Evaluation of the anticancer properties of the predicted hBaxBH3-mimetic compound 2-hydroxy-3,5-dinitrobenzamide in a mammary carcinogenesis-induced rat model

Article abstract of DOI:10.1039/c5ra23005e

Designing small molecular prototypes having potential to disrupt binding interfaces of pro-apoptotic-anti-apoptotic/BH3-only proteins is a promising strategy in cancer chemotherapy. Several natural and synthetic chemical compounds have been reported as in

Full text of DOI:10.1039/c5ra23005e

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DOI: 10.1039/C5RA23005E.
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Evaluation of the anticancer properties of the predicted hBaxBH3-mimetic compound
-hydroxy-3,5-dinitrobenzamide in a mammary carcinogenesis-induced rat model
2
a
b
Dakshinamurthy Sivakumar , Krishna Mohan Surapaneni , Ponnachipudhur Chinnaswamy
c
d
d
e
Prabu , Natarajan Hari , Ponnusamy Thiruvasagam , Muthu Rajasekaran and
a
Thirunavukkarasu Sivaraman *
a
Structural Biology Lab, Department of Bioinformatics, School of Chemical and Biotechnology,
SASTRA University, Thanjavur – 613 401, Tamil Nadu, India.
b
Department of Biochemistry, Saveetha Medical College & Hospital, Saveetha University,
Chennai, Tamil Nadu, India.
c
Central Animal Facility, SASTRA University, Thanjavur – 613 401, Tamil Nadu, India.
d
e
Department of Chemistry, Department of Biotechnology, School of Chemical and
Biotechnology, SASTRA University, Thanjavur – 613 401, Tamil Nadu, India.
*To whom correspondence should be addressed.
Tel: +91 4362 264101 Ext.2319
Fax: +91 4362 264120
Eꢀmail: sivaram@scbt.sastra.edu

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Abstract
Designing small molecular prototypes having potential to disrupt binding interfaces of
proꢀapoptotic – antiꢀapoptotic/BH3ꢀonly proteins is a promising strategy in cancer
chemotherapy. Several natural and synthetic chemical compounds have been reported as
inhibitors or BH3ꢀmimetics to antiꢀapoptotic proteins of the Bclꢀ2 family playing essential roles
in apoptosis. In the present study, chemical synthesis, structural characterizations and antiꢀcancer
effect of 2ꢀhydroxyꢀ3,5ꢀdinitrobenzamide (HDNB), a chemical compound identified on the basis
of pharmacophoric features of hBaxBH3 peptide have been systematically reported.
Chemopreventive effect of the HDNB was assessed on NꢀNitrosoꢀNꢀmethylureaꢀinduced Wistar
female rats of mammary gland carcinogenesis for the study period of 18 weeks. The
carcinogenesis Wistar rats were subjected to oral administration of lowꢀdose (8 mg/kg b.w.),
mediumꢀdose (40 mg/kg b.w.) and highꢀdose (200 mg/kg b.w.) of the HDNB in three different
experimental setups. Strikingly, mediumꢀdose (40 mg/kg b.w.) of the HDNB showed appreciable
potential to reverse the abnormalities of various biochemical parameters in blood samples and as
well in breast tissues to the values as close as to that of healthy control or to that of standard
(Tamoxifen) control. The chemopreventive potential of the HDNB could be chiefly attributed to
its ability of restoring antioxidants and as well apoptosis inducing properties in Wistar animal
models.
Key words: Acute toxicities; antiꢀcancer potential; apoptotic proteins; breast cancer; chemo
preventive; HDNB.

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1
. Introduction
Cancer is caused by uncontrolled cell proliferations and as well by abnormally increased
1
cell life due to defects in apoptosis. The two processes are governed by quite a large number of
parallel signaling pathways, which posits great challenges on designing targetꢀbased small
2
, 3
molecular therapeutic agents to the treatments of cancers.
Fortunately, many anticancer
compounds have been shown to regulate apoptotic machinery in one way or other in order to
4
, 5
combat cancerous cells.
In these contexts, designing lead compounds depicting desirable
effects by interfering functions of Bclꢀ2 family proteins has become an attractive strategy in
6
cancer chemotherapy. Though it is well documented that Bclꢀ2 family proteins tightly govern
the apoptotic process by intrinsic or mitochondrial pathway, the mechanism by which the
proteins communicate each other and modulate the process are under debate between two key
7
ꢀ9
models: direct activation model and indirect model. The ‘direct activation model’ proposes
that proꢀapoptotic proteins (Bax and Bak) will be activated by a set of BH3ꢀonly proteins (Bim,
Bid and Puma), which may be abnormally sequestered by antiꢀapoptotic proteins (Bclꢀ2, BclꢀB,
BclꢀW, BclꢀXL, Bflꢀ1 and Mclꢀ1) in cancerous cells, whereas ‘indirect model’ proposes that
functions of proꢀapoptotic proteins will be blocked by antiꢀapoptotic proteins through heteroꢀ
complex formations between the two groups of the Bclꢀ2 family. Interestingly, both models
suggest that chemical molecules that are capable of acting as antagonists to the antiꢀapoptotic
proteins can act as efficient lead antiꢀcancer compounds, in general.
Impairment in apoptosis is one of hallmarks of cancer diseases and successively
overexpression of antiꢀapoptotic proteins (either all or a few of them) have been documented in
1
0
most types of cancers, if not all. To date, six types of antiꢀapoptotic proteins have been well
characterized in mammalian cells and all the proteins have similar threeꢀdimensional (3D)

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architectures. However, biological activities and as well specificities of the antiꢀapoptotic
proteins to interact with various BH3ꢀonly proteins are not identical to each other. Many natural
products (antimycin A, chelerythrine, obatoclax, purpurogallin and gossypol) and synthetic
chemical compounds (HA14ꢀ1, BH3Iꢀ1, isooxazolidine derivatives, gossypol derivatives, ABTꢀ
737 and ABTꢀ263) have been reported as inhibitors to the antiꢀapoptotic proteins and compounds
such as ABTꢀ263, obatoclax, gossypol and isooxazolidine are right now under various levels of
4
clinical trials. Interestingly, of the many molecules mentioned above, the ABTꢀ737 and ABTꢀ
263 have only been authenticated as true antagonists/BH3ꢀmimetics to the antiꢀapoptotic proteins
as cytotoxic activities of the compounds could be well correlated with cellular concentrations of
4
, 11, 12
the antiꢀapoptotic proteins.
/BclꢀXL have also been well studied at atomic level resolutions. However, the two
compounds were found to be weak antagonists to Mclꢀ1 and as well the compounds were
Moreover, structural interactions of the compounds with Bclꢀ
11
2
1
3
inactive against cancerous cells overꢀexpressing the antiꢀapoptotic protein, Mclꢀ1. Ostensibly,
designing/identifying specific antagonists to each antiꢀapoptotic protein and likewise common
antagonists showing high affinities to all of the 6 antiꢀapoptotic proteins have been yet
challenging tasks in cancer chemotherapy.
We have recently reported 2ꢀhydroxyꢀ3,5ꢀdinitrobenzamide (HDNB) as a lead inhibitor
1
4
to human BclꢀB (hBclꢀB) by means of peptidodynmimetic method designed in our laboratory.
The method is useful to define pharmacophoric features of small peptides bound with large
protein molecules under dynamic conditions. Using the peptidodynmimetic method,
pharmacophoric residues of human BaxꢀBH3 (hBaxꢀBH3) peptide bound with hBclꢀB were
mapped out and the HDNB was identified as novel lead inhibitor to the hBclꢀB upon a database
screening followed by high throughput virtual screening on the basis of the pharmacophoric

