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Chemico-Biological Interactions 172 (2008) 195–205

Available online at www.sciencedirect.com

Topically applied vitamin E prevents massive cutaneousinflammatory and oxidative stress responses induced by double

application of 12-O-tetradecanoylphorbol-13-acetate(TPA) in mice

Shakilur Rahman a, Kanchan Bhatia a, Abdul Quaiyoom Khan a, Manpreet Kaur a,Firoz Ahmad a, Hina Rashid a, Mohammad Athar b, Fakhrul Islam a,

Sheikh Raisuddin a,∗a Department of Medical Elementology and Toxicology, Jamia Hamdard, Hamdard University,

New Delhi 110 062, Indiab Department of Dermatology, College of Physicians and Surgeons, Columbia University,

630 West 168th Street VC15-204, New York, NY 10032, USA

Received 11 October 2007; received in revised form 13 November 2007; accepted 27 November 2007Available online 4 January 2008

bstract

Vitamin E (�-tocopherol) is a promising chemopreventive and pharmacologically safe agent, which can be exploited or testedgainst skin cancer. It is an established antioxidant with an ability to ameliorate the UV-induced skin damage and chemically inducednflammation in lungs. However, there are some conflicting reports about its role as a modulator of chemically induced promotion.

e evaluated its efficacy in preventing the inflammatory and oxidative stress responses in a double 12-O-tetradecanoylphorbol-3-acetate (TPA) application tumor skin promotion protocol. Double application of TPA was undertaken to produce massivenflammatory and oxidative stress responses. Topical TPA treatment adversely altered many of the marker responses of stage Ikin tumor promotion. Vitamin E application 30 min prior to TPA treatment (10 nmol) inhibited induction of hydrogen peroxide,

ltered antioxidants of mouse skin. Histological examination also revealed marked improvement. These results confirm the efficacyf vitamin E against early inflammatory and oxidative stress responses, which are hallmark of tumor promotion and provide rationalasis for chemopreventive action of vitamin E in skin cancer.

2007 Elsevier Ireland Ltd. All rights reserved.

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∗ Corresponding author at: Department of Molecular and Envi-onmental Bioscience, Graduate School, Hanyang University, Seoul33-791, South Korea. Tel.: +82 2 2220 0769/+91 11 26059688;ax: +82 2 2229 9450/+91 11 26059663.

E-mail address: sheikhraisuddin@yahoo.com (S. Raisuddin).

009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.cbi.2007.11.017

idative stress; Inflammatory response; Vitamin E; Chemoprevention

1. Introduction

The promotion is the most important stage in the mul-

ed.

ogical I

events during promotion are frequently targeted fordevelopment of preventive strategy for skin cancer [2].The main reason for this strategy relates to the fact thattumor promotion is a reversible process at least in theearly stages and requires repeated and prolonged expo-sure to a promoting agent. Furthermore, the long latencyperiod between the initiation and promotion stages ofcancer offers a window of opportunity for interventionbefore malignant tumor develops [3,4]. Therefore, theintervention of cancer at the promotion stage seems tobe an appropriate and practical strategy. Several attemptshave been made to test the efficacy of synthetic andnatural products or combinations of both to alter thepromotional events [5,6].

12-O-tetradecanolyphorbal-13-acetate (TPA) is themost widely used promoting agent to experimentallystudy the events of skin carcinogenesis [7]. Topical appli-cation of TPA to mouse skin is a well-known modelfor induction of oxidative stress or reactive oxygenspecies (ROS) production, cutaneous inflammation andsubsequently occurring hyperplasia [8]. Stimulation ofinflammatory cells by TPA and other tumor promoterscauses release of ROS [9]. These changes are also knownas stage I tumor promotion markers.

