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Acute cytogenetic effect of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2, a food preservative) on rat bone marrow cells in vivo
Mutation Research, 31 (1975) 247-254 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands
247
R E L A T I O N S H I P B E T W E E N E X P E R I M E N T A L R E S U L T S IN MAMMALS AND MAN I. C Y T O G E N E T I C ANALYSIS OF BONE MARROW I N J U R Y I N D U C E D BY A S I N G L E DOSE OF C Y C L O P H O S P H A M I D E
P. GOETZ, R. J. ~R~M AND J. DOHNALOV2~
Research Institute of Child Development, Faculty of Pediatrics, Prague, Institute of Hygiene and Epidemiology, Prague and Department of Radiology. Teaching Hospital, Prague-Motol (Czechoslovakia) (Received August I4th, 1974) (Revision received February I8th, 1975)
SUMMARY
The extrapolation of experimental results to man was studied by cytogenetic bone marrow analysis and micronucleus test in mice, rats and Chinese hamsters. Furthermore, the frequency of chromosomal aberrations was compared with the frequencies of polychromatic erythrocytes containing micronuclei. Cyclophosphamide (CY) was given intraperitoneally at the doses of 5, io, 20, 4 ° and 80 mg/kg b.w. to ICR mice and Wistar rats and at the doses of IO, 20, 4 o, 80, 12o and 16o mg/kg b.w. to Chinese hamsters. Five patients with various types of malignancies until then medically untreated, were i.v. administered 40 mg CY/kg b.w. Bone marrow cells were examined 24 h after the administration. CY induced in all rodents a clear-cut dose-effect relationship in the frequency of breaks, abnormal metaphases as well as in the frequency of micronuclei in polychromatic erythrocytes. When comparing the results in rodents and man at the dose of 40 mg CY/kg b.w., the sensitivity pattern of species was mice > rats > Chinese h a m s t e r s > man. From this aspect the possible differences in the metabolism of CY in analysed species are discussed. The presented results tend to a conclusion that micronucleus testing m a y be a very suitable method used for screening purposes, however, the method of classical cytogenetic analysis, especially the evaluation of breaks, still remains the most exact and reliable technique.
INTRODUCTION
The extrapolation of experimental results from laboratory rodents to man represents the most important and difficult problem in evaluating the mutagenic activity of chemical compounds. This should be taken into account also in cytogenetic analysis of chromosome aberrations, since it m a y be assumed that there is a variability of
248
e. GOETZ el al.
aberrations induced in closely related mammalian species as well as in phylogenetically more remote species, e.g. rodents and man. A possible interspecies relationship in the sensitivity of mice, rats, Chinese hamsters and man was studied by comparing the frequency of chromosome aberrations induced in bone marrow cells after a single dose of CY. This alkylating agent commonly used in medical therapy was chosen for its repeatedly proved nmtagenic activity",7,v', 14. Results of micronucleus test and chromosolnal analysis were compared in all mammals used and the feasibilitv of the micronucleus test for the nmtagenicity testing of chemicals was evaluated. MATERIAL AND METHODS
CY (Germed, GDR) was given intraperitoneally in doses of 5, IO, 20, 4 ° and 8o mg/kg b.w. to ICR mice and Wistar rats and in doses of IO, 2o, 4 o, 8o, 12o and 16o mg/kg b.w. to Chinese hamsters. Five patients with various types of malignancies (excluding hematological ones), until then medically untreated, were i.v. administered 4 ° mg CY/kg b.w. Each experimental as well as control group consisted of 5 individuals. The micronucleus test technique of SCHMID and coworkers2,1°, ~a was used in the experiment. IOOOOerythroeytes were examined in each experimental and control group. Micronuclei were recorded in polychromatic as well as in normochromatic erythrocytes. Mutagenic activity of the drug was evaluated according to SCHMIOTM as the frequency of micronuclei in polychromatic erythrocytes. Slides for chromosomal analysis were prepared according to a modification of the classical TJIO AND W H A N G ' S methodlL 25 ° metaphases of bone marrow cells were examined 24 h after the administration; a similar scheme was used also in the control groups. The results from the cytogenetic analysis of human bone marrow were compared with those obtained by sternal puncture performed before the administration of CY. Gaps, breaks and exchanges wine evaluated. Cells bearing some of these chromosomal abnormalities were considered abnormal. Cells with more than IO aberrations were counted separately. RESULTS
The results of micronuclei test and cytogenetic analysis are indicated in Tables I--IV and Fig. i. A dose-effect relationship (in the micronucleus test) was found in mice, rats and Chinese hamsters. The increase in the frequency of polychromatic erythrocytes with micronuclei was marked in mice, especially with a higher dose. In all studied species, however, the highest dose induced a decreased frequency of micronuclei. A clear dose-effect relationship was also found when analysing chromosome aberrations. This relationship was evident in the frequency of abnormal metaphases as well as in the yield of chromosome breaks. A steeper slope was similarly observed in mice, especially with higher doses. The dose of 80 mg CY/kg b.w. in rats and 16o mg/kg b.w. in Chinese hamsters severely suppressed the bone marrow activity, so that no sufficient number of cells in metaphase could be found. Nevertheless, an increased frequency of chromosome aberrations is quite apparent even with lower doses.
TABLE
I
o.44 0.46 o.46 o.63 1.82 0.56
II
0.37 0.45 0.45 o.61 1.5o o.52
e W h e n ceils w i t h m o r e t h a n
IO a b e r r a t i o n s
are counted
a N, N u m b e r o f a n a l y s e d cells. bG[C, B[C, E[C, g a p s , b r e a k s , e x c h a n g e s p e r cell.
