Chromosomal aberrations in cultured human lymphocytes treated with Marshal and its effective ingredient Carbosulfan

Mutation Research, 319 (1993) 103-111 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00

103

MUTGEN 01918

Chromosomal aberrations in cultured human lymphocytes treated with Marshal and its effective ingredient Carbosulfan Mehmet Topakta§ and Eyyiip Renciizo~ullar ~ukurova University, Faculty of Arts and Sciences, Department of Biology, 01330Adana, Turkey (Received 7 October 1992) (Revision received 14 April 1993) (Accepted 14 April 1993)

Keywords: Marshal; Carbosulfan; Human lymphocytes;Chromosomal aberrations

Summary The aim of this study was to investigate the ability of Marshal (insecticide/nematocide) and its effective ingredient Carbosulfan to induce chromosomal aberrations (CA) and other chromosomal abnormalities in human peripheral lymphocytes. Carbosulfan induced the formation of CA at all concentrations (10 -6, 5 x 10 -6, 10 -5, 5 x 10 -5 v / v ) and treatment times. Marshal significantly induced the formation of CA at the two highest concentrations (10 -5 and 5 x 10 -5 v / v ) at all treatment times. The extent of damage was greatest with Carbosulfan.

The extensive use of pesticides has resulted in environmental pollution. Pesticides have been used extensively in the ~ukurova region, which is one of the most important agricultural regions in Turkey. An estimated 2,878,623 kg of pesticides were used in this region in 1991. Carbamates were the most extensively used pesticides, with 328,827 kg used in 1991. The test substance Marshal is a carbamate pesticide (insecticide and nematocide) of which 125,147 kg was used in the ~ukurova region during 1991.

Correspondence: Dot Dr. M. Topakta§, (~ukurovaUniversity, Faculty of Arts and Sciences, Department of Biology,01330 Adana, Turkey.

Carbamate pesticides are positive in short-term mutagenicity assays in different strains of Salmonella typhimurium, Saccharomyces cerevisiae and in Escherichia coli and also in other microorganisms (Seiler, 1972; Blevins et al., 1977b; Kappas and Bridges, 1981; Nelson et al., 1981; Speakman and Nirenberg, 1981; Gentile et al., 1982; Klopman et al., 1985; Albertini, 1989). It has also been reported that methyl-2-benzimidazole c a r b a m a t e ( M B C ) (Bavistin = Carbendazim), which is known as a carbamate fungicide, induced the formation of micronuclei and gave a positive result with the spot test in mice (Seiler, 1976; Fahrig and Seiler, 1979). P r o p o x u r , an insecticide, induced the formation of micronuclei in human lymphocytes (Cid et al.,

104

1990). Urethane (ethyl carbamate) caused mitotic recombination in Drosophila (Fr6hlich and Wfirgler, 1990). Wiedenmann et al. (1990) reported that Carbosulfan, the effective ingredient of Marshal, was not mutagenic in strains TA97, TA98, TA100, TA102 of S. typhimurium and strain D7 of S. cerevisiae. However, it caused mitotic aneuploidy in the D61.M strain of S. cerevisiae. Furthermore, it has been determined that some of the carbamate pesticides increased the formation of chromosomal aberrations (CA), DNA single-strand breaks and mitotic abnormalities in different test systems (Amer, 1965; Blewins et al., 1977a; Cid and Matos, 1987; Rao et al.,

1988; Adhikari and Grover, 1988; Cid et al., 1990; Zelesko et al., 1990). At the present time, the effects of Marshal pesticides on CA in human lymphocytes have not been investigated. For this reason, the aim of this study was to investigate the effect of Marshal and its effective ingredient Carbosulfan on the formation of CA and other chromosomal abnormalities in human peripheral lymphocytes. Materials and methods

Human peripheral blood was used as the test system, and Marshal and its effective ingredient

TABLE 1 MI, STRUCTURAL AND NUMERICAL S U L F A N F O R 12, 24 A N D 48 h * Time (h)

Concentration

MI

(%) 12

24

48

CHROMOSOME

ABNORMALITIES

IN CULTURES

Structural and numerical chromosome abnormalities B'

B"

