Pesticide Biochemistry and Physiology 93 (2009) 8–12

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Pesticide Biochemistry and Physiology

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Cytogenetic effects of commercially formulated atrazine on the somatic cellsof Allium cepa and Vicia faba q

Khyati Srivastava, Kum Kum Mishra *

Plant Genetic Unit, Botany Department, Lucknow University, University Road, Lucknow 226007, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 December 2007Accepted 14 August 2008Available online 28 August 2008

Keywords:Allium cepaVicia fabaAtrazineMitotic IndexChromosome aberrations

0048-3575/$ - see front matter � 2009 Published bydoi:10.1016/j.pestbp.2008.08.001

q Cytogenetic effects of atrazine on plants.* Corresponding author.

E-mail address: dr_kumkummisra@rediffmail.com

In the present study cytogenetic effects of atrazine herbicide, were examined on the root meristem cellsof Allium cepa and Vicia faba. Test concentrations were chosen by calculating EC50 values of formulatedatrazine against both the test systems which determined to be 30 mg l�1 for A. cepa and 35 mg l�1 forV. faba, respectively. For cytogenetic effects root meristem cells of A. cepa were exposed to 15, 30 or60 mg l�1 whereas V. faba to 17.5, 35 or 70 mg l�1 for 4 or 24 h. Roots exposed for 4 or 24 h, after sam-pling, were left in water for 24 h recovery and sampled at 24 h post-exposure. A set of onion bulbs orseedlings of V. faba exposed to DMSO (0.3%) was run parallel for negative control. Treatment of atrazinesignificantly and dose-dependently inhibited the mitotic index (MI) and induced micronucleus formation(MN) chromosome aberrations (CA) and mitotic aberrations (MA) in both the test systems at 4 or 24 h.Root meristem cells examined at 24 h post-exposure also revealed significant (p < 0.001) frequencies ofMN, CA or MA despite considerable decline. Chromosome breaks and fragments were found to be majorCA whereas C-metaphase, chromosome bridges and laggards were prevalent MA. Results of our study,indicate that atrazine may produce genotoxic effects in plants. Further, both the plant bioassays foundto be sensitive indicators for the genotoxicity assessment as the outcome of majority of in vivo/in vitromammalian tests are comparable.

� 2009 Published by Elsevier Inc.

1. Introduction

Atrazine [CAS No. 1912-24-9; Chemical name 6-chloro-N-ethyl-N0 (1 methy1ethy1) 1,3,5-triazine-2,4-diamine] is a selective tri-azine herbicide indiscriminately used for the control of broad leafweeds and grasses and also in non-agricultural areas [1]. Atrazineis a major water contaminant. Toxic levels of atrazine has been de-tected in ground and well water in Wisconsin and several coastalwaters of European countries whereas maximum acceptable val-ues (MAV) in different regions such as USA, New Zealand, Australiaand Canada were determined between 0.5 and 3 lg l�1 [2]. Atra-zine was listed in the Annexure one of the EC Council Directive91/414/EEC and its use has been forbidden in the EU members[3,4].

The LD50 of atrazine in mice is 1900 mg/kg, indicating low acutetoxicity to mammals, though it produces severe health effects.Atrazine is believed to be an endocrine disruptor in human beingsand wild life [5–7]. Atrazine has also been reported to cause seri-ous health hazards like cancer, behavioral changes and reproduc-tive abnormalities [8]. In addition, life time exposure of atrazineinduced mammary tumors in rats and also suspected to induce

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(K.K. Mishra).

prostate cancer in workers [9]. Data available on the genotoxic po-tential of atrazine and its commercial formulation in humans androdents show conflicting results. Atrazine showed negative resultsin Ames Test and chromosomal aberrations assay in male mice butcaused structural alterations in female mice [10,11]. Low doses ofatrazine, available in ground and well water of American cities,showed negative effect in mice bone marrow and human lympho-cytes, yet induced significant frequencies of CA in combination ofother compounds like alachlor or linuron [12,13].

On the other hand highest dose of atrazine that significantlyinhibited the MI (15 lg l�1) failed to induce CA, SCE and micronu-cleus formation in cultured human lymphocytes [14]. Recentlyanuron larvae exposed to various concentrations of atrazine andthe nuclear heterogeneity analyzed by the flow cytometer revealedno genetic damage [15]. However, low concentrations (6.25 lg l�1

onwards) of atrazine found to induce micronucleus induction, nu-clear shape abnormalities and DNA damage examined through co-met assay in Oreochromis niloticus which necessitates its thoroughassessment in other organisms [16].

