Naphthofurans induced chromosomal aberrations detected in metaphase, anaphase and telophase V79 Chinese hamster cells

Mutation Research,

157 (1985) 53-62

53

Elsevier MTR 00980

Naphthofurans induced chromosomal aberrations detected in metaphase, anaphase and telophase V79 Chinese hamster cells * W . V e n e g a s 1,2, C. L a s n e 1 . . , R. L o w y a, j . . p . B u i s s o n 3 a n d I. C h o u r o u l i n k o v l lnstitut de Recherches Scientifiques sur le Cancer, P.O. Box 8, 94802- Villejuif C~dex (France), 2Departamento de Biologia Molecular, Facultad de Ciencias Biologicas, Unioersidad de Concepcion (Chile), and 31nstitut Curie, 26, rue d'Ulm, 75231- Paris C~dex 05 (France)

(Received25 April 1984) (Revision received17 January 1985) (Accepted 18 January 1985)

Summary The mutagenic activities of 5 newly synthesized naphthofurans were analysed in two in vitro cytogenetic assays: the metaphase chromosomal aberration assay and the anaphase telophase bridge-fragment assay. Both assays were conducted using V79 Chinese hamster cells. The compounds included: 2-nitro-7methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitro-naphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The cells were treated with 3 concentrations (0.1, 0.2 and 0.4 # g / m l ) of each compound, in the dose range already tested in studies on the mutagenic properties of the same compounds realised with other systems. The highest concentration, only, was used in the anaphase-telophase assay. In the first approach, compounds A, B and C were active while compounds D and E did not increase significantly the aberration frequency above that of the DMSO controls. The results were confirmed in the second approach. They demonstrated that the two studies were complementary. Based on their genotoxic activities, the 5 compounds were ranked in the following decreasing order of potency: A = B >> C > D = E = DMSO; which is comparable to the ranking order obtained in different in vitro mutagenic and carcinogenic assays. All these activities are closely related to the highly specific molecular structure of each compound, particularly to the nature and position of the different substituents introduced on the skeleton.

Nitrofurans exhibit antibacterial properties, which make them useful as human and veterinary medicinals and food additives. These effects are amplified in the nitrobenzofurans, while the naphthofurans are more active still. These antibacterial * This investigation was supported in part by the European Communities Commission: Contract No. CEE-ENV, 545 F(RS), by the Association de RecherchesScientifiquesPaul Newman, and by the Department of Molecular Biology, University of Concepcion(Chile). ** To whom correspondenceshould be adressed.

properties have been shown to vary according to the position of substituents on the molecular skeleton of each nitrofuran derivative. In order to contribute to the elucidation of the structure-activity relationship, we have chosen 5 newly synthesized naphthofuran derivatives (Royer and Buisson, 1980a, b) (see Fig. 1). The mutagenic activities of some of these compounds have been studied already in bacteria, in fungi and in some in vitro mammalian assays. 3 of the chemicals are efficient inducers of in vitro micronuclei (MN) and sisterchromatid exchanges (SCE) (Lasne et al., 1984)

0165-1218/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)

54

CH30~NO2~NO2 2-nltro-7-methoxy-naphtho I 2,1.blfuran (RT00O).

2-nit r o- 8 -me t h o x y - n a p h t h o I 2.~-b~furan,

A

B

~NO2 BRUNO2 CH30"~

2-nltro-na p t h t h o l

using the traditional metaphase and anaphasetelophase analysis, compared with their other biological activities, and on the relationship between all of the properties studied and the molecular structure of each compound.

2 ,l-b] fur~n,

2 _nit ro_ 7 .bromo_ n ~ p h t h ° ~ 2,1.b ~furan.

C

D

7 - m e t h o x y ~ n a p h t h o i 2,1- b Ifuran,

E

Fig. 1. Structure of naphthofurans.