1.39 2.62 3.6o 5.o 4 6.28 5.61
ioooo IOOOO ioooo ioooo ioooo IOOOO
(%)
OF RAT BONE MARROW
Controls 5 io 2o 4° 80
(%)
25o 250 25o 25o 250 250
6.0 14.8 22. 4 26.0 20.8 24. 4
o.88 0.240 0.324 o.516 o.288 o.368
G/C b o. 4 6.8 16.o 12. 4 I 7 .2e 24.8d
Breaks % o.oo 4 o.116 o.316 o.400 o.456e o.652°
B]C b
.
2.4 9.6 9.6 12.o 19.6 .
0.024 o.132 o.124 o.196 0.324
G /C b
.
. . 3.2 7.6 2o.4e .
Breaks % . .
o.o56 O.lO4 o.512e .
B ]Cb . .
t h e v a l u e o f cells w i t h b r e a k s i n % is 2o.8 a n d
250 250 250 25 ° z5o .
Chromosomes Na Gaps %
a r e c o u n t e d t h e v a l u e o f c e l l s w i t h b r e a k s i n 0/o is e 2 1 . 6 a n d
Micronuclei Na Polychromatic Normochromatic erythrocytes erythrocytes
TREATMENT
lO a b e r r a t i o n s
Dose (mg]kg b.w.)
CYCLOPHOSPHAMIDE
TABLE
W h e n ceils w i t h m o r e t h a n
a N, N u m b e r of a n a l y s e d cells. b GIC, B]C, E/C, g a p s , b r e a k s , e x c h a n g e s p e r cell.
1.41 2.13 3.3 o 4.21 8.54 5.06
ioooo ioooo ioooo ioooo 1oooo IOOOO
(%)
Controls 5 io 20 4° 8o
(%)
Chromosomes Na Gaps %
OF M O U S E B O N E MARROW"
Micronuclei Na Polychromatic Normochromatic erythrocytes erythrocytes
TREATMENT
Dose (mg/kg b.w.)
CYCLOPHOSPHAMIDE
0.056 o.o4o
.
. .
B[C is o . 5 5 2 .
.
1.2 8.8
. .
o.o12 o.152
Exchanges % E /C b
.
Abnormal cells
2.4 3o.8
Abnormal cells
o. 4
2.4 9.6 12.o 17.2 33.6
tions(%) (%)
> zo aberra-
6. 4 20.o 32.o 33.2 39.6 66.0
tions (%) (%)
> zo aberra-
B]C 0 . 8 9 6 , a 5 8 . o a n d B/C 3-7.
4.4 3.6
Exchanges % E]C b
4~ ~D
z
0q
O~
~d X >
t~
III
TREATMENT
OF CHINESE
HAMSTER
ioooo
16o
4.22
0.55 1.26 2.28 2.73 4.07 4.29
0.26
°.14 o.16 0.28 o.29 0-35 0.34
(%)
MARROW
25 ° 250 250 250 250 250
2.8 15.6 20.0 25.2 28.8 20.0
o.o28 1.18o 0.276 0.328 o.360 0.268
Chromosomes Na Gaps - °'o G /Cb
BONE
3. 6 3.6 io.o i2.8 e i8.4d io.4 e
Breaks oo o.o36 0.036 o.172 o.232e o.372d o.i32e
B /Cb 1.6 1.2 2.0 1.2 0.020 0.020 o.o28 o.o16
Exchanges o,° E /CI~ 0.8 1.6 0.4
4-4 17"2 27 .6 32.6 42.8 27.6
> zo Abnormal aberracells tions(Oo) (°~0)
IV
ioooo ioooo
o.31 o.71
o)
o/
OF HUMAN
BONE
(%)
0.22 0.3o
250 250
1.2 lO.8
oo
Chromosomes Na Gaps
M A R R O x'V
Micronuclei Na Polychromatie Normochromatic erythrocvtes erythrocytes -
TREATMENT
a N, N u m b e r of a n a l y s e d cells. b G/C, B/C, E/C, g a p s , b r e a k s , e x c h a n g e s p e r cell.
Controls 4°
Dose (mg/kg b.w.)
CYCLOPHOSPHAMIDE
TABLE
e Io.8Oo a n d B / C o.172.
o.o12 o.o12
G/C b
0.4 6. 4
Breaks oo
0.4 0.072
B/C b - -
0.8
0.008
----
Exchanges Oo E/C b
-
16.8
1.6
> Io Abnormal aberracells tions('!o) (0o)
W h e n cells w i t h m o r e t h a n i o a b e r r a t i o n s a r e also c o u n t e d t h e v a l u e of cells w i t h b r e a k s m ° o is e 13.6 a n d B / C o . 3 1 2 , d 20.o0. o a n d B / C 0.532,
a N, N u m b e r of a n a l y s e d cells. b G/C, B/C, E/C, g a p s , b r e a k s , e x c h a n g e s p e r cell.
ioooo ioooo ioooo ioooo ioooo 10000
(%)
Micronuclei Na Polychromatic Normochromatie erythrocytes erythrocytes
Controls io 20 4° 80 12o
Dose (mg/kg b.w.)
CYCLOPHOSPHAMIDE
TABLE
C
b~
b~
©
E X P E R I M E N T A L R E S U L T S IN MAMMALS A N D MAN. I
251
The frequency of chromosomal aberrations after the dose of 16o mg CY/kg b.w. was lower in Chinese hamsters as compared with the dose of 80 mg CY/kg b.w., as was the case in the micronuclei test. After the highest doses there was increased variability of the chromosome aberration frequency in all animal species used in the experiment. The curves of CY effect show the most pronounced dose-effect relationship in the yield of chromosome breaks. Curves indicating the frequency of aberrant cells with gaps, breaks and exchanges as well as curves of polychromatic erythrocytes with micronuclei are of similar pattern, but not so steep. The sensitivity of man and experimental mammals to the dose of 40 mg CY/kg b.w., analysed by both methods is shown in Fig. 2. From all the species involved in the experiment, mice and rats were the most sensitive animals for cytogenetic analysis as well as for micronuclei testing. The sensitivity of Chinese hamsters was lower and the most significantly decreased sensitivity was found in man. DISCUSSION
Contrary to some authors who administered CY per os in their experiments, we used an intraperitoneal application since the drug is therapeutically administered to man intravenously. The same method of drug adnfinistration used in this experiment, and the same method by which the same tissue (bone marrow) was taken and processed, permit a comparison between the interspecies variations as well as the two methods used. CY is a drug whose mutagenic activity is dependent on the presence of an active metabolite. CY does not, e.g., induce any mitotic gene conversion if there is a direct °/o
10. Micronucleus test 9-
Mouse L
Ch hamster
Rat
~b ~E4
._v E
£
37
jg~62b~
jg 1 6 2 b ~
C
C
Dose r n g / k g b.w.
