Risalah Seminar Nasional Pengawetan Makanan Dengan Iradiasi, Jakarta, 6 - 8 Juni 1983

MUTAGENICITY STUDIES ON ALCOHOL EXTRACTS FROMGAMMA-IRRADIATED POTATOES

Akira Matsuyama *

ABSTRAK - ABSTRACT

Studi mutagenisitas ekstrak alkohol dati kentang iradiasi. Suatu penelitian gabungan telahdilakukan di Jepang untuk mempelajari keamanan kentang iradiasi dengan menggunakan peng­ujian kimia dan biologis. Contoh uji dibuat dengan cara mengekstraksi dengan alkohol kentangvarietas Danshaku yang telah disimpan selama 3 bulan dalam ruangan gelap dengan suhu dibawah lOoC, lalu diiradiasi 150 Gy dengan menggunakan 2 macam laju dosis, yaitu 450 Gy/jamdan 57 Gy/jam. Hasil pemeriksaan kimia dengan HPLC, kromatografi kertas dan pengujianenzim tidak mendeteksi adanya o-kuinon atau "radiotoksin" dalam ekstrak alkohol kentangiradiasi. Hasil pemeriksaan biologis pada sistem sel bakteri dan mamalia dengan menggunakanpengujian mutasi, "inductest", uji kerusakan kromosom dan uji mikronukleus menunjukkanbahwa tidak ada aktivitas mutagen dalam ekstrak alkohol kentang iradiasi yang baru dibuat.

Mutagenicity studies on alcohol extracts from gamma-irradiated potatoes. A joint researchhas been carried out in Japan to study the wholesomeness of irradiated potatoes using chemicaland_ biological assays. Test samples were prepared by extraction with alcohol from potatoesof Danshaku variety which had been stored in the dark for 3 months after harvest at tempera­ture below lOoC, and irradiated at 150 Gy using 2 different dose rates, i.e. 450 Gy/h and 57Gy/h. Result of chemical analyses by HPLC, paper chromatography and enzyme assays did notdetect the presence of o-quinone or "radiotoxin" in the alcohol extract of irradiated potatoes.Result of biological evaluations using mutation assays, inductest, chromosomal aberrations testand micronucleus test in bacterial and mammalian cell systems showed no mutagenic activityin the freshly prepared alcohol extract of irradiated potatoes.

INTRODUCTION

Feasibility studies on the inhibition of sprouting in potatoes by ionizing radia-"tions have revealed that very low doses less than 150 Gy are sufficiently effective,and various types of safety tests of irradiated whole tubers of potatoes have in­dicated no adverse effect for human consumption attributable to the irradiationtreatment (1). On the other hand, only a series of papers to provoke a question onthe wholesomeness aspects of potato irradiation technology has been presentedby KUZIN and his coworkers, in which they have described on "radiotoxins" foundin alcohol extracts prepared from irradiated potatoes (2, 3). According to KUZINet aI., radiotoxins are the oxidation products from polyphenols in the tuber tissue,which are responsible for the radiation-induced mutagenic effects of alcoholextracts from raw potatoes. However, other authors (4,5) failed to find out themutagenic activity of the alcohol extracts from uncooked or cooked irradiatedpotatoes.

In order to determine whether the mutagenic activity is found in alcoholextracts from freshly irradiated potatoes, the committee on food irradiation re­search was set up at..Jhe Japan Radioisotope Association and the results obtainedby the cooperation of the committee members were published in thret originalpapers (6, 7, 8). The present review paper will introduce the outline of the experi­mental results described in these three papers.

• Institut of Physical and Chemical Research, Wako-shi Saitama-ken 351, Japan .

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MATERIALS AND METHODS

Potatoes used throughout these studies were those of the "Danshaku" varietysupplied from Hokkaido, Japan. The tubers were stored in the dark for threemonths after the harvest at temperature below 10°C and irradiated by 60Co-gammarays with 150 Gy at two different dose rates, 450 Gy/h (H) and 57 Gy/h (L).