and are mutagenic in Salmonella typhimurium (Weill-Thevenet et al., 1981, 1982), in Saccharomyces cerevisiae (Averbeck et al., 1982), and in mammalian V79 Chinese hamster cells (Venegas et al., 1984). Moreover, they are probably carcinogenic as they induced cell transformation and gave positive results in the in vivo mouse short-term skin test (Venegas et al., 1984). The considerable variation in the mutagenic activities was found to be closely related to the specific molecular structure displayed by these derivatives. As cytogenetic assays have widespread use in mutagenicity testing (Abe and Sasaki, 1977; Ishidate and Odashima, 1977; Hollstein et al., 1979; Preston et al., 1981), we have analysed the genotoxic activities of the 5 compounds in the metaphase aberration assay. This assay detects structural chromosomal changes (Evans, 1976; I nui and Taketomi, 1977; Evans and O'Riordan, 1977; Ishidate et al., 1981), the relevance of which is well established (Nicoloff and Gecheff, 1976; Savage, 1980; Latt et al., 1981; Scott et al., 1983). To support the results obtained in the metaphase analysis, we have also employed the anaphasetelophase-bridge-fragment assay, which in addition to detecting structural aberrations, is used to assess mitotic poisons (Nichols et al., 1977; Dulout et al., 1982; Parry et al., 1982). This paper reports on the clastogenic activity of 5 naphthofurans in V79 Chinese hamster cells,

Materials and methods

Chemicals The compounds 2-nitro-7-methoxynaphtho[2,1b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitro-naphtho[2,1-b]furan (C), 2-nitro-7bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E) were provided by Dr. Royer (Fig. 1). Their synthesis, purity and other physico-chemical characteristics have been described in detail (Royer and Buisson, 1980a; Duquesne et al., 1983). All compounds were added to the medium as a solution in dimethyl sulfoxide (DMSO) to give a final concentration of 0.50%. In order to compare the order of potency of the 5 compounds 0.1, 0.2 and 0.4 # g / m l were the concentrations chosen per each compound, within the dose range tested previously in the other tests. Cells The V79 Chinese hamster lung cells were obtained from IARC (Lyon, France). For each experiment, cryopreserved cells from the same batch were seeded in 100-mm plastic petri dishes (Falcon) using 10 ml Eagle's MEM (Eurobio, Paris, France) supplemented with 10% fetal calf serum (Gibco, Flobio, Courbevoie, France) and 1% antibiotics from a 5000 units/ml penicillin-streptomycin solution (Gibco). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO 2 in air, then subcultured once before use. Metaphase preparations. After subculturing, one million cells were seeded per 100 mm X 20 mm plastic petri dishes (Falcon) in 10 ml complete medium. 18 h later, two sets of actively growing cells were submitted to I of the 3 concentrations of each compound or solvent DMSO alone for 13 h. This period covers one cell cycle as estimated previously (Renault et al., 1982). Another set of cells was treated in G2. For this purpose, the compounds A, B and C as well as solvent alone were added to the cells at a concentration of 0.4 # g / m l for 1 h prior to sampling. Colchicine (Col-

55 chineos, Houdr, France) at a final concentration of 5/~g/ml in the medium was added 1.5 h before the cells were collected by trypsinization and centrifugation. Then, the cells were treated for 15 min at 37°C to a hypotonic treatment consisting of fetal calf serum in distilled water ( 1 : 6 v/v). Next, the cells were fixed in acetic acid-methanol (1 : 3) for 10 min, then spread on slides and stained with Giemsa (4% in phosphate buffer M / 1 0 pH 6.7). For each substance tested, 100 metaphases per concentration were scored without knowledge of the chemical treatment. For aberration analysis, only cells with 22 centromeres were examined. All aberrations were scored separately, including gaps which were taken into account in the calculation of the total number of chromosome aberrations, and were subdivided into chromosome-,:ype and chromatid-type (Evans and O'Riordan, 1977; Savage, 1977; Preston et al., 1981; Scott et al., 1983). The frequency of the different aberration types and the total frequency of aberrations were determined per 100 metaphases observed. The frequency of metaphases with aberrations was also determined as well as the distribution of the aberrations in each metaphase. The mitotic index (M.I.) was determined for the same preparations by scoring the number of dividing cells among 1000 cells. A naphase-telophase (A • T) preparations. Cells were grown directly on coverslips in 30 mm x 10 mm petri dishes. 2 x 105 cells in 2 ml complete medium were seeded per plate containing a 22 m m x 22 mm sterile coverslip. 18 h later, the cells were treated in their logarithmic growth phase, with the highest concentration (0.4 # g / m l ) of each compound tested in the metaphase aberration assay and with solvent alone. After 13 h of incubation at 37°C, cultures were fixed by adding an equal volume of acetic acid (1:3) to the culture medium for 10 min. Then, the cells were rinsed twice in fresh fixative, the coverslips removed, air-dried, and stained as previously described, before being mounted onto slides with a drop of Eukitt (Kindler, Germany) mounting medium. Two slides were prepared for each compound concentration, and 100 A . T cells per slide were scored without knowledge of the treatment to determine the frequency of the different aberration types, the total frequency of aberrations and the number of

cells with aberrations. Bridges, acentric fragments and multipolar figures were recorded separately even when a cell contained more than one abnormality. The same preparations were also used for determination of the M.I. by counting 2000 cells. The repartition of the different mitotic stages: prophase, metaphase, anaphase and telophase was scored by counting 500 mitotic cells for each compound treatment.

