Mutagenicities of protein pyrolysates

Cancer Letlers, 2 (1977) 335--340 © Elsevh.~r/North-HollandScientific Publishers Ltd.

335

MUTAGEN'ICITIES OF PROTEIN PYROLYSATES*

M1NAKO NAGAO,MASAKOHONDA, YUKO SEINO, TAKIE k-AHAGI,TAKASHI KAWACHIand TAKASHISUGIMURA Biochemistry Dw~ston, Nahonal Cancer Canter Research institute, Tsukilt, Chuo-ku, Tokyo 104 (Japan)

(Becelved ,2~ December 1976) (Rev,sed ver:,mnreceived 26 Jz.nuary 1977)

SUMMARY Smoke condensate obtmned by pyrolysis of prol;e~s, such as lysozyme and hls'~one, was shown ~o be mutagemc to Salmonella typhirnurmm TA100 and TA98. In vitro metabolic activatmn by a mammahan postmitochondriat enzyme preparation (S-9 Mix) was required. Smoke condensates obtained by pyrolysis of DNA, B.NA, starch and vegetable off were shghlly mutagemc, whereas those from pyrolysis of L- and D-tryptophan and 5-hyctroxy-D,Ltryptophan were very strongly mutagenic with metabolic actlva,2on by S-9 Mix. Because of the high correlatmn between rnutagenicity and carcinogenictty, it is theorized that the c o o i n g of proteinaceous foods m~ght be an important cause of human cancers.

INTRODUCTION Chemicals m the enwronment are a welJ -known cause of human ,cancers It has been shown by use of a modification [9] of the Ames' method [1] that S typhimurium TA100 and TA98 are effective in screening fo~ euvlronrnental carcinogens. More than 400 chemmals were tested m our laboratory for mutageniclty, and a high correlation between mutagens and cz~clnogens was demonstrated [10--12], confirming a report by McCann et al. [7]. Much attention has been paid to possible carcinogens m food, such as ~Tlus work was supported in part by grants from the Mimstry of Hetflt,h and Welfare, the Ministry of Education, Science and Culture, and NzssanScm;.ce Foundatmn, J~pan Address correspondence to. Mmako Nagao, Bmehermstry Division, Natmnal Cancer Conter Research Institute, TJsukl]l5-1, Chuo-ku, Tokyo 104, Japan

33~} nitaosamines, benzo(a)pyrene, mycotoxins, cycasm [2] and pyrrolizidine alk~loids [3]. We reported previously t h a t ~he charred surface of broiled fish a n d beefsteak was mutagenm to S. typhimurium TA98 m the presence of a rat liver postmitochondrial e n z y m e preparatmn (8-9 Mix) i~ vitro [8., 10] Smoke condensates produced by broiling fish were also mutagenic to TA~8 with S-9 Mix [8, .'[0]. One report indicated that the cham'ed surface o f gas broiled h s h contained only 0.25 u g benzo(a)pyrene/kg fish [5], and beefsteak conteaned only 8--50 ~g benzo(a}pyrene/kg meat [4]. Calculations base,2 on our restdts revealed that the mutagenic activity of a dimethyl su][foxide (DMSO) extract of the charred surface of 1 kg fish was rough]7 eqmvalent to the activity of 3--5 m g benzo(a)pyrene [8, 10], and that of 1 kg beef was equivalent to that of a b o u t 4.5 m g benzo(a)pyrene [8, 10]. This indicates clearly ~hat the major m u t a g e m c prinmple in the charted materi~fi and ~;moke condensate was n o t benzo(a)pyrene [8,10]. In this study, we demonstrated that smoke condensates obtained by p:~rolysis o f proteins and amino acids were mutagenic. MATI~RIALS AND METHODS Ct,.emicals. Chicken egg white lysozyme, calf t h y m u s histone (type II L calf 1,hymus DNA and 5-hydroxy-D,L-tryptophan were purchased from Sigma Chemmal Co., St. Louis, Mo. Yeast R N A was from K o k o k u Rayon and Pulp Co. Ltd.., Tokyo. Potato starch, L-tryptophan, D-trypt,ophan and kynuxenic acid were from Wako Pure Chen~'Lcal Industry Ltd., Osaka. Vegetable oil (trade n ~ e Crisco) was f r o m Procl~,er and Gamble Co., Ohio. DMSO was spectmphotometric grade material from Waldo Pure Chemical Industry Ltd., Osaka. S m o k e condensate: One h u n d r e d milligrams of each material was heated in a crucible over a gas burner and the smoke was collected by the vacuum action of a vacuum p u m p through an inverted funnel on glass fiber paper (Whitman GF/C) m o u n t e d in a millipore microsyringe filter hotder held 15 cm above the top of the crucible. The glass fiber paper was dipped in DMSO (100 ul solvent per mg smoke condensate). After thorough extractmn with DMSO, t h e glass fiber paper was removed by centnfugation at 2500 rev/mm for 5 nun at 25°C. The preparation was immedmtely subjected ~o the mu~at m n ,est. In most cases, about 10 mg o f smoke condensate was obtained. M~croblal strains. 8. typhimurium T A 1 0 0 and TA98 [1] were kindly supplied by Dr Bruce N. Ames, University of California, Berkeley, Califo Ovenlight cultures of bacteria m n u t r i e n t broth were used for mutation assay My.tartan assay. Assay was carried o u t ~ls described by Ames et al. [I]1 with :~ome modifications [9]. The tes~ substance, S-9 Mix or 0.1 M phosphate buffer (pH 7.4). and bacteria were preiacubated at 37°C for 20 miit and then poured into a plate o~' m i m m a l glucose agar. Revertant colonies

