Sister-chromatid exchanges and chromosomal aberrations in cultured chinese hamster cells treated with pesticides positive in microbial reversion assays

177

Mutation Research, 78 (1980) 177--191

© Elsevier/North-HollandBiomedicalPress

SISTER-CHROMATID EXCHANGES AND CHROMOSOMAL ABERRATIONS IN CULTURED CHINESE HAMSTER CELLS TREATED WITH PESTICIDES POSITIVE IN MICROBIAL REVERSION ASSAYS

H. TEZUKA,N. ANDO, R. SUZUKI,M. TERAHATA,M. MORIYA and Y. SHIRASU Toxicology Division, Institute of Environmental Toxicology,,2-772, Suzuki-cho, Kodaira-shi, Tokyo 187 (Japan)

(Received 5 November 1979) (Revision received 15 January 1980) (Accepted 16 January 1980)

Summary The induction of sister~hromatid exchanges (SCEs), chromosomal aberrations and polyploids was investigated in cultured Chinese hamster cells treated with pesticides or a related compound positive in microbial reversion assays. The chemicals tested were captan, captafol, 1,2~libromoethane (EDB), 1,2-dibromo-3 ~hloropropane (DBCP), 5-nitro-l-naphthonitrile (NNN), p-dimethylaminobenzenediazo sodium sulfonate (DAPA), 2-hydrazinoethanol (HEH), vamidothion, dichlorvos (DDVP), N-nitroso~thylenethiourea (N-nitroso-ETU), and 2,4~linitrophenyl thiocyanate (NBT). A significant and dose~lependent increase in the frequency of SCEs and chromosomal aberrations was observed in the cell cultures treated with captan, captafol, EDB, DBCP, HEH, DDVP, vamidothion, DAPA or N-nitroso-ETU. Neither NNN nor NBT induced SCEs or chromosomal aberrations. Treatment with EDB, NNN, vamidothion, DDVP, N-nitroso-ETU or NBT produced a significant increase in the frequency of polyploid cells, whereas the other agents did not. When compared with results from microbial reversion assays, a close correlation was observed between the ability to induce SCEs or chromosomal aberrations and the mutagenic potency in bacteria (r: 0.71-0.84).

Various kinds of pesticide have been widely used in the world for a long time and will be in the future. Some of them have potent adverse effects such as mutagenicity, teratogenicity and carcinogenicity, and become potential hazards to human health. Investigation o f the safety of the pesticides and their related compounds should be done systematically by sensitive and reliable methods for detecting their harmful effects (Committee 17, 1975; Latt, 1974; Perry and Evans, 1975; Solomon and Bobrow, 1975).

178 In previous reports from our institute (Kada et al., 1974; Shirasu, 1975; Shirasu et al., 1976, 1977), nearly 200 pesticides were assayed for their mutagenicities in bacterial test systems; 17 of them were found to be mutagens. From them, 8 were selected for the present study in mammalian test systems in vitro because of their potent activities in inducing mutation. The chemicals studied were captan, captafol, NNN, DAPA, HEH, vamidothion, DDVP and NBT. There have been several cytogenetic studies on the 2 chemicals, captan and DDVP. Positive (Legator e t a l . , 1969) and negative (Tezuka et al., 1978) results were obtained on the cytogenetic effects of captan. No genetic effects were found with DDVP in mammalian cells in culture (Dean, 1972; Nicholas et al., 1978; Wild, 1975). The other 6 compounds have not been investigated for their clastogenicity in mammalian test systems. In addition to the 8 described above, 3 additional chemicals were tested. The first, N-nitroso-ETU, is the nitrosation product of ETU. ETU, a weak mutagen listed in the above 17 chemicals, showed no cytogenetic effects on cultured cells and animals (Teramoto et al., 1977). However, this compound, when nitrosated, turned out to have a strong mutagenic activity in bacteria and a clastogenicity in mammalian systems in vivo (Seiler, 1977; Shirasu et al., 1977; Teramoto et al., 1978) and to induce pulmonary and lymphocytic tumors in mice (Moriya et al., 1979). Its genetic effects in cell cultures in vitro have remained unknown. The other 2 chemicals tested were nematocides, EDB and DBCP. These 2 chemicals, not included in the above 17 compounds, were recently shown to induce point mutations in microorganisms (Brem et al., 1974; Rozenkranz, 1975; Scott et al., 1978) and stomach cancer in rats and mice (Olson et al., 1973). Cell mutation studies on EDB in the mouse lymphoma system (Clive et al., 1979) and dominant lethal studies on DBCP in rats (Teramoto et al., 1980) also gave positive results. However, there have been no cytogenetic studies available in mammalian test systems in vitro. In the present paper we report the results on the ability of these 11 chemicals to induce SCEs, chromosomal aberrations and polyploids in cultured Chinese hamster cells. The possible correlation between the mutagenic potency of the chemicals in bacteria and their ability to induce SCEs or chromosomal aberrations in cultured cells is discussed. Materials and methods Chemicals The chemicals were obtained from the following sources: Captan, purity 99.9%, Nishio Industry Co. Ltd., Japan; Captafol and DDVP, purity >98.0%, Wako Pure Chemicals Co. Ltd., Japan; NNN, NBT and vamidothion, standard samples, the Agricultural Chemicals Inspection Station of the Ministry of Agriculture, Forestry and Fisheries, Kodaira-shi, Japan; EDB, purity >99%, HEH, purity >95%, and DAPA, quality for special use, Tokyo Kasei Industries Co. Ltd., Japan. DBCP and N-nitroso-ETU were prepared in this institute. The methods of synthesizing these 2 compounds have been described elsewhere (Teramoto et al., 1978, 1980). No impurities were found by ECD gas-chromatographic analysis in the 2 chemicals. The structural formulae of these chemicals are shown in Fig. 1. Uses of the pesticides are as follows: captan,

179

Captan

(301) O II

O I!

Cl

q,.UJ

-s-c-c1

,

l

(188)

DBCP

Br--C--C--Br I

H HEH

NO

j i CI C1

H

H

H

I I

! [

! [

H Br H Vamidothion

(287)

CH 30\~ O[! sH P--S--CH 2CH 2SCHCN..

