ORIGINAL ARTICLE

Comparative evaluation of promutagens o-PDA, m-PDAand MH for genotoxic response in root cells of Allium cepa L.

Manosij Ghosh & Jit Paul & Sonali Sinha &

Anita Mukherjee

Received: 15 January 2010 /Published online: 23 December 2010# Archana Sharma Foundation of Calcutta 2010

Abstract Awide number of chemicals from different sourcesare released into the environment, some ofwhich uponmetabolicactivation are converted to mutagenic products. These chemicalsmight hence possess threat to public health. Genotoxic potentialof three promutagens – o-phenylenediamine (o-PDA), m-phenylenediamine (m-PDA) and maleic hydrazide (MH) wasevaluated using Allium cepa as the test system. Followingtreatment the genotoxic endpoints measured in the root tissuewere mitotic index, cells with mitotic or chromosome aberrationand micronuclei. DNA damage in root cells was evaluated bycomet assay. EC50 determined on the basis of root growth for o-PDA, m-PDA, MH were between 7–8 mg/l, 65–70 mg/l and 9–10 mg/l respectively. All the three agents induced genotoxicitysignificantly, that followed a dose response. While o-PDA andm-PDA did not induce DNA damage significantly, induction ofDNA damage by MH was significant and dose dependent. Thestudy identifies the genotoxic potential and re-establishes thefact that Allium cepa could ideally be used for evaluation ofchromosome aberration and DNA damage.

Keywords Chromosome aberrations . Comet assay .

DNA damage .Micronuclei . Maleic hydrazide

AbbreviationsMH Maleic hydrazideo-PDA Ortho phenylenediaminem-PDA Meta phenylenediamineOTM Olive tail momentMN MicronucleiMI Mitotic index

Introduction

Chemicals with genotoxic potential from anthropogenicsources are released into the environmental realm influencingbiota through effluent and gaseous emissions that might enterfood chain threatening human and environmental health.Study of such chemical agents and their interaction hasbecome increasingly important owing to their coexistence innature. Promutagens are such agents, which upon metabolicactivation are converted to yield products that are mutagenic[1]. The activation of such chemicals into mutagens by greenplants is an added concern that could affect public health [2].The formation of high molecular weight conjugates formedwithin plants might be released as and when consumed byanimals and humans, posing genotoxic risk [1, 2].

Maleic hydrazide (MH), o-phenylenediamine (o-PDA) andm-phenylenediamine (m-PDA) are some such economicallyimportant and widely used promutagens. MH (l,2-dihydropyridazine-3,6-dione) a promutagen [3–5]; sinceits discovery has been of growing importance as anherbicide with uses including inhibition of sprouting invegetables and stored root crops, prevention of suckerproduction in tobacco plants and growth control of grass,and foliage [6]. o-PDA and m-PDA are promutagenicmonocyclic arylamines/aromatic amine used in the prepara-tion of various polymers, as an important constituent of hairdyes and dyes for leather and textiles [7].

Considering the wide use of these promutagens in a largenumber of products their genotoxicity needs to be evaluated.Furthermore a convenient system needs to be developed tostudy the impact of environmental exposure to such promu-tagens. In this work we intend to evaluate the genotoxiceffects of these three promutagens in Allium cepa roots. Theadvantage of using onion roots is that in the same testsystem, induced DNA damage can be measured by the

M. Ghosh : J. Paul : S. Sinha :A. Mukherjee (*)Centre of Advanced Study, Cell and Chromosome Research,Department of Botany, University of Calcutta,Kolkata 700 019, Indiae-mail: anitamukherjee28@gmail.com

Nucleus (2010) 53(1–2): 45–50DOI 10.1007/s13237-010-0005-x

comet assay as well as the frequency of induced chromosomeaberrations andmicronuclei [8]. The Allium test is a well knownand convenient method used in laboratories owing to the easeof their storage and germination, and more over can be used asa convenient system for the evaluation of macroscopic (measureof EC50) and microscopic (micronuclei formation, chromosomeaberrations) parameters pertaining to genotoxicity [9]. Apartfrom Allium test, Allium cepa has been used successfully in ourlaboratory as a biomarker of genotoxicity using comet assay[10]. Comet assay is a simple, sensitive and effective techniqueused to quantitatively express the extent of genotoxicity ofmany hazardous agents and has been used widely ingenotoxicity monitoring of the environment.

