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Mutation Research, 40 (1976) 339--348 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
THE MUTAGENICITY OF BENZIMIDAZOLE AND BENZIMIDAZOLE DERIVATIVES. VI. CYTOGENETIC EFFECTS OF BENZIMIDAZOLE DERIVATIVES IN THE BONE MARROW OF THE MOUSE AND THE CHINESE HAMSTER
J.P. SEILER Swiss Federal Research Station, CH-8820 Waedenswil (Switzerland) (Received March 22nd, 1976) (Accepted June 8th, 1976)
Summary Methyl benzimidazole-2-ylcarbamate (MBC) was mutagenic in mice by the micro-nucleus test. Other benzimidazole derivatives, with the exception of the parent c o m p o u n d of MBC, benomyl, and the very closely related substance 2benzimidazolylurea, did n o t produce micro-nuclei in mouse bone marrow. Evidence is presented that MBC acts through inhibition of mitosis and that for this action the carbamoyl group is a necessary b u t not a sufficient condition. It is also demonstrated that for this particular type of mutagenic activity a threshold limit exists, which seems to be in the order of less than 10 pg MBC per ml blood.
Introduction
The mutagenicity of benzimidazole derivatives has been amply demonstrated in several test systems [2,6,7,13,17,19,20]. Tests in mammals, however, have mostly yielded negative results [9,11,14,18]. Thus, dominant-lethal studies in mice and cytogenetic tests in rats with the fungicide thiophanate-methyl, a c o m p o u n d which undergoes in the mammalian organism a rapid transformation to the benzimidazole derivative MBC [ 12], did n o t show any mutagenic activity [11], and neither did dominant-lethal tests in rats with benomyl [18]. However, in view of the importance of benzimidazole c o m p o u n d s as fungicides and of their fungicidal mode of action [4,5], further investigations into the mutagenic potential of benzimidazoles for mammals seemed indicated. As a sensitive and rapid m e t h o d the micro-nucleus test (m.t.) in mouse bone marrow served for the investigation of chromosomal damage induced b y chemical agents,
340
whereas chromosome analysis in a metaphase preparation of Chinese hamster bone marrow cells was used only for a characterization of mutagenic effects. Materials and methods Benzimidazole and benzimidazole derivatives were commercially available from Aldrich Europe (Beerse, Belgium) with the exception of MBC, which was prepared synthetically after the m e t h o d of Loux [10]. [2-~4C]MBC was from The Radiochemical Centre, Amersham (UK). For the m.t. ICR mice about 8 weeks old and weighing 25--30 g were used. Application of test compounds was either by intraperitoneal (i.p.) injection or peroral (p.o.) by garage. For i.p. injection the compounds were dissolved or suspended (by 10 min sonication) in DMSO and made up t o volume with physiological saline. The final DMSO concentration was 5% and the concentration of the c o m p o u n d was calculated to contain the necessary a m o u n t for a 30-g mouse in 0.5 ml. For the p.o. application the substances were dissolved or suspended at the same concentration as above in 2% gum arabic solution. Usually the test compounds were given twice 24 h apart and the animals were killed 30 h after the first treatment. The same regimen was used for chromosome analysis of Chinese hamster bonemarrow cells; here the animals received in addition an i.p. injection of colcemid at 10 mg/kg (Calbiochem, Lucerne, Switzerland) 2 h before they were killed. Bone-marrow smears for the m.t. and metaphase preparations were then made according to Schmid [15,16]. The time-dependent action of MBC was investigated with a single application of this compound. Also the serum level of MBC was measured after a single application. For this purpose [2-~4C]MBC was added to the unlabelled compound. At various time intervals mice were killed and their blood obtained by heart puncture. After centrifugation of the citrate blood, radioactivity was determined in an aliquot of the supernatant by liquid scintillation counting in a toluene--triton X-100--BBOT (Ciba-Geigy Ltd., Basle) cocktail. Results and discussion The first experiments were made with MBC, because this substance occupies a key position in the degradation pathways of several widely used fungicides, besides being used itself as a fungicide, whereas man's exposure to other benzimidazole derivatives is of minor importance. The first results obtained with MBC in the m.t. were negative and thus the genetic consequences of an exposure to such c o m p o u n d s seemed nil. However, on dissection of the intraperitoneally treated mice a deposit of a solid substance, mass spectrometrically identified as MBC, was evident, and this immediately posed the question of the availability of this c o m p o u n d to the organism when given i.p. These preliminary experiments were therefore repeated using p.o. application. It was hoped that the better solubility in an acid environment would enhance the uptake of the c o m p o u n d , and that thus an effect, if present, would be more easily recognizable. Indeed, the evaluation of these experiments revealed an increased number of micro-nucleated erythrocytes. These preliminary experiments were subsequently expanded in order firstly to obtain a dose-response relationship and
341
secondly to compare MBC with other benzimidazole derivatives. As can be seen from Table I, the number of micro-nucleated erythrocytes showed a dose-dependent increase, when mice were treated orally with MBC, whereas neither benzimidazole nor other benzimidazole c o m p o u n d s were active at the concentrations tested. This seems to indicate that the micro-nuclei-forming process is highly dependent on chemical structure, since very closely related derivatives such as benzimidazolylurea or benzimidazolecarbamonitril had less or even no activity in the m.t. Some peculiar features in the preparations of bone marrow were evident, however, and led to further investigations. Among these were the unusually high frequency of micro-nucleated normochromatic erythrocytes, the sometimes rather large micro-nuclei (see Fig. la,b) and the large number of mitotic figures in the nucleated cells. As the fungicidal m o d e of action of MBC involves disturbancies in the mitotic process [4,5] the hypothesis of a similar action in bone-marrow cells leading to micro-nuclei had to be tested. The first evidence for such an action has already been referred to above. Normochromatic erythrocytes are older than about 24 h, and only such
TABLE I MICRO NUCLEATED ERYTHROCYTES (PER 1000 POLYCHROMATIC ERYTHROCYTES) FROM MOUSE BONE MARROW AFTER TREATMENT WITH SEVERAL BENZIMIDAZOLE COMPOUNDS Compound
Benzimidazole
Application
Formula
J~'~Jl~l ~ ' ~
Amount mg/kg
Micronucleated polychromatic erythrocytes
Micronucleated normochromatic erythrocytes
i.P.
