This article was downloaded by: [University of Connecticut] On: 01 January 2014, At: 14:12 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tcar20 Genotoxicity of an organochlorine pesticide dichlorophene by micronucleus and chromosomal aberration assays using bone marrow cells of Rattus norvegicus Mohammad Iqbal Lone a , Nazia Nazam a , Sibhghatulla Shaikh a & Waseem Ahmad a a Gene-Tox laboratory, Division of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh, 202002, UP, India Published online: 06 Nov 2013. To cite this article: Mohammad Iqbal Lone, Nazia Nazam, Sibhghatulla Shaikh & Waseem Ahmad (2013) Genotoxicity of an organochlorine pesticide dichlorophene by micronucleus and chromosomal aberration assays using bone marrow cells of Rattus norvegicus, Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 66:4, 296-303, DOI: 10.1080/00087114.2013.852344 To link to this article: http://dx.doi.org/10.1080/00087114.2013.852344 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
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This article was downloaded by: [University of Connecticut]On: 01 January 2014, At: 14:12Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK
Caryologia: International Journal of Cytology,Cytosystematics and CytogeneticsPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tcar20
Genotoxicity of an organochlorine pesticidedichlorophene by micronucleus and chromosomalaberration assays using bone marrow cells of RattusnorvegicusMohammad Iqbal Lonea, Nazia Nazama, Sibhghatulla Shaikha & Waseem Ahmada
a Gene-Tox laboratory, Division of Genetics, Department of Zoology, Aligarh MuslimUniversity, Aligarh, 202002, UP, IndiaPublished online: 06 Nov 2013.
To cite this article: Mohammad Iqbal Lone, Nazia Nazam, Sibhghatulla Shaikh & Waseem Ahmad (2013) Genotoxicity of anorganochlorine pesticide dichlorophene by micronucleus and chromosomal aberration assays using bone marrow cells ofRattus norvegicus, Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 66:4, 296-303, DOI:10.1080/00087114.2013.852344
To link to this article: http://dx.doi.org/10.1080/00087114.2013.852344
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose ofthe Content. Any opinions and views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be reliedupon and should be independently verified with primary sources of information. Taylor and Francis shallnot be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and otherliabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Genotoxicity of an organochlorine pesticide dichlorophene by micronucleus and chromosomalaberration assays using bone marrow cells of Rattus norvegicus
Mohammad Iqbal Lone, Nazia Nazam, Sibhghatulla Shaikh and Waseem Ahmad*
Gene-Tox laboratory, Division of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh, 202002, UP, India
The aim of this study was to evaluate possible genotoxic damage of dichlorophene stress in rats by chromosomalaberration (CA), micronucleus (MN) and mitotic index (MI) assays in bone marrow cells. The study was carried outin vivo using three sublethal concentrations, 66.9 mg, 133.8 mg and 200.7 mg kg–1 body weight of rat of dichloropheneadministered intraperitoneally. The bone marrow cells were evaluated in each of the three treated groups at multipledurations. The MN and CA frequencies were increased significantly. A positive time- and dose-response relationship inall exposures was observed. However, the MI significantly decreased at each concentration compared to normal control.The results confirm the cytotoxic and genotoxic damage in Rattus norvegicus, and the suitability of the parameters forthe screening of the genotoxicant is further discussed.
The threat of some chemicals is so serious that theirroutine use may be mutagenic to human population(Saghir et al. 2001). Studies on industrial workers andfarmers have shown that exposure to such hazardouschemicals cause somatic as well as hereditary mutations(Antonelli et al. 2003; Farah et al. 2003). A few studies onmammals demonstrated the mutagenic potential of chemi-cal compounds can be assessed by using CAs as an indica-tor of mutagenic potential (Sharma et al. 2000). However,studies determining the genotoxic potential of organochlo-rine pesticides in mammals using CAs are rare. A surveyof the literature showed no studies investigating the geno-toxicity of dichlorophene in mammalian system.