F

Control C. + e t h a n o l 10 6 5 X 10 - 6 10 - 5 5X10 -5

3.13 2.52 2.66 2.36 2.19 1.85

6 8 13 16 17 15

1 2 1

4 5 7 6 5 4

Control C. + e t h a n o l 10 - 6 5 × 10 - 6 10 5 5 × 10- 5

3.31 2.33 2.88 1.95 1.57 0.82

3 6 12 12 13 24

1 2 1 2

6 6 11 13 11 15

Control C. + e t h a n o l 10 - 6 5 X 1 0- 6 10 - 5 5X10 -5 **

2.98 2.95 2.64 2.27 1.67 0.22

5 5 11 14 17

1 2 1

5 6 10 9 11

R

SU

-

1

-

1

1 1

1 -

1

-

-

1

--

1

DS

T

TR

TT

P

E

TREATED

Number of

Total number

abnor, cells

of CA

WITH CARBO-

Number of C A / c e l l _ + Sx

2 1

1 -

9 11 22 21 19 20

10 13 24 22 24 22

0.10+0.033 0.13 + 0.039 0.24_+0.049 0.22_+0.043 0.24_+0.053 0.22_+0.048

1 2 2 -

1

11 13 22 22 23 32

12 14 27 28 25 42

0.12-+0.035 0.14 + 0 . 0 3 7 0.27 -+ 0 . 0 5 4 0.28+0.058 0.25+0.044 0.42 -+ 0.071

---

9 9 19 25 26

10 13 23 28 29

0.10-+ 0.033 0.13 _+0.044 0.23-+0.050 a 0.28 _+0.053 ca' 0.29_+0.051 cb'

2 2 --

-

1

-

b a a a

ca' ba' aa' rid'

* 100 cells w e r e s c o r e d p e r d o s e g r o u p . * * N o t s c o r a b l e d u e to excessive toxicity. B ' , c h r o m a t i d b r e a k ; B " , c h r o m o s o m e b r e a k ; F, f r a g m e n t ; R , r i n g ; S U , sister u n i o n ; D S , d i c e n t r i c c h r o m o s o m e ; T, t r a n s l o c a t i o n ; T R , t r i p l o i d cell; T I ' , t e t r a p l o i d cell; P, p e n t a p l o i d cell; E, e n d o r e d u p l i c a t i o n . a S i g n i f i c a n t f r o m c o n t r o l at 0.05 (t-test). a' S i g n i f i c a n t f r o m a l c o h o l c o n t r o l at 0.05 (t-test). b S i g n i f i c a n t f r o m c o n t r o l a t 0.02 (t-test). b' S i g n i f i c a n t f r o m a l c o h o l c o n t r o l at 0.02 (t-test). c S i g n i f i c a n t f r o m c o n t r o l at 0.01 (t-test). c' S i g n i f i c a n t f r o m a l c o h o l c o n t r o l at 0.01 (t-test). d S i g n i f i c a n t f r o m c o n t r o l at 0.001 (t-test). d' S i g n i f i c a n t f r o m a l c o h o l c o n t r o l at 0.001 (t-test).

105

~ O CH~

Fig.

CH 3 CH 3

2

]

1. 2,3-Dihydro-2,2-dimethyl-7-benzofuranyl [(dibutylamino)thio] methyl carbamate (CAS No. 55285-14-8).

Carbosulfan as test substances. The chemical structure and formula of Carbosulfan is shown in Fig. 1. Whole blood (0.1 ml) from a healthy male donor (age 26, non smoker) was added to 2.5 ml Chromosome Medium B (Biochrom). The cultures were incubated at 37°C for 72 h. The cells were treated with 10 -6, 5 x 10 -6, 10 -5 and 5 x 10 -5 (v/v) final concentrations of Marshal and Carbosulfan for 12, 24 and 48 h. In addition, a normal control and solvent control (80% ethanol) were present at each treatment time. Colchicine