NH2

(221)

CHsO~

DAPA

(225)

/

(236)

H

CH,O%~P--O--CH=CCI2 CH~O I

NBT

II 0

I

H[ H[ /H HO--C--C--N%

DDVP

N--S--C--CH

Br--C--C--C--CI

(76)

H| HI

C1 Cl

i

<..~c

Cl

II 0

EDB

(349)

Captafol

NNN

SCN NO2

~Hs

CHs

(251) CH,k~N=NSOsNa CH3 /

(198)

N-nitroso-ETU

(131)

i

H-- - - N ~ _ s H-C-NI I H

I H

Fig. I . Stzuetttval formulae of chemicals. The number in parentheses is the value of the molecular weight

of the chemical.

captafol, N N N , NBT and DAPA as fungicides; EDB and DBCP as nematocides and soil fumigants; HEH as a plant-growth regulator; DDVP and vamidothion as insecticides. EDB and D D V P are also used as space fumigants.

Cell cultures Because various karyotypes and numbers of chromosomes (20--22) were lffresent in the original Chinese hamster V 7 9 cell line, a clone of No. 15 (22 chromosomes) was selected and' routinely cultured at 37°C in an atmosphere having 5% CO2. There were no significant differences between the cells of this clone and the original cells with respect to the following parameters: population doubling time (14--15 h), plating efficiency (95--100%), frequency of chromosomal aberrations (0--0.05 per cell) and polyploid cells (0--3%). The growth medium consisted of Eagle's MEM supplemented with 2 mM giutamine and 10% fetal bovine serum, and it contained no antibiotics.

Cytogenetic study A b o u t 24 h after 3 × l 0 s cells were seeded into the cultures (~ 60 m m plate, Falcon), test chemicals freshly dissolved and diluted with dimethylsulfoxide (DMSO) were added to the cultures so that the final concentration of DMSO

180 was 1% irrespective of the concentration of the chemical being tested. 5-Bromodeoxyuridine (BrdUrd) at a final concentration of 2 /~M was added immediately to each culture under a yellow safety lamp, and the cultures were incubated for 26.5 h in complete darkness. With N-nitroso-ETU , a further experiment was performed to examine whether this agent could induce both SCEs and chromosomal aberrations during a 16-h exposure. For most doses of each chemical, duplicate cultures were set up. Control cultures containing DMSO and BrdUrd were prepared for each series of experiments. Colcemid was added to a concentration of 0.1 pM at 2 h before the cells were harvested. Chromosome preparations were made according to the routine air-drying m e t h o d after h y p o t o n i c treatment with 0.075 M KC1 for 8 min and subsequent fixation with methanol--acetic acid (3 : 1). The m e t h o d of differentiating sister chromatids was essentially that described by Perry and Wolff (1974) and Wolff and Perry (1974). The n u m b e r of SCEs per cell was examined in cells with all the chromatids clearly differentiated. Scoring of chromosomal aberrations followed the m e t h o d of Cohen and Hirschhorn (1971). Cells with gaps alone were n o t evaluated as cells with chromosomal aberrations. As a rule, 50 metaphases were scored for detecting SCEs, and 100 metaphases for chromosomal aberrations at each concentration. Frequencies of SCEs per cell in treated cultures w e r e compared with those in the control b y t-test. Fisher's exact probability test or chi-square test was used for the frequencies of cells with chromosomal aberrations and for those of polyploid cells. Induced SCEs per cell per /~M was calculated from the formula: (SCEs per cell in test cultures minus SCEs per cell in control cultures) divided b y (concentration of the chemical in/~M). The induced frequency of cells with chromosomal aberrations per #M was calculated from the formula: (frequency of cells with aberrations in test cultures minus frequency of cells with aberrations in control cultures) divided by (concentration of the chemical in pM). Results

The results of the study on the induction of SCEs, chromosomal aberrations and polyploids in cell cultures exposed to the chemicals for 26.5 h are shown in Tables 1 and 2. The mean + standard error of the frequency of SCEs was 5.2 + 0.1 per cell and the range was from 4.1 to 6.7 per cell in 22 control cultures of 10 independent experiments. The frequency of cells with chromosomal aberrations in control cultures ranged from 0.8% to 4.5% and the chromosomal aberrations observed were essentially of the chromatid type, gaps and breaks. The frequency of polyploid cells, mostly tetraploids, did n o t exceed 2.6%. Marked increases in the frequency of SCEs, chromosomal aberrations and polyploids were observed in some of the cultures treated with the chemicals for 26.5 h, and the induced aberrations were again essentially of the chromatid type, including chromatid and isochromatid gaps and breaks, and chromatid exchanges. A small n u m b e r of cells with stickiness was observed in cell cultures treated with captan or DBCP, b u t the frequency was n o t dose related. Many cells with stickiness were observed in cell cultures treated with vamidothion at 1 X 10 -2 M. Cells with stickiness are those with chromosomes exhibiting different morphology and that have adhesions between t w o or more chromo-

181

TABLE 1 FREQUENCY OF SIS~ER-CHROMATID CHEMICALS FOR 26,5 h

EXCHANGES

IN CELL C U L T U R E S

TREATED

SCEs/eell ( m e a n ± SE)

WITH

Induced SCEs/ cell//~M

Expt. No.

Chemical

Concentration (M)

