Mutation Research, 240 (1990) 203-208 Elsevier

203

MUTGEN 01524

Genotoxicity studies on the organophosphorus insecticide chloracetophone A. K a p p a s l, R. V a c h k o v a

2, S.

L a l c h e v 3, M . T z o n e v a ~ a n d M . M a r k a k i

1

I Institute of Biology, National Research Center 'Democritus', Athens 153 10 (Greece), 2 Institute of Gastroenterology and Nutrition, Medical Academy, Sofia (Bulgaria) and 3 Chair of Medical Genetics, Medical Academy. Sofia (Bulgaria) (Received 6 July 1989) (Revision received 23 October 1989) (Accepted 24 October 1989)

Kevwords: Chloracetophone; Microbial test systems; Mammalian test systems; Genotoxicity

Summary Chloracetophone (O,O-dimethyl-2,2,2-trichloro-l-(chloroacetoxy)phosphonate), a new insecticide of the organophosphorus group of pesticides, was tested for genotoxicity in a variety of systems with different genetic end-points and varying parameters. The test systems included 2 microbial systems, Salmonella and Aspergillus for point mutations and mitotic segregation, respectively, and human lymphocyte cultures and mammalian bone marrow cells (from rats and hamsters treated acutely and subacutely) for chromosomal aberrations and micronuclei. Chloracetophone was negative in Aspergillus at concentrations of 1-500 # g / m l , in human lymphocyte cultures at concentrations of 2.5-40 /~g/ml, in rats at doses of 420-21 m g / k g b.w. and in hamsters at doses of 210-42 m g / k g b.w. for chromosomal aberrations. It did not cause any increase of micronuclei in human lymphocytes and rat bone marrow cells but did cause a significant increase in hamster bone marrow cells. Chloracetophone induced base-pair substitutions in strain TA100 of Salmonella with and without metabolic activation at a concentration range of 2000-6000/~g/plate.

Many organophosphorus-derived chemicals form a class of pesticides widely used in agriculture. Most of these are esters or amide esters of phosphoric acid and, therefore, are also correctly named organophosphates (Wild, 1975). Most of them have been shown to have alkylating properties (Mohn, 1973) and because of that much work has been done on their possible mutagenic activity.

Correspondence: Dr. A. Kappas, Institute of Biology, National Research Center 'Democritus', Athens 153 10 (Greece).

One of the best studied compounds is dichlorvos, which has been shown to be mutagenic in bacterial systems, Drosophila, plant systems and mammalian cell cultures (Hanna and Dyer, 1975; Ramel et al., 1980; Dzwonkowska and Hubner, 1986), but it is still questionable whether it is carcinogenic (Bremmer et al., 1988). Malathion, another widely used organophosphorus pesticide, has also been found to induce several types of genetic damage (Nicholas et al., 1979; Dulout et al., 1982, 1983). Other organophosphorus pesticides also have been found to be positive for chromosomal aberrations in bone marrow cells (Kurinny, 1975) or for sister-chro-

0165-1218/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

204 matid exchange (SCE) in mammalian cells (Tezuka et al., 1980; Chen et al., 1981). In this work we studied the mutagenic activity of chloracetophone, a new insecticide which belongs to the group of organophosphorus pesticides, in Salmonella, Aspergillus, human lymphocytes and mammalian bone marrow cells. Materials and methods

Chloracetophone (O, O-dimethyl-2,2,2-trichloro-l-(chloroacetoxy)phosphonate) was kindly supplied by the Institute of Chemical Industry, Sofia (Bulgaria). The LDs0 for male rats was 421 m g / k g b.w. Stock solutions of pure chemical were made in ethanol or dimethyl sulfoxide (DMSO) and small aliquots were added to the medium to give the appropriate concentrations. In the case of A. nidulans the solvent concentration in the medium, which never exceeded 2% (v/v), had no effect on either growth or segregation in control plates. In the in vivo tests the chemical in its commercial emulsion form (50% pure compound) was diluted in water.

Test procedures Ames test. Strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium were used. TA100 and TA1535 are proper for scoring basepair substitutions, and TA98 and TA1537 for frameshifts. Strains TA98 and TA100 have the plasmid pKM101, which makes them more sensitive in mutagenicity testing (Maron and Ames, 1983). The plate incorporation technique was used as described by the Ames group (Ames et al., 1975; Maron and Ames, 1983). Bacterial cells from stock cultures were grown for 18 h in liquid medium at 37°C. 0.1 ml of cell suspension (2-3 × 10 s cells/ml) was added in 2 ml of top agar medium containing 0.5% agar, 0.6% NaC1 and traces of histidine and biotin. The chemical and the $9 were also added to the top agar, which was then plated on top of minimal medium (MM). Aflatoxin B 1 was used as a positive control. Three p l a t e s / d o s e were used in each experiment. Histidine revertant colonies were counted 48 h after inoculation and incubation at 37 o C. The results are means of 3-5

experiments. The Duncan test was used for statistical analysis.

