DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet...

Ž .Mutation Research 419 1998 79–90

DNA damaging effects of pesticides measured by the single cellž /gel electrophoresis assay comet assay and the chromosomal

aberration test, in CHOK1 cells

C. Vigreux a, J.M. Poul b, E. Deslandes a, P. Lebailly a, T. Godard a,b, F. Sichel a,M. Henry-Amar a,c, P. Gauduchon a,)

a INSERM CJF 96-03 and EA 1772, Laboratoire de Cancerologie Experimentale, Centre Francois Baclesse, Route de Lion-sur-mer, 14076,´ ´Caen Cedex 05, France

b CNEVA Laboratoire des Medicaments Veterinaires, la Haute-Marche-JaÕene 35133, Fougeres, France´ ´ ´ ´c Registre General des Tumeurs du CalÕados, Centre Francois Baclesse, Route de Lion-sur-mer, 14076, Caen Cedex 05, France´ ´

Received 24 March 1998; revised 21 August 1998; accepted 21 August 1998

Abstract

Ž . Ž . ŽOne herbicide isoproturon , two fungicides carbendazim and chlorothalonil and etoposide an effective antitumor agent.used as a positive control , were tested for their ability to induce cytotoxic and genotoxic effects in Chinese Hamster Ovary

Ž . Ž .CHOK1 cells. Etoposide induced DNA damage detectable both by the alkaline Single Cell Gel Electrophoresis SCGEŽ .assay and the chromosomal aberration CA test in absence of noticeable cytotoxicity. With the SCGE assay, a clear

induction of DNA damage was observed for chlorothalonil within a 0.2 to 1 mM concentration range. In the CA test,chlorothalonil gave also positive results, inducing mainly chromosome breaks. In contrast, no DNA damage was observedwith the SCGE assay for carbendazim and isoproturon. In the CA test, carbendazim induced only numerical aberrations inthe concentration range of 25 mM to 100 mM, and isoproturon did not induce any significant increase in CA. In conclusion,chlorothalonil appears genotoxic in proliferative CHOK1 cells, and as expected, the aneugenic compound, carbendazim, didnot induce DNA strand breaks in the SCGE assay. q 1998 Elsevier Science B.V. All rights reserved.

Keywords: SCGE assay; CA test; DNA damage; Pesticide; Etoposide; Cytotoxicity

1. Introduction

In recent years, the use of pesticides in agriculturehas much increased. Although a majority of pesti-cides have not been shown to be mutagenic in vitro,

) Corresponding author. Tel.: q33-2-31-45-50-70; Fax: q33-2-31-45-50-53; E-mail: [email protected]

several of them are clastogenic in vitro or in vivow x1–4 . In addition, various studies have revealed asignificantly elevated risk for particular tumors in

w xfarmers as compared with non-farmers 5–7 , al-though overall mortality by cancer was lower in ruralpopulation. These observations and the lack of stud-ies on the genotoxic effects of these compoundsupon occupationally exposed people led us to con-duct a molecular epidemiological study on the rural

1383-5718r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved.Ž .PII: S1383-5718 98 00126-0

( )C. Vigreux et al.rMutation Research 419 1998 79–9080

Žpopulation in our area Departement du Calvados,´.France to assess the carcinogenic risk associated

with occupational exposure to pesticides or other riskfactors. In one part of this study, biomarkers arecurrently evaluated in farmer volunteers whose en-rolment was based on handling heavily used pesti-cides. Since available data were insufficient for someof this compounds, we are also conducting an invitro study on genotoxic and non-genotoxic effects,which may also contribute to an increased risk forcancer.

Among the methods used for in vitro genotoxicityevaluation of these chemicals, particular attention

Ž .was focused on chromosome aberrations CA testŽand the alkaline comet assay single cell gel elec-

.trophoresis assay, SCGE . The alkaline comet assay,which detects DNA strand breaks, alkali-labile sitesand incomplete excision repair events in individualcells, is a rapid and sensitive method for the detec-

w xtion of DNA damage induced in vitro 8,9 or afterw xenvironmental and occupational exposures 10,11 .

However, further validation of this assay is requiredby application to a wider range of cell types andchemicals and by comparison with classical cytoge-netic methods.

w xIn a previous report 12 , we observed thatchlorothalonil, but not carbendazim induced DNAdamage in human lymphocytes detectable with alka-line SCGE assay. Since these damages could not bedissociated from delayed lethality, we cautiouslyconcluded that chlorothalonil should not be consid-ered as a true genotoxin in quiescent human lympho-cytes. In the present paper, this study on chlo-rothalonil and carbendazim was extended to a prolif-

Ž .erative cell line, Chinese Hamster Ovary CHOK1 ,and to one herbicide, isoproturon. Alkaline SCGEassay and CA test were used in parallel to detect

Ž .DNA damage and etoposide an anti-tumoral agentwas taken as a positive control in both tests.