The preparation procedures of alcohol extracts from irradiated potatoes em·ployed by KUZIN et al. (2, 3) were carefully followed except (l) storage of irra­diated potatoes for 24 h at 4°C or 20°C, (2) the use of glasswared home mixer forthe treatment of potatoes to be extracted with alcohol and (3) the storage of con­centrated alcohol extracts under frozen after the deaeration by argon bubbling inbrown ampoules. The final concentrate of alcohol extracts from potatoes indicatedpH values of 3-4.

Chemical studies were conducted by paper chromatography, high performanceliquid chromatography (HPLC) using two systems and enzyme assays for phenoloxidase, peroxidase, cresolase and catecholase activities. The model experimentwas conducted according to the method of KUZIN et al. in order to find out thequinone complex by HPLC.

Tests for biological activities in bacterial and mammalian cell systems carriedout in these studies are as follows: (l) histidine reverse mutation assays in Sal­monella ryphimurium strains, (2) reverse mutation assays in a streptomycinmutant, Salmonella ryphimurium TA 100-10, (3) Prophage lambda induction tests

using Escherichia coli KI2 mutants, (4) chromosomal aberration tests in vitrowith chinese hamster cells and (5) micro-nucleus tests in ddY-SLC mice, a dominantlethal test after consecutive administrations of potato extracts to male 9-week-oldBDFJ mice. In a dominant lethal test, male mice were orally given with 0.5 mlofthe alcohol extracts per 25 g body weight twice a day for 7 days. Thirty males were

used in both the negative control and sample groups. After administration, eachmale mouse was mated with a 9-week-old virgin female mouse and then to a newfemale successively tit 4.day intervals for 56 days. The frequency of induced do­minant lethals was calculated as follows:

Frequency of induced dominant lethals (%) =

(l _ living implants per pregnant female in the experimental group) x 100living implants per pregnant female in the control group

A statistical analysis was made with the data ·by means of the t test.

RESULTS AND DISCUSSION

The o-quinones or radiotoxins reported by KUZIN e~ al. were not detected inthe alcohol extracts prepared from gamma irradiated potatoes by two systems ofHPLC (Fig. I). Enzyme activities of both peroxidase and phenol oxidase in potatotubers increased linearly with irradiation doses, but the rates of increase were rathersmaller than those reported by KUZIN et al. (Fig. 2). As well, increase of activitiesof catecholase for caffeic acid and cresolase for tyrosine seemed rather small (TableI). In the model experiment to trace that of KUZIN et aI., the typical reaction mix-

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ture was composed of 10 ml of 0.05 M phosphate buffer, pH 6.8, containing caffeicacid, tyrosine, tryptophan, methionine and leucine and 10 ml of the enzyme solu­tion. A steady decrease of caffeic acid was observed (Fig, 3) and only a smallamount of caffeic acid was detected after 2-hour incubation of the reaction mix­ture, whereas the system with boiled enzyme showed no decrease 'in caffeic acideven after 2-hour incubation. A HPLC analysis of the reaction products indicatedno characteristic difference between the irradiated and unirradiated sample system.The amino acid analysis revealed no amino acid consumption in the system. It wasalso found that caffeic acid decreased at the similar reaction rate without amino

acids. The results of chemical studies on the radiation effect on the enzyme acti­vities may be explained by the following scheme:

OH Cresolase OH Catecho lase Y(] 1 0:/OH II III

I ) I ) ---- -) pigments~ Kl ~ K2 ~ K3R R

Monophenol o-Diphenol o-Quinone

In unirradiated potatoes, the reaction rate of process III, k3 will be much largerthan k1 or k2. It is assumed that the steady state concentration of o-quinones inthe unirradiated system is below the detection limit. In the irradiated system of

KUZIN et aI.., k1 increased as much as about three times and k2 increased about30%, resulting in the increase in the steady state concentration of o-quinone, tosuch a degree 'a~' to be detected. In contrast, in the present study, both k1 and k2

increased only about 10 - 20%, and so the steady state concentration of o-quinonewould not go over the detection. It is concluded in these chemical studies thato-quinone or radiotoxins reported by KUZIN et al were not detected even in theirradiated system.

