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In Vivo Micronucleus Assay and GST Activity inAssessing Genotoxicity of Plumbagin in Swiss AlbinoMiceV. SivaKumar a; R. Prakash a; M. R. Murali a; H. Devaraj b; S. Niranjali Devaraj aa Department of Biochemistry, University of Madras, Guindy Campus, Chennai, Indiab Department of Zoology, University of Madras, Guindy Campus, Chennai, India

Online Publication Date: 01 October 2005To cite this Article: SivaKumar, V., Prakash, R., Murali, M. R., Devaraj, H. andDevaraj, S. Niranjali (2005) 'In Vivo Micronucleus Assay and GST Activity inAssessing Genotoxicity of Plumbagin in Swiss Albino Mice', Drug and ChemicalToxicology, 28:4, 499 - 507To link to this article: DOI: 10.1080/01480540500263019

URL: http://dx.doi.org/10.1080/01480540500263019

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In Vivo Micronucleus Assayand GST Activity inAssessing Genotoxicityof Plumbagin in SwissAlbino Mice

V. SivaKumar,1 R. Prakash,1 M. R. Murali,1 H. Devaraj,2 and

S. Niranjali Devaraj1

1Department of Biochemistry, University of Madras, Guindy Campus, Chennai, India2Department of Zoology, University of Madras, Guindy Campus, Chennai, India

Information available on the mutagenicity of a large number of indigenous drugscommonly employed in the Siddha and Ayurveda systems of medicine is scanty. In thiscontext, the current investigation on plumbagin, 5-hydroxy-2methyl-1,4-napthoqui-none, an active principle in the roots of Plumbago zeylanica used in Siddha andAyurveda for various ailments, was carried out; 16 mg/kg b.w. (LD50) was fixed as themaximum dose. Subsequent dose levels were fixed as 50% and 25% of LD50 amountingto 8 mg and 4 mg/kg b.w., respectively, and given orally for 5 consecutive days in 1%Carboxyl methyl cellulose (CMC) to Swiss albino mice weighing 25–30 g. Themicronucleus assay was done in mouse bone marrow. Plumbagin was found to inducemicronuclei at all the doses studied (4 mg/kg, 8 mg/kg, 16 mg/kg b.w.), and it proves tobe toxic to bone marrow cells of Swiss albino mice. Animal treated with cyclophospha-mide (40 mg/kg b.w.) served as positive control. In addition, glutathione S-transferase(GST) activity was observed in control, plumbagin (4 mg, 8 mg, 16 mg/kg b.w.,respectively), and genotoxin-treated experimental group of animals. No significantchange in GST activity was observed with plumbagin dose of 4 mg/kg b.w., whereasGST activity was significantly inhibited by higher doses of plumbagin (8 mg and 16 mg/kg b.w.) and cyclophosphamide.

Keywords Cyclophosphamide, Genotoxicity, Micronucleus test, Plumbagin.

Drug and Chemical Toxicology, 28:499–507, 2005

Copyright D Taylor & Francis Inc.

ISSN: 0148-0545 print / 1525-6014 online

DOI: 10.1080/01480540500263019

Address correspondence to Dr. S. Niranjali Devaraj, University of Madras, GuindyCampus, Chennai 600 025, India; Fax: 91-44-22352494; E-mail: niranjali@yahoo.com

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INTRODUCTION

Many factors in our environment are potential causes of cancer and or

mutagenic risk. These include a broad spectrum of chemicals, both naturally

occurring and synthetic, of both simple and complex structures that are

present in the air we breathe, the water we drink, the food we eat, and the

region in which we work and live (Fishbein, 1983).

Thousands of chemicals including pharmaceutical products, domestic and

food chemicals, pesticides, and petroleum products are present in the envi-

ronment, and new chemicals are being introduced every year. It is important,

therefore, that chemicals to which people are exposed either intentionally

(e.g., therapeutically), in the course of their daily lives (e.g., in domestic prod-

ucts, cosmetics, etc.), or inadvertently (e.g., pesticides) are tested for their

potential to produce cancer and genetic damage (WHO, 1985).

Antineoplastic agents are highly reactive in biological systems, and their

usefulness in malignant disease derives from their toxicity to rapidly pro-

liferating neoplastic tissue. These agents have low therapeutic index, how-

ever, which reflects their ability to damage rapidly proliferating normal tissue

as well. The long-term toxicity of antitumor agents is the subject of increasing

concern (Sieber and Adamson, 1975). Hence, by monitoring the use of the

drugs based on their mutagenicity, it should be possible to minimize their

immediate harmful effects on the genetic material and the consequent long-

term effects leading to another kind of cancer.