Number of cultures

Number o f cells observed f o r SCEs

1

Control

0

4

200

1

Captan

6 1.6 3 4.5 6

X 1 0 -6 × 10 -5 X 1 0 -5 X 10 -5 X 10 -s

2 2 2 2 2

50 50 50 50 50

6.6 11.2 15.0 21.1 24.9

± ± ± ± ±

0.5 0.8 0.7 1.1 1.1

c c e e e

0.43 m 0.34 0.36 0.34

2 5 1 1.5 2

X 10-6 X 10-6 X 10-5 X 10-5 X 10-5

2 2 2 2 2

50 50 50 50 2O

8.8 15.7 28.6 38.2 45.3

± ± ± ± ±

0.6 1.3 1.7 1.9 1.9

c c e c c

2.20 2.39 m 2.23 2.03

c e e e

0.018 m 0.016 0.011 0.0082

1

Captafol

4.7 ± 0 . 2

2

Control

0

4

200

2

EDB

5 1 2 5 1

X 1 0 -4 X 10 -3 X 10 -3 X l O -3 X 1 0 -2

2 2 2 2 2

50 50 50 15

15.2 21.9 28.4 46.9

± ± ± ±

2

DBCP

1 5 1 2 5 1

X 10-5 X 1 0 -$ X 10-4 X 10 -4 X 1 0 -4 × 1 0 -3

2 2 2 2 2 1

50 50 50 50

7.3 15.5 37.2 58.4

± 0,4 b ± 0.7 c ±'i.3 c ± 1.7 c

2

100

2 2 2 2

50 50 50 50

2

100

2 3 3 3

50 75

2

100

2 2 2 2

50 50 50 50

3

Control

0

3

NNN

1 3 1 3

4

Control

0

4

DAPA

5 1 5 1

X 1 0 -5 X 10-5 X 10-4 X 10-4

X 10-5 X 10-4 X 10 -4 X 1 0 -3

5

Control

0

5

HEH

5 1 2 5

X 10 -$ X 10 -4 X 10-4 X 10-4

8

X 10-4

6

Control

0

6

Vamidothion

1 2 5 1

7

Control

0

7

DDVP

1 2 5 1

0.6 0.8 0.9 2.2

0.19 0.31 m 0.26

4.3 ± 0.2 4.0 5.5 5.2 4.5

± ± ± ±

0.3 0.3 b 0.3 a 0.3

4.8 ± 0.2 7.3 ± 0 . 4 c 11.5 ± 0.5 c

0.067 m

5.3 ± 0 . 2 6.7 7.9 13.0 16.8

± ± ± ±

0.4 0.5 0.8 0.7

b c c c

0.039 m 0.023

2 2

I00

1 0 -3 l O -3 10 -3 10 -2

2 2 2 2

50 50 32

2

100

X 10-4 X 10-4 X lO-4 X l O -3

2 2 2 2

50 50 50

X X X X

6.1 ± 0 . 2

4.8 ± 0 . 2 12.4 ± 0.6e 19.9 ± 0.7 c 3 9 . 9 ± 1,7 c

0.0076 m 0.0076 0.0070

5.1 ± 0 . 2 8.6 ± 0 . 4 c 12.0 ± 0.5 c 24.7 ± 0.8 e

0.035 0,039 m

182

TABLE 1 (continued) Expt. No.

Chemical

Concentration (M)

Number of cultures

Number o f cells observed for SCEs

SCEs/cell ( m e a n _+ S E )

Control

0

2

100

8

N-Nitroso-ETU

5 1 2

× 10 -5 X 10 -4 × 10-4

2 2 2

50 50 50

5

X 10-4

2

--

--

1

X 10 -3

2

--

--

4.1 • 0 . 2 7.3 + 0 . 4 c 11.4 ± 0.6 c 19.5 + 1.5 c

9

Control

0

2

100

9

NBT

5 1

× 10 -5 X 10 -s

2 2

50 50

2

X I 0 -s

2

--

--

5

× 1 0 -5

2

--

--

2

100

6 . 0 _+ 0 . 3

× 10-5 X 10-5 X 1 0 -5 X 10 -s

2 2 3 2

50 50 75

5.5 ± 0 . 4 5.8 + 0 . 4 6 . 8 -+ 0 . 4

Control

0

10

NBT

2 5 1 2

--

SCEsl cell//~M

8

10

Induced

0.073 0.077 m

6.7 + 0.2 6.4 + 0.4 8.6 ± 0.5 c

--

a,b a n d c S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l at P < 0 . 0 5 , P < 0 . 0 1 a n d P < 0 . 0 0 1 , resp. m M a x i m a l value o f i n d u c e d S C E s / c e l l / D M .

somes. The polyploid cells induced were triploids and tetraploids. A chemical was evaluated as an inducer of SCEs, chromosomal aberrations or polyploids when the frequency of these indices was dose-related and more than twice the frequency in the control. Taking the sample size into consideration, the following values were thought to be positive: (i) more than 10.4 per cell in the frequency of SCEs, (ii) more than 9.0% in the frequency of cells with chromosomal aberrations, and (iii) more than 5.2% in the frequency of polyploids. The maximal values of induced SCEs per cell and of induced frequencies of cells with aberrations were calculated from these values. According to the criteria mentioned above, captan, captafol, EDB, DBCP, DAPA, HEH, vamidothion and DDVP were inducers of both SCEs and chromosomal aberrations. Although a slight increase in the frequency of SCEs and aberrations was observed in NBT-treated cells, no reproducibility was observed. N N N induced neither SCEs nor aberrations. N-Nitroso-ETU was also an inducer of SCEs. However, the sample size of cells with 22 chromosomes in some of the test cultures was too small for the detection o f a significant increase in the frequency of cells with aberrations because of a marked increase in the frequency of tetraploids in cultures treated with N-nitroso-ETU for 26.5 h at the concentration of 5 × 10 -2 M. Because dicentrics and acentric fragments were observed in induced tetraploid cells, a further experiment on the induction of chromatid aberrations by N-nitroso-ETU in cells with an intact number of chromosomes was performed in cell cultures added with the compound 10.5 h after exposure to BrdUrd and then incubated for an additional 16 h in the presence of BrdUrd. The results of the experiment are shown in Table 3. A significant and dose-

183

dependent increase in the incidence of SCEs and chromosomal aberrations was found. The aberrations induced were essentially chromatid gaps, breaks and exchanges. N-Nitroso-ETU was also an inducer of b o t h SCEs and chromosomal aberrations. In the present study on the 11 test compounds, the chemicals that induced SCEs also induced chromosomal aberrations. A linear dose--response relationship was observed between the logarithm of the frequency of SCEs and that of the initial concentration of the chemical, as shown in Fig. 2. When the relative ability to induce SCEs and chromosomal aberrations was compared by logarithm of their maximal value per pM, a close correlation was observed (r: 0.96), as shown in Table 4. When these results were compared with the results obtained from microbial reversion assays, a close correlation was also observed between the logarithm of maximal value of the appropriate parameters of the chemicals (r: 0.71--0.84), as shown in Table 4. A significant increase in the frequency of polyploid cells was observed in cell cultures treated with EDB, NNN, vamidothion, DDVP, N-nitroso-ETU and NBT for 26.5 h. A significant increase in the frequency of polyploids was observed in some of the captafol-treated cultures, b u t the frequency was n o t dose-related. The other c o m p o u n d s did n o t induce polyploids. The inducer of polyploids may have no relation to the inducer of SCEs or chromosomal aberrations. Discussion

All 9 chemicals that induced both SCEs and aberrations, namely captan, captafol, EDB, DBCP, DAPA, HEH, vamidothion, DDVP and N-nitroso-ETU, were base-pair-substituting mutagens (Brem et al., 1974; Rosenkranz, 1975;

loo 70 50

•

o~ ' - ~ /

.