In mutagenicity tests in bacterial and mammalian cell systems, the negative re­sults were obtained from all test systems mentioned above. In addition, no diffe­rence in the mutagenic activities was found between extracts prepared from irradia­ted and unirradiated potatoes, suggesting that no mutagenic substance was pro­duced in potatoes following the gamma-irradiation treatment (Tables 2 - 7).

In a dominant lethal test in mice, no significant difference was observed in thenumber of living implants between the groups treated with alcohol extracts pre­pared from irradiated and unirradiated potatoes (Table 8).

In conclusion, no experimental evidence for the mutagenic activity of alcoholextracts prepared from gamma irradiated potatoes was obtained by the chemicalanalysis and biological assays in the present studies,

AKCNOWLEDGEMENTS

The author wishes to express his thanks to Japan Radioisotope Association,especially to the late Prof. Dr. F. Yamazaki and Prof. Dr. H. Yoshikawa for theirencouragements and contributions to the studies, He is also indebted to National

Food Research Institute, Japan, for kind arrangements of samples supply and ad-

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vices and to the members of the Committee on Food Irradiation Research and

their associates for their cooperation throughout the studies.

REFERENCES

1. Joint FAOfIAEAfWHO Expert Cummittee on the Wholesomeness of Irradiated Food,WHO Technical Report Series No. 604 (1977) and No. 659 (1981).

2. KOPYLOV, V.A., OSIPOVA, J.N., and KUZIN, A.M., Radiobiologiya 12 (1972) 524.3. KUZIN, A.M., KOPYLOV, V.A., and OSIPOVA, J.N., USSR Academy of Science, Push-

chino-na-Oke (1974).4. LEVINSKY, H.V., and WILSON, M.A., Food Cosmet. Toxicol. 13 (1975) 243.5. HOSSAIN, M.M., HUISMANS, J.W., and DIEHL, J.F., Toxicology 6 (1976) 243.6. SHINOZAKI, Y.,HOGETSU, D., OKUYAMA, N., MANABE, T., SASAGAWA, T.,MINA­

MIKAWA, T., and MATSUYAMA, A., Radioisotopes 30 (1981) 655.7. ISHIDATE, M. Jr., YOSHIKAWA, K., SOFUNI, T., IWAHARA, S., SHIBUYA, T., MU­

RAMATSU, S., KITAYAMA, S., and MATSUYAMA, A., Radioisotopes 30 (1981) 622.8. SHIBUYA, T., MUROTA, T., IWAHARA, S., HASHIMOTO, K., MINEGISHI, A., HO­

GETSU, D., and MATSUYAMA, A., Radioisotopes 31 (1982) 74.

Table 1. Effect of irradiation on phenol oxidase activity estimated with various sub·strates.

Relative enzyme activity (%)*Substrate

TyrosineCaffeic acid

Unirradiated

100 (100)100 (100)

Irradiated (H)

111 (283)118 (126)

*) Numbers in parenthesis are the normalized activity cited from KUZIN et al. (3)

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Table 2. Reverse mutation assays in histidine requirement (Ames test) with Salmo­. nella typhimurium strains on alcohol extract of irradiated potatoes.