Materials and methods

Chemicals Test chemicals, o-phenylenediamine (o-PDA,CAS No. 95-54-5), m-phenylenediamine (m-PDA, CASNo. 108-45-2) and maleic hydrazide (MH, CAS No. 123-33-1) were purchased from Sigma chemical Co. (St. Louis,Missouri, USA). Ethyl methanesulphonate (EMS, CAS No.62-50-0), Normal melting point agarose (NMPA), lowmelting point agarose (LMPA), di-sodium salt of EDTA,Tris buffer, ethidium bromide (EtBr) were also purchasedfrom Sigma Chemical Co. (St Louis, Missouri, USA).Phosphate buffered saline (Ca++, Mg++free; PBS) waspurchased from Hi-Media Ltd., Mumbai, India. All otherchemicals like sodium hydroxide, sodium chloride, acetic acid,orcein were obtained locally and were of analytical grade.

Test system Equal-sized bulbs, from a population of a localmarket variety of Allium cepa L. (2n=16) were used. Theloose outer scales were removed and the dry bases werescraped to expose the root primordia, and the onions wereallowed to germinate on sand taken in earthen pots [11, 12]at room temperature (28±1°C) under a 12 h light/darkcycle. The germinated bulbs and the roots were used forfurther assays.

Root growth inhibition test and determination of EC50 Theroot growth inhibition test was performed according toFiskesjö [13] and Rank [14] with slight modifications. TheAllium cepa bulbs were allowed to germinate on sand for48 h. The root lengths (longest and smallest five roots perbulb) were recorded prior to treatment with the testreagents. The bulbs were then exposed to different concen-trations (1, 5, 10, 40 and 80 mg/l) of test chemicals (pH 7.4)diluted in double distilled water. The test solutions werereplaced by fresh solutions every 24 h with final rootmeasurement taken at the end of 48<h of exposure. TheEC50 (50% inhibition of root length) was obtained byinterpolation from the graph with concentration against root

length expressed as percent of control. The appropriateconcentrations to be used for the genotoxicity test werecalculated from the EC50 values thus obtained.

Allium test and anaphase- telophase chromosome aberra-tion assay The germinated bulbs were exposed to differentconcentrations (0, 1, 5 and 10 mg/l) of test chemicals (pH7.4) diluted in single distilled water. After 48 h of exposure,root meristems (10–15 in number), chosen at random from5 bulbs per exposure, were excised and fixed immediatelyfor 3 h in acetic acid/ethanol (1:3) and the remaining rootswere used for the comet assay. The excised root tips werehydrolyzed and stained in 9: 1, 2% aceto-orcein - (N) HClmixture. Slides were prepared from each of the rootmeristem following the squash technique of Sharma andSharma [15], and coded to prevent observer bias. At least5,000 cells from about 5 root meristems per exposure werescored. The mean values for mitotic index, binucleated cell,micronucleus, chromosome breaks at each point werescored and the standard deviation was calculated accordingly[11, 16]. Allium anaphase- telophase chromosome aberrationassay was done according to Rank [14]. At least 500anaphase and telophase cells from about 5–10 rootmeristems per exposure were scored, except in concen-trations where MI was very low. The anaphase andtelophase cells were observed for aberrations and werecategorized as fragments, vagrants, bridges and others.Mean values for each aberration were calculated perconcentration of exposure and were statistically correlated.The experiments were repeated at least once in order toestablish the reproducibility of the results.