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residues of the hBaxꢀBH3. Interestingly, the lead compound (HDNB) also showed comparable
binding affinities on the BH3ꢀbinding grooves of all the antiꢀapoptotic proteins but with BclꢀW,
appreciable bioavailability and desirable pharmacokinetic properties as predicted by using an
1
4, 15
array of computational strategies.
In the present study, we report chemical synthesis,
structural characterizations (using ultraviolet visible (UV) spectroscopy, Fourier transform
infrared (FTꢀIR) spectroscopy, Gas chromatography ꢀ mass spectrometry (GCꢀMS) and proton
1
13
15
(
H), carbon ( C) and nitrogen ( N) nuclear magnetic resonance (NMR) spectroscopy) and
chemopreventive potential of the HDNB in NꢀNitrosoꢀNꢀmethylurea (NMU) ꢀ induced Wistar
mammary gland carcinogenesis in detail. Comprehensive analyses of data obtained from the
experiments suggested that the HDNB is a promising lead depicting antiꢀcancer potential and the
HDNB would also be perhaps used as a seed molecule to develop highly efficient de novo
antagonists to the antiꢀapoptotic proteins.
2
. Results and Discussion
Understanding relationships between small bioꢀmolecules and macromolecules
synthesized by cells is crucial to make ‘central dogma’ of life to be entirely descriptive one.
Natural and nonꢀnatural small chemical molecules are also key elements, powerful probes and
1
6, 17
drugs in biological systems.
In these connections, designing small molecules having
potential especially to disrupt protein – protein interfaces has intrigued researchers in the past
decade. However, designing/identifying chemical molecules binding to proteinꢀprotein interfaces
1
8, 19
is an uphill task as surface areas of the interfaces are large and flat, in general.
Interestingly,
hotspots (subset of residues contributing most of the free energy of binding) of proteins
constituting interfaces could be mapped out by using siteꢀdirected mutational studies as
2
0
demonstrated for several proteins. The hotspots of the proteins could be used as ‘footholds’ for

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designing of small molecules interacting on the interfaces of protein complexes. Computational
methods such as fragmentꢀbased lead designing and peptidomimetics have also been shown as
2
1
promising strategies for designing leads to interact on proteinꢀprotein interfaces. In these series
of in silico tools, a recent peptidodynmimetic method has also been shown to define
14
pharmacophoric residues of the hBaxBH3 bound with hBclꢀB under dynamic conditions. Using
the pharmacophoric residues of the peptide, MMsINC database (as represented in the original
2
2
article ) consisting of 17 million conformers of about 4 million diverse molecules was screened
through pepMMsMIMIC interface in order to identify lead chemical compounds mimicking the
1
4, 22
interaction of the hBaxBH3 on the BH3ꢀbinding groove of the hBclꢀB.
After subjecting a
few hundred compounds screened using the ‘peptododynmimetic strategy’ to high throughput
virtual screening followed by pharmacokinetic evaluations, the HDNB was identified as a
prototypic chemical molecular inhibitor to the hBclꢀB. Furthermore, binding affinities of the
HDNB towards the 6 antiꢀapoptotic proteins from human beings have also been recently
scrutinized by means of in silico methods and differential binding modes of the HDNB with the
antiꢀapoptotic proteins could be chiefly attributed to differences in the chemical properties of
1
5
BH3ꢀbinding grooves of the proteins.
2.1 Synthesis and structural characterizations of the HDNB
The HDNB was not commercially available during course of the present studies and even
till now. Synthetic methodology of the HDNB is outlined in Figure S1 and also described in the
‘Method’ section. Structural characterizations of the HDNB as examined by UV, FTꢀIR and GC
ꢀMS spectrometric techniques have been elaborately delineated in the supplementary material
(Figure S2 and Table S1) and comprehensive analyses of the data could consistently represent
the structure of the HDNB. In addition, elemental composition of the HDNB (C H N O ) as
7
5
3
6

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determined by using 2400 series CHNS Elemental analyzer (PerkinElmer, Wellesley, USA) were
in good agreement with elemental composition of the compound calculated out theoretically:
percentage compositions of C, H & N were 34.87%, 2.50% & 16.95% respectively, as
determined by the experimental method and the theoretic values were 37.02%, 2.22% & 18.50%
respectively, for the elements. Moreover, nuclear magnetic frequencies of protons, carbons and
nitrogen present in the compound were also recorded using various 1D and 2D NMR methods.
1
1
D H NMR spectra of the 3,5ꢀdinitiro salicylic acid (DNSA, the starting compound) and HDNB
are shown in Fig. 1A and 1B, respectively. The DNSA showed two dispersed resonances at δ
th
th
8
.67 and δ 8.71 for aromatic protons located at 4 and 6 positions, respectively and phenolic
OH resonance of the compound was observed as a weakly resolved peak at δ 9.0. The HDNB
also showed two peaks for its aromatic protons at δ 8.65 and δ 8.70. In addition, the compound
showed three sharp singlet resonances at δ 7.04, δ 7.21 & δ 7.38 and a weak broadened peak at δ
+
1
4.16. The former and later resonances were assigned to protonated amide group (CONH ) and
3
phenolic OH group of the compound. The resonance assignments were further authenticated by
1
acquiring 1D H NMR for the HDNB for which labile protons could be exchanged to solvent
1
deuterium and the deuterated HDNB showed two H resonances for its two nonꢀlabile aromatic
protons only (Fig. 1C). Moreover, Distortionless Enhancement by Polarization Transfer (DEPT)
1
3
experiments carried out for the HDNB is shown in Fig. 1D and the data depicted two
C
resonances at δ 125.9 and δ 131.5 indicating that the compound has only two tertiary carbon
atoms (C4 and C6, which are free of substitutions in the aromatic ring). Thus, the DEPT and 1D
1
H NMR data on the HDNB endorsed each other and confirmed the resonance assignments. We
1
5
also performed 2D Heteronuclear Single Quantum Correlation (HSQC) ꢀ N experiments for the
HDNB and the data is shown in Fig. 1E. The HSQC spectrum depicted a single cross peak at δ