2. Materials and methods

2.1. Cells

Chinese hamster ovary cells were obtained fromICN Biomedical. To maintain karyotypic stability,the cells were used at no more than 15 passages aftercloning. They were routinely maintained from stocksstored in liquid nitrogen and transferred twice aweek.

Chinese hamster ovary cells were grown at 378Cin a humidified atmosphere at 5% CO in air, in2

HAM’S F12 medium supplemented with 10% fetalŽ . Ž .calf serum FCS , penicillin 100 unitsrml andŽ .streptomycin 100 mgrml . Cultures were set up the

day before treatment at a uniform cell density.

2.2. Chemicals

ŽChlorothalonil tetrachloroisophtalonitrile, CASŽ .No. 1897-45-6, molecular weight MW 265.9, )

. Ž98.8% purity , carbendazim methyl benzimidazol-2-ylcarbamate, CAS No. 10605-21-7, MW 191.2,

. Ž)99% purity and isoproturon CAS No. 34123-59-.6, MW 206.3, )99.6% purity were purchased from

Ž .Cluzeau Info Labo Ste Foy La Grande, France .Ž .Etoposide MW 588.6 was obtained as Vepesideq

Ž .Sandoz, Rueil Malmaison, France . Structures ofthese compounds are given in Fig. 1.

All compounds were dissolved in dimethyl sulfox-Ž .ide DMSO , etoposide excepted, which was directly

soluble in culture medium. The final concentration ofDMSO in culture medium was 1.25%.

Fig. 1. Molecular structures.

( )C. Vigreux et al.rMutation Research 419 1998 79–90 81

Ž . Ž . Ž . ŽFig. 2. Individual CHOK1 cells with various degrees of DNA damage: a, b intact cell IC and slightly damage cells SCD tail moment. Ž . Ž . Ž . Ž . Ž . Ž .0–5 ; c damage cell DC tail moment 5–60 and d highly damage cell HDC tail moment was not calculated .

2.3. Cytotoxicity assays

In parallel to the assessment of genotoxicity, cellviability was measured immediately after treatmentusing the Trypan blue exclusion method. Cell viabil-ity was expressed as proportion of living cells. Each

value represents the mean of at least two experi-ments.

Cytotoxicity of each concentration of a givencompound was also evaluated 2 days after treatment

Žby measuring cellular reduction of XTT 2,3 bisŽ . wŽ .2-methoxy-4-nitro-5-sulfophenyl -5 phenylamino

Table 1Cytotoxicity and genotoxicity of etoposide

a b cŽ .mM Cytotoxicity SCGE assay CA test

Ž . Ž . Ž .1 h 4 h % DC % HDC % Aberrant cellsŽ .excluding gap

Ž . Ž .0 100 "5.8 100 "3.9 8 0 9Ž . Ž .0.2 94.8 "2.3 93.7 "3.9 40 4 22)

Ž . Ž .1 91.7 "9.4 90.2 "6.3 36 0 40)

Ž . Ž .2 80 "11 78.3 "3.4 68 12 52)

aCytotoxicity was determined by the XTT in vitro assay 2 days after treatment and expressed as percentage of control absorbance.b Effect of increasing concentration of etoposide on DNA migration tested by using the one-sided non-parametric Jonckeere–Terpstra test. A

Ž .statistically significant p-0.05 dose–response relationship was observed.c Effect of increasing concentration of etoposide on percentage of aberrant cells tested by using the one-tailed Fisher’s exact test.

Ž .)Indicates statistically significant difference from the concurrent control p-0.05 .


x .carbonyl -2 H-tetrazolium hydroxyde in the pres-Ž .ence of PMS phenazine methosulfate according to

w xScuderio et al. 13 . In brief, CHOK1 cells wereplated at 104rwell in 96 well tissue-culture mi-crotiter plates and grown for 24 h in HAM’S F12medium supplemented with 10% FCS. Cells werethen treated for 1 h or 4 h in the absence of FCS,washed twice with HAM’S F12 without serum, andpost-incubated in fresh medium supplemented with10% FCS for 2 days at 378C. An amount of 50 ml ofa solution containing XTT at 1 mgrml and phenazinemethosulfate at 7.65 mgrml in HAM’S F12 mediumwas then added to each well. After incubation for 4 hat 378C, the ratio of optical density at 450 nm vs.650 nm was measured using a microplate readerŽ .Molecular Devices . Each value represents the meanof four replicate determinations. One representativeexperiment of at least three is shown.

