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Toxicology 302 (2012) 266– 274

Contents lists available at SciVerse ScienceDirect

Toxicology

j ourna l ho me pag e: www.elsev ier .com/ locate / tox ico l

oy isoflavones (daidzein & genistein) inhibit2-O-tetradecanoylphorbol-13-acetate (TPA)-induced cutaneous inflammationia modulation of COX-2 and NF-�B in Swiss albino mice

bdul Quaiyoom Khan, Rehan Khan, Muneeb U. Rehman, Abdul Lateef, Mir Tahir,arrah Ali, Sarwat Sultana ∗

ection of Molecular Carcinogenesis and Chemoprevention, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi10062, India

r t i c l e i n f o

rticle history:eceived 17 July 2012eceived in revised form 23 August 2012ccepted 24 August 2012vailable online 4 September 2012

eywords:soflavonesnflammationroinflammatory cytokines

a b s t r a c t

It is well established that aberrant production of inflammatory mediators has been associated with mostthe toxic manifestations and the genesis of different chronic diseases including cancer. The basic aimof the present study is to investigate the effects of soy isoflavones (SIF) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cutaneous inflammatory responses and to explore the underlying molecularmechanisms. We have studied the protective effects of SIF against TPA induced oxidative stress, pro-inflammatory cytokines level, activation of NF-�B, expression of COX-2 and ki-67 in mouse skin. Animalswere divided into five groups I–V (n = 6). Groups II, III and IV received topical application of TPA at the doseof 10 nmol/0.2 ml of acetone/animal/day, for 2 days. Animals of the groups III and IV were pre-treated withSIF 1.0 �g (D1) and 2.0 �g (D2) topically 30 min prior to each TPA administration, while groups I and V

F-�B were given acetone (0.2 ml) and SIF (D2), respectively. We have found that SIF pretreatment significantlyinhibited TPA induced oxidative stress, proinflammatory cytokines production and activation of NF-�B.SIF also inhibited the expression of COX-2 and ki-67. Histological findings further supported the protectiveeffects of SIF against TPA-induced cutaneous damage. Thus, our results suggest that inhibitory effect of SIFon TPA-induced cutaneous inflammation includes inhibition of proinflammatory cytokines, attenuationof oxidative stress, activation of NF-�B and expression of COX-2.

. Introduction

Inflammation, one of the important universal physiologicalesponses associated with the process of carcinogenesis, catal-ses the conversion of pre-malignancy to the malignancy. Bothcute inflammation and chronic inflammation act as mediatorsf various degenerative disorders including skin cancer. Improper

egulation of redox sensitive signal transduction pathways inducedy various inflammatory stimuli has been implicated in tumourromotion and carcinogenesis. Exposure to various physical and

Abbreviations: BSA, bovine serum albumin; CAT, catalase; D1, dose one1.0 �g); D2, dose two (2.0 �g); DTNB, 5,5′-dithio bis-[2-nitrobenzoic acid]; GSH,educed glutathione; IL, interleukin; NF-�B, nuclear factor-kappa B; NO, nitricxide; ROS, reactive oxygen species; RNS, reactive nitrogen species; SOD, super-xide dismutase; TBA, thiobarbituric acid; TNF, tumour necrosis factor; TPA-12-O,etradecanoylphorbol-13-acetate.∗ Corresponding author at: Jamia Hamdard, Department Of Medical Elementology

nd Toxicology, New Delhi 110062, India.el.: +91 11 26054685x5565/5566/26089688; fax: +91 11 26059663.

E-mail address: sarwat786@rediffmail.com (S. Sultana).

300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.tox.2012.08.008

© 2012 Elsevier Ireland Ltd. All rights reserved.

chemical agents are known to induce different biological eventslike infiltration of neutrophils which are key producers of reactiveoxygen species (ROS) and reactive nitrogen species (RNS), cytokinesand prostaglandins (Murakami et al., 2000). So for evidence fromvarious approaches reveals the critical role of proinflammatorycytokines such as TNF-�, IL-6, IL1-�, cycloxygense-2 and NF-�B in the inflammation driven carcinogenesis (Allavena et al.,2008).

