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Genetic and related effects of Vinca rosea alkaloids
31
Mutation Research, 55 (1978) 31--42
© Elsevier/North-Holland Biomedical Press
G E N E T I C A N D R E L A T E D E F F E C T S O F V i n c a rosea A L K A L O I D S
N. DEGRAEVE Laboratoire de G~n~tique, Universitd de Liege, Rue Forgeur 15, B-4000 Liege (Belgique)
(Received 26 April 1978} (Revision received 11 July 1978) (Accepted 20 July 1978)
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antitumor properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stathmokinetic effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on DNA and RNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on gametogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Teratogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
31 32 33 33 34 36 37 38 39 40 40
Introduction In t h e e n d o f t h e n i n e t e e n fifties, t w o g r o u p s o f researchers, o n e in C a n a d a a n d t h e o t h e r in t h e U n i t e d States, s t u d i e d i n d e p e n d e n t l y t h e p r o p e r t i e s o f e x t r a c t s o f p e r i w i n k l e , V i n c a r o s e a L. (syn.: L o c h n e r a rosea R e i c h e n b a c h , C a t h a r a n t h u s r o s e u s D o n . ) , a p l a n t t h a t has e n j o y e d a p o p u l a r r e p u t a t i o n as an oral h y p o g l y c e m i c . A l t h o u g h t h e results o b t a i n e d in this field w e r e n o t pertinent, the Canadian researchers on screening experiments observed a peripheral g r a n u l o c y t o p e n i a a n d a b o n e - m a r r o w d e p r e s s i o n in t h e r a t [ 2 0 ] w h e r e a s t h e A m e r i c a n g r o u p n o t e d a p r o l o n g a t i o n o f life in t h e m o u s e w h i c h h a d b e e n given i m p l a n t s o f an a c u t e l y m p h o c y t i c n e o p l a s m , t h e P - 1 5 3 4 l e u k e m i a [ 4 1 ] . A f t e r t h e s e p r e l i m i n a r y results, e x t e n s i v e w o r k was carried o u t to i d e n t i f y t h e active p r i n c i p l e s o f t h e e x t r a c t s . T h e a c t i v i t y was f o u n d t o be e n t i r e l y l o c a t e d in t h e alkaloidal f r a c t i o n [ 4 1 ] . A great m a n y alkaloids w e r e i s o l a t e d , p u r i f i e d a n d d e t e r m i n e d c h e m i c a l l y [ 4 0 ] . Several o f t h e m h a d an a n t i t u m o r e f f e c t ; b u t s o o n t h e e f f o r t s w e r e c o n c e n t r a t e d on t h e t w o m o s t active o f t h e m : v i n b l a s t i n e ( V i n c a l e u k o b l a s t i n e , V e l b a n , Velbe, V L B ) a n d vincristine ( L e u r o cristine, O n c o v i n , V C R ) . T h e s e m o l e c u l e s a p p e a r t o be d i m e r i c i n d o l e - - d i h y -
32 H C ~
HC"
I
HC_ ' ~ C
H H C~C ~H H~ ,C N
C
II
II
C
~ N
C
Jl
.
H
R= CH3
VINBLASTINE I
..Clio
V,.C.,~,N~
HCH HCH C~CH \ ~ ~H H C ~ C
\'
.C
~
C
H_ 0 HC H
\k C__C
I II c~/c.
-~
I
C H
II
CH
I HC
/\/
N C I / ~ R 0 H
C:H
H
C__C
H
I
o, .
HC
"
O__C__CH
H C__O--CH II H 0
Fig. 1. S t r u c t u r e o f v i n b l a s t i n e a n d v i n e r i s t i n e
droindole c o m p o u n d s in which the indole part is linked by a C--C bond to the aromatic ring of the dihydroindole moiety of the molecule (see formula). The double molecules are c o m p o s e d of catharanthine and vindoline moieties with minor molecular modifications. The difference lies in the substitution of a methyl group b o u n d to the anilino nitrogen in the vindoline moiety (vinblastine) by a CHO group (vincristine) [58]. Antitumor properties The researches achieved on the antitumor properties of the t w o alkaloids showed positive effects, b u t differences were recorded in their toxicity and in the specificity of successfully treated cancers. Despite only a minor difference in their chemical structures, the clinical effects of vinblastine and vincristine differ considerably and surprisingly, there is no clinical evidence of cross-resistance b e t w e e n them. A great variety of cancers has n o w been treated, and the most favorable indications for using Velbe (vinblastine sulfate) are actually some lymphosarcomas, choriosarcomas, monocytic leukemias and mainly Hodgkin's disease. Oncovin (vincristine sulfate) is used for treating lymphosarcomas, Hodgkin's disease and especially for acute lymphoblastic leukemias in children [7,30,38]. Alkaloids are generally used as a treatment combined with endoxan, prednisone, methotrexate, bleomycin or daunomycin [7,58]. The dose requirements differ markedly: 0.1--0.3 mg/kg i.v. weekly for vinblastine; 0.01--0.05 mg/kg i.v. weekly for vincristine in adults. The doses used are higher for an optimal remission of acute childhood leukemias. Fortunately, leukemic children tolerate large doses of vincristine better than adults. The factors limiting the use of higher doses are the occurrence of neuromuscular
33 abnormality and constipation with vincristine and the appearance of leukopenia with vinblastine [42]. This latter effect results from the action on myeloid leukocytes while the frequency of megakaryocytes and derived platelets is normal [21,78]. Yet it is noted that vinblastine does not accumulate in marrow. After injection, it is rapidly and efficiently metabolized, probably in the liver [4]. Stathmokinetic effect At the cellular level, vinblastine and vincristine produce effects similar to those observed after colchicine treatment: accumulation of mitotic figures, arrested metaphases with highly coiled chromosomes, multipolar anaphases with lagging chromosomes in the interpolar region, tendency to polyploidy by doubling of the chromosomes but failure of chromatid separation, and, at high dose, pycnosis. This C-mitotic effect was found in higher plants [23,25,26,37], animal cells in vitro [13,18,19,33,70], in vivo [13,14,19,52,69,74], normal and malignant human cells in vitro [ 54,60,70] and in patients treated with alkaloids [28,30,76]. This effect could sometimes be reversed after treatment by several amino acids, including glutamic acid and tryptophan [19,76]. However, the fact that optimal doses of vinblastine were n o t affected suggests that amino acids play a role of only minor importance in alkaloid action [40]. Prolonged exposures to relatively high concentrations of vincristine does not produce gross morphological or cytochemical changes in the nucleus or cytoplasm of resting cells and does not prevent these cells from initiating normal prophases. The moving forward through GI, S and G2 is not altered, and vincristine does n o t inhibit the onset of mitosis [33]. Whereas alkaloids have no evident action on the structure of centrioles or kinetochores, spindle tubules are lacking in arrested metaphases. It seems that these substances interfere with the normal assembly of spindle-fiber proteins into an oriented tubular structure. Without organization of structural units, functional spindle tubules are not found and normal chromosome movements are prevented [33]. Ultrastructural investigations have demonstrated the proliferation of 80- to 100-A filaments and the presence of crystalline inclusion bodies in the cytoplasm of various types of cell. This structure is not only observed in mammals both in vivo and in cell cultures, but also after treatment of solutions of pure microtubule protein (tubulin) [5,6,8,53,79]. These crystals are composed of microtubule proteins precipitated by the alkaloids. A similar effect has been obtained with colchicine but the binding sites are different with vinblastine, vincristine and colchicine [9]. The ratio of molecular binding of colchicine is always twice that of vinblastine and vincristine [ 59]. Effects on DNA and R N A syntheses
