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Inhibition of protein pyrolysate mutagenicity by retinol (Vitamin A)
F,/ Chm.
Tmtc.
Vol. 20. pp. 535 lo 539. 19llZ
0278~6915/82/050535-05103.00/0
prtnted in Great Britain. All rights rescrved
INHIBITION
Copyright 0 1982 Pergsmon Press Ltd
OF PROTEIN PYROLYSATE BY RETINOL (VITAMIN L. BusK,*t, U. G. AHLBORG~ l
SUnit
and L.
MUTAGENICITY A)
ALBANUS*
Luhorutory, National Food Administration, Box 622. S-751 26 Uppsala tDepartment o/Genetics, University of Uppsalu, S-750 07 Uppsala o/ Toxicology, National Institute of Environmental Medicine. Karolinsku Institrrte, S-104 VI Stockholm, Sweden To.ricology
(Receioed
I5 February
1982)
Abstract--The mutagenicity in the Ames Salmonella-microsome test of four protein pyrolysate products, formed during the cooking of meat, (Trp-P-l, Trp-P-2, Glu-P-I and Glu-P-2) was found to be inhibited by the addition of vitamin A in vitro in the form of retinol. The effect is interpreted as an inhibition of the metabolic activation of the mutagens to their respective ultimate mutagenic forms since retinal has been shown to have no eRect on the survival of the Salmonella cells, no effect on directly acting mutagens and no effect on the formation of NADPH in the test system. The results demonstrate the need for an increased understanding of the interaction of dietary components in evaluating mutagenic/ carcinogenic risks from processed food.
ferent groups in our society we undertook the present
INTRODUCTION
Short-term tests for mutagenicity, in particular the Ames Salmonella-microsome test, have been used to an increasing extent to screen for potential mutagens and carcinogens in the environment. While the qualitative correlation between mutagenicity in the Ames test and carcinogenicity in uioo is fairly good (McCann, Choi, Yamasaki & Ames, 1975),the quantitative correlation is less good (Ashby & Styles, 1978) and is dependent on the multistep nature of carcinogenesis; the initiating genotoxic damage, recognized by the mutagenicity assay, may be changed by e.g. DNA-repair processesand promotor or antipromotor agents.However, the initiation processitself may also be influenced by a number of modifying factors such as. for example, vitamin A. The proposal that vitamin A may affect the initiating step stems from the observation that retinol acts as a modifier of the mutagenicity of compounds requiring metabolic activation (Busk & Ahlborg, 1980 I!%1982a,bl. The processing of foods has been found to produce a number of mutagenic compounds (Sugimura & Nagao, 1979)someof which have been shown to have carcinogenic properties in animal studies (Hosaka, Matsushima, Hirono & Sugimura, 1981; Matsukura, Kawachi, Morino et al. 1981). However, these coniPounds seemto be less carcinogenic than would have beenexpected from their mutagenicity. Since diet is regarded as a major contributor to the aetiology of cancer and vitamin A is an essential dietary component ingested in different amounts by difA& = 2-Amino-9H-pyrido(2,3-b)indole; Glu-P-I = 2-amino&methvldioyrido( I .2-a :3’,2’-d)imidazol;Glu-P-2 = 2-aminodipyrido(l,2-a:3’,2’-d)imidazol; W-P-1 = 3-amino-3,4-dimethyl-SH-pyrido(4,3-b)indole; Trp-P-2 = 3-amino-l-methyl-SH-pyrido(4,3-b)indole.
Ahhreviations:
investigation to elucidate the possible influence of vitamin A on the mutagenicity of some compounds formed during the cooking of meat. EXPERIMENTAL Animals. Liver homogenate was prepared from male Sprague-Dawley rats (200g) obtained from Anticimex, Sollentuna, and maintained on Astra Ewos R3 diet, supplied by Ewes AB, Sijdertllje, containing 12,000IU vitamin A/kg. The rats were injected ip with Aroclor 1254(500mg/kg) dissolved in arachis oil 5 days before they were killed. The animal5 were killed by decapitation and the livers were removed aseptically. All subsequent steps were performed under sterile conditions and at +4”C. The tissue was minced and homogenized in three vol. 0.15M-KC1 with four strokes of the pestle at lOOOrev./min in a Potter-Elvenhjelm homogenizer. After centrifugation at 9000g for 10min the supernatants (S-9 fractions) from five rats were pooled, divided into aliquots, immediately frozen to -90°C. and stored at this temperature prior to the start of the experiments (max 2 months). The protein concentration, measured by the method of Hartree (1972), of the S-9 fraction was 25.5mg/ml. The final preparation of the metabolizing system(the S-9 mix) was carried out by the method of Ames, McCann & Yamasaki (1975). The amount of cofactors added to each plate was constant although the amount of S-9 fraction was varied. Chemicals. Trp-P-l, Trp-P-2, Glu-P-l and Glu-P-2 were a gift from Professor T. Sugimura, National Cancer Center ResearchInstitute. Tokyo, Japan. Retinol (all trans), NADP and glucose-6-phosphatewere obtained from Sigma Chemical Company, St. Louis, MO, USA. All other chemicals were purchased from Kebo Grave, SpBnga.