C
~Ol, O. O.9 0.8 0.7-
120
hromosomal abnormalities
Mouse
Ch hamster
Rat
/;"
°/o
]
6o~
(?.60.50.4
,~
/
50 E
3o~
(~30.2
20<
0.17
I~ 16 ~b4bso
is ~o 2o4o
110 20 40 80 120
C
C
Dose mg/kg b,w.
Fig. i . I n t e r s p e c i e s v a r i a t i o n s a f t e r c y c l o p h o s p h a m i d e t r e a t m e n t . M i c r o n u c l e u s t e s t : - - , e x p e r i m e n t a l g r o u p s ; . . . . , c o n t r o l s , C h r o m o s o m a l a b n o r m a l i t i e s : - - , aberrant m e t a p h a s e s ; . . . . , b r e a k s ; C, c o n t r o l s .
1~. GOETZ et al.
252 .1.0
10.
0.9 9"Z
.0.8 0.7 0.6
U E:
0.5~
Eo~4 . o IL
2
~Z -//,
0.4
0.3 m 0.2 0.1
Rat Mon Ch. hamster Mouse Fig. 2. I n t e r s p e c i e s v a r i a t i o n s in f r e q u e n c y of micronuclei a n d c h r o m o s o m a l b r e a k s w i t h cyclop h o s p h a m i d e 4 ° m g / k g b.w.[~., P o l y c h r o m a t i c e r y t h r o c y t e s w i t h m i c r o n u c l e i ; [3, b r e a k s per cell.
effect on Saccharomyces cerevisiae, but if a host-mediated assay is used, positive results are observed for CY, tooS, 7. It has been proved that the CY biotransformation in all species examined (mouse, rat, Chinese hamster, rabbit, sheep, dog, monkey, man) originates in the liver microsomes due to the effect of oxidase enzyme activity. However there is no precise knowledge on CY metabolic pathways and the biological role of CY metabohtes. It is generally accepted that the mutagenic activity of CY is determined by its active alkylating metabolites, especially by aldophosphamide 18. The results of studies on the dynamics of CY-metabolite concentrations in the blood and urine after CY administration in all species tested show species-dependent differences in the metabolic activation and excretion rates. Laboratory rodents used in our experiments showed a very fast CY activation. A presence in the blood of alkylating metabolites was observed in mice within i o - i 5 min following the administration of CY, and in rats and hamsters within I5-3o min. Mice excreted in their urine 6o% of the CY in 24 h, elimination in rats is similar. The excretion rate depends on the dose. The most rapid urine excretion of the drug was recorded in hamsters. The highest plasma levels of CY alkylating metabolites in man were reached 2-3 h after the intravenous administration of CY, but the values corresponded to 1/22/~ of values found in rats after equivalent doses. The concentration of alkylating metabolites declined only very slowly ; 8 h after the administration of CY it was still about 77% of the maximum leveP 8. SIEBERT15,16 found that there appear metabolites of similar alkylating capacity in the urine of rats and man. The interval of 24 h was chosen between the administration of the drug and the final comparison of cytogenetic analysis and micronuclei test, since this period seems to be quite convenient because of the maximum frequency of chromosomal aberrations e and the maximum frequency of erythrocytes with micronucleP ° found in bone marrow cells after a single dose of CY and Trenimon. Metabolic experiments show that the difference in the frequency of chromosomal aberrations 24 h after treatment between rodents and man after the same dose of CY may be due to a different rate of metabolic activation. Although there is practically no difference in the metabolism and excretion rates between mice and rats given i.v.
EXPERIMENTAL RESULTS IN MAMMALSAND MAN. I
253
4 ° mg CY/kg b.w. we found a higher frequency of chromosome breaks in mice. A lower chromosome break frequency found in hamsters m a y result from a CY metabolite excretion rate which is higher than in mice and rats, although the metabolic activation in all three species is similar. CY activation in man is slower than in laboratory rodents. BROCK et al. 4 estimated the ratio of CY activation between the rat and man as 2 : I. On the other hand they found a slower excretion of metabolites in man as compared to rats. However, in our experiments we found the ratio of chromosome breaks between rat and man reaching the value of 8:1. Even though the differences in the chromosome break frequency seems more pronounced in our experiments, as would correspond to metabolic interspecies variations, it will be, however, necessary to study this question also from the aspect of the different duration of the mitotic cell cycle in the species in question. ABRAHAMSON et al. 1 and BREWEN et al. 3 expected a similar sensitivity between mice and man as to the mutations induced b y ionizing radiation. BREWEN et al. ~ suggested that human sensitivity is twofold as far as the induction of translocations is concerned but it is equal with respect to the induction of deletions. However, this conclusion was not confirmed in experiments with CY. Differential ability of CY to induce different frequencies of chromosomal abnormalities m a y indicate a probable difference between the spectrum of genetic changes induced by alkylating agents and by ionizing radiation. Five doses of CY were administered to each species of rodents to determine doseeffect relationship. After the highest dose it was observed that the frequency of aberrations in the studied categories was decreasing. It is assumed in the micronucleus test that the more serious mutagen-induced injury of bone marrow m a y increase the relative number of normochromatic erythrocytes, which m a y result in a decreased count of polychromatic erythrocytes with micronuclei 1°. Similarly, the change in mitotic activity m a y also result in a decreased number of chromosomally aberrant cells (3-4 times more slides have to be analysed to get a reasonable number). The results presented show that the most sensitive species is probably the mouse because of a distinctly increased number of aberrations after small doses and because of a steep dose-effect relationship curve. Similar conclusions concerning sensitivity to CY and 6-mercaptopurine were published by ROHRBORN ee al. 12 and FROHBERG AND SCHULZTE SCHNECKING 8. However, MATTER AND SCHMID 11 did not find any differences when comparing the sensitivity of six mammalian species to Trenimon. It m a y be expected that interspecies variations are not determined only by a karyologic characteristic of a certain species, but they m a y be also affected by a pharmacokinetic drug effect in the organism. A complete understanding of the relationship between the species-specific sensitivities to genetic damage is made difficult by the fact that we are still unable to analyse fully all induced changes, as it is still impossible to determine properly the frequency of gene mutations in mammals. So it cannot even be excluded that CY induces a different ratio of gene mutations and chromosomal aberrations in rodents and man, which means that the differences found in cytogenetic analysis need not necessarily be connected with the spectrum of induced genetic changes. The comparison of the results obtained by cytogenetic analysis of chromosomal