100 300
4.3 3.1
2.8 2.6
i.p. p.o.
500 50 100 500 1000
4.5 4.2 13.4 19.7 26.8
1.8 2.9 6.5 12.3 18.2
p.o.
500 1000
7.2 16.3
3.7 5.0
p.o.
500
3.5
2.5
~"~,,~-N, ~Nk~ N H2
p.o.
100 500 1000
3.2 4.1 2.8
3.0 1.9 2.5
~ x ~
p.o.
4.2 12.6
3.0 5.6
3.6
2.5
N
H MBC
~ N , NH COOCH 3 N
H 2-Benzimidazoly]urea
~ N . I ~ NX) NH C O N H 2 H
Benzimidazolecarbamonitril
2-Aminobenzimidazole Benomyl
i~Nk~ NH C N ~.~'N-
H
NHCOOCH3
500 1000
N M ~'4P!9
--
I N o n e (control)
CO
342
Fig. 1. M i c r o - n u c l e i f r o m m o u s e b o n e m a r r o w . (a) N o r m a l size m i c r o - n u c l e u s : (b) g i a n t m i c r o - n u c l e u s o b tained by MBC treatment.
agents capable of interfering with the mitotic process will be able to enhance the frequency of micro-nucleated cells of this stage. Also, only mitotic poisons will be capable of generating very large micro-nuclei, because only through such agents will whole chromosomes be lost during mitosis in appreciable numbers and subsequently be incorporated into large micro-nuclei. Three kinds of experiment were used for the evaluation of this mode of action. The first of these was the investigation of potential chromosome breakage in Chinese hamster bone-marrow cells induced by orally administered MBC. Although positive control experiments with trenimon were successful, MBC did not induce chromosome breakage even at the highest doses, where micro-nuclei induction was very prominent. One single chromatid break was observed in a total of 500 metaphases from 4 animals given MBC (1000 mg/kg) twice orally in the same time intervals as for the micro-nuclei experiments. On the other hand, treatment with trenimon yielded chromatid breaks and exchanges in amounts comparable to the values given by Schmid [15]. One must conclude, then, that MBC is n o t a chromosome-breaking agent; its micro-nuclei-producing mode of action must involve some kind of influence on the mitotic process. Positive evidence for this kind of mutagenic action was then to be given by the t w o other experimental designs. A re-examination of the mouse bone-marrow smears, this time not for micronuclei, but for mitotic figures, yielded the results summarized in Table II. Although such phenomena as bridge formation and lagging pieces of chromatin material (see Fig. 2a,b) in anaphases are n o t quite convincing evidence of spindle inhibition, their appearance in the preparations examined strengthens the belief that MBC acts as a spindle poison. Much more indicative for such an action though, were cells, as shown in Fig. 2c,d, where unequal chromatin distribution in the anaphase or tripolar anaphases can be seen. Such abnormalities can only be explained by an inhibitory action of MBC on the spindle apparatus. The experiments designed to evaluate the time course of the action of MBC pointed in the same direction. By preparing bone-marrow smears every 2 h af-
343 T A B L E II O C C U R R E N C E O F A B E R R A N T M I T O T I C F I G U R E S IN M O U S E B O N E - M A R R O W C E L L S , A F T E R TWO ORAL TREATMENTS WITH MBC AT 1000 mg~g, PER 1000 NUCLEATED CELLS
Mitotic figures Metaphase with external chromatin material a Anaphase with lagging chromatin material a Anaphase with bridge Anaphase, tripolar Anaphase, unequal ehromatin distribution
MBC
Control
70 7.0 3.6 0.8 2.4 5.0
20 0 0 0 0 0
a In t h i s k i n d o f p r e p a r a t i o n n o d i s t i n c t i o n is p o s s i b l e bet~veen a c e n t r i c f r a g m e n t s a n d w h o l e c h r o m o s o m e s . T h e m o r e n e u t r a l t e r m " c h r o m a t i n m a t e r i a l " is t h e r e f o r e u s e d t o d e s c r i b e t h e e f f e c t .
Fig. 2. A b n o r m a l i t i e s in m i t o t i c figures f r o m b o n e m a r r o w o f m i c e t r e a t e d w i t h M B C . (a) A n a p h a s e w i t h l a g g i n g f r a g m e n t ; (b) a n a p h a s e w i t h b r i d g e f o r m a t i o n ; (c) t r i p o l a r a n a p h a s e ; (d) u n e q u a l d i s t r i b u t i o n o f c h r o m a t i n in t h e d a u g h t e r cells.
344
ter the application of MBC and counting micronuclei the values in Table III were obtained. In contrast with a chromosome-breaking agent, e.g. Trenimon, the onset of the action of MBC is earlier, and this, too, demonstrates that the action of MBC is on mitosis. Alkylating agents must act long before mitosis, i.e. in or before the S-phase of the cell cycle, to let chromatid breaks become apparent [3]. Thus, aberrations based on the action of alkylating agents will be expected to be recognizable later than those caused by spindle poisons. We can therefore safely assume that MBC produces micro-nuclei by inhibiting the mitotic process, interfering somehow with spindle formation or spindle function. Two issues now needed some further clarification. The first concerned structure-effect relationships, whereas the second was to explain the apparent difference between the two modes of application. From the data compiled in Table I it is evident that the methylcarbamate group plays the significant role in the spindle inhibitory action. We were considering the question whether the presence of this group would be a sufficient condition for the activity of a substance or whether the benzimidazole moiety would also be a necessary condition. We therefore investigated the micro-nuclei-producing potential of a herbicide, propham (N-phenyl-methylcarbamate), which is a known spindle poison [ 1 ]. Our experiments with this substance, however, failed to show, at any concentration, elevated numbers of micro-nucleated erythrocytes. Although these results indicate, that, at least in mammals, the inactive part of the molecule, the benzimidazole, is also needed to produce the spindle poison activity, further studies with other carbamates will be needed fully to elucidate these effects of structure. The last question remaining to be settled was the differential effect of intraperitoneal and peroral application. Whereas by intraperitoneal injection no mutagenic effect was observed, the incidence of micro-nucleated erythrocytes could be considerably enhanced by peroral dosage. An explanation for this difference in genetic toxicity could be that MBC is practically insoluble in aqueous media around pH 7, but is readily dissolved in acids. Therefore MBC might have been dissolved in the stomach of the mouse and then taken up at the be-
TABLE
IIi
TIME COURSE OF APPEARANCE OF MICRO-NUCLEI IN MOUSE BONE-MARROW ERYTHROCYTES (PER 1000 POLYCHROMATIC ERYTHROCYTES) AFTER A SINGLE PERORAL DOSE OF MBC AT 500 mg/kg, COMPARED WITH THE VALUES OBTAINED AFTER A SINGLE DOSE OF TRENIMON AT 0.250 mg/kg (VALUES FROM VON LEDEBUR AND SCHMID [8]).
