Risalah Seminar Nasional Pengawetan Makanan Dengan Iradiasi, Jakarta, 6- 8 Juni 1983 MUTAGENICITY STUDIES ON ALCOHOL EXTRACTS FROM GAMMA-IRRADIATED POTATOES Akira Matsuyama * ABSTRAK - ABSTRACT Studi mutagenisitas ekstrak alkohol dati kentang iradiasi. Suatu penelitian gabungan telah dilakukan di Jepang untuk mempelajari keamanan kentang iradiasi dengan menggunakan peng- ujian kimia dan biologis. Contoh uji dibuat dengan cara mengekstraksi dengan alkohol kentang varietas Danshaku yang telah disimpan selama 3 bulan dalam ruangan gelap dengan suhu di bawah lOoC, lalu diiradiasi 150 Gy dengan menggunakan 2 macam laju dosis, yaitu 450 Gy/jam dan 57 Gy/jam. Hasil pemeriksaan kimia dengan HPLC, kromatografi kertas dan pengujian enzim tidak mendeteksi adanya o-kuinon atau "radiotoksin" dalam ekstrak alkohol kentang iradiasi. Hasil pemeriksaan biologis pada sistem sel bakteri dan mamalia dengan menggunakan pengujian mutasi, "inductest", uji kerusakan kromosom dan uji mikronukleus menunjukkan bahwa tidak ada aktivitas mutagen dalam ekstrak alkohol kentang iradiasi yang baru dibuat. Mutagenicity studies on alcohol extracts from gamma-irradiated potatoes. A joint research has been carried out in Japan to study the wholesomeness of irradiated potatoes using chemical and_ biological assays. Test samples were prepared by extraction with alcohol from potatoes of Danshaku variety which had been stored in the dark for 3 months after harvest at tempera- ture below lOoC, and irradiated at 150 Gy using 2 different dose rates, i.e. 450 Gy/h and 57 Gy/h. Result of chemical analyses by HPLC, paper chromatography and enzyme assays did not detect the presence of o-quinone or "radiotoxin" in the alcohol extract of irradiated potatoes. Result of biological evaluations using mutation assays, inductest, chromosomal aberrations test and micronucleus test in bacterial and mammalian cell systems showed no mutagenic activity in 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 in- dicated no adverse effect for human consumption attributable to the irradiation treatment (1). On the other hand, only a series of papers to provoke a question on the wholesomeness aspects of potato irradiation technology has been presented by KUZIN and his coworkers, in which they have described on "radiotoxins" found in alcohol extracts prepared from irradiated potatoes (2, 3). According to KUZIN et aI., radiotoxins are the oxidation products from polyphenols in the tuber tissue, which are responsible for the radiation-induced mutagenic effects of alcohol extracts from raw potatoes. However, other authors (4,5) failed to find out the mutagenic activity of the alcohol extracts from uncooked or cooked irradiated potatoes. In order to determine whether the mutagenic activity is found in alcohol extracts from freshly irradiated potatoes, the committee on food irradiation re- search was set up at..Jhe Japan Radioisotope Association and the results obtained by the cooperation of the committee members were published in thret original papers (6, 7, 8). The present review paper will introduce the outline of the experi- mental results described in these three papers. • Institut of Physical and Chemical Research, Wako-shi Saitama-ken 351, Japan . 129
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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 .
129
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-
130
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-
131
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-
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)
132
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
The mutation frequency was calculated from the number of revertants per via-ble cells; - no induced mutants.
134
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
135
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
136
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.