Evaluation of Genotoxic and Cytotoxic Properties of Pesticides Employed in Italian Agricultural Practices

Environmental Research Section A 83, 311}321 (2000) doi:10.1006/enrs.2000.4057, available online at http://www.idealibrary.com on

Evaluation of Genotoxic and Cytotoxic Properties of Pesticides Employed in Italian Agricultural Practices A. De Marco,* R. De Salvia,* S. Polani,* R. Ricordy,* F. Sorrenti,* P. Perticone,* R. Cozzi,- C. D’Ambrosio,? C. De Simone,? M. Guidotti,A T. Albanesi,B G. Duranti,B F. Festa,B G. Gensabella,B and M. OwczarekB *Centro di Genetica Evoluzionistica, CNR, Rome, Italy; -Dipartimento di Biologia, Universita` ‘‘Roma Tre,’’ Rome, Italy; ?Istituto Sperimentale Studio e Difesa del Suolo, Rieti, Italy; APresidio Multizonale di Prevenzione, Rieti, Italy; and BResearch contracts from Ministero Lavoro, Rome and Rieti, Italy Received July 30, 1999

sensitive than those referring to cytotoxicity.

 2000

Academic Press

In a program coordinated by the Italian Ministry of Works, we tested in vitro four pesticides widely employed in a developed agricultural region of central Italy. The four commercial agents were chosen on the basis of their diffusion in agricultural practice, knowledge of their active principle(s), and scant availability of data concerning their toxic and genotoxic activity. The agents were Cirtoxin, Decis, Tramat Combi (TC), and Lasso Micromix (LM). All substances were tested in three in vitro systems: Chinese hamster ovary (CHO) cells, a metabolically competent hamster cell line (Chinese hamster epithelial liver; CHEL), and root tips of Vicia faba (VF). The cytotoxic and genotoxic end points challenged were micronuclei and root tip length (RTL) in VF and mitotic index (MI), proliferation index (PI), cell survival (CS), cell growth (CG), cell cycle length (CCL), sister chromatid exchanges, chromosomal aberrations, and single-cell gel electrophoresis, or comet assay, in CHEL and CHO cells. Tested doses ranged from the Aeld dose up to 200ⴛ the Aeld dose to take into account accumulation effects. On the whole, tested agents appear to induce genotoxic damage only at subtoxic or toxic doses, indicating a low clastogenic risk. MI, PI, CS, CG, RTL, and CCL appear to be the less sensitive end points, showing no effects in the presence of a clear positive response in some or all of the other tests. Using cytogenetic tests, we obtained positive results for TC and LM treatments in CHO but not in CHEL cells. These data could be accounted for by postulating a detoxifying activity exerted by this cell line. However, cytogenetic end points appear to be more

Key Words: Cirtoxin; Decis; Tramat Combi; Lasso Micromix; alaclor; clopyralid; ethofumesate; lenacil; deltamethrine; comet assay; cell cycle; cell growth; cell survival; micronuclei; root tips length; sister chromatid exchanges; mitotic index; proliferation index; chromosomal aberrations; CHO cells; CHEL cells; Vicia faba.

INTRODUCTION

Despite the increasing use of pesticides in recent decades, not only in the so-called ‘‘developed countries,’’ available literature data do not cover the concentration and use of all the more widely used agents. This appears to be detrimental to the health not only of the workers actually involved but also of the human population as a whole, in view of the presence of residues in commercially available foodstuffs (Pang et al., 1999; Molinari et al., 1998; Lewis et al., 1998; Gamon et al., 1998). Indeed, even though the majority of the pesticides tested were found not to be mutagenic in vitro, many of them are clastogenic both in vitro and in vivo (Der7eld et al., 1993; Garren et al., 1986; WHO, 1983, 1991), as is con7rmed by the increased risk of tumors in farmers vs nonfarmers (Blair et al., 1985; Morrison et al., 1992; Zohm and Blair, 1992). In the present collaborative study, we focused on a well-developed agricultural region in the center of Italy, ‘‘piana Reatina’’ (Rieti plain), 120 km northeast of Rome (Fig. 1), with intensive cultivars of beet, maize, wheat, and sun8ower. Although not covering a particularly large area (10,000 ha), the region is almost completely given over to agriculture and food processing activities, and, more interesting, health

 To whom reprint requests should be addressed at Centro di Genetica Evoluzionistica del CNR, c/o Dipartimento di Genetica e Biologia Molecolare, Universita` ‘‘La Sapienza,’’ 00185 Rome, Italy. Fax: ##39/064457529. E-mail: [email protected]. 311

0013-9351/00 $35.00 Copyright  2000 by Academic Press All rights of reproduction in any form reserved.

312

FIG. 1.

DE MARCO ET AL.