Time after treatment (b)
0 2 4 6 S 16 24
Micronucleated polychromatie erythrocytes obtained by treatment with MBC
Trenimon
3 5 6 9 15 26 27
3 --3 6 26 72
345 ginning of its passage through the intestine. In order to check this possibility we conducted a small pharmacokinetic experiment. Mice were dosed either intraperitoneally or orally with MBC (containing [2~4C]MBC) at 100 or 500 mg/kg. The radioactivity measurements at intervals of 2 h revealed the picture indicated in Table IV. It was demonstrated that by intraperitoneal injection the solubility of the c o m p o u n d limits the uptake, and thus the serum level of MBC was the same at doses of 100 and 500 mg/kg. Dosing the animals orally, however, resulted in a marked increase in radioactivity of the serum during the first few hours. The uptake of orally administered MBC is here dependent on the dose applied and n o t governed solely by its solubility. Furthermore, a continuous concentration of 8 pg MBC/ml serum is n o t a sufficient a m o u n t to provoke spindle disturbances and formation of micro-nuclei, whereas a short-time rise to 12 pg MBC/ml does lead to a significant increase i n the number of micro-nucleated erythrocytes. The body concentration of 8 #g/ ml might therefore be considered to constitute a threshold limit for this substance. Such a threshold limit could be expected practically from the micro-nuclei counts and theoretically from the mode of action of MBC. As a spindle poison, MBC acts through protein--ligand interactions, where a minimal i n h i b i t o r y concentration must exist. In conclusion, we can summarize our findings as follows. MBC produces micro-nuclei in mouse bone-marrow cells. Its activity is based on the methylcarbamate group, as even slight modifications of this side chain render the molecule inactive. The benzimidazole moiety, however, seems also to be necessary for the activity of this substance in mammals. The very slight solubility o f MBC in water leads to a constant serum concentration of 8 pg/ml upon intraperitoneal application in the mouse; in this case no excess micro-nuclei are produced.
T A B L E IV [2 - 1 4 C ] M B C IN B L O O D S E R U M O F M I C E T R E A T E D O R A L L Y O R I N T R A P E R I T O N E A L L Y ( R A D I O A C T I V I T Y C O U N T S P E R ML S E R U M ) T h e s p e c i f i c a c t i v i t y o f t h e M B C s u s p e n s i o n was 1 . 1 5 ~ C i / m g . c p m • 10 -3
MBC c o n c . ~g/ml serum
4 6
29 20
11.5 8
50O
2 4 6 8 16 24
42 61 53 34 24 27
17 24.5 21 13.5 9.5 11
i.p.
100
4 6
20 19
8 7.5
i.p.
500
2 4 6 8 16 24
20 19 20 21 22 21
8 7.5 8 8.5 9 8.5
Treatment
Amount (mg/kg)
p.o.
100
Time after t r e a t m e n t (h)
346 Upon peroral dosage, however, the serum concentration rises considerably and micro-nuclei are formed owing to the inhibition of spindle formation or spindle function. As this is the only recognizable mutagenic effect of MBC in mammals, a threshold limit for this mode of action of the substance can safely be assumed. Assuming furthermore that (a) absorption, metabolism and excretion of benzimidazole compounds proceed equally in man and mouse, (b) man is no more sensitive genetically than the mouse, and (c) meiotic cells are no more sensitive to the action of MBC than mitotic ones, then we can apply this threshold limit to man also. From such a point of view MBC does not seem to be genetically dangerous for the average man, who is exposed to very minute amounts only. Possible exceptions are factory workers or agricultural spray men, whose blood level should once be monitored exactly in order to obtain information about their chemical load. Exposure to such compounds above the threshold limits, thus leading to the effects described in this paper, might produce a larger number of cells with lethal defects, but also a few cells with mono- or trisomy due to only slight spindle disturbances. Therefore an exact blood content analysis of high risk people should be undertaken as soon as possible. Acknowledgements These investigations were supported by grant No. 3.358-0.74 from the Swiss National Foundation for Scientific Research. I am indebted to Prof. W. Schmid for many helpful discussions regarding the micronucleus test. References 1 A s h t o n , F . M . a n d A . S . C r a f t s , M o d e o f A c t i o n o f H e r b i c i d e s , J o h n Wiley & S o n s , N e w Y o r k , 1 9 7 3 , p. 205. 2 D a s s e n o y , B. a n d J . A . M e y e r , M u t a g c n i c e f f e c t o f b e n o m y l o n Fusarium o x y s p o r u m , M u t a t i o n R e s . , 21 ( 1 9 7 3 ) 1 1 9 - - 1 2 0 . 