ORIGINAL PAPER

Vanadium as a chemoprotectant: effect of vanadium(III)-L-cysteinecomplex against cyclophosphamide-induced hepatotoxicityand genotoxicity in Swiss albino mice

Abhishek Basu • Arin Bhattacharjee •

Somnath Singha Roy • Prosenjit Ghosh •

Pramita Chakraborty • Ila Das • Sudin Bhattacharya

Received: 17 December 2013 / Accepted: 10 April 2014

� SBIC 2014

Abstract Vanadium is an essential micronutrient for

living systems and has antioxidant and genoprotective

property. In the present study, the protective role of an

organovanadium compound vanadium(III)-L-cysteine (VC-

III) was evaluated against hepatotoxicity and genotoxicity

induced by cyclophosphamide (CP) (25 mg/kg b.w., i.p.) in

Swiss albino mice. Treatment with VC-III (1 mg/kg b.w.,

p.o.) mitigated CP-induced hepatic injury as indicated by

reduction in activities of alanine transaminase, aspartate

transaminase, alkaline phosphatase by 1.57-, 1.58- and

1.32-fold in concomitant treatment schedule and by 1.83-,

1.77- and 1.45-fold in pretreatment schedule, respectively,

and confirmed by histopathological evidences. Parallel to

these changes, VC-III ameliorated CP-induced oxidative

stress in liver by 1.46-, 1.26-, 1.32- and 1.42-fold in con-

comitant treatment group and by 1.95-, 1.40-, 1.46- and

1.73-fold in pretreatment group at the level of H2O2,

superoxide, nitric oxide and lipid peroxidation, respec-

tively. VC-III also enhanced activities of antioxidant

enzymes such as superoxide dismutase, catalase, glutathi-

one peroxidase, glutathione S-transferase and glutathione

(reduced) level in mice liver by 1.46-, 1.37-, 1.29-, 1.44-

and 1.45-fold in concomitant treatment schedule and by

1.64-, 1.65-, 1.42-, 1.49- and 1.57-fold in pretreatment

schedule, respectively. In addition, the organovanadium

compound could efficiently attenuate CP-induced chro-

mosomal aberrations, DNA fragmentation and apoptosis in

bone marrow cells and DNA damage in lymphocytes by

1.49-, 1.43-, 1.48- and 1.59-fold in concomitant treatment

group and by 1.76-, 1.92-, 1.99- and 2.15-fold in pre-

treatment group, respectively. Thus, the present study

showed that VC-III could exert protection against CP-

induced hepatotoxicity and genotoxicity.

Keywords Organovanadium � Cyclophosphamide �Oxidative stress � Genotoxicity � Comet assay

Abbreviations

ALT Alanine aminotransaminase

ALP Alkaline phosphatase

AST Aspartate aminotransaminase

BUN Blood urea nitrogen

CP Cyclophosphamide

RNS Reactive nitrogen species

ROS Reactive oxygen species

VC-III Vanadium(III)-L-cysteine

Introduction

Cyclophosphamide (CP) is one of the most commonly used

alkylating agents [1] indicated for different types of hae-

matological and solid malignancies and other medical

conditions [2]. However, clinical use of CP is often limited

due to its side effects and toxicity [3]. Hepatotoxicity is one

of the major adverse effects of CP [4] as the biotransfor-

mation of CP principally takes place within the hepatocytes

[5]. About 43 % of patients experience mild to severe form

of hepatic dysfunction after CP therapy [6]. Experimental

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-014-1141-6) contains supplementarymaterial, which is available to authorized users.

A. Basu � A. Bhattacharjee � S. S. Roy � P. Ghosh �P. Chakraborty � I. Das � S. Bhattacharya (&)

Department of Cancer Chemoprevention, Chittaranjan National

Cancer Institute, 37, S. P. Mukherjee Road,

Kolkata 700 026, West Bengal, India

e-mail: sudinb19572004@yahoo.co.in

123

J Biol Inorg Chem

DOI 10.1007/s00775-014-1141-6

studies suggest that oxidative stress is mainly responsible

for CP-induced hepatotoxicity [7]. Another severe toxicity

of CP is genotoxicity which is related to DNA binding

characteristics of CP [8]. CP-induced nucleic acid damage

may lead to DNA mutations that result in cytotoxicity [9],

carcinogenicity [10] and teratogenicity [11]. The Interna-

tional Agency for Research on Cancer (IARC) evaluated

CP as carcinogenic to humans (Group 1) [10].

Chemoprotectants like amifostine, mesna and dex-

razoxane were used to suppress the toxic effects of cancer

chemotherapy but these drugs are not approved for wide

clinical use due to their lack of efficacy and side effects

[12]. So, limitations to such conventional therapy have

spurred real need for such agents which can suppress CP-

induced toxic effects to prolong its clinical use and

outcome.

Vanadium, a dietary micronutrient and an ultra trace

element, recently has received attention of researchers

because of its wide range of biological activities [13]. A

number of studies have proved that vanadium possesses

antioxidant efficacy [14] and protects against oxidative

stress induced by different xenobiotics [15, 16]. Addi-

tionally, vanadium has a profound role in DNA mainte-

nance reactions and also prevents genomic instability that

leads to cancer [17, 18]. However, use of inorganic vana-

dium compounds are often restricted owing to their toxicity

[19]. Vanadium complexes with organic ligands to improve

their bioavailability and toxicity profile are of great inter-

est. In this regard, an organovanadium compound, viz.,

vanadium(III)-L-cysteine complex (VC-III) was used in

this study. The purpose of the present work is to investigate

the probable protective role of VC-III against CP-induced

hepatotoxicity and genotoxicity in Swiss albino mice. The

present study is the first of its kind to evaluate the che-

moprotective role of vanadium compound against cancer

chemotherapy-induced toxicity in vivo.

Materials and methods

Experimental animals

Adult (5–6 weeks) Swiss albino female mice (25 ± 2 g),

bred in the animal colony of Chittaranjan National Cancer

Institute (Kolkata, India) were used for this study. The

mice were maintained at controlled temperature and

humidity under alternating light and dark conditions.

Standard food pellets (EPIC rat and mice pellet from

Kalyani Feed Milling Plant, Kalyani, West Bengal, India)

and drinking water was provided ad libitum. The experi-

ments were carried out strictly following the guidelines of

Institutional Animal Ethics Committee [Committee for the

Purpose of Control and Supervision of Experiment on

Animals (CPCSEA Registration. No. 175/99/CPCSEA),

India].

