Mutagenicity of BCNU and related chloroethylnitrosoureas in drosophila

297

Mutation Research, 57 (1978) 297--305 © Elsevier/North-HollandBiomedicalPress

MUTAGENICITY OF BCNU AND RELATED CHLOROETHYLNITROSOUREAS IN DROSOPHILA

M.J.H. KORTSELIUS Department of Radiation Genetics and Chemical Mutagenesis, Sylvius Laboratories, State University of Leiden, Wassenaarseweg 72, Leiden (The Netherlands)

{Received 5 October 1977) {Revision received 31 January 1978) (Accepted 7 February 1978)

Summary BCNU and 10 related chloroethylnitrosoureas were tested for their ability to induce sex-linked recessive lethals in Drosophila spermatozoa. All chloroethylnitrosoureas tested were potent mutagens. Among the substances with one chloroethylnitrosourea group, chlorozotocin, BCNU and methanesulfonyloxyethyl chloroethylnitrosourea exhibited the strongest mutagenic effects. Two hydroxyalkyl chloroethylnitrosoureas behaved as potent mutagens too, although the mutation frequencies obtained were one order of magnitude lower relative to the other substances. Among the compounds with two chloroethylnitrosourea groups, bisCNUethane and bisCNU-diphenylmethane were most active. When the interconnecting polymethylene chain was elongated from 2 methylene groups (bisCNUethane} to 6 methylene groups (bisCNU-hexane), the mutagenic activity decreased by a factor of 2. The mutagenic activity of polymethylene bischloroethylnitrosoureas with connecting chains of intermediate length was not different from bisCNU-hexane. Differences in mutagenic activity were supposed to reflect different concentrations reaching the target cells, possibly in part as a result of differences in transportability of the substances.

A b b r e v i a t i o n s : BCNU, 1,3-bis(2-chloroethyl)-l-nitrosourea; bisCNU-diphenylmethane, l,l'-diphe-

nyhnethylene bis-3-(2-chloroethyl)-3-nitrosourea; bisCNU-ethane to bisCNU-hexane, 1,1'-polymethylenebis-3-(2-chloroethyl)-3-nitrosoureas; chlorozotocin, 1-(glucopyranos-2-yl)-3-(2-chloroethyl)-3-nitrosotwea; CNU-ethanol and CNU-propanol, 1-(w-hydroxyalkyl)-3-(2-chloroethyl)-3nitrosoureas; ENU, 1-ethyl-l-nitrosottrea; MNU, 1-methyl-l-nitrosourea; MSO-CNU, 1-(2-methanesulfonyloxyethyl)-3-(2-ch]oroethyl)-3-nitrosourea.

298 Introduction

Chloroethylnitrosoureas are an important class of anti-tumor agents. BCNU, a representative of this class, is particularly useful against tumor cells in the central nervous system [23], since like other nitrosoureas, it can easily pass the brain-blood barrier [ 15 ]. The mode of decomposition of BCNU has been extensively studied by Montgomery and co-workers [14]. Although BCNU contains two chloroethyl groups (Table 1) and thus in its structure resembles nitrogen mustard, the mechanism of alkylation differs on account of the rapid decomposition of BCNU in vivo [14,24]. Mutagenic activity of nitrosoureas has been established in a variety of organisms [6,7,12,13] including Drosophila [2,16--18]. These studies concern MNU and ENU, but little information exists on chemotherapeutically active nitrosoureas like BCNU [4]. The recent synthesis of a series of analogs of BCNU [5] stimulated mutagenicity studies in Saccharomyces cerevisiae [19], mouse lymphoma cells in vitro [9], mouse spermatocytes and bone marrow in vivo [20,21] and the present study on the induction of sex-linked recessive lethals in Drosophila melanogaster. For the compounds tested in the present study it is presumed, that the chloroethylnitrosourea group is the directly acting mutagenic entity. The aim TABLE 1 STRUCTURE OF THE 3-(2-CHLOROETHYL)-3-NITROSOUREAS TESTED

BCNU and its water-soluble analogs BCNU CNU - ethanol CNU- propanol chlorozotocin MSO- CNU

0

II

ClCHzCHzXN/C-,,,,N/- R

R: R' R: R: R:

--CHzCH2C| --CH2CH20H -CH2CH2CH20H

-glucose --CH2CH2-O-SO2CH 3

analogs of BCNU with two 3-( 2-chloroethyl )- 3- nitrosourea functions

0

0

II

II

CtCH2CH2,~N<~%/R,~N~C~N/CH2 CH2CI

bisCNU - ethane bisCNU- propane bisCNU- butane bisCNU- pentane bisCNU- hexane

R: R: R: R: R:

bisCNU- diphenylrnethane

-CH2CH 2 -CH2CH2CH2 -CH2CH2CH2CH2 -CH2CH2CH2CH2CH2~ -CH2CH2CH2CH2CH2CH2-

299 of this work has been to determine whether or not the mutagenic activity of the chloroethylnitrosourea group is influenced b y N-1 substitutions. Since the nature of the substitutions at the non-nitrosated nitrogen atom affects rate of decomposition, lipid solubility and transport across cell membranes, the various chloroethylnitrosoureas could differ considerably in their mutagenic activity, particularly in a complex in vivo system such as Drosophila. Material and methods

Chemicals Analogs of BCNU were synthesized by Dr. G. Eisenbrand of the Institute of Toxicology and Chemotherapy of the German Cancer Research Centre in Heidelberg, FRG [ 5]. Chemical structures are given in Table 1. The polymethylene bischloroethylnitrosoureas contain 2 chloroethylnitrosourea groups connected by a polymethylene chain. The number of methylene groups in the connecting chain varies between 2 in bisCNU~thane and 6 in bisCNU-hexane. In bisCNU-diphenylmethane both CNU-groups are connected by a diphenylmethylene group. The water-soluble chloroethylnitrosoureas differ in the sidechains substituted at the non-nitrosated nitrogen. In chlorozotocin, the chloroethylnitrosourea group is attached to the C-2 position of glucose.

Treatment procedure The test substances were fed to adult Drosophila males in aqueous sucrose solution according to the technique described by Vogel and Liiers [22]. A mixture of equal parts of ethanol and of the polyethoxylated vegetable oil Cremophor EL (Sigma No. C-5135) was used to dissolve the bifunctional analogs of BCNU. Ethanol was used to dissolve BCNU. The final contents in the test solution were 1% Cremophor/ethanol mixture and 4% ethanol, unless indicated differently in the tables. Under our conditions of tests none of the chloroethynitrosoureas was toxic to the Drosophila males.

Genetical tests The compounds were tested for the induction of sex-linked recessive lethals by means of the Basc-technique. 2-Day~ld wild-type males of our tester strain Berlin K were treated for 1 or 2 days. Treated males were mated individually with virgin females of the genetic constitution In(1)scSILscSR+S, scSlscSwaB (Basc, ref. 11) in a ratio of 1 d : 3 ~ for 3 days. With this scheme, the germ cells tested represent mainly treated mature spermatozoa [3]. When mutation induction in different germ cell stages [3] was studied, each treated male was mated with 3 or 4 fresh Basc virgins per 2 or 3~lay brood. Sex-linked recessive lethals were scored in the F2-generation according to standard procedures [1]. Results

Table 2 shows results of experiments that were carried out with CNU-ethanol. After exposure to a concentration of 0.5 mM CNU-ethanol, an average recessive lethal frequency of 0.7% was observed (broods I--V). At a 10 times

300 TABLE 2 I N D U C T I O N OF SEX-LINKED RECESSIVE L E T H A L S IN S U B S E Q U E N T MALE G E R M S T A G E S BY C N U - E T H A N O L W H E N F E D T O D R O S O P H I L A M A L E S F O R 2 D A Y S Concentration (mM)

Brood

Days after treatment

n chromosomes tested

n lethals

0.5

I II III IV V I--V

1--2 3--5 6--7 8--9 10--12 1--12

510 600 441 344 386 2281

4 4 5 2 0 15

0.8 0.7 1.1 0.6 -0.7

± ± ± ±

5

I II In iv v I--V

1--2 3---5 6-7 8-9 10---12 1--12

310 605 60 34 104 1113

32 49 5 1 0 87

10.3 S.1 8.3 2.9 7.8

± ± + ±

CELL

Recessive l e t h a l f r e q u e n c y (%) 0.4 0.3 0.5 0.4

± 0.2 1.7 1.1 3.6 2.9

± 0.8

higher concentration a 10-fold higher induction of sex-linked recessive lethals was observed in postmeiotic stages (an average of 8.9% in broods I and II). In broods III and IV the fertility was strongly reduced as indicated by the low numbers of chromosomes tested. Broods III and IV sample cells which mainly represent treated meiotic stages. A slight recovery of treated germ cells was noticed in brood V, sampling mainly treated spermatogonia. In contrast with the substantial mutation induction in postmeiotic broods at both concentrations tested, no lethals were found in premeiotic cells (brood V). So, mature spermatozoa appeared to be most sensitive to the induction of recessive lethals by CNU-ethanol as a representative of this class of chemicals, and therefore comparative studies were carried out on mature sperm. Table 3 shows results of experiments with bischloroethylnitrosoureas, which differ in the length c.q. structure of the connecting chain between the two chloroethylnitrosourea moieties of the molecule. All six bischloroethylnitrosoureas tested were mutagenic, whilst the solvent Cremophor EL was not. After treatment with equimolar concentrations of 0.1 mM for 2 4 h , bisCNUdiphenylmethane and bisCNU-ethane appeared to be the most active mutagens of this group. The mutagenic activity of bisCNU-ethane was about twice that of bisCNU-hexane, the compound with the longest polymethylene chain. With an exposure time of 2 days, the mutagenic activity of bisCNU-ethane was again twice as high as that of bisCNU-hexane. The differences are statistically significant in most experiments and on the verge of significance in other experiments, probably due to the small numbers of chromosomes tested. The 3 polymethylene bischloroethylnitrosoureas with connecting chains of intermediate length did not differ from bisCNU-hexane in mutagenic activity, and were less effective than bisCNU-ethane. From these observations it is clear, that in Drosophila bisCNU-ethane is more active than any of the other polymethylene bischloroethylnitrosoureas tested. Table 4 shows the mutagenic action of BCNU and its water-soluble analogs. BCNU and 1-(2-methanesulfonyloxyethyl)-3-(2~hloroethyl)-3-nitrosourea

301 TABLE3 INDUCTION SOUREAS

OF

SEX-LINKED

Compound

RECESSIVE

Concentzation (raM)

LETHALS

n chromosomes tested

BY

BIS-3-(2-CHLOROETHYL)-3-NITRO-

n lethals

Recessive lethal frequency (%)

3 28 8 7 13 11 18

0.11 2.61 1.32 1.16 1.25 1.41 2.94

-+ 0 . 0 7 ± 0.49 + 0.46 ± 0.43 -+ 0 . 3 5 ± 0.42 -+ 0 . 6 8

1 29 14 6 24

0.07 5.0 14.0 2,05 7,5

± 0.07 -+ 0.9 ± 3.5 -+ 0 . 8 3 ± 1.5

p a

Exposure time 24 h; solvent 1% Cremophor/ethanol control bisCNU-ethane bisCNU-propane bisCNU-butane bisCNU-pentane bisCNU-hexane bisCNU-diphenylmethane

0.1 0.1 0.1 0.1 0.1 0.I

2627 1072 605 605 1037 791 613

~0.06 (0.04 (0.02 ~0.05

}~0.01

Exposure time 2 days; solvent 10% Cremophor/ethanol control bisCNU-ethane

0.1 1 0.1 1

bisCNU-hexane

1373 577 100 292 318

<0.03 <0.06

a A s i g n i f i c a n t l y IQwez recessive l e t h a l f r e q u e n c y t h a n w i t h b i s C N U - e t h a n e [ 8 ] .

(MSO-CNU) produced 12.0% and 14.1% recessive lethals when I mM of these compounds was fed for 24 h. At lower concentrations the lethal frequency decreased with concentration. Chlorozotocin, in which chloroethylnitrosourea is attached to glucose, produced 7.9% recessive lethals when 1 mM was fed for 24 h, and 5.570 already with 0.1 mM solution. This lack of correlation in effectiveness with increasing concentration coincided with a strong reduction of the fertility when males were exposed to 1 mM of chlorozotocin. Such a reduction of fertility was not observed at 0.1 mM chlorozotocin and therefore this con-

TABLE 4 I N D U C T I O N O F S E X - L I N K E D R E C E S S I V E L E T H A L S BY B C N U A N D W A T E R - S O L U B L E A N A L O G S Compound