Atrazine is a pre- and post-emergence herbicide mainly ab-sorbed through roots, translocate upwards and accumulate ingrowing tips [17]. It primarily affects photosynthesis in thylakoidmembrane as binds with polyphenoloxidase of complex B of Pho-tosystem II thus affects the Hill Reaction [18,19]. Apart from pho-totoxic effects, exposure to atrazine can also cause genotoxic

K. Srivastava, K.K. Mishra / Pesticide Biochemistry and Physiology 93 (2009) 8–12 9

effects in non-target plants. In several plant species induction of CAhas been reported with the treatment of atrazine [11]. Exposure ofhigher concentrations of atrazine has been shown to induce CA inthe root meristem cells of Hordeum vulgare and Vicia faba [20,21],meiotic cells of H. vulgare [22], microsporocytes of Sorghum vulgare[23,24]. Plants of Z. mays exposed to commercial grade of atrazinerevealed gene mutation at the wx locus [25]. However, in thesestudies plants were exposed to very high concentrations (200–600 or 500–1500 ppm) of atrazine that are not possible in the envi-ronment. Further, none of these studies reported the effect of atra-zine on the cells undergone recovery.

As this compound is indiscriminately used in India and reportedto be major water contaminant the assessment of genotoxicitypotential of this compound is important. Among the higher plantsAllium cepa and V. faba root meristem assays are well accepted formonitoring the genotoxicity potential of environmental chemicals.These two plant models were also included in collaborative studyconducted by International Programme on Chemical Safety andfound to be suitable for the genotoxicity assessment of environ-mental chemicals [26].Still 580 tonnes of atrazine is manufacturedand consumed in India and the results of earlier studies carried outon various in vivo and in vitro system are inconclusive, it is neces-sary to evaluate this compound, particularly on plants as they areprimary recipients of these chemicals. Present study reports thecytogenetic effects of atrazine on the root meristem cells of A. cepaand V. faba safely.

2. Materials and methods

2.1. Chemicals

Commercial formulations of atrazine (Gesaprim 50% EC) wasprocured from Northern Minerals Limited, Gurgaon, Haryana, In-dia. Stains and other chemicals bought from BDH, England orGlaxo, India.

2.2. Stock solution

Test compound was dissolved in DMSO (dimethyl sulfoxide)and the desired test concentrations (based on active ingredient)were made by diluting the stock with tap water.

2.3. EC50 determination

2.3.1. Allium cepaEC50 of atrazine against the Allium root growth was determined

according to the method described earlier [27,28]. In brief, cleanand healthy onion bulbs, locally obtained, were placed over the testtubes filled with test concentrations of atrazine (0.5, 1.0, 5, 10, 15,25, 30, 35 and 40 mg l�1). A set of five bulbs was also exposed to0.3% DMSO and considered as negative control. The test concentra-tions were renewed every 24 h after during the experiment. On the5th day root lengths were measured from each group-control aswell as atrazine exposed bulbs. Taking root lengths of controlgroups as 100%, lengths of experimental groups were plottedagainst test concentrations and the point showed 50% growthwas designated as EC50 concentration.

2.3.2. Vicia fabaSeeds locally bought were cleaned by 0.5% sodium hypochlorite

solution for 5 min, thoroughly washed and soaked in tap water for10 h. Seed coats were removed gently and seeds were spread inpetridishes containing cotton bed moistened with test concentra-tions (0.5, 1.0, 5, 10, 15, 25, 30, 35 and 40 mg l�1). For each concen-tration three sets were used and each petridish had 10 seeds. A set

of 10 seeds was treated with tap water containing 0.25% DMSO fornegative control. On seventh day growth of radicals were mea-sured from each test concentration. Taking control as standardgrowth mean lengths of treated roots were plotted on graph sheetand the point showed 50% growth was designated as EC50.

2.4. Cytogenetic assay

2.4.1. Allium cepa CA assay2.4.1.1. Test concentrations and exposure schedule. Onion bulbs,rooted in tap water, were exposed to 15, 30 or 60 mg l�1 of atra-zine. A minimum of five bulbs were exposed to each test concen-tration. For positive control bulbs were exposed to 100 mg l�1 ofEMS (ethyl methonate sulfonic acid) for 24 h in which recoverystudies were not carried out whereas for negative control bulbswere exposed to 0.3% DMSO and run parallel to experimentals.Two sets of bulbs were employed. One set was exposed for 4 hwhereas another for 24 h. After fixing few roots from each concen-tration bulbs with intact roots were washed and placed on testtubes filled tap water to sample the roots at 24 h post-exposure.Roots were fixed in Carnoy’s fixative (Ethanol + acetic acid; 3:1)and stained with hematoxylin method [29].