Chemicals

CP was obtained from Cadila Pharmaceuticals, Bhat, Ah-

medabad, India. Vanadium(III) trichloride, L-cysteine,

1-chloro-2,4-dinitrobenzene (CDNB), ethylenediaminete-

traacetic acid (EDTA), reduced glutathione (GSH), pyro-

gallol, 5,50-dithio-bis(2-nitrobenzoic acid) (DTNB), sodium

dodecyl sulphate (SDS), b-nicotinamide adenine dinucleo-

tide phosphate (NADPH), glutathione reductase, bovine

serum albumin (BSA), sodium nitrite, N-(1-napthyl) ethy-

lenediamine dihydrochloride (NEDD), normal melting

agarose, low melting point agarose, ethidium bromide,

sodium azide (NaN3), HEPES, 20,70-dichlorodihydro-

fluorescein diacetate (DCFH-DA), dihydroethidium (DHE),

Histopaque, Triton X-100, Giemsa, colchicine, diphenyl-

amine and phenylmethanesulfonyl fluoride (PMSF),

2-vinylpyridine, oxidized glutathione (GSSG) were obtained

from Sigma-Aldrich Chemicals Private Limited, Bangalore,

India. Methanol, xylene, sulphanilamide and Folin-phenol

reagent were purchased from Sisco Research Laboratories

Private Limited Mumbai, India. Rectified spirit and absolute

alcohol were obtained from Bengal Chemicals Pharmaceu-

ticals Limited, Kolkata, India. Serum alanine transaminase

(ALT), aspartate transaminase (AST), alkaline phosphatase

(ALP), urea and creatinine assay kits were obtained from

Span Diagnostics Ltd., Udhna, Surat, India. In situ cell death

detection kit, AP was purchased from Roche Diagnostics

India Private Limited. All other chemicals not specified were

obtained from Merck (India) Limited, Mumbai, India.

Synthesis of VC-III

VC-III was synthesized by following the literature proce-

dure of Papaioannou et al. [20] (Fig. S1). Vanadium(III)

chloride (500 mg, 3.17 nM) was dissolved in dry methanol

(25 ml) and stirred using a magnetic stirrer at an ambient

temperature under nitrogen atmosphere. Solid L-cysteine

(1.16 g, 9.57 nM) was added to the solution in one portion.

Upon stirring for another 3 h, a greenish brown precipitate

was formed. The precipitate was collected by filtration and

washed with chilled methanol and diethyl ether and dried

in vacuo over phosphorus pentoxide. Yield: 820 mg

(48.8 %). IR (KBr, cm-1): v 3,408 (OH), 1,621, 1,446

(C=O), 588 (V–O), 455 (V–N); kmax in distilled water:

432 nm (emM = 20); leff: 1.74 lB (Fig. S2a-c).

Stability study of VC-III

To study the chemical stability of the compound, VC-III

was dissolved in phosphate buffer saline (PBS), pH 7.4 and

J Biol Inorg Chem

123

kept at 37 �C. The absorption spectra of vanadium(III)-L-

cysteine solution, was taken at various time points up to

24 h.

Experimental design

For dose selection of the compound, VC-III was adminis-

tered by oral gavage at different doses of 1, 2, 3 and 5 mg/

kg b.w. for 28 days in Swiss albino mice (n = 6). The

compound manifested no toxicity up to the dose of 2 mg/

kg b.w. based on pathological examination (Table 1).

Additionally, VC-III produced optimum antioxidant effi-

cacy at a dose of 1 mg/kg b.w., per orally in vivo (Table 2).

In the present study, the animals were divided into six

groups containing six animals (n = 6) in each group

(Fig. 1). All the experiments were performed twice.

• Group I (vehicle control group): each animal was given

distilled water orally and intraperitoneally for 10 days.

• Group II (only VC-III-treated group): each animal was

treated only with VC-III at a dose of 1 mg/kg. b.w.

orally throughout the experimental period.

• Group III (CP-treated group): each animal was injected

with CP intraperitoneally at a dose of 25 mg/kg b.w. in

water for 10 days.

• Group IV (CP ? L-cysteine-treated group): L-cysteine

was administered orally at a dose of 0.88 mg/kg b.w.

(comparable with the amount of ligand content in the

complex at the dose of 1 mg/kg b.w.) in water for

10 days and CP was given as in Group III.

• Group V (concomitant treatment group): VC-III was

administered orally at a dose of 1 mg/kg b.w. in water

for 10 days and CP was given as in Group III.

• Group VI (pretreatment group): VC-III was adminis-

tered orally at a dose of 1 mg/kg b.w. in water 7 days

prior to CP treatment and then continued along with CP

for 10 days.

The mice were killed on day 11, and the parameters

described below were studied.

Determination of serum ALT, AST, ALP, blood urea

nitrogen (BUN) and creatinine

Blood samples were collected from mice by retro-orbital

puncture under anesthesia and centrifuged at 2,000g for

10 min for serum separation. Then the ALT, AST, ALP

activity and BUN, creatinine level were measured spec-

trophotometrically by standard methods using commercial

kits [21–24].

Table 2 Effect of different doses of VC-III on hepatic GSH levels and GST, GPx, SOD, CAT activities in different groups after 28 days in

Swiss albino mice

Groups GSH

(nM/mg protein)

GST (CDNB-GSH

conjugate/min/mg protein

GPx (lM NADPH

utilized/min/mg protein

SOD

(U/mg protein)

CAT

(U/mg protein)

Vehicle control 54.87 ± 3.06 289.59 ± 13.65 0.449 ± 0.044 231.40 ± 13.40 34.82 ± 3.07

VC-III at 1 mg/kg b.w. 68.16 ± 4.10* 303.52 ± 16.42 0.459 ± 0.029 238.16 ± 23.61 35.17 ± 2.39

VC-III at 2 mg/kg b.w. 60.18 ± 4.19 292.60 ± 25.99 0.445 ± 0.038 232.65 ± 14.45 33.96 ± 1.55

VC-III at 3 mg/kg b.w. 56.25 ± 2.95 284.49 ± 15.75 0.444 ± 0.024 229.16 ± 16.42 33.53 ± 2.60

VC-III at 5 mg/kg b.w. 43.72 ± 3.84* 275.68 ± 14.79 0.433 ± 0.034 215.10 ± 6.43 32.08 ± 2.89

Data were represented as mean ± SD, n = 6

* Significantly (P \ 0.05) different from vehicle control mice

Table 1 Effect of different doses of VC-III on body weight, hepatic LPO, serum ALT, AST activities and BUN, creatinine levels in different

groups after 28 days in Swiss albino mice

Groups Body

weight (g)

LPO

(nmTBARS/mg protein)

ALT

(IU/ml)

AST

(IU/ml)

BUN

(mg/dl)