254
P. GOETZ et al.
aberrations and by the micronuclei test technique confirmed a relationship between the frequency of chromosome aberrations and the frequency of polychromatic erythrocytes containing micronucleP. Our results seem to indicate that the most sensitive and reliable te:~t for the evaluation of chromosome aberrations, preferably breaks, in bone marrow cells remains a clasical cytogenetic analysis. The gap counting in tile evaluation of chromosomal abnormalities may be considered problematical. Nevertheless, some authors show that gaps represent a type of abnormality very characteristic for chemical inutagens '~. The micronuclei test is held as a method useful preferably for screening chemica[ mutagens. Possible drawbacks may be due to observer's bias when analysing polychromatic erythrocytes with micronuclei as; well as the lack of evidence about the lnechanism of lnicronuclei origin, which would confirm their direct relationship to chromosomal aberrations. R E F E RI{NCES I ABRAHAMSON, S., 5I. A BENDER, A. 1). CONGER AND S. WOLFF, Uniformity of radiation induced m u t a t i o n rates a m o n g diffrent species, Nature (l.ondon), 245 (I973) 46o 402. 2 BOLLER, K., AND \~Q •CIIMID, Chemische Mutagenese beinl SS,uger. Das K u o c h e m a r k des Chinesischen H a m s t e r s also in vivo-Testsystem. Hiimatologische Befundc nach B e h a n d l u n g mit Trenilnon, Humangenetik, 11 (i97 o) 35 54. 3 BREWEN, J. G., R. J. PRESTON, K. P. JoNEs a.xD D. G. GOSSLI~E, Genetic hazards of ionizing radiations: cytogenetic e x t r a p o l a t i o n s from mouse to man, Mutation Res., 17 {~973) 245 -'54, 4 BROCK, N., R. (;ROSS, 1t. J. HOHORST, I-{. (). KLEIN AND B. SCHNEIDER, Actiwttion of cyelop h o s p h a l n i d e in m a n and animals, Cancer (Brussels), 27 (i97 I) 151"2 1529. .5 I~;RUSICK, I). J., AND V. \¥. MAYER, New d e v e l o p m e n t s in m u t a g e n i c i t y screening techniques with yeast, Environ. Hea!th Per@., 6 (1973} 83 96. 6 ])ATTA, P. l~., AND }~. SCHLEIERMACHER, The effects of c y t o x a n on the c h r o m o s o m e s of mous¢ bone m a r r o w , Mutatio~z Res., 8 (i969) 623 628. 7 lZAHRIG, R., Metabolic activation of m u t a g e n s in m a m m a l s . H o s t - m e d i a t e d assay utilizing the induction of mitotic gene conversion in Saccharomyces cerevisiae, Age~ts and Actions, 3 (t973) 99 t l o . N FROHBERG, H., AND M. SCHULZE SCHNECKING, Recent findings concerning dose response relationship in m u t a g e n i c i t y t'~!sting of chemicals, Arch. Toxicol., 32 (t974) i 17. 9 (;EBttART, l{., Experiln:mtalc t3eitr/~ge zum P r o b l e m der Iokalen Achromasien (Gaps), Hulnangenelik, 13 (I97I) 98-1o 7. to YON LEDEBUR, M,, ANt~ W. SCHMU~, The micronucleus test. Methodological aspects, Mutation Res., 19 (t973) ~o9 ii 7. I l MATTER, 13. AND W. So'minD, T r e n i m o n - i n d u c e d c h r o m o s o m a l d a m a g e in b o n e - m a r r o w cells of six m a m m a l i a n species, evaluated by the micronucleus test, Mutation lies., t 2 ( t 971 ) 417-425 . I2 I~fiHRBORN, O., A. HERWIG, P. PROPPING AND W. BUSELMEIER, Chemical constitution and lnutagenic activity of three c y c l o p h o s p h a m i d e s in three m a m m a l i a n test systenls, Mutation Res., 21 (5973) 46. 47I 3 SCltMII), \¥., Chemical n m t a g e n testing on i~7 rive somatic m a m m a l i a n cells, Agents arid Actions, 3 (I973) 77 85. 14 ~CHMID, \¥., 1). T. ARAKAKI, N. A. BRESLAU AND J. C. CULI3ERTSON, Chenlicai mutagencsis. The Chinese h a m s t e r bone m a r r o w as an in rive test systenl, I. Cytogenetic results on basic aspects of the methodology, o b t a i n e d with alkylating ageuts, Humangenetik, I I (197 t) 103 -118. 15 SIEm~RT, D., A new m e t h o d for testing genetically active metabolites. U r i n a r y assay with c y c l o p h o s p h a m i d e (Endoxan, Cytoxan) and Saccharomyces ceYevisiae, ,'llutation Res., 17 (1973) 3o7-314. 16 SIEBERT, I)., AND U. ,
247
R E L A T I O N S H I P B E T W E E N E X P E R I M E N T A L R E S U L T S IN MAMMALS AND MAN I. C Y T O G E N E T I C ANALYSIS OF BONE MARROW I N J U R Y I N D U C E D BY A S I N G L E DOSE OF C Y C L O P H O S P H A M I D E
P. GOETZ, R. J. ~R~M AND J. DOHNALOV2~
Research Institute of Child Development, Faculty of Pediatrics, Prague, Institute of Hygiene and Epidemiology, Prague and Department of Radiology. Teaching Hospital, Prague-Motol (Czechoslovakia) (Received August I4th, 1974) (Revision received February I8th, 1975)
SUMMARY
