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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.
REFERENCES
Butler, J., Dodd, N. J. F., Land, E. J., Swallow, A. J. (1987). Twenty-first Patersonsymposium: bioactivation of anti-tumour agents. Br. J. Cancer 55:327–330.
Chandra sekaran, B., Nagarajan, B. (1981). Metabolism of echitamine and plumbaginin rats. J. Biol. Sci. 3:395–400.
Chasseaud, L. F. (1979). The role of glutathione and glutathione S-transferase in themetabolism of chemical carcinogens and other electrophilic agents. Adv. CancerRes. 29:175–274.
Farr, S. B., Natvig, D. O., Kogoma, T. (1985). Toxicity and mutagenicity of plumbaginand the induction of possible new DNA repair pathway in E. coli. J. Bacteriol.165(3):1309–1316.
Fishbein, L. (1983). Mutagens and carcinogens in the environment as in genetics: newfrontiers. In: Chopra, V. L., Joshi, B. C., Sharma, R. C., Bansal, H. C., eds.Proceedings of the XV International Congress of Genetics. Genetics and Health,New Delhi: Oxford and IBH Publ., pp. 1–22.
Fuji, N., Yamashita, Y., Arima, Y., Nagashima, M., Nakano, H. (1992). In-duction of topoisomerase II mediated DNA cleavage by the plant naptho-quinones plumbagin and shikonin. Antimicrob. Agents Chemother. 36(12):2589–2594.
Habig, W. H., Pabst, M. J., Jakoby, W. B. (1974). Glutathione-S-transferase, the firstenzymatic step in mercapturic acid formation. J. Biol. Chem. 249:7130–7139.
Hart, J. W., Engberg-Pederson, H. (1983). Statistics of the mouse bone marrowmicronucleus test: counting, distribution and evaluation of results. Mutat. Res.111:195–207.
Hayashi, M., Tice, R. R., Mac Gregor, J., Aderson, D., Blakey, D. H., Krish-Volders, M.,Vannier, B. (1994). In vivo rodent erythrocyte micronucleus assay. Mutat. Res.312:293–304.
Imlay, J., Fridovich, I. (1992). Exogenous quinones directly inhibit the respiratoryNADH dehydrogenase in E. coli. Arch. Biochem. Biophys. 296(1):337–346.
Jaiswal, A. S., Bloom, L. B., Narayan, S. (2002). Long-patch base excision repair of
SivaKumar et al.506
Dow
nloa
ded
By:
[IN
FLIB
NE
T, In
dia
orde
r 200
5] A
t: 18
:05
10 A
pril
2008
apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast celllines treated with plumbagin involvement of cyclin-dependent kinase inhibitorp21 Waf-1/Cip-1. Oncogene 21(38):5912–5922.
Ketterer, B., Meyer, D. J. (1989). Glutathione transferases: a possible role in thedetoxification and repair of DNA and lipid hydroperoxides. Mutat. Res. 45:1–8.
Krishnaswamy, M., Purushothaman, K. K. (1980). Plumbagin: a study of itsanticancer, antibacterial, antifungal properties. Indian J. Exp. Biol. 18:876–877.
Lowry, O. H., Rosenbrough, N. J., Farr, A. I., Randall, R. J. (1951). Proteinmeasurement with Folins reagent. J. Biol. Chem. 193:265–275.
MacGregor, J., Heddle, J. A., Hite, M., Margolin, B. H., Ramel, C., Salamone, M. F.,Tice, R. R., Wild, D. (1987). Guidelines for the conduct of micronucleus assay inmammalian bone marrow erythrocytes. Mutat. Res. 189:103–112.
Martinez, A., Urios, A., Felipo, V., Blanco, M. (2001). Mutagenicity of nitric oxide-releasing compounds in Escherichia coli: effects of superoxide generation andevidence for two mutagenic mechanisms. Mutat. Res. 497(1–2):159–167.
Mitchell, J. B., Rosso, A. (1987). The role of glutathione in radiation and drug inducedcytotoxicity. Br. J. Cancer 55:99–104.
Ono, T., Nunoshiba, T., Nishioka, H. (1991). Sensitivity and adaptive response ofmutants lacking active oxygen defense systems against different oxygen species.Mutat. Res. 253(3):271.
Premkumar, K., Pachiappan, A., Santhiya, S. T., Gopinath, P. M., Ramesh, A.,Abraham, S. K. (2001). Effect of Spirulina fusiformis on cyclophosphamide andmitomycin C induced genotoxicity and oxidative stress in mice. Fitoterapia72:906–911.
Prieto-Alamo, M. J., Abril, N., Pueyo, C. (1993). Mutagenesis in Escherichia coli K-12mutants defective in superoxide dismutase or catalase. Carcinogenesis 14:282–284.
Santhakumari, G., Saralamma, P. G., Radhakrishnan, N. (1980). Effect of plumbaginon cell growth and mitosis. Indian J. Exp. Biol. 18(3):215–218.
Schmid, W. (1975). The micronucleus test. Mutat. Res. 31:9–15.
Sieber, S. M., Adamson, H. (1975). Toxicity of antineoplastic agents in man:chromosomal aberrations, antifertility effects, congenital malformations andcarcinogenic potential. Adv. Cancer Res. 22:57–144.
Singh, U. V., Udupa, N. (1997). Reduced toxicity and enhanced antitumour efficacy ofbeta cyclodextrin plumbagin inclusion complex on mice bearing Ehrlich ascitescarcinoma. Indian J. Physiol. Pharmacol. 11(2):171–175.
Sparins, V. L., Veegas, P. L., Watterbrg, L. W. (1982). Glutathione-S-transferaseactivity by compounds inhibiting chemical carcinogenesis and by dietaryconstituents. J. Natl. Cancer Inst. 68:493–496.
Wattenburg, L. W. (1985). Chemoprevention of cancer. Cancer Res. 45:1–8.
WHO (1985). Environmental Health Criteria 51. Guide to Short Term Tests forDetecting Mutagenic and Carcinogenic Chemicals. Environmental Program,Series 51, WHO, ILO and UN.
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