2.4.2. Vicia faba CA assay2.4.2.1. Test organism/culture condition. Vicia faba cytogenetic assaywas carried out as described by Kanaya et al. [30]. In brief, seeds ofV. faba were treated with 0.5% sodium hypochlorite. Treated seedswere thoroughly washed with tap water and soaked for 10 h. Seedcoats were gently removed and seeds were left in petridishes forgermination. After 4 days when the primary roots were 3–4 cmtheir tips were cut to facilitate lateral roots. Seedlings were trans-ferred in aerated tap water and after 5 days when the lateral rootsbecame 1–2 cm seedlings were exposed to test concentrations(17.5, 35 and 70 mg l�1).For positive control seeds were exposedto 1000 mg�l of EMS for 24 h in which recovery studies were notcarried out. Exposure periods (4 or 24 h), recovery and fixationwere carried out as it was done for A. cepa.

2.5. Mitotic index determination/scoring of CA and MA

Determination of MI and scoring of CA/MA was carried out asdescribed by Chauhan et al. [30]. Slides were randomly codedand scored blind. For MI, dividing cells were counted out from5000 to 6000 interphase cells and the data were expressed in per-cent. For CA 200–300 well spread metaphase cells and for MA 500–600 dividing cells were scored.

2.6. Statistical analysis

The data were expressed in percent and the prevalence of sig-nificance was determined by ANOVA or Chi-Square test.

3. Results

Growth response curves obtained between the concentrationsof atrazine and root lengths determined the EC50 at 30 and35 mg l�1 in A. cepa and V. faba, respectively. During the 5 daystreatment roots appeared to be yellowish and stiff but did notshow any other sign of toxicity.

Table 1 summarizes the effect of atrazine on MI and micronu-cleus induction in the root meristem cells of A. cepa and V. faba ex-posed for 4 or 24 h. Treatment of atrazine inhibited the mitoticindex dose-dependently and induced MN in both the test systems,however, the significant (p < 0.01) level of inhibition in A. cepa andV. faba was observed at 30 and 17.5 mg l�1, respectively. At 24 h

Table 1Inhibition of mitotic index and induction of micronucleus formation in the root meristem cells of Allium cepa and Vicia faba exposed to atrazine for 4 or 24 h

Test plant andConcentrations (mg l�1)

Exposuretime

Mitoticindexa

% Micronucleatedcellsa,b

% Micronucleated cells at24 h post-exposure

% recovery inmicronucleated cells

A. cepaControl 4 9.06 ± 1.20 ND ND —

24 9.25 ± 0.72 0.06 0.06 —15 4 7.15 ± 0.87 0.03 0.04 —

24 5.02 ± 0.63 0.26* 0.08 69.2330 4 7.30 ± 0.85 0.05 0.03 40.00

24 5.90* ± 0.55 0.39** 0.13* 66.6660 4 7.01 ± 0.88 0.52** 0.22* 57.69

24 3.03* ± 0.36 0.97** 0.45** 53.60EMS 100 24 5.84 ± 0.85 0.41 — —

V. fabaControl 4 10.65 ± 0.85 ND ND —

24 11.02 ± 0.86 0.05 0.07 —17.5 4 10.23 ± 1.08 0.07 0.05 40.00

24 8.71 ± 0.62 0.31** 0.16* 48.3835 4 8.36 ± 0.78 0.08 0.06 25.00

24 7.47 ± 0.49 0.43** 0.27* 37.2170 4 6.29 ± 0.65 0.32** 0.36** —

24 3.33** ± 0.18 1.26** 0.93** 26.19EMS1000b 24 6.46 ± 0.57 0.28 — —

Significance calculated by ANOVA. Data expressed in standard mean ± SE, v2 test.a Data obtained from 5000 to 6000 interphase cells.b Concentrations lower than 1000 mg l�1 did not show comparable results.* p < 0.05.

** p < 0.01.

10 K. Srivastava, K.K. Mishra / Pesticide Biochemistry and Physiology 93 (2009) 8–12

post-exposure decrease in the percentage of MN was observed buthigher concentrations still showed significant frequencies of MNno matter exposed for 4 or 24 h.

