Toxicology, 8 (1977) 213--222 ©Elseviei/North-Holland Scientific Publishers, Ltd.
CHROMOSOME STUDIES ON BONE MARROW CELLS OF CHINESE HAMSTERS FED A RADIOSTERILIZED DIET
H.W. RENNER Institut fiir Biochemie der Bundesforschungsanstalt fiir Erniihrung, Engesserstrasse 20, D-7500 Karlsruhe (F.R.G.)
(Received January 27th, 1977) (Revision received April 12th, 1977 ) (Accepted April 28th, 1977)
SUMMARY Metaphase preparations of chr om osom es from bone m a r r o w cells of Chinese hamsters were examined for mutagenic effects following the feeding o f a radiosterilized diet. No increase in the incidence of structural chromosomal aberrations was observed. As far as numerical aberrations were concerned, the p r o p o r t i o n o f cells with polyploidy increased to between 4 to 5 times the control level, irrespective of the moisture c o n t e n t of the diet. This p o ly p lo id y effect occurred very early, being detectable within 24 h, if the diet fed had been irradiated with an absorbed dose of 4.5 • 106 rad. The incidence of pol ypl oi dy remained below 0.5%, however, nor did it rise with higher radiation doses. When the feeding of the irradiated diet was stopped, the p r o p o r t i o n of polyploid cells returned to t he control level within a maximum o f 6 weeks. If the diet was stored (initially) for 6 weeks following irradiation before being fed to the animals no increase in the n u m b e r of polyploid cells was noted. These results are not interpreted as a mutagenic effect of the irradiated diet.
INTRODUCTION
Whilst toxicological tests of irradiated food have never disclosed any adverse effects, possible mutagenic effects following ingestion of irradiated f o o d have repeatedly been conjectured and m a n y studies to investigate this problem have been carried out (for review studies, see refs. 1--3). It is now generally recognized, particularly when testing weak or doubt ful mutagens, that: (a) only those mutagenicity tests are relevant which take into a c c o u n t mammalian metabolism [ 4 ] ; (b) only the use of several test meth o d s fulfilling these requirements allows careful extrapolation of the results to man, because no single m e t h o d exists which is capable of detecting all types of mutational events [5]. Over the past few years our study group
213
has carried o u t m u t a g e n i c i t y tests on irradiated f o o d and animal feeds by means o f the d o m i n a n t - l e t h a l test, the h o s t - m e d i a t e d assay, and the micronucleus test, in every case with negative results [ 6 - - 9 ] . O u r f u r t h e r studies in this field, which i n v o l v e d the e m p l o y m e n t o f c h r o m o s o m a l analysis, are r e p o r t e d in this paper. MATERIALS AND METHODS A n i m a l selection and maintenance T h e Chinese hamsters used in these studies were o b t a i n e d from the Zoological I n s t i t u t e o f the D a r m s t a d t T e c h n i c a l University, f r o m a c o l o n y i n b r e d over f o r t y generations and m a i n t a i n e d in t h e c o n v e n t i o n a l way. In o u r laboratories a c o l o n y o f these r o d e n t s in sufficient n u m b e r for o u r purposes was built up f r o m 10 pairs o f r a n d o m l y selected breeding animals; the animals were k e p t in a fully a i r - c o n d i t i o n e d e n v i r o n m e n t (air t e m p e r a t u r e 22 ± I°C, relative h u m i d i t y 55 -+ 5%, 15 air changes/h, air filtration and light-dark cycle o f 12 h). The h a m s t e r c o l o n y was free f r o m disease at all times. T h e animals used in the e x p e r i m e n t were aged b e t w e e n 3 and 5 m o n t h s and weighed b e t w e e n 23 and 43 g. G r o u p s o f 25 or 26 animals were used as controls or test groups, the p r o p o r t i o n s o f males and females being equal in each group. Diet T h e animals were fed ad lib. a b r e e d i n g d i e t f o r hamsters ( A l t r o m i n Z*) in a pelletized f o r m . T h e test diet was irradiated a t w e e k l y intervals at the lnstitute's e l e c t r o n linear a c c e l e r a t o r with 10 MeV e l e c t r o n s in o p e n a l u m i n u m trays and t h e n fed e i t h e r for 6 weeks or f o r 1 day. Once a week all animals were given vitamin s u p p l e m e n t s in their drinking w a t e r ( M u l t i b i o n t a * * ) . Preparation o f c h r o m o s o m e s 2 m g / k g of C o l c e m i d ( C a l b i o c h e m ) was injected into t h e animals 2.5 h b e f o r e sacrifice. The f e m o r a were r e m o v e d and the b o n e m a r r o w was flushed o u t by means o f a 1% s o d i u m citrate s o l u t i o n c o n t a i n i n g added heparin. T h e cell suspension was f u r t h e r processed in a c c o r d a n c e with the i n s t r u c t i o n s c o n t a i n e d in a r e c e n t h a n d b o o k o f c y t o g e n e t i c m e t h o d s [ 1 0 ] . T h e slide preparations (2/animal) were stained with a 2% a c e t o - o r c e i n solution. This technique furnished g o o d t o e x c e l l e n t m e t a p h a s e p r e p a r a t i o n s . Evalua tio n T h e slide p r e p a r a t i o n s were evaluated blind b y two investigators w o r k i n g i n d e p e n d e n t l y . O n l y well spread m e t a p h a s e s w i t h o u t recognisable superimposed c h r o m o s o m e s were c o u n t e d . This criterion was also applied t o polyploid cells. In a d d i t i o n , the a r r a n g e m e n t o f the p o l y p l o i d c h r o m o s o m e s was t a k e n into a c c o u n t (Figs. 1 and 2).
**
Altromin GmbH, D-4937 Lage/Lippe. Trademark registered by E. Merck, Darmstadt.
214
x Fig. 2. Metaphase
of a polyplaid
cell (n = 88) of Chinese
hamster.
x
600.