Comet assay The DNA damage studies were carried outusing Comet assay, that is, single cell gel electrophoresis(SCGE) according to Singh [17] with slight modification[10, 18]. Roots from the same bulbs as has been used forAllium test and anaphase- telophase chromosome aberrationassay were used for comet assay following 48 h treatment.For positive control 2 mM of EMS was used. For isolationof nuclei, the roots were finely sliced using a new razorblade. Taking the nuclear suspension in 1% low meltingpoint agarose, slides were prepared in duplicates perconcentration. The slides were placed in alkaline electrolysisbuffer (300 mMNaOH and 1 mMEDTA; pH>13) for 15 minto allow unwinding of the DNA followed by electrophoresisat 4°C for 20 min at 26 V adjusted to 300 mA. Slides wereneutralized in 0.4 M Tris (pH 7.5) for 5 min and finally rinsedin water. Each experiment was repeated twice. The slides werethen stained with ethidium bromide (Et Br; 20 μg/ml) andrinsed in cold water to wash off excess stain. Slides werescored using image analysis system (Kinetic imaging; AndorTechnology, Nottingham, UK) attached to a fluorescencemicroscope (Leica, Wetzlar, Germany) equiped with appro-

46 Nucleus (2010) 53(1–2): 45–50

priate filters (N2.1). The microscope was connected to acomputer through a charge- coupled device (CCD) camera totransport images to software (Komet 5.5) for analysis. Thefinal magnification was 100X. The parameters studied werethe Olive tail moment (arbitrary units), tail DNA (%), taillength (μm) and tail extent moment (μm) which gave us a clearindication of the extent of DNA damage induced by the testchemicals. Images of 75 cells per concentration were analysedtaking 25 images/slide. The median values of each concentra-tion with respect to the comet parameters were calculated.

Statistical analysis For all statistical tests Sigma Stats.3software (SPSS Inc., Chicago, Illinois, USA) was used. Thelevel of significance was established at P≤0.05. For mitoticindex, anaphase-telophase aberrations and micronuclei,students ‘t’ test was performed. For DNA damage, oneway analysis of variance (ANOVA) test was done.

Results

Root growth inhibition test and determination of EC50 TheEC50 values of MH and o-PDA as obtained from root growthinhibition test were between 9–10 mg/l and 7–8 mg/l respec-tively. Root growth inhibition test with m-PDA revealed ahigh EC50 value between 65–70 mg/l. From the EC50 valuesthus obtained and also maintaining uniformity for furthergenotoxicity analysis the test concentrations (0, 1, 5 and10 mg/l) were selected.

Allium test and anaphase- telophase chromosome aberra-tion assay The clastogenic effect of the three promutagenswere evaluated on the basis of Allium test results (mitoticindex, micronucleus, chromosome breaks) and also

anaphase- telophase chromosome aberration assay (bridges,fragments, vagrants etc.). Though all the three promutagensshowed mito-depressive properties, the variation in MI wasnot significant for MH. m-PDA showed mito-depressiveactivity, with significant decrease in MI observed at1 mg/l (t- test; P<0.001) and 10 mg/l (t test; P=0.014) ascompared to control set. o-PDA treatment also resulted ina decrease of MI at all the concentration, with significantdecrease being observed at concentrations 5 mg/l (t- test;P=0.002) and 10 mg/l (t test; P<0.001) (Table 1).Genotoxic potential of the promutagens, evaluated on the basisof number of micronuclei per 1000 cells demonstrated a dosedependent increase in the number. MH showed a significantdose dependent increase in the number of micronuclei atconcentrations 1 mg/l (t test; P=0.020), 5 mg/l (t test; P=0.029) and 10 mg/l (t test; P=0.002) as compared to controlcells (Table 1). Treatment with o-PDA also resulted in asignificant increase in the number of micronuclei at concen-trations 5 mg/l (t test; P=0.006) and 10 mg/l (t test; P=0.021)(Table 1). A similar dose dependent increase in number ofmicronuclei was observed after treatment with m-PDA, withsignificant difference from control set (Table 1). A fewabberations as observed, has been represented in Fig. 1.

Anaphase- telophase chromosome aberration assay revealsthe genotoxic potential of the three promutagens in Allium.Treatments with different concentrations (0, 1, 5, and 10 mg/l)of MH, m-PDA and o-PDA have shown a significant dosedependent increase in% of chromosome aberration (Table 1).