535
536
L. BUSK. U. G. AHLBORG and L. ALBANUS 700
(b)
(a)
Retinal, pg /plate Fig. 1. The mutagenicity of (a) Trp-P-l at levels of 40 (V) and 80 (0) of 5 (V) and 10 (0) rig/plate towards Salmonella ryphimurium strain amounts of retinol. Each point represents the mean of two experiments
Bacterial tester strain. The Salmonella typhimrcrium histidine auxotroph strain TA98 was kindly provided by Professor B. N. Ames, University of California, USA, and was checked in our laboratory for proper genetic characteristics by the method of Ames et al. (1975). Mutagenicity assay. The procedure of Ames et al. (1975)with the modification of Yahagi, Nagao, Seino et al. (1977) was followed. The S. typhimurium cells were grown for 14 hr at 37°C with continuous shaking. The test compounds were pre-incubated with the S-9 mix at 37°C 20 min prior to the addition of bacteria and top agar. To avoid oxidation, retinol was dissolved in absolute ethanol and added to the liver homogenate from freshly prepared solutions shortly before the addition of the S-9 mix to the test compounds. All retinol solutions were kept on ice and in the dark. The final concentration of ethanol (0.6%) was the samein all S-9 mixes. The addition of ethanol in concentrations varying from 0-1.0% did not affect the mutagenicity of any of the pyrolysate products under the experimental conditions used. The test compounds were dissolved in water and added to the plates in 100PI portions. Measurement of NADPH generation. The S-9 mix was prepared as described for the mutagenicity assays with 10~1 liver homogenate/ml. The rate of NADPH generation was measured as an increase in absorbance at 340nm in a Beckman M24 spectrophotometer. The blank contained the complete reaction mixture with the exception of glucose-6-phosphate. Retinol as a freshly prepared ethanol solution was added to the reaction mixture and the blank as described for the mutagenicity assays. RESULTS
The mutagenic potency of amino acid pyrolysates in the Ames assay is known to vary with the amount of S-9 added to the plates. The optimal amounts of S-9 for all four test compounds were determined in order to conduct the inhibition experiments in a sys-
Retinal, pg /plate q/plate and (b) Trp-P-2 at levels TA98 in the presence of different with three plates/concentration.
tem with maximal sensitivity. The optimal amounts of S-9 were found to be 8, 10, 15 and 30~1 for Trp-P-l, Trp-P-2, Glu-P-l and Glu-P-2, respectively. To elucidate the effect of vitamin A on the mutagenicity of the four amino-acid pyrolysates, vitamin A, in the form of retinol, was added to the test system at dosesranging from 2 to lSOpg/plate. Figure 1 shows that retinol strongly inhibits the mutagenicity of Trp-P-l and Trp-P-2 towards strain TA98. Both compounds were tested at two different concentrations, Trp-P-l at 40 and 80 rig/plate and Trp-P-2 at 5 and long/plate. The inhibition was readily detected even at the lowest concentration of the vitamin, 2pg/plate, regardless of the dose of the mutagen and at concentrations of 5O-1OOpgretinal/ plate the inhibition was almost complete. The spontaneous mutation frequency was 25 + 5 (mean + SEM) revertants/plate. Similar results were obtained with Glu-P-I at concentrations of 7.5 and I5 rig/plate and Glu-P-2 at 500 and lOOOng/plate (Fig. 2). The inhibition was evident at 2pg retinal/plate and was almost complete at 50-100 pg retinal/plate. As different amounts of S-9 were used for the detection of mutagenicity of the various amino acid pyrolysates, the inhibition curves in Figs 1 and 2 are not directly comparable. In Fig. 3, the Ames test data from the highest dose of each mutagen expressed as his+-revertants/plate are plotted against the amount of retinol per mg protein to facilitate comparison of the inhibition at a constant ratio between retinol and the metabolizing enzymes. To find out whether the inhibition by retinol was due to a depletion of NADPH, a cofactor generated in the test system and vital for the activity of the metabolizing enzymes, the formation of NADPH in the S-9 mix was recorded in the presenceof different amounts of retinol. Table 1 shows that retinol at levels of up to 820pg/mg protein had no effect on the formation of NADPH. The rate of formation varied between 95 and 100% of the control with no added retinol. The highest dose of retinol used in the inhibition experiments was about 750 pg/mg protein (Fig.