2.4. Genotoxicity assays

Genotoxicity assay doses were adjusted so thatcytotoxicity, evaluated 2 days after treatment, did notexceed 50% at the highest dose retained.

2.4.1. Alkaline SCGE assayThe SCGE assay was performed immediately af-

w xter the 1-h treatment as described previously 12 .For each experiment, a set of 15 slides was pro-cessed simultaneously, including a negative or sol-vent control and positive control. After incubationwith the tested compound for 1 h in the absence ofFCS, cells were collected and suspended in 70 ml

Žprewarmed LMP agarose 0.5% in phosphate-Ž .buffered saline PBS without calcium and magne-

sium and 65 ml of the suspension was deposited on aŽfully frosted microscope slide Touzard et Matignon,

. ŽFrance precoated with 80 ml normal agarose 0.8%. Žin PBS and covered with a coverglass 24=32

.mm . After gelling 5 min at 08C, coverglass wasgently removed and a third layer of 80 ml LMP

Ž .agarose 0.5% in PBS was added and covered againwith a coverglass. After gelling at 08C, the cover-glass was removed and the slide was put into a tank

Žfilled with the lysis solution 2.5 M NaCl, 0.1 MEDTA, 10 mM Tris adjusted to pH 10, 10% dimethyl

.sulfoxyde and 1% Triton X-100 both freshly addedfor 1 h at room temperature. To allow DNA unwind-ing, the slide was incubated in fresh electrophoresis

Ž .buffer 0.3 M NaOH and 1 mM EDTA, pH 13.6 for40 min at room temperature. The slide was then

Žplaced into a horizontal electrophoresis tank Hoeffer. Ž .HE 99 Xq and exposed to 0.7 Vrcm 300 mA for

24 min at room temperature. After electrophoresis,the slide was washed twice in fresh neutralisation

Žbuffer 0.4 M Tris adjusted to pH 7.5 with concen-.trated HCl before staining with 50 ml ethidium

Ž .bromide solution 20 mgrml and observation at a250= magnification using a Dialux fluorescencemicroscope.

Twenty-five randomly selected cells per slide werevisually analysed and submitted to image analysis.

Ž .Cells were eye-graded Fig. 2 into three categories

Ž . Ž .Fig. 3. Effect of isoproturon A , carbendazim B andŽ .chlorothalonil C on CHOK1 cells growth measured by XTT

Ž . Ž .method 48 h after 1-h – I – or 4-h – v – exposure. Eachpoint represents the mean of four replicate wellsrgroup. Onerepresentative experiment of at least three is shown.


Ž .depending on DNA migration level: intact cells ICŽ .and slightly damaged cells SDC , damaged cells

Ž . Ž .DC , and highly damaged cells HDC . These HDC,characterized by a small head and exceeding DNA

w xfragmentation, may represent apoptotic cells 14 .For this reason, they were considered as an indepen-dent class. In order to know if these 25 cells taken atrandom for image analysis were representative of allthe slides, an eye-grading evaluation was performedon 200 additional cells per slide and the two groupsare compared by using the chi2 test. When no differ-ence was observed between the two groups, the 25cells were retained for image and statistical analysis.In the few cases where a variation was detected, theexperiment was rejected. Image analysis was per-formed using an original software, developed in ourlaboratory, that provides a wide range of quantitativeparameters to describe the densitometric and geomet-

Ž .ric properties of the comet. Tail moment TM ,defined by the product of the distance between thetwo barycentres of the head and the tail by theproportion of DNA in the tail, was used to evaluatethe extent of DNA migration. The HDC are scoredamong the 25 cells but are not submitted to imageanalysis. For this reason, results are presented interms of cell distribution into classes of increasingtail moment but not in terms of mean response forTM.

2.4.2. CA testOne day after culture initiation, the cells were

exposed to the test chemical, negative and positivecontrol for 4 h in the absence of FCS, and post-in-cubated in complete growth medium for 18 h. Thiscollection time corresponded to the appearance offirst generation metaphase cells. At the end of thepost-incubation time, cells were treated by 0.6mgrml colcemid for 2 h and collected by mitotic

Žshake-off. After incubation with hypotonic KCl 75.mM at room temperature, cells were fixed in

Ž .ethanolrglacial acetic acid 3:1, VrV , dropped ontoslides and air-dried.