This could partly be because dichlorophene is arelatively recently introduced compound. Chemically, itis represented as 2,2′-methylenebis(4-chlorophenol), ahalogenated phenolic compound with wide applications.It is used as a fungicide, bactericide and antiprotozoan(Gemmel and Johnston 1981; Kintz et al. 1997). Dichlor-ophene spray also has therapeutic use in the diseasedigital dermatitis (Ghashghaei 2007). In guinea pigs, afew studies obtained mixed results in dicholorophenesensitization tests (Yamarik 2004). Some derivatives,such as chlorinated bisphenol, used as antibacterial andantifungal agents, are indicated to be potent inhibitors ofglucose-6-phosphate dehydrogenase in yeast. There havebeen other studies of related compounds includingdichlorophen (Wang and Buhler 1981; Kintz et al. 1997)but research on dichlorophene is lacking.
We propose to evaluate the in vivo mutagenic poten-tial of dichlorophene in Rattus norvegicus by using CAsto look for breaks, gaps, rings, translocations and
multiple aberrations, MN induction and MI using bonemarrow cells (BMCs).
Materials and methods
Specimens
The animals were procured from Central Drug ResearchInstitute (CDRI), Lucknow, India, and acclimatized for aweek. The regular feed included commercial standardfood and water ad libitum. All rats were 8–10 weeks ofage, had an average weight of 100 ± 10 g, and were keptin controlled conditions (12 h dark and light period;temperature 22 ± 2°C; and humidity 70–80%). Of thefive groups, two groups served as controls (positive andnormal) and three groups received treatments with aspecific concentration of dichlorophene for a specifiedtime. The sacrifice of rats was in compliance with theethical regulations formulated by the Ethical Committeeof the Aligarh Muslim University, Aligarh.
Chemicals
Bearing CAS No. 97-23-4, 99.6%, dichlorophene wassupplied by Sigma-Aldrich Laborchemikalein,Niedersachsen, Germany. Chemicals used in variousother formulations were also of the highest purityanalytical grade. These were potassium chloride, metha-nol, colchicine, glacial acetic acid, foetal bovine serum,Giemsa stain, May–Grunwald stain, ethanol, potassiumdihydrogenphosphate and disodium hydrogen phosphate.
Treatment
A stock solution was prepared by dissolving dichloroph-ene in distilled water. Sublethal concentrations were
Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 2013Vol. 66, No. 4, 296–303, http://dx.doi.org/10.1080/00087114.2013.852344
prepared on the basis of LD50 values of dichlorophene669 mg kg–1 body weight (bw) in rat. Sublethalconcentrations of 66.9, 133.8 and 200.7 mg kg–1 bwwere administered intraperitoneally, comprising fiveanimals per treatment. Bone marrow flushed and thecells were screened after completion of specifieddurations. Concurrently positive control cyclophospha-mide (0.02 mg g–1) and normal control (distilled water)
runs were also made and evaluated in the same manner.A comparative analysis of the data was prepared.
Slide preparation
Two hours prior to sacrificing of rats, colchicine, 0.004mg kg–1 bw was injected intraperitoneally. This wasbased on prior runs for scoring the optimal aberrations.
Table 1. Micronuclei scoring in bone marrow cells of Rattus norvagicus treated in vivo with different doses of Dicholorophene
GroupTime(h)
Total PCEsScored
Total NCEsScored
Total number ofMNPCEs
Mean frequency of MN per 1000PCEs ± S.E
PCE/NCE
Normal control 24 1979 4011 2 0.29±0.09 0.3348 2016 3972 2 0.27±0.09 0.3372 2009 3982 2 0.27±0.09 0.33
Normal control (distilled water); positive control (cyclohosphamide); B (chromatid break); G (chromatid gap); B′ (Chromosomal break); G′(Chromosomal gap); MA (Multiple aberrations); DCP1 (66.9 mg/kg b.wt); DCP2 (133.8 mg/kg b.wt); DCP 3 (200.7 mg/kg b.wt);*statistically significant values at 0.05. (MWU – Test), compared to normal control.