Creatinine

(lg/ml)

Vehicle control 27.75 ± 1.19 0.080 ± 0.008 43.74 ± 2.95 129.94 ± 7.75 20.98 ± 1.44 5.70 ± 0.56

VC-III at 1 mg/kg b.w. 28.71 ± 1.68 0.073 ± 0.005 43.66 ± 1.68 129.12 ± 8.10 18.01 ± 1.18* 5.60 ± 0.18

VC-III at 2 mg/kg b.w. 28.24 ± 2.39 0.079 ± 0.004 44.81 ± 2.31 131.62 ± 9.77 20.42 ± 1.74 5.67 ± 0.38

VC-III at 3 mg/kg b.w. 27.60 ± 1.44 0.094 ± 0.009* 46.85 ± 1.79 130.48 ± 6.90 20.87 ± 1.55 6.04 ± 0.41

VC-III at 5 mg/kg b.w. 26.92 ± 1.97 0.108 ± 0.009* 48.91 ± 3.08* 135.69 ± 11.08 22.21 ± 1.01 7.30 ± 0.63*

Data were represented as mean ± SD, n = 6

* Significantly (P \ 0.05) different from vehicle control mice

J Biol Inorg Chem

123

Measurement of hepatic ROS production

ROS measurement in liver tissue homogenate was done

following two simplified protocol with slight modification

using two probes, DHE [25, 26] and DCFH-DA [27]. DHE

is a nonfluorescent dye and freely permeable to the cell.

Upon oxidation by superoxide anions (O2-) forms the red

fluorescent product ethidium [28]. Liver tissues were

homogenized in HEPES buffer (pH 7.4, containing 25 mM

HEPES, 1 mM EDTA and 0.1 mM PMSF) to yield a 4 %

w/v homogenate. 150 ll of tissue homogenate in whole

was taken and then loaded with 10 lM DHE to make a

final volume of 3 ml. The samples were incubated in dark

for 30 min to allow the formation of ethidium and then

analyzed for fluorescence (excitation 475 nm/emission

610 nm) using spectrofluorimeter (Varian Cary Eclipse).

Values were expressed as fluorescence intensity per mg of

protein.

DCFH-DA is a nonfluorescent probe that is hydrolyzed

by mitochondrial esterase to form 20,70-dichlorodihydro-

fluorescein (DCFH). DCFH upon oxidation by H2O2 forms

the fluorescent compound 20,70-dichlorofluorescein (DCF)

[29]. Liver tissues were homogenized in Locke’s buffer

(pH 7.4, containing 140 mM NaCl, 5 mM KCl, 10 mM

HEPES, 1 mM CaCl2, 1 mM MgCl2 and 10 mM dextrose)

to yield a 10 % w/v homogenate. 250 ll of tissue

homogenate in whole was taken and then loaded with

10 lM DCFH-DA to make a final volume of 3 ml. The

samples were incubated in dark for 45 min to allow the

formation of DCF and then analyzed for fluorescence

(excitation 485 nm/emission 530 nm) using spectrofluo-

rimeter (Varian Cary Eclipse). Values were expressed as

fluorescence intensity per mg of protein.

Measurement of hepatic nitric oxide (NO) production

NO production in liver tissue homogenate was determined

by estimating the level of stable NO metabolites, viz.,

nitrate (NO3-) and nitrite (NO2

-) ions [30, 31]. The nitrate

content in tissue homogenate was reduced to nitrite by

VCl3. The reaction is followed by colorimetric detection of

nitrite as an azo dye product of the Griess Reaction. Liver

tissues were homogenized with phosphate buffer (pH 7.5,

containing 137 mM NaCl, 27 mM KCl, 10 mM Na2HPO4

and 1.8 mM KH2PO4) to yield a 20 % w/v homogenate.

The total nitrite level was determined by reaction with

Griess reagent (1 % sulphanilamide, 5 % phosphoric acid

and 0.1 % NEDD) using sodium nitrite as standard. The

absorbance was taken at 545 nm and expressed as lM

nitrite per mg of protein.

Estimation of lipid peroxidation (LPO) level

LPO level was estimated in liver microsomal fraction. The

level of lipid peroxides formed was measured using TBA

(0.8 % w/v) and expressed as nM of thiobarbituric acid

reactive substances (TBARS) formed per mg of protein

using an extinction coefficient of 1.56 9 105 M-1 cm-1 at

532 nm [32].

Estimation of reduced glutathione (GSH) and oxidized

glutathione (GSSG) level

Both GSH and GSSG levels were estimated from the same

liver cytosol spectrophotometrically. Reaction of GSH with

DTNB produced oxidized glutathione–TNB adducts. The

rate of formation of TNB measured at 412 nm was

Fig. 1 The basic experimental

treatment schedule. CP

cyclophosphamide, VC-III

vanadium(III)-L-cysteine

complex

J Biol Inorg Chem

123

proportional to the concentration of GSH in the sample.

The level of GSH was expressed as nM GSH per mg of

protein [33].

The GSSG reductase recycling method was used for

determination of the level of GSSG by monitoring NADPH

spectrophotometrically. The tissue homogenate was treated

with 2-vinylpyridine, which covalently reacts with GSH

(but not GSSG). The excess 2-vinylpyridine was neutral-

ized with triethanolamine. The level of GSSG was

expressed as nM GSSG per mg of protein [33].

Estimation of glutathione S-transferase (GST) activity

GST activity in the liver cytosol was determined from the

increase in absorbance at 340 nm with CDNB (30 mM) as

the substrate and specific activity of the enzyme was

expressed as formation of CDNB–GSH conjugate per min

per mg of protein [34].

Estimation of glutathione peroxidase (GPx) activity

GPx activity in liver cytosol was determined by NADPH

oxidation using a coupled reaction system consisting of

GSH, glutathione reductase, and H2O2 [35]. Briefly,

100 ll of enzyme sample was incubated for 10 min with

800 ll reaction mixture (0.25 M potassium phosphate

buffer containing 2.5 mM EDTA and 2.5 mM NaN3,

10 mM GSH, 2.5 mM NADPH and 2.4 units of gluta-

thione reductase). The reactions started on adding 100 ll

H2O2 and follow the decrease in NADPH absorbance at

340 nm for 3 min. The enzyme activity was expressed as

lM NADPH utilized per min per mg of protein using

extinction coefficient of NADPH at 340 nm as

6,200 M-1 cm-1.