Semmel [68] has studied the reactions of vinblastine and vincristine with poly-A and poly-U chains in vitro. Alkaloids can bind with nucleic acids, b u t the bonds disappear at high ionic strength indicating that they are weak electrostatic bonds. In thymus cells in vitro, vinblastine interferes with some metabolic reactions
34 related to DNA synthesis. The incorporation of labeled precursors into DNA purines seems to be particularly perturbed. This inhibition occurs after the synthesis of the purine ring [64]. A comparable result was obtained in Musca [55]. Vinblastine inhibited the incorporation of [14C]glycine into DNA, and the analysis of the bases revealed that the specific activities of DNA purines were decreased more than those of DNA thymine. In Tetrahymena, vinblastine decreased the rate of synthesis of DNA b u t did not inhibit the possibility for the cells to synthesize new DNA [67]. In Vicia root tips, autoradiographic techniques after [3H]thymidine showed that the number of labeled nuclei remains normal b u t the number of silver grains per nucleus is decreased [71]. Nevertheless, in mouse t u m o r cells, even if incorporation of [3H]thymidine into DNA is decreased by high vinblastine doses in vitro, it is not modified after therapeutic doses of this c o m p o u n d in vivo [16]. In cell cultures, both alkaloids inhibit R N A synthesis. Separation of the R N A into soluble, ribosomal and rapidly labeled fractions indicates that the primary effect is on soluble RNA. The uptake of [3H]uridine into soluble R N A of Ehrlich ascites cells recovered from mice treated with vinblastine is depressed by a b o u t 60% compared with the control value. Since the specific activity of both uridine and cytidine is reduced, it appears that the alkaloid inhibits the synthesis of the whole chain of soluble R N A [16]. At higher doses minor changes are produced in the synthesis of ribosomal R N A [17]. According to Wagner and Roizman [77], there is a preferential inhibition of 28S r R N A synthesis, and t R N A synthesis is less affected. Pretreatment with glutamic acid prevents to various extents the inhibition of R N A synthesis but the doses required to reverse the action of the Vinca alkaloids are so massive that it is improbable that there is any direct reaction with the alkaloids themselves. A possible explanation is that the function of the glutamate is to bring a b o u t a general elevation in amino acid levels at least in certain specific ones that are particularly involved in cell division [17]. At doses where the C-mitotic effect is observed, R N A synthesis is not affected, and inhibition of R N A synthesis parallels the direct c y t o t o x i c effect. Hence it seems that the effects are independent of each other, b u t the basic mechanism might be the same and related to precipitation or crystallization of spindle fibers, microtubular protein or other protein [62]. Effects on chromosomes The induction of chromosome damage by vinblastine was studied in higher plants after treatment of root tips. In Hordeum and in Vicia, no effect was reported in metaphases as well as in anaphases. However, in Nigella, cells entering anaphase showed a number of chromosome bridges and fragments higher than the control number. The maximum of aberrations was scored immediately, or only a short time, after treatment, and the number decreased quickly with the recovery period [23]. In metaphase, the chromosomal aberrations mainly consist in breaks giving rise to acentric fragments. The a m o u n t of such aberrations is very low as compared with that obtained for anaphases. These results suggest that the anaphase aberrations are induced subsequently to the metaphase. They could be related to disturbances of the spindle apparatus and
35 their origin could be the same as that of multipolar anaphases [23]. Treatments of dry seeds of Nigella did n o t result in meiotic aberrations [22]. In Triturus, Sentein [69] demonstrated the antimitotic efficiency of vinblastine and stated that it was w i t h o u t direct action on chromosomes. Some secondary breaks may be f o u n d but t h e y are a mere consequence of the spindle disorganization. In rat-cell culture, Cutts [19] could not induce chromosome breakage. This result was confirmed in different hamster cell lines [1,51,61]. The percentage of structural aberrations was not increased even at doses higher than the therapeutic range. In Chinese hamster, Palyi and Palyi [61] observed a large number of tetraploid and octoploid cells and 11% of the metaphases contained endoreduplicated diplochromosomes. Vinblastine also induced various chromosome alterations: thin unreplicated chromosomes and chromosomal segments and chromosome pulverization of various extents. Also, after treatment of normal and neoplastic h u m a n cells with vinblastine, there was no evidence of chromosome damage [60]. After treatment of h u m a n l y m p h o c y t e s with vincristine (1.25--5.0 pg/ml), Gebhart et al. [32] observed chromosome erosion, gaps, fragments and only a small number of exchanges. No dose--effect relationship was obtained in this experiment for the a m o u n t of abnormal metaphases or the n u m b e r of aberrations per metaphase. On the other hand, in the same material, the data of Kucerova and Polivkova [46] indicated that both vinblastine and vincristine (10 -3 to 10 -7 mg/ml) induce only few, if any, mutagenic effects even at high concentrations. The same authors, using the fluorescence plus Giemsa technique noted that at 10 -7 mg/ml, the frequency of sister-chromatid exchanges in cultured l y m p h o c y t e s was not increased. At a higher dose of vincristine (5 • 10 -s mg/ml), a significant increase was reported by Raposa [63]. Unfortunately, only few cells were analyzed in this experiment. After t r e a t m e n t of mice in vivo, vinblastine and vincristine gave a positive response in the micronucleus test [36,51]. For vincristine, the dose--effect curve obtained had a peculiar shape, with a peak at 0.2 mg/kg and a very low effect after the highest doses. Concurrently, tetraploid polychromatic erythrocytes began to appear after a dose of 0.2 mg/kg. These findings are best explained by the assumption that low doses cause a partial impairment of the spindle apparatus. Single chromosomes or groups of chromosomes lag at anaphase and give rise to micro-nucleated daughter cells. At high concentrations, spindle formation is completely blocked and the chromosomes reassemble into a tetraploid nucleus [51]. In Chinese hamster, vincristine gave positive results n o t only in the micronucleus test [56] but also in the nucleus anomaly test, a m e t h o d which takes into account 5 different criteria {single Jolly bodies, erythrocytes with nuclear debris or micronuclei, micronuclei in leukopoietic cells, polyploid cells) [49]. However, the effect on chromosomes in bone marrow of a hamster treated with vincristine remained negative even at high doses (1/3 of the LDs0) [56]. A similar result was obtained in rat [10] and in mouse [ 56] spermatogonia. These animals received 5 doses on 5 consecutive days and were killed one day after the last injection. The doses used were 1/24, 1/12 and 1/6 of the LDs0. In the mouse, a positive effect was observed in spermatocytes I and II when vincristine (0.4 and 0.8 mg/kg) was given 12, 10, 9, 7 and 3 days before the killing [56]. As compared with the cumulative a m o u n t of different