Inhibition of protein pyrolysate mutagenicity by retinol
537
(b)
(a)
II 1 IO 20
1 50
I loo
1 150
Retinal, pg/piote Fig. 2. The mutagenicity of (a) Glu-P-I at levels of 7.5 (V) and 15 (0) rig/plate and (b) Glu-P-2 at levels ryphirtwiw strain TA98 in the presence of different of 500 (V) and 1000(0) rig/plate towards Su/rt~o~~ulku amounts of retinal. Each point represents the mean of two experiments with three plates/concentration. 3). However, when retinol was tested, at 1536pg/mg protein, a 24% reduction in NADPH formation was recorded. The availability of NADPH does not appear to be responsible for the recorded inhibition of mutagenicity under the experimental conditions used. DISCUSSION
The present data demonstrate that retinol inhibits the mutagenic effect of a series of amino acid pyroly sates. This inhibition is not due to a cytotoxic effect of retinol on the Salmonella cells (Baird & Birnbaum, 1979; Busk & Ahlborg, 1980). It has also been demonstrated that retinol does not inhibit the mutagenic effects of direct-acting mutagens (Baird & Birnbaum, 1979; Busk & Ahlborg, 1980). As in earlier studies with the indirect mutagens aflatoxin B, (Busk & Ahlborg, 1980) and o-aminoazotoluene (Busk & Ahlborg, 1982b) the inhibitory effect of retinol presented here is interpreted as the result of an inhibition of the metabolic activation of the test compounds. 800
r
The metabolism of the pyrolysis products of tryptophan and glutamic acid has not been elucidated in detail. The results of Yamazoe, Ishii, Kamataki et al. (1980) suggest that, in the case of Trp-P-2, N-hydroxylation is the first step in the metabolic activation and is followed by oxidation to a nitrosocompound. Both the N-hydroxy- (M3) and the nitroso- (M4) metabolites (see Fig. 4) are direct-acting mutagens, but recent studies suggest that further conjugation reactions with serine also produce active metabolites (Yamazoe, Ishii. Mita ef crl. 1981). It is therefore difficult to assess which step(s) in the activation of the pyrolysis products are affected by retinol. The proposal that N-hydroxylation is inhibited by retinol is supported by our own finding that retinol also inhibits the mutagenicity of o-aminoazotoluene (Busk & Ahlborg, 1982b). which is believed to be metabolized via an N-hydroxylation (Ames, Durston, Yamasaki & Lee, 1973; Sugimura. Sato. Nagao er ul. 1976). However we have also found that, under the conditions described in this paper, retinol enhances the mutagenicity of two aromatic amines. 2-aminofluorene and 2-acetylaminofluorene (Busk & Ahlborg, 1982a); the metabolism of both of these compounds is known to involve N-hydroxylation as the first step (Aune, Dyb-
NADPH generated Retinal added (&mg protein) I
! I*
I
200
I
300
1
400
Retinal, p9W
1
500
’
600
’
TOO
J
a00
protein
Fig. 3. The mutagenicity of 80 ng Trp-P-l (. . . .). IO ng Trp-P-2 (-), 15 ng Glu-P-l (-.-) and 1OOOngGlu-P-2 (---) towards ~&I~~w//~ I ~yhirmhtr~~ strain TA98 in the presenceof diRerent relativk concentrations of retinal and S-g-mix protein.