Estimation of superoxide dismutase (SOD) activity

SOD activity in liver cytosol was determined by quantifi-

cation of pyrogallol auto-oxidation inhibition and expres-

sed as unit per mg of protein. One unit of enzyme activity

is defined as the amount of enzyme necessary for inhibiting

the reaction by 50 %. Auto-oxidation of pyrogallol

(20 mM) in Tris–HCl buffer (50 mM, pH 7.5) is measured

by increase in absorbance at 420 nm [36, 37].

Estimation of catalase (CAT) activity

CAT activity in liver cytosol was determined spectropho-

tometrically at 240 nm and expressed as unit per mg of

protein, where the unit is the amount of enzyme that lib-

erates half the peroxide oxygen from H2O2 in second at

25 �C [38].

Estimation of protein

Total protein content in tissue homogenate during bio-

chemical analysis was measured through Lowry method

using Folin-Phenol reagent [39]. The absorbance of the

color was measured against the colorless blank sample at

660 nm using the TECAN Infinite� 200 PRO Multimode

Reader.

Histopathological studies

Liver tissues were fixed in 10 % neutral buffered formalin

for 24 h. The tissue samples were dehydrated in ascending

concentrations of ethanol, cleared in xylene and embedded

in paraffin to prepare the blocks. Liver tissues were sec-

tioned at 5 lm, mounted on slides and stained with hae-

matoxylin–eosin. Stained sections were evaluated by

observing the arrangement of hepatic architecture with a

light microscope (Leica DM 1000) at 4009 magnification.

Photomicrographs were taken with the software Las EZ.

Apoptosis in the liver section were examined using the

terminal deoxynucleotidyl transferase (TdT)-mediated

dUTP nick end labeling (TUNEL) method with the help of

in situ cell death detection kit, according to the manufac-

turer’s instructions. The slides were analyzed under fluo-

rescence microscope (Leica DM 4000B) and

photomicrographs (Leica FW 4000) were taken at 4009

magnification [40].

Chromosomal aberration study

Mice were injected intraperitoneally with 0.03 % colchi-

cine (1 ml/100 g b.w.) 90 min before sacrifice. Marrow of

the femur was flushed in 1 % sodium citrate solution at

37 �C and fixed in acetic acid/ethanol (1:3). Slides were

prepared by the conventional flame drying technique [41]

followed by Giemsa staining for scoring bone marrow

chromosome aberrations. Chromosome aberrations of var-

ious natures like stretching, terminal association, break,

fragment, constriction, ring formation etc. were analyzed.

A total of 300 bone marrow cells were observed, 50 from

each of 6 mice of a set.

Determination of DNA fragmentation

by diphenylamine (DPA assay)

DNA fragmentation in bone marrow cells were carried out

according to Zhivotovsky et al. [42]. Briefly, about

5 9 106 bone marrow cells were lysed in lysis buffer (pH

8.0 containing 5 mM Tris–HCl, 20 mM EDTA and 0.5 %

Triton X-100) for 30 min at 4 �C. The cell lysate were

centrifuged at 15,000g for 15 min at 4 �C. Then, the

J Biol Inorg Chem

123

supernatant containing small DNA fragments was sepa-

rated from the pellet containing large pieces of DNA. The

supernatant and pellet were resuspended in 10 and 5 % of

trichloroacetic acid (TCA), respectively, and kept over-

night. Then both samples were heated at 100 �C for 15 min

and centrifuged at 2,500g for 5 min to remove proteins.

Supernatant fractions were reacted with diphenylamine

(DPA) for 4 h at 37 �C and the developing blue color was

measured at 600 nm. DNA fragmentation in samples was

expressed as percentage of total DNA appearing in the

supernatant fraction.

Fragmented DNA (%) ¼ Absorbance of the supernatant

Absorbance of supernatantþ pellet

� 100:

Immunocytochemical detection of apoptosis by terminal

deoxynucleotidyl transferase dUTP nick end labeling

(TUNEL) technique

Apoptosis of bone marrow cells were determined using the

terminal deoxynucleotidyl transferase (TdT)-mediated

dUTP nick end labeling (TUNEL) method with the help of

in situ cell death detection kit, according to the manufac-

turer’s instructions. The slides were analyzed under a

fluorescence microscope (Leica DM 4000B) and photo-

micrographs (Leica FW 4000) were taken at 2009 mag-

nification. The apoptotic cells were identified by green

fluorescence [40]. Randomly selected 80–100 cells from 5–

6 zones/slide were counted to determine the number of

apoptotic cells. The apoptotic index (AI) was determined

by following formula:

AI ð%) ¼ Number of labeled cells

Total number of cells counted� 100:

Detection of DNA damage by comet assay

Possible DNA damage induced by CP was detected using

the alkaline single-cell gel electrophoresis (Comet assay)

following a simplified protocol with slight modification

[43, 44]. The mice were sacrificed and blood was collected

from each mouse of all groups. Lymphocytes were isolated

from blood samples by standard centrifugation over a

cushion of Histopaque, washed with isotonic solution and

centrifuged. The pellet was resuspended in isotonic phos-

phate buffer saline solution (pH 7.4). The cell viability in

each group was measured and approximately 104 cells/slide

were taken for the assay. An aliquot of 10 ll of freshly

prepared single-cell suspension was mixed with 1 % low

melting point agarose and layered on the half frosted slides

precoated with normal melting agarose. A third layer of

0.5 % low melting point agarose was layered on the top of

the second layer. The cells were lysed for overnight at 4 �C

in lysing solution (pH 10, containing 2.5 M NaCl, 100 mM

EDTA, 10 mM Tris buffer, 10 % DMSO, 1 % Triton X-

100). After lysis, the slides were subjected to electropho-

resis in electrophoresis buffer (pH 13.1, containing 1 mM

EDTA, 0.3 M NaOH) for 30 min. After electrophoresis,

the slides were neutralized with neutralizing buffer (pH

7.5, containing 0.4 M Tris buffer). The microscopic slides

were carefully dried at room temperature and stained with

ethidium bromide in water (20 lg/ml; 80 ll/slide). The

slides were examined at 4009 magnification under a

fluorescence microscope (Leica DM 4000B) with imaging

system. Komet 5.5 software was used to take the photo-

micrograph of cells and to analyze various parameters of

the comet. 50–100 randomly cells in each slide were ana-

lyzed (2 slides/animals in each group). Cell that formed

distinct comet tail was identified as damaged cell. The

damaged cell (%) was calculated using the following

formula:

Damaged cell ð%)¼ Number of damged cells

Total number of cells counted� 100:

The parameters analyzed for the lymphocytes were

damaged cell (%) in each group, head DNA (%), tail DNA

(%), tail length [migration of the DNA from the nucleus

(lm)] and Olive tail moment [product of tail length and the

fraction of total DNA in the tail (arbitrary units)].