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1
5
1
3
58.1 of N dimension and δ 7.18 of H dimension confirming the presence of an amide group
in the compound. Taken together, the data presented above could unambiguously confirm the
structure of the HDNB as represented in the Fig. S1.
2.2 Acute oral toxicity (AOT) studies on the HDNB
The HDNB consists of two nitro groups, which could rise up skeptical concerns on
toxicity and adverse effects of the compound in human system. Since compounds consisting of
nitro groups become electrophilic in nature, it is generally considered that the nitro compounds
may cause some deleterious changes by interacting with biological nucleophilic molecules such
2
3
as nucleic acids and proteins. Unless until the nitro compounds damage healthy normal cells,
they may be of safe to use as drugs in the human system. Interestingly, several drugs with nitro
groups are already in market. For instance, drugs such as chloramphenicol (as an antibiotic),
nitrazepam (as a tranquilizer), pyrrolnitrin (as fungicide), nitridazole (as schistosonicide),
parathion (as insecticide) and azathioprine (as antineoplastic) are popular examples for the
2
3
category of the compounds. In addition, ABTꢀ737, a wellꢀknown antagonist to antiꢀapoptotic
4
proteins, has a nitro group and the compound is right now under Phase I clinical trials.
However, toxicities of small chemical molecules are not predictable in a straightforward manner,
since about 40% of lead molecules fail either in preꢀclinical or clinical trials as suggested by
2
4, 25
recent surveys on toxicities of small molecules.
In these backgrounds, irrespective of
literature evidences on nonꢀtoxic/toxic properties of compounds possessing nitro group(s), we
have thoroughly evaluated toxicity of the HDNB by using female Wistar rats.
As per OECD (Organization for Economic Cooperation and Development) 425
guidelines (OECD, 2001), ‘Main test’ (5 Wistar female rats were used in the test: first and
second rats were administrated with 175 mg/kg b.w. and 550 mg/kg b.w., respectively; other

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three rats were administrated with 2000 mg/kg b.w.) procedure was strictly followed to
determine ‘lethal dose’ (LD ) of the HDNB. All the 5 rats treated with various doses of the
5
0
HDNB were periodically monitored for 14 days from the treatments and the HDNBꢀtreated
animals did not develop any toxicity signs such as tremors, convulsions, salivation, diarrhea,
lethargy, sleep and coma. Only the animals administrated with 2000 mg/kg b.w. seemed to be
lethargic in the first 20 minutes of treatment periods and thereafter they become active as healthy
animals. All the five HDNBꢀtreated Wistar rats survived AOT time span of 14 days and at
completion of the treatment periods, all the 5 Wistar rats were sacrificed and subjected to
detailed necropsy studies. Fig. 2 shows histopathology of different organs (such as brain, heart,
lung, liver, kidney and intestine) of the Wistar female rats treated with 2000 mg/kg b.w. of the
HDNB. Examination on the histology of the organs suggested that there were no abnormal or
pathological changes due to oral administration of the HDNB to the test animals:
cerebral/cerebellar hemispheres of brains, myocytes of heart section and hepatocytes of liver
were characterized to be as normal as in the healthy rats; lung section showed normal alveolar /
bronchiolar epithelium with no infiltrates; kidney section showed the normal cortex and medulla
with glomeruli and tubules showing their normal microꢀarchitecture; there were no necrotic
2
6, 27
lesions of the epithelium in the intestinal section.
Thus, the AOT studies revealed that the
HDNB administrated orally to the Wistar rats was not toxic at dose level of 2000 mg/kg b.w. and
LD of the compound should also be at least greater than 2000 mg/kg b.w.
5
0
2
.3 Chemopreventive effect of the HDNB using animal models
.3.1 Cancer induction and treatment
2
Chemopreventive effect of the HDNB was tested on breast cancer animal models on the
basis of results obtained from in vitro MTT (3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]2,5ꢀdiphenyltetrazolium

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bromide) assays carried out for the HDNB. Cell growth inhibition potential of the HDNB was
examined against MCFꢀ7 (human breast cancer cells), HeLa (cervical cancer cells) and MGꢀ63
(
human osteosarcoma cells) cell lines and results of the assays are shown in Fig. 3. The cell lines
were treated with various concentrations of the HDNB at constant treatment time of 48 h
Materials and Methods). By fitting the doseꢀdependent inhibition potential of the HDNB against
(
growth of the cell lines to an appropriate nonꢀlinear regression reveled that IC50 values of the
HDNB against MCFꢀ7, MGꢀ63 and HeLa cell lines were 574 ± 3 µM, > 2 mM and > 5 mM,
respectively. The MTT assay suggested that the HDNB strongly inhibited growth of breast
cancer cells than that of bone/cervical cancer cells used in the study. Hence, chemopreventive
effect of the HDNB on NMUꢀinduced breast cancer animal models was examined using 42
female Wistar rats for the time span of 18 weeks. The experimental animals were divided into 7
groups with 6 animals in each group (Table 1): control group, vehicle group, diseased group,
standard group and 3 HDNBꢀtreated groups (groups treated with low, medium and high doses of
the HDNB were denoted as lowꢀdose group, mediumꢀdose group and highꢀdose group,
respectively). The animals of the control group and vehicle group received only normal saline (1
ml) and corn oil (1 ml) as treatment doses, respectively, every alternative day throughout the
experimental period. The breast cancer inductions and treatments were started simultaneously by
administrating the NMU at concentration 75 mg/kg intraperitoneally (i.p) to animals of all the
groups except animals belonging to the control and vehicle groups. The NMU dose of 75 mg/kg
st
rd
i.p was given to the animals two times at 1 and 63 days of experimental timelines in the
present study. In these contexts, it should also be mentioned that dose levels (50 mg/kg –75
mg/kg), number of doses (1ꢀ3 times) and administrating time intervals (6 ꢀ 9 weeks) of the NMU
were found to be different from each other in quite a large studies on the NMUꢀinduced

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2
8ꢀ34
cancerous animal models as reported in literature.
After the NMU administration, treatments
regime of the groups were as follows: animals of the diseased group received only usual food
and water throughout the experiments; animals of the standard group received 20 mg/kg dose of
Tamoxifen every alternative day throughout the course of the experiments; similarly, the lowꢀ
dose, mediumꢀdose and highꢀdose groups received the HDNB at concentration of 8 mg/kg, 40
mg/kg and 200 mg/kg (the doses were calculated based on results obtained from the AOT
studies), respectively. At end of the experimental timeline, blood samples (serum and plasma)
and breast tissues from all 6 animals of each group were collected, processed and preserved as
per standard procedures. The samples were then subjected to hematology, histopathology,
biochemical and immunohistochemical analysis and outcomes of the studies have been described
in the following sections.
It is worthy of mentioning that all animals used in the chemopreventive studies were
survived for the whole period of 18 weeks and the animals did not develop any toxic symptoms
such as tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma as inferred from
protocols used to monitor the animals in a systematic manner (refer to Methods). The body
weights of the different experimental groups were also monitored in regular intervals for 18
weeks and average body weights of animals belonging to seven different groups i.e. from Group
I to VII were found to be 186 ± 3 g, 187 ± 2 g, 188 ± 4 g, 190 ± 4 g, 187 ± 3 g, 189 ± 5 g and 188
±
5 g, respectively. There were no significant differences in the body weights between the
HDNBꢀtreated groups and any other groups (control/vehicle/standard/diseased groups) as
determined from ANOVA (Analysis of Variance) statistical analysis of the data. Moreover, these
results suggested that dose levels of the HDNB used in the chemopreventive studies did not
cause any apparent behavioral changes and as well toxic symptoms to the animal models.