In order to permit a more satisfactory statistical analysis of the expected results it was considered preferable to increase the number of animals per group instead of examining a larger number of metaphases per animal. RESULTS
In our initial investigations we used 6 Chinese hamsters (12 weeks old, 3 dd, 3 90) treated with the cytostatic Trenimon” as positive controls employing method and classification described in 1971 [ll] (2 i.p. injections of 0.25 mg/kg each of Trenimon at 24 h intervals, sacrificing the animals 6 h after the second injection). Between the two injections the plexus of the orbital vein was punctured to reduce the bone marrow depression and to stimulate the mitotic index. A total of 100 metaphases was counted per animal and the results are presented in Table I. These show that t.he results of other authors [ 11,121 could be reproduced satisfactorily. The design and the results of our main experiment are given in Table II. The mitotic index of 4.7-6.6% was of the same order in both the controls and the test groups fed the diet irradiated at a dose of 4.5 Mrad + 5%.** Our results in the control animals confirmed the extremely low spontaneous aberration rate in bone marrow cells of Chinese hamsters reported in other studies [ll, 121. Moreover, we were able to confirm that only one type of aberration occurred, namely chromatid breaks. Karyograms were made of approx. 10 animals in each of the groups to ensure as far as possible that no chromosomal anomalies had been missed. -* Trademark
**
registered
by Farbenfabriken
rad = radiation absorbed dose. = 10.’ J I rad= lOOerg*g”
l
kk-‘;
Bayer AG, Leverkusen.
1 Mrad (Megarad)
= lo6 rad.
215
TABLE I CHROMOSOMAL ANALYSIS ON BONE MARROW CELLS OF CHINESE HAMSTERS Positive control following Trenimon treatment (2 × 0.25 mg/kg at 24 h intervals), n, 6 animals; 100 metaphases/animal were scored. Normal cells 1--2 aberrations Multiple aberrations Pulverized
13.0% a 16.3% 65.0% 5.7%
(break and/or interchanges) (/> 3, all types) (unscorable chromosomes)
Total, abnormal cells
87.0%
aNo. of scored metaphases = 100% TABLE II CHROMOSOMAL ANALYSIS ON BONE MARROW CELLS OF CHINESE HAMSTERS Results after feeding irradiated diets. Metaphases evaluated
Chromatid gaps
Chromatid breaks
Polyploid cells
No.
%a
No.
%a
No.
%a
Control group n = 25 animals (unirradiated diet)
7500
56
0.75
21
0.28
5
0.06
Test group (a) n = 26 animals (4.5 Mrad irradiated diet for 6 weeks)
7800
74
0.95
16
0.21
25
0.32
Test group (b) n = 25 animals (4.5 Mrad irradiated diet for 1 day, administered after 1 day of starvation)
7500
60
0.80
22
0.29
20
0.27
(300 metaphases were evaluated per animal. Most of the polypioid cells had n = 88 chromosomes. ) aNo. of evaluated metaphases = 100%
T h e f r e q u e n c y o f s t r u c t u r a l a n o m a l i e s ( c h r o m a t i d b r e a k s ) in t h e t w o t e s t g r o u p s w a s s i m i l a r t o t h a t in t h e c o n t r o l g r o u p . I n v e s t i g a t i o n o f t h e n u m e r i c a l a b e r r a t i o n s i n d i c a t e d t h a t cells w i t h 21 c h r o m o s o m e s o c c u r r e d w i t h t h e s a m e a b u n d a n c e in b o t h t h e c o n t r o l a n d t h e t e s t g r o u p s , t h e y w e r e r e g a r d e d
216
as dying cells. However, it appeared that polyploid cells occurred about five times more frequently in those animals which had been fed irradiated diets compared to the control group (statistical probability of error P = 0.01, Wilcoxon test). No differences attributable to sex could be observed througho u t the groups. The ×2 four field test shows that the events (= polyploid cells) f o u n d in the evaluation of 300 mitoses/animal will differ random l y from each o t he r only if their num be r lies between 0 and 6. Since this requiremen t was met, all cells within each animal group were pooled and evaluated independently of the individual animals. The n u m b e r of events in the control group differs from t hat in the pooled test group ((a) and (b)). In order to be able to test the significance of this difference the distributions of the events in both groups were investigated. Th ey could be appr oxi m at ed satisfactorily by Poisson distributions. Accordingly, the significance test was carried o u t by the distribution-free U-test according to Wilcoxon, Mann and Whitney. Table III presents a survey of additional studies carried o u t to define more accurately the conditions responsible for an increased incidence of polyploidy. DISCUSSION The absence, in these experiments, of any effect of irradiated diets on the incidence o f structural anomalies (point mutations are not detectable in this test system) in c h r o m o s o m e s of bone marrow cells discounts the likelihood o f mutagenic activity of these treated diets. On the other hand, there is doubtless a clear effect upon the polyploidy incidence. It is surprising to find that this effect is of similar magnitude as early as 24 h after the onset of feeding o f the irradiated diet as that seen after 6 weeks o f feeding of the same diet. The dose-dependent increase in this effect within the range of 1--4.5 Mrad indicates that irradiation must have produced one or m ore substances in the diet giving rise to this effect. These are, however, unstable products, because feeding a diet stored for 6 weeks produced no such effect. In 1968 Bugyaki et al. [13] reported meiotic studies in mice fed for life a diet consisting of 50% freshly irradiated (5 Mrad) wheat flour. T h e y found increased numbers of chromosomal aberrations (translocations, bridges and fragments) o f the male germ cells in all meiotic stages. The authors used descendants o f the C 57 BL strain of mice with a spontaneous mutation. These animals were e xt r em e l y sensitive to the spontaneously increased occurrence of translocations and ot he r chromosomal anomalies. According to Vijayalaxmi and Sadasivan [14] the administration of a wheat flour diet irradiated with 75 krad increased the n u m b e r of structural chromosomal aberrations in the bone marrow cells of Wistar rats. The same effect was obtained by feeding a diet with low protein c o n t e n t (5%). The polyploidy rate was not increased by a low protein diet but rose after feeding an irradiated diet. It was later reported [15] that, at the radiation dose emp lo y ed , the normal fraction of polyploid bone marrow cells rose from ap p r o x imatel y 0.04% to some 0.4% after only 10 weeks. If flour was stored
217
t~ 00
S t a r v a t i o n for 36 h, t h e n feeding u n i r r a d i a t e d diet for 24 h No s t a r v a t i o n period, t h e n feeding irradiated diet for 3 days (4.5 Mrad)
Can starving per se initiate p o l y p l o i d y ?