Statistical analysis For chromosomal aberrations scored in metaphase preparations, a common linear regression technique was used to demonstrate dose-response relationships. The slope found for each compound was also compared to that of the others. A chi-square analysis was employed to analyse the influence of the various compounds on the mitotic division stages, the distribution of abnormalities, and the mitotic index. Finally, the total number of abnormalities per cell, under the influence of the various compounds, was studied using Student's t-test. All these statistical methods can be found in Snedecor and Cochran (1967). Results Table 1 gives the results of the chromosomal aberration analysis in metaphase preparations. The scoring categories included deletions, rings, dicentrics, exchanges and gaps (Fig. 2). In the control group, 5% of the cells exhibited chromatid gaps only, which are generally treated separately in chromosomal aberration analysis. The compounds A, B and C produced chromosome-type and chromatid-type aberrations, all kinds of chromatid-type aberrations being produced by these 3 compounds. In all cases, the n u m b e r of chromatid-type aberrations was higher than the number of chromosome-type aberrations. As shown in Fig. 3, the naphthofurans A, B and C produced a significant dose-responsive increase in metaphase aberrations (for A: p < 0.02; B: p < 0.01; and C: p < 0.05), compounds A and B being more effective inducers of chromosomal aberrations than compound C. The number of aberrations per abnormal metaphase was dependent on the derivatives and concentrations employed (Fig. 4). As shown in Table 1, compound D produced

~

~I~ ~

57 TABLE 1 TYPES AND FREQUENCIES OF CHROMOSOME ABNORMALITIES INDUCED IN V79 METAPHASE CELLS BY 5 NAPHTHOFURANS Compounds

Concentration ( # g/ml)

M.I. a

Chromosome type Gb D R

DMSO

0.5%

4.5

R-7000 A

0.1 0.2 0,4

3.4 2.4 1.8

2 7 32

R-6998

0.1 0.2 0.4

3.2 2.3 1.45

5 7 16

0.1 0.2 0.4

3.8 3.1 3.7

R-7160 D

0.1 0.2 0.4

3.8 3.9 3.7

R-7187 E

0.1 0.2 0.4

4.4 4.0 4.2

B

R-6597 C

Di

Chromatid type G D E 5

5

5

31 95 249

22 60 88

3

1

23

8

39 90 214

32 56 80

1

22 42 53

22 34 39

8 15 23

08 15 21

5 7 8

4 7 8

1 5 35

31 78 161

3

2 6 8

19 34 43

1 2 1

1

8 13 20

2 2

2

5 7 6

2 2

5

1

1

Cells (%) with C.A. c

1 4 4

27 75 163

1 13

Aberrations per 100 metaphases

For all compounds, 100 metaphase cells were scored for each concentration. a M.I., mitotic index, % dividing cells/2000 cells scored for each concentration. b G, gaps; D, deletion; R, ring; Di, dicentrics; E, exchanges. c C.A., chromosome aberrations.

o n l y c h r o m a t i d - t y p e aberrations, the great m a j o r ity of them being gaps, which are r e s p o n s i b l e for the d o s e - r e s p o n s e relationship ( p < 0.01) o b served. G a p s are n o t well a c c e p t e d as criteria for genotoxic effects a n d are generally c o n s i d e r e d as u n r e l i a b l e i n d i c a t o r s of real d a m a g e (Evans a n d O ' R i o r d a n , 1977; Preston et al., 1981); conseq u e n t l y we c o n c l u d e d that c o m p o u n d D was n o t clastogenic. C o m p o u n d E d i d n o t increase signific a n t l y the f r e q u e n c y of m e t a p h a s e a b e r r a t i o n s over the solvent control level. In c o m p a r i n g the slopes of the d o s e - r e s p o n s e relationships, using the n u m b e r of a b e r r a t i o n s p e r 100 cells (Fig. 3), it is clear that c o m p o u n d s A a n d B, on the one hand, a n d c o m p o u n d s D a n d E on the other, d i d not significantly differ from each other. In a d d i t i o n , the values f o u n d for increasing c o n c e n t r a t i o n s of the D a n d E c o m p o u n d s were n o t different from that f o u n d for the D M S O control. Lastly, the slopes o f c o m p o u n d s A a n d B were very significantly different ( p < 0.01) f r o m

that f o u n d for c o m p o u n d C, a n d the slope of c o m p o u n d C was significantly different ( p < 0.05) f r o m the zero slope values o b t a i n e d with c o m p o u n d s D a n d E. Thus, the five n a p h t h o f u r a n s m a y be r a n k e d in the following o r d e r of p o t e n c y : A = B >> C > D = E = D M S O . A s o b s e r v e d in T a b l e 1, the M.I. was progressively r e d u c e d in a c o m p o u n d - a n d - c o n c e n t r a t i o n d e p e n d e n t m a n n e r . A clear d o s e - r e s p o n s e relationship was o b s e r v e d with c o m p o u n d s A a n d B which gave the m o s t toxic effects, the r e d u c t i o n in M.I. being associated with an increase in clastogenic activities. Results of a n a p h a s e - t e l o p h a s e analysis are presented in T a b l e 2. T h e types of a n a p h a s e - t e l o p h a s e a b e r r a t i o n s that c o u l d be identified i n c l u d e d acentric f r a g m e n t s which c o r r e s p o n d to single or p a i r e d c h r o m o s o m e fragments, acentric f r a g m e n t s which are a t t a c h e d to the m a i n b o d y b y an a t t e n u a t e d fragment, single o r d o u b l e bridges, bridges plus acentric f r a g m e n t s a n d m u l t i p o l a r spindles with or

58 250

~A

"B

-~ 200

o 150 o~

:=- 1oo

~, 50

"

"~

D

0.1

0.2

pg/ml

0,4

Fig. 3. Chromosome aberrations induced by 5 naphthofurans in V79 cells. Abnormalities included chromosome- and chromatid-type aberrations. Data from 100 metaphases were scored. Straight lines were fitted by linear regression analysis, r = 0.955 (A); 0.979 (B) and 0.962 (C) with p < 0.05; r = 0.878 (D and E) not significant.

without bridges (Fig. 2). Compounds A and B were the most active, followed by compound C, then by compounds D and E. Whether the data are expressed as the number of chromosome

abnormalities per 100 cells or as the number of abnormal cells, the effects produced by compounds A and B did not differ significantly from each other (p > 0.05)• Nevertheless, compound A included more acentric fragments and less bridges than compound B (p < 0.01). Compared to the activity induced by compound C, the difference was highly significant (p < 0.001). However, the total number of aberrations produced by compound C was significantly higher than that produced by the control treatment (p < 0.001). As expected, the M.I. was affected particularly (p < 0.001) by compounds A and B. Concomitantly to the mitotic inhibition, the frequency of mitotic division stages was affected, as shown in Fig. 5. In comparison to the solvent control, compounds A and B affected significantly the different mitotic division stages (p < 0.01), increasing significantly the telophase frequency (p <0.01) at the expense of the metaphase and prophase frequencies (p < 0.01), while the number of anaphase cells remained almost constant. The effects produced by these two compounds A and B did not differ significantly (p > 0.05) from each other• Compounds C, D and E did not affect signifi-

~ug/ml •

,

,

.

.

.

.

.

.

.

.

.

.

.

L

o; 301

0,4

~ug/ml

20.

. . . . . . . .

,

.

,

.

LLLL

=~ o,2 ~ ~g/ml

4O

.

20

oL

30

C

20

0t

ot

30

D

20

30

E

ao

T

10

1'2"3'4

•

No. of a b n o r m a l i t i e s p e r metaphase Fig. 4. Distribution of metaphase aberrations in 100 abnormal metaphases from control and naphthofuran-treated V79 cells.

59 TABLE 2 TYPES A N D F R E Q U E N C I E S O F A N A P H A S E - T E L O P H A S E ( A . T ) C H R O M O S O M E A B N O R M A L I T I E S I N D U C E D IN V79 CELLS BY 5 N A P H T H O F U R A N S

Compounds

M.I. a

0.4/tg/ml

Control

4.7

N u m b e r of A . T cells

A - T abnormalities %

scored

bB

Ac

100

0 0

100

Mean number of abnormalities %

CL

MF

3 3

0 0

0 0

3

3

10 14.5

A

1.6

100 100

12 8

32 35

1 2

3 3

B

1.3

100 100

14 15

26 28

0

5

2

3

B

Ac

Total A - T

abnormalities mean (%)

A - T cells (%) cells

abnormalities

CL

MF

0

0

3

3

33.5

1.5

3

48

41

27

1

4

46.5

40.5

14.5

0.5

0.5

20.5

18.5

C

3.5

100 100

6 4

13 16

1 0

0 1

5

D

3.7

100 100

1 0

2 4

0 0

0 0

0.5

3

0

0

3.5

3.5

E

4.1

100

0

3

0

0

0

2.5

0

0

2.5

2.5

100

0

2

0

0

a M.I., mitotic index: % dividing cells per 2000 cells scored. b B, bridge; Ac, acentric fragments: CL, chromosome lagging; MF, multipolar figures: tripolar and tetrapolar figures with/without Ac or B.

cantly the level of either frequencies ( p > 0.05). The decrease in prophase and metaphase cells reflects the toxic effect of the substances and the accumulation of telophase cells could be explained by an increasing delaying effect of the compounds on the cell cycle, the duration of the telophase division stage being lengthened.

so

L. 2S

Z I

DM~O

A

B

C

D

E

Fig. 5. Incidence and distribution of mitotic division stages in V79 cells treated with 5 naphthofurans. Incidence is expressed as the percentage of cells in each division stage. For each compound A, B, C, D, E and controls (DMSO), each bar represents the percentage of cells in prophase ( P ) B , metaphase (M)I---L anaphase (A) ~ and telophase (T) [ I ~ , 500 mitotic cells were scored for each treatment.

Discussion Our results demonstrate that chromosome damage could be induced by some naphthofurans and detected both in the traditional metaphase aberration assay as well as in the anaphasetelophase aberration assay, as the results of both assays were in very good agreement. The 5 naphthofuran derivatives gave the same ranking response in the 2 assays employed to estimate their clastogenic activities. Compounds A and B were the most clastogenic substances, followed in decreasing order of potency by compound C then by compounds D and E, which were considered negative in both methods at the concentrations tested. Consequently, under our experimental conditions, 3 of the 5 nitrofuran derivatives induced chromosomal aberrations. Moreover, they were mitotic poisons, since they produced abnormal mitotic division stages, multipolar figures and chromosome lagging. Although the aim of this study was not to compare the two methods for scoring chromosomal aberrations, it is clear that each assay has particular advantages. The first approach measures the chromosome

60 "FABLE 3 C H R O M A T I D TYPE ABERRATIONS I N D U C E D IN G 2 PERIOD BY 3 N A P H T H O F U R A N S Compound

Treatment (/~ g / m l )

D a

G b

Total aberrations c

Number of cells with aberrations

% cells with aberrations

R-7000 A

0.4

16

74

90

48

0.9

R-6998 B

0.4

12

66

78

40

0.78

R-6597 C

0.4

0

21

21

19

0.21

DMSO

0.5%

0

2

2

2

0.02

a D, deletion. b G, gap. c 100 metaphases were scored for each compound.

TABLE 4 SUMMARY OF THE BIOLOGICAL ACTIVITIES OF 5 N A P H T H O F U R A N S DETECTED IN D I F F E R E N T MAMMALIAN ASSAYS Compound

Mutagenesis

Carcinogenesis

V79 cells C.A.

HGPRT

SCE

MN

Human Chinese lymphocytes(G0) hamster B.M.

Cell transformation

Mouse skin test

SCE

MN

SHE

C3H10T1/2

SGI.

Ep.Hy.

+

++

-

+

Met.

A.T

A

+++

+++

++

+++

++

++

+++

B

+++

+++

+++

+++

++

+++

NT

++

++

+(+)

++

C

+

+

+

+ +

+

+

+(+)

NT

NT

+ +

+ + +

D

-

NT

NT

NT

-

(+)

E

.

NT

NT

-

(+)

.

.

(+) .

(+) .

.

.

B.M., bone marrow; C.A., chromosome aberrations; Met., metaphase aberrations; A.T, anaphase-telophase aberrations; HGPRT, gene locus mutation; SCE, sister-chromatid exchanges; MN, micronucleus; SHE, Syrian hamster embryo cells; C3H10T1/2, mouse cell line; S.GI., sebaceous glands; Ep.Hy., epidermal hyperplasia; NT, not tested.

number and identifies various categories of acute breaks as chromosome and chromatid deletions and exchanges. The results, obtained at the highest concentration of 0.4/~g/ml of each of the 5 naphthofurans (Tables 1 and 2) in both assays, are in agreement with this statement that the metaphase assay is more sensitive than the other. The A - T aberration analysis, though not as definitive, allowed the analysis of the frequency of the different mitotic division stages, and of mitotic abnormalities. Some of these events may have resulted from disorganization of the centrioles and/or from depolymerisation of mitotic apparatus tubulin (Parry et al., 1982).

The reduction in the prophase figures would suggest that some of the cells were destroyed by the action of the compounds. Others, as suggested by Hsu et al. (1982) may have reverted to interphase. Naphthofurans were also suggested to render the cells fragile by modifying the proteins which form an integral part of the chromosomes and of the spindle apparatus (Hsu et al., 1982), thus leading to a blockage of cells in telophase. Events detected in one system sometimes escape detection in a different system. For example, the symmetrical exchanges are observable in the metaphase preparations, only. In contrast, isochromatid breaks with proximal sister reunion are not

61

accurately detected in this approach, whereas they always give rise to bridges detected in the A. T procedure. This difference in ascertainment can explain partly the appreciable number of unexpected bridges observed. Furthermore, multipolar spindles and lagging chromosomes which can result in abnormalities of chromosome number are easily visualized in anaphase. However, to quantify the frequency of aberrations it is necessary to employ the metaphase aberration analysis. For these reasons, it is concluded that both assays give complementary results. The nitrofurans, which have been studied before naphthofuran synthesis, cause essentially chromatid-type breaks in V79 cells (Klemencic and Wang, 1978) and were suggested to damage cells during the S phase of the cycle (Ebringer, 1972; Olive, 1978; Klemencic and Wang, 1978; McCalla, 1983) in a manner similar to that of alkylating agents (Natarajan and Obe, 1978; Natarajan et al., 1983). Naphthofurans which have a similar chemical structure might be expected to act in a similar manner. Thus, the significant formation of chromosome-type aberrations suggests that the latter compounds are S-independent clastogenic agents (Evans and O'Riordan, 1977; Hsu et al., 1982). As compounds A, B and C lengthened the cell cycle (Lasne et al., 1984) and as the S phase is more extended than G 1, we can deduce that the majority of the chromosome-type aberrations were produced during early S rather than in G 1. Chromatid-type aberrations were certainly produced during S, the most sensitive period of the cell cycle; G 2 is also implied, since compounds A, B and C induced chromatid-type aberrations in V79 cells treated for 1 h prior to sampling, as presented in Table 3. Moreover, we have demonstrated that these chemicals induce SCEs with a dose-response relationship in human lymphocytes treated in G O (manuscript in preparation). Thus, it seems that naphthofurans may induce chromosomal aberrations during the entire cell cycle. From Table 4, which summarizes the biological activities of the 5 naphthofurans, it is clear that the clastogenic activities strongly correlate with their genotoxic activities previously studied in V79 cells. The reported results confirm that the biological properties of compounds A, B, C, D and E are strongly dependent on their molecular structure

(Cavier et al., 1981). In fact, these genotoxic activities are related to the nitro group in position 2 (compound C). They are enhanced by the addition on the skeleton of a methoxyl group in position 7 or 8 (compounds A and B) and suppressed if the methoxy is replaced by a bromine in position 7 (compound D). The methoxy group is an "electro-donor" and the bromine an "electro-attractor" (Cavier et al., 1981). Compound E, the only one which does not carry a NO 2 group, was inactive in all assays. Thus, the presence of a 2-nitro group was demonstrated to be essential for the induction of all the genotoxic properties by this family of substances. The absence of the nitro group is associated with loss of antibacterial effects (Ichikawa, 1978). This is true for the furans, benzofurans and naphthofurans synthesized to date, and which do not carry a nitro group. They all fail to produce therapeutic effects and do not exhibit genotoxic and oncogenic properties. In conclusion, the mutagenic response measured in cytogenetic assays varied with the nature and the position of the substituents on the skeleton.

Acknowledgements We are grateful to Dr. R. Royer for critical discussion and for helpful advice. We wish to thank M.T. Maunoury for help with statistics, and F. Maugain and L. Guglielmi for preparation of the manuscript.

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