent by all animal and human preparations. The hamster was the only animal species which readily activated 2-naphthylamine. In general the hamster was the only animal species which effectively activated all the compounds studied. In the standard Ames test employing an activating system derived from the Aroclor 1254-pretreated rat, benzidine is a weak mutagen (Garner, 1975; McCann et al., 1975). In the present study the rat and all animal species, with the exception of the hamster, activated this carcinogen poorly. Pretreatment of animal with monooxygenase inducers failed to enhance the mutagenicity of this carcinogen suggesting that the major forms of cytochrome P-450 are not involved in its activation (Tucker and Tang, 1979). In addition, we were unable to demonstrate activation of this carcinogen to mutagens using highly purified preparations of the major forms of cytochrome P-450 induced in the rat by administration of phenobarbital and fl-naphthoflavone (H. Masson, G.G. Gibson and C. Ioannides, unpublished observations). However, chronic ethanol administration, which induces a specific form of cytochrome P-450 involved in the production of hydroxy radicals (Ingelman-Sundberg and HagbjiSrk, 1982), also enhanced the mutagenicity of benzidine in both hamster and rat (C.M. Steele, C.E. Phillipson and C. Ioannides, unpublished observations). It is possible that this ethanol-induced isozyme and cytochrome P-450 (Koop et al., 1982) may also catalyse the activation of benzidine. No correlation could be obtained in this study between the degree of activation of the aromatic amines and either mono-oxygenase or mixed-function amine oxidase activities. This is hardly surprising since the contribution of these two systems in N-hydroxylation may vary among aromatic amines. 2-Aminothracene and 2aminofluorene which were extensively activated by purified mixed-function amine oxidase (Pelroy and Gandolfi, 1980) were also activated by the pig, an animal possessing high mixed-function amine oxidase activity but low mixed-function oxidase activity, while in contrast 2-acetylaminofluorene and 2-naphthylamine were activated by neither the purified enzyme nor the pig. It is of interest that the hamster, the only animal species to readily activate all aromatic amines studied, always exhibited the highest cytochrome P-448 activity, as exemplified by the O-deethylation of ethoxyresorufin, an enzyme catalysed exclusively by this isozyme of the cytochrome (Burke and Mayer, 1975; Ioannides et al., 1983). Experimental evidence indicates that cytochrome P-448 may facilitate the N-hydroxylation of aromatic amines more readily than the phenobarbital-induced cytochrome P-450 (Lotlikar and Zalesky, 1975; Norman et al., 1979; Johnson et al., 1980; Razzouk et al., 1982). Furthermore, the hamster also possessed the highest dimethylaniline N-oxidase activity, being catalysed by cytochrome P-448 in addition to the mixed-function amine oxidase (Hlavica and Hiilsmann, 1979). In conclusion we have demonstrated that the hamster exhibits higher capacity than the rat and other animals to activate aromatic amines to mutagens and the use of this species in routine mutagenicity studies merits further investigation.

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Acknowledgements T h e a u t h o r s w o u l d like to t h a n k M i s s . C . M . S t e e l e f o r e x p e r t t e c h n i c a l a s s i s t a n c e , the Medical Research Council for financial support of this work and for the Studentship for C.E.P., and Dr. Frantz and Dr. Kreiss, Paris-Transplant, for the provision of the human liver samples.

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