Aspergillus nidulans. The diploid strain of A. nidulans used for detecting somatic segregation has been described previously (Kappas, 1978, 1983). It was prototrophic and heterozygous for nutritional requirements and for the white and yellow conidia color mutations. The diploid strain produced green conidia and had the genotype suA1 ad E20, yA2, ad E20, pabaA1, biAl(I); AcrA1, wA3, thiA4, cnxE16 (lI) (Kappas, 1978). Minimal (MM) and complete (CM) media were used according to the routine culture techniques of Pontecorvo et al. (1953). Dilute suspensions of conidia from the diploid strain were spread on M M and incubated overnight. Blocks of inocula (1 m m 2) were then transferred to plates of CM containing the chemical under test. At least 100 colonies were tested in each chemical concentration. The diploid strain forms green conidia, and somatic segregation was detected by the appearance of sectors with either white or yellow conidia. The numbers of segregants on control plates and on treated plates were scored after 6 days' incubation at 38°C. For toxicity of the chemicals the colony diameter was measured 3 days after inoculation. The segregants were thoroughly scored and included all the macroscopically visible areas of white and yellow appearing as sectors, patches or spots. Conidia from all these areas were picked up and transferred onto slants of CM for further use and tests. Haploid and diploid segregants were distinguished by their phenotype according to the genetic assay described previously (Kappas, 1978). Metabolic activation system. Wistar rats weighing 200-250 g were injected intraperitoneally with Aroclor (500 m g / k g b.w.) 5 days before sacrifice. The livers obtained were washed and homogenized, and the homogenate was centrifuged at 9800 × g for 20 min. The procedure followed was based upon Ames et al. (1975) and Frantz and Malling (1975). The $9 fraction was kept at - 8 0 ° C . The $9 mix contained in a final volume of 1 ml, 0.05 ml $9 fraction and cofactors: glucose 6-phosphate 7.4 mM, N A D P 1.8 mM, G - 6 - P D H 1

205

unit, MgC12. 6 H 2 0 1.8 raM, Tris 16 mM, and sucrose 39 mM.

The micronucleus test was performed according to Schmid (1975). The slides were prepared 24 h after the acute treatment. 2000 polychromatic erythrocytes were scored from each animal. Cyclophosphamide was used as the positive control. Student's t test was employed in all cases at experiments with mammalian systems.

Human lymphocyte cultures. Tests in vitro were performed in human lymphocyte cultures according to the microculture technique (Evans, 1984). Culture medium RPMI-1640 with added calf serum and phytohemagglutinin was used. The chemicals under test were added in the culture medium. 0.2 #g of colchicine/ml was added for 2 h, 70 h after plating. Then the lymphocytes were harvested, fixed properly and stained with Giemsa. A solvent control (alcohol) was also used. For the micronucleus test the lymphocytes were prepared according to the technique of Hogstedt (1984). For chromosomal aberrations at least 50 cells at metaphase were scored, and for mitotic index (MI) or micronuclei 1000 cells from each treatment were scored.

Results and discussion

Table 1 shows the effect of chloracetophone in strains TA98, TA100, TA1535 and TA1537 of S. typhimurium with and without $9 mix. The chemical was mutagenic only in strain TA100 at concentrations from 2000 to 6000/zg/plate. The rather weak mutagenicity in strain TA100 became slightly stronger when $9 was added to the medium. In all other strains used chloracetophone was negative (Table 1). Several pesticides of the organophosphorus group have been shown to induce point mutations in microbial systems. These include the wellstudied insecticide dichlorvos for which alkylation of D N A bases in Escherichia coli has been shown (Wild, 1975). Dichlorvos is one of the metabolic products of chloracetophone. Dichlorvos, as well as other organophosphorus compounds, is known to have alkylating properties (Mohn, 1973). This may explain our results which show that chloracetophone induces mutations of the base-pair substitution type. The chemical was effective at rather high concentrations, which is in accordance with the

Bone marrow cells. Experiments in vivo were done in Syrian hamsters and Wistar rats. The chemical under test was administered to the animals either acutely or in short-term treatments. For chromosomal aberrations 2 h before killing the animals were injected with 3 mg colchicine/kg b.w. intraperitoneally. 5 - 9 animals were used for each treatment taken at different intervals. Slides from bone marrow specimens were prepared according to Legator et al. (1969) and stained with Giemsa. About 50-100 cells at metaphase were scored for chromosomal aberrations and about 1000 cells for MI.