Information available on the mutagenicity of a large number of indigenous

drugs commonly employed in the Siddha and Ayurveda systems of medicine

is rather scanty. It is in this context that the current investigation on

plumbagin, an active principle in Plumbago zeylanica, was planned and

carried out.

Plumbagin (Figure 1) is 5-hydroxy-2methyl-1,4-napthoquinone and is

present in the roots of the plants belonging to Plumbaginaceae family.

Antitumor activity of this compound has been demonstrated in experimental

animal tumors (Krishnaswamy and Purushothaman, 1980). Plumbagin was

reported to have many side effects, including diarrhea, skin rashes, increase

in WBC, and neutrophil counts, increase in serum phosphatase and acid

Figure 1: Structure of plumbagin.

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phosphatase levels, and hepatic toxicity (Chandra and Nagarajan, 1981; Singh

and Udupa, 1997).

Plumbagin was shown to induce mitotic arrest and nuclear anomalies in

fibroblast cultures of chick embryo (Santhakumari et al., 1980). Cytogenetic

effects as a measure of genotoxicity of this drug in mammalian system em-

ploying micronucleus test to determine the dose-related toxicity in Swiss

albino mice was carried out. In addition to it, assay for glutathione S-trans-

ferase (GST) was carried out, as glutathione conjugation (GSH) is general

protection system against electrophilic compounds and metabolites that

induce genotoxic/carcinogenic effects (Chasseaud, 1979; Ketterer and Meyer,

1989; Mitchell and Rosso, 1987). GST catalyzes the conjugation of GSH with a

variety of reactive electrophiles and it takes on considerable importance as a

mechanism for carcinogen detoxification and cellular protection (Ketterer and

Meyer, 1989). Agents inducing GSTs in cells are generally known to inhibit

genotoxicity and carcinogenicity (Sparins et al., 1982; Wattenburg, 1985).

Therefore in this investigation, we included an assay for evaluating changes

in GST activity after treating with various doses of plumbagin (4 mg, 8 mg,

16 mg/kg b.w.) and with a known genotoxin, cyclophosphamide 40 mg/kg b.w.

MATERIALS AND METHODS

ChemicalsPlumbagin was obtained from Sigma Chemical Co. (CAS Reg. no. P7262),

and all other chemicals were of analytical grade.

AnimalsMice (Swiss albino strain) were procured from Kings Institute of

Preventive Medicine (Guindy, Chennai, India). They were maintained in the

animal house of the department and were fed with animal feed and water ad

libitum. Animals of either sex about 3 months old and of weight 20–25 g were

used in the experiment.

Selection of DosageIn a study conducted by Krishnaswamy and Purushothaman (1980), it was

reported that 16 mg/kg b.w. is the LD50 in mice for plumbagin given

intraperitoneally and orally. In this study, 16 mg/kg b.w. was fixed as the

maximum dose. Subsequent dose levels were fixed at 50% and 25% of the LD50

values amounting to 8 mg/kg and 4 mg/kg weight. The carboxy methyl ether of

cellulose (1% CMC) is readily soluble in hot or cold water and is used as a

vehicle in our study.

501Genotoxicity of Plumbagin

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Experimental ProtocolThe animals were divided into 6 groups (groups A–F) of six animals each.

Group A: Normal control received only the standard diet.

Group B: Positive control, received a single dose of cyclophosphamide (Cp) (Cp

40 mg/kg i.p.) in saline (10 mL/kg)

Group C: Administered 1% CMC for 5 days.

Group D: Administered plumbagin (4 mg kg�1 day�1 for 5 days in 1% CMC by

gavage)

Group E: Administered plumbagin (8 mg kg�1 day�1 for 5 days in 1% CMC by

gavage)

Group F: Administered plumbagin (16 mg kg�1 day�1 for 5 days in 1% CMC by

gavage)

At the end of the experimental period, animals were sacrificed by cervical

dislocation 30 h after dosing. Both the femora were removed and cleaned with

gauze by removing all the adhering muscle and tissue and subjected to

micronucleus assay. Livers were excised and GST activity was assessed.

Micronucleus TestThe mouse bone marrow test was carried out according to Schmid (1975)

for evaluating chromosomal damage. The bone marrow cells from both

femurs of each animal were flushed in the fine suspension into centrifuge

tubes containing fetal bovine serum. This cell suspension was centrifuged at

Table 1: Frequency of micronucleated PCE and NCE induced byplumbagin at various concentrations.