S. fYphimurium strains

Sample

T A 100TA 1535TA98TA 1537- - S -: 9 '+!S - 9 - S - 9 + S - 9 - S - 9 + S - 9 - S - 9 + S - 9

Phosphate buffer

140130 372947531117129

127 332443601219

Unirradiated

143190 483042691025145

159 29254884212648

3072851824

/372060701319

Irradiated (L)

156165 525253671434156

173 36566386192543

376374213040

4163801421

Irradiated (H)

151156 352952611320133

154 34244848182240

224477121742

3640701520

Positive control

563* 321 *2136 *373 *4137 *5180 *2106 * 693 *4602

31512465125163127104

*AF-2, O.Ol,ugjplate; *2 2-Aminoanthracene, 0.5 ,ugjplate;*3 ENNG, 5,ugjplate;

*4 2-Amino-anthracene, 1.0 ,ugjplate; *5 AF-2, 0.02 ,ugjplate; *6 9-Aminoacri-dine, 50 ,ugjplate.

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Table 3. Mutation assays in streptomycin dependence with Salmonella typhimur;"

um TA 100-10 (SMd) strain on alcohol extract of the irradiated potatoes.

Sample

Dose (ltg/ml)Viable cells (107 /ml) Revertants/plate Induced

-S-9

+S-9-S-9+S-9mutation

frequency •Unirradiated

03.93.1738250

3.83.03281100

3.13.12185200

2.7.2.21040500

0.41.9 015

Irradiated (L)

02.52.46168100

3.33.41039200

2.82.51139500

0.0040.000100

Irradiated (H)

02.92.6657850

3.63.06423100

3.23.06110200

3.43.01217500

2.41.82911

Positive control (MNNG)

03.0 7710 (ltg/ml)

2.5 17317.lx10-4

(2-Aminoanthracene)07.0141

2(ltg/ml)5.63513.64x 10 - 6

*

The mutation frequency was calculated from the number of revertants per via-ble cells; - no induced mutants.

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Table 4. Inductests on alcohol extract of the irradiated potatoes with or without acombination with mitomycin C.

Number of plaques/plateSample

Mitomycin C-S-9

+S-9

None (Control)

028 16None

2 J1g449 280Unirradiated

( 25J11)0 16

( 50J1l)

0 20

(100J1l )

0 .52

Irradiated (L)( 25J1!)0 16

( 50J1l)

0 16

(100J11 )

0 26Unirradiated

(100J1l )2 J1g 181

Irradiated (L)(1 00J1! )2 J1g 236

Table 5. Inductests on alcohol extracts of the irradiated potatoes.

Number of plaques/plate

SampleAflatoxin B1

-S-9+S-9

None

0134

None5 J1g585

Unirradiated( 10 J1Q)0 105

( 25 J1Q)

0 II6

( 50 J1Q)

0 210

(100 J1Q)

0 25I

( 50 J1Q)

10 J1g 18

(100 J1Q)

10 J1g 26

Irradiated (L)

( 10 J1Q)0 IS4

( 25 J1Q)

0 252

( 50 J1Q)

0 130

(100 J1Q)

0 190

( 50 J1Q)

10 J1g 29

(100 J1Q)

10 J1g 24

Irradiated (H)

( 25 J1Q)0 02

( 50 J1Q)

0 I6

(100 J1Q)

0 0I

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Table 6. Chromosomal aberration tests in vitro with Chinese hamster cells on alcohol

extracts of irradiated potatoes.

Chromosomal aberrations (%)Sample

Dose (pJ./ml) Polyploid*.cells (%)

24 hr48 hrJudge**

None (Control)

-1.03.02.0

Unirradiated

3.131.Q,3.00.06.25

0.02.00.012.50

2.01.01.0

Irradiated (L)

3.133.00.00.06.25

0.03.02.012.50

1.00.01.0

Irradiated (H)

1.571.00.01.06.25

1.01.01.012.50

4.02.00.0

* The incidence of polyploid cells at 48 h;** The final judgement: Negative (-);less than 5.0%.

Table 7. Micronucleus tests in ddY·SLC mice on alcohol extracts of the irradiated

potatoes.