Statistical analysis

All data were presented as mean ± SD, n = 6 mice per

group. One-way ANOVA followed by Dunnett’s Multiple

Comparison Test using GraphPad Prism (version 5.0) was

performed for comparisons among groups. Significant

difference was indicated when the P value was \0.05.

Results

Synthesis of VC-III

As indicated in Fig. S1, the organovanadium complex, VC-

III was synthesized in one step. After filtration and washing

by diethyl ether the compound was obtained in pure form

with an overall yield of 48.8 %. The compound was

characterized by infrared (IR), UV–visible and electron

paramagnetic resonance (EPR) spectroscopy. The IR

spectrum showed a medium intensity broad band at

3,408 cm-1, which was designated to v(OH) arising from

the lattice water molecules [20]. The S–H stretching

vibration developed a weak shoulder at around 2,600 cm,

and this indicated that the S–H proton is probably involved

in H-bonds with oxygen atoms in water. The vas(COO-)

and vs(COO-) bands appeared at 1,621 and 1,446 cm-1,

respectively, which is an indication of a monodentate

J Biol Inorg Chem

123

carboxylate coordination [20, 45]. The vV–O and vV–N

bands were observed at 588 and 455 cm-1 confirming the

complex formation of the compound [46] (Fig. S2a). The

UV–visible spectrum showed a distinct band at 432 nm,

which is the characteristic absorption band of this com-

pound [20] (Fig. S2b). In EPR spectra, VC-III exhibited an

axial spectrum in solid form. This was because of that the

V3? is EPR silent. V3? does not have any unpaired electron

in its outer orbital, which is the origin of an EPR signal. So

the spectrum confirmed that the investigated compound

VC-III has a d2 electronic structure V3? [47] (Fig. S2c).

Stability of VC-III

The absorption spectra of VC-III at different time points

were shown in Fig. 2. From the spectrum, it is clear that

VC-III was relatively stable in aqueous buffer solution of

physiological pH and temperature. However, slow pro-

gressive decrease of intensity of the characteristic band was

observed during 24 h.

Dose selection

Effective dose of VC-III was determined on the basis of

some safety (Table 1) and efficacy (Table 2) endpoints.

Treatment with VC-III at 3 mg/kg b.w. caused significant

(P \ 0.05) increase in hepatic LPO level whereas treat-

ment with VC-III at 5 mg/kg b.w caused significant

(P \ 0.05) increase in hepatic LPO level, serum ALT

activity and serum creatinine level. These findings rendered

these two doses to be toxic. But treatment with VC-III at

1 mg/kg b.w. had no adverse effects on liver and kidney

function markers. On the other hand, among the four doses

VC-III at 1 mg/kg b.w. showed most profound antioxidant

efficacy indicated by its significant (P \ 0.05) increase in

GSH level along with numerical (P [ 0.05) increase in

activities of other antioxidant enzymes. So, the dose of

1 mg/kg b.w. p.o. was selected for rest of the study.

Modulation of serum ALT, AST and ALP activity

Organovanadium compound, VC-III did not produce any

significant change in these parameters when administered

alone as compared to the Group I. The liver was severely

damaged by CP administration, as indicated by considerable

increase in serum ALT, AST and ALP activities (Fig. 3a–c).

Serum ALT, AST and ALP activities were significantly

(P \ 0.05) enhanced by 2.37-fold, 1.88-fold and 1.56-fold,

respectively, in Group III compared to Group I. Treatment

with the ligand L-cysteine could not provide any protection

against CP-induced hepatic damage. Concomitant treatment

with organovanadium compound significantly (P \ 0.05)

reduced the increased activities of ALT, AST and ALP by

1.57-fold, 1.58-fold and 1.32-fold, respectively, in compari-

son to Group III. Administration of VC-III in pretreatment

schedule reduced ALT, AST and ALP activities by 1.83-fold,

1.77-fold and 1.45-fold, respectively, compared to Group III.

Attenuation of ROS level

ROS level in liver tissue homogenate was estimated by

using DHE and DCFH-DA to quantify the level of super-

oxide and H2O2, respectively. Administration of CP

resulted in a significant (P \ 0.05) increase in ROS level

by 1.78-fold and 2.64-fold in liver of Group III mice in

comparison to the vehicle control group (Fig. 4a, b) when

estimated by DHE and DCFH-DA, respectively. Con-

comitant treatment with VC-III resulted in significant

(P \ 0.05) reduction in ROS level by 1.26-fold and 1.46-

fold in comparison to the CP-treated group when quantified

by DHE and DCFH-DA, respectively. In case of pretreat-

ment group the reduction in ROS level was found to be 1.4-

fold and 1.95-fold compared to Group III mice upon esti-

mation by DHE and DCFH-DA, respectively. However,

administration of L-cysteine in Group IV failed to reduce

(P [ 0.05) hepatic ROS level.

Reduction of NO level

The level of NO metabolites in liver of CP-treated mice

increased (P \ 0.05) significantly by 1.95-fold as com-

pared to Group I mice (Fig. 4c). Concomitant treatment

and 7 days pretreatment with the vanadium compound

significantly (P \ 0.05) reduced the elevated level of NO

by 1.32- and 1.46-fold, respectively.

Inhibition of LPO level

Intraperitoneal administration of CP significantly

(P \ 0.05) elevated the hepatic LPO level by 2.06-fold in

Fig. 2 Stability study of VC-III in PBS measured at various time

point up to 24 h. [VC-III] = 20 mM, pH = 7.4, T = 37 �C

J Biol Inorg Chem

123

Group III mice compared to Group I (Fig. 4d). The test

compound inhibited the hepatic LPO level by 1.42-fold in

case of concomitant treatment group and by 1.73-fold in

case of pretreatment group compared to Group III.

Increase of GSH level

GSH content in liver depleted significantly (P \ 0.05) by

2.06-fold after CP administration in Group III animals

compared to Group I (Fig. 5a). Concomitant treatment and

pretreatment with VC-III increased GSH level in hepatic

tissues by 1.45- and 1.64-fold, respectively, in comparison

to Group III.

Reduction of GSSG level

The level of GSSG in liver of CP-treated mice increased

(P \ 0.05) significantly by 1.96-fold as compared to Group

I mice (Fig. 5b). Concomitant treatment and 7 days pre-

treatment with the vanadium compound significantly

(P \ 0.05) reduced the elevated level of GSSG by 1.44-

fold and 1.57-fold, respectively.

Enhancement of GST activity

The activity of GST in liver of mice treated with CP

showed a significant (P \ 0.05) decrease by 1.51-fold

Fig. 3 VC-III reduced hepatotoxicity in mice after CP administration

as evident from a ALT activity, b AST activity and c ALP activity in

mice serum. Data were represented as mean ± SD, n = 6.