0 82 205 410 820
I536
(nmol/min mg protein) 333 316 321 332 323 253
(4, of control) 100 95 96
100 97 76
538
L. BUSK, U. G. AHLBORG
and L. ALBANUS REFERENCES
Ames, B. N.. Durston. W. E.. Yamasaki, E. & Lee, F. D. (1973). Carcinogens are mutagens: A simple test system TRP-P-2 M3 M4 combining liver homogenates for activation and bacteria for detection (frameshift mulagens/aflatoxin/benzo(a)pyrFig. 4. Proposed metabolic activation of Trp-P-2. rne~acetylaminofluorene). Proc. nurn. Acud. Sci. U.S.A. 70. 228 I. Ames, B. N.. McCann. J. & Yamasaki, E. (1975). Methods ing & Thorgeirsson, 1980; Sakai, Reinhold, Wirth ef for detecting carcinogens and mutagens with the Salmoal. 1978). nella/mammalian-microsome mutagenicity test. MuraA number of epidemiological studies have indicated rim Rm. 31, 347. that vitamin A may act as an anticarcinogenic agent Arimoto, S.. Ohara, Y., Namba, T.. Nagishi, T. & Hayatsu, H. (1980). Inhibition of the mutagenicity of amino acid in man (Basu, Donaldson, Jenner er al. 1976; Bjelke, pyrolysis products by hemin and other biological pyrrole 1975: Mettlin & Graham. 1979; Shekelle, Kiu, Raypigments. Biocltem. hiophjrs. Rex Convnw~. 92, 662. nor et a/. 1981; Wald, Idle & Boreham, 1980). The Ashby. J. & Styles, J. A. (1978) Does carcinogenic potency mechanism is not known but it has been suggested correlate with mutagenic potency in the Ames assay? that promotion could be influenced by the vitamin Ntrture. Lortd. 271, 452. (Sporn & Newton, 1979). Our present data further Aune, T., Dybing, E. & Thorgeirsson. S. S. (1980). Developsupports the hypothesis that vitamin A can also affect ment pattern of 3-methylcholanthrene-inducible mutainitiation. genic activation of N-2-fluorenylacetamide, 2-fluorenamine and 2.4-diaminoanisole in the rabbit. J. rmrn. A number of factors other than vitamin A have Cancer Ins:. 64, 765. been found to modify the mutagenicity of amino acid pyrolysates. Borek & Ong (1981) have shown that Baird, M. B. & Birnbaum, L. S. (1979). Inhibition of 2-fluorenamine-induced mutagenesis in Salmonella rrphiX-rays have a synergistic effect on the oncogenic muritrm by vitamin A. J. )~utrr. Cuncer Inst. 63, 1093. action of Trp-P-2 on golden hamster embryo cells. Basu. T. K.. Donaldson. D.. Jenner, M.. Williams, D. C. & Yoshida, Matsumoto & Okamoto (1979) found that Sakula, A. (1976). Plasma vitamin A in patients with amino-a-carboline (A&) had a synergistic effect on bronchial carcinoma. Br. J. Concur 33, 119. Trp-P-2 induced mutagenesis in Salmonella. Pyrrole Bjelke. E. (1975). Dietary vitamin A and human lung pigments such as hemin, biliverdine, chlorophyllin cancer. fnr. J. Cancer 15, 561. Borek. C. & Ong, A. (1981). The interaction of ionizing and protoporphyrin act as inhibitors of the mutagenradiation and food pyrolysis products in producing icity of some amino acid pyrolysates as shown by oncogenic transformation in vitro. Ccmcer Lerf. 12, 61. Aromoto, Ohara, Namba et al. (1980). Tsuda. Takahashi, Nagao et al. (1980) presented evidence that Busk, L. & Ahlborg, U. G. (1980). Retinal (vitamin A) as an inhibitor of the mutagenicity of aflatoxin B,. Toxicoloy~ nitrite in acidic solution deaminates Trp-P-l, Trp-P-2 ht. 6, 243. and Glu-P-1 and thereby prevents the formation of Busk, L. & Ahlborg. U. G. (1982a). Retinal (vitamin A) as a the ultimate mutagenic forms of these substances. modifier of 2-aminofluorene and 2-acetylaminofluorene They have also shown that nitrite converts AaC to a mutagenesis in the Salmonella/microsome assay. Arch Toxicol. 49. 169. non-mutagenic hydroxy derivative but that prolonged Busk. L. & Ahlborg. U. G. (1982b). Retinoids as inhibitors nitrite treatment gave a 2-hydroxy-3-nitro- compound of the orthoaminoazotoluene induced mutagenesis in the that was a direct-acting mutagen (Tsuda, Nagao, HirSalmonella/microsome test. Mrrrctriorr Res. Lert. 104. 225. ayama & Sugimura, 1981a). Inoue, Morita & Kada (1981) have isolated and purified an inhibitor of Hartree. E. F. (1972). Determination of protein: A moditication of the Lowry method that gives a linear photoTrp-P-2 mutagenicity, probably a haemo protein, metric response. Anctlr~. Biochmt. 