Induction of DNA damaging effects as analyzed by cometassay Following treatment with MH, m-PDA and o-PDA,isolated nuclei from Allium roots were analyzed using thealkaline (pH≥13) comet assay. In general, the DNAmigration increased in the comet assay in a concentrationdependent manner, represented in terms of percentage of

Table 1 Effects of concentrations of MH, o-PDA and m-PDA on mitotic cells of Allium cepa L.

Test compound (mg/l) No. of cellsexamined

Cells inmitosis

Mitotic index(±SD) a

Cells withmicronuclei

Micronucle/1000 cells

No. of anaphase-telophase analyzed

% aberrant cells (±SD) withanaphase- telophase aberrationb

Control 5131 400 7.83±0.927 0 0 516 0.97±1.178

MH 1 5056 304 6.020±7.118 57 11.27** 35 14.29**

5 5076 280 5.48±2.934 150 29.43** 48 18.75**

10 5009 392 7.85±3.184 147 28.98** 42 30.95**

m-PDA 1 5009 138 2.79±0.610** 2 0.39 37 8.11**

5 5052 315 6.22±1.099 10 1.96* 533 13.88±3.947**

10 5030 251 5.03±0.883* 14 2.74* 256 12.5±3.951**

o-PDA 1 5030 344 6.194 ±1.695 2 0.04 510 3.704±2.549*

5 5001 221 4.480±1.331* 10 0.198* 502 5.802±3.940*

10 5084 265 5.212±0.356** 10 0.196* 500 5.667±3.429**

a data represents mean values±SD for 5 samples; b indicates bridges, fragments, vagrants and certain other uncategorized aberrations as analyzed for Alliumanaphase -telophase chromosome aberration assay; * significant at p<0.05, ** significant at p<0.001

Nucleus (2010) 53(1–2): 45–50 47

DNA in the Tail [% Tail DNA] (Fig. 2) and Olive TailMoment [OTM] (Fig. 3). Positive effect in comet assay wasestablished with 2 mM EMS. The values of% Tail DNA andOTM in MH treated roots of Allium demonstrated asignificant increase in the DNA damage at 5 mg/l andabove. Non-significant increase in% tail DNA and OTMvalues were observed in nuclei, from o-PDA and m-PDAtreated Allium root cells.

Discussion

In this study three different promutagens (o-PDA, m-PDAand MH) were evaluated for their possible genotoxicity in

plant system Allium cepa. The study also evaluated the useof Allium cepa as a standard plant test system forgenotoxicity evaluation using both Allium test and cometassay as genotoxic endpoints.

The herbicide MH was evaluated for genotoxicity inAllium, and different macroscopic (measure of EC50) andmicroscopic (micronuclei formation, chromosome aberra-tions) parameters pertaining to genotoxicity were analyzed.MH has been reported to be toxic to humans, inducingcarcinogenicity, reproductive and developmental toxicity,neurotoxicity, and acute toxicity [19]; selectively toxic toplants but not to bacteria, fungi, rodents or dogs [20]. It has,however been found to be mutagenic to Drosophilamelanogaster [21], Bacillus megatherium [22]; genotoxicto human lymphocytes [23] and produces cytotoxicity,

Fig. 1 Microphotographs showing nuclear aberrations following treat-ment with MH, m-PDA and o-PDA in Allium cepa root tip cells; a–d,Interphasic cell with nuclear bud; b–c, binucleate cell; e, Telophase with

bridge and vagrant; f, Telophase with bridge; g–h, Anaphase with earlyseparation; i, Interphasic cell with micronuclei