For scoring of aberrations, slides were stained inGiemsa. One hundred metaphase cells were scoredper dose. They were selected for structural aberrationscoring on the basis of good morphology and achromosome number of 19"2. The structural aber-rations analysed included chromatid-type and chro-mosome-type breaks and exchanges. Gaps were de-

Žfined as discontinuities along the chromosome in-.cluding one or both chromatid without displace-

Žment. Numerical aberrations observed among the.100 metaphase cells were scored in three categories:

polyploidy, endoreduplication and hyperdiploidy.Each experiment represents the mean of duplicate

determinations and two independent experimentswere performed.

Fig. 4. Effects of increasing concentration of chlorothalonil on DNA migration measured by SCGE assay. Each histogram is a singleŽ . Ž .representative experiment. A statistically significant p-0.05 dose–response relationship was observed in each experiment Fig. 5A, B .

HDC: highly damaged cells.


2.5. Statistical analysis

Effects of increasing concentrations of etoposide,chlorothalonil, carbendazim or isoproturon on tail

Ž .moment distribution SCGE assay were tested usingthe one-sided non-parametric Jonckeere–Terpstra testw x15 . This test exploits the double ordering so as tohave good power against alternative hypotheses inwhich an increase in the dose level leads to anincrease in the DNA migration level, expressed bytail moment values. Tail moment values were cate-gorized into twelve classes including HDC. Theproportion of structural aberrations, numerical aber-rations and total aberrant cells observed with increas-ing concentrations of etoposide, chlorothalonil, car-bendazim or isoproturon was compared to that ob-served in the control group using the one-tailedFisher’s exact test. The StatXact 3 software was used

Žfor analysis StatXact 3 for Windows, Cytel soft-.ware, Cambridge, USA, Fifty ed. 1997 .

2.6. PositiÕe control

For both tests, 1 mM etoposide was chosen as apositive control. At this concentration, etoposide wasable to induce both DNA damage and CA in CHOK1cells in absence of noticeable delayed cytotoxicity

Ž .measured by the XTT method Table 1 .

3. Results

3.1. Choice of doses

To define genotoxicity assay doses, delayed cyto-Žtoxic effects, induced by a 1-h protocol of treatment

. Žfor SCGE assay or a 4-h protocol of treatment for.CA test exposure with chlorothalonil, carbendazim

or isoproturon in CHOK1 cells, were measured 2Ž .days after treatment using the XTT method Fig. 3 .

Doses were adjusted so that cytotoxicity did notexceed 50% at the highest dose retained. Thus, forchlorothalonil, the higher doses tested were, respec-

Ž .tively, 1 mM alkaline SCGE assay and 0.5 mMŽ .CA test . With carbendazim and isoproturon, cellgrowth was not significantly affected in the range ofconcentrations tested which correspond to the limitof solubility of drugs in the experimental condition.

3.2. Alkaline SCGE assay

The DNA damaging effects of a 1-h treatmentwith chlorothalonil, carbendazim or isoproturon inCHOK1 cells were investigated using the SCGEassay.

A clear induction of DNA damage was observedŽ .for chlorothalonil Fig. 4 . Within a 0.2 to 1 mM

concentration range, DNA damage was revealed byŽ .an elevation of the proportion of damaged cells DC

as illustrated in Fig. 4. When tail moment distribu-

Ž . Ž .Fig. 5. Effect of isoproturon A , carbendazim B andŽ .chlorothalonil C on cell viability determined by the Trypan blue

Ž . Ž .exclusion test immediately after 1-h – ^ – or 4-h – ' –exposure. Results were expressed as the percentage of living cellsand each value represents the mean of at least two experiments.


Fig. 6. Effects of increasing concentration of carbendazim on DNA migration measured by SCGE assay. Each histogram is a singleŽ .representative experiment. In each experiment A, B , no statistically significant dose–response relationship was observed. HDC: highly

damaged cells.

Žtion was analysed, a statistically significant p-.0.05 dose–response relationship was observed. The

increase of DC was also accompanied by an eleva-Ž .tion of the mean image length data not shown . This

happened in the absence of Trypan blue permeabilityŽ .measured immediately after treatment Fig. 5 . In-

crease of chlorothalonil concentrations from 2 mMto 10 mM led to a dose-related increase in the

Ž .proportion of HDC data not shown . At these doses,immediate loss of cell viability was not observed, butcell growth measured 2 days after treatment was

Ž .considerably affected Fig. 3 .Ž . Ž .For carbendazim Fig. 6 and isoproturon Fig. 7 ,

the proportion of DC remained very low in the rangeof concentrations tested and no statistically signifi-cant increase of tail moment was detected.

Fig. 7. Effects of increasing concentration of isoproturon on DNA migration measured by SCGE assay. Each histogram is a singleŽ .representative experiment. In each experiment A, B , no statistically significant dose–response relationship was observed. HDC: highly

damaged cells.