Rieti Plain (darker zone) is located in central Italy, 120 km north east of Rome. The approximate extension is 10,000 ha.

monitoring structures for farmers are present (Presidio Multizonale di Prevenzione), as well as of7cial national structures depending on the Italian Agriculture Ministry (Experimental Institute for Soil Defense and Study, Regional Institute for Environmental Defense) from which a complete list of the pesticides used, including doses and quantities used per kilogram of soil, was obtained. On the basis of these background data, we surveyed the available literature to choose a group of agents that satis7ed the following criteria: (i) they were in wide use; (ii) the literature contained few or contradictory data on their genotoxicity; and (iii) we had knowledge of the active principles. In the 7rst approach, our attention was focused on the following commercial pesticides: Cirtoxin (CX), Decis (DS), Tramat Combi (TC), and Lasso Micromix

(LM).Characteristics, 7eld doses used, CAS numbers, available literature data, and active principles with extended chemical names and formulas are given in Fig. 2. As detailed in the next section, three cell systems were used: root tips of Vicia faba (VF), Chinese hamster ovary (CHO) cells, and a Chinese hamster epithelial liver cell line (CHEL). The latter is a metabolically competent cell line, able to activate/detoxify certain procarcinogens/promutagens. Ten end points, both cytotoxic and genotoxic, were used to investigate the effects of these pesticides. In our opinion the combination of 10 end points in three cell systems produces a suf7ciently clear picture of the agents investigated. Data presented here will be integrated with epidemiological and biomonitoring results from other groups working on

TOXICITY OF PESTICIDES IN VITRO

FIG. 2.

313

Chemical data on tested pesticides.

the coordinated project and subsequently disseminated among those working in this 7eld. MATERIALS AND METHODS

Agents Tested Cirtoxin (Dow-Elanco SpA), Decis (Roussel Hoechst Agrovet SpA, No. 7393), Tramat Combi (Agrevo Italia SpA, No. 6032), and Lasso Micromix (Monsanto Italia, No. 8068) were tested in this work. Please refer to Fig. 2 for details on chemical names,

formulas, molecular weights and CAS numbers of the active principles. For the VF experiments, commercial products were administered to the cells as suggested by the factories; for details please refer to the appropriate section below. In all other cases the commercial products were dissolved in PBS, through magnetic stirring at room temperature, immediately before use. The indicated dilutions refer to the whole commercial product. To calculate the amount (%) of the active principle(s), please refer to Fig. 2 for the

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DE MARCO ET AL.

molecular weights. To compare 7eld doses with doses used in mammalian cell experiments, we considered 1;103 ml medium as approximately equivalent to 1;103 g soil. Using this approximation, we can compare the range of tested doses, in all investigated systems, both within them and with probable effects in the practice (in the 7eld). Moreover, VF tests let us use doses unambiguously related to those used in the 7eld, because of the nature of the test (plants growing in soil). Mammalian Cell Cultures CHO cells were routinely cultured in Ham’s F-12 medium (Flow, U.S.A.) supplemented with 10% FBS (Gibco, U.S.A.), 1% L-glutamine, penicillin (5000 IU/ml), and streptomycin (5000 lg/ml). Under these growth conditions, the average generation time was 12 h, as measured by bromodeoxyuridine (BrdUrd) incorporation (Ivett and Tice, 1982). Chinese hamster epithelial liver cells established by Turchi et al. (1987) were cultivated in Williams E. medium supplemented with 10% FBS (Gibco, U.S.A.), 2 mM L-glutamine, and antibiotics (complete Williams medium). Cells were seeded at density of 1;106 cells/10 ml medium. Under our experimental conditions, more than 60% of cells exhibited 24 chromosomes; only cells with this modal number were scored in experiments. For all other experimental conditions, please refer to the CHO cell section. Vicia faba Root Tips Seeds of Vicia faba var minor from the same stock, homogeneous in size, and stored in a cold chamber were used. The main physicochemical features of the soil used were sand, 89.9%; silt 8.7%; clay, 1.5%; total CaCO3, 15.5%; pH (H2O), 8.72; pH (KCl), 7.79; total organic carbon, 0.21%, and organic matter, 0.36%. Particle size distribution was determined by pipette analysis with dispersion with sodium hexametaphosphate. Soil pH, in both H2O and 1 N KCl, was determined by using a glass electrode at 2.5 : 1 liquid : soil ratio (v/v). Total organic carbon (TOC) was analyzed using the Walkley}Black method and the organic matter was calculated by multipying the TOC by 1.724. Determination of total CaCO3 was carried out by using a De Astis calcimeter. Seeds (50 for each experimental point) were set to germinate in aluminum basins containing 485 g of sandy soil with 110 ml of deionized H2O. After 3 days they were transplanted into other basins in which the different pesticides were dissolved. For treatments and collec-

tion of samples, please refer to MN and RTL sections below.