3 D a t t a , P . K . , H. F r i g g e r a n d E. S c h l e i e r m a c h e r , T h e e f f e c t o f c h e m i c a l m u t a g e n s o n t h e m i t o t i c c h r o m o s o m e s o f the m o u s e , in vivo, in F. V o g e l a n d G. R ~ h r b o r n (eds.) C h e m i c a l M u t a g e n c s i s in M a m m a l s a n d M a n , S p r i n g e r , Berlin, 1 9 7 0 , p. 2 1 1 . 4 Davidse0 L.C., A n t i m i t o t i c a c t i v i t y o f m e t h y l b e n z i m i d a z o l - 2 - y l c a r b a m a t e ( M B C ) in Aspergillus nidulans, Pest. B i o c h e r n . P h y s i o l . , 3 ( 1 9 7 3 ) 3 1 7 - - 3 2 5 . 5 D a v i d s e , L.C., A n t i m i t o t i c a c t i v i t y o f m e t h y l b e n z i r n i d a z o l - 2 - y l c a r b a r n a t e in f u n g i a n d its b i n d i n g t o c e l l u l a r p r o t e i n , in M. B o r g e r s a n d M. de B r a b a n d e r (eds.) M i c r o t u b u l e s a n d M i c r o t u b u l e I n h i b i t o r s , N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 5 , p. 4 8 3 - - 4 9 5 . 6 Hastic, A.C., Benlate-induced instability of Aspergillus diploids, Nature, 226 (1970) 771. 7 K a p p a s , A., S.G. G e o r g o p o u l o s a n d A . C . H a s t i e , O n t h e g e n e t i c a c t i v i t y o f b e n ~ i m i d a z o l c a n d t h i o p h a n a t e f u n g i c i d e s o n dip]Did Aspergillus nidulans, M u t a t i o n R e s . , 26 ( 1 9 7 4 ) 1 7 - - 2 7 . 8 V o n L e d e b u r , M. a n d W. S c h r n i d , T h e m i c r o n u c l e u s t e s t - - m e t h o d o l o g i c a l a s p e c t s , M u t a t i o n R e s . , 19 (1973) 109--117. 9 L ~ o n a r d , A., R . V a n d e s t e c n e a n d R. M a r s b o o r n , M u t a g e n i c i t y t e s t s w i t h r n e b e n d a z o l e in t h e m o u s e , Mutation Res., 26 (1974) 427--430. 1 0 L o u x , H . M . , P r o c e s s for m a n u f a c t u r e o f c e r t a i n a l k y l e s t e r s o f b c n z i r n i d a z o l e c a r b a m i c a c i d , U.S. Pat e n t N o . 3 , 0 1 0 0 9 6 8 ( N o v . 28, 1 9 6 1 ) . 11 M a k i t a , T., Y. H a s h i m o t o a n d T. N o g u c h i , M u t a g e n i e , c y t o g e n i e a n d t e r a t o g e n i e s t u d i e s o n t h i o phanate-methyl, Toxicol. Appl. Pharmacol., 24 (1973) 206--215. 12 N o g u c h i , T., K. O h k u r n a a n d S. K o s a k a , C h e m i s t r y a n d m e t a b o l i s m o f t h i o p h a n a t e f u n g i c i d e s , in A.S. T a h o r i (ed.) P e s t i c i d e T e r m i n a l R e s i d u e s , B u t t e r w o r t h s , L o n d o n , 1 9 7 1 , p. 2 5 7 - - 2 7 0 . 13 N o v i c k , A,, M u t a g e n s a n d a n t i r n u t a g e n s , B r o o k h a v e n S y r n p . Biol., 8 ( 1 9 5 6 ) 2 0 1 - - 2 1 5 . 1 4 R u z i c s k a , P., S. P e t e r a n d C. Czeizel, S t u d i e s o n t h e c h r o m o s o m a l m u t a g e n i e e f f e c t o f b e n o r n y l in rats and humans, Mutation Res., 29 (1975) 201.
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1 5 S c h m i d , W., D . T . A r a k a k i , N.Y. Breslau a n d J.C. C u l b e r t s o n , C h e m i c a l m u t a g e n e s i s - - t h e C h i n e s e h a m s t e r b o n e m a r r o w as a n in vivo t e s t s y s t e m , I. C y t o g e n e t i c r e s u l t s o n b a s i c a s p e c t s o f t h e m e t h o d o l o g y , o b t a i n e d w i t h a l k y l a t i n g a g e n t s , H u m a n g e n e t i k , 11 ( 1 9 7 1 ) 1 0 3 - - 1 1 8 . 16 S c h m i d , W., C h e m i c a l m u t a g e n t e s t i n g o n i n vivo s o m a t i c m a m m a l i a n ceils, A g e n t s a n d A c t i o n s , 3 (1973) 77--85. 17 Seller, J . P . , T h e m u t a g e n i c i t y o f b e n z i m i d a z o l e a n d b e n z i m i d a z o l e d e r i v a t i v e s . I. F o r w a r d a n d reverse m u t a t i o n s in S a l m o n e l l a t y p h i m u r i u m c a u s e d b y b e n z i m i d a z o l e a n d s o m e o f its d e r i v a t i v e s , M u t a t i o n Res., 1 5 ( 1 9 7 2 ) 2 7 3 - - 2 7 6 . 1 8 S h e r m a n , H., R . C u l i k a n d R . A . J a c k s o n , R e p r o d u c t i o n , t e r a t o g e n i c , a n d m u t a g e n i c s t u d i e s w i t h benomyl, Toxicol. Appl. Pharmacol., 32 (1975) 305--315. 19 S t y l e s , J . A . a n d A. G a r n e r , B e n z i m i d a z o l e c a r b a m a t e m e t h y l e s t e r - - e v a l u a t i o n o f its e f f e c t s in vivo a n d in v i t r o , M u t a t i o n R e s . , 26 ( 1 9 7 4 ) 1 7 7 - - 1 8 7 . 2 0 S z y b a l s k i , W., S p e c i a l m i c r o b i o l o g i c a l s y s t e m s . IL O b s e r v a t i o n s o n c h e m i c a l m u t a g e n e s i s in m i c r o o r g a n i s m s , A n n . N . Y . A c a d . Sci., 76 ( 1 9 5 8 ) 4 7 5 - - 4 8 9 .