48 Nucleus (2010) 53(1–2): 45–50

causes mitotic inhibition and unbalanced growth in culturedmammalian cells [6]. MH is believed to induce nucleolaralteration, inhibiting biosynthetic activity of the nucleolus[24].The chromosome- breaking effects of MH in plantshave been confirmed for a number of plant species [forreview see Swietlinska and Zuk] [25] and the frequency ofchromosomal aberration depends strongly on pH andtemperature of treatment [26]. In the present studytreatment with different concentrations of MH did notreveal any significant changes in MI. At concentrations1 mg/l and above MH gave a significant increase in thenumber of micronuclei and anaphase- telophase aberrations.Treatment with MH at concentrations of 5 mg/l and above,revealed significant increase in DNA fragmentation asanalyzed from the comet parameters -% tail DNA andOTM. Based on our result, the clastogenicity and DNAdamage could be correlated in the Allium test system.Previous studies have shown that MH can cause chromo-some breakage, inhibition of cell division, retardation ofplant growth, and inhibition of nucleic acid synthesis incorn [27]. Much like our findings, MH has also shown toinduce DNA damage in Nicotiana tabacum var. xanthi [22]unlike as has been reported by Gichner et al. [28].According to Gichner et al. [28] MH induces highfrequency of somatic mutations in Nicotiana tabacum var.xanthi but failed to induce DNA migration in comet assay.The mutagenicity and clastogenicity of MH are well knownand our observations were consistent with similar reports ofgenotoxicity in Vicia faba [29] and Allium [30].

Treatment with the arylamines m-PDA and o-PDA leadto a mitodepressive response and a significant increase inanaphase - telophase aberrations, at all doses as comparedto control in Allium cepa roots. When analyzed for

micronuclei m-PDA and o-PDA treatment resulted in asignificant increase in number of micronuclei at doses5 mg/l and above. Aberrations such as anaphasic andtelophasic bridges and fragments as have been observed incells treated with both m-PDA and o-PDA establishes thatthese aromatic arylamines are clastogenic in nature.Aberrations such as binucleate cells scored in m-PDA treatedcells arise as a result of inhibition of cell plate formation oreven mitotic irregularities [31]. Presence of micronuclei (atinterphase), following o-PDA and m-PDA treatment, aremanifestation of chromosome fragmentation that occurred inthe previous cell cycle. Despite significant amount ofclastogenic effect, the genotoxic potential of m-PDA ando-PDAwas not detectable using comet assay. The clastogenicproperties as analyzed by Allium test and DNA damagingpotential as analyzed by comet assay could not be correlated.Previous study of hair dye components reveals varying extentof mutagenicity of phenylenediamine including o-PDA andm-PDA [32]. Certain reports also suggest that o-PDA andsome of its derivatives are carcinogenic and have attributedthe carcinogenicity to their structure [33]. Amo et al. [34] inanother study on (C57BL/6 x C3H/He) F1 (B6C3F1) micereported no carcinogenic potential of m-PDA. o-DA andm-PDA were mutagenic in Salmonella strains howevercarcinogenicity studies were positive in mouse and rat liverfor o-PDA [35, 36]. It has been reported that o-PDA and notm-PDA is genotoxic in Nicotiana tabacum var. xanthi, andthat the extent of DNA damage induced by o-PDA isproportional to the intensity of light exposed and henceo-PDA mediated genotoxicity is light dependent [1].

From the study thus far all the three promutagens appear tobe genotoxic in Allium as evaluated using anaphase- telophase

Fig. 2 Genotoxicity of MH, m-PDA and o-PDA in Allium cepa rootsas analyzed by comet parameter-% Tail DNA; * significant at p<0.05

Fig. 3 Genotoxicity of MH, m-PDA and o-PDA in Allium cepa rootsas analyzed by comet parameter-Olive tail moment (OTM);* significant at p<0.05

Nucleus (2010) 53(1–2): 45–50 49

chromosome aberration assay. The promutagens have shownsignificant increase in the number of interphase micronuclei.The study however does not give a comparable result betweenAllium test and comet assay, with the exception of MH. Thestudy reveals that Allium cepa could be used as a model testsystem for the evaluation of genotoxicity of promutagens andthat both the classical Allium test and comet assay needs to bedone so as to evaluate the genotoxic potential of thepromutagens.

Acknowledgements The authors are grateful to the coordinatorCAS, Cell and Chromosome Research, University of Calcutta forlaboratory facility and the University Grants Commission, New Delhifor funding.

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