Cytotoxic and Genotoxic End Points Micronuclei (MN) and root tips length (RTL). After 2 days of treatment the seedlings were removed, their primary root lengths were measured, and the root tips were 7xed in ethyl alcohol : acetic acid, 3 : 1 (v/v), and prepared for microscopic observation according to a previously described technique (De Marco et al., 1992). All samples were kept in a climatic chamber at 20$13C. Experiments, performed in duplicate, were repeated at least two times. At each point the genotoxic effects were assessed by studying the frequency of micronucleated cells in 20,000 cells (10 root tips, 2000 cells per root tip). The toxic effects were evaluated by analyzing the growth of the primary roots in 25 seedlings per experimental point at the end of the treatment. Data were analyzed by a one-way variance analysis, and the means were compared by Duncan’s multirange test at P"0.05 level using SPSS software (SPSS Corp.). To obtain greater homogeneity among variances (Cochran’s test), the data on micronucleated cell frequency were submitted to square-root transformation before the analysis. Mitotic and proliferation indices (MI and PI). The mitotic index was calculated as the number of mitoses (c mitosis) in 100 cells. The proliferation index was calculated for 100 BrdUrd-treated cells from the relative frequencies of M1, M2, M3 cells, following the formula PI"(M3#1)!M1. Data were derived from at least three separate experiments for each point considered. Cell survival (CS). Both CHEL and CHO cells were counted from a suspension in a hemocytometer, and 100 cells were seeded into a petri dish. The cells were treated with the appropriate concentration of tested substances and incubated for 1 week. Colonies were counted and the surviving fraction was calculated as colonies counted/cells seeded;(PE/100), where PE is the planting ef7ciency, calculated as the percentage of cells seeded that are able to grow into colonies. Experiments were repeated at least three times. Cell growth (CG). Both CHEL and CHO cells were counted, seeded into 8asks (300 cells/8ask), and treated with the appropriate concentration of tested substances. After 24, 48, and 72 h, cells were trypsinized and counted to evaluate the rate of

315

TOXICITY OF PESTICIDES IN VITRO

growth. Experiments were repeated at least three times. Cell cycle length (CCL). Both CHEL and CHO cells were treated with the appropriate concentrations of tested substances and analyzed after 24, 48, 72, and 96 h. Cells were treated with a pulse of BrdUrd (10 lM) 15 min before 7xing. BrdUrd detection was carried out by incubating, in 0.5% Tween 20, 0.1 mg mouse anti-BrdUrd (Ylem) plus 5 ll serum for 30 min at room temperature. Flow cytometric analysis was carried out on a FACStar Plus (Becton-Dickinson) equipped with a 5-W Innova-90 argon-ion laser (Coherent), using dual 8uorescence excitation at 488 nm. Experiments were repeated at least three times. Sister chromatid exchanges (SCEs). Harvesting and preparation of slides were performed, with both cell lines, according to the air-drying method. The usual Giemsa plus Hoechst technique (Perticone et al., 1987) was used for differential staining of sister chromatids. A total of 40 metaphases from coded slides were scored for each point and each treatment. Data analyzed were derived from at least three separate experiments. Means, standard deviations, and standard errors were determined. For each harvesting time, control and treated cultures were compared with the t test and the P value was determined. Chromosomal aberrations (CA). Harvesting and preparation of slides were carried out as for SCEs. Slides were stained with 3% Giemsa in Sorensen’s buffer and then made permanent with Eukitt. At least two slides per dose level were coded and examined under oil immersion at 1250;. Two hundred well-spread metaphases were analyzed at each dose. Data were derived from at least three separate experiments for both cell lines. The number of aberrant cells was used for statistical analysis with Fisher’s exact test. Comet assay (SCGE). The assay was performed basically according to Singh et al. (1988), with some modi7cations. Brie8y, 20 ll of cell suspension (10,000}20,000 cells approximatively) was mixed in 380 ll of 0.7% low-melting agarose in PBS at 373C and immediately pipetted onto frosted glass microscope slides precoated with a layer of 1% normalmelting-point agarose, similarly prepared in PBS. The agarose was allowed to set at 43C for the necessary time, and the slides were incubated in lysis solution (2.5 M NaCl, 10 mM Tris}HCl, 100 mM Na2EDTA, NaOH to pH 10, 1% Triton, and 10%

DMSO). After lysis, slides were placed on a horizontal electrophoresis unit containing fresh buffer (1 mM Na2EDTA, 300 mM NaOH, pH 13). Electrophoresis was conducted for 15 min at 25 V (300 mA) at 43C. Slides were then washed gently in neutralization solution for 5 min (0.4 M Tris}HCl, pH 7.5), 7xed in 100% fresh methanol for 3 min, and stained with 90 ll ethidium bromide. Stained nucleoids were scored visually using a 8uorescence microscope (Leica) with a Cohu camera. Fifty comets on each slide were acquired using the ‘‘IAS’’ software automatic image analysis system purchased from Delta Sistemi (Italy). Parameters considered were as follows: (i) Tail length was calculated from either the center or the trailing edge of the cell. (ii) Tail moment, as described by Olive et al. (1990), better quanti7es the amount of DNA damage, as it describes not only the distance the DNA has migrated but also the amount of DNA that has migrated from the head region. It was calculated as the percentage of DNA in the tail multiplied by the distance between centers of mass. (iii) Tail inertia, as de7ned by Helmann et al. (1995), provides an index of damage induced, considering both the migration of DNA and the relative amount of DNA in the tail. It seems to be a more sensitive indicator of heavily fragmented/damaged DNA. Data are the means of at least three separate experiments. RESULTS