Table 2Chromosome aberrations of Chinese hamster ovary cells induced by chlorothalonil

Ž . Ž . Ž .mM Aberrations types % Aberrant cells excluding gapa b cGap Chromosome Chromatid MA P Structural Numeric Total

Breaks Exchanges Breaks Exchanges

0 0 4 0 0 0 0 0 4 0 40.25 4 10 1 1 1 0 0 13) 2 15)

0.5 5 11 0 2 6 0 1 15) 0 15)

Total of 100 metaphase cells were counted for each treatment.a Multiple aberrations.b Pulverization.c Polyploidy, endoreduplication, hyperdiploidy.


Table 3Chromosome aberrations of Chinese hamster ovary cells induced by isoproturon



0 0 0 0 0 0 0 0 0 4 41 0 0 0 0 0 0 0 0 2 2

10 1 1 0 0 0 0 0 1 4 5Ž . Ž . Ž .100 1 1 0 1 0 1 0 3 NS 7 NS 10 NS

Total of 100 metaphase cells were counted for each treatment.a Multiple aberrations.b Pulverization.c Polyploidy, endoreduplication, hyperdiploidy.NS: non-statistically different from the concurrent control.

Table 4Chromosome aberrations of Chinese hamster ovary cells induced by carbendazim



0 0 1 0 0 0 0 0 1 2 325 0 0 0 0 0 0 0 0 12) 12)

50 0 0 0 1 0 0 0 1 23) 24)

100 0 1 0 0 0 0 0 1 19) 20)

Total of 100 metaphase cells were counted for each treatment.a Multiple aberrations.b Pulverization.c Polyploidy, endoreduplication, hyperdiploidy.



3.3. CA

The ability of chlorothalonil, carbendazim andisoproturon to induce CA in CHOK1 cells was inves-tigated. The results are shown in Tables 2–4.

Although in every case, few or no chromosomalgaps appeared, a significant increase of total CA was

Ž .observed for chlorothalonil Table 2 . Chlorothalonilproduced structural CA and more particularly, chro-mosome breaks. Chromatid-type exchanges were alsoobserved at the highest concentration tested.

For isoproturon, the structural CA observed at anon-significant level were essentially chromatid andchromosome-breaks without chromosomal ex-

Ž .changes Table 3 .Concerning numerical CA, mainly carbendazim

Ž .Table 4 and high concentrations of isoproturoncaused the appearance of polyploid cells. In contrast,chlorothalonil did not induce any numerical CA.

4. Discussion

ŽIn our geographical area Departement du Calva-´.dos , isoproturon, chlorothalonil, and carbendazim

are among the most commonly used agriculturalchemicals and have been applied for a long time inlarge-scale farming. To get more information aboutthe potential genotoxicity of these chemicals, theirgenotoxicity was investigated by means of the SCGEand CA assays.

Evaluation of the genotoxicity of chemicals needs,in parallel, an adequate assessment of their cytotoxic-ity. In many in vitro studies using SCGE assay,cytotoxicity is evaluated in terms of decrease in cellviability immediately after treatment, using most fre-quently the Trypan blue exclusion test. Less fre-quently are delayed cytotoxic effects evaluated, forinstance by colony formation assay. We have re-

w xcently reemphasized 12 that measure of cell viabil-ity immediately after treatment may be insufficientto detect a cytotoxic effect. In the present study, thecytotoxicity of each compound was systematicallyevaluated 2 days after treatment. This culture dura-tion, corresponding to four cell doubling times, pro-vides sufficient time to detect reduced kinetics ofproliferation or delayed cell death processes—twomanifestations of cytotoxicity.

Chlorothalonil is a heavily used agricultural ornon-agricultural fungicide with multiple cellular tar-gets. Its effects on fungal respiration are particularly

w xlinked to its ability to bind sulfhydryl 16,17 .Ž .Chlorothalonil has been ranked in category 3 by

ŽIARC i.e., insufficient available data to evaluate.carcinogenicity to humans . It causes kidney carcino-

w xmas in rats and to a lesser extent in mice 18 . Thisoncogenicity might be linked to the formation inkidney of nephrotoxic thiol metabolites, through amechanism that seems specific to rodents. Cytotoxic-ity may be followed by compensatory cell prolifera-tion and hyperplasia, that may eventually progress toneoplasia. In dogs exposed to chlorothalonil, no such

w xproliferative changes in kidney were observed 19 .Chlorothalonil gave negative results in vivo withbone marrow CA and micronucleus tests in rat,mouse and Chinese hamster, after a single dose. Aprevious report that repeated exposure induced CA

w xin Chinese hamster 1 was not confirmed in moreŽrecent studies J.C. Killeen, personal communica-