(5 X 10 -7 mol final concentration) was present for the last 2 h of culture. To collect cells, the cultures were centrifuged (1000 rpm, 10 min), treated with hypotonic solution (0.4% KC1) for 23 min at 37°C, and then fixed in fixative (methanol:glacial acetic acid 3:1) for 20 min at room temperature. The treatment with fixative was repeated 3 times. The cells were spread on glass slides and air dried. One day old slides were stained with 5% Giemsa stain (pH 6.72) prepared in Sorensen buffer solution, for 15 min, washed in distilled water, dried and mounted with Entellan. A total of 100 well spread metaphases per dose were examined at 1000 x magnification for the occurrence of different types of CA. Various numerical and structural chromosomal abnormalities within each metaphase were recorded. These data were used to determine the percentage of cells with abnormalities and the total number of CA. Also, in these ceils the number of gaps were scored. The criteria for distinguishing between

Fig. 2. Chromatid break (10 -5 Carbosulfan 12 h). x 1000.

106

gaps and chromatid breaks were according to Kauderer et al. (1991) based on Preston (1987). Because cells with chromosome contractions were present in the cultures treated with 5 x 10 -5 ( v / v ) concentration of Marshal and Carbosulfan, the number of cells with chromosome contractions was determined for cultures of this dose. The chromosomes of cells with chromosome contraction are shorter and thicker than normal cell chromosomes. A total of 3,000 cells were scored for mitotic index (MI) and the percentage of cells in mitotic division was determined. The significance between m e a n CA and its controls was determined with the t-test.

TABLE

Carbosulfan increased the total number of abnormalities and the number of cells that had abnormalities at all treatment times and concentrations (Table 1). In the 12 h treatment time, an increased number of cells with aberrations was observed but the dose response was not dose-dependent. This increase was significant from control only. However, abnormalities increased due to alcohol control but not significantly. In the cultures treated for 24 and 48 h, both the total number of abnormalities and the number of cells which included abnormalities increased in a dose-dependent

2

MI, STRUCTURAL SHAL

Results

FOR

AND

12, 24 A N D

NUMERICAL

CHROMOSOME

ABNORMALITIES

IN CULTURES

TREATED

WITH

MAR-

48 h *

Time

Concen-

Structural and numerical

Number

Total

Number

of

(h)

tration

chromosome

of

number

CA/cell

+ Sx

(%)

B'

abnor, cells

of CA

12

MI

R

SU

DS

3.24

6

-

2

-

-

C. + e t h a n o l 10 - 6

3.00 3.56

7 7

-

3 2

-

1

-

-6

T

TR

1

-

TT

P

E

2

10

8

0.08 + 0 . 0 2 7

-

11 10

11 10

0.11+0.031 0.10+0.031

2.99

5

-

7

-

-

2

2.82

9

-

4

-

1

-

6

1.99

17

-

5

-

1

-

-

Control

3.63

7

-

2

C. + ethanol 10 - 6

3.02 2.80

7 6

-

3 1

1

-

-

-

5×10 -6

2.72

5

-

2

-

1

-

-

8

8

0.08-t-0.027

10 - 5

2.46

10

-

6

-

-

13

16

0.16-t-0.044

5 x 10-5

1.07

21

-

9

-

-

24

31

0.31+0.061

Control

2.79

8

-

3

2

13

12

0.12+0.035

C. + ethanol

3.23

6

-

6

1

-

13

14

0.14 + 0.035

10 - 6

2.47

7

1

6

1

-

15

15

0.15+0.038

5 x 10- 6 10 - 5 5xlO -5 **

2.64 1.62 0.32

9 13

1 -

4 10

1 1

-

14 20

15 24

0.15 + 0 . 0 3 8 0.24+0.53 a

10 - 5 5x10

48

F

Control

5x10

24

B"

abnormalities

-5

-

-

* 100 c e l l s w e r e s c o r e d . ** Not scorable due to excessive toxicity. For abbreviations

s e e T a b l e 1.

a

S i g n i f i c a n t f r o m c o n t r o l a t 0.05 ( t - t e s t ) .

a' b

S i g n i f i c a n t f r o m a l c o h o l c o n t r o l a t 0.05 ( t - t e s t ) . Significant from control at 0.02 (t-test).

c'

S i g n i f i c a n t f r o m a l c o h o l c o n t r o l a t 0.01 ( t - t e s t ) .

d

S i g n i f i c a n t f r o m c o n t r o l a t 0.001 ( t - t e s t ) .