Data are corrected in Tables 1}4. For each agent tested, the tables contain six parts (A}F), except for Table 2 (Decis), where the data on MN, RTL, SCE, CA, MI, and PI where pooled in three parts. Each table summarizes data obtained from all applied tests, in all cell systems, and for each commercial agent tested. Part A of each table addresses MN frequency and RTL in Vicia faba. In both cases data were challenged with Duncan’s multirange test. Control values were obtained by the use of H2O. Doses range from the 7eld dose up to a dose 200; greater. Parts B and C contain data for CS and CG in both CHO and CHEL, when available, and CCL in both CHO and in CHEL. Part D of each table contains data on SCGE in CHEL and CHO; tail length, moment, and inertia are also indicated. CA after both 12- and 18-h pulses and SCE, MI, and PI are listed in Parts E and F, respectively. In the following, four sections, the results for each pesticide are described.

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DE MARCO ET AL.

TABLE 1

TABLE 1EContinued

Cirtoxin A. Micronuclei and root tips length in Vicia fabaa MN RTL Dose (g/kg soil)

% of MN

SD

Length

Control 0.016 (fd) 0.16 0.32 1.66 3.32

0.09 0.08 0.09 0.22 0.18 2.11

0.04 0.09 0.03 0.12 0.21 1.99*

34.2 31.3 18.7 14.3 12.7 11.6

SD 2.4 5.6 4.1* 3.6* 2.8* 2.06*

B. Cell survival in CHEL cellsb Dose (mg/ml)

Plate A

Plate B

Plate C

Plate D

Mean

82 84 79 72

83 75 80 88

76 79 74 76

73 78 71 84

78.5 79 76 80

Control 0.016 (fd) 0.080 0.160

C. Cell growth in CHO and CHEL cellsc CHO CHEL Dose (mg/ml) Control 0.016 (fd) 0.080 0.160

0h

24h

48h

0h

24h

48h

500 500 500

1200 1500 1100

2090 1910 1730

300 300 300 300

200 320 345 270

550 645 552 595

T. length

Control H2O2 0.016 (fd) 0.16

13.06 40.15** 2.91 5.39

Control 0.010 (fd) 0.10 1.04 2.08

8.09 9.01 11.9 14.03 13.67

0.31 0.46 0.22* 1.35* 1.20*

4.6 4.04 4.5 2.1 5.3

1.0 0.95 1.0 0.93 0.94

10.10 10.03 14.91 17.83

0.33 0.83 1.25* 2.01*

3.6 4.3 5.1 2.6

1.03 1.0 0.83 0.65

Note. fd, Field dose. a Controls were performed in tap water. * P:0.01, Duncan’s multirange test. MN, % of micronucleated cells/20,000 cells. RTL, mean length of the primary root (mm)/25 roots. b Each dish plated with 100 cells. Cells treated 24 h after plating and 7xed 120 h later. c All values are intended ;1000. Treatments 3.5 h after seeding of cells. 0 to 72 h, harvesting after time treatments. d H2O2"10\4 M as positive control. For a description of tail length, moment, and inertia, please refer to Materials and Methods. * P:0.05 and **P:0.001, t test. e Chromosomal aberrations in 100 cells. Treatment pulses of 2 and 18 h * P:0.05, Fisher’s exact test. % abnor., % abnormal cells. Ctid exs and Cme exs, respectively, chromatid and chromosome exchanges. f Mitotic index of 100 cells. * P:0.01, t test.

72h 485 500 515 530

D. Single-cell gel elecrophoresis in CHO and CHEL cellsd CHO CHEL Dose (mg/ml)

F. Mitotic and proliferation indices and sister chromatid exchanges in CHO and CHEL cellsf CHO CHEL Dose (mg/ml) SCE SE MI PI SCE SE MI PI

T. moment

T. inertia

T. length

T. moment

T. inertia

8.23 35.55** 0.8 1.74

551.6 3457.1** 57.74 113.23

4.08 14.3** 6.8 5.28

1.15 7.3** 4.19 2.02

122.48 723.1** 429.11 236.51

Cirtoxin?Field Dose: 0.016 mg/ml (Table 1) Available data do not include CCL for both mammalian cell lines and CS for CHO and CA for CHEL. The agent on the whole appears not to affect CS, MI, and PI. In contrast a signi7cant effect on RTL appears at doses 20; above the 7eld dose (fd). MN are signi7cantly increased only at a dose 200; above the fd. SCE appear to be affected at doses 10; above the fd in CHEL but not in CHO. CA appear to increase starting at a dose 60;greater in CHO cells. SCGE is affected in CHO but not in CHEL cells. Decis?Field Dose: 0.002 mg/ml (Table 2)

E. Chromosomal aberrations in CHO cellse Chromosomal aberrations Dose (mg/ml) 2-h pulse Control 0.01 (fd) 0.63 2.11 6.9 22.62 18-h pulse 0.19 0.63 2.11

% abnor.