$Significantly (P \ 0.05) different from Group I and #significantly

(P \ 0.05) different from Group III

Fig. 4 VC-III inhibited a H2O2

level, b superoxide level, c NO

level and d LPO level in mice

liver after CP administration.

Data were represented as

mean ± SD, n = 6.$Significantly (P \ 0.05)

different from Group I and#significantly (P \ 0.05)

different from Group III

J Biol Inorg Chem

123

compared to vehicle control group (Group I) (Fig. 5c).

GST activity elevated significantly (P \ 0.05) by 1.44- and

1.49-fold in case of concomitant treatment and pretreat-

ment with VC-III, respectively.

Elevation of GPx activity

Intraperitoneal administration of CP significantly

(P \ 0.05) reduced the GPx activity by 1.62-fold in Group

III compared to Group I (Fig. 5d). Organovanadium com-

pound significantly enhanced the GPx activity by 1.29- and

1.42-fold in case of concomitant treatment group and

pretreatment group, respectively, compared to Group III.

Amelioration of SOD activity

CP administration caused a significant (P \ 0.05) reduction

in hepatic SOD activity by 1.74-fold in Group III compared

to Group I (Fig. 5e). Treatment with VC-III resulted in

significant (P \ 0.05) rise in hepatic SOD activity by 1.46-

fold and 1.55-fold in concomitant and pretreatment sche-

dule, respectively, in comparison to Group III.

Increase of CAT activity

CAT activity in liver was decreased significantly

(P \ 0.05) by 2.02-fold in Group III following

administration of CP in comparison to Group I (Fig. 5f).

Concomitant treatment and pretreatment with test com-

pound increased the CAT activity by 1.37- and 1.65-fold,

respectively, in comparison to Group III.

Histopathological examination

The microphotograph of liver section of vehicle control

group showed a normal architecture of hepatocytes

(Fig. 6a). Liver section of only VC-III-treated group also

showed normal hepatocyte structure (Fig. 6b). But CP

treatment resulted in marked diffuse swelling of hepato-

cytes, congestion of blood vessels accompanied by leu-

kocyte infiltration (Fig. 6c, d). The liver section of CP-

treated mice also showed dilatation of central vein and

portal vein along with disruption of wall of the vein.

Treatment with L-cysteine could not ameliorate CP-

induced hepatic damages (Fig. 6e). Concomitant treatment

with organovanadium compound effectively attenuated

CP-induced pathological changes as characterized by mild

swelling of hepatocytes, minute central vein dilatation and

no portal vein dilatation (Fig. 6f, g). In case of pretreat-

ment group, there are minimal central vein dilatation, no

portal vein dilatation and quite normal hepatocytes

architecture (Fig. 6h, i).

Immunohistochemical staining of liver sections with

TdT and fluorescein-dUTP showed null or very rare

Fig. 5 VC-III restored a GSH level, b GSSG level, c GST activity,

d GPx activity, e SOD activity and f CAT activity in mice liver after

CP administration. Data were represented as mean ± SD, n = 6.

$Significantly (P \ 0.05) different from Group I and #significantly

(P \ 0.05) different from Group III

J Biol Inorg Chem

123

presence of TUNEL-positive hepatocytes in liver sections

of different groups (Fig. 7a–e).

Prevention of CP-induced chromosomal aberrations

Animals treated with CP showed significantly (P \ 0.05)

high proportion of chromosomal aberrations of 46.83 %

(Fig. 8a–c) compared to relatively low frequency of chro-

mosomal aberrations of 11.52 % in vehicle control group.

The frequency of chromosomal aberrations was 31.29 and

26.47 %, respectively, in case of concomitant and pre-

treatment group, which were significantly (P \ 0.05) much

lower compared to the CP-treated group.

Attenuation of CP-induced DNA fragmentation

Genomic DNA fragmentation in bone marrow cells was

found 10.71 % in Group I (Fig. 8d). CP administration

caused a significantly (P \ 0.05) greater rate (52.74 %) of

DNA fragmentation in Group III mice. In case of con-

comitant and pretreatment, the percentages of DNA frag-

mentation were reduced to 36.69 % and 27.68 %,

respectively.

Reduction of CP-induced apoptosis

AI in the bone marrow cells of vehicle control group was

found to be 5.12 % (Fig. 8e, 9a), which was increased

markedly due to cytotoxic effect of CP in Group III to

38.93 % (Fig. 8e, 9c). The test compound significantly

(P \ 0.05) inhibited CP-induced apoptotic cell death to

26.29 % in concomitant treatment schedule and to 19.47 %

in pretreatment schedule (Fig. 8e, 9e, f).

Protection from CP-induced DNA damage

Comet assay was carried out to examine DNA damage in

lymphocytes (Fig. 10a–f) and for this purpose damaged

Fig. 6 Photomicrographs of liver section of mice of different groups,

H&E, 9400 magnification. a Normal architecture of hepatocytes

radiating from central vein (brown star) in Group I, b liver histology

showing normal structure of hepatocytes and sinusoids in Group II

c diffused swelling of hepatocytes, central vein (brown star) dilation

and vein wall disruption in Group III, d portal vein (green star)

dilatation and leukocyte infiltration (blue arrow) in Group III,

e marked central vein (brown star) dilatation and hepatocyte swelling

in Group IV, f minute swelling of hepatocytes and minimal central

vein (brown star) dilatation in Group V, g no portal vein (green star)

dilatation found in Group V. h nominal central vein (brown star)

dilatation and normal hepatocyte architecture in Group VI, i portal

vein (green star) dilatation not found in Group VI

J Biol Inorg Chem

123

cell (%), head DNA (%), tail DNA (%), average tail length

(lm) and Olive tail moment were analyzed (Fig. 11a–e).

Large round head and no tail was observed in the lym-

phocytes of Group I mice. But CP treatment resulted in

long comet tail formation due to DNA damage in large

number of cell population. DNA with diffused head and

scattered tail was also observed. Concomitant and pre-

treatment with oraganovanadium compound significantly

Fig. 7 Photomicrographs of TUNEL assay performed in the liver sections of different groups, 9400 magnification. No TUNEL-positive

hepatocytes were found in a Group I, b Group II, c Group III, d Group IV, e Group V and f Group VI

Fig. 8 Metaphase complements

of bone marrow cells showing

a stretching (STR), terminal

association (TA); b constriction

(CON), break (B), acentric

fragment (AF), and ring

formation (R); 91,000

magnification. VC-III

attenuated CP induced

c chromosomal aberration,

d DNA fragmentation, and

e apoptotic index in mice bone

marrow cells after CP

administration. Data were

represented as mean ± SD,

n = 6. $Significantly (P \ 0.05)

different from Group I and#significantly (P \ 0.05)

different from Group III

J Biol Inorg Chem

123

reduced % of damaged cells, comet tail length and Olive

tail moment. Also VC-III administration normalized head

DNA–tail DNA ratio in lymphocytes. But treatment with L-

cysteine could not protect lymphocytes against CP-induced

DNA damage.