TABLE 1 MUTAGENICITY

OF THE INSECTICIDE

CHLORACETOPHONE

IN

Salmonella typhimurium

Concentra-

R e v e r t a n t s (his + ) p e r p l a t e

tion (/~ g / p l a t e )

TA98 - $9

+ $9

- $9

+ $9

- $9

+ $9

- $9

+ $9

0 1000 2000 3000 4000 5000 6000

30 27 25 23 21 20 14

41 40 38 32 31 21 23

107 122 144" * 166 * * 175 * * 182 * * 182 * *

114 140 161 173 195 205 193

34 25 20 16 10 7 6

34 23 21 18 15 7 10

23 28 24 27 19 18 10

27 27 27 29 23 30 12

* * P < 0.01.

TA100

TA1535

** ** ** ** **

TA1537

206 TABLE 2 TEST OF THE INSECTICIDE CHLORACETOPHONE FOR I N D U C E D M I T O T I C S E G R E G A T I O N I N Aspergillus nidulans

Concentration ( ~t g / m l )

Toxicity ~

Number of mitotic segregants per 100 colonies

0 1 10 50 100 200 500 750 b

-

27 36

1

39

3 5 8 9 _

18 19 14 13 _

Percentage reduction of colony size, measured 3 days after inoculation. Average of 3 colonies per treatment. b A t concentrations above 750 ~ g / m l the chemical affected the green color of the colonies, making it impossible to score for color sectors.

results of Bridges et al. (1973) and Green et al. (1974) who suggested that the lack of detectable mutagenic effects at low concentrations is due to a sensitivity threshold of the individual test system, not to the existence of a principal no-effect concentration. Table 2 shows that chloracetophone did not cause any increase of mitotic segregants in A. nidulans either with or without $9 mix. It was not possible to use higher concentrations than those shown in Table 2 because the chemical affected

the green color of the colonies, thus making it impossible to score for color sectors. The results with Aspergillus indicate that chloracetophone had no aneugenic effect, at least at the concentration used, and we are not aware of any work with organophosphorus pesticides showing interference with cell division. Although chloracetophone did not show any effect either on chromosomal aberrations or on micronuclei in human lymphocyte cultures (Table 3) when used in vivo in bone marrow cells from hamsters chloracetophone induced micronuclei formation at doses of 60-210 m g / k g b.w. (Table 4) but not any chromosomal aberrations (Table 4). It is noted that the MI tended to decrease with increased doses and in particular with the doses of 20 and 40 b~g/ml (Table 3). The chemical was also negative for chromosomal aberrations in bone marrow cells of rats exposed to single and repeated oral doses (Table 5). The negative effect of chloracetophone on chromosomal aberrations suggests that the formation of micronuclei in rats was not due to chromosomal breaks but could possibly be due to an effect on cell division. Such an effect of course is not supported by our finding that chloracetophone did not induce any mitotic segregation in Aspergillus. On the other hand the negative results of other investigators in the micronucleus test in rat bone marrow cells (Hadzitodorova-Maseva and Andreev, 1987) could be attributed to differences

TABLE 3 EFFECT OF CHLORACETOPHONE

Dose

Cytogenetic analysis

(/zg/ml)

Number of cells scored

ON HUMAN

LYMPHOCYTE

CULTURES

Micronucleus test Cells with aberrations

Number of cells scored

n

7c 3.83 3.38 3.24

40 20 10 5 2.5 0

235 325 370 300 300 415

9 11 12 3 4 11

2.65

6000 6000 6000 6000 6000 6000

Solvent control

295

14

4.75

6000

1.00 1.33

Mitotic index

Cells with micronuclei

(%)

n

%o

5 13 13 12 6 0

0.83 2.16 2,16 2,00

0

1.07 1.77

0

2.12 2.70 3.12 2.12

0

1.38

1.00

207 TABLE 4 EFFECT OF CHLORACETOPHONE Compound

ON HAMSTER

BONE MARROW

Dose

Number of

Cytogenetic analysis

M i c r o n u c l e u s test

(mg/kg)

animals

Number of

Cells w i t h

Number of

Cells with

index

cells s c o r e d

aberrations

cells s c o r e d

micronuclei

(%)

n

Chloracetophone

Cyclophosphamide *P