Gaps

Breaks

Ctid exs Cme exs

CA-gaps

4 3 10 9 10 6

3 2 7 4 2 2

3 3 3 7 8 6

0 0 1 0 1 0

0 0 2 0 0 0

3 2 6 7 9 6

9 17 No mitoses

7 11

5 13

0 0

0 0

5 13*

Available data do not include SCGE, CCL, CM, CS, and CG for both mammalian cells and CA for CHEL. MI, PI, and RTL show the absence of cytotoxic effects. MN indicate a slight (but not signi7cant) increase only at highest dose, whereas CA appears to be dramatically affected starting at an 8; dose, and SCE show an increase at a 100; dose only in CHO cells. Tramat Combi?Field Dose: 0.016 mg/ml (Table 3) Available data do not include SCE, PI, and MI for CHEL cells.

317

TOXICITY OF PESTICIDES IN VITRO

TABLE 2

TABLE 3

Decis

Tramat Combi

A. Micronuclei and root tips length in Vicia fabaa MN RTL

A. Micronuclei and root tips length in Vicia fabaa MN RTL

Dose (g/kg soil)

% of MN

SD

Length

SD

Control 0.002 (fd) 0.025 0.25 2.5 5.0

0.09 0.10 0.14 0.12 0.15 0.25

0.04 0.07 0.16 0.08 0.05 0.14

34.2 33.2 31.5 33.8 31.6 33.6

2.4 7.91 5.56 9.77 9.47 10.41

Dose (g/kg soil)

% of MN

SD

Length

SD

Control 0.016 (fd) 0.16 0.32 1.66 3.32

0.09 0.10 0.12 0.10 0.18 0.12

0.04 0.05 0.08 0.06 0.11 0.09

34.2 30.3 28.1 20.9 21.3 19.8

2.4 2.4 2.8 2.0* 1.7* 1.2*

B. Chromosomal aberrations in CHO cellsb Chromosomal aberrations Dose (mg/ml) 2-h pulse Control 0.002 (fd) 0.02 0.40 1.60 18-h pulse 0.02 0.04 0.16 0.40

% abnor.

Gaps

Breaks

Ctid exs Cme exs

3 11 23 20 24

2 8 19 13 12

2 3 8 11 11

0 0 0 0 0

0 0 0 0 1

2 3 8 11* 12*

12 14 19 40

8 16 13 34

4 2 11 32

0 0 0 0

0 0 0 0

4 2 11* 22**

CA-gaps

C. Mitotic and proliferation indices and sister chromatid exchanges in CHO and CHEL cellsc CHO CHEL Dose (mg/ml) SCE SE MI PI SCE SE MI PI Control 0.002(fd) 0.02 0.20

9.05 9.75 10.10 13.21

0.71 0.37 0.53 1.35*

4.6 3.8 4.5 3.3

1.0 0.98 0.95 0.84

10.5 12.85 11.90

0.42 0.48 0.75

3.6 4.2 2.5

CS in CHO cells Dose (mg/ml) Control 0.016 (fd) 0.064 0.128

Controls were performed in tap water. fd, 7eld dose. * P:0.01, Duncan’s multirange test. MN, % of micronucleated cells/20,000 cells. RTL, mean length of the primary root (mm)/25 roots. b Chromosomal aberrations in 100 cells. Treatment pulses of 2 and 18 h. * P:0.05 and ** P:0.01, Fisher’s exact test. % abnor., % abnormal cells. Ctid exs and Cme exs, respectively, chromatid and chromosome exchanges. c Mitotic index of 100 cells. * P:0.01, t test.

The agent appears able to affect RTL at a dose 20; the 7eld dose. CCL shows a clear decrease in G1 cells at all times tested after the treatment in CHO cells alone, whereas in CHEL, cell cycle parameters do not differ from controls. SCGE appears to be affected only in CHO cells. CS is only slightly affected in CHO cells, CG is affected in a dose-dependent way in CHO and is more evident in CHEL cells. MI and PI in CHO cells are decreased only at the higest dose. CA are increased more clearly than SCE in CHO and CHEL, whereas both MI and PI are especially affected in 24-h treatments. No increase is evident in the MN test.