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Chromosomal aberrations
The slide preparation and the staining followed theprotocol of Preston et al. (1987). In brief: both femurs ofeach rat were extracted and bone marrow was flushedusing a syringe having 0.56% KCl solution. The KClserved as a hypotonic solution incubated for 30 m at37°C, following centrifugation of 10 m at 1500 rpm.The contents were fixed in glacial acetic acid:methanol(1:3 v/v). These cells were then prepared for microscopicexamination by pouring 3–4 drops of cell suspension onpre-cleaned, chilled, ethanol-dipped slides, flame- andair-dried and stained with 5% Giemsa for 25–30 m.
Micronucleus test
The micronucleus test was carried out according toSchmid (1975) on the animals treated with dichloropheneand the control groups. The flushing of BMCs from boththe femurs was collected as a fine suspension into a tubecontaining 1 ml foetal bovine serum (FBS). The
Figure. 1 Multiple concentration and duration-dependent profiles of (MN) by dichlorophene at different intervals in Rattusnorvegicus along with their standard percent error depicted by error bars. Normal control (distilled water); positive control(cyclohosphamide); DCP1 (66.9 mg); DCP2 (133.8 mg); DCP 3 (200.7 mg); *Statistically significant values at 0.05.
Table 3. Incidence of in vivo chromosomal aberrationsrecorded in the bone marrow cells of Rattus norvegicusexposed to multiple doses of dichlorophene
Group24 h (Mean% MI ±SE)
48 h (Mean% MI ±SE)
72 h (Mean% MI ±SE)
Normal control(NC)
6.02±1.67 5.18±1.47 4.90±1.41
Positive control(PC) (40 mg/kgb. wt.)
4.34±1.31* 3.79±1.21* 3.50±1.15*
DCP 1 (66.9mg/kgb.wt.)
5.73±1.58* 4.90±1.41* 4.63±1.36
DCP 2 (133.8mg/kg b.wt.)
4.90±1.41* 4.34±1.31 4.05±1.26
DCP 3 (200.7 mg/kg b.wt.)
5.18±1.47* 4.34±1.31 4.34±1.31
Normal control (distilled water); positive control (cyclohosphamide);DCP1 (66.9 mg/kg b.wt); DCP2 (133.8 mg/kg b.wt); DCP 3 (200.7mg/kg b.wt);*Statistically significant values at 0.05. (Chi Square Test), compared tonormal control.
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Figure. 2 Multiple concentrations and duration-dependent profiles of (CAs) by dichlorophene at different intervals in Rattus norvegi-cus along with their standard percent error depicted by error bars. Normal control (distilled water); positive control (cyclohosphamide);DCP1 (66.9 mg/kg bw); DCP2 (133.8 mg/kg bw); DCP 3 (200.7 mg/kg bw); *Statistically significant values at 0.05.
Figure. 3 Multiple concentrations and duration-dependent profiles of (MI) by dichlorophene at different intervals in Rattus norvegicusalong with their standard percent error depicted by error bars. Normal control (distilled water); positive control (cyclohosphamide);DCP1 (66.9 mg/kg bw; DCP2 (133.8 mg/kg bw); DCP 3 (200.7 mg/kg bw); *Statistically significant values at 0.05.
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Figure 4 (Color online) Photomicrograph showing polychromatic erythrocytes (a) and micronucleated cell (b) in the bone marrowcells of Rattus norvegicus treated in vivo with different doses of dicholorophene intraperitoneally and metaphase plates of bonemarrow cells for different types of chromosomal aberrations with dicholorophene (c–h); (c) Normal metaphase; (d) Dicentric; (e)Acentric fragment and Gap; (f) Break; (g) Polyploidy; (h) Ring (100X oil immersion lens).