337 were counted after mcubation for 2 days. S-9 MLx contained 150 gl S-9 fraction which was prepared as described by Ames et al. [1] from rat pretreated with polychlonnated biphenyl, 50/~mol sodium phosphate buffer (pH 7.4)~ 4 umol MgC12, 16.5 umol KC1, 2.5 ~mol glucose-6.phosphate, 2.5 ~mol ATP, 2 umol NADH and 2 umol NADPH to a total of 0.5 ml. RESULTS AND DISCUSSION As shown in Table 1, smoke conden~ates obtained by pyrolysis of lysCzyme and histone showed strong mutagenic activity on TA98 with metabolic activation. The condensates were also mutagenic to TA100 with metabolic activation. Smoke condensates obtained from DNA and RNA showed low mutagemc actnrity with metaboU c activatmn. Condensate obtained from starch showed a weak base-substituting mut~gemc activity ~uthout metabohc activatkm. 7n th;s case, a rat liver enzyme preparation reduced tts mu tageniciLy to onefifth. TABLE 1 M U T A G E N I C ACTIVIT,IES O F S M O K E C O N D E N S A T E S OF BIOMACROMOLECULES

Lysozyme Histone DNA RNA Starch Vegetable oil

OBTAINED BY P YROLYSIS

TA100 + S-9 Mix

-- S-9 Mix

TA98 ~ S-9 l~hx

-- S-9 Mix

2319a 1311 170 0 70 85

0 0 0 0 338 0

8311 50,12 278 83 0 0

0 0 0 0 0 0

aNumber of revertant colomes/mgsmoke condensate per plate. S2?ntaneous revertaut colonies were not included Numbers were calculated from 3 experiments to determine hnear dose responsiblerange Smoke condensate from Cnsco (a processed vegetable oil which is widely used as shortening) sho wed a slight mutagemc activity to TA100 with rnetab olic activation. It should be pomted out that only protein can produce a highly mutagenic product(s) by a pyrolytic process. Based on these resurts, it can be assumed that charred parts of fish and beef broiled at high temperatures contain mutagenic potentlal resulting from pyrolysm of their prel.ems. Consequently, smoke eondensates generated by pyrolysm of various amino acres were subjected to mutation tests. Tryptophan yielded the most poten~ mu tagemc ~ctivity. Tabl,e 2 lists mutagenic act~lvltms of smoke condensates obtained by pyrolysis of tryp~l,ophan and its related compounds L- and

338 TABLE 2 ":t '

t

"~i(

'

"

;

.,,,,,)

,

. ()%

j- ~:~ ,. ~r

i '){ ¢,*':'",)

O']" %'~'-.D -Iv-,-'p,O L Y S I S O F

~ I )"

TAI00

L-Tryp~ophan I)-Tryu ,ophan 5-Hydroxy-D,L-tryptophan Xynuremc acid

TA98

+ S-9 Mix

-- S-9 Mix

+ S-9 Mix

-- S-9 MIx

1960 a 2460 8095 0

0 0 0 126

22,000 26,000 19,047 0

O 0 0 0

aRevert~.nt colomes/mg smoke condensate per plate as described in Table 1 D-Trypl.ophan produced compound(s) highly mutagenm to TA98 w,ith metabolic activation. Smoke condensate from 5-hydxoxy-D,L-tryptophan showed a specific mutagemc activity on TA98 with S-9 Mix similar to t ha t from Lor D-try,~tophan. Smoke condensates fzom D- and L-tryptophan and 5-hydroxy-D L-tryptoph~a showed less mutagenicity to TA100 than to TA98. Kynulenic acid, a metabolic product of L tryptophan, yielded almost no mutagemc pro duct by pyrolysis. L-trypLophan, D-tryptophan, 5-hydroxyD,L-tryp~,ophan and kynurenic acid themselves were n o t mutagenic to either strain. Recently, Matsumoto et al. [6] also found t h a t pyrolytic products o£ D,L-tryptophan were mutagenic to TA98 w~th S-9 Mix. Chicken egg white lysozyme contains 4.65% L-tryptophan whereas calf t h y m u s h~stone c o n t ~ u s almost none. However, smoke condensates from both were strongly mutagenic, indmatmg t h a t the pyrolytic products of amino acids o Lher than L.tryptophan also play a role in the mutagenic activity of smoke condensates from proteins. I t is likely th at mutagens are produced by pyrolysis of proteins in ot[r environment. In vivo and in vitro carcinogenicity experiments are beiJag conducted m our laboratory. Pyrolysate from proteins may be another cause of human cancers. Studies ~o elucidate the mutagenic principles of pyrolytic products of LtlTptophan and L-phenylalanine are also underway. It is qa[te plausible t h a t the pimciples from L-tryptophan are 7-carboline derivatives. REFERENCES I Ames, B N., McCann, J. and Yarnasaki, E. (1975) Methods for detecting carcinogens and mut~tgenswith Saimonella/rnammalian-mmrosome mutagemci~y test Muta~. Res., 31,347--364. 2 Berg, J W. (1975) Diet. In' Persons at High Risk of Cancer, pp 201--224. Editor: J F. Fraumeni, Jr. Academic Press, New York. 3 IARC Monographs on t,Se Evaluation of the Carcinogenic Risk of Chemicals to Mran (1976). Some naturally occurring substances In- Pyrrellzldine Alllaloid~, VoL 10, pp. 265-327. iARC, Lyon.

339

4 Lljmsky, W. and Shublk, P. (1964) Benzo(a)pyrene and other polynuctear hydro., , •,, ,i ¢c" 5 " ~. "'. : ' .. ,.'~ ": ,.' P~" ~ " *matmhydrocarbons . . . . . ,, ' ',v , .* h : ~, *yu, and cmamel. Gann, b'/, '133--142'. 6 Matsumoto, T., Yoshlda, D., Mlzusakz, S. and Okamoto, [I (1976) Mutagenic activities of amino acid p~rolysates. Proc. Jpn. Environ. Murat Soc. 5th Ann. Meeting, 6. 7 :MeCanrL,J., Chc4 D., Yamasaki, E. and Ames, B.N. (1975) Detechon of carcinogens as mtttagens in the Selmonella/mmrosome test Assay of 300 chemicals Prcc. Natl. Acad So: USA, 72, 5135---5J 39 8 Nagso M., Honda, M., Seino, Y., Yahagi, T. and Sug~r0ara, T. Mutagemcltm.,~of smoke condensates and the charred surface of fish and meat Cancer Left., m press. 9 Nagao, IvL,Yahag4 T., Semo, Y., Suglmura, T. and Itc, N. Mutagemeltms of qumohne and ;is derivatives. Mutat Res, m press. I0 Suglmura, T., Nagao, M., Kawachi, T., Honda, M., Yahagl, T , Semo, Y , Matsushlma, T.~ Shiraz, A., Sawamma, M., Sato, S , Matsumoto, H and Matsukura, N Mutagencmcinogens in food with speeml reference to highly mutagenm pymlytm products m broiled foods. In: Ongms of Human Cancer Editors' J.D Watson and H Hlatt Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y., m press. 11 Suglmura, T., Sato, S., Nagao, M, Yahagl, T , Matsushima, T , Semo, Y , Take achl, M and Kawachh T (1978) Overlapping of carcinogens and mutagens. In Fundamentals m Cancer Prevention, Proc. 6th Internatl Syrup. Princess Takamatsu Cance- Res Fund, pp. !9_'.--215 Editors P N Magee, S Takayama, T. Suglmura and T M,d.sushima. Umvers~ty Tokyo Press, T o k y o 1/` Suglmura, T., Yahag4 T, Nagao, M., Takeuch4 M., K a w a c h 4 T., Hara, K., Y a m .sakl, E, Matsushlma, T, Hashimoto, Y. and Okada, M. (1976) Validity of mutagemclty tes1~susing microbes as a rapid screening method for environmental carcinogens IA]~C Scmntif:c Pub., N o 12, pp. 81--101.