Mean

Dose (mg/ml)

0h

24 h

48 h

72 h

280 267 246 236

Control 0.016 (fd) 0.064 0.128

500 500 500 500

1650 1390 1420 1240

3730 3550 2800 2000

3820 4450 3320 3240

C. Cell cycle length in CHO and CHEL cellsc Phase 24 h 48 h 72 h

96 h

Control

TC

1.03 0.93 0.99

a

B. Cell survival and cell growthb CG in CHEL cells

G  S G 

35}33 52}38 13}28

50}47 42}12 8}40

81}41 13}13 2}45

88}48 2}6 5}45

G  S G 

76}36 9}32 6}31

62}51 16}12 6}36

63}47 8}11 8}41

59}50 4}2 9}48

D. Single-cell gel electrophoresis in CHO and CHEL cellsd CHO CHEL Dose (mg/ml)

T. length

Control H2O2 0.016 (fd) 0.16

13.06 40.15** 2.91* 5.39

T. moment

T. inertia

T. length

T. moment

T. inertia

8.23 35.55** 0.8* 1.74

551.6 3457.1** 57.74* 113.23

4.08 14.3** 6.8 5.28

1.15 7.3** 4.19 2.02

122.48 723.1** 429.11 236.51

E. Chromosomal aberrations in CHO and CHEL cellse Chromosomal aberrations Dose (mg/ml) 2-h pulse Control 0.016 (fd) 0.07 0.23 0.70 18-h pulse 0.006 0.016

% abnor.

Gaps

Breaks

Ctid exs Cme exs

CA-gaps

2}3 16}14 10}22 12}10 12}8

2}1 2}0 0}9 0}0 0}0

1}2 10}21 7}12 16}9 13}0

0}0 6}4 6}10 3}2 3}8

0}0 6}0 0}2 0}2 2}0

1}2 22** 25** 13*}24** 19**}13* 18**}8

8}2 22}10

0}0 2}0

13}2 33}8

1}0 0}4

0}0 0}0

14**}2 33**}12*

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DE MARCO ET AL.

TABLE 3EContinued

TABLE 4 Lasso Micromix

F. Mitotic and proliferation indices and sister chromatid exchanges in CHO cellsf Dose (mg/ml) SCE SE MI PI Control 0.016 (fd) 0.16 1.60 3.20

9.33 10.65 16.7 12.8 11.0

1.04 1.03 2.19* 1.03 0.55

4.6 4.1 4.7 4.4 2.7

1.0 0.89 0.94 0.89 0.87

Note. fd, Field dose. a Controls were performed in tap water. * P:0.01, Duncan’s multirange test. MN, % of micronucleated cells/20,000 cells. RTL, mean length of the primary root (mm)/25 roots. b Each of four dishes was plated with 200 cells. Cells treated 24 h after plating and 7xed 96 h later. All values are intended ;1000. Treatments 3.5 h after seeding of cells. 0 h to 72 h, harvesting time after treatments. c Values are the % of cells in that phase of the cell cycle. Underlined values"CHEL. Dose"0.16 mg/ml. d H2O2"10\ M as positive control. For a description of tail length, moment, and inertia, please refer to Materials and Methods. * P:0.05 and **P:0.001, t test. e Chromosomal aberrations in 100 cells. Treatment pulses of 2 and 18 h * P:0.05 and ** P:0.01, Fisher’s exact test. % abnor., % abnormal cells. Ctid exs and Cme exs, respectively, chromatid and chromosome exchanges. Underlined values"CHEL cells. f Mitotic index of 100 cells. * P:0.01, t test.

Lasso Micromix?Field Dose: 0.041 mg/ml (Table 4) Available data do not include SCE, PI, and MI for CHEL cells. A clear cytotoxic effect on RTL is evident at the higest dose (200;). Cell cycle parameters, such as CCL, MI, and PI, appear not to be affected. Conversely, CS and CG are signi7cantly affected. SCGE is clearly affected only in CHO cells, as in the case of SCE, which present a dose}effect relationship. CA are dramatically increased in CHEL but not in CHO cells. MN show a slight increase only at the strongest dose. DISCUSSION

A. Micronuclei and root tips length in Vicia fabaa MN RTL Dose (g/kg soil)

SD

Length

SD

0.09 0.13 0.14 0.22 0.18 0.29

0.04 0.02 0.01 0.07 0.09 0.13

34.2 32.1 33.0 26.6 25.8 19.4

2.4 2.7 1.8 1.6 1.3 1.2*

Control 0.04(fd) 0.40 0.80 4.00 8.00

B. Cell survival and cell growthb CG

CS

Dose Mean Mean Dose (mg/ml) CHEL CHO (mg/ml) Control 0.04 (fd) 0:40

87 73.5 0.5

0h

24 h

48 h

72 h

194.5 Control 300}300 480}860 1600}223 1400}2300 160.5 0.04(fd) 300}300 435}770 1580}220 1530}2600 28.75 0.2 300 440 545 710 0.40 300}300 395}640 505}1170 915}1400