48, 422. from cabbage and Hayatsu. Arimoto, Togawa & Hayatsu, H.. Arimoto, S., Togawa. K. & Makita, M. (1981). Makita (1981) have shown that ether extracts of Inhibitory effect of the ether extracts of human feces on human faeces can inhibit the mutagenicity of Trp-P-l, activities of mutagens: inhibition by oleic and linoleic Glu-P-1 and AaC. The inhibitors in the faeces acids. Mtrtutiort Res. 81, 287. extracts were found to be oleic and linoleic acid. Hosaka, S.. Malsushima. T., Hirono, 1. & Sugimura, T. (1981). Carcinoaenic activitv of 3-amino-I-methyl-SHFurthermore Tsuda, Wakabayashi. Hirayama et al. pyrido[4,3-b]indole (Trp-P-2). a pyrolysis product of (1981b) have shown that chlorinated tap water tryptophan. Cmrcer Lerl. 13, 23. strongly inhibits the mutagenicity of both Trp-P-l Inoue, T., Morita, K. & Kada. T. (1981). Purification and and Trp-P-2. properties of a plant desmutagenic factor for the mutaThe occurrence of both endogenous and exogenous genic principle of tryptophane pyrolysate. Agric. biol. compounds that can modify the mutagenicity of Cl1em. 45. 345. amino-acid pyrolysates makes it difficult to assess the Matsukura, N., Kawachi. T., Morino, K., Ohgaki, H., Sugiextent to which they pose a health hazard to man. mura. T. & Takayama. S. (1981). Carcinogenicity in mice of mutagenic compounds from a tryptophane pyrolysate. However, the mere existe&e of them, in food ingested Science, N.K 213, 346. by man, and their biological activity in mode) test McCann, J., Choi. E.. Yamasaki. E. & Ames, B. N. (1975). systems, make them highly suspect as a group of comDetection of carcinogens as mutagens in the Sa/mot~cllo/ pounds that may be involved in the observed relationmicrosome test: assay of 300 chemicals. Proc. natn. Acad. ship between diet and cancer incidence. Sci. U.S.A. 72. 5 135. Ack,lo,s/edy~menrs-This work was supported by grants Mettlin, C. & Graham, S. (1979). Dietary risk factors in Medical Research Council from Swedish the human bladder cancer. Am. J. Epidemic/. 3 (1 IO), 225. (381-03X-05923-01) and from the National Board for Sakai, S., Reinhold, C. E., Wirth. P. J. & Thorgeirsson, S. S. Laboratory Animals (80-126). The skilful technical assist(1978). Mechanism of in vitro mutagenic activation and ance of C. Nordensten and K. Podra is appreciated. covalent binding of N-hydroxy-2-acetylaminofluorene in
Inhibition
of protein
pyrolysate
isolated liver cell nuclei from rat and mouse. Cancer Rrs. 38, 2058. Shekelle, R. B.. Kiu. S., Raynor, W. J., Jr, Lepper. M.. Maliza, C. & Rossof, A. H. (1981). Dietary vitamin A and risk of cancer in the Western Electric study. Lancer II. 1185. Sporn, M. B. & Newton. D. L. (1979). Chemoprevention of cancer with retinoids. Fedrr Proc. Fvdn Am. Sots esp. Biol. 38. 2528. Sugimura, T. & Nagao. M. (1979). Mutagenic factors in cooked foods. CRC Crir. Rw. Tosicol. 6, 189. Sugimura. T.. Sate. S.. Nagao, M., Yahagi. T.. Matsushima. T.. Seino. Y.. Takeuchi. M. & Kawachi, T. (1976). Ovcrlapping of carcinogens and mutagens. In Fw~drtrnmru/s in Concrr Prewnrion. Edited by P. N. Magee cr cl/. p. 191. University of Tokyo Press/University Park Press. Baltimore. MD. Tsuda, M.. Takahaski. Y.. Nagao, M.. Hirayama, T. & Sugimura, T. (1980). Inactivation of mutagens from pyrolysates of tryptophan and glutamic acid by nitrite in acidic solution. Mururion Rrs. 78, 331. Tsuda. M., Nagao, M.. Hirayama, T. & Sugimura. T. an indirect (1981a). Nitrite converts 2- amino-a-cdrboline. mutagen, into 2-hydroxy-a-carboline, a non mutagen. and 2-hydroxy-3-nitroso-%-carboline. a direct mutagen. Mura~ion Rrs. 83. 61.
.
mutagenicity
by retinol
539
Tsuda, M., Wakabayashi. K.. Hirayama. T., Kawachi, T. & Sugimura. T. (1981b). Inactivation of the pyrolysate mutagens TRP-P-2, GLU-P-l and IQ by chlorinated tap water. Presentation at the 3rd International Conference on Environmental Mutagens. Tokyo, Japan. Wald, N., Idle. M. & Boreham, J. (1980). Low serumvitamin-A and subsequent risk ofcancer. Lancer II. 813. Yahagi, T.. Nagao. M.. Seino, Y.. Malsushima. T., Sugimura. T. & Okada. M. (1977). Mutagenicities of N-nitrosamines on Salmonella. Mururion Res. 48, 121. Yamazoe, Y.. Ishii, K.. Kamataki. T.. Kate, R. & Sugimura. T. (1980). Isolation and characterization of active tryptophane-pyrolysate mutagen, metabolites of TRP-P-2. formed by rat liver microsomes. Chnico-hiok Inrerocrio~u 30, 125. Yamazoe. Y., Ishii. K.. Mita. S.. Kamataki. T. & Kato. R. (1981). Studies on the active metabolite of 3-amino-lmethyl-5H-pyrido(4.3-b)indole (TRP-P-2). Presentation at the 3rd International Conference on Environmental Mutagens. Tokyo. Japan. Yoshida. D., Matsumoto, T. & Okamoto, H. (1979). Interaction between amino-a-carboline and amino-)I-carboline on mutagenicity in Sol~nonella fyphirnwium Mtrrariot1 Rex 68, 175.