Table 2 shows induction of CA in the root meristem cells of A.cepa and V. faba exposed to atrazine for 4 or 24 h. Both the plantsshowed concentration-related increase in the frequencies of CA,however, significant percentage of aberrations was observed at30 mg l�1 in A. cepa and 35 mg l�1 in V. faba. Chromosome breaks

Table 2Chromosome aberrations in Allium cepa and Vicia faba root meristem cells exposed to atra

Test plant andConcentration (mg l�1)

Exposureperiod

Breaks Fragments Ringchromosom

Allium cepaControl 4 — 2 —

24 2 3 —15 4 2 4 —

24 8 4 130 4 2 5 —

24 12 3 160 4 5 7 —

24 9 10 —EMS100 24 5 4 —

Vicia fabaControl 4 — 2 —

24 2 1 —17.5 4 — 3 —

24 2 7 —35 4 2 4 —

24 2 11 —70 4 4 7 —

24 2 15 —EMS1000 24 7 5 —

ND, not detected.EMS.

a Data obtained from 200 to 300 metaphase cells.b Data expressed in percent.* p < 0.01

and fragments were observed to be frequent aberrations in bothplants. Roots meristem cells examined at 24 h post-exposureshowed decrease in percent aberrations ranging from 40.62% to56.81% in A. cepa and 34–71.39% in V. faba. Significant frequenciesof CA were still observed in the root meristems exposed to highestconcentrations—60 and 70 mg l�1 of atrazine.

Table 3 presents the frequencies of MA observed with the treat-ment of atrazine in A. cepa and V. faba root meristem cells. Expo-

zine

esTotalaberrationsa

% aberrationsb %aberrationsat 24 hpost-exposure

% recoveryof aberrations

2 0.85 0.90 ND5 1.60 0.95 40.626 1.85 1.05 —

13 4.33 1.87 56.817 2.86 0.87 —

16 5.33* 2.53 52.5312 3.55* 2.07 —19 6.34* 3.27* 48.22

9 3.6 — —

2 0.96 0.75 —3 1.00 0.66 34.003 1.65 1.05 —9 3.00 0.96 68.006 1.98 1.25 —

13 4.30* 1.23 71.3911 3.86* 2.40 —17 5.66* 3.00* 46.9912 4.00 — —

Table 3Mitotic aberrations in Allium cepa and Vicia faba root meristem cells exposed to atrazine

Plant system and concentrationsof compound (mg l�1)

Exposureperiod (h)

C-metaphase Stickiness Bridges Laggard Unequalseparation

Polyploid Aberrant cellsa

(% aberration)% aberration24 h post-exposure

% recoveryof aberration

Allium cepaControl 4 — 2 — — — — 2 (0.84) 0.86 —

24 — 3 — — — 1 3 (1.64) 1.86 13.4115 4 — 4 — — — — 4 (0.63) 0.75 —

24 5 — 6 1 — 1 13 (3.52) 2.89 17.0030 4 — 5 — 2 — — 7 (2.02) 1.65 —

24 4 3 4 3 1 1 16 (5.00*) 3.96* 20.860 4 3 6 4 2 2 1 18 (5.8**) 3.20* —

24 2 8 4 1 1 4 20 (9.86**) 5.41** 21.02EMS100 24 4 3 7 4 — — 18 (3.27) — —

Vicia fabaControl 4 — 3 — — — — 3 (0.95) 0.75 —

24 — 3 — — — — 3 (1.02) 1.89 6.4317.5 4 — 4 — — — — 4 (1.50) 1.05 —

24 3 2 3 1 2 — 11 (0.74) 2.07 24.4535 4 — 6 — 2 1 — 9 (3.59*) 2.68 —

24 9 — 3 1 2 1 15 (5.68**) 4.47* 5.4970 4 3 7 — 2 1 2 13 (6.13*) 4.03* —

24 9 6 3 4 2 2 25 (9.65**) 5.00* 28.77EMS1000 24 1 4 8 3 — — 16 (3.09) — —

a Data obtained from 500 to 600 metaphase cells and expressed in percent.* p < 0.05.