/

--~/

3O

.~

20

s.<,/

/I

/8 /

/

xo~,

zo 7 ~

/

~

s

I

i~ /

C~./

T

,?~ I

/

~

/"

~"

~

Control range

2

_

I

L

I

i I Is,I

10-5

,

,

s

L

,

,x,I

10 -4

,

~

,

, ,,,,i

,

,

,

, ,,,

10 -3

Concentration (M) Fig. 2. Dose--response relationships b e t w e e n the frequency of SCEs in cultured cells a n d the Initial concentration of each chemical. The lines in the figure were drawn by eye. Each point represents the mean ± standard error.

Control

NNN

Control

DAPA

3

4

4

DBCP

2

3

Control

Captafol

1

EDB

Captan

1

2

Control

1

2

Chemical

Expt. No.

5 1

0

1 3 1 3

0

1 5 1 2 5 1

5 1 2 5 1

0

2 5 1 1.5 2

6 1.5 3 4.5 6

0

10-5 1 0 -5 10 -4 10-4

1 0 -5 10-5 10 -4 10-4 10-4 1 0 -3

1 0 -4 10 -3 10 -3 1 0 -3 1 0 -2

1 0 -.6 10 -6 10-5 10-5 10-5

× 10-5 X 10-4

X X X X

X X X X X X

X X X X X

X X X X X

X 1 0 --6 X 1 0 -`5 X 10-5 X 1 0 -5 X 1 0 --5

Coneentration (M)

100 150

200

100 100 100 100

400

100 100 100 100 100 50

100 100 100 68 -- g

400

100 100 100 100 50

100 100 100 100 100

400

Total numbez o f cells observed

1 3

3

2 2 3 2

3

6 6 9 14 50 46

.

d • (1) f • e

1 5 16 e 48 e .

7

2 3 24 e 28 • 24 e

1 4 7 2 3 e (5) f 28 e (2) !

14

Number

1.0 2.0

1.5

2.0 2.0 3.0 2.0

0.8

6.0 6.0 9.0 14.0 50.0 92.0

1.0 5.0 16.0 70.6 .

1.8

2.0 3.0 24.0 28.0 48.0

1.0 4.0 7.0 23.0 28.0

3.5

(%)

0 0

1

0 0 0 0

0

0 0 0 0 4 32

0 0 0 26 .

0

0 0 8 12 24

0 0 2 16 13

0

Cells with 10 or more aberrations (%)

.

4 1

4

4 5 2 1

5

1 2 8 11 33 33

3 3 2 16

9

2 0 6 5 5

5 4 4 2 14

6

.

edg

0 0

0

0 2 0 0

1

1 1 1 2 6 2

0 1 5 4

4

0 1 3 5 0

0 1 1 0 3

1

.

icdg

1 3 1 14 .

1

0 1 7 9 8

2 0 1 4 11

5

cdb

1 2

1

0 1 0 0

0

4 4 1 5 19 30

Cbcomatid type

Aberrations b

0 0

2

1 1 4 2

3

3 1 5 6 38 23

0 0 7 13 .

4

2 2 9 14 7

0 3 5 3 10

6

icdb

0 0

0

0 0 1 0

0

1 2 2 0 43 56

0 2 8 44

0

0 0 8 9 5

0 0 4 6 18

1

cdx

0 2

3

1 0 0 0

0

0 1 2 5 1 0

0 1 1 1

8

0 0 2 4 0

0 1 0 1 1

4

Chromosome type

0.061 0.096 m 0.090

0.007 0.014 m

2.05 1.63 2.23 m

0.43 m 0.41

Induced freq. o f cells w i t h chromosomal aberrations (%)/#M

A N D P O L Y P L O I D C E L L S IN C E L L C U L T U R E S T R E A T E D W I T H C H E M I C A L S F O R 2 6 . 5 h

Cells w i t h chromosomal aberrations a

FREQUENCY OF CHROMOSOMAL ABERRATIONS

TABLE 2

428 d 788 565 c 509 125

461 489 441 402 409

441 404 519 ,'45 451 214

418 438 416 232 e

3/ 452

3/ 474

11/ 824 2011140 41/ 412 e 46/ 911e

12/ 636

4/ 9/ 5/ 51 7/ 11

91 9/ 4/ 22/

21/1391

13/ 10/ 14/ 10/ 1/

8/ 5/ 7/ 10/ 8/

16/1426

Number

0.7

0.6

1.3 1.8 10.0 14.8

1.9

0.9 2.2 1.0 1.1 1.6 0.5

2.2 2.1 1.0 9.5

1.5

3.0 1.3 2.5 2.0 0.8

1.7 1.0 1.6 2.5 2.0

1.1

(%)

Polyploid cells/ t o t a l cells observed

NBT

Control

NBT

9

I0

10

2 5 1 2

0

5 1 2

0

5 1 2 5

0

1 2 5 1

0

1 2 5 1

0

5 1 2 5 8

0

X 1 0 -6 X 10-6 X 1 0 -5 X 10 "~

X 10 -6 X 10 -5 x l 0 -s

X 10 -5 X 10 -4 X 10 .4 X 1 0 -4

× 10-4 X 10-4 X 10-4 X 10 -3

X 1 0 -3 X 1 0 -3 X 10-3 X l 0 -2

X 1 0 -5 X 10-4 X 10 -4 × 10-4 × 10-4

× 10 -4 × 10 -3

100 100 150 -- g

200

100 100 --g

200

100 100 100 2

200

100 100 100 -- g

200

100 100 100 5

200

100 100 100 100 62

200

56 32

2 6 14 c .