Tmtc.
Vol. 20. pp. 535 lo 539. 19llZ
0278~6915/82/050535-05103.00/0
prtnted in Great Britain. All rights rescrved
INHIBITION
Copyright 0 1982 Pergsmon Press Ltd
OF PROTEIN PYROLYSATE BY RETINOL (VITAMIN L. BusK,*t, U. G. AHLBORG~ l
SUnit
and L.
MUTAGENICITY A)
ALBANUS*
Luhorutory, National Food Administration, Box 622. S-751 26 Uppsala tDepartment o/Genetics, University of Uppsalu, S-750 07 Uppsala o/ Toxicology, National Institute of Environmental Medicine. Karolinsku Institrrte, S-104 VI Stockholm, Sweden To.ricology
(Receioed
I5 February
1982)
Abstract--The mutagenicity in the Ames Salmonella-microsome test of four protein pyrolysate products, formed during the cooking of meat, (Trp-P-l, Trp-P-2, Glu-P-I and Glu-P-2) was found to be inhibited by the addition of vitamin A in vitro in the form of retinol. The effect is interpreted as an inhibition of the metabolic activation of the mutagens to their respective ultimate mutagenic forms since retinal has been shown to have no eRect on the survival of the Salmonella cells, no effect on directly acting mutagens and no effect on the formation of NADPH in the test system. The results demonstrate the need for an increased understanding of the interaction of dietary components in evaluating mutagenic/ carcinogenic risks from processed food.
ferent groups in our society we undertook the present
INTRODUCTION
Short-term tests for mutagenicity, in particular the Ames Salmonella-microsome test, have been used to an increasing extent to screen for potential mutagens and carcinogens in the environment. While the qualitative correlation between mutagenicity in the Ames test and carcinogenicity in uioo is fairly good (McCann, Choi, Yamasaki & Ames, 1975),the quantitative correlation is less good (Ashby & Styles, 1978) and is dependent on the multistep nature of carcinogenesis; the initiating genotoxic damage, recognized by the mutagenicity assay, may be changed by e.g. DNA-repair processesand promotor or antipromotor agents.However, the initiation processitself may also be influenced by a number of modifying factors such as. for example, vitamin A. The proposal that vitamin A may affect the initiating step stems from the observation that retinol acts as a modifier of the mutagenicity of compounds requiring metabolic activation (Busk & Ahlborg, 1980 I!%1982a,bl. The processing of foods has been found to produce a number of mutagenic compounds (Sugimura & Nagao, 1979)someof which have been shown to have carcinogenic properties in animal studies (Hosaka, Matsushima, Hirono & Sugimura, 1981; Matsukura, Kawachi, Morino et al. 1981). However, these coniPounds seemto be less carcinogenic than would have beenexpected from their mutagenicity. Since diet is regarded as a major contributor to the aetiology of cancer and vitamin A is an essential dietary component ingested in different amounts by difA& = 2-Amino-9H-pyrido(2,3-b)indole; Glu-P-I = 2-amino&methvldioyrido( I .2-a :3’,2’-d)imidazol;Glu-P-2 = 2-aminodipyrido(l,2-a:3’,2’-d)imidazol; W-P-1 = 3-amino-3,4-dimethyl-SH-pyrido(4,3-b)indole; Trp-P-2 = 3-amino-l-methyl-SH-pyrido(4,3-b)indole.
Ahhreviations:
investigation to elucidate the possible influence of vitamin A on the mutagenicity of some compounds formed during the cooking of meat. EXPERIMENTAL Animals. Liver homogenate was prepared from male Sprague-Dawley rats (200g) obtained from Anticimex, Sollentuna, and maintained on Astra Ewos R3 diet, supplied by Ewes AB, Sijdertllje, containing 12,000IU vitamin A/kg. The rats were injected ip with Aroclor 1254(500mg/kg) dissolved in arachis oil 5 days before they were killed. The animal5 were killed by decapitation and the livers were removed aseptically. All subsequent steps were performed under sterile conditions and at +4”C. The tissue was minced and homogenized in three vol. 0.15M-KC1 with four strokes of the pestle at lOOOrev./min in a Potter-Elvenhjelm homogenizer. After centrifugation at 9000g for 10min the supernatants (S-9 fractions) from five rats were pooled, divided into aliquots, immediately frozen to -90°C. and stored at this temperature prior to the start of the experiments (max 2 months). The protein concentration, measured by the method of Hartree (1972), of the S-9 fraction was 25.5mg/ml. The final preparation of the metabolizing system(the S-9 mix) was carried out by the method of Ames, McCann & Yamasaki (1975). The amount of cofactors added to each plate was constant although the amount of S-9 fraction was varied. Chemicals. Trp-P-l, Trp-P-2, Glu-P-l and Glu-P-2 were a gift from Professor T. Sugimura, National Cancer Center ResearchInstitute. Tokyo, Japan. Retinol (all trans), NADP and glucose-6-phosphatewere obtained from Sigma Chemical Company, St. Louis, MO, USA. All other chemicals were purchased from Kebo Grave, SpBnga.