Development of cancer is one of the highly complex heterogenicand multistage, viz., initiation promotion and progression, process.Promotion, a reversible and long term process, appears to be prac-tically best for the intervention strategies than at the initiationstage.

Topical application of phrobol ester induces inflammatoryresponses, mediated by different cytokines and regulatory factors,which are closely associated with promotion phase of carcinogene-sis. In response to proinflammatory stimuli, activated inflammatory

cells generate ROS and RNS; over production of ROS leads to oxida-tive stress, a condition which is critically associated with most of thehuman diseases, and plays important role in inflammation-drivencarcinogenesis (Cerutti and Trump, 1991).

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Promotion phase of carcinogenesis is a complex step thatnvolves cell proliferation, inflammation and oxidative stress-

ediated signal transduction. TPA is the most widely usedistinguished tumour promoting agent to understand the molec-lar and cellular alterations associated with carcinogenesis as it

nduces oxidative stress, ROS production, inflammation and hyper-lasia (Riehl et al., 2010; Ha et al., 2006). Research studies haveevealed that it is a well known model to understand the role ofOS, inflammation and hyperplasia in the promotion stage of car-inogenesis (Khan et al., 2012; Ha et al., 2006).

In recent years, attention has been given to the use of naturallyccurring compounds and their formulations for the prevention ofifferent chronic diseases (Sarkar and Li, 2007; Anthony et al., 1998;arnes, 1998), particularly those with inflammatory mechanism

n which reactive moieties are formed. Epidemiological findingseveal that use of natural compounds is a well promising approachor the chemoprevention and management of human cancers (Zhaot al., 2010; Nakachi et al., 1996). Chemoprevention is a strategy ofancer control by administration of one or more naturally occur-ing and or synthetic compounds to block initiation or suppress orelay promotion/progression of carcinogenesis (Surh, 2003).

Isoflavones are some of them and exert protective effectsgainst a series of cancer modules in vitro and in vivoMessina et al., 1994). Soybeans, most widely used in Asianountries, are the rich source of biologically active isoflavones,uch as genistein (4,5,7-trihydroxyisoflavone) and daidzein (4,7-ihydroxyisoflavone) known to have spectrum of biologicalctivities (Tikkanen et al., 1998; Setchell and Adlercreutz, 1988).pidemiological observations reveal that use of soy products asso-iated with reduced incidence of breast and prostate cancers, heartisease, etc. (Clarkson et al., 1995).

On the basis of above facts we hypothesise that interventiontrategies targeting inflammatory pathways may prevent TPA-nduced early tumour promotional changes and provide somensights to understand the underlying mechanism of action of soysoflavones. So we have assessed the protective effects of SIF pre-reatment on TPA induced cutaneous oxidative stress, epidermalyperplasia and inflammation.

. Materials and methods

.1. Chemicals and reagents

Griess reagent, thiobarbituric acid (TBA), pyrogallol, poly-l-lysine, bovineerum albumin (BSA), Mayer’s haematoxylin, 5,5′-dithio-bis-[2-nitrobenzoic acid]DTNB), 12-O-tetradecanolyphorbal-13-acetate (TPA) etc., were purchased fromigma–Aldrich Co. (St. Louis, MO, USA). Hydrogen peroxide, sulphosalicylic acid,richloroacetic acid (TCA), tween-20, Folin–Ciocalteau reagent (FCR), tri-sodiumitrate (di-hydrate), di-sodium hydrogen phosphate, sodium di-hydrogen phos-hate and sodium hydroxide were purchased from E. Merck Limited, India. All thether reagents used were of highest purity and commercially available.

.2. Animals

Eight weeks old female Swiss albino mice (25–30 g), free from infection, werebtained from the Central Animal House facility of Hamdard University, New Delhi,ndia. Animals were housed in a well-ventilated room at 25 ◦C under a light–darkycle in polypropylene cages and have free access to standard laboratory feed (Hin-ustan Lever Ltd., Bombay, India) and water. Present study protocols were approvedy the Animal Ethics Committee (IAEC) of the university and animals were under-one to experiment with the approved ethical guidelines.