36 control groups {1800 spermatocytes I and 1800 spermatocytes II), the number of aberrations was significantly increased. They consisted exclusively of fragments. At the 0.2 mg/kg dose no effect was obtained. Studies of chromosomal aberrations have also been carried out in patients treated with vinblastine and vincristine. The results are usually difficult to interpret. The n u m b e r of patients is often small, doses used as well as duration of treatments are rather variable and the treatments are often combined with the dosage of other chemicals. Furthermore, there is the problem of controls: should the results obtained be compared with those of healthy persons or those who are ill and not y e t treated? Several reports have described numerical changes of chromosomes, i.e. aneuploidy and hyperdiploidy [12,45,47]. In l y m p h o c y t e s of 5 patients treated with vinblastine, Hartwich et al. [35] noted the absence of effect in three cases {including a patient who received 110 mg) and only a slight increase of the percentage of aberrations in the others. No dose--effect relationship was reported. The authors concluded that there is no indication of a chromosomebreaking ability of vinblastine. Another report [66] dealing with 67 patients under treatment combining alkylating agents (endoxan, myleran, nitrogen mustard), cytostatic antibiotics (actinomycin D, bleomycin, daunomycin) and antimetabolites (methotrexate, mercaptopurine, azathioprine, fluorouracil, cytosine arabinoside, L-asparaginase) concludes that 19 o u t of the 20 patients receiving only alkaloids and antimetabolites had an aberration rate comparable to the control. On the other hand, in patients treated with alkaloids and alkylating agents or antibiotics, the chromosome damage was increased. In 20 patients suffering from Hodgkin's disease or chronic myelosis and treated with Velbe, Kiibock et al. [47] observed some nuclear abnormalities, breaks and dicentric chromosomes. This effect could be related to a prolonged arrest in metaphase rather than to a direct action on chromosomes. We should notice that the majority of these patients were previously submitted to radiotherapy, endoxan or myleran. In one paper a significant increase of chromosome damage after treatment with vincristine has been reported. Unfortunately, these data were collected from only 4 patients. One showed no increase and another one:had previously been submitted to radiotherapy. Thus, the positive effect actually concerns only 2 persons. Besides figures such as C-mitosis, polyploidy, endoreduplication, Gebhart et al. [32] observed gaps and breaks and rather few exchanges. Effects on gametogenesis Under specific conditions, it seems that Vinca alkaloids can interfere with spermatogenesis. Thus, after vincristine treatment the incorporation of [3H]thymidine, [3H]uridine and [3H]leucine by spermatogonia, early and late elongated spermatids, respectively, is highly decreased in mouse [50]. These results indicate that the alkaloid inhibits n o t only the proliferating spermatogonia b u t also the non-replicating spermatids. Fertility, measured by the serialmating technique, decreases for at least 8 weeks, and all spermatogenic cell types with the possible exception of mature spermatozoa are affected. Delayed recovery of fertility suggests a significant reduction in the number of stem cells
37 [50]. This effect is confirmed for vinblastine-treated rats. Cell necroses correlate better with the e x t e n t of cell differentiation than with the mitotic indices. Thus the more differentiated In and B spermatogonia are less affected than A4 although the former have higher mitotic indices [10,11]. These results were obtained after t r e a t m e n t at sub-lethal doses. Similar conclusions can be drawn from observations of sperm abnormalities in mouse [36,81]. Only the groups in which some animals died showed a percentage of abnormal spermatozoa significantly higher than the control. In a sub-acute treatment (5 daily injections) a total dose of more than 10 mg/kg was necessary to produce an increase o f 10 abnormal sperm per 1000 cells scored above the spontaneous frequency. The increase was very small a week after the t r e a t m e n t but it rose after a 4-week recovery period. The effective doses are 10 times higher than those used in the micronucleus test [36]. Folded-head type abnormality observed after vinblastine treatment might well be an example of non-genetic damage since this type of abnormality is n o t seen at longer periods after t r e a t m e n t and since vinblastine is known to interact with microtubules t h a t are present during sperm development [ 81]. In mouse oogenesis, after treatment with vinblastine and vincristine, spindle inhibition at first meiotic metaphase was induced in vitro and in vivo. A stathmokinetic effect with cleavage abnormalities and formation of binucleated blastomeres was described [34]. In vivo, the minimal effective dose for vincristine was 1 mg/kg s.c., and in vitro, 4 • 10 -7 pg/ml for 14 h of incubation [39]. Since, in the mouse oocytes, the first meiotic metaphase can be inhibited in vivo, one could reasonably expect heteroploidy arising from fertilization. However, examination of ova that had recovered from inhibition revealed normal orientation of spindle fibers and of chromosomes [39]. In Musca domestica, vinblastine (0.1% in food) caused sterility in the female flies but not in the males [55]. Mutagenicity All the experiments attempting to demonstrate a mutagenic activity of the two alkaloids yielded negative results. This is true for vincristine in the Ames' test using Salmonella with and w i t h o u t the microsomal fraction carried out on 5 different strains and also for the point-mutation assay with mouse l y m p h o m a cells (L5178Y) performed in vitro, and in the host-mediated assay (Fischer test) [56]. Negative results were also obtained for vinblastine in the Ames' test using the TA98, TA100, TA1534 and TA1537 strains of Salmonella [36], in forward mutations with adenineless ad6 and ad7 in Schizosaccharomyces pombe and in back mutations arginine-1 in Chlamydomonas reinhardi [22]. The serum from orally treated rats was used in a host-mediated assay with histidine m u t a n t s of Salmonella. The frequency of back mutations was n o t significantly increased after treatment with each alkaloid [72]. In the dominant-lethal m u t a t i o n test in the mouse after a single dose (1/9 and 1/3 of the LDs0) followed by 8 mating periods of one week's duration, vincristine produced early fetal deaths and preimplantation losses within control limits [56]. In another experiment, male mice were injected i.p. with vinblastine at 1 mg/kg. The numbers o f corpora lutea and implants were normal, and the distribution of live fetuses showed no