535
536
L. BUSK. U. G. AHLBORG and L. ALBANUS 700
(b)
(a)
Retinal, pg /plate Fig. 1. The mutagenicity of (a) Trp-P-l at levels of 40 (V) and 80 (0) of 5 (V) and 10 (0) rig/plate towards Salmonella ryphimurium strain amounts of retinol. Each point represents the mean of two experiments
Bacterial tester strain. The Salmonella typhimrcrium histidine auxotroph strain TA98 was kindly provided by Professor B. N. Ames, University of California, USA, and was checked in our laboratory for proper genetic characteristics by the method of Ames et al. (1975). Mutagenicity assay. The procedure of Ames et al. (1975)with the modification of Yahagi, Nagao, Seino et al. (1977) was followed. The S. typhimurium cells were grown for 14 hr at 37°C with continuous shaking. The test compounds were pre-incubated with the S-9 mix at 37°C 20 min prior to the addition of bacteria and top agar. To avoid oxidation, retinol was dissolved in absolute ethanol and added to the liver homogenate from freshly prepared solutions shortly before the addition of the S-9 mix to the test compounds. All retinol solutions were kept on ice and in the dark. The final concentration of ethanol (0.6%) was the samein all S-9 mixes. The addition of ethanol in concentrations varying from 0-1.0% did not affect the mutagenicity of any of the pyrolysate products under the experimental conditions used. The test compounds were dissolved in water and added to the plates in 100PI portions. Measurement of NADPH generation. The S-9 mix was prepared as described for the mutagenicity assays with 10~1 liver homogenate/ml. The rate of NADPH generation was measured as an increase in absorbance at 340nm in a Beckman M24 spectrophotometer. The blank contained the complete reaction mixture with the exception of glucose-6-phosphate. Retinol as a freshly prepared ethanol solution was added to the reaction mixture and the blank as described for the mutagenicity assays. RESULTS
The mutagenic potency of amino acid pyrolysates in the Ames assay is known to vary with the amount of S-9 added to the plates. The optimal amounts of S-9 for all four test compounds were determined in order to conduct the inhibition experiments in a sys-
Retinal, pg /plate q/plate and (b) Trp-P-2 at levels TA98 in the presence of different with three plates/concentration.
tem with maximal sensitivity. The optimal amounts of S-9 were found to be 8, 10, 15 and 30~1 for Trp-P-l, Trp-P-2, Glu-P-l and Glu-P-2, respectively. To elucidate the effect of vitamin A on the mutagenicity of the four amino-acid pyrolysates, vitamin A, in the form of retinol, was added to the test system at dosesranging from 2 to lSOpg/plate. Figure 1 shows that retinol strongly inhibits the mutagenicity of Trp-P-l and Trp-P-2 towards strain TA98. Both compounds were tested at two different concentrations, Trp-P-l at 40 and 80 rig/plate and Trp-P-2 at 5 and long/plate. The inhibition was readily detected even at the lowest concentration of the vitamin, 2pg/plate, regardless of the dose of the mutagen and at concentrations of 5O-1OOpgretinal/ plate the inhibition was almost complete. The spontaneous mutation frequency was 25 + 5 (mean + SEM) revertants/plate. Similar results were obtained with Glu-P-I at concentrations of 7.5 and I5 rig/plate and Glu-P-2 at 500 and lOOOng/plate (Fig. 2). The inhibition was evident at 2pg retinal/plate and was almost complete at 50-100 pg retinal/plate. As different amounts of S-9 were used for the detection of mutagenicity of the various amino acid pyrolysates, the inhibition curves in Figs 1 and 2 are not directly comparable. In Fig. 3, the Ames test data from the highest dose of each mutagen expressed as his+-revertants/plate are plotted against the amount of retinol per mg protein to facilitate comparison of the inhibition at a constant ratio between retinol and the metabolizing enzymes. To find out whether the inhibition by retinol was due to a depletion of NADPH, a cofactor generated in the test system and vital for the activity of the metabolizing enzymes, the formation of NADPH in the S-9 mix was recorded in the presenceof different amounts of retinol. Table 1 shows that retinol at levels of up to 820pg/mg protein had no effect on the formation of NADPH. The rate of formation varied between 95 and 100% of the control with no added retinol. The highest dose of retinol used in the inhibition experiments was about 750 pg/mg protein (Fig.