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centrifugation was carried out for 10 min at 1000 rpm.The pellet was resuspended in FBS. The suspension wassmeared onto pre-cleaned and air-dried slides. Followingfixation in 100% methanol for 5 min, staining was carriedout in May–Grunwald and Giemsa. The clearing of slidesfor both CA and micronucleus test was done in xyleneand they were permanently mounted in DPX DibutylPhathalate Xylene. An appropriate number of slides weresorted. The selection of slides was made on the basis ofstaining quality, and they were coded and scoredrandomly. A maximum of 6000 cells in each group wereexamined at 10× and 100× magnifications. Identificationof MN followed Schmid’s recommendations (1975).
Mitotic indices
The method of Hedges et al. (1995) was followed. TheMI was calculated from a total of 2000 cells scored ineach concentration category. A separate lot saved fromslides as prepared for CA assessment was used for MIestimation. More than 500 cells per animal in all individ-ual groups were used to evaluate the potential toxicity ofthe test chemical using the formula: MI = total no. ofdividing cells × 100/total no. of cells observed. Inthese calculations interphase, prometaphase and othersub-stages were excluded.
Statistical analysis
All the scorings were done from slides under code. TheMann–Whitney U test was applied on CA and MN datato calculate the percentage of frequencies of CAs andMN and the mean percentage, standard error and to seethe difference (significant or non-significant) for requiredgroups computed with the help of Statistical Package forSocial Sciences (SPSS) version 16.0. For the comparisonof proportions of mitotic indices between the control andexposed groups, the chi-square test was applied by usingMed Calc version 12.0.
Results
A summary of MN and CA counts, mean frequency andstandard deviation is provided in Tables 1 and 2. Theadministration of intraperitoneal doses of dichlorophenedisplayed a significant induction of MN. A high value ofMN induction occurs in PCEs polychromatic erythrocytesat 200.7 mg kg–1 bw concentration for 24 h duration,registering a mean frequency of 1.05 ± 0.50 with 16 mi-cronucleated cells. The frequency of MN in PCEs is in aconcentration and duration dependent manner. However,at 24 h post-administration the frequency of MN forma-tion declined so that significantly low incidences wereobserved in subsequent durations. The concentration andduration dependent profiles of MN are more conspicuousin Figure 1.
The PCE/NCE (NCE - Normochromatic erythrocytes)ratio was also studied using this compound. In each
category of concentration the ratio showed a decreasingtrend from 0.38 to 0.33 for respective concentrations asthe time elapsed following administration of pesticide.
Chromosomal aberrations for this compound werealso observed. With a maximum at 24 h duration, asignificant decrease in mean frequency was observed asthe duration increased. The results are presented inTable 2. Most conspicuous is the maximum effect at thehigh concentration: chromosomal damage was recordedas 6.30 ± 1.69 No, they dont need units, which is close tothe severity of cyclophosphamide. In the remaining twoconcentrations the injurious effect measured by CA is less.The studies of discrimination between chromatid and chro-mosomal aberrations showed a marginal increase in favourof chromatid type errors. Hence, the multiple aberrationswere found in significant numbers. The breaks, gaps,translocations, stickiness and pulverization were notunique for any treatment group qualitatively; however, adose-dependent response for total aberrations wastypically observed. CA revealed a concentration-depen-dant increase and the maximal response in test chemicalwas observed at 24 h treatment in all the concentrations.The profiles of CAs are illustrated in Figure 2.
The results of the MI assay are summarized in Table 3.Although the trend indicated a dose-dependent inhibition,it was less convincingly time-dependent. A minimumvalue (4.05 ± 1.26) was observed for 133.8 mg kg–1 bw;as opposed to the normal value. The maximum decreasewas seen at 72 h in a concentration of 133.8 mg kg–1 bw.This pattern is shown in Figure 3.