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2.3.2 Histopathological analysis of breast tissues
All six Wistar rats of the control group showed no abnormality in cellular architectures
of their breast tissue sections (Fig. 4A). Animals of the vehicle group also showed that there
were no abnormalities in their breast tissues as compared to that of control group indicating the
vehicle (corn oil) did not cause any adverse effect to the animals. On contrary, histopathological
results clearly confirmed developments of mild dysplasia in the breast tissues of all six animals
belonging to the diseased group (Fig. 4B) and the observations are in line with the literature
3
5,36
reports on the NMUꢀinduced cancerous animal models.
rats belonging to Group IV (lowꢀdose group), Group V (mediumꢀdose group) and Group VI
highꢀdose group), respectively showed breast tissues as similar as to that of control group. It is
Interestingly, 20%, 80% and 50% of
(
also worthy of mentioning that the rest of 80% rats in Group IV were characterized to have mild
dysplasia in their breast tissues, while the rest of 20% and 50% of rats in Group V and Group VI
depicted basophilia in alveolar and ductal epithelium. In the standard group, breast tissues of
50% of rats were found to be normal and rest of them were found to have basophilia in alveolar
and ductal epithelium. Thus, the histopathological results suggested that the mediumꢀdose (40
mg/kg b.w.) of the HDNB exhibited significant chemopreventive potential by suppressing
abnormal cell proliferation occurring in the NMUꢀinduced breast carcinoma of female Wistar
rats (Fig. 4C).
2.3.3 Analyses of hematological parameters
Hematology studies were carried out to investigate alterations that may occur in standard
levels of WBC (white blood cells) count, lymphocytes, neutrophils, monocytes, eosinophil,
basophils, RBC (red blood cells) count and Hb (hemoglobin) of the animal models used in the
present studies. The results of the studies have been enumerated in Table 2. The data revealed

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that the diseased group showed twoꢀfold increase of WBC, lymphocytes and neutrophils as
compared to that of control group. From a review of data in Table 2, it is interesting to note that
values of the hematological parameters of the mediumꢀdose and highꢀdose groups showed no
statistically significant differences to that of control group implying levels of WBC, lymphocytes
and neutrophils could be restored in these groups as compared to the diseased group. Moreover,
there were no significant differences between the medium/high dose groups and standard group
with respect to these parameters. The same trend was also observed in the case of basophils of
animal models, while eosinophils showed no significant alterations among all the seven groups.
Concentration of hemoglobin, RBC and monocytes were found to be decreased in the diseased
group relative to that of the control group. Though there were no statistically significant
differences between all the 3 HDNBꢀtreated groups and standard group with respect to these
parameters, the mediumꢀdose group restored the hemoglobin, RBC and monocytes in a
noteworthy manner as compared to that of other 2 groups.
Taken together, remarkably decreased levels of RBC and increased levels of lymphocytes
in Wistar rats of the diseased group are indications of developing anemia and lymphocytosis,
respectively in those animal models. These types of inflammatory responses have also been
3
7, 38
reported in animals with mammary carcinoma as demonstrated in literature.
In these
contexts, the results described above are in line with those literature reports. In addition,
development of leukaemia in the NMUꢀadministered rats was also evidenced as per a few reports
3
9, 40
in the literature.
Interestingly, the present study suggested that oral administration of the
HDNB (40 mg/kg b.w.) brought back the status of WBC, RBC, lymphocytes and Hb to near
normal range in the NMUꢀtreated Wistar rats (Group V).

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2.3.4 Analyses of biochemical end-products
Biochemical endꢀproducts such as total protein, albumin, creatinine, urea, alanine
transferase (ALT), aspartate transferase (AST), gluthathione reductase (GR) and glutathioneꢀSꢀ
transferase (GST) in blood serum/plasma were used to assess biological effect of the HDNB in
the NMUꢀinduced mammary carcinogenesis. Table 3 enumerates levels of these biochemical
parameters as estimated using standard assays (refer to Methods) for all seven different
experimental Wistar groups of the present study. The status of total protein and albumin were
significantly decreased in Wistar rats of the diseased group comparing to that of the control
group. Oral administration of medium dose of the HDNB (40 mg/kg b.w.) to the NMUꢀinduced
Wistar rats (Group V) significantly restored status of the total protein and albumin to near values
of the respective parameters estimated for the control group (Table 3).
Aspartate transferase (AST) and alanine transferase (ALT) are generally being used as
hepatic markers and significant alterations of these markers in blood plasma are probable
indications of liver dysfunction and/or liver damage. The levels of AST and ALT were found to
be significantly increased and decreased, respectively in the diseased group as compared to that
of the control group. Among the lowꢀ, mediumꢀ and highꢀdose groups, Wistar rats of mediumꢀ
dose group depicted AST and ALT levels, which were in good agreement with that of the
control/standard groups (Table 3). These results indicated that the HDNB could restore liver
functions by probably interfering with metabolic activations in the NMUꢀtreated Wistar rats. The
concentration of urea and creatinine, which serve as markers to examine renal functions, showed
no significant alterations among all the seven experimental Wistar groups studied in the present
study (Table 3). It should be mentioned that neither the hepatic markers nor renal markers
mentioned above were abnormally estimated in both mediumꢀdose and highꢀdose groups (Group

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V & VI) suggesting 18 weeks oral administration of the HDNB at the dose concentration (40/200
mg/kg b.w.) in the NMUꢀinduced mammary Wistar rats did not cause any severe adverse effects
on the liver and kidney tissues. These findings lend credence to the reports from the AOT studies
on the HDNB demonstrated in previous section of the work.
The GST and GR are phase II detoxification enzymes and they have been shown to be
4
1
significantly altered in cancerous animal models. The GST is reported to actively play critical
roles in antioxidant defense mechanisms and detoxification of electrophilic/hydrophobic
4
2
chemical compounds. The enzyme is also known to be an important preꢀneoplastic marker in
4
3
assessing the carcinogenꢀinduced free radical damage. The GR is a critical cellular antioxidant
4
4
enzyme which regulates the intracellular ratio of oxidized and reduced glutathione level. While
increased levels of the GST and GR facilitate excretion of carcinogenic metabolites either by
conjugation with reduced glutathione or by destroying the reactive centers of carcinogen,
activities of the enzymes are shown to be drastically decreased in cancerous animal models
4
5, 46
comparing that of healthy counterparts.
In the present study, activations of both enzymes
were significantly decreased in the diseased group as compared to that of the control group and
the results corroborate the literature reports discussed above. Oral administration of mediumꢀ
dose HDNB increased the GST and GR concentration on par with the standard drug, tamoxifen
used in the present study, for bringing back the status of the enzymes to near normal range in
blood plasma (Table 3). The data thus imply that the HDNB is capable of restoring the phase II
detoxification enzymes and consequently stimulating excretion of carcinogenic metabolites that
may be generated in the NMUꢀinduced mammary carcinogenesis.