Discussion

In the present study, one vanadium complex with amino

acid L-cysteine, as a ligand, was synthesized. Generally, the

toxicity of vanadium compounds increases as the oxidation

state increases [48]. Keeping this in mind, an organova-

nadium complex in reduced oxidation state, i.e., ?3, was

synthesized. An available strategy for improving biological

functionality of vanadium compounds is to include sulphur

coordination for potentially useful enzyme binding in vivo

[49]. The ligand L-cysteine was chosen to enhance the

absorption and enzyme-binding affinity of the complex.

Inorganic vanadium compounds in ?3 state are very

unstable due to its rapid oxidation to ?4 state. So, the

reaction was carried out under nitrogen environment in

dark condition. The stability of the compound was deter-

mined in isotonic physiological buffer at physiological pH

and temperature. The result of the stability study showed

that the vanadium complex was reasonably stable in

physiological buffer. The results were in concordance with

previously reported literatures [20, 50]. To study the effect

of the vanadium compound on Swiss albino mice one

group of mice (Group II) was fed only with VC-III at a

dose of 1 mg/kg b.w. No significant changes were observed

in the antioxidant, genotoxic and cytotoxic markers in this

group compared to Group I mice.

The most critical obstacle in cancer chemotherapy is the

non-specific cytotoxic effect on both tumor and healthy

normal cells [51]. CP, a bifunctional alkylating agent, is

extensively prescribed as a treatment regimen in a number

of leukemic and lymphoproliferative disorders, certain

solid tumors and many autoimmune diseases [52]. The

wide use of CP among other alkylating agents for treatment

of malignancies is influenced by several factors; (1) being a

bifunctional agent, CP can cause interstand cross-linkages

in DNA. As a result, cancer cells require more complex

mechanisms to repair, so develop less resistance to CP

[53]. This is an advantage of CP over monofunctional

alkylating agents; (2) apart from DNA alkylation, CP can

induce caspase 9-dependent apoptosis in tumor cells [54].

This is a unique feature of CP among other alkylating

agents; (3) CP also possesses anti-angiogenic property and

can cause damage to the vasculature of the tumor [55]; (4)

the most important point is that CP is a very economical

drug compared to new-generation alkylating agents. In

lower-middle-income countries and low-income countries,

a large number of patients receive this drug as the first line

of treatment.

The primary requirement for pharmacological and tox-

icological effects of CP is bioactivation by hepatic

microsomal cytochrome P450 mixed function oxidase

system. Metabolic activation through several pathways

ultimately forms the DNA-crosslinking agent, phosphora-

mide mustard and an equimolar amount of toxic byproduct,

acrolein [56]. Biotransformation of CP to these metabolites

Fig. 9 Photomicrographs of TUNEL assay performed in bone

marrow cells of different groups, 9200 magnification. a No apoptosis

found in Group I, b no apoptotic cell death in Group II, c high

proportion of TUNEL label cells found in Group III, d high rate of

apoptotic death in Group IV, e lower proportion of apoptosis in Group

V, f very low rate of apoptosis in Group VI

J Biol Inorg Chem

123

leads to the formation of high level of free radicals [5] and

compromised antioxidative state in liver cells [7]. Any

pathological state that leads to increased production and/or

ineffective scavenging of ROS may advance to tissue

injury [57].

CP induces hepatic injury, as a consequence of which

the hepatotoxicity marker enzymes (ALT, AST and ALP)

leach out of hepatocytes leading to their increased activity

in the systemic circulation [58]. In the present study, mice

treated with CP displayed a significant increase in activities

of ALT, AST and ALP in serum, which obviously indi-

cated damage in the morphological integrity of liver [59].

Supplementation with VC-III significantly decreased the

activities of these enzymes in serum, as compared to only

CP-treated animals. This observation suggested the hepa-

toprotective role of VC-III against CP-induced liver

damage.

Previously, the prooxidant nature of CP was discussed.

In this study, the ROS level in liver tissue was measured

spectrofluorometrically by the use of DHE and DCFH-DA.

The oxidation of DHE to fluorescent compound ethidium is

mainly modulated by superoxide anion [28], whereas the

conversion of DCFH-DA to fluorescent DCF is mainly

regulated by H2O2 [29]. In this study, CP intoxication

substantially elevated superoxide and H2O2 levels in liver

tissue of mice as measured by DHE and DCFH-DA,

respectively. Organovanadium complex VC-III prevented

CP-induced ROS generation which could be attributed to

free radical scavenging activity of the test compound.

NO is a short lived and highly reactive free radical.

Nitrite is a stable metabolite of NO and can be used as an

indicator of the overall formation of NO in vivo [60]. The

hepatic tissue of CP-treated mice showed significant rise in

NO2- level. Treatment with VC-III significantly reduced

hepatic nitrite level suggesting protective role of the

compound against nitrosative stress induced by CP.

LPO is one of the major markers of oxidative damage

initiated by ROS and its effect has been linked to the

altered membrane structure and enzyme inactivation [61].

Corroborating with the previous finding of raised ROS and

RNS level, administration of CP resulted in a significant

increase in LPO level (measured by the level of TBARS) in

liver. The organovanadium compound VC-III-treated mice

showed significant reduction in TBARS level which is in

line with its protective role against free radicals found in

this study. The protective efficacy of VC-III against CP-

induced free radical generation was found to be more

pronounced in the pretreated group.

In the present study, CP-induced alteration of cellular

antioxidant and detoxification status was also observed.