1

-

1

1

-

1

-

-

-

1

1 1

-

8

12

0.12+0.055

19

14

0.14+0.040

17

23

0.23+0.054

9

9

0.09 + 0.032

10 7

11 7

0.11 + 0 . 0 3 9 0.07+0.025

ha'

ac'

107 manner. The increases of abnormalities at these two treatment times were significant from both controls. The most common types of abnormalities observed were chromatid breaks and fragments (Fig. 2). Marshal, on the other hand, significantly induced the formation of CA at the highest concentration (5 x 10 -5 v / v ) only and at two treatment times (12 and 24 h) (Table 2). This increase was significant from both controls. At the 48 h treatment time, the 10 -5 ( v / v ) concentration increased abnormalities significantly from control only. As with Carbosulfan, chromatid breaks and fragments were the most common abnormalities observed (Fig. 3). Both test ingredients also decreased the MI dose-dependently for all treatment times (Tables 1 and 2). Both test substances increased the number of gaps, but no relation was established between the number of gaps and increasing treatment times and concentrations. Also ethanol moderately in-

creased the number of gaps compared to the normal control. Neverthless, Carbosulfan induced an increase in the number of gaps at all concentrations compared to its controls, while Marshal effectively induced an increase primarily at the two highest concentrations (Table 3). Proportions of cells which contained chromosome contractions were increased at the highest concentration (5 x 10 -5 v / v ) of Carbosulfan and Marshal. During the 12 and 24 h treatment times, both test substances caused chromosome contractions. The percentage of cells which had chromosome contractions after the 12 h treatment with Carbosulfan and Marshal was higher than after treatment for 24 h (Table 4 and Fig. 4). Discussion

In our study, the test substances induced the formation of chromosomal aberrations. The resuits are similar to those obtained by Cid and Matos (1987), who showed that Aldicarb, a carba-

-! I-

Fig. 3. Two fragments (10 -5 Marshal 24 h). × 1000.

t

!

108

Fig. 4. (A) Cells with chromosome contraction (5 × 10 -5 Carbosulfan 12 h). (B) Chromosomes of cells of control cultures. × 1000.

109 TABLE 3 T H E F O R M A T I O N OF GAPS IN C U L T U R E S T R E A T E D W I T H V A R I O U S C O N C E N T R A T I O N S OF CARBOSULFAN A N D M A R S H A L F O R 12, 24 A N D 48 h * Time Concen-

Carbosulfan

Marshal

(h)

tration

Number

Number

(%)

of cells number of cells number with gaps of gaps with gaps of gaps

12

Control C. + ethanol 10 -6 5 x 10-6 10 -5 5 x 1 0 -5

45 53 58 58 68 60

50 74 104 98 118 113

36 51 50 44 62 58

50 76 71 63 91 104

24

Control C. + ethanol 10 -6 5 x 10 -6 10 -5 5 x 10 -5

30 31 47 51 40 51

33 44 77 72 61 85

46 48 51 48 63 64

71 78 80 71 101 116

48

Control C.+ethanol 10 -6 5 x 10-6 10 -5 5 x 10-5 **

31 37 43 56 54

36 52 62 84 87

44 43 58 52 52

63 70 79 78 86

Total

Total

* 100 cells were scored. ** Not scorable due to excessive toxicity.

TABLE 4 T H E P E R C E N T A G E OF T H E CELLS T H A T H A D CHROMOSOME CONTRACTIONS TREATED WITH 5 x 10 -5 F I N A L C O N C E N T R A T I O N O F C A R B O S U L F A N A N D M A R S H A L F O R 12 A N D 24 h * Substance

Concentration (%)

Treatment time (h)

Percentage of cells with chrom. contraction

Carbosulfan

5 x 10 -5

12 24 48 **

89 53

Marshal

5 × 10-5

12 24 48 **

70 34

* 100 cells were scored. ** Not scorable due to excessive toxicity.