.tion . Thus, chlorothalonil appears as a non-geno-toxic carcinogen in rodents. Concerning in vitrostudies, chlorothalonil induced neither reverse muta-

ww xtions in Salmonella typhimurium 3 ; unpublishedxresults from our laboratory nor CA and micronuclei

in the mammalian V79 and BALBrc 3T3 cell linesw x20 . However, it was positive in the CHO aberrationassay and weakly positive in the L5178Y mouse

w xlymphoma assay 1 .In the present study, chlorothalonil appeared

highly cytotoxic in CHOK1 cells and gave positiveresults with SCGE and CA tests. In SCGE assay,DNA damage was observed in absence of immediatepermeability to Trypan blue. DC appeared at concen-trations that did not induced noticeable delayed cyto-toxic effects, whereas the presence of HDC wasassociated with cytotoxicity. These results on rodentproliferative cells contrast with our previous studyon non-activated human peripheral blood lympho-

w xcytes 12 in which we could not detect genotoxiceffects in absence of cytotoxicity. Our data on CAtest are in agreement with previous in vitro studies.Chlorothalonil induced essentially chromosome-typeaberrations that were dissociable from cytotoxic ef-fects only in a narrow concentration range. Accord-

w xing to Yamano and Morita 21 , chlorothalonil couldexert its cytotoxic effects on mammalian cells through


its depleting effect on cellular sulfhydryls. Highlyreactive towards intracellular thiols, it may affect theactivity of essential macromolecules and the level oflow molecular weight thiol compound, such as glu-tathione. After thiol compound depletion,chlorothalonil stimulates lipid peroxidation, althoughin hepatocytes this effect is not related to cytotoxic-

w xity 21 . One can speculate that the cytotoxicity ofchlorothalonil in CHOK1 cells is based on a similarmechanism: continued generation of thiol conjugatesof chlorothalonil would result in sufficient ATP de-pletion to finally lead to death. Unbalance in oxygenmetabolism induced by thiol capture may also berelated to the production of DNA strand breaksdetectable at non- or weakly cytotoxic concentra-tions, as well as to epigenetic effects of chlorothalonilobserved in Syrian hamster embryo cells preexposed

Ž . w xto low concentrations of benzo a pyrene 20 . Alter-natively, DNA damage detected by the SCGE inalkaline conditions and CA may be related to theformation of DNA adducts by chlorothalonil or someof its cell metabolites. Available data do not, how-ever, favor this hypothesis. First, in a rat study withradiolabelled chlorothalonil, radiolabel was not found

w xassociated with DNA 22 . Secondly, Daconil, acommercial preparation of chlorothalonil, was unableto form DNA adducts with calf thymus DNA in vitrow x23 .

Isoproturon is a well-known herbicide that be-longs to the family of substituted phenyl ureas. It iswidely used against grassy and non-grassy weedsbecause of its broad spectrum herbicidal activity andhigh effectiveness. However, little information aboutits genotoxic effects in mammalian test systems isavailable. Only one study has shown genotoxic ef-fects of isoproturon using in vivo CA, micronucleus

w xand sperm-shape abnormality assays 24 . A signifi-cant dose-dependent mutagenic effect in CA andsperm-shape abnormality tests was observed after24-h treatment. With the micronucleus assay, theeffect was only significant at the highest dose tested.In the present study, isoproturon was not found to betoxic and did not induce DNA damage in the SCGEassay. Only weak positive results were observed atthe highest concentration in the CA test. These re-sults suggest that isoproturon itself is not genotoxicand that positive results detected in vivo were proba-bly related to the presence of active metabolites as

w xproposed by Behera and Bhunya 24 . In order toconfirm this hypothesis, it would be interesting tostudy the in vitro induction of DNA damage in the

Ž .presence of exogenous activation fraction S -mix .9

Moreover, in vivo studies on time- and dose-depen-dent induction of DNA damage by isoproturon arecurrently in progress in our laboratory, and will helpus to evaluate the role of metabolic activation.