NumberMicronucle-PCEs with

Sample

of miceated/PCEsmicronucleusused

observed *(0/00) ± S.D.-

None (Control)

411/80001.4±0.3Unirradiated

411/80001.4±0.3**

Irradiated (L)

49/80001.1 ±0.5**

Irradiated (H)

412/80001.5 ±0.6**

* PCE: Polychromatic erythrocytes** Not significant at 5% level

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Table 8. Dominant lethal test after consecutive administrations of irradiated potatoextract to male mice.

No. of preg-

No. (per pregnant female) oraInducedDay of

Groupnant females/ dominant

mating

No. of fema- Corpora-hnp1antsLiving1ethals (%)

1esmated

1utea implants

1 "'4

Cont.28/3010.8 ±1.39.4±2.19.1±2.2-VIP

29/3010.5 ±l.39.8±1.49.0±2.01.10

IP·28/3010.8:!O.99.7±2.09.1 ±2.30

DMBA19/2010.8±1.59.3 ±2.68.1 ±3.610.99

5"'8

Cont.28/3010.8 ±1.110.1 ±2.19.5±2.5

VIP26/3011.1 ±1.09.8±2.99.5±2.7-0

IH28/3011.2±1.49.9±2.09.7±2.1-2.11

DMBA

18/2011.2±1.15.4±2.2c3.6±2.1 c 62.119"'12

Cont.28/3011.2±1.010.8±1.610 .3 ±1.8

VIP24/3011.0±1.210.5±1.59.9±1.93.89

IP28/3011.1±1.310.6±1.59.9±2.63.89

DMBA18/201O.8±1.29.8B.Ob9.1 ±1.9b 11.65

13 "'16

Cont.25/3011.3±1.31O.8±2.010.5±2.2

VIP29/3011.4±1.310.5±2.21O.0±2.44.76

IP28/3011.9±1.311.1 ±1.610.2±2.72.86

DMBA18/2011.l±1.59.8±3.09.3 13 .011.43

17 '" 20

Cont.28/301O.6±1.09.9±1.59.6±1.5

VIP29/3010.8±1.l10.1 ±1.69.5±2.01.04

IP28/3011.0±1.21O.6±1.49.9±1.6- 3.13

DMBA20/2011.0±1.l1O.4±1.49.8 ±1.l- 2.08

21 "'24

Cont.28/3010.4±o.99.9±1.59.5 ±1.6

VIP27/3011.0±1.l1O.2±1.09.4±1.71.05

IP28/3011.1 ±o.810.3±2.09.5±2.20

DMBA16/2010.8±1.09.7±1.99.1 ±2.04.21

25 '" 28

Cont.29/3010.9±1.41O.0±2.19.5±2.4

VIP30/3010.7±1.29.8 ±1.59.4 ±1.81.05

IP28/3011.0±1.010.2±1.39.6±1.3- 2.01

DMBA19/201O.8±1.110.3 ±1.l9.4 ±1.21.05

29 '" 32

Cont.27/3010.7±1.91O.3±2.210.1 ±2.3VIP

26/3011.l±1.l10.8 ±1.31O.4±1.6- 2.97

IP26/3010.9±1.010.4±1.710.0£.01.00

DMBA18/2010.8±1.09.3±2.18.6±2.4b 14.85

33 '" 36

Cont.26/3011.3 ±1.010.9±1.810.3 ±2.3VIP

25/3011.3:!O.911.0:!O.910.2±1.40.93

IP25/301O.8±1.710.0±2.79.6+2.76.80

DMBA16/201O.6:!O.8b7.3±2.8c7.0±2.8c 32.04

37 '" 40

Cont.24/3011.0±1.010.2±1.79 .5±1.8

137

UIP29/3011.2±1.3lO.0±2.29.7±2.3- 2.11IP

29/3010.8::!:O.9lO.3±1.09.9+1.5 - 4.21DMBA

14/2010.9±1.25.9±3.8c- c5 .4±3.3 43.16

41. "'44

Cont.28/30lO.6±1.09.7±2.49.2±2.4DIP

29/30lO.4::!:O.89.8 ±1.89.5 ±1.9- 3.26IP

27/3010.3±1.09.8±1.89.4 ±1.5- 2.17DMBA

8/2010.4±o.86.8±3.8 c6.5±3.5c 29.3545 '" 48

Cont.23/30lO.9±o.910.0±1.79.4±2.2DIP

28/3011.0±1.1lO.5±1.710.1±1.9- 7.45IP

29/3010.7±1.l10.2±2.29.9±2.2- 5.32DMBA

15/209.8::!:O.77.0±3.4 c6.7±3.2 c 28.7249 '" 52

Cont.26/3010.1::!:O.79.8::!:O.99.5±1.lDIP

30/3010.5±o.99.9±1.59.6±1.2- 1.05IP