6

1 5 .

6

2 4 4 2

2

4 4 34 • .

9

5 4 16 e 5(22) f

8

2 5 12 d 12 d 15 e

5

7 e 13 e

.

.

.

2.0 6.0 9.3

3.0

1.0 5.0

3.0

2.0 4.0 4.0 --g

1.0

4.0 4.0 34.0

4.5

5.0 4.0 16.0 --g

4.0

2.0 5.0 12.0 12.0 24.2

2.5

10.9 40.6

.

.

.

.

0 0 0

0

0 1

0

0 0 0

0

0 0 0

0

0 0 0 .

0

0 0 0 0 2

0

1 0

.

.

.

.

2 3 3

1

4 4

6

1 1 3

6

0 0 55

6

10 16 29 .

15

6 4 6 2 7

1

5 20

.

.

.

.

0 1 3

1

1 0

1

0 0 0

0

0 0 1

2

1 1 4 .

0

1 3 3 0 2

2

1 0

.

1 0 2 .

1

0 0 .

1

0 1 0 .

1

2 1 33 .

2

0 2 9

8

0 2 6 2 4

2

4 23

.

1 5 8

4

1 2

5

1 2 3 .

1

3 2 12

6

1 2 7

3

2 '1 5 7 14

3

8 4

.

.

.

.

.

0 0 0

0

0 0

0

0 0 0

0

0 2 11

0

4 0 5

1

0 2 2 3 4

0

1 5

.

.

.

.

0 1 1

1

0 2

0

1 1 1

0

0 0 0

1

3 0 0

0

0 0 1 0 0

0

1 0

.

.

.

.

.

.

0.059 m .

0.0024 m .

0.048 m 0.019 0.027

0.019 0.039 m

56 32

437 370 350 426 63

600 800 300 e 101 e

5/ 412 10/ 456 202/ 617 e /

8/ 401

35/1000 d 89/ 400e /

14/1000

14/ 16/ 29/ 99/

26/1000

36/ 306 e 3 5 / 223 e 31/ 334 e /

11/ 446

8/ 322 18/ 392c 22/ 242e /

12/ 602

4/ 5/ 6/ 8/ 1/

4/ 552

1/ 0/

1.2 2.2 32.7

2.0

3.5 22.3

1.4

1.8 2.0 9.7 98.0

2.6

11.8 15.7 9.3

2.5

2.5 4.6 9.1

2.0

0.9 1.4 1.7 1.9 1.6

0.7

1.8 0

A b b r e v i a t i o n s : cdg, c h r o m a t i d g a p s ; i c d g , i s o c h r o m a t i d g a p s ; c d b , c h x o m a t i d b r e a k s ; i c d b , i s o c h r o m a t l d b r e a k s ; c d x , c b x o m a t i d e x c h a n g e s . a Cells w i t h gaps a l o n e w e r e e x c l u d e d . b T h e n u m b e r o f a b e r r a t i o n s l i s t e d is t h e v a l u e f r o m cells w i t h 9 o r less c h r o m o s o m a l a b e r r a t i o n s . A c e n t r i c f r a g m e n t s n o t a s s o c i a t e d w i t h d i c e n t r i c s a n d r i n g s w e r e s c o r e d as i s o c b x o m a t i d b r e a k s . C h r o m o s o m e - t y p e a b e r r a t i o n s i n c l u d e d d i e e n t r i c s , rings a n d r e c i p r o c a l t r a n s l o c a t i o n s . c,d a n d e Significantly d i f f e r e n t f r o m c o n t x o l a t p ~ 0 . 0 5 , p <~ 0 . 0 1 a n d p ~ 0 . 0 0 1 , r e s p . f T h e v a l u e in p a r e n t h e s e s is t h e n u m b e r o f cells w i t h s t i c k i n e s s ; it is n o t i n c l u d e d i n t h e n u m b e r o f cells w i t h c h r o m o s o m a l a b e r r a t i o n s . g D a t a n o t available. m k f ~ r l m a l v a l u e o f i n d u c e d f r e q u e n c y o f cells w i t h c h r o m o s o m a l a b e r r a t i o n s (%)//JM.

Control

DDVP

9

Control

7

7

Control

Vamidothion

6

N-NitrosoETU

Control

6

6

HEH

5

8

Control

5

5 1

2

0

1 × 10 -4 2 X 10-4 5 X 10-4 1 × 10 -3

Control

N-Nitroso-ETU

-- f

50 50 75

100

Number o f cells observed f o r SCEs

7.6 _+ 0 . 5 c 11.4 ± 0.7 d 24.1 ± 1.0 d

6 . 2 ~- 0.3

SCEs/cell ( m e a n ± SE)

I00 100 150

200

Number o f cells observed for aberrations

2 2 3 6 d,e

2 2.0 2.0 24.0

1.0 2 3 43

8

0 0 3

0

icdg

cdg

No.

(%)

Chromatid type

Aberrations b

CeiLs w i t h chromosomal aberrations a

0 0 34

0

cdb

A b b r e v i a t i o n s : cdg, e h r o m a t i d g a p s ; i c d g , i s o c b x o m a t i d g a p s ; c d b , c b x o m a t i d b r e a k s ; i c d b , i s o c h r o m a t i d b r e a k s ; c d x , e h r o m a t i d e x c h a n g e s . a Cells w i t h gaps a l o n e w e r e e x c l u d e d . b See f o o t n o t e b i n T a b l e 2. c a n d d Significantly d i f f e r e n t f r o m c o n t r o l a t p ~ 0 . 0 5 a n d p <~ 0 . 0 0 1 , r e s p . e C o n t a i n e d a cell w i t h pulve14zation. f D a t a n o t available.