Mutation Research, 40 (1976) 339--348 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
THE MUTAGENICITY OF BENZIMIDAZOLE AND BENZIMIDAZOLE DERIVATIVES. VI. CYTOGENETIC EFFECTS OF BENZIMIDAZOLE DERIVATIVES IN THE BONE MARROW OF THE MOUSE AND THE CHINESE HAMSTER
J.P. SEILER Swiss Federal Research Station, CH-8820 Waedenswil (Switzerland) (Received March 22nd, 1976) (Accepted June 8th, 1976)
Summary Methyl benzimidazole-2-ylcarbamate (MBC) was mutagenic in mice by the micro-nucleus test. Other benzimidazole derivatives, with the exception of the parent c o m p o u n d of MBC, benomyl, and the very closely related substance 2benzimidazolylurea, did n o t produce micro-nuclei in mouse bone marrow. Evidence is presented that MBC acts through inhibition of mitosis and that for this action the carbamoyl group is a necessary b u t not a sufficient condition. It is also demonstrated that for this particular type of mutagenic activity a threshold limit exists, which seems to be in the order of less than 10 pg MBC per ml blood.
Introduction
The mutagenicity of benzimidazole derivatives has been amply demonstrated in several test systems [2,6,7,13,17,19,20]. Tests in mammals, however, have mostly yielded negative results [9,11,14,18]. Thus, dominant-lethal studies in mice and cytogenetic tests in rats with the fungicide thiophanate-methyl, a c o m p o u n d which undergoes in the mammalian organism a rapid transformation to the benzimidazole derivative MBC [ 12], did n o t show any mutagenic activity [11], and neither did dominant-lethal tests in rats with benomyl [18]. However, in view of the importance of benzimidazole c o m p o u n d s as fungicides and of their fungicidal mode of action [4,5], further investigations into the mutagenic potential of benzimidazoles for mammals seemed indicated. As a sensitive and rapid m e t h o d the micro-nucleus test (m.t.) in mouse bone marrow served for the investigation of chromosomal damage induced b y chemical agents,
340
whereas chromosome analysis in a metaphase preparation of Chinese hamster bone marrow cells was used only for a characterization of mutagenic effects. Materials and methods Benzimidazole and benzimidazole derivatives were commercially available from Aldrich Europe (Beerse, Belgium) with the exception of MBC, which was prepared synthetically after the m e t h o d of Loux [10]. [2-~4C]MBC was from The Radiochemical Centre, Amersham (UK). For the m.t. ICR mice about 8 weeks old and weighing 25--30 g were used. Application of test compounds was either by intraperitoneal (i.p.) injection or peroral (p.o.) by garage. For i.p. injection the compounds were dissolved or suspended (by 10 min sonication) in DMSO and made up t o volume with physiological saline. The final DMSO concentration was 5% and the concentration of the c o m p o u n d was calculated to contain the necessary a m o u n t for a 30-g mouse in 0.5 ml. For the p.o. application the substances were dissolved or suspended at the same concentration as above in 2% gum arabic solution. Usually the test compounds were given twice 24 h apart and the animals were killed 30 h after the first treatment. The same regimen was used for chromosome analysis of Chinese hamster bonemarrow cells; here the animals received in addition an i.p. injection of colcemid at 10 mg/kg (Calbiochem, Lucerne, Switzerland) 2 h before they were killed. Bone-marrow smears for the m.t. and metaphase preparations were then made according to Schmid [15,16]. The time-dependent action of MBC was investigated with a single application of this compound. Also the serum level of MBC was measured after a single application. For this purpose [2-~4C]MBC was added to the unlabelled compound. At various time intervals mice were killed and their blood obtained by heart puncture. After centrifugation of the citrate blood, radioactivity was determined in an aliquot of the supernatant by liquid scintillation counting in a toluene--triton X-100--BBOT (Ciba-Geigy Ltd., Basle) cocktail. Results and discussion The first experiments were made with MBC, because this substance occupies a key position in the degradation pathways of several widely used fungicides, besides being used itself as a fungicide, whereas man's exposure to other benzimidazole derivatives is of minor importance. The first results obtained with MBC in the m.t. were negative and thus the genetic consequences of an exposure to such c o m p o u n d s seemed nil. However, on dissection of the intraperitoneally treated mice a deposit of a solid substance, mass spectrometrically identified as MBC, was evident, and this immediately posed the question of the availability of this c o m p o u n d to the organism when given i.p. These preliminary experiments were therefore repeated using p.o. application. It was hoped that the better solubility in an acid environment would enhance the uptake of the c o m p o u n d , and that thus an effect, if present, would be more easily recognizable. Indeed, the evaluation of these experiments revealed an increased number of micro-nucleated erythrocytes. These preliminary experiments were subsequently expanded in order firstly to obtain a dose-response relationship and
341
secondly to compare MBC with other benzimidazole derivatives. As can be seen from Table I, the number of micro-nucleated erythrocytes showed a dose-dependent increase, when mice were treated orally with MBC, whereas neither benzimidazole nor other benzimidazole c o m p o u n d s were active at the concentrations tested. This seems to indicate that the micro-nuclei-forming process is highly dependent on chemical structure, since very closely related derivatives such as benzimidazolylurea or benzimidazolecarbamonitril had less or even no activity in the m.t. Some peculiar features in the preparations of bone marrow were evident, however, and led to further investigations. Among these were the unusually high frequency of micro-nucleated normochromatic erythrocytes, the sometimes rather large micro-nuclei (see Fig. la,b) and the large number of mitotic figures in the nucleated cells. As the fungicidal m o d e of action of MBC involves disturbancies in the mitotic process [4,5] the hypothesis of a similar action in bone-marrow cells leading to micro-nuclei had to be tested. The first evidence for such an action has already been referred to above. Normochromatic erythrocytes are older than about 24 h, and only such
TABLE I MICRO NUCLEATED ERYTHROCYTES (PER 1000 POLYCHROMATIC ERYTHROCYTES) FROM MOUSE BONE MARROW AFTER TREATMENT WITH SEVERAL BENZIMIDAZOLE COMPOUNDS Compound
Benzimidazole
Application
Formula
J~'~Jl~l ~ ' ~
Amount mg/kg
Micronucleated polychromatic erythrocytes
Micronucleated normochromatic erythrocytes
i.P.