The chromatid and chromosomal aberrations includedpermanent features of damage such as breaks and gaps.Other aberrations such as rings, dicentrics, polyploidy,stickiness, acentric fragments and pulverization, scored asthe multiple aberrations class (MA). Figure 4 showsselected micrographs of above bone marrow ell withmicronucleus and metaphase chromosome (BMCh).
Discussion
To the best of our knowledge, only one study has been car-ried out on the cytotoxic and genotoxic effects ofdichlorophene on plant systems (Shaikh et al. 2012),despite the fact that plant test systems are efficient materialfor cytogenetic studies (Dixit et al. 2013; Frescura et al.2013).
Likewise, only a few reports are available in themammalian system on the genotoxic effect of dichlor-ophene; the present investigation on Rattus norvegicus istherefore important. Our results show that this pesticideinduces significant cytogenetic damage in BMCs,resulting in an increase in MN induction and CAs. Thetests adopted in the study are standard one and havereceived much attention as parameters for genotoxicassessment (Avishai et al. 2002; Klobucar et al. 2003).Other studies also recommend using CAs for qualitativeand quantitative assessment to detect clastogenic activity(Prasad et al. 2009). The MN assay is advocated for
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assessing clastogenic effects and damage to the mitoticapparatus with aneugenic consequences (Yoshioka et al.2007; Dimitrov et al. 2006). A combination of these twotests is preferred (Brzovic et al. 2009). The time periodchosen in the present study, i.e. 24, 48 and 72 h, isjustified in the light of earlier studies (Nazam et al.2013; Nabeel et al. 2008), as it allows a sufficientwindow period to detect clastogen and spindle poisons.
During our observations on CAs, as also in an earlierstudy on rat (Bird et al. 1982), metaphase analysis showsmore chromatid breaks than chromosome breaks,pointing to DNA strand damage in the late S phase. Thetype of breaks indicates that dichlorophene is active inthe G1 and S phase of the cell cycle; a similarconclusion was arrived at by Arzt et al. (1989). Thephenomenon of a decline in the frequency of aberrationsin later intervals could be due to factors such as elimina-tion of chemicals or metabolites from the body, repairingof the damaged genetic material, and elimination ofchromosomes with damaged genetic material (Shyamaand Rahiman 1993).
Induction of micronuclei in the PCE of bone marrowcells has been regarded as a sensitive bioassay formutagenic toxicity of candidate compounds (Hammamand Foda 2004). The frequency of dichlorophene-induced micronucleated polychromatic erythrocytes(MNPCEs) increases, especially at 24 h post treatment,but gradually decreases. The PCE/NCE ratio and thepercentage of polychromatic cells show a little variationfor this chemical. Generally the ratio is regarded as anindicator of inhibition of nucleated erythropoeitic celldivisions, and therefore, important for MNT. Suchchanges are suggested because of unbalanced changes innumber of PCEs and NCEs (Suzuki et al. 1989). Thealtered ratio points to acceleration of differentiation oferythrocytes from erythroblasts, inhibition of erythroblastdivision or because of recovery of erythroblast division(Suzuki et al. 1993).
The MI assay can help to characterize proliferatingcells, another standard way of identifying compoundsthat inhibit or induce mitotic progression. Such an inhibi-tion could indicate a possible cellular death or delay inthe cell proliferation kinetics (Öcal and Eroglu 2012).This trend is not very obvious in our case, as theresponse to dichlorophene seems not to follow the time–response manner. Could lower concentrations of thischemical stimulate the rate of cell division (Kalchevaet al. 2009)? This has to be verified.
AcknowledgementsThe research grants of University Grants Commission, NewDelhi; 40-355/2011(SR), 40-3 (M/S)/2009(SA-III-MANF) andCouncil of Science and Technology, U.P.; CST/D-598/2011 areacknowledged. The authors are also grateful to the Chairman,Department of Zoology, Aligarh Muslim University, Aligarhfor providing the necessary laboratory facilities.
Declaration of interest
The authors declare no potential conflicts of interest withrespect to the research, authorship, and/or publication of thisarticle.
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