.3. Treatment protocol

Animals were divided into five groups (I–V) with six animals in each (n = 6).orsal skin of all the animals was shaven with the electric clipper 2 days prior to

he start of the experiment. Only mice that showed no signs of hair regrowth were

ncluded in the experiments. All the treatments were done topically onto the shavedrea of dorsal skin.

Group I Animals of this group were given topical application of vehicle (0.2 mlacetone) only and served as vehicle control group.

302 (2012) 266– 274 267

Group II Mice were treated with the topical application of only TPA (10 nmol) in0.2 ml acetone.

roup III Animals of the group III were given pretreatment of SIF at the dose of 1.0 �g(D 1) in 0.2 ml acetone, 30 min before TPA [(10 nmol) in 0.2 ml acetone]application.

roup IV Animals of the group IV were given pretreatment of SIF at the dose of 2.0 �g(D 2) in 0.2 ml acetone, 30 min before TPA [(10 nmol) in 0.2 ml acetone]application.

Group V Animals of this group were given topical application of higher dose (D2)of SIF only.

The Nova Soy isoflavone Product yields 33 mg of genistein and 67 mg of daidzeinin 100 mg product therefore, the dose 1 amounts to 0.33 �g and 0.67 �g of genisteinand daidzein, respectively, while dose 2 corresponds to about 0.66 �g and 1.34 �gof genistein and daidzein, respectively.

The treatments were carried out for 2 days at an interval of 24 h. Animals of theall the group were sacrificed by cervical dislocation 1 h after the last TPA treatmentand skin tissue was processed for the evaluation of different parameters.

2.4. Tissue processing

Animals were sacrificed 1 h after the second TPA application by cervical disloca-tion and skin tissue was processed for sub cellular fractionation. A piece of skin waspreserved in 10% neutral buffered formalin for histological observations. 10% tissuehomogenates were prepared in chilled phosphate buffer (0.1 M, pH 7.4) using Poly-tron homogeniser (Kinematica, Inc., Switzerland). The homogenised tissues werecentrifuged at 10,500 × g for 30 min at 4 ◦C to obtain the post mitochondrial super-natant (PMS).

2.4.1. Oedema measurementEffect of SIF on TPA induced skin oedema was assessed by using the method

of Afaq et al. (2005). Skin punch (1 cm diameter, free from extraneous materials)was weighed before and 24 h after drying at 50 ◦C and the loss of water contentwas determined. Increase in the mass of skin punch is directly proportional to thedegree of inflammation. The extent of skin oedema was determined by using thedifferences in the water gain between control and treated groups.

2.4.2. Nitric oxide (NO) estimationProduction of NO, after SIF and TPA application in the skin tissue, was evalu-

ated by measuring the level of nitrite (an indicator of NO) in the tissue supernatantwith the help of Griess reagent (Green et al., 1982). Briefly, 150 �l of tissue super-natant was mixed with 150 �l Griess reagent [0.1% N-(1-naphthyl) ethylenediaminedihydrochloride, 1% sulphanilamide and 2.5% H3PO4]. After incubation at room tem-perature in the dark for 10 min, absorbance was measured with a microplate ELISAreader (BioRAD, U.S.A.) at 540 nm.The assay for lipid peroxidation was done by themethod of Wright et al. (1981), with slight modifications

2.4.3. Lipid peroxidation assayThe assay for lipid peroxidation was done by the method of Wright et al. (1981),

with slight modifications. Reaction mixture in a total of 3 ml containing 1 ml of (10%tissue homogenate), 1.0 ml of TCA (10%), and 1.0 ml TBA (0.67%). Reaction mixturewas incubated in boiling water for 45 min. After cooling the tubes were centrifugedat 2500 × g for 10 min. Absorbance of the supernatant was taken at 525 nm. Rate oflipid peroxidation was assessed by measuring MDA content as nmol MDA formed/gtissue at 37 ◦C by using a molar extinction coefficient of 1.56 × 105 M−1 cm−1.

2.4.4. Estimation of reduced glutathioneGSH content of the skin tissue was determined by the method of Jollow et al.,

1974. Briefly 1 ml of PMS fraction (10%) was mixed with 1.0 ml of sulphosalicylic acid(4%). The samples were incubated at 4 ◦C for 1 h and then centrifuged at 1200 × gfor 15 min at 4 ◦C. The assay mixture (3 ml) consists of 0.4 ml supernant, 2.2 mlphosphate buffer (0.1 M, pH 7.4) and 0.4 ml DTNB (4 mg/1 ml). The yellow colourdeveloped was read immediately at 412 nm. The GSH concentration was calculatedas nmol DTNB conjugate formed/g tissue.

2.4.5. Assay for catalase activityCatalase activity was assayed by the method of Claiborne (1985). Briefly the

reaction mixture consists of 1.95 ml phosphate buffer (0.1 M, pH 7.4), 1.0 ml hydro-gen peroxide (0.019 M) and 0.05 ml of tissue PMS in a final volume of 3 ml. Changesin absorbance were recorded at 240 nm. Catalase activity was calculated as nmolH2O2 consumed per min per mg protein.

2.4.6. Measurement of superoxide dismutase (SOD) activityThe SOD activity was measured by the method of Marklund and Marklund

(1974). The reaction mixture consisted of 2.875 ml Tris–HCl buffer (50 mM, pH 8.5),pyrogallol (24 mM in 10 mM HCl) and 100 �l of tissue PMS in a total volume of3 ml. The enzyme activity was measured at 420 nm and was expressed as units/mgprotein. One unit is defined as the enzyme activity that inhibits auto-oxidation ofpyrogallol by 50%.

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.4.7. Measurement of proinflammatory cytokinesThe level of TNF-�, IL-6 and IL1-� was measured by enzyme-linked immunosor-

ent assay (ELISA) kit (eBioscience, Inc., San Diego, USA). Samples were prepared inhosphate buffered saline (1× PBS, pH 7.4) containing protease inhibitor cocktail.nalysis was performed according to the manufacturer’s instruction.

.5. Histological investigation

For histopathology study, the skin was removed and immediately fixed in freshlyrepared 10% neutral buffered formalin. Then, the skin was embedded in paraffinax. A vertical section of skin (5 �m thick) was cut and stained with haematoxylin

nd eosin (H & E). The leucocytes infiltration and epidermal thickness were observeds an indicator of histological changes with microscope (Fluorescent Microscope,lympus) at least in six different regions.

.6. Immunohistochemical detection of NF-�B (p65), COX-2 and Ki-67

The dorsal skin tissues were first fixed in formalin and then paraffinised. Sec-ions of 5 �m thickness were cut onto poly-lysine coated glass slides. Sectionsere deparaffinised three times (5 min each) in xyline fallowed by dehydration in

raded ethanol and finally rehydrated in running tap water. For antigen retrievalections were boiled in 10 mM citrate buffer (pH 6.0) for 5–7 min. Sections werencubated with hydrogen peroxide for 15 min to minimise nonspecific stainingnd then washed three times (5 min each) with 1× TBST (0.05% Tween-20). Sec-ions were incubated first with power block for 10 min and then for overnightith the primary antibodies at 4 ◦C. Further processing was done by using Ultraision plus Detection System Anti-Polyvalent, HRP/DAB (Ready-To-Use) stainingit from Thermo Scientific System. The peroxides complex was visualised with 3,3-iaminobenzidine. Lastly the slides were counterstained with haematoxylin. Slidesere then cleaned in xyline, gradually dehydrated with ethanol, after DPX mountingicroscopic (Fluorescent Microscope, Olympus) analysis was done. Primary anti-

odies, rabbit anti-COX-2 (dilution 1:200, Santa Cruz), rabbit anti-NF-�B (1:300,iolegend) and ki-67 (dilution 1:200, Thermo Scientific).

.7. Protein estimation

Protein content in the skin tissue was estimated by the method of Lowry et al.1951), using bovine serum albumin (BSA) as standard.

.8. Statistical analysis

The data from individual groups are presented as the mean ± standard error ofhe mean (SEM). Differences between groups were analysed by using one way anal-sis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons test andinimum criterion for statistical significance was set at p < 0.05 for all comparisons.

ig. 1. Effect of SIF pre-treatment on TPA induced cutaneous: (a) oedema formation, (b) lipn = 6). ***p < 0.001, **p < 0.01 shows significant difference in group II [TPA (10 nmol) in 20he significant difference in the group III [SIF (1 �g) + TPA (10 nmol)] and IV [SIF (2 �g) + T

302 (2012) 266– 274

3. Results

3.1. Effect of SIF (daidzein & genistein) on oedema

Application of TPA leads to massive cutaneous inflammation andwas assessed by oedema formation. We have found that double TPAapplication in the group II resulted in massive oedema formation(p < 0.001) in comparison with the acetone treated group I. How-ever, pre-treatment with SIF in the groups III and IV significantly(p < 0.01, p < 0.05) inhibits oedema formation as compare to groupII. There was no significant difference observed between groups Iand V, Fig. 1(a).

3.2. Effect of SIF (daidzein & genistein) on TPA induced oxidativestress in mouse skin

SIF inhibits lipid peroxidation caused by TPA application interms of TBARS (MDA) formation, a well known biomarker of oxida-tive stress. Double application of TPA causes significant elevationin the level of MDA in group II as compared to that of the acetonetreated group I (p < 0.001). Pre-treatment with soy isoflavones ingroup III and IV half an hour before TPA application was found sig-nificantly (p < 0.01, p < 0.001) effective in the amelioration of MDAlevel. There was no significant change observed in the level ofMDA formation between control and only SIF (D2) treated animals,Fig. 1(b).

3.3. Effect of SIF (daidzein & genistein) on the cutaneous NOproduction

Topical application of TPA at the dose of 10 nmol resulted in

the elevated NO production in group II when compared with theacetone treated group I (p < 0.01). We have observed that pre-treatment with SIF was significantly effective in the reducing theNO production in groups III and IV (p < 0.05) when compared with

id peroxidation and (c) nitric oxide production. Values are expressed as mean ± SEM0 �l acetone] when compared with group I. #p < 0.05, ##p < 0.01, ###p < 0.001 showsPA (10 nmol)] when compared with group II.

A.Q. Khan et al. / Toxicology 302 (2012) 266– 274 269

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ig. 2. Effect of SIF pre-treatment on endogenous anti-oxidants: (a) GSH level, (b) SOhows significant difference in group II [TPA (10 nmol) in 200 �l acetone] when comII [SIF (1 �g) + TPA (10 nmol)] and IV [SIF (2 �g) + TPA (10 nmol)] when compared w

he group II. There was no significant difference observed betweenroups I and V as far as NO production is concerned, Fig. 1(c).

.4. Effect of SIF (daidzein & genistein) on the cutaneous GSHontent

TPA application causes significant depletion in the level of GSHontent in group II when compared with group I (p < 0.001). Pre-reatment with SIF in groups III and IV had shown a significantp < 0.05, p < 0.01) increase in the level of GSH content as comparedith group II. There was no significant difference in the GSH content

etween groups I and V, Fig. 2(a).

.5. Effect of SIF (daidzein & genistein) on the antioxidantnzymes

The effect of SIF pre-treatment on TPA induced alteration in thectivity of antioxidant enzymes (SOD and CAT) was assayed andhe results were shown in Fig. 2(b) and (c), respectively. We havebserved that there was a significant (p < 0.001) depletion in thectivity of both the antioxidant enzymes in group II as comparedo group I. However, pretreatment with SIF in the group III and IVefore TPA application leads to the significant (p < 0.05) restoration

n the activity of antioxidant enzymes when compared with thePA treated group II. There was no significant difference observedetween the groups I and V as far as the activity of antioxidantnzymes was concerned.

.6. Effect of SIF (daidzein & genistein) on cutaneousroinflammatory cytokines

We have assessed the effect of topical application of SIF on TPAnduced cutaneous TNF-�, IL-6 and IL1-� production, as shown in

ig. 3(a)–(c), respectively. We found that there was a significantp < 0.001) increased in the level of all the three cytokines in TPAreated group II as compared to only vehicle treated group I. Pre-reatment with SIF has shown significant inhibition of cytokines

vity and (c) catalase activity. Values are expressed as mean ± SEM (n = 6). ***p < 0.001 with group I. #p < 0.05 and ##p < 0.01 shows the significant difference in the groupoup II.

production in the group III and IV when compared with the TPAtreated group II (p < 0.05, p < 0.01, p < 0.001). There was no signifi-cant difference found between groups I and V.

3.7. Effect of SIF (daidzein & genistein) on the TPA-inducedcutaneous immunohistochemical expression of NF-�B, COX2 andki-67

Cutaneous expression of the NF-�B, COX2 and ki-67 has beenshown in Figs. 4–6, respectively. The intensity of the brown colourin the only TPA treated animals (group-II) clearly indicates the morenumber of cells having NF-�B, COX-2 and ki-67 expression. Pre-treatment with SIF in the groups III and IV shows less numberof cells having the expression of NF-�B, COX-2 and ki-67. How-ever, there was no significant difference observed in the expressionof these proteins in the group V as compared to group I. Forimmunohistochemical analyses, brown colour indicates specificimmunostaining of NF-�B, COX-2 and ki-67 and light blue colourindicates haematoxylin staining. Original magnification: 40× and100×.

3.8. Effect of SIF (daidzein & genistein) on the cutaneoushistological alterations

Effect of topically applied SIF was further observed on the skinhistological alteration caused by TPA application with reference toneutrophil infiltration and epidermal hyperplasia as shown in Fig. 7.We have found that TPA application leads to considerable increasein the leucocyte infiltration and epidermal thickening in group II(Fig. 7B) as compared with the group I (Fig. 7A). Pre-treatmentwith SIF in the groups III (Fig. 7C) and IV (Fig. 7D) diminishes the

TPA induced leucocyte infiltration as well as hyperplasia. There wasno distinguished change observed between the groups I (Fig. 7A)and V (Fig. 7E) as far as leucocytes infiltration and hyperplasia isconcerned. Original magnification: 10×.

270 A.Q. Khan et al. / Toxicology 302 (2012) 266– 274

Fig. 3. Effect of SIF pre-treatment on TPA induced cutaneous proinflammatory cytokines: (a) TNF-�, (b) IL-6 and (c) IL1-�. Values are expressed as mean ± SEM (n = 6).***p < 0.001 shows significant difference in only TPA treated group II [TPA (10 nmol) in 200 �l acetone] when compared with only vehicle treated group I. #p < 0.05, ##p < 0.01,###p < 0.001 shows the significant difference in the groups III [SIF (1 �g) + TPA (10 nmol)] and IV [SIF (2 �g) + TPA (10 nmol)] when compared with group II.

Fig. 4. Effect of SIF pretreatment on TPA-induced cutaneous expression of NF-�B activation. Representative photomicrographs (magnification 40×), (A) only vehicle (groupI) (B) TPA only (group II) (C) SIF (D1) + TPA (group III) (D) SIF (D2) + TPA (group IV) (E) SIF only (group V). Brown colour indicates NF-�B specific staining and blue colourindicates haematoxylin staining. TPA treated group (group II) shows more NF-�B immunopositive staining (arrows) as compared with vehicle treated group (group I). SIFpretreatment (groups III and IV) reduces NF-�B expression as compared to group II. However there was no significant difference in the NF-�B immunostaining in group V ascompared to group I. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

A.Q. Khan et al. / Toxicology 302 (2012) 266– 274 271

Fig. 5. Effect of SIF pretreatment on TPA-induced cutaneous expression of COX-2 expression. Representative photomicrographs (magnification 100×), (A) only vehicle (groupI) (B) TPA only (group II) (C) SIF (D1) + TPA (group III) (D) SIF (D2) + TPA (group IV) (E) SIF only (group V). Brown colour indicates COX-2 specific staining and blue colourindicates haematoxylin staining. TPA treated group (group II) shows more COX-2 immunopositive staining (arrows) as compared with vehicle treated group (group I). SIFp Howec end,

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retreatment (groups III and IV) reduces COX-2 expression as compared to group II.ompared to group I. (For interpretation of the references to colour in this figure leg

. Discussion

Skin, being the largest organ of the body, provides first line ofefense against different harmful exogenous exposures. So far, itas been observed that consumption of natural food has benefi-ial effects on human health including skin diseases. One of thepproaches to prevent the malignancies including that of skin ishemoprevention. Chemoprevention is a means of cancer controlhat is based on the use of specific natural or synthetic chemi-al substances that can suppress, retard or reverse the process ofarcinogenesis. Polyphenols are the most promising group of natu-al compounds possessing anti-inflammatory, immunomodulatorynd antioxidant properties and have shown considerable protec-ion against array of human diseases including skin cancer (Nicholsnd Katiyar, 2010).

Carcinogenesis consists of three distinct stages initiation, pro-otion and progression. Promotion stage has gained attention for

he intervention of cancer development by cellular and molecu-ar approaches. In this study we have examined the attenuativeffects of soy isoflavones with reference to TPA induced inflamma-ory responses which are critically associated with the promotionhase of cancer development.

Inflammatory responses, play important roles in skin tumour

romotion and infiltration of neutrophils, and are the pivotal sourcef ROS production leading to oxidative stress which is closely asso-iated with carcinogenesis. Our findings also show the antioxidantffects of soy isoflavones. We have found that application of TPA

ver there was no significant difference in the COX-2 immunostaining in group V asthe reader is referred to the web version of the article.)

causes oxidative stress because of massive ROS production that ulti-mately leads to membrane damage which is evident from enhancedlipid peroxidation. Level and activity of different antioxidants werealso found distorted that shows the presence of milieu crucial forthe promotion stage of tumour development. Pre-treatment withisoflavones shows protective response as the result implicates.Present study outcomes are in accord with previous research on soyisoflavones (Khan and Sultana, 2004). These effects may be directlybecause of the molecule’s phenolic ring (Dugas et al., 2000), whichprotects against lipid peroxidation, or to its ability to up-regulateanti-oxidant genes (Vina et al., 2007; Siow et al., 2007).

It has been observed from the present study findings that soyisoflavones have strong anti-inflammatory potential. Proinflam-matory cytokines are the important mediators of inflammationand have distinguished roles in cancer development. TNF-�, IL-6and IL1-� have been implicated in various cellular and molecularmechanisms associated with most of the inflammation associatedchronic human diseases including cancer. They along with NOact as cell-to-cell messenger and help in the production of dif-ferent moieties including activation of NF-�B (Kundu and Surh,2008). Their association with inflammation driven tumerogenesiswas confirmed by the previous findings that mouse deficient inTNF-�, IL-6 and IL1-� are resistant to skin tumour formation and

chronic inflammation, neoplasia and tumour metastasis, respec-tively (Moore et al., 1999; Tricot, 2000; Vidal-Vanaclocha et al.,2000). Further, studies on human and mouse show that the proin-flammatory cytokines play an important role in the development

272 A.Q. Khan et al. / Toxicology 302 (2012) 266– 274

Fig. 6. Effect of SIF pretreatment on TPA-induced cutaneous expression of ki-67. Representative photomicrographs (magnification 40×), (A) only vehicle (group I) (B) TPAonly (group II) (C) SIF (D1) + TPA (group III) (D) SIF (D2) + TPA (group IV) (E) SIF only (group V). Brown colour indicates ki-67 specific staining and blue colour indicatesh tive st( e wasg er is r

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aematoxylin staining. TPA treated group (group II) shows more ki-67 immunoposigroup III and IV) reduces ki-67 expression as compared to group II. However, therroup I. (For interpretation of the references to colour in this figure legend, the read

f microenvironment for tumour progression (Smith et al., 1998).he data of the present study evidently support that soy isoflavonesffectively suppress the production of cytokines which implicatesheir anti-inflammatory and chemopreventive potential.

COX, one of the important proinflammatory enzymes, catalyseshe formation of prostaglandins from arachidonic acid, and playsital role in inflammation (Dubois et al., 1998). Two well knownorms of the COX, viz., COX-1 and COX-2 have been identified. Thexpression of COX-1 is ubiquitous in most of the cells and involvedn normal homeostasis of prostaglandins. While, expression ofOX-2 is inducible and was found to increase in response to varioustimuli like growth factors, cytokines and oncogenes (Smith et al.,000). Substantial role of COX-2 has been recognised as it critically

nvolved inflammation, tumour promotion and carcinogenesis (Lait al., 2007). A number of stimuli like pro-oxidant, proinflammatoryytokines, anomalous regulation of signalling regulated by kinasesnd transcription factors may lead to abnormal expression of COX-

(Chun et al., 2004). Different approaches for cancer preventionave demonstrated the critical importance of COX-2 in tumour pro-otion and so the targeted inhibition of COX-2 emerges as one

f the promising approaches to inhibit inflammation and carcino-enesis (Philip et al., 2004). Since the aberrant expression of COX-2s recognised as a connecting link for the tumour promotion thendings of the current study reflect the anti-inflammatory and anti-

umour property of soy isoflavones which attenuates TPA inducedutaneous expression of COX-2 in mouse.

NF-�B, one of the ubiquitous redox sensitive transcriptionactors, regulates many biological processes including cellular

aining (arrows) as compared with vehicle treated group (group I). SIF pretreatment no significant difference in the ki-67 immunostaining in group V as compared toeferred to the web version of the article.)

proliferation, differentiation and inflammation. The main inducibleform of NF-�B is heterodimeric consisting of the p50/p65 subunits.In resting cells NF-�B resides along with its cytosolic repressorinhibitory protein I�B. Exposure with different stimuli includingoxidative stress and phorbol esters causes the phosphorylation anddegradation of I�B by cytoplasmic I�B kinase (IKK) which results inthe nuclear translocation of NF-�B. In the nucleus, it regulates thetranscription of array of target genes including proinflammatorymediators, such as iNOS, COX-2, various cytokines, chemokines,and adhesion molecules (Pahl, 1999; Baeuerle and Baltimore, 1996;Chao et al., 2010). Previous findings implicate that activation ofNF-�B triggers transcriptional up-regulation of COX-2 and proin-flammatory cytokines, such as IL-6, IL1-� and TNF-� (Wu et al.,2008). The present study findings show that cutaneous applicationof TPA leads to the activation of NF-�B in mouse which was stronglysuppressed by soy isoflavones pretreatment suggesting the stronganti-inflammatory and therefore anti-tumour promoting potentialof soya isoflavones.

Immunohistological staining of ki-67 also supports the anti-proliferative effects of soy isoflavones on TPA induced cutaneoustumour promotional events. The above mentioned findings corrob-orated with the histological data which exhibited the protectiveeffects of soy isoflavones against TPA induced cellular anomaliessuch as disorganisation of epithelium, presence of necrotic cells

and focal proliferative areas with epidermal thickness, neutrophilinfiltration and intercellular oedema.

In conclusion the biochemical, molecular and histological find-ings of present study reveal the antioxidant, anti-inflammatory

A.Q. Khan et al. / Toxicology 302 (2012) 266– 274 273

Fig. 7. Effect of SIF pretreatment on TPA-induced cutaneous histological alterations. Representative photomicrographs (magnification 10×), (A) only vehicle (group I) (B)TPA only (group II) (C) SIF (D1) + TPA (group III) (D) SIF (D2) + TPA (group IV) (E) SIF only (group V). There was marked infiltration of neutrophils and epidermal thicknessi als [gri als (gt anim

aitai

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R

A

A

A

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n TPA treated animals [group II, (B)] as compared with only vehicle treated animnduced cutaneous alterations significantly as compared with only TPA treated animhe animals only treated with SIF [group V, (E)] as compared to only vehicle treated

nd anti-proliferative potential of soy isoflavones against TPAnduced early tumour promotional changes in mouse skin. Inhibi-ion of COX-2 expression, production of proinflammatory cytokinesnd activation of NF-�B provide the molecular basis for the anti-nflammatory potential of soy isoflavones.

onflict of interest statement

The authors declare that they have no conflict of interest.

cknowledgement

The author (Sarwat Sultana) is thankful to University Grantsommission (UGC), Government of India, for providing funds and

ellowship to the first author to carry out this work.

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