2

2 2 3

Number of cultures

Concentration (M)

Chemical

2 2 14

2

icdb

0 1 12

0

cdx

0 0 1

0

Chromosome type

F R E Q U E N C Y OF S I S T E R - C H R O M A T I D E X C H A N G E S A N D C H R O M O S O M A L A B E R R A T I O N S IN C E L L C U L T U R E S T R E A T E D WITH N - N I T R O S O - E T U F O R 16 h

TABLE 3

0.48 2.39 0.018 0.31 0.067 0.089 0.0076 0.089 0.077

Captan Captafol EDB DBCP DAPA HEH Vamidothion DDVP N-Nitroso-ETU

was calculated.

a Microbial mutapnicity

0.43 2.23 0.014 0.096 0.039 0.048 0.0024 0.059 --

---0.37 0.35 --1.85 --1.02 --1.40 --1.32 --2.62 --1.23 --

l O g l 0 M2

r between log M 1 and log M4 : 0.71 r between log M 2 and log M 3 : 0.84

--0.37 0.38 --1.74 --0.51 --1.17 --1.41 --2.12 --1.41 --1.11

Max~reml v a l u e of induced f r e q . o f cells with chromosomal aberzat/ons (%)/~M (M2)

EFFECTS

2.28 3.40 3.60 -0.72 1.43 4.39

1.9 2.52 3.94 -5.2 2.7 2.46

r between log M 2 and log M4 : 0.81

X 10 X 104

X 102 X 103 × 103

5.00 - -

1.01 X 10 s

logl0 M 3

- -

Maximal value of induced revertants in TA10Ol~mol (M 3) a

OF CHEMICALS IN CULTURED

2.18 3.84 7.7 1.9 1.36 5.5 0.65 8.0 4.42

× 103

X 10 X 103 X 10

X 104 X 103

Maximal value of induced revertants in E. eoli W P 2 h c r / ~tmol (M4) a

ACTIV-

4.33 3.58 0.89 1.28 3.13 1.74 ---0.19 0.90 3.65

logl0 M4

CELLS AND MUTAGENIC

t e s t i n g w i t h o u t 8 9 m i x w a s p e r f o r m e d a c c o r d i n g t o t h e p r e v i o u s r e p o r t ( T e r a m o t o et al., 1 9 7 7 ) , a n d t h e v a l u e ~ f i n d u c e d r e v e r t a n t s / / ~ m o l

C o r r c i a t / o n c o e f f i c i e n t (r), r between log M 1 and log M 2 : 0.96 r between log M 1 and log M 3 : 0.88

Maximal value of induced SCEs/ecil/ ~M (M 1 )

Chemical

l o g l 0 M1

BETWEEN THE INDUCING POTENCY OF CYTOGENETIC

ITIES IN BACTERIA

CORRELATION

TABLE 4

Q0 .-3

188

Seiler, 1977; Shirasu et al., 1976). However, the reason why the p o t e n t frameshift-type mutagens NNN and NBT (Shirasu et al., 1976} did n o t induce SCEs or aberrations remains unknown. A further study is now in progress on the induction of SCEs and chromosomal aberrations by various kinds of frameshiftt y p e mutagen. In the previous investigation (Tezuka et al., 1978), we reported no induction of cytogenetic effects in cultured human cells and in intact animals exposed to captan. In cultured human cells treated with captan, no metaphases were observed at a higher concentration than 4.0 #g/ml. However, in the present study with cultured Chinese hamster cells, metaphases were still observed at 4.5 pg/ml and above, and the induction of SCEs and chromosomal aberrations was observed. This positive result corresponds with the finding by Legator et al. (1969). In the cytogenetic study on captan, a discrepancy of the results was also observed in the investigations on DDVP. DDVP did n o t induce SCEs in cultured human l y m p h o c y t e s or fibroblasts (Nicholas, 1978} or chromosomal aberrations in cultured human l y m p h o c y t e s (Dean, 1972). Nevertheless, the induction of both SCEs and aberrations was observed in the present study. This is the first study reporting the cytogenetic effects of DDVP. The divergence between the results of the experiments on captan and DDVP might be due to the different sensitivities of the test:systems. In this study N-nitroso-ETU also induced SCEs, chromosomal aberrations and polyploids in cultured cells. This c o m p o u n d gave positive results in all the test systems including microbial reversion assays (Seiler, 1977; Shirasu et al., 1977), mammalian test systems in vitro and in vivo (Seiler, 1977; Shirasu et al., 1977; Teramoto et al., 1978) and a carcinogenicity test (Moriya et al., 1979). On the genetic and toxicological effects of a halogenated soil fumigant DBCP, Torkelson et al. first reported the spermicidal action of DBCP in animals as early as 1961. Aspermia in male workers of a DBCP-manufacturing plant was reported recently (Whorton et al., 1977). Histological studies on these workers revealed a decrease or an absence of spermatogenic activity (Biava et al., 1978). Another recent study by Kapp Jr. et al. (1979) indicated a significant increase in the frequency of non-disjunction of the Y-chromosome in workers exposed to DBCP. In a dominant lethal study on DBCP, Teramoto et al. (1980) observed an obvious dominant lethal mutation-inducing effect in rats. EDB, a fumigant similar to DBCP, was reported to be effective in mutation induction in a cultured mouse l y m p h o m a system (Clive et al., 1979). Both DBCP and EDB were p o t e n t mutagens in microbial studies (Brem et al., 1974; Buselmaier et al., 1972; Rosenkranz, 1975; Scott et al., 1978), induced SCEs and chromosomal aberrations in this test system and induced cancer in rats and mice (Olson et al., 1973; Weisburger, 1977). These reports on the genetic and toxicological effects of DBCP and EDB were supported by studies on the metabolism, detoxication and reactivity of the chemicals. A series of studies on the biological reactivity of DBCP was p e r f o r m e d b y Kato et al. (1979a,b; 1980a,b), and evidence was obtained that this chemical was enzymically metabolized to an active intermediate b y microsomes, in vitro and in vivo, that became bound to cellular macromolecules such as DNA and protein. A biochemical investigation on the metab-

189 olism of EDB in rats was done by Hill et al. (1978) who found that EDB also became bound to cellular protein, RNA and DNA. All these reports on the genetic and toxicological effects and the biological reaction of DBCP and EDB indicate that both pesticides may be hazardous to human health. As for the other pesticides than DBCP and EDB, Moriya et ah (1978) investigated the effects of cysteine, blood and a metabolic activation system ($9 mix) on the activity in vitro of the 7 mutagenic pesticides, captan, captafol, NBT, NNN, DDVP, HEH and vanmidothion. These authors reported that captan, captafol and NBT were extremely sensitive to liver homogenate, cysteine and blood, indicating non-enzymic inactivation of the mutagenicity of the 3 pesticides. The 2 pesticides captan and captafol may be too unstable in conditions in vivo to show an apparent carcinogenicity in chronic feeding studies in mammals (FAO/WHO, 1970; Innes et al., 1969; WHO, 1974). The other 4 compounds appeared not to be so sensitive to $9 mix and cysteine as the 3 mentioned above. The mutagenicity of NNN increased immediately after the addition of $9 mix and gradually decreased in the course of incubation. Cysteine did n o t reduce the mutagenicity of NNN. This result suggests t h a t NNN may not be bound to protein but may be enzymically metabolized to mutagens of greater and lesser potencies. After the addition of $9 mix, the mutagenicity of DDVP for E. coli B/r WP2 remained unchanged but that for S. typhimurium TA1535 was reduced markedly, suggesting that there might be 2 mutagenic ingredients in the DDVP samples studied. No marked change in the mutagenicity of HEH or vamidothion was observed after the addition of, and during the incubation with, $9 mix. In studies in vivo, Innes et al. (1969) found that the incidence of hepatomas increased significantly in HEH-treated mice. However, there have been no long-term studies on the carcinogenicity of the 4 chemicals, NBT, NNN, DDVP and vamidothion. Further studies on the genetic and toxicological effects, including biochemical investigations such as binding studies, should be done on all mutagenic pesticides in various screening systems in vitro and in vivo for better assessment of risks against human health and genome of the populations. Acknowledgement We are grateful to Dr. Y. Kuroda, National Institute of Genetics, for his gift of the original cells of cultured Chinese hamster V79 cell line. We also acknowledge helpful advice from Dr. M. S. Sasaki, Kyoto University, and Dr. T. Kada, National Institute of Genetics. This study was supported in part by grants from the Ministry of Agriculture, Forestry and Fisheries, and the Ministry of Education, Science and Culture. References Biava, C ~ . , E.A. Smu ckler and D. Whorton (1978) The testicular m o r p h o l o g y of individuals e xpos e d to d i b r o m o c h l o r o p r o p a n e , Exp. Mol. Pathol., 2 9 , 4 4 8 - - 4 5 8 . Brem, H., A.B. Stein and H.S. Rosenk~anz (1974) The m u t a g e n i c i t y and D N A - m o d i f y i n g effect of haloalkanes, Cancer Res., 34, 2576--2579. Bridges, B.A. (1975) The mutagenieity o f captan and related fungicides, Mutation Res., 32, 3--34. Buselmaier, M., G. R 6 h r b o r n und P. Propping (1972) Mutagenit~ts-Untersuehungen mit Pestiziden i m host-mediated Assay u nd mit d e m d o m i n a n t e n Lethaltest an der Maus, Biol. Zbl., 9 1 , 3 1 1 - - 3 2 5 .

190 Clive, D., K.O. Johnson, J.F.S. Spector, A.G. Batson and M.M.M. Brown (1979) Validation and char acterization of the L 5 1 7 8 Y / T K + / - mouse l y m p h o m a m u t a g e n assay system, Mut a t i on Res., 59, 61--108. Cohen M.M., and K. Hirschhorn (1971) Cytogenetic studies in animals, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for Their Detection, Vol. 2, Plenum, New York, pp. 515--534. C o m m i t t e e 17 (1975) E n v i r o n m e n t a l m u t a g e n i c hazards, Science, 187, 503--514. Dean, B.J. (1972) The effect of Dichlorvos on cultured h u m a n l y m p h o c y t e s , Arch. Toxikol., 30, 75--85. FAO/WHO (1970) 1969 Evaluation of Some Pesticide Residues in F ood, R ome . Hill, D.L., T.-W. Shin, T.P. J o h n s t o n and R.F. Struck (1978) Macromolecular binding and m e t a b o l i s m of the carcinogen 1,2-dibromoethane, Cancer Res., 38, 2438--2442. Innes, J.R.M., B.M. Ulland, M.G. Valerio, L. Petrucelli, L. Fishbein, E.R. Hart, A.J. Pallotta, R.R. Bates, H.L. Falk, J.J. Gast, M. Klein, I. Mitchell and J. Peters (1969) Bioassay of pesticides a nd industrial chemicals for t u m o r i g e n i c i t y in mice: a preliminary note, J. Natl. Cancer Inst., 42, 1 1 0 1 - - 1 1 1 4 . Kada, T., M. Moriya and Y. Shirasu (1974) Screening of pesticides for DNA i nt e ra c t i ons by " R e c -a s s a y" and mutagenesis testing, and f~ameshift m u t a g e n s d e t e c t e d , M ut a t i on Res., 26, 243--248. Kapp Jr., R.W., D.J. Picciano and C.B. Jacobson ( 1 9 7 9 ) Y - c h r o m o s o m a l non-disjunction in dibromochloropropane-exposed w o r k m e n , M u t a t i o n Res., 64, 47--51. Kato, Y., K. Sato, S. Makt, O. Matano and S. Goto (1979a) Metabolic fate of 1,2-dibromo-3-chloropr opane (DBCP) in rats, J. Pesticide Sci., 4, 195--203. Kato, Y., O. Matano and S. Goto (1979b) Covalent binding of DBCP t o prot e i ns in vitro, Toxicol. Lett., 3, 299--302. Kato, Y., K. Sato, O. Matano and S. Goto (1980a) Metabolic fate of DBCP in rats, 2. A l k y l a t i o n of cellular m a c r o m o l e c u l e s b y reactive m e t a b o l i c i n t e r m e d i a t e s of DBCP, J. Pesticide Sci., 5, 45--53. Kato, Y., K. Sato, T. Harada, S. Maki, O. Matano and S. Goto (1980b) Metabolic fate of DBCP in rats, 3. Correlation b e t w e e n m a c r o m o l e c u l a r binding of reactive m e t a b o l i t e s of DBCP a nd p a t h o g e n i c i t y of necrosis, J. Pesticide Sci., 5, 81--88. Latt, S.A. (1974) Sister c h r o m a t i d exchanges , indices of h u m a n c h r o m o s o m e damage a nd repair: d e t e c t i o n by fluorescence and i n d u c t i o n by m i t o m y c i n C, Proc. Natl. Acad. Sci. (U.S.A.), 71, 3 1 6 2 - 3166. Legator, M.S., F.J. Kelly, S. Green and E.J. Oswald (1969) Mutagenic effects of captan, Ann. N.Y. Acad. Sci., 1 6 0 , 3 4 4 - - 3 5 1 . Moriya, M., K. Kato and Y. Shirasu (1978) Effects of cysteine and a liver m e t a b o l i c a c t i va t i on s ys t e m on the activities of m u t a g e n i c pesticides, M u t a t i o n Res., 5 7 , 2 5 9 - - 2 6 3 . Moriya, M., K. Mitsumori, K. Kato, T. Miyazawa and Y. Shirasu (1979) Carcinogenicity of N-nitrosoe t h y l e n e t h i o u r e a in female mice: a preliminary study, Cancer Lett., 7 , 3 3 9 - - 3 4 2 . Nicholas, A.H., M. Vienne and H.V.D. Berghe (1978) Sister c h r o m a t i d exchange frequencies in c ul t ure d h u m a n cells exposed to an o r g a n o p h o s p h o r u s insecticide: dichlorvos, Toxicol. Lett., 2, 271--275. Olson, W.A., R.T. Habermann, E.K. Weisburger, J.M. Ward and J.H. Weisburger (1973) I n d u c t i o n of s t o m a c h cancer in rats and mice b y halogenated aliphatic fumigants, J. Natl. Cancer Inst., 51, 1 9 9 3 - 1995. Perry, P., and H~I. Evans (1975) Cytological d e t e c t i o n of muta ge n--c a rc i nogc n exposure by sister chrom a t i d exchange, Nature (London), 258, 121--125. Perry, P., an d S. Wolff (1974) New Giemsa m e t h o d for the differential staining of sister chromatids, Nature (L o ndon), 2 5 1 , 1 5 6 - - 1 5 8 . R o s e n k r a n z , H.S. (1975) Genetic activity of 1,2-dibromo-3-chloropropane, a widely-used fumigant, Bull. Environ. Contain. Toxicol., 14, 8--12. Scott, B.R., A.H. Sparrow, S.S. S c h w e m m e r and L.A. Schairer (1978) Plant m e t a b o l i c activation of 1,2d i b r o m o e t h a n e (EDB) to a m u t a g e n of greater p o t e n c y , Mutat i on Res., 49, 203--212. Seiler, J.P. (1 977) Nitrosat ion in vitro and in vivo by s o d i u m nitrite, and m u t a g e n i c i t y of n i t r o g e n o u s pesticides, M u t a t i o n Res., 4 8 , 2 2 5 - - 2 3 6 . Shirasu, Y. (1975) Significance of m u t a g e n i c i t y testing on pesticides, in: F. Coulston and F. Korte (Eds.), E n v i r o n m e n t a l Quality and Safety, Vol. 4, Thieme, Stuttga rt , and Academic Press, New York, pp. 226--231. Shirasu, Y., M. Moriya, K. Kato, A. Furuhashi and T. Kada (1976) Mutagenicity screening of pesticides in the m i c r o b i a l sy stem, Mutation Res., 40, 19--30. Shirasu, Y., M. Moriya, K. Kato, F. Lienard, H. Tezuka, S. T e r a m o t o and T. Kada (1977) Mutageulcity screening on pesticides and m o d i f i c a t i o n p r o d u c t s : a basis of carcinogenicity evaluation, in: H.H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Cold Spring Harbor Conferences on Cell Proliferation, Vol. 4, Cold Spring Harbor Laboratory, pp. 267--286. S o l o m o n , E., and M. Bobrow (1975) Sistcr-chromatid exchanges -- a sensitive assay of agents damaging h u m a n chro mo so mes, Mutation Res., 30, 273--278. Teramoto, S., M. Moriya, K. Kato, H. Tezuka, S. Nakamura, A. Shingu and Y. Shirasu (1977) Mutagenicity testing on e t h y l e n e t h i o u r e a , M u t a t i o n Res., 56, 121--129. Ter amoto, S., A. Shingu and Y. Shirasu (1978) I n d u c t i o n of do mi na nt -l e t ha l m u t a t i o n s after administra-

191 t i o n of e t h y l e n e t h i o u r e a in c o m b i n a t i o n wit• nitrite or of N-nitroso-ethylenethiottrea in mice, Mutat i o n Res., 5 6 , 3 3 5 - - 3 4 0 . Teramoto, S., R. Saito, H. A o y a m a and Y. Shirasu (1980) D o m i n a n t lethal m u t a t i o n i nduc e d in male rats by 1,2-dibromo-3-chloropropane, M u t a t i o n Rcs., 77, 71--78. Tezuka, H., S. Teramoto, M. Kancda, R. Henmi, N. Murakami and Y. Shirasu (1978) Cytogenetic and d o m i n a n t lethal studies on eaptan, M u t a t i o n Res., 57, 201--207. Torkelson, T.R., S.E. Sadek, V.K. Rowe, J.K. Kodama, H.H. Anderson, G.S. Loguvam a nd C.H. Hine (1961) Toxicologic investigations of 1,2-dibromochloropropane, Toxicol. Appl. Pharmacol., 3, 549-559. WeisbtLrger, E.K. (1977) Carcinogenicity studies on halogenated hydroc a rbons , Environ. Health Perspect., 21, 7--16. WHO (1974) 1973 Evaluation of Some Pesticide residues in Food, Geneva. Whorton, D., R.M. Krauss, S. Marshall and T.H. Milby (1977) Infertility in male pesticide workers, Lancet, fl, 1259--1261. Wild, D. (1975) Mutagenicity studies on organophosphorus insecticides, M u t a t i o n Rcs., 32, 133--150. Wolff, S., and P. Perry (1974) Differential Giemsa staining of sister c hroma t i ds and t he s t udy of sister c h r o m a t i d exchanges w i t h o u t autoradiography, Chromosoma, 48, 341--353.