100 300
4.3 3.1
2.8 2.6
i.p. p.o.
500 50 100 500 1000
4.5 4.2 13.4 19.7 26.8
1.8 2.9 6.5 12.3 18.2
p.o.
500 1000
7.2 16.3
3.7 5.0
p.o.
500
3.5
2.5
~"~,,~-N, ~Nk~ N H2
p.o.
100 500 1000
3.2 4.1 2.8
3.0 1.9 2.5
~ x ~
p.o.
4.2 12.6
3.0 5.6
3.6
2.5
N
H MBC
~ N , NH COOCH 3 N
H 2-Benzimidazoly]urea
~ N . I ~ NX) NH C O N H 2 H
Benzimidazolecarbamonitril
2-Aminobenzimidazole Benomyl
i~Nk~ NH C N ~.~'N-
H
NHCOOCH3
500 1000
N M ~'4P!9
--
I N o n e (control)
CO
342
Fig. 1. M i c r o - n u c l e i f r o m m o u s e b o n e m a r r o w . (a) N o r m a l size m i c r o - n u c l e u s : (b) g i a n t m i c r o - n u c l e u s o b tained by MBC treatment.
agents capable of interfering with the mitotic process will be able to enhance the frequency of micro-nucleated cells of this stage. Also, only mitotic poisons will be capable of generating very large micro-nuclei, because only through such agents will whole chromosomes be lost during mitosis in appreciable numbers and subsequently be incorporated into large micro-nuclei. Three kinds of experiment were used for the evaluation of this mode of action. The first of these was the investigation of potential chromosome breakage in Chinese hamster bone-marrow cells induced by orally administered MBC. Although positive control experiments with trenimon were successful, MBC did not induce chromosome breakage even at the highest doses, where micro-nuclei induction was very prominent. One single chromatid break was observed in a total of 500 metaphases from 4 animals given MBC (1000 mg/kg) twice orally in the same time intervals as for the micro-nuclei experiments. On the other hand, treatment with trenimon yielded chromatid breaks and exchanges in amounts comparable to the values given by Schmid [15]. One must conclude, then, that MBC is n o t a chromosome-breaking agent; its micro-nuclei-producing mode of action must involve some kind of influence on the mitotic process. Positive evidence for this kind of mutagenic action was then to be given by the t w o other experimental designs. A re-examination of the mouse bone-marrow smears, this time not for micronuclei, but for mitotic figures, yielded the results summarized in Table II. Although such phenomena as bridge formation and lagging pieces of chromatin material (see Fig. 2a,b) in anaphases are n o t quite convincing evidence of spindle inhibition, their appearance in the preparations examined strengthens the belief that MBC acts as a spindle poison. Much more indicative for such an action though, were cells, as shown in Fig. 2c,d, where unequal chromatin distribution in the anaphase or tripolar anaphases can be seen. Such abnormalities can only be explained by an inhibitory action of MBC on the spindle apparatus. The experiments designed to evaluate the time course of the action of MBC pointed in the same direction. By preparing bone-marrow smears every 2 h af-
343 T A B L E II O C C U R R E N C E O F A B E R R A N T M I T O T I C F I G U R E S IN M O U S E B O N E - M A R R O W C E L L S , A F T E R TWO ORAL TREATMENTS WITH MBC AT 1000 mg~g, PER 1000 NUCLEATED CELLS
Mitotic figures Metaphase with external chromatin material a Anaphase with lagging chromatin material a Anaphase with bridge Anaphase, tripolar Anaphase, unequal ehromatin distribution
MBC
Control
70 7.0 3.6 0.8 2.4 5.0
20 0 0 0 0 0
a In t h i s k i n d o f p r e p a r a t i o n n o d i s t i n c t i o n is p o s s i b l e bet~veen a c e n t r i c f r a g m e n t s a n d w h o l e c h r o m o s o m e s . T h e m o r e n e u t r a l t e r m " c h r o m a t i n m a t e r i a l " is t h e r e f o r e u s e d t o d e s c r i b e t h e e f f e c t .
Fig. 2. A b n o r m a l i t i e s in m i t o t i c figures f r o m b o n e m a r r o w o f m i c e t r e a t e d w i t h M B C . (a) A n a p h a s e w i t h l a g g i n g f r a g m e n t ; (b) a n a p h a s e w i t h b r i d g e f o r m a t i o n ; (c) t r i p o l a r a n a p h a s e ; (d) u n e q u a l d i s t r i b u t i o n o f c h r o m a t i n in t h e d a u g h t e r cells.
344
ter the application of MBC and counting micronuclei the values in Table III were obtained. In contrast with a chromosome-breaking agent, e.g. Trenimon, the onset of the action of MBC is earlier, and this, too, demonstrates that the action of MBC is on mitosis. Alkylating agents must act long before mitosis, i.e. in or before the S-phase of the cell cycle, to let chromatid breaks become apparent [3]. Thus, aberrations based on the action of alkylating agents will be expected to be recognizable later than those caused by spindle poisons. We can therefore safely assume that MBC produces micro-nuclei by inhibiting the mitotic process, interfering somehow with spindle formation or spindle function. Two issues now needed some further clarification. The first concerned structure-effect relationships, whereas the second was to explain the apparent difference between the two modes of application. From the data compiled in Table I it is evident that the methylcarbamate group plays the significant role in the spindle inhibitory action. We were considering the question whether the presence of this group would be a sufficient condition for the activity of a substance or whether the benzimidazole moiety would also be a necessary condition. We therefore investigated the micro-nuclei-producing potential of a herbicide, propham (N-phenyl-methylcarbamate), which is a known spindle poison [ 1 ]. Our experiments with this substance, however, failed to show, at any concentration, elevated numbers of micro-nucleated erythrocytes. Although these results indicate, that, at least in mammals, the inactive part of the molecule, the benzimidazole, is also needed to produce the spindle poison activity, further studies with other carbamates will be needed fully to elucidate these effects of structure. The last question remaining to be settled was the differential effect of intraperitoneal and peroral application. Whereas by intraperitoneal injection no mutagenic effect was observed, the incidence of micro-nucleated erythrocytes could be considerably enhanced by peroral dosage. An explanation for this difference in genetic toxicity could be that MBC is practically insoluble in aqueous media around pH 7, but is readily dissolved in acids. Therefore MBC might have been dissolved in the stomach of the mouse and then taken up at the be-
TABLE
IIi
TIME COURSE OF APPEARANCE OF MICRO-NUCLEI IN MOUSE BONE-MARROW ERYTHROCYTES (PER 1000 POLYCHROMATIC ERYTHROCYTES) AFTER A SINGLE PERORAL DOSE OF MBC AT 500 mg/kg, COMPARED WITH THE VALUES OBTAINED AFTER A SINGLE DOSE OF TRENIMON AT 0.250 mg/kg (VALUES FROM VON LEDEBUR AND SCHMID [8]).