C. Cell cycle length in CHO and CHEL cellsc 24 h 48 h 72 h

Phase Control G1 S G2 TC G1 S G2

96 h

35}64 52}16 13}19

50}43 42}15 8}41

81}43 13}12 2}44

88}42 2}4 5}40

65}52 23}19 6}27

45}62 38}4 9}33

56}59 25}5 5}35

72}53 3}3 9}32

D. Single-cell gel electrophoresis in CHO and CHEL cellsd CHO CHEL Dose (mg/ml)

T. length

Control H2O2 0.04 (fd) 0.4

Background The use of pesticides/herbicides and their spreading in the environment should be accompanied by a profound knowledge of their action, taking into account that several are known to cause alterations in the genetic material of different biological systems (Der7eld et al., 1993; Hrelia et al., 1994; IARC, 1991. In the past few years new pesticides that are more selective and ef7cient and possibly ‘‘safe’’ for nontarget organisms have been produced; so far, they have coexisted in farming practice with agents in

% of MN

T. moment

13.6 8.23 40.15** 35.55** 17.19 12.51 43.63** 30.32**

T. inertia

T. length

T. moment

T. inertia

551.6 3457.1** 1043.3 3164**

4.8 14.3** 5.7 6.8

1.15 7.3** 2.95 3.22

122.48 723.1** 287.7 286.56

E. Chromosomal aberrations in CHO and CHEL cellse Chromosomal aberrations Dose (mg/ml) 2-h pulse Control 0.04 (fd) 0.120 0.360 18-h pulse 0.01 0.04

% abnor.

Gaps

Breaks Ctid exs Cme exs CA - gaps

2}4 14}8 16}22 10}12

1}2 9}2 17}6 7}2

2}2 5}2 5}15 4}5

0}0 0}5 0}6 0}5

0}0 0}0 0}6 0}5

2}2 5}7 5}27** 4}15*

13 10

5 6

7 6

0 0

0 0

7 6

TOXICITY OF PESTICIDES IN VITRO

TABLE 4EContinued F. Mitotic and proliferation indices and sister chromatid exchanges in CHO cellsf CHO Dose (mg/ml) SCE SE MI PI Control 0.04 (fd) 0.20 0.40

6.54 9.6 11.0 10.6

0.49 0.76* 0.69* 0.76*

4.6 4.9 5.1 5.2

1.0 0.98 0.99 0.91

Note. fd, Field dose. a Controls performed in tap water. * P:0.01, Duncan’s multirange test. MN, % of micronucleated cells/20,000 cells. RTL, mean length of the primary root (mm)/25 roots. b Each of four dishes was plated with 100 (CHEL) and 200 (CHO) cells. Cells treated 24 h after plating and 7xed 96 h later. All values are intended ;1000. Treatments 3.5 h after seeding of cells. 0 h to 72 h, harvesting time after treatments. Underlined values"CHO. c h, harvesting times after the treatment. Values are the % of cells in that phase of the cell cycle. Underlined values"CHEL. Dose"0.40 mg/ml. d H O "10\ M as positive control. For a description of tail length,   moment, and inertia, please refer to Materials and Methods. * P:0.05 and **P:0.001, t test. e Chromosomal aberrations in 100 cells. Treatment pulses of 2 and 18 h. * P:0.05 and **P:0.01, Fisher’s exact test. % abnor., % abnormal cells. Ctid and Cme exs, respectively, chromatid and chromosome exchanges. Underlined values"CHEL. f Mitotic index of 100 cells. Doses expressed in mg/ml. * P:0.01, t test.

which one or more active principles have been found to be genotoxic and cytotoxic to various systems. In the present study two main goals were pursued: (i) to start a collaborative study on cytotoxic and genotoxic risks associated with agricultural practices in a controlled region (health monitoring, knowledge of working situation in terms of doses of commercial agents used, time of exposure, etc.) and (ii) to devise a set of sensitive, reliable, rapidly executable in vitro cytogenetic tests that can be applied in further studies. In the following sections we discuss our data in this light, taking both the activity of the pesticides tested and the sensitivity of the tests used into account.

Pesticides Four pesticides were investigated: CX, DS, TC, and LM. As previously stated, these agents were chosen because of their widespread use in the test region and the absence of available data in the literature. Described here in more detail are the characteristics of use and the availability of data (research performed in May 1999 by ENTREZ (NCBI)