The extrapolation of experimental results to man was studied by cytogenetic bone marrow analysis and micronucleus test in mice, rats and Chinese hamsters. Furthermore, the frequency of chromosomal aberrations was compared with the frequencies of polychromatic erythrocytes containing micronuclei. Cyclophosphamide (CY) was given intraperitoneally at the doses of 5, io, 20, 4 ° and 80 mg/kg b.w. to ICR mice and Wistar rats and at the doses of IO, 20, 4 o, 80, 12o and 16o mg/kg b.w. to Chinese hamsters. Five patients with various types of malignancies until then medically untreated, were i.v. administered 40 mg CY/kg b.w. Bone marrow cells were examined 24 h after the administration. CY induced in all rodents a clear-cut dose-effect relationship in the frequency of breaks, abnormal metaphases as well as in the frequency of micronuclei in polychromatic erythrocytes. When comparing the results in rodents and man at the dose of 40 mg CY/kg b.w., the sensitivity pattern of species was mice > rats > Chinese h a m s t e r s > man. From this aspect the possible differences in the metabolism of CY in analysed species are discussed. The presented results tend to a conclusion that micronucleus testing m a y be a very suitable method used for screening purposes, however, the method of classical cytogenetic analysis, especially the evaluation of breaks, still remains the most exact and reliable technique.
INTRODUCTION
The extrapolation of experimental results from laboratory rodents to man represents the most important and difficult problem in evaluating the mutagenic activity of chemical compounds. This should be taken into account also in cytogenetic analysis of chromosome aberrations, since it m a y be assumed that there is a variability of
248
e. GOETZ el al.
aberrations induced in closely related mammalian species as well as in phylogenetically more remote species, e.g. rodents and man. A possible interspecies relationship in the sensitivity of mice, rats, Chinese hamsters and man was studied by comparing the frequency of chromosome aberrations induced in bone marrow cells after a single dose of CY. This alkylating agent commonly used in medical therapy was chosen for its repeatedly proved nmtagenic activity",7,v', 14. Results of micronucleus test and chromosolnal analysis were compared in all mammals used and the feasibilitv of the micronucleus test for the nmtagenicity testing of chemicals was evaluated. MATERIAL AND METHODS
CY (Germed, GDR) was given intraperitoneally in doses of 5, IO, 20, 4 ° and 8o mg/kg b.w. to ICR mice and Wistar rats and in doses of IO, 2o, 4 o, 8o, 12o and 16o mg/kg b.w. to Chinese hamsters. Five patients with various types of malignancies (excluding hematological ones), until then medically untreated, were i.v. administered 4 ° mg CY/kg b.w. Each experimental as well as control group consisted of 5 individuals. The micronucleus test technique of SCHMID and coworkers2,1°, ~a was used in the experiment. IOOOOerythroeytes were examined in each experimental and control group. Micronuclei were recorded in polychromatic as well as in normochromatic erythrocytes. Mutagenic activity of the drug was evaluated according to SCHMIOTM as the frequency of micronuclei in polychromatic erythrocytes. Slides for chromosomal analysis were prepared according to a modification of the classical TJIO AND W H A N G ' S methodlL 25 ° metaphases of bone marrow cells were examined 24 h after the administration; a similar scheme was used also in the control groups. The results from the cytogenetic analysis of human bone marrow were compared with those obtained by sternal puncture performed before the administration of CY. Gaps, breaks and exchanges wine evaluated. Cells bearing some of these chromosomal abnormalities were considered abnormal. Cells with more than IO aberrations were counted separately. RESULTS
The results of micronuclei test and cytogenetic analysis are indicated in Tables I--IV and Fig. i. A dose-effect relationship (in the micronucleus test) was found in mice, rats and Chinese hamsters. The increase in the frequency of polychromatic erythrocytes with micronuclei was marked in mice, especially with a higher dose. In all studied species, however, the highest dose induced a decreased frequency of micronuclei. A clear dose-effect relationship was also found when analysing chromosome aberrations. This relationship was evident in the frequency of abnormal metaphases as well as in the yield of chromosome breaks. A steeper slope was similarly observed in mice, especially with higher doses. The dose of 80 mg CY/kg b.w. in rats and 16o mg/kg b.w. in Chinese hamsters severely suppressed the bone marrow activity, so that no sufficient number of cells in metaphase could be found. Nevertheless, an increased frequency of chromosome aberrations is quite apparent even with lower doses.
TABLE
I
o.44 0.46 o.46 o.63 1.82 0.56
II
0.37 0.45 0.45 o.61 1.5o o.52
e W h e n ceils w i t h m o r e t h a n
IO a b e r r a t i o n s
are counted
a N, N u m b e r o f a n a l y s e d cells. bG[C, B[C, E[C, g a p s , b r e a k s , e x c h a n g e s p e r cell.
1.39 2.62 3.6o 5.o 4 6.28 5.61
ioooo IOOOO ioooo ioooo ioooo IOOOO
(%)
OF RAT BONE MARROW
Controls 5 io 2o 4° 80
(%)
25o 250 25o 25o 250 250
6.0 14.8 22. 4 26.0 20.8 24. 4
o.88 0.240 0.324 o.516 o.288 o.368
G/C b o. 4 6.8 16.o 12. 4 I 7 .2e 24.8d
Breaks % o.oo 4 o.116 o.316 o.400 o.456e o.652°
B]C b
.