GSH plays an important role in protecting cells from oxi-

dative injury via regulation of various cellular functions

[62]. Within the cells, GSH exists in two different forms:

the reduced sulfhydryl form (GSH) and the oxidized glu-

tathione disulfide (GSSG) form [33]. In this present study,

the observed significant depletion in GSH level after CP

exposure might be attributed to the direct conjugation of

CP and its metabolites with free or protein bound -SH

groups [63]. Parallel to this change, CP also caused sig-

nificant increase in GSSG level owing to ROS-mediated

oxidation of GSH in liver; thereby minimizing cellular

antioxidative functions. Treatment with VC-III restored the

GSH and GSSG level and normalized the condition. GST

detoxifies the electrophilic species including the toxic

metabolites of CP via a spontaneous enzyme-catalyzed

Fig. 10 Representative photomicrographs of DNA damage in mice

lymphocytes, 9400 magnification. a no significant DNA damage in

Group I, b no significant DNA migration in Group II, c highly

damaged DNA with scattered tail migration in Group III, d higher

value of tail length with less percentage of DNA in the head and

greater in the tail in Group IV e less migration of DNA and short tail

length in Group V, f very less migration of DNA in Group VI

J Biol Inorg Chem

123

conjugation reaction to protect the cells against peroxida-

tive damage. The decreased activity of GST in CP-treated

group observed in this study may be partly due to the lack

of its substrate (GSH) and also because of oxidative

modification of its protein structure [64]. VC-III adminis-

tration also enhanced the GST activity which further con-

tributed to its hepatoprotective efficacy.

Cellular antioxidant defences involving the enzymes

SOD, CAT and GPx which detoxify the oxygen free radi-

cals and the consequent oxidative burst. SOD catalyzes

dismutation of the superoxide anion to H2O2 and O2. The

harmful H2O2 is further detoxified to water by CAT and

GPx [65]. Therefore, the activities of these antioxidant

enzymes confer a vital protection against oxidative stress.

In our study, the activities of the antioxidant enzymes

SOD, CAT and GPx, in the mice liver were significantly

reduced by CP administration indicating pronounced oxi-

dative stress. SOD activity was drastically reduced which

was corroborated with elevated superoxide anion level in

the liver of CP-treated mice. On the other hand, depletion

of the activities of CAT and GPx resulted in enhanced level

of H2O2 found in this study. Administration of VC-III

significantly elevated SOD, CAT and GPx activities, sug-

gesting that it had the ability to restore the activities of

antioxidant enzymes.

The toxic effect of CP was ascertained by the assess-

ment of histopathological alterations of liver. Histological

study revealed diffused swelling of hepatocytes, leukocyte

infiltration along with central vein and portal vein dilata-

tion after CP treatment. These pathological changes were

resulted from CP-induced free radical generation and

diminished antioxidant status. However, CP did not induce

apoptosis in mice liver as evident from TUNEL assay. The

elevation of ALT, AST and ALP level in mice serum might

be due to CP-induced damage in cell membrane structure

via lipid peroxidation. In comparison, liver sections of

vanadium-treated mice showed significant attenuation of

CP-induced pathological changes attributed to its antioxi-

dant and cytoprotective capacity.

Genotoxicity is another severe toxic effect induced by

CP which leads to mutagenicity (IARC), teratogenicity

[11] and carcinogenicity [10]. Phosphoramide mustard, the

active metabolite of CP forms DNA crosslinks which lead

to DNA strand breaks and subsequently to chromosomal

breaks [66]. Acrolein, the other metabolite interferes with

tissue antioxidant defence mechanism, through producing

highly reactive oxygen free radicals that further react with

DNA causing its damage [67]. Hence, the genoprotective

efficacy of the test compound against CP-induced genetic

damage was evaluated.

CP affects the cell populations that typically exhibit

rapid cell turnover, such as bone marrow cells [53]. In this

study chromosomal aberration, percentage DNA fragmen-

tation assay and TUNEL assay were performed in murine

bone marrow cells. The percentage of chromosomal aber-

rations and extent of DNA damage in bone marrow cells

Fig. 11 VC-III prevented CP-induced DNA damage as evident from

a damaged cell (%), b head DNA (%), c tail DNA (%), d comet tail

length and e Olive tail moment. Data were represented as

mean ± SD, n = 6. $Significantly (P \ 0.05) different from Group

I and #significantly (P \ 0.05) different from Group III

J Biol Inorg Chem

123

were increased due to CP administration. The common

types of aberration observed in this study were chromatid

breaks, chromosomal stretching and terminal association.

Treatment with the test compound reduced the percentage

of chromosomal aberrant cells and percentage of DNA

fragmentation in bone marrow cells. DNA fragmentation is

generally resulted in apoptotic cell death, if not repaired

[68]. TUNEL assay has been designed to detect apoptotic

cells that undergo extensive DNA degradation during the

late stages of apoptosis [69]. Here, we observed that

administration of CP caused a significant proportion of

TUNEL-positive bone marrow cells in Group III mice.

Treatment with the vanadium compound was able to

diminish this apoptotic cell death and conferred cytopro-

tection to the host.

The extent of DNA damage was estimated by comet

assay in murine lymphocytes considering lymphocytes as a

representation of surrogate cells [70]. In the present study,

the extent of DNA damage was significantly increased in

lymphocytes of mice treated with CP. Supplementation

with VC-III substantially reduced CP-induced DNA dam-

age in murine lymphocytes. The antigenotoxic potential of

the vanadium compound may be partly resulted from its

ability to inhibit CP-induced ROS generation. In addition,

vanadium-mediated stimulation of the DNA repair mech-

anism could possibly take place [71].

To observe the effect of the complexation, the ligand L-

cysteine was given to CP-treated mice concomitantly

(Group IV). The dose of L-cysteine was calculated

according to the L-cysteine content in the organovanadium

complex. However, L-cysteine alone could not able to give

any protection against CP-induced hepatic and genetic

damages. This proves the specificity of vanadium in pro-

viding the protective efficacy towards CP-induced

damages.

The present study showed that the test compound VC-III

quite adeptly reduced the hepatotoxicity and genotoxicity

induced by subsequent CP injection. The protective effi-

cacy conferred by VC-III may be because of the—(1)

direct inhibition of ROS and RNS like H2O2, superoxide

and NO; (2) significant reduction in the lipid peroxidation

level; (3) enhancement of the activities of various antiox-

idant and detoxifying enzyme systems like SOD, CAT,

GPx and GST which, in turn, detoxify free radicals pro-

duced by CP metabolites. Furthermore, the role of the

vanadium compound in the regulation of GSH and GSSG

might be of significant value in the restoration of redox

homeostasis of the host and minimization of cellular

damage due to ROS. The results furnished in this study

may provide a beneficial therapeutic use of vanadium

compounds as an adjunct in cancer chemotherapy to defend

the toxic side effects of anticancer drugs.

Acknowledgments This work was supported by Grant from Indian

Council of Medical Research (ICMR) (no. 3/2/2/58/2011/NCD-III),

New Delhi, India. Abhishek Basu gratefully acknowledges ICMR for

Senior Research Fellowship. Arin Bhattacharjee also gratefully

acknowledges ICMR for Senior Research Fellowship (no. 45/36/

2008/PHA-BMS).

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