Time after treatment (b)
0 2 4 6 S 16 24
Micronucleated polychromatie erythrocytes obtained by treatment with MBC
Trenimon
3 5 6 9 15 26 27
3 --3 6 26 72
345 ginning of its passage through the intestine. In order to check this possibility we conducted a small pharmacokinetic experiment. Mice were dosed either intraperitoneally or orally with MBC (containing [2~4C]MBC) at 100 or 500 mg/kg. The radioactivity measurements at intervals of 2 h revealed the picture indicated in Table IV. It was demonstrated that by intraperitoneal injection the solubility of the c o m p o u n d limits the uptake, and thus the serum level of MBC was the same at doses of 100 and 500 mg/kg. Dosing the animals orally, however, resulted in a marked increase in radioactivity of the serum during the first few hours. The uptake of orally administered MBC is here dependent on the dose applied and n o t governed solely by its solubility. Furthermore, a continuous concentration of 8 pg MBC/ml serum is n o t a sufficient a m o u n t to provoke spindle disturbances and formation of micro-nuclei, whereas a short-time rise to 12 pg MBC/ml does lead to a significant increase i n the number of micro-nucleated erythrocytes. The body concentration of 8 #g/ ml might therefore be considered to constitute a threshold limit for this substance. Such a threshold limit could be expected practically from the micro-nuclei counts and theoretically from the mode of action of MBC. As a spindle poison, MBC acts through protein--ligand interactions, where a minimal i n h i b i t o r y concentration must exist. In conclusion, we can summarize our findings as follows. MBC produces micro-nuclei in mouse bone-marrow cells. Its activity is based on the methylcarbamate group, as even slight modifications of this side chain render the molecule inactive. The benzimidazole moiety, however, seems also to be necessary for the activity of this substance in mammals. The very slight solubility o f MBC in water leads to a constant serum concentration of 8 pg/ml upon intraperitoneal application in the mouse; in this case no excess micro-nuclei are produced.
T A B L E IV [2 - 1 4 C ] M B C IN B L O O D S E R U M O F M I C E T R E A T E D O R A L L Y O R I N T R A P E R I T O N E A L L Y ( R A D I O A C T I V I T Y C O U N T S P E R ML S E R U M ) T h e s p e c i f i c a c t i v i t y o f t h e M B C s u s p e n s i o n was 1 . 1 5 ~ C i / m g . c p m • 10 -3
MBC c o n c . ~g/ml serum
4 6
29 20
11.5 8
50O
2 4 6 8 16 24
42 61 53 34 24 27
17 24.5 21 13.5 9.5 11
i.p.
100
4 6
20 19
8 7.5
i.p.
500
2 4 6 8 16 24
20 19 20 21 22 21
8 7.5 8 8.5 9 8.5
Treatment
Amount (mg/kg)
p.o.
100
Time after t r e a t m e n t (h)
346 Upon peroral dosage, however, the serum concentration rises considerably and micro-nuclei are formed owing to the inhibition of spindle formation or spindle function. As this is the only recognizable mutagenic effect of MBC in mammals, a threshold limit for this mode of action of the substance can safely be assumed. Assuming furthermore that (a) absorption, metabolism and excretion of benzimidazole compounds proceed equally in man and mouse, (b) man is no more sensitive genetically than the mouse, and (c) meiotic cells are no more sensitive to the action of MBC than mitotic ones, then we can apply this threshold limit to man also. From such a point of view MBC does not seem to be genetically dangerous for the average man, who is exposed to very minute amounts only. Possible exceptions are factory workers or agricultural spray men, whose blood level should once be monitored exactly in order to obtain information about their chemical load. Exposure to such compounds above the threshold limits, thus leading to the effects described in this paper, might produce a larger number of cells with lethal defects, but also a few cells with mono- or trisomy due to only slight spindle disturbances. Therefore an exact blood content analysis of high risk people should be undertaken as soon as possible. Acknowledgements These investigations were supported by grant No. 3.358-0.74 from the Swiss National Foundation for Scientific Research. I am indebted to Prof. W. Schmid for many helpful discussions regarding the micronucleus test. References 1 A s h t o n , F . M . a n d A . S . C r a f t s , M o d e o f A c t i o n o f H e r b i c i d e s , J o h n Wiley & S o n s , N e w Y o r k , 1 9 7 3 , p. 205. 2 D a s s e n o y , B. a n d J . A . M e y e r , M u t a g c n i c e f f e c t o f b e n o m y l o n Fusarium o x y s p o r u m , M u t a t i o n R e s . , 21 ( 1 9 7 3 ) 1 1 9 - - 1 2 0 . 