2.4 9.6 9.6 12.o 19.6 .
0.024 o.132 o.124 o.196 0.324
G /C b
.
. . 3.2 7.6 2o.4e .
Breaks % . .
o.o56 O.lO4 o.512e .
B ]Cb . .
t h e v a l u e o f cells w i t h b r e a k s i n % is 2o.8 a n d
250 250 250 25 ° z5o .
Chromosomes Na Gaps %
a r e c o u n t e d t h e v a l u e o f c e l l s w i t h b r e a k s i n 0/o is e 2 1 . 6 a n d
Micronuclei Na Polychromatic Normochromatic erythrocytes erythrocytes
TREATMENT
lO a b e r r a t i o n s
Dose (mg]kg b.w.)
CYCLOPHOSPHAMIDE
TABLE
W h e n ceils w i t h m o r e t h a n
a N, N u m b e r of a n a l y s e d cells. b GIC, B]C, E/C, g a p s , b r e a k s , e x c h a n g e s p e r cell.
1.41 2.13 3.3 o 4.21 8.54 5.06
ioooo ioooo ioooo ioooo 1oooo IOOOO
(%)
Controls 5 io 20 4° 8o
(%)
Chromosomes Na Gaps %
OF M O U S E B O N E MARROW"
Micronuclei Na Polychromatic Normochromatic erythrocytes erythrocytes
TREATMENT
Dose (mg/kg b.w.)
CYCLOPHOSPHAMIDE
0.056 o.o4o
.
. .
B[C is o . 5 5 2 .
.
1.2 8.8
. .
o.o12 o.152
Exchanges % E /C b
.
Abnormal cells
2.4 3o.8
Abnormal cells
o. 4
2.4 9.6 12.o 17.2 33.6
tions(%) (%)
> zo aberra-
6. 4 20.o 32.o 33.2 39.6 66.0
tions (%) (%)
> zo aberra-
B]C 0 . 8 9 6 , a 5 8 . o a n d B/C 3-7.
4.4 3.6
Exchanges % E]C b
4~ ~D
z
0q
O~
~d X >
t~
III
TREATMENT
OF CHINESE
HAMSTER
ioooo
16o
4.22
0.55 1.26 2.28 2.73 4.07 4.29
0.26
°.14 o.16 0.28 o.29 0-35 0.34
(%)
MARROW
25 ° 250 250 250 250 250
2.8 15.6 20.0 25.2 28.8 20.0
o.o28 1.18o 0.276 0.328 o.360 0.268
Chromosomes Na Gaps - °'o G /Cb
BONE
3. 6 3.6 io.o i2.8 e i8.4d io.4 e
Breaks oo o.o36 0.036 o.172 o.232e o.372d o.i32e
B /Cb 1.6 1.2 2.0 1.2 0.020 0.020 o.o28 o.o16
Exchanges o,° E /CI~ 0.8 1.6 0.4
4-4 17"2 27 .6 32.6 42.8 27.6
> zo Abnormal aberracells tions(Oo) (°~0)
IV
ioooo ioooo
o.31 o.71
o)
o/
OF HUMAN
BONE
(%)
0.22 0.3o
250 250
1.2 lO.8
oo
Chromosomes Na Gaps
M A R R O x'V
Micronuclei Na Polychromatie Normochromatic erythrocvtes erythrocytes -
TREATMENT
a N, N u m b e r of a n a l y s e d cells. b G/C, B/C, E/C, g a p s , b r e a k s , e x c h a n g e s p e r cell.
Controls 4°
Dose (mg/kg b.w.)
CYCLOPHOSPHAMIDE
TABLE
e Io.8Oo a n d B / C o.172.
o.o12 o.o12
G/C b
0.4 6. 4
Breaks oo
0.4 0.072
B/C b - -
0.8
0.008
----
Exchanges Oo E/C b
-
16.8
1.6
> Io Abnormal aberracells tions('!o) (0o)
W h e n cells w i t h m o r e t h a n i o a b e r r a t i o n s a r e also c o u n t e d t h e v a l u e of cells w i t h b r e a k s m ° o is e 13.6 a n d B / C o . 3 1 2 , d 20.o0. o a n d B / C 0.532,
a N, N u m b e r of a n a l y s e d cells. b G/C, B/C, E/C, g a p s , b r e a k s , e x c h a n g e s p e r cell.
ioooo ioooo ioooo ioooo ioooo 10000
(%)
Micronuclei Na Polychromatic Normochromatie erythrocytes erythrocytes
Controls io 20 4° 80 12o
Dose (mg/kg b.w.)
CYCLOPHOSPHAMIDE
TABLE
C
b~
b~
©
E X P E R I M E N T A L R E S U L T S IN MAMMALS A N D MAN. I
251
The frequency of chromosomal aberrations after the dose of 16o mg CY/kg b.w. was lower in Chinese hamsters as compared with the dose of 80 mg CY/kg b.w., as was the case in the micronuclei test. After the highest doses there was increased variability of the chromosome aberration frequency in all animal species used in the experiment. The curves of CY effect show the most pronounced dose-effect relationship in the yield of chromosome breaks. Curves indicating the frequency of aberrant cells with gaps, breaks and exchanges as well as curves of polychromatic erythrocytes with micronuclei are of similar pattern, but not so steep. The sensitivity of man and experimental mammals to the dose of 40 mg CY/kg b.w., analysed by both methods is shown in Fig. 2. From all the species involved in the experiment, mice and rats were the most sensitive animals for cytogenetic analysis as well as for micronuclei testing. The sensitivity of Chinese hamsters was lower and the most significantly decreased sensitivity was found in man. DISCUSSION
Contrary to some authors who administered CY per os in their experiments, we used an intraperitoneal application since the drug is therapeutically administered to man intravenously. The same method of drug adnfinistration used in this experiment, and the same method by which the same tissue (bone marrow) was taken and processed, permit a comparison between the interspecies variations as well as the two methods used. CY is a drug whose mutagenic activity is dependent on the presence of an active metabolite. CY does not, e.g., induce any mitotic gene conversion if there is a direct °/o
10. Micronucleus test 9-
Mouse L
Ch hamster
Rat
~b ~E4
._v E
£
37
jg~62b~
jg 1 6 2 b ~
C
C
Dose r n g / k g b.w.