Carbendazim did not appear to be cytotoxic inCHOK1 cells at a concentrations as high as 250 mMŽhigher concentrations could not be tested due to

.insolubility in DMSO and did not induce any DNAdamage detected by SCGE assay or CA test. Incontrast, significant positive results were obtained interms of formation of numerical CA in the absenceof cytotoxicity. These results are in agreement withprevious in vivo or in vitro studies which haveshown a low toxicity of the benzimidazole pesticides

w xin acute, sub-acute and chronic studies 25 despite aclear aneugenic effect through their ability to bindb-tubulin and to disrupt the normal formation of

w xmicrotubules 26 . It has been suggested that theinhibition of chromosomal migration during mitosismay be responsible for the known testicular atrophyand male infertility observed in rats exposed to

w xcarbendazim 27 .In summary, chlorothalonil appears genotoxic in

proliferative CHOK1 cells, isoproturon itself is notgenotoxic and as expected carbendazim did not in-duce DNA strand breaks in the SCGE assay.

w xMonteith and Vanstone 28 have demonstratedthat genotoxicants routinely used as positive con-

Ž .trols, such as cyclophosphamide and benzo a pyrene,induced DNA migration in SCGE assay under cir-cumstances where an increase in DNA damage wasdetected by other genotoxicity endpoints includingCA test. In the present study, we also observed thatSCGE assay results were in good agreement withthose obtained with the more classical CA test, anobservation that strengthens the interest of this rapidand simple test for genotoxicity evaluation.

Acknowledgements

We are grateful to Professor J.F. Heron, Director´of Centre Francois Baclesse, and Pr. J.Y.Le Talaer,¨

ŽDirector of EA 1772 Groupe Regional d’Etude sur´


.le Cancer, Universite de Caen for their constant´support. This work was supported by the French

ŽLeague against Cancer Comites du Calvados, de´.l’Orne et de la Manche . C. Vigreux and P. Lebailly

are recipients of a fellowship from the French LeagueŽ .against Cancer Comites de l’Orne et de la Manche .´

T. Godard is a recipient of a fellowship fromCNEVA. The technical assistance of A. Mourot andS. Huet was highly appreciated. Dr. C. Staedel andPr. E. Bar are gratefully acknowledged for readingthe manuscript. We thank Dr. Killeen and Dr. Blanc-

Ž .quaert ISK Biosciences for helpful discussions.

References

w x1 K.L. Dearfield, H.F. Stack, J.A. Quest, R.J. Whithing, M.D.Waters, A survey of EPArOPP and open literature data onselected pesticide chemicals tested for mutagenicity: I. Intro-

Ž .duction and first ten chemicals, Mutat. Res. 297 1993197–233.

w x2 N.E. Garrett, H.F. Stack, M.D. Waters, Evaluation of thegenetic activity profiles of 65 pesticides, Mutat. Res. 168Ž .1986 301–325.

w x3 World Health Organization, Miscellaneous pesticides, IARCMonograph on Evaluation of Carcinogenic Risk of Chemi-cals to Humans, IARC Scientific Publication No. 30, WorldHealth Organization, Lyon, 1983, 430 pp.

w x4 World Health Organization, Occupational exposures in insec-ticide application and some pesticides, IARC Monograph onEvaluation of Carcinogenic Effects on Human, IARC Scien-tific Publication No. 53, World Health Organization, Lyon,1991, 618 pp.

w x5 A. Blair, H. Malker, K.P. Cantor, L.F. Burmeister, K. Wik-lund, Cancer among farmers, Scand. J. Work Environ. Health

Ž .11 1985 397–407.w x6 H.I. Morrison, K. Wilkins, R. Semenciw, Y. Mao, D. Wigle,

Herbicides and cancer: a review, J. Natl. Cancer Inst. 84Ž .1992 1866–1874.

w x7 S.H. Zahm, A. Blair, Pesticides and non-Hodgkin’s lym-Ž .phoma, Cancer Res. 52 1992 5485s–5488s, Suppl.

w x8 N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, Asimple technique for quantitation of low levels of DNA

Ž .damage in individual cells, Exp. Cell. Res. 175 1988184–191.

w x9 V.J. Mc Kelvey-Martin, M.H.L. Green, P. Schmezer, B.L.Pool-Zobel, M.P. De Meo, A. Collins, The single cell gel´

Ž .electrophoresis assay comet assay : a European review,Ž .Mutat. Res. 288 1993 47–63.

w x10 R.J. Albertini, J.A. Nicklas, J.P. O’Neill, Future researchdirections for evaluating human genetic and cancer risk fromenvironmental exposures, Environ. Health Perspect. 104Ž .1996 503–510.

w x11 T. Leroy, P. van Hummelen, D. Anard, P. Castelain, M.Kirsh-Volders, R. Lauwerys, D. Lison, Evaluation of three

methods for the detection of DNA single-strand breaks inhuman lymphocytes: alkaline elution, nick translation, andsingle-cell gel electrophoresis, J. Toxicol. Environ. Health 47Ž .1996 409–422.

w x12 P. Lebailly, C. Vigreux, T. Godard, F. Sichel, J.Y. LeTalaer,¨M. Henry-Amar, P. Gauduchon, Assessment of DNA dam-age induced in vitro by etoposide and two fungicidesŽ .carbendazim and chlorothalonil in human lymphocytes with

Ž .the comet assay, Mutat. Res. 375 1997 205–217.w x13 D.A. Scuderio, R.H. Shoemaker, K.D. Paull, A. Monks, S.