** p < 0.01.

K. Srivastava, K.K. Mishra / Pesticide Biochemistry and Physiology 93 (2009) 8–12 11

sure of atrazine induced MA in a dose-dependent manner. The per-centage of MA was found to be higher than that of CA. Root meri-stem cells exposed for 24 h and left in water for 24 h revealedconcentration-dependent decrease in the percentage of MA at24 h post-exposure. Both the plants showed significant percentageof MA at 24 h exposure of atrazine.

Further, decline in the frequency of CA was found to be higherthan that of MA; such as 46.99–71.39% decline was observed inthe frequencies of CA whereas 5.49–28.77% of MA (Tables 2 and 3).

4. Discussion

Inhibition of MI in this study indicate the cytotoxic potential ofatrazine, which persisted even at 24 h recovery. Stickiness and C-mitosis are known spindle-related abnormalities but are attributedto be indicative of cytotoxicity [31] such inhibition of MI has alsobeen reported in A. cepa and mice bone marrow cells earlier [3,14].

Significant increase in the frequencies of chromosome breaksand fragments as observed in the present study indicate the clas-togenicity of atrazine. Our results are in agreement with the earlierstudies reported similar aberrations in A. cepa, V. faba and H. vulg-are root meristem cells [3,11,21,23]. However, earlier studies onplants except Bolle et al. [3] reported these effects at very high con-centrations that are not found in the environment [20–22]. Induc-tion of significant frequencies viz. chromosome bridges andlaggards, in consistent to earlier studies, suggest the effect of atra-zine on mitotic spindles of cells [3]. Induction of laggards is consid-ered important aberration from genotoxicity point of view as itleads to aneuploidy. The impairment of mitotic spindles leads tothe formation of mitotic aberrations which is a frequent effect ofherbicidal chemicals on plant cells reported with several herbi-cides [28,32]. Similar effects of atrazine like inhibition of MI, induc-tion of CA and SCE have been reported in human lymphocytes andthe cause of genotoxicity was attributed to be due to the increasein glucose-6-phosphate dehydrogenase which acts as a pro-oxi-dant [33]. However, mechanism of atrazine induced genotoxicityis not still clear.

Conversely, many studies in vivo/in vitro employed on mice andhuman lymphocytes either showed no genotoxicity or modest ef-fect to very high concentrations of atrazine. Kligerman et al. [14]exposed mice to atrazine and did not observe any significant in-crease in CA or aneuploidy. Administration of atrazine (1500 and2000 mg/kg bd. wt.) in mice through oral gavage has been demon-strated to induce dominant lethal mutations and chromatid breaksin mice [34]. Sprague–Dawley rats exposed through gastric intuba-tion using single dose (875 mg/kg) or 15 daily doses of 350 mg/kgshowed modest increase in DNA damage in the liver, kidney andstomach [35]. DNA damage analyzed by single cell gel electropho-resis (comet assay) in atrazine treated cells revealed mild effect onDNA at extremely high concentrations [36] but in O. niloticus signif-icant DNA damage at low concentrations environmentally compat-ible concentrations [16]. Blood samples collected from the tadpolesexposed to various concentrations of atrazine through aquaticmedium and the DNA damage was measured by SCG assay re-vealed the concentrations equal to or greater than 4.8 mg l causesignificant DNA damage [37].

Present findings, along with the results of earlier studies on A.cepa, suggest that atrazine is capable of inducing genotoxicity inplants. Further, data generated from mammalian in vivo andin vitro test models show that higher doses of atrazine that are be-yond the environmental concentrations can induce genotoxicity.Lack of genotoxicity with the lower concentrations in these sys-tems made the results inconclusive. However, to detect the geno-toxicity potential of atrazine testing the low concentrations thatare environmentally relevant is more important. We tend to agreethat plants are sensitive test systems which respond to environ-ment concentrations [38]. Plant cells are capable of activating atra-zine into promutagen that can cause more risk to human beingswhereas in human peripheral lymphocytes exposed in vitro to atra-zine treated with S9 microsomal fraction and examined the cul-tured lymphocytes for CA, SCE and MN revealed no genotoxicity[39]. As atrazine is more prevalent contaminant of ground waterand Environmental Protection Agency’s Office of Pesticide Program(OPP) Peer Review Committee concluded triazine herbicidesin Group C-possible human carcinogens [40] there is a great

12 K. Srivastava, K.K. Mishra / Pesticide Biochemistry and Physiology 93 (2009) 8–12

requirement to examine the toxicity of this compound in variousother systems to establish safe limits. However, contribution ofadjuvants that are used in the commercial formulation cannot beruled out in this study.

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