29/3010.3±1.l9.6±1 .99.1 ±2.04.21DMBA

18/2010.3±o.89.3 ±1.58.8±1.57.3753 '" 56

Cont.27/3010.8±1.210.1±1.09.8±1.lUIP

29/30l1.l±1.llO.4±1.99.9±2.0-1.02IP

29/30lO.8::!:O.910.0±1.79.6±1.62.04DMBA

19/2011.2±1.110.8±1.l10.7±1.29.18

a : Mean ± S.D.band c denote results significantly different from the control, at p <0.05 and p <0.01, respectively. Abbreviations: UIP; Unirradiated potato, IP; Irradiated potato,DMBA; 7, l2-dimethylbenz (a) anthracene.

138

A

h g~

f+

B

c

Fig. 1. HPLC chromatograms (System II) of potato extracts under various condi­tions. Column: Stainless column, 0.8 cmct> x 25 cm, packed with Lichro­sorb (KNAUR), 5J.1 particles. Detector: Spectro flow monitor (Schoffel)and fluorescence detector (Shimazu FLD-l). Solvent: 0.1 M NaH2P04­

10% 2-propanol. Flow rate: 1.0ml/min.---- Unirradiated Irradiated (H) A: UV absorbance at 280 nm;B: Fluorescence intensity at 400 nm excited at 350 nm; a: Dopa; b: Tyro­sine; c. Chlorogenic acid; d: Tryptophan; e: Caffeic acid; f: Esculetin; g:Scopoletin; h: Umbelliferone.

2

.-' ~:::~._ __ -.•..•.....- _ .

200 400

Dose (Gy)

600

Fig. 2. Effect of irradiation on peroxidase and phenol oxidase activities in potatotubers. (1 Gy = 100 rad)- 0 - Peroxidase ...•... Phenoloxidase

139

Fig. 3. HPLC patterns of the model system with the enzyme solution extractedfrom potatoes irradiated at higher dose rate (Irradiated (H)) Pressure: 40kg/cm2; Solvent; 0.1 M NaH2P04-10% 2-propanol; Sample volume: SOpi.A: 0 min; B: 90 min.

140

DISKUSI

SUMPENO PUTRO:

You only tried alcohol extract for mutagenicity assay. Since the solvent for extrac­tion, especially in Ames Test, is very crucial, I am very interested in knowingwhether you attempted to use another solvent, and compare the result with your al­cohol extract?

A. MATSUYAMA:

Safety test for food extracts are not always necessary. Wholesomeness studies onirradiated food may be done under the specified condition of food treatments forpractical application. Our purpose in the mutagenicity tests on alcohol extractsfrom irradiated potatoes was to trace the experiments of KUZIN and his coworkers.

RD. ESTI W.:

Are there any irradiation done on microbial products such as baker's yeast. In thiscase, irradiation only affect microbes which might contaminate a certain productand not the primary yeast/microbes such as Saccharomyces cerevisiae in the case ofbaker's yeast.

A. MATSUYAMA:

Although 1 am not sure on this matter, there might be useful references available.Inside the microbial cell mass, there would be some anaerobic condition whichmakes cell-inactivation effects of radiation a little lower.

141