6 weeks o f irradiated diet (4.5 Mrad), t h e n 6 weeks of unirradiated diet
Irradiated diet (4.5 M r a d ) s t o r e d for 6 weeks, t h e n fed for 24 h a f t e r a s t a r v a t i o n break of 1 day
How long will polyploidy persist w h e n an u n i r r a d i a t e d diet is fed again?
Will irradiated diet, s t o r e d a f t e r irradiat i o n , still be able to initiate p o l y p l o i d y ?
H o w quickly will polyploidy o c c u r w i t h o u t a s t a r v a t i o n period between feeding unirrad i a t e d or irradiated diets?
Treatment
Problem
14 7000
15 7500
11 5500
10 5000
n animals, metaphases
6
5
13
4
No.
Polyploid cells
0.08
0.06
0.24
0.08
%
No p o l y p l o i d y e f f e c t a f t e r storage
P o l y p l o i d y n o longer detectable after 6 weeks
E f f e c t w i t h i n 3 days
No e f f e c t o f starvation
Result
T y p e s and results o f f u r t h e r e x p e r i m e n t s c o n c e r n i n g t h e i n c i d e n c e of p o l y p l o i d cells. 5 0 0 M e t a p h a s e s / a n i m a l were e v a l u a t e d ; No. o f evaluated m e t a p h a s e s = 100%
C H R O M O S O M A L I N V E S T I G A T I O N S ON B O N E M A R R O W C E L L S OF C H I N E S E H A M S T E R S F E D I R R A D I A T E D DIETS
T A B L E III
t,0 q~
14 7000
U n i r r a d i a t e d diet + 3 x 0.5 ml o f 0.3% H202 a d m i n i s t e r e d by gavage at 8 h intervals
Could H202 be the cause o f t h e increased incidence of polyploidy?
a D a t a t a k e n f r o m Table II " T e s t g r o u p ( b ) " (300 m e t a p h h s e s / a n i m a i ) .
11
8 4000
13
22
20
25 7500
14 7000
15
15 7500
Irradiated diet {4.5 Mrad) s o a k e d in water, t h e n fed to animals for 24 h after 1 day of starvation
13
15 7500
Is t h e r e a p o l y p l o i d y e f f e c t also a f t e r f e e d i n g a moist, irradiated diet?
6
15 7500
(a) Diet i r r a d i a t i o n dose 1 Mrad 1 day s t a r v a t i o n period, 24 h feeding (b) Diet irradiation dose 2 Mrad O t h e r w i s e as u n d e r (a) (c) Diet irradiation dose 3 Mrad O t h e r w i s e as u n d e r (a} (d) Diet irradiation dose 4.5 Mrad O t h e r w i s e as u n d e r (a) (e) Diet irradiation dose 10 Mrad O t h e r w i s e as u n d e r (a)
Is the p o l y p l o i d y o c c u r r i n g within one day a f u n c t i o n o f the dose?
0.19
0.31
0.28
0.27
0.20
0.17
0.08
Increased incidence o f polyploidy
Full p o l y p l o i d y e f f e c t
Polyploidy does not rise any f u r t h e r with higher radiation d o s e s
Full p o l y p l o i d y a effect
Beginning increase in polyploidy
No p o i y p i o l o y
for 12 weeks after irradiation, it caused no polyploidy. However, another Indian research group [16], on repeating these experiments, was unable to confirm an increase in the number of polyploid cells in the bone marrow of rats fed an irradiated diet. Differences in the cytogenetic methods employed by the two research teams should be taken into account when assessing these divergent results. Bhaskaram and Sadasivan [ 17 ] fed 3 groups each of 5 hospitalized children, all suffering from severe protein deficiency, either unirradiated or freshly irradiated wheat flour or wheat flour stored for 3 months following irradiation. Leukocyte cultures were started every two weeks. These children when given freshly irradiated flour developed polyploid cells and cells with other ch r o mo s o ma l aberrations, the longer t h e y ate irradiated flour. Surprisingly enough, no p o l y p l o i d y at all was observed in the control group. After this diet had been stopped, the incidence of p o l y p l o i d y fell to zero in the children who had co ns um ed irradiated flour. The c h r o m o s o m a l studies so far carried out in relation to the ingestion of dry irradiated f ood have thus p r o d u c e d c o n t r a d i c t o r y results. In some cases no ef f ect was found, in ot he r cases there was an increased incidence of polyploidy or even an increased incidence of aberrations. The latter finding was n o t seen in our results, and an increased incidence of pol ypl oi dy was only f o u n d at 2 Mrad and above. The physiological significance of an increased incidence of polyploidy in bone mar r o w cells o f our test animals, observed within 24 h of feeding an irradiated diet, is not clear. In man, a variable incidence of pol ypl oi dy is normally f o u n d in osteoblasts, liver cells, cells of the respiratory epithelium etc. [ 1 8 ] . Similar observations have been made for various l aborat ory mammalian species [ 1 6 ] . Polyploid metaphases with 4 n, 8 n or even 16 n in bone marrow are mainly m e g a k a r y o c y t e s [ 1 0 ] . Finally a higher incidence of p o ly p lo id y is seen in certain diseases, mainly neoplasia, and after whole b o d y irradiation. It is also known t hat colchicine and other alkaloids can induce polyploidy. However, this does not militate against the use of colchicine as a mitosis inhibitor for the p r o d u c t i o n o f metaphase chromosomes. F o r purposes of comparison it is preferable to use incidence rates of polyploidy observed in controls and test groups of the same study rather than socalled spontaneous incidence rates of polyploidy characteristic of the test species e m p l o y e d . The methodological difficulties and the subjective factors entering into the det er m i nat i on of p o l y p l o i d y introduce a large element of u n cer tain ty into the c o m p u t a t i o n a l use of spontaneous pol ypl oi dy incidence rates [10, 1 9 ] . The majority of in vivo observations point to spontaneous incidence rates varying from 0.1% to approx. 1%. Th e question arises w he t he r the feeding of irradiated diets could result in a shift o f hematopoiesis, granulopoiesis, and the p r o d u c t i o n o f l y m p h o c y t e s and o t h e r reticulo-endothelial cells in the bone marrow o f Chinese hamsters. Such a shift might be related to the proliferation of polyploid cells. In earlier studies [20] we fed Sprague-Dawley rats for life a diet containing 35% of a highly irradiated (4.5 Mrad) feed. No shift was detected into the newly f o r m e d cells in the femoral marrow o f these animals. 220
The cells of the RES {megakaryocytes) in particular did not show any striking abnormalities. Similarly, no changes in the differential cell count were found when the peripheral blood was examined. In the same long-time feeding experiment extending over 2.5 years we ensured a thorough classification, localization and enumeration of all observed tumors. The results did not indicate any carcinogenic effect due to the feeding of the irradiated diet (4.5 Mrad) and thus appear to confirm the absence of any mutagenic activity in the irradiated diet. These findings and the studies represented in Table III allow the following interpretation: Feeding a standard diet freshly irradiated with high doses (> 3 Mrad) has a transitory effect in the bone marrow of Chinese hamsters as evidenced by an increased incidence of polyploid cells. The observed incidence in the test groups is greater by a factor of 4 to 5 than that in the controls, but is still well below 0.5%. When the irradiated diet is no longer fec~, the incidence of polyploidy returns to the pretreatment level., No increased incidence of other chromosomal aberrations was observed. Under these experimental conditions, there is no evidence for any mutagenic effect being produced as a result of feeding an irradiated diet. The cause of the increased incidence of polyploidy remains the subject of conjecture. Free radicals are unlikely to be the reason because, in our experiments, a diet completely soaked in water immediately after irradiation also caused the same effect. Free radicals can however exist for a prolonged period of time following irradiation only in dry matter. Starch irradiation with a dose of 1 Mrad has been found to contain hydrogen peroxide in a concentration of about 1 mg/100 g [21]. The administration of 0.3% H202 by gavage resulted in a prompt increase in the polyploidy rate (bottom of ,Table III). This effect corresponds approximately to the effect observed after feeding a diet irradiated with 2 Mrad. This does not prove that the H202 content of a freshly irradiated diet is responsible for the increased polyploidy, but for the time being this conjecture can serve as a useful hypothesis. Because of the possibility of the application of irradiation as a method for insect disinfestation, it should be pointed out that the dose required for disinfestation is less than 100 krad. It should be noted that in the studies described here, no effects on polyploidy incidence were seen at doses below 2 Mrad. ACKNOWLEDGEMENTS Thanks are due to Prof. Dr. W. Schmid, Genetics Department of the University Children's Hospital, Z//rich, Switzerland, for his advice on problems related to this work. The author should also like to express his gratitude to Prof. Dr. H.G Miltenburger, Zoological Institute of the Darmstadt Technical University, and to Dr. E. Gebhart, Institute of Human Genetics and Anthropology of the University of Erlangen, for their instructions in the preparation and evaluation of chromosomal preparations, and to Dr. Th. Grfinewald, Federal
221
Research Center for Nutrition, for the statistical evaluation. M y c o w o r k e r s , in p a r t i c u l a r Mr. M. K n o l l , h a v e s i g n i f i c a n t l y c o n t r i b u t e d to the g e n e r a t i o n of e x p e r i m e n t a l results by their careful and c o n s c i e n t i o u s technical assistance. REFERENCES 1 P.S. Chauhan, M. Aranvindakshan, A.S. Aiyar and K. Sundaram, Food Cosmet. Toxicol., 13 (1975) 433. 2 J. Schubert, Bull. WHO, 41 (1969) 873. 3 A.N. Zaytsev, Ju. I. Shillinger and Z.M. Kamaldinova, Food Irradiat. Inform., 5 (1975) 43. 4 WHO Techn. Rep. Ser., No. 482 (1971) Geneva. 5 Deutsche Forschungsgemeinschaft, Kommission ffir Mutagenit~itsfragen, Mitteilung III: Mutagenit~/tspriifung 1975. 6 H.W. Renner, Th. Grfinewald and W. Ehrenberg-Kieckebusch, Humangenetik, 18 (1973) 155. 7 Ruth Mfinzner and H.W. Renner, Int. J. Radiat. Biol., 27 (1975 ) 371. 8 H.W. Rennet, Food Cosmet. Toxicol., 13 (1975) 427. 9 M.M. Hossain, J.W. Huismans, J.F. Diehl, Toxicology, 6 (1976) 243. 10 H.G. Schwarzacher and U. Wolf, Methods in human cytogenetics, Springer, BerlinHeidelberg-New York, 1974. 11 W. Schmid, D.T. Arakaki, N.A. Breslau and J.C. Culbertson, Humangenetik, II (1971) 103. 12 W. Schmid and G.R. Staiger, Mutat. Res., 7 (1969) 99. 13 L. Bugyaki, A.R. Deschreider, J. Moutschen, M. Moutschen-Dahmen. A. Thijs et A. Lafontaine, Atompraxis, 1,i (1969) 112. 14 Vijayalaxmi and G. Sadasivan, Int. J. Radiat. Biol., 27 (1975) 135. 15 Vijayalaxmi, Int. J. Radiat. Biol., 27 (1975) 283. 16 K.P. George, R.C. Chaubey, K. Sundaram and A.R. Gopal-Ayengar, Food Cosmet. Toxicol., 14 (1976) 289. 17 C. Bhaskaram and G. Sadasivan, Am. J. Clin. Nutr., 28 (1975) 130. 18 H.A. Hienz, Chromosomenfibel, Gg. Thieme, Stuttgart {1971). 19 H. Frohberg and M. Schulze-Schencking, Arch. Toxicol., 33 (1975) 209. 20 H.W. Rennet und D. Reichelt, Zbl. Vet. Med., B 20 (1973) 648. 21 G. Berger et L. Saint-LSbe, C.R. Acad. Sci. Paris, Set. D, 272 (1971) 1455.
222
CHROMOSOME STUDIES ON BONE MARROW CELLS OF CHINESE HAMSTERS FED A RADIOSTERILIZED DIET
H.W. RENNER Institut fiir Biochemie der Bundesforschungsanstalt fiir Erniihrung, Engesserstrasse 20, D-7500 Karlsruhe (F.R.G.)
(Received January 27th, 1977) (Revision received April 12th, 1977 ) (Accepted April 28th, 1977)
SUMMARY Metaphase preparations of chr om osom es from bone m a r r o w cells of Chinese hamsters were examined for mutagenic effects following the feeding o f a radiosterilized diet. No increase in the incidence of structural chromosomal aberrations was observed. As far as numerical aberrations were concerned, the p r o p o r t i o n o f cells with polyploidy increased to between 4 to 5 times the control level, irrespective of the moisture c o n t e n t of the diet. This p o ly p lo id y effect occurred very early, being detectable within 24 h, if the diet fed had been irradiated with an absorbed dose of 4.5 • 106 rad. The incidence of pol ypl oi dy remained below 0.5%, however, nor did it rise with higher radiation doses. When the feeding of the irradiated diet was stopped, the p r o p o r t i o n of polyploid cells returned to t he control level within a maximum o f 6 weeks. If the diet was stored (initially) for 6 weeks following irradiation before being fed to the animals no increase in the n u m b e r of polyploid cells was noted. These results are not interpreted as a mutagenic effect of the irradiated diet.
INTRODUCTION
Whilst toxicological tests of irradiated food have never disclosed any adverse effects, possible mutagenic effects following ingestion of irradiated f o o d have repeatedly been conjectured and m a n y studies to investigate this problem have been carried out (for review studies, see refs. 1--3). It is now generally recognized, particularly when testing weak or doubt ful mutagens, that: (a) only those mutagenicity tests are relevant which take into a c c o u n t mammalian metabolism [ 4 ] ; (b) only the use of several test meth o d s fulfilling these requirements allows careful extrapolation of the results to man, because no single m e t h o d exists which is capable of detecting all types of mutational events [5]. Over the past few years our study group
213
has carried o u t m u t a g e n i c i t y tests on irradiated f o o d and animal feeds by means o f the d o m i n a n t - l e t h a l test, the h o s t - m e d i a t e d assay, and the micronucleus test, in every case with negative results [ 6 - - 9 ] . O u r f u r t h e r studies in this field, which i n v o l v e d the e m p l o y m e n t o f c h r o m o s o m a l analysis, are r e p o r t e d in this paper. MATERIALS AND METHODS A n i m a l selection and maintenance T h e Chinese hamsters used in these studies were o b t a i n e d from the Zoological I n s t i t u t e o f the D a r m s t a d t T e c h n i c a l University, f r o m a c o l o n y i n b r e d over f o r t y generations and m a i n t a i n e d in t h e c o n v e n t i o n a l way. In o u r laboratories a c o l o n y o f these r o d e n t s in sufficient n u m b e r for o u r purposes was built up f r o m 10 pairs o f r a n d o m l y selected breeding animals; the animals were k e p t in a fully a i r - c o n d i t i o n e d e n v i r o n m e n t (air t e m p e r a t u r e 22 ± I°C, relative h u m i d i t y 55 -+ 5%, 15 air changes/h, air filtration and light-dark cycle o f 12 h). The h a m s t e r c o l o n y was free f r o m disease at all times. T h e animals used in the e x p e r i m e n t were aged b e t w e e n 3 and 5 m o n t h s and weighed b e t w e e n 23 and 43 g. G r o u p s o f 25 or 26 animals were used as controls or test groups, the p r o p o r t i o n s o f males and females being equal in each group. Diet T h e animals were fed ad lib. a b r e e d i n g d i e t f o r hamsters ( A l t r o m i n Z*) in a pelletized f o r m . T h e test diet was irradiated a t w e e k l y intervals at the lnstitute's e l e c t r o n linear a c c e l e r a t o r with 10 MeV e l e c t r o n s in o p e n a l u m i n u m trays and t h e n fed e i t h e r for 6 weeks or f o r 1 day. Once a week all animals were given vitamin s u p p l e m e n t s in their drinking w a t e r ( M u l t i b i o n t a * * ) . Preparation o f c h r o m o s o m e s 2 m g / k g of C o l c e m i d ( C a l b i o c h e m ) was injected into t h e animals 2.5 h b e f o r e sacrifice. The f e m o r a were r e m o v e d and the b o n e m a r r o w was flushed o u t by means o f a 1% s o d i u m citrate s o l u t i o n c o n t a i n i n g added heparin. T h e cell suspension was f u r t h e r processed in a c c o r d a n c e with the i n s t r u c t i o n s c o n t a i n e d in a r e c e n t h a n d b o o k o f c y t o g e n e t i c m e t h o d s [ 1 0 ] . T h e slide preparations (2/animal) were stained with a 2% a c e t o - o r c e i n solution. This technique furnished g o o d t o e x c e l l e n t m e t a p h a s e p r e p a r a t i o n s . Evalua tio n T h e slide p r e p a r a t i o n s were evaluated blind b y two investigators w o r k i n g i n d e p e n d e n t l y . O n l y well spread m e t a p h a s e s w i t h o u t recognisable superimposed c h r o m o s o m e s were c o u n t e d . This criterion was also applied t o polyploid cells. In a d d i t i o n , the a r r a n g e m e n t o f the p o l y p l o i d c h r o m o s o m e s was t a k e n into a c c o u n t (Figs. 1 and 2).
**
Altromin GmbH, D-4937 Lage/Lippe. Trademark registered by E. Merck, Darmstadt.
214
x Fig. 2. Metaphase
of a polyplaid
cell (n = 88) of Chinese
hamster.
x
600.
In order to permit a more satisfactory statistical analysis of the expected results it was considered preferable to increase the number of animals per group instead of examining a larger number of metaphases per animal. RESULTS
In our initial investigations we used 6 Chinese hamsters (12 weeks old, 3 dd, 3 90) treated with the cytostatic Trenimon” as positive controls employing method and classification described in 1971 [ll] (2 i.p. injections of 0.25 mg/kg each of Trenimon at 24 h intervals, sacrificing the animals 6 h after the second injection). Between the two injections the plexus of the orbital vein was punctured to reduce the bone marrow depression and to stimulate the mitotic index. A total of 100 metaphases was counted per animal and the results are presented in Table I. These show that t.he results of other authors [ 11,121 could be reproduced satisfactorily. The design and the results of our main experiment are given in Table II. The mitotic index of 4.7-6.6% was of the same order in both the controls and the test groups fed the diet irradiated at a dose of 4.5 Mrad + 5%.** Our results in the control animals confirmed the extremely low spontaneous aberration rate in bone marrow cells of Chinese hamsters reported in other studies [ll, 121. Moreover, we were able to confirm that only one type of aberration occurred, namely chromatid breaks. Karyograms were made of approx. 10 animals in each of the groups to ensure as far as possible that no chromosomal anomalies had been missed. -* Trademark
**
registered
by Farbenfabriken
rad = radiation absorbed dose. = 10.’ J I rad= lOOerg*g”
l
kk-‘;
Bayer AG, Leverkusen.
1 Mrad (Megarad)
= lo6 rad.
215
TABLE I CHROMOSOMAL ANALYSIS ON BONE MARROW CELLS OF CHINESE HAMSTERS Positive control following Trenimon treatment (2 × 0.25 mg/kg at 24 h intervals), n, 6 animals; 100 metaphases/animal were scored. Normal cells 1--2 aberrations Multiple aberrations Pulverized
13.0% a 16.3% 65.0% 5.7%
(break and/or interchanges) (/> 3, all types) (unscorable chromosomes)
Total, abnormal cells
87.0%
aNo. of scored metaphases = 100% TABLE II CHROMOSOMAL ANALYSIS ON BONE MARROW CELLS OF CHINESE HAMSTERS Results after feeding irradiated diets. Metaphases evaluated
Chromatid gaps
Chromatid breaks
Polyploid cells
No.
%a
No.
%a
No.
%a
Control group n = 25 animals (unirradiated diet)
7500
56
0.75
21
0.28
5
0.06
Test group (a) n = 26 animals (4.5 Mrad irradiated diet for 6 weeks)
7800
74
0.95
16
0.21
25
0.32
Test group (b) n = 25 animals (4.5 Mrad irradiated diet for 1 day, administered after 1 day of starvation)
7500
60
0.80
22
0.29
20
0.27
(300 metaphases were evaluated per animal. Most of the polypioid cells had n = 88 chromosomes. ) aNo. of evaluated metaphases = 100%
T h e f r e q u e n c y o f s t r u c t u r a l a n o m a l i e s ( c h r o m a t i d b r e a k s ) in t h e t w o t e s t g r o u p s w a s s i m i l a r t o t h a t in t h e c o n t r o l g r o u p . I n v e s t i g a t i o n o f t h e n u m e r i c a l a b e r r a t i o n s i n d i c a t e d t h a t cells w i t h 21 c h r o m o s o m e s o c c u r r e d w i t h t h e s a m e a b u n d a n c e in b o t h t h e c o n t r o l a n d t h e t e s t g r o u p s , t h e y w e r e r e g a r d e d
216
as dying cells. However, it appeared that polyploid cells occurred about five times more frequently in those animals which had been fed irradiated diets compared to the control group (statistical probability of error P = 0.01, Wilcoxon test). No differences attributable to sex could be observed througho u t the groups. The ×2 four field test shows that the events (= polyploid cells) f o u n d in the evaluation of 300 mitoses/animal will differ random l y from each o t he r only if their num be r lies between 0 and 6. Since this requiremen t was met, all cells within each animal group were pooled and evaluated independently of the individual animals. The n u m b e r of events in the control group differs from t hat in the pooled test group ((a) and (b)). In order to be able to test the significance of this difference the distributions of the events in both groups were investigated. Th ey could be appr oxi m at ed satisfactorily by Poisson distributions. Accordingly, the significance test was carried o u t by the distribution-free U-test according to Wilcoxon, Mann and Whitney. Table III presents a survey of additional studies carried o u t to define more accurately the conditions responsible for an increased incidence of polyploidy. DISCUSSION The absence, in these experiments, of any effect of irradiated diets on the incidence o f structural anomalies (point mutations are not detectable in this test system) in c h r o m o s o m e s of bone marrow cells discounts the likelihood o f mutagenic activity of these treated diets. On the other hand, there is doubtless a clear effect upon the polyploidy incidence. It is surprising to find that this effect is of similar magnitude as early as 24 h after the onset of feeding o f the irradiated diet as that seen after 6 weeks o f feeding of the same diet. The dose-dependent increase in this effect within the range of 1--4.5 Mrad indicates that irradiation must have produced one or m ore substances in the diet giving rise to this effect. These are, however, unstable products, because feeding a diet stored for 6 weeks produced no such effect. In 1968 Bugyaki et al. [13] reported meiotic studies in mice fed for life a diet consisting of 50% freshly irradiated (5 Mrad) wheat flour. T h e y found increased numbers of chromosomal aberrations (translocations, bridges and fragments) o f the male germ cells in all meiotic stages. The authors used descendants o f the C 57 BL strain of mice with a spontaneous mutation. These animals were e xt r em e l y sensitive to the spontaneously increased occurrence of translocations and ot he r chromosomal anomalies. According to Vijayalaxmi and Sadasivan [14] the administration of a wheat flour diet irradiated with 75 krad increased the n u m b e r of structural chromosomal aberrations in the bone marrow cells of Wistar rats. The same effect was obtained by feeding a diet with low protein c o n t e n t (5%). The polyploidy rate was not increased by a low protein diet but rose after feeding an irradiated diet. It was later reported [15] that, at the radiation dose emp lo y ed , the normal fraction of polyploid bone marrow cells rose from ap p r o x imatel y 0.04% to some 0.4% after only 10 weeks. If flour was stored
217
t~ 00
S t a r v a t i o n for 36 h, t h e n feeding u n i r r a d i a t e d diet for 24 h No s t a r v a t i o n period, t h e n feeding irradiated diet for 3 days (4.5 Mrad)
Can starving per se initiate p o l y p l o i d y ?
6 weeks o f irradiated diet (4.5 Mrad), t h e n 6 weeks of unirradiated diet
Irradiated diet (4.5 M r a d ) s t o r e d for 6 weeks, t h e n fed for 24 h a f t e r a s t a r v a t i o n break of 1 day
How long will polyploidy persist w h e n an u n i r r a d i a t e d diet is fed again?
Will irradiated diet, s t o r e d a f t e r irradiat i o n , still be able to initiate p o l y p l o i d y ?
H o w quickly will polyploidy o c c u r w i t h o u t a s t a r v a t i o n period between feeding unirrad i a t e d or irradiated diets?
Treatment
Problem
14 7000
15 7500
11 5500
10 5000
n animals, metaphases
6
5
13
4
No.
Polyploid cells
0.08
0.06
0.24
0.08
%
No p o l y p l o i d y e f f e c t a f t e r storage
P o l y p l o i d y n o longer detectable after 6 weeks
E f f e c t w i t h i n 3 days
No e f f e c t o f starvation
Result
T y p e s and results o f f u r t h e r e x p e r i m e n t s c o n c e r n i n g t h e i n c i d e n c e of p o l y p l o i d cells. 5 0 0 M e t a p h a s e s / a n i m a l were e v a l u a t e d ; No. o f evaluated m e t a p h a s e s = 100%
C H R O M O S O M A L I N V E S T I G A T I O N S ON B O N E M A R R O W C E L L S OF C H I N E S E H A M S T E R S F E D I R R A D I A T E D DIETS
T A B L E III
t,0 q~
14 7000
U n i r r a d i a t e d diet + 3 x 0.5 ml o f 0.3% H202 a d m i n i s t e r e d by gavage at 8 h intervals
Could H202 be the cause o f t h e increased incidence of polyploidy?
a D a t a t a k e n f r o m Table II " T e s t g r o u p ( b ) " (300 m e t a p h h s e s / a n i m a i ) .
11
8 4000
13
22
20
25 7500
14 7000
15
15 7500
Irradiated diet {4.5 Mrad) s o a k e d in water, t h e n fed to animals for 24 h after 1 day of starvation
13
15 7500
Is t h e r e a p o l y p l o i d y e f f e c t also a f t e r f e e d i n g a moist, irradiated diet?
6
15 7500
(a) Diet i r r a d i a t i o n dose 1 Mrad 1 day s t a r v a t i o n period, 24 h feeding (b) Diet irradiation dose 2 Mrad O t h e r w i s e as u n d e r (a) (c) Diet irradiation dose 3 Mrad O t h e r w i s e as u n d e r (a} (d) Diet irradiation dose 4.5 Mrad O t h e r w i s e as u n d e r (a) (e) Diet irradiation dose 10 Mrad O t h e r w i s e as u n d e r (a)
Is the p o l y p l o i d y o c c u r r i n g within one day a f u n c t i o n o f the dose?
0.19
0.31
0.28
0.27
0.20
0.17
0.08
Increased incidence o f polyploidy
Full p o l y p l o i d y e f f e c t
Polyploidy does not rise any f u r t h e r with higher radiation d o s e s
Full p o l y p l o i d y a effect
Beginning increase in polyploidy
No p o i y p i o l o y
for 12 weeks after irradiation, it caused no polyploidy. However, another Indian research group [16], on repeating these experiments, was unable to confirm an increase in the number of polyploid cells in the bone marrow of rats fed an irradiated diet. Differences in the cytogenetic methods employed by the two research teams should be taken into account when assessing these divergent results. Bhaskaram and Sadasivan [ 17 ] fed 3 groups each of 5 hospitalized children, all suffering from severe protein deficiency, either unirradiated or freshly irradiated wheat flour or wheat flour stored for 3 months following irradiation. Leukocyte cultures were started every two weeks. These children when given freshly irradiated flour developed polyploid cells and cells with other ch r o mo s o ma l aberrations, the longer t h e y ate irradiated flour. Surprisingly enough, no p o l y p l o i d y at all was observed in the control group. After this diet had been stopped, the incidence of p o l y p l o i d y fell to zero in the children who had co ns um ed irradiated flour. The c h r o m o s o m a l studies so far carried out in relation to the ingestion of dry irradiated f ood have thus p r o d u c e d c o n t r a d i c t o r y results. In some cases no ef f ect was found, in ot he r cases there was an increased incidence of polyploidy or even an increased incidence of aberrations. The latter finding was n o t seen in our results, and an increased incidence of pol ypl oi dy was only f o u n d at 2 Mrad and above. The physiological significance of an increased incidence of polyploidy in bone mar r o w cells o f our test animals, observed within 24 h of feeding an irradiated diet, is not clear. In man, a variable incidence of pol ypl oi dy is normally f o u n d in osteoblasts, liver cells, cells of the respiratory epithelium etc. [ 1 8 ] . Similar observations have been made for various l aborat ory mammalian species [ 1 6 ] . Polyploid metaphases with 4 n, 8 n or even 16 n in bone marrow are mainly m e g a k a r y o c y t e s [ 1 0 ] . Finally a higher incidence of p o ly p lo id y is seen in certain diseases, mainly neoplasia, and after whole b o d y irradiation. It is also known t hat colchicine and other alkaloids can induce polyploidy. However, this does not militate against the use of colchicine as a mitosis inhibitor for the p r o d u c t i o n o f metaphase chromosomes. F o r purposes of comparison it is preferable to use incidence rates of polyploidy observed in controls and test groups of the same study rather than socalled spontaneous incidence rates of polyploidy characteristic of the test species e m p l o y e d . The methodological difficulties and the subjective factors entering into the det er m i nat i on of p o l y p l o i d y introduce a large element of u n cer tain ty into the c o m p u t a t i o n a l use of spontaneous pol ypl oi dy incidence rates [10, 1 9 ] . The majority of in vivo observations point to spontaneous incidence rates varying from 0.1% to approx. 1%. Th e question arises w he t he r the feeding of irradiated diets could result in a shift o f hematopoiesis, granulopoiesis, and the p r o d u c t i o n o f l y m p h o c y t e s and o t h e r reticulo-endothelial cells in the bone marrow o f Chinese hamsters. Such a shift might be related to the proliferation of polyploid cells. In earlier studies [20] we fed Sprague-Dawley rats for life a diet containing 35% of a highly irradiated (4.5 Mrad) feed. No shift was detected into the newly f o r m e d cells in the femoral marrow o f these animals. 220
The cells of the RES {megakaryocytes) in particular did not show any striking abnormalities. Similarly, no changes in the differential cell count were found when the peripheral blood was examined. In the same long-time feeding experiment extending over 2.5 years we ensured a thorough classification, localization and enumeration of all observed tumors. The results did not indicate any carcinogenic effect due to the feeding of the irradiated diet (4.5 Mrad) and thus appear to confirm the absence of any mutagenic activity in the irradiated diet. These findings and the studies represented in Table III allow the following interpretation: Feeding a standard diet freshly irradiated with high doses (> 3 Mrad) has a transitory effect in the bone marrow of Chinese hamsters as evidenced by an increased incidence of polyploid cells. The observed incidence in the test groups is greater by a factor of 4 to 5 than that in the controls, but is still well below 0.5%. When the irradiated diet is no longer fec~, the incidence of polyploidy returns to the pretreatment level., No increased incidence of other chromosomal aberrations was observed. Under these experimental conditions, there is no evidence for any mutagenic effect being produced as a result of feeding an irradiated diet. The cause of the increased incidence of polyploidy remains the subject of conjecture. Free radicals are unlikely to be the reason because, in our experiments, a diet completely soaked in water immediately after irradiation also caused the same effect. Free radicals can however exist for a prolonged period of time following irradiation only in dry matter. Starch irradiation with a dose of 1 Mrad has been found to contain hydrogen peroxide in a concentration of about 1 mg/100 g [21]. The administration of 0.3% H202 by gavage resulted in a prompt increase in the polyploidy rate (bottom of ,Table III). This effect corresponds approximately to the effect observed after feeding a diet irradiated with 2 Mrad. This does not prove that the H202 content of a freshly irradiated diet is responsible for the increased polyploidy, but for the time being this conjecture can serve as a useful hypothesis. Because of the possibility of the application of irradiation as a method for insect disinfestation, it should be pointed out that the dose required for disinfestation is less than 100 krad. It should be noted that in the studies described here, no effects on polyploidy incidence were seen at doses below 2 Mrad. ACKNOWLEDGEMENTS Thanks are due to Prof. Dr. W. Schmid, Genetics Department of the University Children's Hospital, Z//rich, Switzerland, for his advice on problems related to this work. The author should also like to express his gratitude to Prof. Dr. H.G Miltenburger, Zoological Institute of the Darmstadt Technical University, and to Dr. E. Gebhart, Institute of Human Genetics and Anthropology of the University of Erlangen, for their instructions in the preparation and evaluation of chromosomal preparations, and to Dr. Th. Grfinewald, Federal
221
Research Center for Nutrition, for the statistical evaluation. M y c o w o r k e r s , in p a r t i c u l a r Mr. M. K n o l l , h a v e s i g n i f i c a n t l y c o n t r i b u t e d to the g e n e r a t i o n of e x p e r i m e n t a l results by their careful and c o n s c i e n t i o u s technical assistance. REFERENCES 1 P.S. Chauhan, M. Aranvindakshan, A.S. Aiyar and K. Sundaram, Food Cosmet. Toxicol., 13 (1975) 433. 2 J. Schubert, Bull. WHO, 41 (1969) 873. 3 A.N. Zaytsev, Ju. I. Shillinger and Z.M. Kamaldinova, Food Irradiat. Inform., 5 (1975) 43. 4 WHO Techn. Rep. Ser., No. 482 (1971) Geneva. 5 Deutsche Forschungsgemeinschaft, Kommission ffir Mutagenit~itsfragen, Mitteilung III: Mutagenit~/tspriifung 1975. 6 H.W. Renner, Th. Grfinewald and W. Ehrenberg-Kieckebusch, Humangenetik, 18 (1973) 155. 7 Ruth Mfinzner and H.W. Renner, Int. J. Radiat. Biol., 27 (1975 ) 371. 8 H.W. Rennet, Food Cosmet. Toxicol., 13 (1975) 427. 9 M.M. Hossain, J.W. Huismans, J.F. Diehl, Toxicology, 6 (1976) 243. 10 H.G. Schwarzacher and U. Wolf, Methods in human cytogenetics, Springer, BerlinHeidelberg-New York, 1974. 11 W. Schmid, D.T. Arakaki, N.A. Breslau and J.C. Culbertson, Humangenetik, II (1971) 103. 12 W. Schmid and G.R. Staiger, Mutat. Res., 7 (1969) 99. 13 L. Bugyaki, A.R. Deschreider, J. Moutschen, M. Moutschen-Dahmen. A. Thijs et A. Lafontaine, Atompraxis, 1,i (1969) 112. 14 Vijayalaxmi and G. Sadasivan, Int. J. Radiat. Biol., 27 (1975) 135. 15 Vijayalaxmi, Int. J. Radiat. Biol., 27 (1975) 283. 16 K.P. George, R.C. Chaubey, K. Sundaram and A.R. Gopal-Ayengar, Food Cosmet. Toxicol., 14 (1976) 289. 17 C. Bhaskaram and G. Sadasivan, Am. J. Clin. Nutr., 28 (1975) 130. 18 H.A. Hienz, Chromosomenfibel, Gg. Thieme, Stuttgart {1971). 19 H. Frohberg and M. Schulze-Schencking, Arch. Toxicol., 33 (1975) 209. 20 H.W. Rennet und D. Reichelt, Zbl. Vet. Med., B 20 (1973) 648. 21 G. Berger et L. Saint-LSbe, C.R. Acad. Sci. Paris, Set. D, 272 (1971) 1455.
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