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2.3.5 Analyses of lipid peroxidation and antioxidant markers
Oxidative stress occurring due to imbalance between oxidant and antioxidant levels plays
4
7
pivotal roles in various stages of carcinogenesis. Overproduction of reactive oxygen species
ROS) under the oxidative stress causes damages to biomacromolecules such as nucleic acids,
(
proteins and lipids and subsequently altering cellular architectures and proliferations. Thus, endꢀ
products of oxidative stress, especially lipid peroxidation (oxidative degradation of lipids)
byproducts such as malondialdehyde (MDA), tumor necrosis factor – alpha (TNFꢀα) and nitic
4
8, 49
oxide are considered as markers in many inflammatory diseases and as well in cancers.
The
MDA affects DNA functions by forming DNAꢀMDA adduct and TNFꢀα plays an important role
in tumor progression by promoting expression of matrix metalloproteinases (MMP) and
5
0, 51
endothelial adhesion molecules to produce DNA damage.
While NO (Nitric Oxide) acts as
an antioxidant at low level, it is capable of damaging DNA at elevated concentrations and as well
5
2, 53
inducing alterations in cell functions to stimulate cell growth.
In contrast, antioxidants play a
major role in safeguarding the cells against the free radical induced toxicity. Hence, homeostasis
of antioxidants and proꢀoxidants are vital for normal cellular functioning and impairment in the
5
4ꢀ56
balance would trigger the pathoꢀphysiological events causing damages to vital organs.
In the present study, abnormalities of lipid peroxidation byproducts such as MDA, TNFꢀα
and NO and as well antioxidant markers such as superoxide dismutase (SOD), catalase,
glutathione peroxidase (GPx), reduced glutathione (GSH), vitamin C and vitamin E have been
analyzed in blood samples of the experimental Wistar animal models. The status of MDA, TNFꢀ
α and NO were found to be significantly elevated in the diseased group comparing to that of the
control group and the results are in conformity to lipid peroxidation effect on cancerous animal
4
9, 57
models reported in the literature.
A comparative analysis of the status of the lipid

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peroxidation byproducts in the HDNBꢀtreated groups and diseased group revealed that lowꢀdose
group showed no significant changes, whereas both mediumꢀdose and highꢀdose groups showed
significant variations (Table 4). Of the two groups, the mediumꢀdose group depicted better
significant variations than the highꢀdose group as compared to the diseased group with respect to
levels of the MDA, TNFꢀα and NO in those animal models. Moreover, there were no significant
differences between estimated values of the parameters from the mediumꢀdose group and the
control/standard group indicating elevated levels of the MDA, TNFꢀα and NO were reversed to
the normal levels in the NMUꢀinduced Wistar mammary carcinogenesis upon subjecting the
animal models to the HDNB mediumꢀdose (40 mg/kg b.w.) treatments for 18 weeks.
Concentration of enzymatic antioxidants such as catalase, SOD and GPx and nonꢀ
enzymatic antioxidants such as reduced glutathione and vitamin C were found to be significantly
declined in the diseased group as compared to that of the control group. However, there were no
significant changes in the status of vitamin E between the control group and other six groups
(Table 4) implying metabolic activations in the NMUꢀtreated Wistar rats had no effect on
vitamin E status of blood plasma as inferred from the present study. However, significantly
decreased level of vitamin E from the reference range was demonstrated in several animal
5
8
models possessing cancer diseases due to chemical carcinogens as reported in the literature and
further discussion on the discrepancy is beyond the scope of this present study. Comprehensive
analysis of the data on enzymatic and nonꢀenzymatic antioxidants (GSH and vitamin C) obtained
from the control and other six experimental groups revealed following information: both types of
antioxidants could not be restored in lowꢀdose HDNBꢀtreated Wistar rats; though the
antioxidants could be restored near to reference levels in both medium/highꢀdose HDNBꢀtreated
Wistar rats, efficacy of the mediumꢀdose was found to be more satisfactory on reversing the

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antioxidants levels near to the standard/control ranges than that of highꢀdose group (Table 4).
Lowered levels of the antioxidants in blood plasma of the diseased group could presumably be
due to excess amount of antioxidants utilizations by cancerous tissues either to meet their
5
9
nutrient demand or to scavenge excessively generated reactive oxygen species. In general, SOD
removes the superoxide radical by converting it to hydrogen peroxide and molecular oxygen,
whereas catalase decomposes hydrogen peroxides to water and dioxygen and as well protects
60, 61
tissues from the highly reactive hydroxyl radicals.
The antioxidant glutathione peroxidase
(GPx) plays an important role in peroxyl scavenging and as well maintaining the integration of
6
2
the cell membranes. Nonꢀenzymatic antioxidants like reduced glutathione (GSH), vitamin C
and vitamin E are acting as a second line defense agents to scavenge residual free radicals
eluding from primary defense mechanisms by enzymatic antioxidants. As demonstrated above,
oral administration of the mediumꢀdose HDNB (40 mg/kg b.w.) to the NMUꢀtreated Wistar rats
was found to restore the antioxidants and lipid peroxidation byproducts to the level of the control
group suggesting the HDNB may probably prevent cancer progression in the animal models by
restoring the biomarkers scavenging the free radicals and consequently protecting cellular
architectures. Strikingly, it is also obvious to note from the studies that the capabilities of the
HDNB on restoring the antioxidants could supersede the standard drug, tamoxifen, used in the
present study (Table 4).
2.3.6 Immunohistochemical studies on cancer biomarkers
Modulating effect of the HDNB on expression pattern of cell proliferative markers (Kiꢀ
7, p53) and as well an apoptotic (Bclꢀ2) marker have been investigated and described herein.
6
Overexpression of Kiꢀ67 in all proliferating cells is reported in the literature. Moreover,
overexpression of Kiꢀ67 in more than 50% of the breast cancer cells have been shown to be at

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high risk of recurrent disease and thus it is also considered as a specific biomarker of breast
6
3ꢀ65
cancers.
Tumor suppressor gene, p53 plays an important role in the prevention of cancers by
inducing apoptosis, DNA repair and cellꢀcycle arrest in response to different types of cellular
6
6
stress. However, mutated forms of the p53 were shown to conferring a proliferative advantage
to the neoplasm. Overexpression and as well active roles of p53 mutants have been well
6
7, 68
documented in various cancer types including breast carcinoma.
Bclꢀ2, an antiꢀapoptotic
protein, promotes cell survival by inhibiting apoptosis and the protein is well characterized to be
6
9
overexpressed in various cancers, including breast cancer. In general, all six antiꢀapoptotic
proteins characterized to date are considered as crucial biomarkers as they are shown to be
overexpressed in most of cancers, if not all, though overexpression pattern of each antiꢀapoptotic
protein is unique with respect to cancer types. Overexpression of the antiꢀapoptotic proteins in
cancer is due to impairment of apoptosis regulating a delicate balance of the proꢀapoptotic and
7
0
antiꢀapoptotic proteins.
Immunohistochemical studies carried out on detecting the three molecular markers, Kiꢀ
7, p53 and Bclꢀ2 in breast tissues of female Wistar rats belonging to the diseased group
6
apparently revealed overexpression of the markers. Immunostaining of proliferative markers Kiꢀ
6
7 and p53 variants and an apoptotic marker, Bclꢀ2 for the diseased group are depicted in Fig.
B, Fig. 5E and Fig. 5H, respectively and overexpression of the molecular markers are
5
represented by ‘arrow’ symbols in the respective pictorial illustrations. Abnormal expression
patterns of the three molecular biomarkers in the female Wistar rats of the diseased group in the
present study are in line with literature reports on status of the markers in animal models of
6
5, 67, 71
different types of carcinogenesis.
Oral administration of the HDNB at a dose of 40 mg/kg
b.w. to the NMUꢀtreated Wistar rats significantly restored the three molecular markers to near

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normal levels (Fig. 5C, 5F & 5I). Expression patterns of the Kiꢀ67, p53 and Bclꢀ2 in the breast
tissues of the control group are also shown for sake of comparison in Fig 5A, 5D & 5G,
respectively. This particular efficaciousness of the HDNB on restoring the expressions of the
molecular markers to normalcy justifies not only the protective effect of the HDNB in the NMUꢀ
induced Wistar mammary carcinogenesis and as well supports that the HDNB is a promising
lead molecule to be developed as an efficient antiꢀcancer drug in near future.
3
. Concluding remarks
In the present study, 2ꢀhydroxyꢀ3,5ꢀdinitro benzamide (HDNB), a chemical compound
identified by means of peptidodynmimetic computational approach, was synthesized and
structurally characterized by using molecular spectrometric techniques such as UV, FTꢀIR, GCꢀ
MS, 1D and 2D NMR. To our best knowledge, biological properties of the compound have not
yet been well documented in the literature. We have herein demonstrated the antiꢀcancer effect
of the HDNB in animal models. The AOT studies suggested that LD of the compound must be
5
0
at least greater than 2000 mg/kg b.w. The biological roles of the HDNB in NMUꢀinduced Wistar
mammary carcinogenesis were assessed by systematically analyzing the levels of various
hematological parameters (WBC, RBC, Hb, lymphocytes, monocytes, neutrophils, basophils and
eosinophils), biochemical end products (total protein, albumin, creatinine, urea ALT, AST, GST
and GR), lipid peroxidation byproducts (MDA, TNFꢀα and NO), antioxidants (vitamin C,
vitamin E, GSH, catalase, SOD and GPx), proliferative markers (Kiꢀ67 and p53) and an
apoptotic molecular marker (Bclꢀ2) by using established procedures. Mediumꢀdose (40 mg/kg
b.w.) of the HDNB showed appreciable potential to reverse the abnormalities of various
biochemical parameters in blood samples and as well in breast tissue samples to the values as
close as to that of the control/standard (Tamoxifen) groups. The chemopreventive effect of the

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HDNB could be primarily attributed to its ability of restoring antioxidants and as well apoptosis
inducing properties during the NMUꢀinduced mammary carcinoma in Wistar animal models. The
above results suggest that the HDNB is a promising seed molecule for developing prototypic
chemical inhibitors to the antiꢀapoptotic proteins by using computational strategies such as ‘core
1
4, 25
hopping’ and de novo drug designing approaches in near future.
4
. Materials and Methods
4.1 Chemicals
Thionyl chloride, 3,5ꢀdinitrosalicylic acid (DNSA), liquid ammonia (ammonium
hydroxide solution), tetrahydofuran (THF), dimethyl sulfoxide (DMSO), deuterium oxide (D O),
2
NꢀnitrosoꢀNꢀmethyl urea (NMU) were obtained from SigmaꢀAldrich chemicals Pvt. Ltd.,
Bangalore, Karnataka, India. Corn oil (brand name ꢀ Avita Rꢀgold) was purchased from J.S. oils,
Thanjavur, TamilNadu, India. 2,4ꢀdinitrophenylhydrazine (DNPH), FolinꢀCiocalteau reagent,
5
,5’ꢀdithiobis (2ꢀnitro benzoic acid) (DTNB), trichloroacetic acid (TCA), hydrogen peroxide,
heparin, phosphate buffer, reduced glutathione, epinephrine, ethylenediamine tetraꢀacetic acid
EDTA), 1ꢀchloroꢀ2,4 dinitro benzene (CDNB), thiobarbituric acid (TBA), diphenylꢀ1,10
(
phenonthroline, griess reagent, sodium nitroprusside were purchased from Hiꢀmedia laboratories,
Mumbai, Maharashtra, India. P53, Kiꢀ67 and Bclꢀ2 primary antibodies and corresponding
secondary antibodies conjugated with horseradish peroxidase were purchased from Dako,
Carprinteria, CA, USA. All the other chemicals/solvents used in the present study were also of
analar grade.
4.2 Synthesis of the 2-hydroxy-3,5-dinitrobenzamide (HDNB)
A mixture of 3,5ꢀdinitro salicylic acid (16.3 g, 0.05 mol) and thionyl chloride (120 ml)
was refluxed with constant stirring until the suspension mixture was converted into a clear

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solution. Excess amount of thionyl chloride added to the mixture was then removed under
reduced pressure. The acid chloride of the parent compound obtained upon cooling was
immediately dissolved in dry tetrahydrofuran (THF) and the resultant solution was added
dropwise manner into a fresh ammonia solution kept in ice bath (≈ 273 K) with constant stirring.
Target compound, 2ꢀhydroxyꢀ3,5ꢀdinitro benzamide (HDNB), was formed as yellow precipitate
in the reaction mixture. The HDNB was filtered (using Grade 1 Whatman paper), excessively
washed with double distilled water to remove the acid impurity and then was dried in vacuum
oven at 353 K. The HDNB was a bright yellow solid (5.7 g, 35% yield).
4.3 Structural characterizations of the HDNB
4.3.1 UV Spectroscopy
Absorption spectra of the HDNB and DNSA were recorded at 298 K in the wavelength
range from 400 nm to 550 nm using double beam Lambda25 UV/Vis spectrophotometer
PerkinElmer, Wellesley, USA). The compounds were dissolved in DMSO and concentrations
(
were 1.5 mM for both test solutions. Scan speed and slit width were set as 100 nm/minute and 1
nm, respectively. Absorption spectra for the compounds were also appropriately corrected to
solvent effect.
4.3.2 FT-IR Spectroscopy
FTꢀIR spectra of the HDNB and DNSA were recorded on a Spectrum one FTꢀIR
spectroscopy (PerkinElmer, Wellesley, USA). Sample preparation was carried out by thoroughly
mixing HDNB/DNSA and KBr in the ratio of 1:3 (w/w) and pellet of the sample mixture was
made using hydraulic press. Percentage of transmittances of the test samples was measured at
scan speed of 0.2 cm/second and then the data were Fourierꢀtransformed to generate spectra
ꢀ1
ꢀ1
ꢀ1
(
covering wave number from 400 to 4000 cm ) with resolution of 2 cm . FTꢀIR (KBr, cm ) for

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ꢀ1
the DNSA: 824, 1534, 1677, 2442, 2516, 2596, 2854, 2925, 3104 & 3448. FTꢀIR (KBr, cm ) for
the HDNB: 805, 1564, 1602, 3135 & 3446.
4
.3.3 GC-MS analysis
Molecular mass of the HDNB was determined by using Clarus 500 GCꢀMS
PerkinElmer, Wellesley, USA). Eliteꢀ5MS capillary Column (5% Phenyl, 95%
(
dimethylpolysiloxane) of 30 meter in length was used at 453 K. 2 µL of the HDNB dissolved in
DMSO at concentration of 1mg/100 µL (about 44 mM) was used for the analysis. Helium gas
was used as carrier gas and flow rate was maintained as 1ml/minute. Sample could be
fragmented and ionized by using electron energy of 70 ev in ‘Electron Ionization’ mode. Mass
range for detecting various fragments of the HDNB upon electron bombardment was set to be
from 20 to 600 amu. Total ion chromatogram of the HDNB showed two fragment species
depicting molecular mass of 44 Da and 184 Da and the generated mass/charge data were used to
search against NIST 2005 library in order to identify the structures of the HDNB (refer to
supplementary material).
1
13
15
4
.3.4 H, C and N NMR studies
1
1
One dimensional H NMR (1D H NMR) studies for the DNSA and HDNB dissolved in
DMSOꢀd were carried out using a 300 MHz AVANCE II (Bruker Biospin, Fallanden,
6
Switzerland) NMR spectrometer equipped with a 5 mm BBO probe. Sample concentration was 2
1
mM in both cases. 1D H NMR spectra for the test samples were acquired using zg30 standard
pulse sequence with a relaxation delay of 1.0 sec at 298 K (temperature of the probe was
7
2
calibrated using methanol standard ) and the acquired time domain data were then processed by
1
Topspin 1.3 program. 1D H NMR data of the HDNB dissolved in DMSOꢀd :D O solvents
6
2
1
mixture (3:1, v/v) was also acquired at identical conditions as mentioned above. In all the 1D H

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NMR experiments, tetramethylsilane (TMS) was used as an internal standard for referencing
chemical shifts of various protons of the DNSA and HDNB. DEPT and twoꢀdimensional HSQCꢀ
1
5
N experiments for HDNB were carried out using a 400 MHz NMR spectrometer equipped with
a 5 mm AutoSW probe (Agilent Technologies, CA, USA). Default standard parameter settings of
the programs as defined in the standard pulse sequence library of VnmrJ 2.2C were used except
the following acquisition parameters: transients and t increments were set as 4 and 512,
1
respectively, for recording the HSQC spectrum; transients and a relaxation delay were set as
1
024 and 1 second, respectively, for recording DEPT spectrum. TMS and CH NO were used as
3 2
1
3
15
internal standards for referencing chemical shifts of C and N resonances of the HDNB,
1
respectively. H NMR (300 MHz, DMSOꢀd ) of the HDNB: δ 8.65 (s, 1H, CH), 8.70 (s, 1H,
6
1
3
15
CH); DEPT C NMR (400 MHz, DMSOꢀd ) of the HDNB: δ 125.9, 131.5; HSQC ꢀ N (400
6
1
MHz, DMSOꢀ d ) of the HDNB: δ 358.1; H NMR (300 MHz, DMSOꢀd ) of the DNSA: δ 8.67
6
6
(s, 1H, CH), 8.71 (s, 1H, CH).
4
.4 In vitro cytotoxicity potential of the HDNB by MTT assay
The cancer cell lines (MGꢀ63, MCFꢀ7 and HeLa) were purchased from National Centre
for Cell Science (NCCS), Pune, India and were grown in Eagles Minimum Essential Medium
EMEM), supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 °C in a 5% CO2
atmosphere. The monolayer cells were detached with trypsinꢀethylene diamine tetra acetic acid
EDTA) to make single cell suspensions and viable cells were counted by trypan blue exclusion
(
(
assay using a hemocytometer. The cell suspensions were seeded in 96ꢀwell plates at a density of
4
1
x 10 cells/well and treated with serial concentrations (ranging from 0.2 to 1000 µM) of the
HDNB dissolved in neat DMSO. The plates were then incubated for an additional 48 h at 37 °C
in a 5% CO atmosphere. After the incubation, 15 µl of MTT (3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]2,5ꢀ
2

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diphenyltetrazolium bromide) dissolved at concentration of 5 mg/ml in phosphate buffered saline
PBS) was added to each well and the plates were further incubated for another 4 h. Formazan
(
crystals formed were solubilized in 100 ꢁl DMSO and absorbance was measured at 570 nm using
a microplate reader. The medium containing without the HDNB was served as control and all the
experiments were performed in triplicate. The percentage of cell inhibition caused by the HDNB
7
3
was determined using standard procedure and the data were analysed by using GraphPad Prism
software 5.01 (GraphPad Software, CA, USA).
4.5 Acute oral toxicity studies
A total of 5 Wistar female rats of about 8 weeks old with average weight of 175 ± 5 g
were used as test animals for the acute toxicity studies and the animals were housed in standard
polypropylene cages. Temperature, relative humidity and lighting of animal room were
maintained at 25 ºC, 30ꢀ70% and 12/12 light/dark cycles, respectively throughout the
experiments. The rats were provided with rodent laboratory diets (M/s Provimi Pvt. Ltd.,
Bangalore, Karnataka, India) and potable water purified by reverse osmosis ad libitum. The
studies on the Wistar rats were conducted at Animal House, SASTRA University, using
protocols approved by the Institutional Animal Ethics Committee of SASTRA University
(CPCSEA – Committee for the Purpose of Control and Supervision of Experiments on Animals,
Ministry of Environment and Forest, Government of India ꢀ ethical clearance for the animal
studies is 227/SASTRA/IAEC/RPP).
Acute toxicity of the HDNB was tested in the animal models using ‘Main test’ procedure
7
4
as per the OECD guidelines 425. The HDNB was dissolved in corn oil and various doses (175
mg/kg, 550 mg/kg and 2000 mg/kg b.w.) of the compound were administrated using an oral
gavage needle to the rats. It should be mentioned that single ordered dose progression wherein

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the rats were dosed one at a time, at a minimum of 48 h intervals was followed and volume of
doses was constantly maintained to be 1 ml irrespective of dose levels (dose progression factor
was 3.2) and body weights of animals. Apparent changes on the physical systems and behavior
patterns of the test animals were keenly noticed every 30 minutes for first 6 h and thereafter the
animals were periodically monitored every 12 h for the experimental time span of 14 days. After
14 days, all the experimental animals were sacrificed and gross necropsies were done to examine
any abnormalities or lesions in the internal organs such as brain, heart, lungs, liver, kidney and
intestines.
4.6 In vivo studies using Wistar rats
A total of 42 female Wistar rats of about 8 weeks old with average body weight of 180 ±
g were used as test animals for the in vivo studies and the animals were housed in standard
6
polypropylene cages. Temperature, relative humidity and lighting of animal room were
maintained at 25 ºC, 30ꢀ70% and 12/12 light/dark cycles, respectively throughout the
experiments. The rats were provided with rodent laboratory diets (M/s Provimi Pvt. Ltd.,
Bangalore, Karnataka, India) and potable water purified by reverse osmosis ad libitum. The 42
female Wistar rats were divided into seven groups (Group I – VII) and each group was consisted
of six animals. The total study period was set as 18 weeks and doses (saline/corn
oil/HDNB/Tamoxifen) were administered to animals belonging to various groups at every
alternate day throughout the study period as mentioned below herein. Wistar rats of Group I
served as ‘control group’ and the rats received only physiological saline as an oral dose
throughout the experimental period. Group II rats received only vehicle (1ml of corn oil was
orally given using stainless steel ball tipped oral intubation needle) and acted as ‘vehicle group’.
Rats in groups III to VII were subjected to induction of mammary carcinogenesis by providing