mate, used widely as an insecticide in the ~ukurova region, induced CA in human lymphocytes. Also Cid et al. (1990) reported that Propoxur and its nitroso derivative NO-propoxur increased the formation of micronuclei in human lymphocytes. Some reports indicated that methyl2-benzimidazole carbamate (MBC) (Bavistin, Carbendazim), which is a carbamate fungicide, did not increase the number of CA but did induce tetraploidy and octoploidy, and caused the formation of micronuclei and c-mitosis in human peripheral blood (Banduhn and Obe, 1985). Also it has been reported that MBC induced lagging chromosomes at anaphase I, II and telophase I, increased the number of univalent chromosomes at meiosis division, and induced chromosome bridges and the formation of micronuclei at telophase II in Capsicum annuum (Prakash et al., 1988). Furthermore, it has been demonstrated that various carbamate pesticides induced the formation of CA and micronuclei in various animal and plant cells (Amer, 1965; Nelson et al., 1981; Adhikari and Grover, 1988; Zelesco et al., 1990). As shown from the results of both our study and other investigations, carbamate pesticides induce the formation of CA. Also, some of them induced c-mitosis and polyploidy, and increased the formation of micronuclei. Fahrig (1990) emphasized that pesticides which were mutagenic to somatic cells could affect the generative cells. The results mentioned above show that some pesticides cause both structural and numerical alterations in chromosomes of eukaryotic cells. For this reason, carbamate pesticides have a genotoxic risk for the hereditary structure. In this study, the number of gaps was increased in the treated groups compared to the control. However, no relation was established between the number of gaps and increasing concentrations (Table 3). Mace et al. (1978) from their electron microscopy study reported that a chromatid break is characterized by the complete absence of chromatin fibers between the severed parts and that a chromosomal gap has a loss of chromatin material. However, continuity within the chromatid still remains. As a result, it is our opinion that gaps represent spiral unravellings but not CA. For this reason, we evaluated the gaps apart from CA. However, some investigators

110

have included both types of abnormalities (gaps and CA) in the same catagories in their analysis (Honeycombe, 1978; Basler, 1980; Basler and R6hrborn, 1980; Cid and Matos, 1987; Jablonicka et al., 1989; Kauderer et al., 1991). In our investigations, chromosome contractions were evident in the cultures treated with the highest concentration (5 × 10 -5 v/v) of Carbosulfan and Marshal (Table 4). The percentage of cells which had chromosome contractions was higher at 12 h of treatment than at 24 h. The contractions may have arisen from effects on the histone proteins. In the longer treatment periods, the cclls could decrease the effects of the pesticides with their own repair mechanisms. It has been observed that isoprophyl-N-(3-chlorophenyl) carbamate (CIPC) caused chromosome contractions in root tip meristems of Ficia faba (Yoshida et al., 1983) and Benomyl (a carbamate fungicide) in root tip meristems of Hordeum vulgare (Nicoloft and Kappas, 1987). Similar to this and other investigations, some carbamate pesticides also caused chromosome contractions such as are induced by colchicine or colcemid. We noted in this study that Carbosulfan and Marshal increased the number of CA while in another study Carbosulfan did not induce mutations in strains TA97, TA98, TA100, TA102 of S. typhimurium and strain D7 of S. cerevisiae, but caused mitotic aneuploidy in strain D61.M of S. cerevisiae (Wiedenmann et al., 1990). Carbosulfan is transformed into Carbofuran within plants (Clay and Fucuto, 1984). According to Moriya et al. (1983), Carbofuran is mutagenic in strains TA98 and TA1538 of S. typhimurium. NO-carbofuran and its two derivatives (3-hydroxynitroso carbofuran and 3-ketonitroso carbofuran) induced mutations in strains TA98 and TA100 of S. typhimurium. These substances induced the formation of CA in Chinese hamster and its only derivative increased the number of sister-chromatid exchanges (SCE) moderately (Nelson et al., 1981). Because of these results it can be concluded that Carbosulfan is genotoxic in animals and plants. Before determining whether it definitely poses a genotoxic risk or not, it must be determined whether it induces SCE and CA using metabolic activation and in vivo assay conditions.

Acknowledgements This study was supported by the t~ukurova University research fund and carried out in the Genetics Laboratories, Physiology Department, Medical Faculty of t~ukurova University. We thank Prof. Dr. Tuncay OzgiJnen, Dean of the Medical Faculty and Yrd. Do~. Dr. Giilay Lo~o~lu, head of the Physiology Department. And we also extend our thanks to the personnel of the research fund of t~ukurova University.

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