Groupsa MnPCE � 103/total PCE PCE/NCE

A 2.17 ± 0.75 1.19 ± 0.02B 65.50 ± 5.64* 0.33 ± 0.02*C 1.50 ± 0.55 0.73 ± 0.01*D 4.67 ± 2.16y 0.45 ± 0.02*E 10.83 ± 2.79*,y 0.36 ± 0.01*F 14.50 ± 4.04*,y,z 0.22 ± 0.04*,z

Values are mean ± SD (n = 6). PCE, polychromatic erythrocyte; NCE, normo-chromatic erythrocyte.aGroup A, control; group B, positive control (Cp 40 mg/kg. i.p); group C, vehiclecontrol; group D, plumbagin 4 mg/kg; group E, plumbagin 8 mg/kg; group F,plumbagin 16 mg/kg.*p < 0.05 group A compared with groups B, C, D, E, and F.yp < 0.05 group B compared with groups D, E, and F.zp < 0.05 group D compared with groups E and F.

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1000 rpm for 10 min and the supernatant was removed. The pellet was

resuspended in a drop of serum before being used for preparing the slide.

The air-dried slides were stained with Maygrunwald stain and Giemsa.

From each slide, 1000 PCE (polychromatic erythrocyte) and corresponding

NCE (normochromatic erythrocyte) were scored. With conventional staining

techniques, PCE stain bluish purple because of high content of ribonucleic

acid in cytoplasm. NCEs stain reddish to yellow. The micronucleus test was

performed in mice as recommended (Hayashi et al., 1994; MacGregor et al.,

1987). The scored elements were micronucleated cells and not the number

of micronuclei. After the completion of scoring, all slides were decoded

and the data were computed for the control and test groups and were stat-

istically analyzed.

Assessment of Hepatic GlutathioneS-Transferase ActivityWhen the experimental animals were sacrificed after the duration of the

treatment, the livers were excised and used for determining GST activity

according to Habig et al. (1974). The general cytosolic GST activity was

determined using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The

specific activity was expressed as n moles of CDNB–GSH conjugate formed

per mg of protein. Protein content of each sample was determined according to

Lowry et al. (1951) using BSA as standard.

Table 2: Assessment of hepatic GST activity in plumbagin-treatedanimals at various concentrations.

GroupsaGST activity (nmol CDNB–GSH conjugateformed per min per mg protein)

A 1.08 ± 0.03B 0.77 ± 0.05*C 1.05 ± 0.03D 1.67 ± 0.03y

E 0.90 ± 0.03*,y,z

F 0.76 ± 0.05*,z,x

Values are mean ± SD (n = 6). GST, glutathione S-transferase; CDNB, 1-chloro-2,4-dinitrobenzene; GSH, reduced glutathione.aGroup A, control; group B, positive control (Cp 40 mg/kg i.p.); group C,vehicle control; group D, plumbagin 4 mg/kg; group E, plumbagin 8 mg/kg; group F, plumbagin 16 mg/kg.*p < 0.05 group A compared with groups B, C, D, E, and F.yp < 0.05 group B compared with groups D, E, and F.zp < 0.05 group D compared with groups E and F.xp < 0.05 group E versus F.

503Genotoxicity of Plumbagin

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Statistical AnalysisAll the grouped data were evaluated with SPSS/7.5 software. Hypothesis

testing methods included one-way analysis of variance (ANOVA) followed by

least significant difference (LSD) test. p values of < 0.05 were considered to

indicate statistical significance. All the results were expressed as mean ± SD

for six animals in each group.

RESULTS

The highest incidence (65.5/1000) of micronucleated PCE was observed in the

positive control group. In plumbagin-treated animals, micronucleated poly-

chromatic erythrocyte (MnPCE) was significantly higher than that of either

vehicle control or negative control. This induction is dose-dependent. A maxi-

mum induction of MnPCEs (14.5/1000) was observed for the dose of 16 mg/kg

b.w. A 2 1/2-fold increase in incidence of micronucleated PCE was observed for

an increase in concentration of plumbagin from 4 mg to 8 mg/kg. Nevertheless,

this increase was not all that appreciable for a similar increase in dose from

8 to 16 mg/kg.

The proportion of normochromatic to polychromatic erythrocyte (PCE/

NCE) was found to be decreased in plumbagin-treated animals when

compared with either negative control or vehicle control (Table 1).