3 D a t t a , P . K . , H. F r i g g e r a n d E. S c h l e i e r m a c h e r , T h e e f f e c t o f c h e m i c a l m u t a g e n s o n t h e m i t o t i c c h r o m o s o m e s o f the m o u s e , in vivo, in F. V o g e l a n d G. R ~ h r b o r n (eds.) C h e m i c a l M u t a g e n c s i s in M a m m a l s a n d M a n , S p r i n g e r , Berlin, 1 9 7 0 , p. 2 1 1 . 4 Davidse0 L.C., A n t i m i t o t i c a c t i v i t y o f m e t h y l b e n z i m i d a z o l - 2 - y l c a r b a m a t e ( M B C ) in Aspergillus nidulans, Pest. B i o c h e r n . P h y s i o l . , 3 ( 1 9 7 3 ) 3 1 7 - - 3 2 5 . 5 D a v i d s e , L.C., A n t i m i t o t i c a c t i v i t y o f m e t h y l b e n z i r n i d a z o l - 2 - y l c a r b a r n a t e in f u n g i a n d its b i n d i n g t o c e l l u l a r p r o t e i n , in M. B o r g e r s a n d M. de B r a b a n d e r (eds.) M i c r o t u b u l e s a n d M i c r o t u b u l e I n h i b i t o r s , N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 5 , p. 4 8 3 - - 4 9 5 . 6 Hastic, A.C., Benlate-induced instability of Aspergillus diploids, Nature, 226 (1970) 771. 7 K a p p a s , A., S.G. G e o r g o p o u l o s a n d A . C . H a s t i e , O n t h e g e n e t i c a c t i v i t y o f b e n ~ i m i d a z o l c a n d t h i o p h a n a t e f u n g i c i d e s o n dip]Did Aspergillus nidulans, M u t a t i o n R e s . , 26 ( 1 9 7 4 ) 1 7 - - 2 7 . 8 V o n L e d e b u r , M. a n d W. S c h r n i d , T h e m i c r o n u c l e u s t e s t - - m e t h o d o l o g i c a l a s p e c t s , M u t a t i o n R e s . , 19 (1973) 109--117. 9 L ~ o n a r d , A., R . V a n d e s t e c n e a n d R. M a r s b o o r n , M u t a g e n i c i t y t e s t s w i t h r n e b e n d a z o l e in t h e m o u s e , Mutation Res., 26 (1974) 427--430. 1 0 L o u x , H . M . , P r o c e s s for m a n u f a c t u r e o f c e r t a i n a l k y l e s t e r s o f b c n z i r n i d a z o l e c a r b a m i c a c i d , U.S. Pat e n t N o . 3 , 0 1 0 0 9 6 8 ( N o v . 28, 1 9 6 1 ) . 11 M a k i t a , T., Y. H a s h i m o t o a n d T. N o g u c h i , M u t a g e n i e , c y t o g e n i e a n d t e r a t o g e n i e s t u d i e s o n t h i o phanate-methyl, Toxicol. Appl. Pharmacol., 24 (1973) 206--215. 12 N o g u c h i , T., K. O h k u r n a a n d S. K o s a k a , C h e m i s t r y a n d m e t a b o l i s m o f t h i o p h a n a t e f u n g i c i d e s , in A.S. T a h o r i (ed.) P e s t i c i d e T e r m i n a l R e s i d u e s , B u t t e r w o r t h s , L o n d o n , 1 9 7 1 , p. 2 5 7 - - 2 7 0 . 13 N o v i c k , A,, M u t a g e n s a n d a n t i r n u t a g e n s , B r o o k h a v e n S y r n p . Biol., 8 ( 1 9 5 6 ) 2 0 1 - - 2 1 5 . 1 4 R u z i c s k a , P., S. P e t e r a n d C. Czeizel, S t u d i e s o n t h e c h r o m o s o m a l m u t a g e n i e e f f e c t o f b e n o r n y l in rats and humans, Mutation Res., 29 (1975) 201.
347
1 5 S c h m i d , W., D . T . A r a k a k i , N.Y. Breslau a n d J.C. C u l b e r t s o n , C h e m i c a l m u t a g e n e s i s - - t h e C h i n e s e h a m s t e r b o n e m a r r o w as a n in vivo t e s t s y s t e m , I. C y t o g e n e t i c r e s u l t s o n b a s i c a s p e c t s o f t h e m e t h o d o l o g y , o b t a i n e d w i t h a l k y l a t i n g a g e n t s , H u m a n g e n e t i k , 11 ( 1 9 7 1 ) 1 0 3 - - 1 1 8 . 16 S c h m i d , W., C h e m i c a l m u t a g e n t e s t i n g o n i n vivo s o m a t i c m a m m a l i a n ceils, A g e n t s a n d A c t i o n s , 3 (1973) 77--85. 17 Seller, J . P . , T h e m u t a g e n i c i t y o f b e n z i m i d a z o l e a n d b e n z i m i d a z o l e d e r i v a t i v e s . I. F o r w a r d a n d reverse m u t a t i o n s in S a l m o n e l l a t y p h i m u r i u m c a u s e d b y b e n z i m i d a z o l e a n d s o m e o f its d e r i v a t i v e s , M u t a t i o n Res., 1 5 ( 1 9 7 2 ) 2 7 3 - - 2 7 6 . 1 8 S h e r m a n , H., R . C u l i k a n d R . A . J a c k s o n , R e p r o d u c t i o n , t e r a t o g e n i c , a n d m u t a g e n i c s t u d i e s w i t h benomyl, Toxicol. Appl. Pharmacol., 32 (1975) 305--315. 19 S t y l e s , J . A . a n d A. G a r n e r , B e n z i m i d a z o l e c a r b a m a t e m e t h y l e s t e r - - e v a l u a t i o n o f its e f f e c t s in vivo a n d in v i t r o , M u t a t i o n R e s . , 26 ( 1 9 7 4 ) 1 7 7 - - 1 8 7 . 2 0 S z y b a l s k i , W., S p e c i a l m i c r o b i o l o g i c a l s y s t e m s . IL O b s e r v a t i o n s o n c h e m i c a l m u t a g e n e s i s in m i c r o o r g a n i s m s , A n n . N . Y . A c a d . Sci., 76 ( 1 9 5 8 ) 4 7 5 - - 4 8 9 .