319

[http://www.ncbi.nlm.nih.gov/Entrez]) for each agent investigated. E Cirtoxin (active principle (ap): 10% clopyralid). This agent has been subjected to very few investigations (only seven available papers, presenting no cytotoxic and genotoxic data but indicating CX as a suspect teratogen), the last dated 1997. The commercial product is used at a 7eld dose of 2 liters/ha (ap"200 g/ha). E Tramat Combi (ap: 12% lenacil and 30% ethofumesate). This agent was chosen because of the few available data (respectively only 10 and 2 references for the active principles and in both cases no cell data). For ethofumesate the last reference were in 1986. The commercial product is used at a 7eld dose of 2 liters/ha (ap: respectively 240 and 600 g/ha). E Decis (ap: 0.02% deltamethrine) This agent was chosen as positive control because of the vast number of available and recent (1998) data (388 references) indicating a clear reported mutagenicity. The commercial product is used in at 7eld dose of 30 g/ha. E Lasso Micromix (ap: 27% alachlor and 11.2% terbuthylazine). Also in this case the agent was chosen as positive control because of the vast amount of available and recent (1997) data (130 references) with a clear reported mutagenicity. The commercial product is used at a 7eld dose of 5 kg/ha (ap: respectively 1350 and 560 g/ha). Data presented here seem to indicate;for all agents tested;that a slight clastogenic risk is present, as indicated by genotoxicity tests (SCGE, MN, SCE, and CA). In fact, even in the case of positive cytogenetic results, the effects appear at toxic or subtoxic doses (as indicated by comparisons with cytotoxicity tests). Moreover, due to the different sensitivities of the tests used, an equivocal picture appears. The MN test seems to us the easiest to execute with clear indications. Data obtained with this test clearly indicate that all four agents tested appear to be slightly genotoxic (only Cirtoxin showed a signi7cant increase in MN and only when tested at higher doses) even when tested at toxic or subtoxic doses. Tramat Combi and Cirtoxin appear to be toxic (RTL) starting at doses 20; the 7eld dose. CA and SCE appear to be more sensitive than MN, as shown by the positive results obtained with all tested drugs. In the case of Tramat Combi, both tests indicate genotoxicity at doses very near the 7eld dose: this effect appears in the presence of a similar toxic effect (CCL, CS, CG, MI in CHO cells), clear in the case of Tramat Combi, slight in the case of the other agents.

320

DE MARCO ET AL.

The comet assay (SCGE) seems to be a very sensitive test, producing positive results for all agents tested even at doses 10; the 7eld dose. In the light of the present results, and in good agreement with the few available literature data, we can conclude that a slight risk factor is connected with the accumulation of the considered agents. Special attention must be given to Lasso Micromix in light of its positive results in some genotoxicity tests, in the presence of slight toxicity. The agent acts as a direct mutagenic and cytotoxic mixture in CHO cells, but appears to be detoxi7ed in CHEL cells, where DNA damage is reduced or absent. One of the two components of the agent (alachlor) is known in the literature as a direct clastogen (Ribas et al., 1995, 1996) even though it can be metabolized to dialkylquinoneimines, which are genotoxic in human lymphocytes (Hill et al., 1997). Under our experimental conditions, the alachlor present in LM is probably metabolized by CHEL cells to quinoneimines, which are rapidly detoxi7ed to quinones (e.g., through DT-diaphorases). Finally, with regard to the battery of tests employed, we stress that the analysis of cell cycle parameters appears to be fundamental in itself and, even more important, also for correct interpretation of genotoxicity data, for example, in the case of DS, where the absence of a clear dose}response relationship in SCE and CA can account for cell cycle delay and cell mortality. In this respect CCL, CG, and CS appear to be the more informative end points. Genotoxicity tests are obviously of great value in determining potential risks for human beings. All the applied tests appear to be useful and informative for our purposes and all of them, accounting for DNA damage, gave coherent results. With respect to the cell culture tests in vitro, it may be claimed that SCGE and SCE are give positive results at lower doses than CA. CONCLUSIONS

Using a ‘‘battery of tests’’ is a widely accepted and fundamental concept in investigating unknown agents and mixtures, as in the case of pesticides. Recent European Community guidelines (Carere et al., 1995) strongly suggest the use of at least three in vitro assays (two of them in mammalian cells) as the 7rst approach to pesticides. In the event of a single positive result, it is mandatory to proceed to in vivo assays with somatic cells and then to in vivo tests for germ cell effects (e.g., dominant lethal). In the present investigation these proceedures have been con7rmed. Moreover, we emphasize the importance of

(i) coupling cytotoxicity and genotoxicity end points, (ii) considering in vivo and in vitro tests, (iii) testing doses higher than the 7eld dose, to take into account accumulation risks, and (iv) considering bioactivation and detoxi7cation of the tested substances using metabolizing systems. Our data, even if devoted primarily to detection of cytotoxicity and genotoxicity, because of their positivity, strongly suggest a deeper consideration of the investigated agents. The comparison with epidemiological monitoring results expected by other groups involved in this multidisciplinary project prompted us to produce extrapolations and suggestions to regulators and the public. The appropriate dissemination of the results obtained to those working in this 7eld is fundamental to diminishing the health risks associated with agricultural practices. This goal could be achieved, or at least approached, by avoiding wherever possible the use of the more dangerous agents, by choosing the less dangerous agent when different substances with the same agricultural target are available, and 7nally by fully alerting operators to the risks involved and the safety procedures associated with the drugs used. ACKNOWLEDGMENTS Dr. Gino Turchi, from ‘‘Istituto di Mutagenesi e Differenziamento’’ del CNR, Pisa, Italy, is kindly acknowledged for supplying CHEL cells. This work was 7nancially supported by the Italian Ministry of Works. T.A., F.T., G.G., and M.O. are postgraduate students and recipients of scienti7c collaboration contracts from the above-mentioned ministry.

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