GST AssayThe hepatic GST activity was observed in mouse. Plumbagin at 4 mg/kg

b.w. showed no significant increase in GST activity compared to negative

control, whereas plumbagin at 8 mg and 16 mg significantly reduced the GST

activity when compared to the negative control. Effect of known genotoxin

cyclophosphamide (40 mg/kg b.w.) (Premkumar et al., 2001) and plumbagin

(16 mg/kg b.w.) was more or less similar on the GST activity (Table 2).

DISCUSSION

The current study on genotoxicity of plumbagin using micronucleus as an end

point reveals that there is a significant induction of MnPCEs in plumbagin-

treated animals and it is dose-dependent. It induces an alteration in the

proportion of normochromatic to polychromatic erythrocyte in the bone

marrow at all doses studied. These observations suggest the mutagenic or

genotoxic effect of plumbagin in vivo. Plumbagin appears to be toxic to the

proliferating bone marrow cells, which is evident from the decrease in the PCE/

NCE ratio.

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Cyclophosphamide given intraperitoneally (i.p.) at a concentration of

40 mg/kg induced MnPCEs of 65.5 per 1000. It has been reported to be a

positive inducer of micronuclei in Swiss albino mice (Premkumar et al., 2001).

According to Hart and Engberg-Pederson (1983) a dose-related increase in

the incidence of MnPCE is the criterion for a positive effect. So based on this

finding, plumbagin can be considered to be a weak inducer of micronucleus,

which implies cytogenetic damage to bone marrow cells.

Farr et al. (1985) determined the mutagenicity of plumbagin by measuring

the Trp� to Trp+ reversion frequency in actively growing Escherichia coli

AQ654 cells and in stationary phase cells. The results showed that plumbagin

was not mutagenic in stationary phase cells but was moderately mutagenic in

exponential phase cells.

According to Santhakumari et al. (1980), plumbagin arrested the cell

growth and proliferation of chick embryo fibroblast and a decrease in mitotic

index with accumulation of cell in metaphase. These changes were evident at

concentrations as low as 0.1 g, associated with chromosomal aberrations. They

concluded that plumbagin at lower concentration behaves like a spindle poison

by inhibiting the entry of cells into mitosis, but at higher concentrations it also

exhibits radiomimetic, nucleotoxic, and cytotoxic effects. It has been reported

that the oxidative DNA damaging agent plumbagin has arrested the mouse

embryonic fibroblast cells at S-G2/M phase and caused cell death by

decreasing p21-mediated long-patch base excision repair, in both Pol-beta

dependent and independent pathway (Jaiswal et al., 2002).

These reports suggest the mutagenicity and toxicity of plumbagin. In the

current study also, decrease in PCE/NCE ratio due to toxicity induced by

plumbagin was observed at all doses.

Plumbagin at a concentration of 4 mg/kg b.w. induced the GST activity,

whereas higher concentrations (8 mg, 16 mg/kg b.w.) inhibited GST activity.

Plumbagin has already been reported as an oxidant (Fuji et al., 1992; Imlay

and Fridovich, 1992; Ono et al., 1991) and superoxide generator (Martinez

et al., 2001). Napthoquinones have been reported to be reduced by glutathione

to the corresponding semiquinones, with concomitant conjugation to gluta-

thione. Napthoquinones also deplete the net GSH levels in the rat hepatocyte

resulting in the blebbing of the cell (Butler et al., 1987).

Plumbagin has been reported as a weak intercalator of DNA (Fuji et al.,

1992). Mutagenicity of plumbagin has been reported to generate superoxide

radical, which in turn produces OH� by Fenton reaction, thereby damaging

the DNA (Prieto-Alamo et al., 1993). The observed GST inhibition at higher

doses may be due to the increase in oxidative stress. These findings suggest

that even though the plumbagin intercalates with the DNA, before inter-

calation, while traveling through the cytosolic compartment, its generation of

superoxide anions and Reactive Oxygen Species (ROS) and its capability of

505Genotoxicity of Plumbagin

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binding to various cellular targets cannot be ruled out. In brief, plumbagin

appears to induce micronuclei and it proves to be toxic to bone marrow cells. In

line with some of the above findings, our results impose that the concentration-

dependent Genotoxicity of plumbagin, may be due to depletion of the hepatic

GST activity in addition to its DNA intercalating activity. Further exten-

sive work is needed to elucidate the mechanism of action of plumbagin as

a genotoxin.

ACKNOWLEDGMENTS

We acknowledge Dr. S. T. Santhiya, Professor, Department of Biomedical

Genetics, University of Madras, for her kind support throughout the study.

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