38 difference between the treated group and controls. There was no significant difference between the total deaths (early losses plus dead implants) observed in the control group and after treatment. The a m o u n t of dominant-lethal mutations induced in the different stages of spermatogenesis did n o t differ significantly [22]. A similar result was obtained with vinblastine [22] at sub-lethal doses of 1.95 and 4.50 mg/kg which induced 14 and 45% lethality respectively in treated males [27] and reduced the percentage of pregnancy [27]. Teratogenicity Since vinblastine inhibits DNA synthesis in fetuses in utero of pregnant mice [80], it was logically inferred that it has a teratogenic effect. In hamster, after injection into the femoral vein on the 8th day of gestation, vinblastine and vincristine have an embryocidal effect which increases with the doses used. At doses similar to those used in therapeutics, evidence was recorded [29] of gross malformations (microphthalmia, anophthalmia, exencephaly, spina bifida). The most effective teratogenic doses (0.25 mg/kg for vinblastine and 0.10 mg/kg for vincristine) are smaller than the most effective embryocidal ones (0.5--2.6 mg/kg for vinblastine and 0.6--2.3 mg/kg for vincristine). This effect was confirmed for vincristine in mouse [44], rat [24,73] and rhesus m o n k e y [15]. At teratogenic doses, in mice fetuses, an accumulation of metaphases was noted as well as a slight increase of the chromosome damage [43,75]. After treatment with Oncovin, 0.25 mg/kg i.p. on the 9th day of gestation, the percentage of metaphases in fetuses rises from 0.5 to 4 h after the treatment. In anaphases the aberrations consist in a majority of bridges and a few fragments (perhaps lagging chromosomes). Nevertheless, several cases were reported of women suffering from Hodgkin's disease and treated with vinblastine during their pregnancy, who gave birth to normal children [2,3,48,65]. Discussion The mutagenicity o f Vinca extracts was investigated from a particular point of view. As a result of the introduction of these substances, expectation of survival of patients suffering from Hodgkin's disease and from acute leukemia has considerably increased, and even if these alkaloids were strongly mutagenic, their use in such cases could n o t obviously be discarded. It is true that vinblastine and vincristine can partially inhibit DNA and RNA syntheses, but only at doses much higher than the therapeutic ones which have a stathmokinetic effect. Similarly, effects obtained on spermatogenesis, though possibly of importance, were induced only at sublethal doses. As regards the induction of chromosomal aberrations, most experiments yielded negative results. Moreover, the induced lesions may often be related to an indirect action on the spindle rather than to a direct action on chromosomes. This hypothesis is supported by the absence of reaction with the DNA and by the low exchange frequency. The positive results obtained in the micronucleus test could be somewhat anticipated since the stathmokinetic effect which results in multipolar anaphases and lagging chromosomes may generate micronuclei. Finally, all the published data agree about the absence of demon-
39 strable mutagenicity in both microorganisms and mammals. Although this latter point in reassuring, we must not forget the reports of numerical changes (aneuploidy, hyperdiploidy) observed at therapeutic doses. It is obvious that this effect can have serious genetic consequences. However, only experiments simulating the therapeutic use of both alkaloids (long-term treatment at therapeutic doses) could definitely evaluate the genetic risk. Investigations should also be performed on the effects of combined treatments, among others with endoxan, methotrexate and bleomycin, because alkaloids are most frequently used in association with these drugs. The well d o c u m e n t e d teratogenic activity of vinblastine and vincristine must also be pointed out. Although some reports were negative in man, the results obtained in several mammalian species indicate a potential danger. In conclusion, we have to remember that the Vinca alkaloids are only used in the treatment of neoplastic diseases. In this field, the risks from the genetic and teratogenic points of view are surely less important than those presented by the previous therapies, i.e. radiotherapy or treatment by alkylating agents.
Acknowledgements I thank Prof. D. Mfiller for supply of the text of the communications presented by himself and his coworkers at the 2nd International Conference on Environmental Mutagens (Edinburgh, July 1977). I am grateful to Prof. J. Moutschen who read the manuscript and made many helpful suggestions.
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B e e r , S o m e e f f e c t s o f V i n c a a l k a l o i d s o n n u c l e i c a c i d m e t a b o l i s m , L l o y d i a , 2 7 (1964) 346--351. 6 5 R o s e n w e i g , A., Q. C r e w s a n d H. H o p w o o d , V i n b l a s t i n e s u l f a t e in H o d g k i n ' s disease in p r e g n a n c y , Ann. Intern. Med., 61 (1964) 108--112. 6 6 S c h i n z e l , A., a n d W. S c h m i d , L y m p h o c y t e c h r o m o s o m e s t u d i e s i n h u m a n s e x p o s e d t o c h e m i c a l m u t a g e n s , T h e v a l i d i t y o f t h e m e t h o d in 6 7 p a t i e n t s u n d e r c y t o s t a t i c t h e r a p y , M u t a t i o n Res., 4 0 ( 1 9 7 6 ) 139--166. 6 7 S e d g l e y , N., D N A s y n t h e s i s in v i n b l a s t i n e - t r e a t e d T e t r a h y m e n a , J. Cell Biol., 3 9 ( 1 9 6 8 ) 1 2 1 a . 6 8 S e m m e l , M.~ I n t e r a c t i o n i n v i t r o d e s a c i d e s n u c l ~ i q u e s a v e c des a l c a l o i ' d e s e x t r a l t s d e Vinca rosea et l'acide glutamiclue, Biochimie, 53 (1971) 457---460. 6 9 S e n t e i n , P., L ' a c t i o n de la v i n c a l e u c o b l a s t i n e s u r la m i t o s e c h e z Trifurus h e l v e t i c u s R a z . , C h r o m o s o ma, 15 (1964) 416--456. 7 0 Siebs, W., M i t o s e a b l a u f s t ~ r u n g e n , III. Zux W i r k u n g y o n V i n c a l e u k o b l a s t i n e a u f n o r m a l c u n d m a l i g n e Z e l l c n i n v i t r o , Z. Z e l l f o r s c h . , 6 1 ( 1 9 6 3 ) 2 3 1 - - 2 7 5 . 71 S r i v a s t a v a , S., S. R a o a n d V° S h a h , C y t o l o g i c a l a u t o r a d i o g r a p h i c s t u d i e s o f t h e e f f e c t s o f v i n c a l e u k o b l a s t i n e ( V L B ) o n t h e r o o t m e r i s t e m cells o f Vicia faba, C y t o l o g i a , 3 8 ( 1 9 7 3 ) 1 - - 9 . 7 2 S t y l e s , J., C y t o t o x i c e f f e c t s o f v a r i o u s p e s t i c i d e s in vivo a n d in v i t r o , M u t a t i o n R e s . , 21 ( 1 9 7 3 ) 5 0 - 51. 7 3 T a n a k i , M., T. S u g a w a r a , Y. K a n e y a m a a n d U. M u r a k a m i , V i n c r i s t i n e - i n d u c c d m a l f o r m a t i o n s i n t h e r a t e m b r y o , A n n . R e p . I n s t . E n v i r o n . M e d . N a g o y a Univ., 1 5 ( 1 9 6 7 ) 6 1 - - 7 2 . 7 4 T h i l a g a r a t n a m , C., a n d J. M a i n , M i t o s t a t i c a c t i o n o f v i n b l a s t i n e s u l f a t e o n o r a l e p i t h e l i a o f h a m s t e r s , J. O r a l P a t h o l . , 1 ( 1 9 7 2 ) 8 4 - - 8 8 . 7 5 U n g t h a v o r n , S., a n d M. J o n e j a , E f f e c t s o f t e r a t o g e n i c d o s e s o f v i n c r i s t i n e o n m i t o t i c cells i n t h e f e t u s e s o f D B A m i c e , A m . J. A n a t . , 1 2 6 ( 1 9 6 9 ) 2 9 1 - - 2 9 7 . 7 6 V a i t k e v i c i u s , V., R. T a l l e y , J . T u c k e r a n d M. B r e n n a n , C y t o l o g i c a l a n d c l i n i c a l o b s e r v a t i o n s d u r i n g vincaleukoblastine therapy of disseminated cancer, Cancer, 15 (1962) 294--306.
42
7 7 W a g n e r , K . , a n d B. R o i z m a n , E f f e c t s o f t h e V i n c a a l k a l o i d s o n R N A s y n t h e s i s in h u m a n cells i n vitro, Science, 162 (1968) 569--570. 7 8 W a r w i c k , O., J. D a r t e a n d T. B r o w n , S o m e b i o l o g i c a l e f f e c t s o f v i n c a l e u k o b l a s t i n e , a n a l k a l o i d i n V i n c a r o s e a L i n n . i n p a t i e n t s w i t h m a l i g n a n t disease, C a n c e r R e s . , 2 0 ( 1 9 6 0 ) 1 0 3 2 - - 1 0 4 0 . 7 9 W e i s c n b e r g , R . , a n d S. T i m a s h e f f , A g g r e g a t i o n o f m i c r o t u b u l e s s u b u n i t p r o t e i n , E f f e c t s o f d i v a l e n t cations, colchicine and vinblastine, Biochemistry, 9 (1970) 4110--4116. S 0 Williams, J., S e l e c t i v e i n h i b i t i o n o f e m b r y o n i c d e o x y r i b o n u c l e i c a c i d s y n t h e s i s b y v i n b l a s t i n e , Cell Tissue K i n e t . , 3 ( 1 9 7 0 ) 1 5 5 - - 1 5 9 . S1 W y r o b e k , A., a n d W. B r u c e , C h e m i c a l i n d u c t i o n of s p e r m a b n o r m a l i t i e s i n m i c e , P r o c . Natl. A c a d . Sci. (U.S.A.), 72 (1975) 4425--4429.
Mutation Research, 55 (1978) 31--42
© Elsevier/North-Holland Biomedical Press
G E N E T I C A N D R E L A T E D E F F E C T S O F V i n c a rosea A L K A L O I D S
N. DEGRAEVE Laboratoire de G~n~tique, Universitd de Liege, Rue Forgeur 15, B-4000 Liege (Belgique)
(Received 26 April 1978} (Revision received 11 July 1978) (Accepted 20 July 1978)
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antitumor properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stathmokinetic effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on DNA and RNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on gametogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Teratogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
31 32 33 33 34 36 37 38 39 40 40
Introduction In t h e e n d o f t h e n i n e t e e n fifties, t w o g r o u p s o f researchers, o n e in C a n a d a a n d t h e o t h e r in t h e U n i t e d States, s t u d i e d i n d e p e n d e n t l y t h e p r o p e r t i e s o f e x t r a c t s o f p e r i w i n k l e , V i n c a r o s e a L. (syn.: L o c h n e r a rosea R e i c h e n b a c h , C a t h a r a n t h u s r o s e u s D o n . ) , a p l a n t t h a t has e n j o y e d a p o p u l a r r e p u t a t i o n as an oral h y p o g l y c e m i c . A l t h o u g h t h e results o b t a i n e d in this field w e r e n o t pertinent, the Canadian researchers on screening experiments observed a peripheral g r a n u l o c y t o p e n i a a n d a b o n e - m a r r o w d e p r e s s i o n in t h e r a t [ 2 0 ] w h e r e a s t h e A m e r i c a n g r o u p n o t e d a p r o l o n g a t i o n o f life in t h e m o u s e w h i c h h a d b e e n given i m p l a n t s o f an a c u t e l y m p h o c y t i c n e o p l a s m , t h e P - 1 5 3 4 l e u k e m i a [ 4 1 ] . A f t e r t h e s e p r e l i m i n a r y results, e x t e n s i v e w o r k was carried o u t to i d e n t i f y t h e active p r i n c i p l e s o f t h e e x t r a c t s . T h e a c t i v i t y was f o u n d t o be e n t i r e l y l o c a t e d in t h e alkaloidal f r a c t i o n [ 4 1 ] . A great m a n y alkaloids w e r e i s o l a t e d , p u r i f i e d a n d d e t e r m i n e d c h e m i c a l l y [ 4 0 ] . Several o f t h e m h a d an a n t i t u m o r e f f e c t ; b u t s o o n t h e e f f o r t s w e r e c o n c e n t r a t e d on t h e t w o m o s t active o f t h e m : v i n b l a s t i n e ( V i n c a l e u k o b l a s t i n e , V e l b a n , Velbe, V L B ) a n d vincristine ( L e u r o cristine, O n c o v i n , V C R ) . T h e s e m o l e c u l e s a p p e a r t o be d i m e r i c i n d o l e - - d i h y -
32 H C ~
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I
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C
II
II
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~ N
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.
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VINBLASTINE I
..Clio
V,.C.,~,N~
HCH HCH C~CH \ ~ ~H H C ~ C
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.C
~
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/\/
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Fig. 1. S t r u c t u r e o f v i n b l a s t i n e a n d v i n e r i s t i n e
droindole c o m p o u n d s in which the indole part is linked by a C--C bond to the aromatic ring of the dihydroindole moiety of the molecule (see formula). The double molecules are c o m p o s e d of catharanthine and vindoline moieties with minor molecular modifications. The difference lies in the substitution of a methyl group b o u n d to the anilino nitrogen in the vindoline moiety (vinblastine) by a CHO group (vincristine) [58]. Antitumor properties The researches achieved on the antitumor properties of the t w o alkaloids showed positive effects, b u t differences were recorded in their toxicity and in the specificity of successfully treated cancers. Despite only a minor difference in their chemical structures, the clinical effects of vinblastine and vincristine differ considerably and surprisingly, there is no clinical evidence of cross-resistance b e t w e e n them. A great variety of cancers has n o w been treated, and the most favorable indications for using Velbe (vinblastine sulfate) are actually some lymphosarcomas, choriosarcomas, monocytic leukemias and mainly Hodgkin's disease. Oncovin (vincristine sulfate) is used for treating lymphosarcomas, Hodgkin's disease and especially for acute lymphoblastic leukemias in children [7,30,38]. Alkaloids are generally used as a treatment combined with endoxan, prednisone, methotrexate, bleomycin or daunomycin [7,58]. The dose requirements differ markedly: 0.1--0.3 mg/kg i.v. weekly for vinblastine; 0.01--0.05 mg/kg i.v. weekly for vincristine in adults. The doses used are higher for an optimal remission of acute childhood leukemias. Fortunately, leukemic children tolerate large doses of vincristine better than adults. The factors limiting the use of higher doses are the occurrence of neuromuscular
33 abnormality and constipation with vincristine and the appearance of leukopenia with vinblastine [42]. This latter effect results from the action on myeloid leukocytes while the frequency of megakaryocytes and derived platelets is normal [21,78]. Yet it is noted that vinblastine does not accumulate in marrow. After injection, it is rapidly and efficiently metabolized, probably in the liver [4]. Stathmokinetic effect At the cellular level, vinblastine and vincristine produce effects similar to those observed after colchicine treatment: accumulation of mitotic figures, arrested metaphases with highly coiled chromosomes, multipolar anaphases with lagging chromosomes in the interpolar region, tendency to polyploidy by doubling of the chromosomes but failure of chromatid separation, and, at high dose, pycnosis. This C-mitotic effect was found in higher plants [23,25,26,37], animal cells in vitro [13,18,19,33,70], in vivo [13,14,19,52,69,74], normal and malignant human cells in vitro [ 54,60,70] and in patients treated with alkaloids [28,30,76]. This effect could sometimes be reversed after treatment by several amino acids, including glutamic acid and tryptophan [19,76]. However, the fact that optimal doses of vinblastine were n o t affected suggests that amino acids play a role of only minor importance in alkaloid action [40]. Prolonged exposures to relatively high concentrations of vincristine does not produce gross morphological or cytochemical changes in the nucleus or cytoplasm of resting cells and does not prevent these cells from initiating normal prophases. The moving forward through GI, S and G2 is not altered, and vincristine does n o t inhibit the onset of mitosis [33]. Whereas alkaloids have no evident action on the structure of centrioles or kinetochores, spindle tubules are lacking in arrested metaphases. It seems that these substances interfere with the normal assembly of spindle-fiber proteins into an oriented tubular structure. Without organization of structural units, functional spindle tubules are not found and normal chromosome movements are prevented [33]. Ultrastructural investigations have demonstrated the proliferation of 80- to 100-A filaments and the presence of crystalline inclusion bodies in the cytoplasm of various types of cell. This structure is not only observed in mammals both in vivo and in cell cultures, but also after treatment of solutions of pure microtubule protein (tubulin) [5,6,8,53,79]. These crystals are composed of microtubule proteins precipitated by the alkaloids. A similar effect has been obtained with colchicine but the binding sites are different with vinblastine, vincristine and colchicine [9]. The ratio of molecular binding of colchicine is always twice that of vinblastine and vincristine [ 59]. Effects on DNA and R N A syntheses
Semmel [68] has studied the reactions of vinblastine and vincristine with poly-A and poly-U chains in vitro. Alkaloids can bind with nucleic acids, b u t the bonds disappear at high ionic strength indicating that they are weak electrostatic bonds. In thymus cells in vitro, vinblastine interferes with some metabolic reactions
34 related to DNA synthesis. The incorporation of labeled precursors into DNA purines seems to be particularly perturbed. This inhibition occurs after the synthesis of the purine ring [64]. A comparable result was obtained in Musca [55]. Vinblastine inhibited the incorporation of [14C]glycine into DNA, and the analysis of the bases revealed that the specific activities of DNA purines were decreased more than those of DNA thymine. In Tetrahymena, vinblastine decreased the rate of synthesis of DNA b u t did not inhibit the possibility for the cells to synthesize new DNA [67]. In Vicia root tips, autoradiographic techniques after [3H]thymidine showed that the number of labeled nuclei remains normal b u t the number of silver grains per nucleus is decreased [71]. Nevertheless, in mouse t u m o r cells, even if incorporation of [3H]thymidine into DNA is decreased by high vinblastine doses in vitro, it is not modified after therapeutic doses of this c o m p o u n d in vivo [16]. In cell cultures, both alkaloids inhibit R N A synthesis. Separation of the R N A into soluble, ribosomal and rapidly labeled fractions indicates that the primary effect is on soluble RNA. The uptake of [3H]uridine into soluble R N A of Ehrlich ascites cells recovered from mice treated with vinblastine is depressed by a b o u t 60% compared with the control value. Since the specific activity of both uridine and cytidine is reduced, it appears that the alkaloid inhibits the synthesis of the whole chain of soluble R N A [16]. At higher doses minor changes are produced in the synthesis of ribosomal R N A [17]. According to Wagner and Roizman [77], there is a preferential inhibition of 28S r R N A synthesis, and t R N A synthesis is less affected. Pretreatment with glutamic acid prevents to various extents the inhibition of R N A synthesis but the doses required to reverse the action of the Vinca alkaloids are so massive that it is improbable that there is any direct reaction with the alkaloids themselves. A possible explanation is that the function of the glutamate is to bring a b o u t a general elevation in amino acid levels at least in certain specific ones that are particularly involved in cell division [17]. At doses where the C-mitotic effect is observed, R N A synthesis is not affected, and inhibition of R N A synthesis parallels the direct c y t o t o x i c effect. Hence it seems that the effects are independent of each other, b u t the basic mechanism might be the same and related to precipitation or crystallization of spindle fibers, microtubular protein or other protein [62]. Effects on chromosomes The induction of chromosome damage by vinblastine was studied in higher plants after treatment of root tips. In Hordeum and in Vicia, no effect was reported in metaphases as well as in anaphases. However, in Nigella, cells entering anaphase showed a number of chromosome bridges and fragments higher than the control number. The maximum of aberrations was scored immediately, or only a short time, after treatment, and the number decreased quickly with the recovery period [23]. In metaphase, the chromosomal aberrations mainly consist in breaks giving rise to acentric fragments. The a m o u n t of such aberrations is very low as compared with that obtained for anaphases. These results suggest that the anaphase aberrations are induced subsequently to the metaphase. They could be related to disturbances of the spindle apparatus and
35 their origin could be the same as that of multipolar anaphases [23]. Treatments of dry seeds of Nigella did n o t result in meiotic aberrations [22]. In Triturus, Sentein [69] demonstrated the antimitotic efficiency of vinblastine and stated that it was w i t h o u t direct action on chromosomes. Some secondary breaks may be f o u n d but t h e y are a mere consequence of the spindle disorganization. In rat-cell culture, Cutts [19] could not induce chromosome breakage. This result was confirmed in different hamster cell lines [1,51,61]. The percentage of structural aberrations was not increased even at doses higher than the therapeutic range. In Chinese hamster, Palyi and Palyi [61] observed a large number of tetraploid and octoploid cells and 11% of the metaphases contained endoreduplicated diplochromosomes. Vinblastine also induced various chromosome alterations: thin unreplicated chromosomes and chromosomal segments and chromosome pulverization of various extents. Also, after treatment of normal and neoplastic h u m a n cells with vinblastine, there was no evidence of chromosome damage [60]. After treatment of h u m a n l y m p h o c y t e s with vincristine (1.25--5.0 pg/ml), Gebhart et al. [32] observed chromosome erosion, gaps, fragments and only a small number of exchanges. No dose--effect relationship was obtained in this experiment for the a m o u n t of abnormal metaphases or the n u m b e r of aberrations per metaphase. On the other hand, in the same material, the data of Kucerova and Polivkova [46] indicated that both vinblastine and vincristine (10 -3 to 10 -7 mg/ml) induce only few, if any, mutagenic effects even at high concentrations. The same authors, using the fluorescence plus Giemsa technique noted that at 10 -7 mg/ml, the frequency of sister-chromatid exchanges in cultured l y m p h o c y t e s was not increased. At a higher dose of vincristine (5 • 10 -s mg/ml), a significant increase was reported by Raposa [63]. Unfortunately, only few cells were analyzed in this experiment. After t r e a t m e n t of mice in vivo, vinblastine and vincristine gave a positive response in the micronucleus test [36,51]. For vincristine, the dose--effect curve obtained had a peculiar shape, with a peak at 0.2 mg/kg and a very low effect after the highest doses. Concurrently, tetraploid polychromatic erythrocytes began to appear after a dose of 0.2 mg/kg. These findings are best explained by the assumption that low doses cause a partial impairment of the spindle apparatus. Single chromosomes or groups of chromosomes lag at anaphase and give rise to micro-nucleated daughter cells. At high concentrations, spindle formation is completely blocked and the chromosomes reassemble into a tetraploid nucleus [51]. In Chinese hamster, vincristine gave positive results n o t only in the micronucleus test [56] but also in the nucleus anomaly test, a m e t h o d which takes into account 5 different criteria {single Jolly bodies, erythrocytes with nuclear debris or micronuclei, micronuclei in leukopoietic cells, polyploid cells) [49]. However, the effect on chromosomes in bone marrow of a hamster treated with vincristine remained negative even at high doses (1/3 of the LDs0) [56]. A similar result was obtained in rat [10] and in mouse [ 56] spermatogonia. These animals received 5 doses on 5 consecutive days and were killed one day after the last injection. The doses used were 1/24, 1/12 and 1/6 of the LDs0. In the mouse, a positive effect was observed in spermatocytes I and II when vincristine (0.4 and 0.8 mg/kg) was given 12, 10, 9, 7 and 3 days before the killing [56]. As compared with the cumulative a m o u n t of different
36 control groups {1800 spermatocytes I and 1800 spermatocytes II), the number of aberrations was significantly increased. They consisted exclusively of fragments. At the 0.2 mg/kg dose no effect was obtained. Studies of chromosomal aberrations have also been carried out in patients treated with vinblastine and vincristine. The results are usually difficult to interpret. The n u m b e r of patients is often small, doses used as well as duration of treatments are rather variable and the treatments are often combined with the dosage of other chemicals. Furthermore, there is the problem of controls: should the results obtained be compared with those of healthy persons or those who are ill and not y e t treated? Several reports have described numerical changes of chromosomes, i.e. aneuploidy and hyperdiploidy [12,45,47]. In l y m p h o c y t e s of 5 patients treated with vinblastine, Hartwich et al. [35] noted the absence of effect in three cases {including a patient who received 110 mg) and only a slight increase of the percentage of aberrations in the others. No dose--effect relationship was reported. The authors concluded that there is no indication of a chromosomebreaking ability of vinblastine. Another report [66] dealing with 67 patients under treatment combining alkylating agents (endoxan, myleran, nitrogen mustard), cytostatic antibiotics (actinomycin D, bleomycin, daunomycin) and antimetabolites (methotrexate, mercaptopurine, azathioprine, fluorouracil, cytosine arabinoside, L-asparaginase) concludes that 19 o u t of the 20 patients receiving only alkaloids and antimetabolites had an aberration rate comparable to the control. On the other hand, in patients treated with alkaloids and alkylating agents or antibiotics, the chromosome damage was increased. In 20 patients suffering from Hodgkin's disease or chronic myelosis and treated with Velbe, Kiibock et al. [47] observed some nuclear abnormalities, breaks and dicentric chromosomes. This effect could be related to a prolonged arrest in metaphase rather than to a direct action on chromosomes. We should notice that the majority of these patients were previously submitted to radiotherapy, endoxan or myleran. In one paper a significant increase of chromosome damage after treatment with vincristine has been reported. Unfortunately, these data were collected from only 4 patients. One showed no increase and another one:had previously been submitted to radiotherapy. Thus, the positive effect actually concerns only 2 persons. Besides figures such as C-mitosis, polyploidy, endoreduplication, Gebhart et al. [32] observed gaps and breaks and rather few exchanges. Effects on gametogenesis Under specific conditions, it seems that Vinca alkaloids can interfere with spermatogenesis. Thus, after vincristine treatment the incorporation of [3H]thymidine, [3H]uridine and [3H]leucine by spermatogonia, early and late elongated spermatids, respectively, is highly decreased in mouse [50]. These results indicate that the alkaloid inhibits n o t only the proliferating spermatogonia b u t also the non-replicating spermatids. Fertility, measured by the serialmating technique, decreases for at least 8 weeks, and all spermatogenic cell types with the possible exception of mature spermatozoa are affected. Delayed recovery of fertility suggests a significant reduction in the number of stem cells
37 [50]. This effect is confirmed for vinblastine-treated rats. Cell necroses correlate better with the e x t e n t of cell differentiation than with the mitotic indices. Thus the more differentiated In and B spermatogonia are less affected than A4 although the former have higher mitotic indices [10,11]. These results were obtained after t r e a t m e n t at sub-lethal doses. Similar conclusions can be drawn from observations of sperm abnormalities in mouse [36,81]. Only the groups in which some animals died showed a percentage of abnormal spermatozoa significantly higher than the control. In a sub-acute treatment (5 daily injections) a total dose of more than 10 mg/kg was necessary to produce an increase o f 10 abnormal sperm per 1000 cells scored above the spontaneous frequency. The increase was very small a week after the t r e a t m e n t but it rose after a 4-week recovery period. The effective doses are 10 times higher than those used in the micronucleus test [36]. Folded-head type abnormality observed after vinblastine treatment might well be an example of non-genetic damage since this type of abnormality is n o t seen at longer periods after t r e a t m e n t and since vinblastine is known to interact with microtubules t h a t are present during sperm development [ 81]. In mouse oogenesis, after treatment with vinblastine and vincristine, spindle inhibition at first meiotic metaphase was induced in vitro and in vivo. A stathmokinetic effect with cleavage abnormalities and formation of binucleated blastomeres was described [34]. In vivo, the minimal effective dose for vincristine was 1 mg/kg s.c., and in vitro, 4 • 10 -7 pg/ml for 14 h of incubation [39]. Since, in the mouse oocytes, the first meiotic metaphase can be inhibited in vivo, one could reasonably expect heteroploidy arising from fertilization. However, examination of ova that had recovered from inhibition revealed normal orientation of spindle fibers and of chromosomes [39]. In Musca domestica, vinblastine (0.1% in food) caused sterility in the female flies but not in the males [55]. Mutagenicity All the experiments attempting to demonstrate a mutagenic activity of the two alkaloids yielded negative results. This is true for vincristine in the Ames' test using Salmonella with and w i t h o u t the microsomal fraction carried out on 5 different strains and also for the point-mutation assay with mouse l y m p h o m a cells (L5178Y) performed in vitro, and in the host-mediated assay (Fischer test) [56]. Negative results were also obtained for vinblastine in the Ames' test using the TA98, TA100, TA1534 and TA1537 strains of Salmonella [36], in forward mutations with adenineless ad6 and ad7 in Schizosaccharomyces pombe and in back mutations arginine-1 in Chlamydomonas reinhardi [22]. The serum from orally treated rats was used in a host-mediated assay with histidine m u t a n t s of Salmonella. The frequency of back mutations was n o t significantly increased after treatment with each alkaloid [72]. In the dominant-lethal m u t a t i o n test in the mouse after a single dose (1/9 and 1/3 of the LDs0) followed by 8 mating periods of one week's duration, vincristine produced early fetal deaths and preimplantation losses within control limits [56]. In another experiment, male mice were injected i.p. with vinblastine at 1 mg/kg. The numbers o f corpora lutea and implants were normal, and the distribution of live fetuses showed no
38 difference between the treated group and controls. There was no significant difference between the total deaths (early losses plus dead implants) observed in the control group and after treatment. The a m o u n t of dominant-lethal mutations induced in the different stages of spermatogenesis did n o t differ significantly [22]. A similar result was obtained with vinblastine [22] at sub-lethal doses of 1.95 and 4.50 mg/kg which induced 14 and 45% lethality respectively in treated males [27] and reduced the percentage of pregnancy [27]. Teratogenicity Since vinblastine inhibits DNA synthesis in fetuses in utero of pregnant mice [80], it was logically inferred that it has a teratogenic effect. In hamster, after injection into the femoral vein on the 8th day of gestation, vinblastine and vincristine have an embryocidal effect which increases with the doses used. At doses similar to those used in therapeutics, evidence was recorded [29] of gross malformations (microphthalmia, anophthalmia, exencephaly, spina bifida). The most effective teratogenic doses (0.25 mg/kg for vinblastine and 0.10 mg/kg for vincristine) are smaller than the most effective embryocidal ones (0.5--2.6 mg/kg for vinblastine and 0.6--2.3 mg/kg for vincristine). This effect was confirmed for vincristine in mouse [44], rat [24,73] and rhesus m o n k e y [15]. At teratogenic doses, in mice fetuses, an accumulation of metaphases was noted as well as a slight increase of the chromosome damage [43,75]. After treatment with Oncovin, 0.25 mg/kg i.p. on the 9th day of gestation, the percentage of metaphases in fetuses rises from 0.5 to 4 h after the treatment. In anaphases the aberrations consist in a majority of bridges and a few fragments (perhaps lagging chromosomes). Nevertheless, several cases were reported of women suffering from Hodgkin's disease and treated with vinblastine during their pregnancy, who gave birth to normal children [2,3,48,65]. Discussion The mutagenicity o f Vinca extracts was investigated from a particular point of view. As a result of the introduction of these substances, expectation of survival of patients suffering from Hodgkin's disease and from acute leukemia has considerably increased, and even if these alkaloids were strongly mutagenic, their use in such cases could n o t obviously be discarded. It is true that vinblastine and vincristine can partially inhibit DNA and RNA syntheses, but only at doses much higher than the therapeutic ones which have a stathmokinetic effect. Similarly, effects obtained on spermatogenesis, though possibly of importance, were induced only at sublethal doses. As regards the induction of chromosomal aberrations, most experiments yielded negative results. Moreover, the induced lesions may often be related to an indirect action on the spindle rather than to a direct action on chromosomes. This hypothesis is supported by the absence of reaction with the DNA and by the low exchange frequency. The positive results obtained in the micronucleus test could be somewhat anticipated since the stathmokinetic effect which results in multipolar anaphases and lagging chromosomes may generate micronuclei. Finally, all the published data agree about the absence of demon-
39 strable mutagenicity in both microorganisms and mammals. Although this latter point in reassuring, we must not forget the reports of numerical changes (aneuploidy, hyperdiploidy) observed at therapeutic doses. It is obvious that this effect can have serious genetic consequences. However, only experiments simulating the therapeutic use of both alkaloids (long-term treatment at therapeutic doses) could definitely evaluate the genetic risk. Investigations should also be performed on the effects of combined treatments, among others with endoxan, methotrexate and bleomycin, because alkaloids are most frequently used in association with these drugs. The well d o c u m e n t e d teratogenic activity of vinblastine and vincristine must also be pointed out. Although some reports were negative in man, the results obtained in several mammalian species indicate a potential danger. In conclusion, we have to remember that the Vinca alkaloids are only used in the treatment of neoplastic diseases. In this field, the risks from the genetic and teratogenic points of view are surely less important than those presented by the previous therapies, i.e. radiotherapy or treatment by alkylating agents.
Acknowledgements I thank Prof. D. Mfiller for supply of the text of the communications presented by himself and his coworkers at the 2nd International Conference on Environmental Mutagens (Edinburgh, July 1977). I am grateful to Prof. J. Moutschen who read the manuscript and made many helpful suggestions.
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