Inhibition of protein pyrolysate mutagenicity by retinol
537
(b)
(a)
II 1 IO 20
1 50
I loo
1 150
Retinal, pg/piote Fig. 2. The mutagenicity of (a) Glu-P-I at levels of 7.5 (V) and 15 (0) rig/plate and (b) Glu-P-2 at levels ryphirtwiw strain TA98 in the presence of different of 500 (V) and 1000(0) rig/plate towards Su/rt~o~~ulku amounts of retinal. Each point represents the mean of two experiments with three plates/concentration. 3). However, when retinol was tested, at 1536pg/mg protein, a 24% reduction in NADPH formation was recorded. The availability of NADPH does not appear to be responsible for the recorded inhibition of mutagenicity under the experimental conditions used. DISCUSSION
The present data demonstrate that retinol inhibits the mutagenic effect of a series of amino acid pyroly sates. This inhibition is not due to a cytotoxic effect of retinol on the Salmonella cells (Baird & Birnbaum, 1979; Busk & Ahlborg, 1980). It has also been demonstrated that retinol does not inhibit the mutagenic effects of direct-acting mutagens (Baird & Birnbaum, 1979; Busk & Ahlborg, 1980). As in earlier studies with the indirect mutagens aflatoxin B, (Busk & Ahlborg, 1980) and o-aminoazotoluene (Busk & Ahlborg, 1982b) the inhibitory effect of retinol presented here is interpreted as the result of an inhibition of the metabolic activation of the test compounds. 800
r
The metabolism of the pyrolysis products of tryptophan and glutamic acid has not been elucidated in detail. The results of Yamazoe, Ishii, Kamataki et al. (1980) suggest that, in the case of Trp-P-2, N-hydroxylation is the first step in the metabolic activation and is followed by oxidation to a nitrosocompound. Both the N-hydroxy- (M3) and the nitroso- (M4) metabolites (see Fig. 4) are direct-acting mutagens, but recent studies suggest that further conjugation reactions with serine also produce active metabolites (Yamazoe, Ishii. Mita ef crl. 1981). It is therefore difficult to assess which step(s) in the activation of the pyrolysis products are affected by retinol. The proposal that N-hydroxylation is inhibited by retinol is supported by our own finding that retinol also inhibits the mutagenicity of o-aminoazotoluene (Busk & Ahlborg, 1982b). which is believed to be metabolized via an N-hydroxylation (Ames, Durston, Yamasaki & Lee, 1973; Sugimura. Sato. Nagao er ul. 1976). However we have also found that, under the conditions described in this paper, retinol enhances the mutagenicity of two aromatic amines. 2-aminofluorene and 2-acetylaminofluorene (Busk & Ahlborg, 1982a); the metabolism of both of these compounds is known to involve N-hydroxylation as the first step (Aune, Dyb-
NADPH generated Retinal added (&mg protein) I
! I*
I
200
I
300
1
400
Retinal, p9W
1
500
’
600
’
TOO
J
a00
protein
Fig. 3. The mutagenicity of 80 ng Trp-P-l (. . . .). IO ng Trp-P-2 (-), 15 ng Glu-P-l (-.-) and 1OOOngGlu-P-2 (---) towards ~&I~~w//~ I ~yhirmhtr~~ strain TA98 in the presenceof diRerent relativk concentrations of retinal and S-g-mix protein.
0 82 205 410 820
I536
(nmol/min mg protein) 333 316 321 332 323 253
(4, of control) 100 95 96
100 97 76
538
L. BUSK, U. G. AHLBORG
and L. ALBANUS REFERENCES
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Toxicoloy~ nitrite in acidic solution deaminates Trp-P-l, Trp-P-2 ht. 6, 243. and Glu-P-1 and thereby prevents the formation of Busk, L. & Ahlborg. U. G. (1982a). Retinal (vitamin A) as a the ultimate mutagenic forms of these substances. modifier of 2-aminofluorene and 2-acetylaminofluorene They have also shown that nitrite converts AaC to a mutagenesis in the Salmonella/microsome assay. Arch Toxicol. 49. 169. non-mutagenic hydroxy derivative but that prolonged Busk. L. & Ahlborg. U. G. (1982b). Retinoids as inhibitors nitrite treatment gave a 2-hydroxy-3-nitro- compound of the orthoaminoazotoluene induced mutagenesis in the that was a direct-acting mutagen (Tsuda, Nagao, HirSalmonella/microsome test. Mrrrctriorr Res. Lert. 104. 225. ayama & Sugimura, 1981a). Inoue, Morita & Kada (1981) have isolated and purified an inhibitor of Hartree. E. F. (1972). 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Inhibition
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pyrolysate
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