Concentration (mM)

control

n chromosomes tested

n lethals

3480

6

R e c e u i v e lethal f r e q u e n c y (%) 0.17 ± 0.07

BCNU

0.1 1 5

1807 1687 93

34 202 16

1.9 12.0 17.2

± 0.3 ~ 0.8 ± 3.9

chlorozotocin

0.1 1

817 624

45 49

5.5 7.9

± 0.8 ± 1.1

MSO-CNU

0.1 1

611 1070

9 151

1.47 ± 0 . 4 9 14.1 ± 1.1

CNU-ethanol

0.1 1 20

1820 2374 336

3 26 46

0.16 ± 0.10 1.10 + 0.21 1 3 . 7 ± 1.9

CNU-propanol

1 10

569 339

4 16

0.70 ± 0.35 4.7 ± 1.2

302 centration seemed more suitable for comparing these compounds. BCNU and MSO-CNU were equally effective, whereas chlorozotocin was the most active mutagen of all chloroethylnitrosoureas tested. Compared with BCNU, MSO-CNU and chlorozotocin, the mutagenic activity of the hydroxyalkyl chloroethylnitrosoureas (CNU-ethanol and CNU-propanol) was much lower. A concentration of 0.1 mM of CNU:ethanol did not increase the mutation frequency at all, and 1 mM produced only 1.1% recessive lethals. High recessive lethal frequencies could be obtained with such high concentrations as 20 mM of CNU-ethanol (13.7%). CNU-propanol induced almost as many lethals as CNU-ethanol. At equimolar basis the mutagenic activities of both hydroxyalkyl chloroethylnitrosoureas was over one order of magnitude lower than any of the other chloroethylnitrosoureas tested. Discussion

BCNU and the 10 related chloroethylnitrosoureas tested were mutagenic in Drosophila. Since mature spermatozoa were found the stage most sensitive to the induction of sex-linked recessive lethals by CNU-ethanol, which served as a representative of this class of chemicals, all further comparative studies were carried out on mature spermatozoa on the assumption that these substances have the same mechanism of action. Among the substances with two chloroethylnitrosourea functional groups, bisCNU-diphenylmethane and bisCNUethane were most active. When the polymethylene chain was elongated from 2 methylene groups (bisCNU-ethane) to 6 methylene groups (bisCNU-hexane), the mutagenic activity decreased by a factor of 2. But there was no negative correlation between the degree of extension of polymethylene chain and mutagenic activity. Our data (as well as those in yeast, ref. 19) suggest, that elongation of the chain from 2 methylene groups (bisCNU-ethane) to 3 such groups (bisCNU-propane) results in decreased mutagenicity, but that further elongation has no effect on mutagenic activity. Elucidation of the relation between chain elongation and mutagenic activity requires extension of experiments with 2 days exposure. At equimolar concentrations bisCNU-ethane was the most active compound among five polymethylene bischloroethylnitrosoureas tested. Chloroethylnitrosoureas with longer polymethylene bridges between the alkylating centres of the molecule were equally effective among themselves. BisCNU-diphenylmethane was as active as bisCNU-ethane. All substances with one chloroethylnitrosourea functional group were found highly mutagenic; however, there was a marked difference between chlorozotocin, BCNU and MSO-CNU on the one hand and CNU-ethanol and CNUpropanol on the other. Mutagenic activities of BCNU and MSO-CNU were about equal at equimolar concentrations, and, at a concentration of 0.1 mM, chlorozotocin was more active. Although potent mutagens too, hydroxyalkyl chloroethylnitrosoureas were far less effective and, in order to reach the same recessive lethal frequency, a 10-fold higher concentration had to be administered. The mechanism of action of BCNU has been studied extensively because of its use in cancer chemotherapy [14,24,25]. Under physiological conditions

303 chloroethylnitrosoureas decompose rapidly, yielding vinyl diazohydroxide and an isocyanate. Vinyl diazohydroxide can give rise to a vinyl diazonium ion and consequently to a vinyl carbonium ion which can alkylate DNA [14]. Isocyanates are capable of carbamoylating proteins. Further degradation of 2~hloroethyl isocyanate (from BCNU) leads to the formation of 2-chloroethyl amine [14], an efficient alkylating agent in vitro [25]. Chemotherapeutic activity of BCNU is attributed to the alkylating capacity of vinyl carbonium ions [24], in spite of the in vitro activity of chloroethylamine. Negative results obtained w h e n 10 mM of chloroethylamine was fed to Drosophila males for 24 h (0.16% recessive lethals in 614 chromosomes tested, unpublished results) indicate that the formation of chloroethylamine does not contribute much to the mutagenic action of BCNU. This finding fits in with the view that BCNU exhibits its mutagenic activity via the in si~u generation of vinyl carbonium ions from the chloroethylnitrosourea moiety. This mechanism applies to all chloroethylnitrosoureas tested. In Drosophila mutagenicity was demonstrated for 2 nitrosoureas, MNU and ENU [2,16--18], which compounds alkylate via the generation of a methyl ion and an ethyl ion respectively [14]. Ond~ej [16] injected Drosophila males with I mM and 10 mM of ENU and obtained recessive lethal frequencies of 5.7% and 41.5%. His data show a clearcut concentration~lependent induction of recessive lethals by ENU. Similar high frequencies were obtained by Alderson [2] when Drosophila males were fed MNU for 24 h. In his experiments 4.85 mM and 9.7 mM of MNU induced 24.8% and 48.5% recessive lethals. Our present results with chloroethylnitrosoureas are consistent with the cited data. It should be noted, however, that the extremely high lethal frequencies induced by ENU and MNU could not be reached with chloroethylnitrosoureas, although BCNU and chlorozotocin were fed in concentrations which should have been sufficient to induce such high frequencies. Whilst 0.1 mM of chlorozotocin induced 5.5% recessive lethais, a 10-fold increase of the concentration produced only 7.9%. Similarly, I mM of BCNU induced 12.0% recessive lethals, whereas a 5-fold increase in concentration resulted in not more than 17.2%. At the highest concentrations of BCNU (5 raM) and chlorozotocin (1 raM) the egg-hatchability was reduced to 5%. Hence it seems likely, that selective elimination of the most affected germ ceils, via dominant lethality, poses a restriction on the yield of recessive lethals that can be recovered. Chloroethylnitrosoureas were reported active in the test on mitotic gene conversion in yeast [19]. BCNU and CNU-ethanol induced HGPRT-deficient mutants in mouse lymphoma cells in vitro [9], and BCNU produced chromatid aberrations in tobacco mouse lung fibroblasts in vitro [21]. In in vivo tests with the mouse, BCNU and CNU-ethanol induced micronuclei in polychromatic erythrocytes and chromosome aberrations in bone marrow, as well as translocations and chromosome aberrations in spermatogonia [20,21]. In yeast, as in Drosophila, bisCNU-ethane was more effective than any other of the polymethylene bischloroethylnitrosoureas. In contrast to Drosophila, in which chlorozotocin was more active than BCNU, there was no difference in mutagenic effectiveness of these compounds in yeast. The most striking difference in mutagenic effectiveness in Drosophila was found between BCNU and the hydroxyalkyl chloroethylnitrosoureas (CNU-ethanol and CNU-propanol).

304 BCNU and CNU-ethanol were tested in a variety of test systems mentioned above, in which no remarkable differences in mutagenic activities between both compounds were found. Thus, the lower mutagenic effectiveness of hydroxyalkyl chloroethylnitrosoureas appears specific for Drosophila, but not for another complex in vivo system, the mouse. The different mutagenic responses observed with the various chloroethylnitrosoureas probably do not result from different reaction mechanisms for these compounds, but reflect differences in dose (as opposed to exposure, see ref. 10) at the genetic target. Although some differences in the observed genetic response may be due to structural features of these compounds, it seems premature to discuss this aspect in detail on the basis of this limited set of data. But it is pertinent to point out that with equimolar concentrations of diverse substances administered to a complex organism such as Drosophila, the actual dose of active mutagen to the genetic target may be a very complicated function of its structure. In case of polymethylene bischloroethylnitrosoureas, transportability across cell membranes may be hindered by elongation of the interconnecting polymethylene chain, and so lead to differences in actual doses at the target cells and consequently in mutagenic effects observed. Transport problems caused by the alcohol side-chain might explain the observation in Drosophila that hydroxyalkyl chloroethyinitrosoureas produce less mutations, the frequencies of which were one order of magnitude lower when compared to those induced by BCNU at equimolar concentrations. The finding that BCNU and CNU-ethanol are equally effective in a number of test systems based on cell incubation as well as in the mouse in vivo, makes it even more complicated. For this discrepancy no reasonable explanation is yet available. The results described above are a good example to illustrate that, even with mutagens which do not require metabolic activation, small structural changes in the non-mutagenic part of the molecule can result in a different mutagenic activity.

Acknowledgements It is a pleasure to thank Mrs. Marjo van de Broek and Mrs. Jos~ van Helten for technical assistance; Dr. G. Eisenbrand (Heidelberg) for kindly supplying the chemicals; Drs. E Vogel, C.S. Aaron and K. Sankaranarayanan for critical reading of the manuscript. This work was supported by PHS Research Grant No. ESO 1027-01 from the National Institute of Environmental Health Sciences (U.S.A.) and by E.C. Environmental Research Programme, contract no. C30-74-1-ENVN.

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305

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