Tierney, T.H. Nofziger, M.J. Currens, D. Sennif, M.R. Boyd,Evaluation of a soluble tetrazoliumrformazan assay for cellgrowth and drug sensitivity in culture using human and other

Ž .tumor cell lines, Cancer Res. 48 1988 4827–4833.w x14 P.L. Olive, Garnet-Frazer, J.P. Banath, Radiation-induced

apoptosis measured in TK6 human B lymphoblast cells usingŽ .the comet assay, Radiat. Res. 130 1993 130–136.

w x15 M. Hollander, D.A. Wolfe, Non-Parametric Statistical Meth-ods, Wiley, New York, 1973.

w x16 P.G. Vincent, H.D. Sisler, Mechanism of antifungal action of2,4,5,6-tetrachloro-isophtalonitrile, Physiologia Plantarum 21Ž .1968 1249–1264.

w x17 R.W. Tillman, M.R. Siegel, J.W. Long, Mechanism of actionŽanf fate of the fungicide chlorothalonil 2,4,5,6-tetrachloro-

.isophtalonitrile in biological systems: I. Reactions with cellsand subcellular components of Saccharomyces pastorianus,

Ž .Pesticide Biochemistry and Physiology 3 1973 160–167.w x18 World Health Organization, Pesticide residues in food 1992,

Toxicology Evaluations, Joint Meeting of the Fao Panel ofExperts on Pesticide Residues in Food and the Environmentand the WHO Expert Group on Pesticide Residues, Rome,21–30 September 1992.

w x19 C.F. Wilkinson, J.C. Killeen, A mechanistic interpretation ofthe oncogenicity of chlorothalonil in rodents and an assess-ment of human relevance, Regulatory Toxicology and Phar-

Ž .macology 24 1996 69–84.w x20 H. Bessi, C. Rast, G. Nguyenba, P. Vasseur, Chlorothalonil

promotes morphological transformation in hamster embryocells but does not inhibit GAP junctional intercellular com-munication either in SHE cells or in the V79 cell line, Cancer

Ž .J. 7 1994 248–253.w x21 T. Yamano, S. Morita, Effects of pesticides on isolated rat

hepatocytes, mitochondria, and microsomes II, Arch. Envi-Ž .ron. Contam. Toxicol. 28 1995 1–7.

w x22 World Health Organization, IPCS, Environmental HealthCriteria 183, Chlorothalonil, Geneva, 1996.

w x23 R.G. Shah, J. Lagueux, S. Kapur, P. Levallois, P. Ayotte, M.Tremblay, J. Zee, G.G. Poirier, Determination of genotoxic-ity of the metabolites of the pesticides guthion, sencor, lorox,reglone, daconil and admire by 32 P post-labelling, Mol. Cell.

Ž .Biochem. 169 1997 177–187.w x24 B.C. Behera, S.P. Bhunya, Genotoxic effect of isoproturon

Ž .herbicide as revealed by three mammalian in vivo muta-Ž .genic bioassays, Indian J. Exp. Biol. 28 1990 862–867.

w x25 I.M. Somlyay, L.E. Varnagy, Changes in blood plasma bio-chemistry of chicken embryos exposed to various pesticide

Ž .formulations, Acta Veterinaria Hungarica 37 1989 179–183.


w x26 P. Vanhummelen, A. Elhajouji, M. Kirschvolders, Clasto-genic and aneugenic effects of three benzimidazole deriva-tives in the in vitro micronucleus test using human lympho-

Ž .cytes, Mutagenesis 10 1995 23–29.w x27 M. Nakai, R.A. Hess, B.J. Moore, R.F. Guttrof, L.F. Strader,

R.E. Linder, Acute and long-term effects of a single dose of

Žthe fungicide carbendazim methyl 2-benzimidazole carba-.mate on the male reproductive system in the rat, J. Androl.

Ž .13 1992 507–518.w x28 D.K. Monteith, J. Vanstone, Comparison of the microgel

electrophoresis assay and other assays for genotoxicity in theŽ .detection of DNA damage, Mutat. Res. 345 1995 97–103.

DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet...

Documents

Transcript of DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet...