Egypt. J. Comp. Path &Clinic Path. Vol. 29 No.1, 2016 ; 121-136 ISSN 1110-7537

121

Potential Apopreventive Effects of Garlic Oil And

Silymarin on Skin Papilloma Induced in Mouse

Mohamed F. Abou Elazab1, Nagwan M. El- Habashi

2 , Mohamed Abdou

3

and Nora F. Ghanem3

1

Department of Clinical Pathology, Faculty of Veterinary Medicine, Kafr Elsheikh

University, Egypt. 2

Department of Pathology, Faculty of Veterinary Medicine, Kafr Elsheikh University,

Egypt. 3

Department of Animal Biology, Faculty of Science, Kafr Elsheikh University, Kafr

Elsheikh, Egypt.

ABSTRACT— The potential apopreventive effects of garlic oil and silymarin on two-

papilloma in mice initiated by 7-12 dimethyl benz [a] anthracin (DMBA) and promoted with

croton oil were investigated. The highest tumor incidence (100%) and the shortest tumor latency

period (5 weeks) were observed in tumor induced group. In contrast, significant reduction in

tumor incidence to 0 and 20% as well as increase of latency period of papilloma formation (week

10) were observed in garlic oil and silymarin treated groups, respectively. Moreover, significant

increase in reduced glutathione and glutathione peroxidase activities as well as decrease in

malondialdehyde levels were also observed in garlic oil and silymarin treated groups in

comparison with the papilloma induced group. Hematological analysis revealed a significant

decrease in RBCs, Hb, PCV, MCH, and MCHC mean values as well as increase in the total

leukocytic count of papilloma induced group. But, there were no significance differences

between garlic oil and silymarin treated mice and those of vehicle control groups. Also,

histopathological examination revealed significant reduction in papilloma formation and

epidermal hyperplasia in garlic oil and silymarin treated mice when compared with papilloma

induced group.

Keywords: Apoprevention, Garlic oil, Silymarin, DMBA induced skin papilloma, Mice ——————— ——————————

INTRODUCTION

Skin cancer, the most frequently diagnosed

cancer, represents an important public health

problem due to its high incidence and

medical costs (Hara-Chikuma and

Verkman 2008). The incidence of both non-

melanoma and melanoma skin cancers has

been increasing over the past decades.

Currently, between 2 and 3 million non-

melanoma skin cancers and 132,000

melanoma skin cancers occur globally each

year. One in every three cancers diagnosed is

a skin cancer (World Health Organization

2013). Although there are available

treatments such as surgery, radiation therapy

and topical chemotherapy, up to date, still

there is no effective chemopreventive agent

against the development and/or progression

of skin cancer. Increase incidence of skin

cancer due to constant exposure of skin to

environmental carcinogens, provides a strong

basis for chemoprevention with both

synthetic and natural remedies (Gupta and

Mukhtar 2002). Polycyclic aromatic

hydrocarbons (PAHs), organic pollutants, are

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

122

released into the environment in large

quantities mainly due to human activities and

the exposure to such environmental

pollutants is associated with the development

of numerous cancers in human (Neef 1985;

Nebert et al. 2004). Enzymatic activation of

PAHs leads to the generation of active

oxygen species such as peroxides and

superoxide anion radicals, which induce

oxidative stress in the form of lipid

peroxidation. Amongst many PAHs that are

used in induction of skin cancer is 7, 12-

dimethylbenz[a]anthracene (DMBA). It is the

most potent initiator that has ability to

damage DNA and produce cancer in short

time (Slaga 1983). The potent carcinogenic

properties of DMBA is related to its ability to

generate various reactive metabolic

intermediates leading to oxidative stress and

tissue damage (Bishayee et al. 2000).

Moreover, the repeated active oxidative

damage has a great link to all stages of tumor

development including initiation, promotion

and progression (Scandalios et al. 2005;

Visconti and Grieco 2009). A commonly

used two-stage model of skin carcinogenesis

in mice involves initiation by single topical

treatment of the skin with DMBA followed

by promotion through several weeks of

repetitive applications of an effective tumor

promoter such as TPA (Abel et al. 2009).

TPA, the most active phorbol ester present in

croton oil, acts as strong promoter through an

oxygen -mediated mechanism; oxygen

components are the critical components of

the tumor promotion process (Huachen and

Krystyna 1991). Therefore, reducing

intracellular oxidative stress or blocking ROS

generation may represent an effective

strategy for preventing skin carcinogenesis.

Human body is equipped with various

antioxidants such as superoxide dismutase

(SOD), glutathione peroxidase (GPx),

catalase (CAT), glutathione (GSH), ascorbic

acid (Vitamin C), α- tocopherol (Vitamin E),

etc., which can counteract the deleterious

effects of ROS and consequently protect the

body from cellular and molecular damage

(Cotgreave et al. 1988). Antioxidants act as

radical scavengers inhibiting free radical-

mediated processes thereby protecting the

human body from various diseases (Jagetia

and Rao 2006). Amongst various molecules

which can inhibit the formation of free

radicals associated with carcinogenesis are

the bioactive compounds extracted from plant

which have the potential to subside the

biochemical imbalances induced by free

radicals.

Silymarin (SIL), a standardized extract from

the milk thistle Silybum marianum (L), is

composed of many polyphenolic flavonoids,

including silibinin (the major one), isosilybin,

silychristin and silidianin (Polyak et al.

2013). Previously, several studies reported

that consumption of silymarin is safe and

non-toxic in animals and humans

(Wellington and Jarvis 2001; Singh et al.

2002) and there is no known LD50 for

silymarin (Gazak et al. 2007). In addition,

several recent studies have shown the

potential cancer preventive and therapeutic

efficacy of Silybinin in different animal

models and cell culture systems (Singh et al.

2004; Tyagi et al. 2007).

Garlic (Allium sativum), has a long history of

being used as a medicinal plant for over 4000

years for a variety of ailments including

headache, bites, intestinal worms and tumors

(Block 1985). Garlic also possesses many

biological properties including antimicrobial,

antioxidant, anticarcinogenic, antimutagenic,

antiasthmatic, immunomodulatory and

prebiotic activities. Previous report regarded

its anticancer effect to its ability to inhibit

free radical and mutations-mediated DNA

damage (Borek 1997). Dietary intake of

Allium vegetables, such as garlic, may play a

role in reducing the risk of certain cancers

(Kim and Kwon 2009). Allyl sulfides, the

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

123

important organosulfur components of garlic

oil, have been reported to suppress the

growth of multiple cancer cells in culture and

in vivo models (Wu et al. 2004; Powolny

and Singh 2008; Yang et al. 2009). Diallyl

sulfide (DAS), diallyl disulfide (DADS), and

diallyl trisulfide (DATS) are most abundant

in garlic oil (Lawson 1991). DATS has been

reported to be the most potent in inhibiting

carcinogen-induced tumor promotion

(Shrotriya 2010).

Therefore, the aim of the present study was to

investigate the potential chemopreventive

effects of garlic oil and silymarin on DMBA

induced skin papilloma. Tumor incidence

(percentage of papilloma bearing

mice/group), average latent period of tumor

occurrence, clinico-pathological examination

and histopathological examination were used

to achieve the aim of the study.

MATERIALS AND METHODS

1. Chemicals

7, 12-dimethylbenz[a]anthracene and croton

oil were purchased from Sigma-Aldrich Co

(USA). Silymarin powder was purchased

from local pharmaceutical company and was

dissolved in corn oil at a concentration of

10mg/200µl. Garlic oil and corn oil were

purchased from local herbal market. DMBA,

as tumor initiator was dissolved in acetone at

a concentration of 200µg/200µl. Croton oil

which served as tumor promoter was

dissolved in acetone to give 1% croton oil

solution.

2. Animals

Seventy five male Swiss albino mice (6

weeks old) were used in the present study.

The animals were kept in Animal House of

Faculty of Sciences, University of

Kafrelshiekh. They were housed in cages

(with 7-8 mice per cage) under normal

laboratory conditions of humidity,

temperature (25±2ºC) and light (12/12 hour

light dark cycle). The mice were allowed for

2 week adaptation period with free access of

water and food ad libitum. The ethical

clearance had been approved by the ethical

committee of Kafr Elshiekh University.

Three days before the experiment, the dorsal

hair of all the animals was shaved by using

electrical hair clipper (2x2cm) and only the

mice showing no hair growth were chosen for

the study. The inhibition of tumor incidence

by garlic oil and silymarin was evaluated on

two-stage skin carcinogenesis, induced by a

single application of DMBA (as initiator),

and 2 weeks later, promoted by repeated

application of croton oil (as promoter) twice

per week, following the protocol for 12

weeks.

3. Experimental Design

Animals were randomly divided into five

groups each comprising 15 mice and treated

as described below: Group I (papilloma

induced group); mice were received a single

topical application of DMBA in acetone

(200µg/200µl/mouse) over the shaved dorsal

skin area, followed by topical application of

croton oil in acetone (200µl of 1% of croton

oil/mouse) twice a week for 12 weeks. Group

II (treatment group 1); All animal received

the same treatment as Group I and garlic oil

(200µl/mouse) was topically applied on the

shaved dorsal skin area for 7 days prior to 7

days after DMBA (anti-initiation)

application, and further applied 30 minutes

prior to croton oil treatment (twice weekly)

until the end of 12 weeks of promotion

period (anti-promotion). Group III (treatment

group 2); All animal received the same

treatment as Group I, in addition, silymarin

(10mg/200µl corn oil/mouse) applied on the

shaved dorsal skin area for 7 days prior to

7days after DMBA application, and further

applied 30 minutes prior to croton oil

treatment (twice weekly) until the end of 12

weeks of promotion period. Group IV

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

124

(vehicle control 1); All animals were

received topical application of acetone

(200µl/mouse) on the shaved dorsal skin area

throughout the experiment. Group V (vehicle

control 2); All animals were received topical

application of corn oil (200µl/mouse) on the

shaved dorsal skin area throughout the

experiment. Body weight of mice, mortality

rate and tumor incidence were observed at

weekly interval.

4. Blood collection

Blood samples were collected from retro-

orbital venous plexus at the end of the

experiment. Nearly 2 ml of blood were

collected per mouse. Blood samples were

collected on an anticoagulant for

hematological profile and collection of

plasma. Plasma was separated from blood by

centrifugation at 3,000 x g for 15 minutes and

stored at -20 ºC until use.

5. Clinico-pathological examination

5.1. Hematological examination

Red blood cell count (RBC), hemoglobin

(Hb), packed cell volume (PCV), mean

corpuscular volume (MCV), mean

corpuscular hemoglobin (MCH), mean

corpuscular hemoglobin concentration

(MCHC), and total leukocyte count (TLC)

were measured by using an automated

hematologic analyzer for veterinary use and

calibrated for mice.

5.2. Biochemical analysis

5.2.1. Measurement of Malondialdehyde

Malondialdehyde (MDA) was

calorimetrically determined in plasma

according to the method adapted by

Esterbauer et al. (1982). This method is based

on the measurement of MDA as one of the

main end products of lipid peroxidation by

the thiobarbituric acid test. Thiobarbituric

acid reacts with MDA in acidic medium at

95oC for 30 minute to form thiobarbituric

acid reactive product. The absorbance of the

resultant color product measured at 534 nm.

5.2.2. Measurement of Reduced

glutathione

Reduced glutathione (GSH) was determined

in plasma according to the methods of

Beutler et al. (1963). This method is based on

spectrophotometrically measurement of the

yellow color of 2-nitro-5-thiobenzoic acid

which was produced from the following

reaction: Glutathione + 5,5'-dithiobis(2-

nitrobenzoic acid) (DTNB)→2-nitro-5-

thiobenzoic acid + glutathione disulfide

(GSSG).

5.2.3. Measurement of Glutathione

peroxidase

Glutathione peroxidase (GPx) activity was

assayed in plasma according to the method of

Gross et al. (1967). The activity of GPx was

measured directly by determining the amount

of unconsumed GSH remaining at specific

time intervals in the presence of small

amounts of peroxide.

6. Histopathological examination

Animals were sacrificed at the end of the

experiment. Tissue specimen of papilloma, as

well as skin of normal mice were collected in

10% neutral buffered formalin for 24h,

routinely processed and embedded in paraffin

blocks. The 5 m thick sections were stained

with haematoxylin and eosin (Bancroft and

Gamble 2007). The morphological evaluation

of the skin sections was performed under the

light microscope.

7. Statistical analysis

Statistical analysis was carried out by using

the SPSS for windows software, version16

(SPSS Inc., Chicago, IL, USA). Groups data

were compared by one-way analysis of

variance (ANOVA), followed by LSD test.

The statistical significance was accepted at a

level of p<0.05.

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

125

RESULTS 1- Effect of garlic oil and silymarin topical

application on body weight and mortality

rate of DMBA- induced skin papilloma

mouse:

The average initial and final body weight and

mortality rate of mice from all treatment

groups were measured (Table 1). There was a

significant decrease in the average body

weight and increase in the mortality rate of

carcinogenic control group in comparison

with vehicle control groups (p<0.05).

However, there were no significant

differences in the average body weight

between garlic oil and silymarin treated

groups and vehicle control groups (p>0.05).

2- Effect of silymarin and garlic oil topical

application on hemogram of DMBA-

induced skin papilloma mouse:

Some hematological parameters (RBCs, Hb,

PCV, MCV, MCH, MCHC and WBCs) were

measured in all experimental groups (Table

2). The mean values of RBCs, Hb and PCV

were significantly decreased (p<0.05) in

papilloma induced group in comparison with

the vehicle-control groups. However, there

were no significant difference between garlic

oil and silymarin treated groups and vehicle-

control groups (p>0.05). RBC indices (MCH,

MCHC) were also significantly decreased

(p<0.05) in papilloma induced group in

comparison with the vehicle-control groups.

But, the mean values of MCV were

significantly increased in papilloma induced

group in comparison with the vehicle-control

groups. However, there were no significant

difference between garlic oil and silymarin

treated groups and vehicle-control groups

(p>0.05). Total leukocytic counts were

significantly increased in papilloma induced

group in comparison with the vehicle-control

groups (p<0.05). However, there were no

significant difference between garlic oil and

silymarin treated groups and vehicle-control

groups (p>0.05).

3- Effect of silymarin and garlic oil topical

application on GSH and GPx activities and

MDA concentrations in DMBA- induced

skin papilloma mouse:

The activities of GSH and GPx, as well as

MDA concentrations were measured in all

experimental groups (Table 3). There was a

significant decrease in the activities of GSH

and GPx as well as increase in MDA

concentrations in papilloma induced group in

comparison with the vehicle-control groups

(p<0.05). Topical application of garlic oil and

silymarin before and after DMBA application

caused significant increase in GSH and GPx

activities as well as decrease in MDA

concentrations when compared with the

carcinogenic group (p<0.05).

4- Effect of silymarin and garlic oil topical

application on histopathological changes in

DMBA- induced skin papilloma mouse:

Normal skin, uniformly arranged epidermal

and dermal layers with normal layer of

keratin over the epidermis, (Figs. 1) were

observed in all vehicle control groups. Skin

lesions in form of papillomas (Figs. 2),

abnormally thickened epidermis due to an

increase of the layers number: acanthosis,

hypergranulosis and hyperkeratosis with

fibrovascular core were observed, however,

the basement membrane is intact (Fig. 2, 3)

in all DMBA treated animals (100%,

incidence of skin lesions /group). The extent

of lesion was much less in garlic oil and

silymarin treated groups (Fig. 3, 4) when

compared with the papilloma induced group.

Topical application of garlic oil and

silymarin, on skin areas exposed to DMBA,

altered the incidence of skin lesions /group to

0% and 20 % of animals respectively, in

comparison with papilloma induced group

(100% of animals). Microscopically, 80% of

animals co-treated with silymarin showed

normal skin, however, 20% of animals

showed papillomas. Moreover, 60% of

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

126

animals co-treated with garlic showed normal

skin, however, 40% of animals showed focal

thickening of epidermis as well as focal

hyperkeratosis mild congestion and

inflammatory cells infiltration in the dermis

(Fig.4).

DISCUSSION

Skin cancer is the most common malignancy

in the world and also one of the major causes

of death worldwide. The development of skin

cancer is a stepwise process, consisting of

initiation, promotion and progression in

experimental animals and possibly in human

cancer (Meeran et al. 2009). Murine skin

carcinogenesis, an excellent in vivo model

used for screening of new cancer protective

agent, is a classical design for the study of

biological changes during carcinogenesis

(Saha and Hait 2012). A commonly used

two-stage model of skin carcinogenesis in

mice involves initiation by treatment of the

skin with 7, 12-dimethylbenz[a]anthracene

(DMBA) followed by promotion through

treatment with croton oil or its active

principle 12-O-tetradecanoylphorbol-13-

acetate (TPA). DMBA is a polycyclic

aromatic hydrocarbon, considered to be one

of the etiological factors for the mice cancers

as well as human cancers (Das and

Bhattacharya 2004). The croton oil is the

most widely used distinguished promoting

agent to understand the cellular and

molecular alterations associated with

promotion stage and also a well-known

model to understand the role of

inflammation, generation of reactive oxygen

species (ROS), and hyperplasia in cancer

promotion (Yaar 1995; Sharma and

Sultana 2004; Ha et al. 2006).

Currently, apoprevention is an important

strategy for controlling the process of cancer

induction. Therefore, there is a need to

explore medicinal plants or other natural

agents that can work as chemopreventive

agents. Chemoprevention with food

phytochemicals offers a new promise for

reducing the incidence and mortality of

cancer. Phytochemicals have the potential of

chemoprevention through several cellular

mechanisms; oxidative or electrophilic

stresses that can trigger a wide variety of

cellular events from which the increasing in

the expression of detoxifying enzymes and/or

antioxidant enzymes (Surh 2003; Chen and

Kong 2005). Therefore, the present study

was designed to investigate the potential

apopreventive effects of garlic oil and

silymarin on DMBA/croton oil-induced

initial and promotional changes in mouse

skin. For testing this possibility, garlic oil and

silymarin were applied topically one week

before and after the topical application of

DMBA and continued 12 weeks thereafter

prior to croton oil treatment.

Firstly, effect of garlic oil and silymarin

topical application on body weight and

mortality rate of DMBA- induced skin

papilloma mouse were measured. There was

a significant decrease in the average body

weight and increase in the mortality rate of

carcinogenic control group in comparison

with vehicle control groups. However, there

were no significant differences in the average

body weight between garlic oil and silymarin

treated groups and vehicle control groups.

The anti-carcinogenic, antibacterial, anti-

inflammatory, antithrombotic, fibrinolytic,

antioxidant, and wound-healing properties of

garlic have been well-documented in

previous studies (Saifzadeh et al. 2006;

Vazquez-Prieto et al. 2011; Santhosha et

al. 2013). Furthermore, it has been reported

that silymarin having multiple

pharmacological activities including

antioxidants, hepatoprotectant, and anti-

infilammatory agent, antibacterial, anti-

allergic, antiviral and anti-neoplastic

(Pradeep et al. 2007; Toklu et al. 2007).

Hepatoprotection and Enhancement of liver

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

127

functions may be could improve the

metabolic rate, body weight and decrease the

mortality rate.

Then, the effect of silymarin and garlic oil

topical application on hemogram of DMBA-

induced skin papilloma mouse were

measured. Significant decrease in RBCs, Hb,

PCV, MCH, and MCHC mean values as well

as increase in MCV and TLC of papilloma

induced group when compared with vehicle

control groups. But, there were no

significance differences between garlic oil

and silymarin treated mice and those of

vehicle control groups. These results indicate

presence of anemia in papilloma induced

group. Also, an increase in the number of

leukocytes (any or all type) above the normal

range usually part of an inflammatory

reaction. Generally, neoplastic lesions

usually associated with elevated numbers of

neutrophils and monocytes. As, macrophages

and neutrophils are both integrated in the

regulation of the innate and adaptive immune

responses in various inflammatory situations,

including cancer (Mantovani ‎2014).

Also, the effect of silymarin and garlic oil

topical application on some oxidative stress

markers in DMBA-induced skin papilloma

mouse were investigated. There was a

significant decrease in the activities of GSH

and GPx as well as increase in MDA

concentrations in carcinogenic group in

comparison with the vehicle-control groups.

Topical application with garlic oil and

silymarin before and after DMBA

application caused significant increase in

GSH and GPx activities as well as decrease

in MDA concentrations when compared with

the carcinogenic group. Oxidative stress has

long been implicated in cancer development

and progression (Hussain et al., 2003).

Repeated topical application of tumor

promoter croton oil on mouse skin involves

both oxidative burst as well as inflammation

by stimulating the generation of NO, reactive

nitric oxide species (RNS) and reactive

oxygen species (ROS) that plays an

important role in the process of mutagenesis

and carcinogenesis particularly tumor

promotion by increasing membrane lipid

peroxidation and decreasing cellular

antioxidant stores (Reed 2011; Das et al.

2012; Rauchová et al. 2012; Saha and Hait

2012). On the other hand, topical application

of garlic oil and silymarin confers

chemopreventive effects in terms of

reduction of lipid peroxidation and protection

against depletion of GSH and GPx. These

effects may be due to their potent antioxidant

properties.

Histopathological examination results

revealed that skin lesions in form of

papillomas, abnormally thickened epidermis

due to an increase of the layers number:

acanthosis, hypergranulosis and

hyperkeratosis with fibrovascular core were

observed, however, the basement membrane

is intact in all DMBA treated animals. During

the tumor promotion stage, repeated

application of TPA can trigger the production

of squamous papilloma and epidermal

hyperplasia, which is the pre-neoplastic

lesion of skin carcinoma (Shelton et al.

2000; Abel et al. 2009). These features have

been used routinely as quantitative markers

of pre-neoplastic lesions during

carcinogenesis via the measurement of the

level of epidermal thickness and the counting

of papilloma number (Cibin et al. 2011; Ko

et al. 2011). On the other hand, the extent of

lesion was much less in garlic oil and

silymarin treated groups when compared with

the papilloma induced group. Topical

application of garlic oil and silymarine, on

skin areas exposed to DMBA, altered the

incidence of skin lesions /group in

comparison with carcinogenic group. It has

been documented that garlic oil and silymarin

are sources of many anticarcinogenic agents

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

128

and antioxidants, which may be useful for the

prevention of cancer. Lan et al. (2013)

documented that garlic oil has potent anti-

tumor property by inhibiting cells growth via

anti-proliferation and inducing apoptosis.

Also, Seki et al. (2008) reported that

phytochemical garlic-derived diallyl

trisulfide, a major constituent of the garlic

oil, has potent anticancer property. Moreover,

silymarin has a strong antioxidant capability

of scavenging free-radicals (Wellington and

Jarvis 2001) and several short-term studies

have suggested that silymarin may be a

potent anti-carcinogenic agent (Kren and

Walterova 2005).

In conclusion, the results of the present

study exhibits apopreventive potential of

garlic oil and silymarin on DMBA-induced

skin papilloma in male Swiss albino mice.

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

129

Table 1. Effect of garlic oil and silymarin topical application on body weight and mortality

rate of DMBA- induced skin papilloma mouse.

Groups

Body weight (g)

Numbers of mouse

Mortality rate

(%) Initial

Final

Initial

Final

CAR

29.00±0.33

22.95±0.67*

15

5

66.67

CAR+SIL 29.00±0.37

28.35±0.29

15 12 20

CAR+GAR 28.81±0.25

28.91±0.23

15 12 20

CON1 27.55±0.29

29.50±.028

15 13 13.33

CON2 26.50±0.50

27.50±0.44 15 13 13.33

CAR means carcinogenic group (DMBA and croton oil); CAR+SIL means DMBA and croton oil plus silymarin;

CAR+GAR means DMBA and croton oil plus garlic; CON1 means vehicle control group number 1(acetone); CON2

means vehicle control group number 2 (corn oil). Data are expressed as means ± SD. * Indicate significant

difference between the treatments at p<0.05.

Table 2. Effect of silymarin and garlic oil topical application on hemogram of DMBA- induced skin papilloma mouse.

Groups

RBCs

(x106/µl)

Hb

(g/dl)

PCV (%)

MCV (%)

MCH (%)

MCHC

(%)

WBCs

(x103/µl)

CAR 6.35±0.25* 9.80±0.20*

29.03±0.51* 46.00±0.41* 9.70±0.83* 21.30±1.67* 5.40±1.56*

CAR+SIL 8.85±0.33 13.00±0.29

38.88±1.10 44.00±0.71 14.75±0.38 33.23±0.23 2.60±1.5

CAR+GAR 9.01±0.27 13.15±0.34

39.25±0.78 43.75±0.25 14.60±0.33 33.40±0.30 3.03±0.55

CON1 8.54±0.56 11.87±0.69

35.23±1.94 43.00±1.15 14.00±0.43 33.66±0.13 2.95±0.10

CON2 9.04±0.28

13.00±0.21

38.98±0.84 43.50±0.50 14.40±0.23 33.28±0.19 4.00±0.14

CAR means carcinogenic group (DMBA and croton oil); CAR+SIL means DMBA and croton oil plus silymarin;

CAR+GAR means DMBA and croton oil plus garlic; CON1 means vehicle control group number 1(acetone); CON2

means vehicle control group number 2 (corn oil). Data are expressed as means ± SD. * Indicate significant

difference between the treatments at p<0.05.

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

130

Table 3. Effect of silymarin and garlic oil topical application on GSH and GPx activities and MDA concentrations in DMBA- induced skin papilloma mouse.

GPx (U/ml)

GSH (µmol/ml) MDA (nmol/ml) Groups

1.11±0.07* 6.73±0.53* 19.72±0.59* CAR

1.24±0.03* 8.46±0.54* 17.55±1.03* CAR+SIL

1.31±0.10* 10.13±0.83* 17.83±0.48* CAR+GAR

1.26±0.03 9.73±1.48 12.67±2.10 CON1

1.26±0.04 10.92±0.80 10.95±1.69 CON2

CAR means carcinogenic group (DMBA and croton oil); CAR+SIL means DMBA and croton oil plus silymarin;

CAR+GAR means DMBA and croton oil plus garlic; CON1 means vehicle control group number 1(acetone); CON2

means vehicle control group number 2 (corn oil). Data are expressed as means ± SD. * Indicate significant

difference between the treatments at p<0.05

Fig. 1. Skin section in group V (corn oil) showing normal histology with uniformly arranged

epidermal (E) and dermal layers (D) as well as normal layer of keratin (K) over the epidermis.

(H & E, X 100).

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

131

Fig. 2. Skin section in DMBA treated mice (GI) showing papilloma, abnormally thickened

epidermis due to increase of the layers number: acanthosis, hypergranulosis and hyperkeratosis

with intact basement membrane. (H & E, X 100).

Fig. 3. Higher magnification of Fig. 2 showing papilloma, abnormally thickened epidermis,

acanthosis (A) as well as hyperkeratosis (H) along with fibrovascular core (arrow) with intact

basal cells layer (B). (H & E, X 200).

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

132

Fig. 4. Skin section in group II (DMBA+ garlic) showing inflammatory cells infiltration in the

dermis and mild congestion. (H & E, X 100).

REFERENCES Abel, E.L., Angel, J.M., Kiguchi, K. and

DiGiovanni, J. (2009): Multi-stage chemical

carcinogenesis in mouse skin: fundamentals

and applications. Nat Protoc, 4:1350-1362.

Bancroft, J.D. and Gamble, M. (2007): Theory and Practice of Histological

Techniques. 5th Ed; Churchill Livingstone,

London, UK, pp: 125-138.

Beutler, E., Duron, O. and Kelly, B.M.

(1963): Improved method for the

determination of blood glutathione. J Lab

Clin Med. 61: 882-888.

Bishayee, A., Oinam, S., Basu, M. and

Chatterjee, M. (2000): Vanadium

chemoprevention of 7, 12-dimethylbenz (a)

anthracene-induced rat mammary

carcinogenesis: probable involvement of

representative hepatic phase I and II

xenobiotic metabolizing enzymes Breast

Cancer Research Treatment 63:133–145.

Block, E. (1985): The chemistry of garlic

and onions. Scientific American, 252: 114-

119.

Borek, C. (1997): Antioxidants and cancer.

Science & Medicine, 4: 51-62.

Chen, C. and Kong, A.N. (2005): Dietary

cancer-chemopreventive compounds:from

signaling and gene expression to

pharmacological effects. Trends Pharmacol.

Sci., 26: 318–26.

Cibin, T.R., Devi, D.G. and Abraham, A.

(2012): Chemoprevention of two-stage skin

cancer in vivo by Saraca asoca. Integr Cancer

Ther., 11: 279-286.

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

133

Cotgreave, I.A., Moldeus, P. and Orrenius,

S. (1988): Host Biochemical Defense

Mechanisms against Prooxidants. Annual

Review of Pharmacology and Toxicology

28:189–212.

Das, I., Acharya, A., Berry, D.L., Sen, S.,

Williams, E., Permaul, E., Sengupta, A.,

Bhattacharya, S. and Saha, T. (2012):

Antioxidative effects of the spice cardamom

against non-melanoma skin cancer by

modulating nuclear factor erythroid-2-related

factor 2 and NF-κB signalling pathways. Br J

Nutr, 108: 984-97.

Das, R.K. and Bhattacharya, S. (2004): Inhibition of DMBA-Croton oil Two stage

Mouse skin Carcinogenesis by

Diphenylmethyl Selenocyanate through

Modultion of Cutaneous Oxidative Stress and

Inhibition of Nitric Oxide Production. Asian

Pacific J. Cancer Prevention 5: 151-158.

Esterbauer, H., Cheeseman, K.H.,

Danzani, M.U., Poli, G. and Slater, T.F.

(1982): Separation and characterization of

the aldhyde products of ADP/Fe2+C

stimulated lipid peroxidation in rat liver

microsomes. Biochem. J. 208: 129-140.

Gazak, R., Walterova, D. and Kren, V.

(2007): Silybin and silymarin–new and

emerging applications in medicine. Curr Med

Chem 14: 315–338.

Gross, R.T., Bracci, R., Rudolph, N.,

Schroeder, E. and Kochen, J.A. (1967):

Hydrogen peroxide toxicity and

detoxification in the erythrocytes of newborn

infants. Blood, 29: 481–493.

Gupta, S. and Mukhtar, H. (2002):

Chemoprevention of skin cancer: current

status and future prospects. Cancer

Metastasis Rev, 21: 363-380.

Ha, H.Y., Kim, Y., Ryoo, Z.Y. and Kim,

T.Y. (2006): Inhibition of the TPA induced

cutaneous inflammation and hyperplasia by

EC–SOD. Biochem. Biophys Res. Commun.,

348:450–458.

Hara-Chikuma, M. and Verkman, A.S.

(2008): Prevention of skin tumorigenesis and

impairment of epidermal cell proliferation by

targeted aquaporin-3 gene disruption. Mol.

Cell Biol., 28: 326-332.

Huachen, W. and Krystyna, F. (1991): In

vivo formation of oxidized DNA bases in

tumor promoter- treated mouse skin. Cancer

Res., 51: 4443-4449.

Hussain, S.P., Hofseth, L.J. and Harris,

C.C. (2003): Radical causes of

cancer. Nature Reviews Cancer, 3: 276–285.

Jagetia, G.C. and Rao, S.K. (2006): Evaluation of the antineoplastic activity of

Guduchi (Tinospora cordifolia) in Ehrlich

Ascites carcinoma bearing mice. Biological

and Pharmaceutical Bulletin 29: 460-466.

Kim, J.Y. and Kwon, O. (2009): Garlic

intake and cancer risk: an analysis using the

Food and Drug Administration's evidence-

based review system for the scientific

evaluation of health claims. Am J Clin Nutr.

89:257-264.

Ko, J.H., Jung, B.G., Park, Y.S. and Lee,

B.J. (2011): Inhibitory effects of interferon-

gamma plasmid DNA on DMBA-TPA

induced mouse skin carcinogenesis. Cancer

Gene Ther, 18: 646- 654.

Kren, V. and Walterova, D. (2005): Silybin

and silymarin--new effects and applications.

Biomed Pap Med FacUnivPalacky Olomouc

Czech Repub 149:29-41.

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

134

Lan, X., Sun, H., Liu, J., Lin, Y., Zhu, Z.,

Han, X., Sun, X., Li, X., Zhang, H. and

Tang, Z. (2013): Effects of garlic oil on

pancreatic cancer cells. Asian Pac J Cancer

Prev. 14:5905-5910.

Lawson, L. D., Wood, S. G. and Hughes, B.

G. (1991): HPLC analysis of allicin and

other thiosulfinates in garlic clove

homogenates. Planta Med. 57: 263–270.

Mantovani, A. (2014): Macrophages,

Neutrophils, and Cancer: A Double Edged

Sword. New Journal of Science. Volume

2014, Article ID 271940, 14 pages

Meeran, S.M., Vaid, M., Punathil, T. and

Katiyar, S.K. (2009): Dietary grape seed

proanthocyanidins inhibit 12-O-tetradecanoyl

phorbol-13-acetate-caused skin tumor

promotion in 7, 12-

dimethylbenz[a]anthracene-initiated mouse

skin, which is associated with the inhibition

of inflammatory responses. Carcinogenesis,

30: 520-528.

Nebert, D.N., Dalton, T.P., Okey, A.B. and

Gonzalez, F.J. (2004): Role of aryl

hydrocarbon receptor-mediated induction of

the CYP1 enzymes in environmental toxicity

and cancer. The Journal of Biological

Chemistry 279: 23847–23850.

Neef, J.M. (1985): Fundamental of Aquatic

Toxicology. New York: Hamisphere

Publication pp: 416–454.

Polyak, S.J., Ferenci, P., and Pawlotsky,

J.M. (2013): Hepatoprotective and Antiviral

Functions of Silymarin Components in HCV

Infection. Hepatology (Baltimore, Md.), 57:

1262–1271.

Powolny, A.A. and Singh, S.V. (2008): Multitargeted prevention and therapy of

cancer by diallyl trisulfide and related Allium

vegetable-derived organosulfur compounds.

Cancer Lett. 269: 305–314.

Pradeep, K., Mohan, C.V., Gobianand, K.

and Karthikeyan, S. (2007): Silymarin

modulates the oxidants-antioxidant

imbalance during diethylnitrosamine induced

oxidative stress in rats. Eur. J. pharmacol.

560: 110-116.

Rauchová, H., Vokurková, M. and

Koudelová, J. (2012): Hypoxia-induced

lipid peroxidation in the brain during

postnatal ontogenesis. Physiol Res, 24: 89-

101.

Reed, T.T. (2011): Lipid peroxidation and

neurodegenerative disease. Free Radic Biol

Med, 51: 1302-1319.

Saha, D. and Hait, M. (2012): An

ontological design: two stage mouse skin

carcinogenesis induced by DMBA and

promoted by croton oil. Asian J Pharm Sci, 2:

1-3.

Saifzadeh, S., Tehrani, A., Jalali, F.S.S.

and Oroujzadeh, R. (2006): Enhancing

effect of aqueous garlic extract on wound

healing in the dog: clinical and

histopathological studies. J Anim Vet Adv

12:1101–1104.

Santhosha, S.G., Prakash Jamuna S.N.

and Prabhavathi. (2013): Bioactive

components of garlic and their physiological

role in health maintenance: a review. Food

Biosci 3:59–74.

Scandalios, J.G. (2005): Oxidative stress:

molecular perception and transduction of

signals triggering antioxidant gene defenses.

Braz J Med Biol Res, 38: 995-1014.

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

135

Seki, T., Hosono, T., Hosono-Fukao, T.,

Inada, K., Tanaka, R., Ogihara, J. and

Ariga, T. (2008): Anticancer effects of

diallyl trisulfide derived from garlic. Asia

Pac J Clin Nutr. 17:249-252.

Sharma, S. and Sultana, S. (2004):

Modulatory effect of soy isoflavones on

biochemical alterations mediated by TPA in

mouse skin model. Food Chem Toxicol.

42:1669–75.

Shelton, S.D., Cherry, V. and

Manjanatha, M.G. (2000): Mutant

frequency and molecular analysis of in vivo

lacI mutations in the bone marrow of Big

Blue rats treated with 7, 12- dimethylbenz (a)

anthracene. Environ Mol Mutagen, 36: 235-

242.

Shrotriya, S., Kundu, J. K., Na, H. K. and

Surh, Y. J, (2010): Diallyl trisulfide inhibits

phorbol ester-induced tumor promotion,

activation of AP-1, and expression of COX-2

in mouse skin by blocking JNK and Akt

signaling. Cancer Res. 70: 1932–1940.

Singh, R.P., Dhanalakshmi, S., Tyagi,

A.K., Chan, D.C., Agarwal, C. and

Agarwal, R. (2002): Dietary feeding of

silibinin inhibits advance human prostate

carcinoma growth in athymic nude mice and

increases plasma insulin-like growth factor-

binding protein-3 levels. Cancer Res.

62:3063–3069.

Singh, R.P., Mallikarjuna, G.U., Sharma,

G., Dhanalakshmi, S., Tyagi, A.K., Chan,

D.C., Agarwal, C. and Agarwal, R. (2004):

Oral silibinin inhibits lung tumor growth in

athymic nude mice and forms a novel

chemocombination with doxorubicin

targeting nuclear factor kappaB-mediated

inducible chemoresistance. Clin Cancer Res

10: 8641–8647

Slaga, T. J. (1983): Mechanisms involved in

multistage chemical carcinogenesis in mouse

skin. In J. Rydström, editor; J. Montelius,

editor; , and M. Bengtsson, editor. , eds.

Extrahepatic Drug Metabolism and Chemical

Carcinogenesis. Elsevier, New York. Pp:

577-585

Surh, Y.J. (2003): Cancer chemoprevention

with dietary phytochemicals. Nat Rev Cancer

3: 768–780.

Toklu, H., Tunali-Akbay, T., Erkanli, G.,

Yuksel, M., Ercan, F. and Sener, G.

(2007): Silymarin, the antioxidant component

of Silybum marianum, protects against burn-

induced oxidative skin injury. Burns. 33:

908-916.

Tyagi, A., Raina, K., Singh, R.P., Gu, M.,

Agarwal, C., Harrison, G., Glode, L.M.

and Agarwal, R. (2007): Chemopreventive

effects of silymarin and silibinin on N-butyl-

N-(4-hydroxybutyl) nitrosamine induced

urinary bladder carcinogenesis in male ICR

mice. Mol. Cancer Ther. 6:3248–3255.

Vazquez-Prieto, M.A., Lanzi, C.R.,

Lembo, C., Galmarini, C.R. and Miatello,

R.M. (2011): Garlic and onion attenuates

vascular inflammation and oxidative stress in

fructose-fed rats. J Nutr Metab 2011; 475216.

Visconti, R. and Grieco, D. (2009): New

insights on oxidative stress in cancer. Curr

Opin Drug Discov Devel, 12: 240-245.

Wellington, K. and Jarvis, B. (2001):

Silymarin: a review of its clinical properties

in the management of hepatic disorders.

BioDrugs. 15:465-489.

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 29 NO.1, 2016 ; 121- 136

136

World Health Organization. Ultraviolet

radiation and the INTERSUN

Programme. http://www.who.int/uv/faq/

skincancer/en/index1.html (Retrieved 1

March 2013).

Wu, C.C., Chung, J.G., Tsai, S.J., Yang,

J.H. and Sheen, L.Y, (2004): Differential

effects of allyl sulfides from garlic essential

oil on cell cycle regulation in human liver

tumor cells. Food Chem. Toxicol. 42: 1937–

1947.

Yaar, M. (1995): Molecular Mechanism of

Skin Aging. Advances in Dermatology. 10:

63-75.

Yang, J.S., Chen, G.W., Hsia, T.C., Ho, H.

C., Ho, C.C., Lin, M.W., Lin, S.S., Yeh,

R.D., Ip, S.W., Lu, H.F. and Chung, J.G.

(2009): Diallyl disulfide induces apoptosis in

human colon cancer cell line (COLO 205)

through the induction of reactive oxygen

species, endoplasmic reticulum stress,

caspases casade and mitochondrial-dependent

pathways. Food Chem. Toxicol., 47: 171–

179.