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st
rd
intraperitoneal injection of 75 mg of NMU at 1 and 63 days of the experiments. The
carcinogen was freshly prepared prior to administration by dissolving it in 1ml of physiological
saline. Group III rats received no other treatments throughout the experiments and hence the rats
were considered as ‘diseased group’. Group IV, V and VI rats were orally administered with the
HDNB at concentration of 8 mg/kg (lowꢀdose), 40 mg/kg (mediumꢀdose) and 200 mg/kg (highꢀ
dose), respectively. The HDNB doses were also freshly prepared by suspending the finely
grinded compound in corn oil, and the dose volume was adjusted to 1ml with plain corn oil in all
cases. The low/medium/high doses of the HDNB were orally administered to rats of Group IV, V
and VI using stainless steel ball tipped oral intubation needle. Group VII rats were orally
administered with tamoxifen citrate (20 mg/kg body weight, dissolved in 1 ml of physiological
saline) every alternate day and the dose was continued till the end of experimental timeline and
the group served as ‘standard group’.
4.6.1 Histopathology
At end of the study period, the rats were killed using CO inhalation euthanasia and
2
breast tissues and other organs of each animal were dissected out. All tissues were preserved in
0% neutral buffered formalin (NBF). The preserved breast tissues were then cut into thinner
1
section (2ꢀ3 mm thick); processed using automatic Leica tissue processor TP 1020 (Leica
Biosystems, Illinois, USA); embedded in paraffin wax using Leica EG 1150 H&C Embedding
machine (Leica Biosystems, Illinois, USA) and cut into 3ꢀ4 ꢁm sections using Leica RM 21°
RTS (Leica Biosystems, Illinois, USA), which were mounted on clean glass slides. The sections
of the tissues were stained with hematoxylin for 10 minutes followed with 0.5% eosin for
another 5 minutes. The sections were then rapidly rinsed in 95% ethanol and dehydrated in
absolute ethanol for 5 minutes using Leica ST 4040 Linear automatic stainer (Leica Biosystems,

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Illinois, USA). Histological studies were carried out for the breast tissues of 6 animals from each
group. A semiꢀquantitative grading scheme was used to evaluate the extent of the lesions in the
tissues using five grades as follows: no lesion (grade 0); minimal (grade 1); mild (grade 2);
4
4, 45
moderate (grade 3); marked (grade 4) and severe (grade 5).
.6.2 Haematological studies
Prior to necropsy of Wistar rats used in the in vivo experiments, blood samples were
4
collected from the retroꢀorbital sinus of the rats in ethyleneꢀdiamineꢀtetraacetate (EDTA)ꢀcoated
vials. The blood plasma was used for estimations of various hematological parameters by using 5
7
5
Part Mythic 22 Haematology AutoAnalyzer (Orphee, Geneva, Switzerland).
.6.3 Biochemical studies
Plasma was prepared from blood samples collected from the retroꢀorbital sinus of the
experimental rats in the heparinized vials upon subjecting the blood samples to centrifugation at
500 rpm for 10 min. After the centrifugation, the buffy coat was removed and the packed cells
were washed thrice with physiological saline. The erythrocyte suspension was then prepared by
4
1
76, 77
the method of Dodge et al and modified by Quist.
Similarly, serum was prepared by
centrifuging the whole blood collected in sterile test tubes at 1500 rpm for 10 minutes. The
7
8
resultant supernatant was collected and stored separately as described elsewhere. Biochemical
analysis were carried using the plasma/serum prepared from blood samples of 6 animals from
each group and estimated values are expressed as mean ± standard error of the mean.
Lipid peroxide content was determined by thiobarbituric acid (TBA) reaction as assessed
7
9
by Okhawa et al. To the 0.5 ml of plasma, 1.5 ml of 20% acetic acid, 0.2 ml of sodium dodecyl
sulphate (SDS) and 1.5 ml of TBA were added. The mixture was made up to 4.0 ml with distilled
water and then heated for 60 minutes at 95° C using glass ball as condenser. After cooling to

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room temperature, 4.0 ml of butanolꢀpyridine mixture was added, centrifuged and then
absorbance was read at 532 nm. Standard and blank were treated in a similar manner. The lipid
peroxide concentration was expressed as nmoles of malondialdehyde (MDA) formed/mg protein.
8
0
The levels of tumor necrosis factorꢀα (TNFꢀα) was assayed by the method of Konturek et al.
The wells of microtiter plates were coated with 100 µL of blood plasma appropriately diluted in
carbonate buffer. After incubating the plates at 4 °C for 12 h, 100 µL of serially diluted antibody
was added and incubated at 37 °C for another 1 h. The wells were then charged with 100 µL of
antiꢀrabbit IgGꢀconjugated with horseradish peroxidase (diluted 1:5000) at 37 °C for 1 h. The
activity of bound horseradish peroxidase was monitored by addition of 50 µL of substrate (1 µL
of hydrogen peroxide, 0.5 mg oꢀphenylenediamine (OPD) in 1 ml of citrate phosphate buffer, pH
5.0) at room temperature for 20 min in dark and the reaction was then arrested by adding 50 µL
of 2 N sulfuric acid. The TNFꢀα concentration was recorded on ELISA (EnzymeꢀLinked
Immunosorbent Assay) reader at 490 nm and expressed as ng/ml.
The extent of inhibition of nitric oxide radical generation in vitro was followed as per the
8
1
method reported by Green et al. The reaction was initiated by incubating mixture consisting of
.0 ml of sodium nitroprusside, 0.5 ml of PBS and 0.5 ml of plasma at 25 °C for 30 minutes.
2
Griess reagent (0.5 ml) was added to the mixture and incubated for another 30 minutes. The
absorbance was read at 546 nm against the reagent blank (Genesys 10ꢀS, USA) and the values
were expressed as nmole/ml. The activity of catalase, superoxide dismutase (SOD) and
Glutathione peroxidase in plasma were assayed by the method of Sinha, Misra & Fridovich and
8
2ꢀ84
Rotruck et al with some modifications, respectively.
To 0.1 ml plasma, 0.9 ml of phosphate
buffer and 0.4 ml of hydrogen peroxide were added and the reaction mixtures were kept in a
boiling water bath for 10 minutes. The colour developed was read at 530 nm and the activity of