C
~Ol, O. O.9 0.8 0.7-
120
hromosomal abnormalities
Mouse
Ch hamster
Rat
/;"
°/o
]
6o~
(?.60.50.4
,~
/
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3o~
(~30.2
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0.17
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is ~o 2o4o
110 20 40 80 120
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C
Dose mg/kg b,w.
Fig. i . I n t e r s p e c i e s v a r i a t i o n s a f t e r c y c l o p h o s p h a m i d e t r e a t m e n t . M i c r o n u c l e u s t e s t : - - , e x p e r i m e n t a l g r o u p s ; . . . . , c o n t r o l s , C h r o m o s o m a l a b n o r m a l i t i e s : - - , aberrant m e t a p h a s e s ; . . . . , b r e a k s ; C, c o n t r o l s .
1~. GOETZ et al.
252 .1.0
10.
0.9 9"Z
.0.8 0.7 0.6
U E:
0.5~
Eo~4 . o IL
2
~Z -//,
0.4
0.3 m 0.2 0.1
Rat Mon Ch. hamster Mouse Fig. 2. I n t e r s p e c i e s v a r i a t i o n s in f r e q u e n c y of micronuclei a n d c h r o m o s o m a l b r e a k s w i t h cyclop h o s p h a m i d e 4 ° m g / k g b.w.[~., P o l y c h r o m a t i c e r y t h r o c y t e s w i t h m i c r o n u c l e i ; [3, b r e a k s per cell.
effect on Saccharomyces cerevisiae, but if a host-mediated assay is used, positive results are observed for CY, tooS, 7. It has been proved that the CY biotransformation in all species examined (mouse, rat, Chinese hamster, rabbit, sheep, dog, monkey, man) originates in the liver microsomes due to the effect of oxidase enzyme activity. However there is no precise knowledge on CY metabolic pathways and the biological role of CY metabohtes. It is generally accepted that the mutagenic activity of CY is determined by its active alkylating metabolites, especially by aldophosphamide 18. The results of studies on the dynamics of CY-metabolite concentrations in the blood and urine after CY administration in all species tested show species-dependent differences in the metabolic activation and excretion rates. Laboratory rodents used in our experiments showed a very fast CY activation. A presence in the blood of alkylating metabolites was observed in mice within i o - i 5 min following the administration of CY, and in rats and hamsters within I5-3o min. Mice excreted in their urine 6o% of the CY in 24 h, elimination in rats is similar. The excretion rate depends on the dose. The most rapid urine excretion of the drug was recorded in hamsters. The highest plasma levels of CY alkylating metabolites in man were reached 2-3 h after the intravenous administration of CY, but the values corresponded to 1/22/~ of values found in rats after equivalent doses. The concentration of alkylating metabolites declined only very slowly ; 8 h after the administration of CY it was still about 77% of the maximum leveP 8. SIEBERT15,16 found that there appear metabolites of similar alkylating capacity in the urine of rats and man. The interval of 24 h was chosen between the administration of the drug and the final comparison of cytogenetic analysis and micronuclei test, since this period seems to be quite convenient because of the maximum frequency of chromosomal aberrations e and the maximum frequency of erythrocytes with micronucleP ° found in bone marrow cells after a single dose of CY and Trenimon. Metabolic experiments show that the difference in the frequency of chromosomal aberrations 24 h after treatment between rodents and man after the same dose of CY may be due to a different rate of metabolic activation. Although there is practically no difference in the metabolism and excretion rates between mice and rats given i.v.
EXPERIMENTAL RESULTS IN MAMMALSAND MAN. I
253
4 ° mg CY/kg b.w. we found a higher frequency of chromosome breaks in mice. A lower chromosome break frequency found in hamsters m a y result from a CY metabolite excretion rate which is higher than in mice and rats, although the metabolic activation in all three species is similar. CY activation in man is slower than in laboratory rodents. BROCK et al. 4 estimated the ratio of CY activation between the rat and man as 2 : I. On the other hand they found a slower excretion of metabolites in man as compared to rats. However, in our experiments we found the ratio of chromosome breaks between rat and man reaching the value of 8:1. Even though the differences in the chromosome break frequency seems more pronounced in our experiments, as would correspond to metabolic interspecies variations, it will be, however, necessary to study this question also from the aspect of the different duration of the mitotic cell cycle in the species in question. ABRAHAMSON et al. 1 and BREWEN et al. 3 expected a similar sensitivity between mice and man as to the mutations induced b y ionizing radiation. BREWEN et al. ~ suggested that human sensitivity is twofold as far as the induction of translocations is concerned but it is equal with respect to the induction of deletions. However, this conclusion was not confirmed in experiments with CY. Differential ability of CY to induce different frequencies of chromosomal abnormalities m a y indicate a probable difference between the spectrum of genetic changes induced by alkylating agents and by ionizing radiation. Five doses of CY were administered to each species of rodents to determine doseeffect relationship. After the highest dose it was observed that the frequency of aberrations in the studied categories was decreasing. It is assumed in the micronucleus test that the more serious mutagen-induced injury of bone marrow m a y increase the relative number of normochromatic erythrocytes, which m a y result in a decreased count of polychromatic erythrocytes with micronuclei 1°. Similarly, the change in mitotic activity m a y also result in a decreased number of chromosomally aberrant cells (3-4 times more slides have to be analysed to get a reasonable number). The results presented show that the most sensitive species is probably the mouse because of a distinctly increased number of aberrations after small doses and because of a steep dose-effect relationship curve. Similar conclusions concerning sensitivity to CY and 6-mercaptopurine were published by ROHRBORN ee al. 12 and FROHBERG AND SCHULZTE SCHNECKING 8. However, MATTER AND SCHMID 11 did not find any differences when comparing the sensitivity of six mammalian species to Trenimon. It m a y be expected that interspecies variations are not determined only by a karyologic characteristic of a certain species, but they m a y be also affected by a pharmacokinetic drug effect in the organism. A complete understanding of the relationship between the species-specific sensitivities to genetic damage is made difficult by the fact that we are still unable to analyse fully all induced changes, as it is still impossible to determine properly the frequency of gene mutations in mammals. So it cannot even be excluded that CY induces a different ratio of gene mutations and chromosomal aberrations in rodents and man, which means that the differences found in cytogenetic analysis need not necessarily be connected with the spectrum of induced genetic changes. The comparison of the results obtained by cytogenetic analysis of chromosomal
254
P. GOETZ et al.
aberrations and by the micronuclei test technique confirmed a relationship between the frequency of chromosome aberrations and the frequency of polychromatic erythrocytes containing micronucleP. Our results seem to indicate that the most sensitive and reliable te:~t for the evaluation of chromosome aberrations, preferably breaks, in bone marrow cells remains a clasical cytogenetic analysis. The gap counting in tile evaluation of chromosomal abnormalities may be considered problematical. Nevertheless, some authors show that gaps represent a type of abnormality very characteristic for chemical inutagens '~. The micronuclei test is held as a method useful preferably for screening chemica[ mutagens. Possible drawbacks may be due to observer's bias when analysing polychromatic erythrocytes with micronuclei as; well as the lack of evidence about the lnechanism of lnicronuclei origin, which would confirm their direct relationship to chromosomal aberrations. R E F E RI{NCES I ABRAHAMSON, S., 5I. A BENDER, A. 1). CONGER AND S. WOLFF, Uniformity of radiation induced m u t a t i o n rates a m o n g diffrent species, Nature (l.ondon), 245 (I973) 46o 402. 2 BOLLER, K., AND \~Q •CIIMID, Chemische Mutagenese beinl SS,uger. Das K u o c h e m a r k des Chinesischen H a m s t e r s also in vivo-Testsystem. Hiimatologische Befundc nach B e h a n d l u n g mit Trenilnon, Humangenetik, 11 (i97 o) 35 54. 3 BREWEN, J. G., R. J. PRESTON, K. P. JoNEs a.xD D. G. GOSSLI~E, Genetic hazards of ionizing radiations: cytogenetic e x t r a p o l a t i o n s from mouse to man, Mutation Res., 17 {~973) 245 -'54, 4 BROCK, N., R. (;ROSS, 1t. J. HOHORST, I-{. (). KLEIN AND B. SCHNEIDER, Actiwttion of cyelop h o s p h a l n i d e in m a n and animals, Cancer (Brussels), 27 (i97 I) 151"2 1529. .5 I~;RUSICK, I). J., AND V. \¥. MAYER, New d e v e l o p m e n t s in m u t a g e n i c i t y screening techniques with yeast, Environ. Hea!th Per@., 6 (1973} 83 96. 6 ])ATTA, P. l~., AND }~. SCHLEIERMACHER, The effects of c y t o x a n on the c h r o m o s o m e s of mous¢ bone m a r r o w , Mutatio~z Res., 8 (i969) 623 628. 7 lZAHRIG, R., Metabolic activation of m u t a g e n s in m a m m a l s . H o s t - m e d i a t e d assay utilizing the induction of mitotic gene conversion in Saccharomyces cerevisiae, Age~ts and Actions, 3 (t973) 99 t l o . N FROHBERG, H., AND M. SCHULZE SCHNECKING, Recent findings concerning dose response relationship in m u t a g e n i c i t y t'~!sting of chemicals, Arch. Toxicol., 32 (t974) i 17. 9 (;EBttART, l{., Experiln:mtalc t3eitr/~ge zum P r o b l e m der Iokalen Achromasien (Gaps), Hulnangenelik, 13 (I97I) 98-1o 7. to YON LEDEBUR, M,, ANt~ W. SCHMU~, The micronucleus test. Methodological aspects, Mutation Res., 19 (t973) ~o9 ii 7. I l MATTER, 13. AND W. So'minD, T r e n i m o n - i n d u c e d c h r o m o s o m a l d a m a g e in b o n e - m a r r o w cells of six m a m m a l i a n species, evaluated by the micronucleus test, Mutation lies., t 2 ( t 971 ) 417-425 . I2 I~fiHRBORN, O., A. HERWIG, P. PROPPING AND W. BUSELMEIER, Chemical constitution and lnutagenic activity of three c y c l o p h o s p h a m i d e s in three m a m m a l i a n test systenls, Mutation Res., 21 (5973) 46. 47I 3 SCltMII), \¥., Chemical n m t a g e n testing on i~7 rive somatic m a m m a l i a n cells, Agents arid Actions, 3 (I973) 77 85. 14 ~CHMID, \¥., 1). T. ARAKAKI, N. A. BRESLAU AND J. C. CULI3ERTSON, Chenlicai mutagencsis. The Chinese h a m s t e r bone m a r r o w as an in rive test systenl, I. Cytogenetic results on basic aspects of the methodology, o b t a i n e d with alkylating ageuts, Humangenetik, I I (197 t) 103 -118. 15 SIEm~RT, D., A new m e t h o d for testing genetically active metabolites. U r i n a r y assay with c y c l o p h o s p h a m i d e (Endoxan, Cytoxan) and Saccharomyces ceYevisiae, ,'llutation Res., 17 (1973) 3o7-314. 16 SIEBERT, I)., AND U. ,