Because skin cancer is a major problem associ-ated with mortality and morbidity, concerted efforts areneeded to develop novel strategies for its prevention.One such approach is to ameliorate the skin cancerat the initial stages through chemoprevention [10]. Inchemoprevention approach cancer control is attemptedby topical or oral administration of naturally occur-ring or synthetic compound or their mixtures [6,10].The supplementation or topical application of certainchemopreventive agents such as vitamins, retinoids,inhibitors of cycloxygenase, lipoxygenase and plantpolyphenols and flavonoids have been shown to pro-tect skin from skin cancer events [11–13]. Tocopherolsand related compounds are considered to be potentcellular antioxidants capable of trapping the ROS andterminating the free radical chain reactions [14–16].Vitamin E (�-tocopherol), in particular has shown effi-cacy in preventing UV-induced skin DNA damage[17,18]. It has also been observed that vitamin E incombination with other agents show more efficacy inpreventing the cutaneous photodamage [19,20]. Vita-min E also showed its efficacy in preventing chemicallyinduced inflammation in lungs [21]. Overall, it is notwell understood as to whether vitamin E would be

nteractions 172 (2008) 195–205

min E against TPA-induced stage I tumor promotion.We used double topical application of TPA to inducemassive inflammatory and oxidative stress responsesin skin and also performed histological investigationof skin to study effect of vitamin E at the cellularlevel.

2. Materials and methods

2.1. Chemicals

TPA, vitamin E (dl-�-tocopherol), bovine serumalbumin (BSA), butylated hydroxytoluene (BHT),1-chloro, 2,4 dinitrobenzene (CDNB), 1,2dithiobis-nitrobenzoicacid (DTNB), ethylenediaminetetraaceticacid (EDTA), glutathione reductase (GR), horseradishperoxidase (HRPO), nicotinamide adenine dinucleotidephosphate reduced tetra sodium salt (NADPHNa4),ortho-phosphoric acid (OPA), oxidized glutathione(GSSG), phenol red, pyrogallol, reduced glutathione(GSH), sulfosalicylic acid, thiobarbituric acid (TBA),xanthine and xanthine oxidase (XO) were purchasedfrom Sigma–Aldrich Co. (St. Louis, MO, USA). Otherroutine chemicals of high purity were obtained fromHi-Media Labs (Mumbai) and BDH (Mumbai, India).

2.2. Animals

Swiss albino female mice (25 ± 2 g) used in this studywere provided by the Central Animal House Facility ofthe University. The study protocols were approved by theInstitutional Animal Ethics Committee (IAEC, projectno. 266) and animals were treated and sacrificed withthe approved ethical guidelines. Animals were bred andmaintained under standard laboratory conditions (tem-perature 25 ± 2 ◦C; photoperiod of 12 h light:12 h dark)on a commercial pellet diet and water ad libitum.

2.3. Treatment schedule

Animals were divided in six groups I–VI (n = 5)and their dorsal skin was shaved 2 days prior to com-mencement of treatment. Control animals (Group I) weretreated with a single topical application of acetone (Ac,100 �l). Group II animals were treated with 10 nmol ofTPA/100 �l of acetone. Vitamin E (VE, 20 �mol/100 �lof acetone) 30 min before TPA (10 nmol/100 �l of ace-tone) treatment was applied to Group III animals. Group

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0 �mol of vitamin E in 100 �l acetone, respectively.fter 24 h the same doses of vitamin E or acetoneere applied 30 min prior to the second TPA applica-

ion (10 nmol/100 �l of acetone). The skin applicationrea was approximately 6 cm2.

.4. Skin processing and biochemical measurements

Animals were sacrificed by cervical dislocation 1 hfter the second TPA application under mild anesthe-ia. Skin from dorsal area was removed and cleaned freef extraneous tissue. The edema formation in skin ofarious groups of animals was measured using the skinunch method of Huang et al. [22]. A piece of skin wasreserved in 10% neutral buffered formalin for histolog-cal investigation. Skin homogenates were prepared inhilled phosphate buffer (0.1 M, pH 7.4) using polytronomogenizer (PT-3100, Kinematica Inc., USA) and fil-ered through muslin cloth. The homogenized tissue wasentrifuged at 10,500 × g for 30 min at 4 ◦C to obtainhe post-mitochondrial supernatant (PMS). PMS wassed in various biochemical measurements as detailedelow.

.4.1. LPO measurementLipid peroxidation (LPO) was measured using the

rocedure of Uchiyama and Mihara [23]. The reactionixture consisted of 10 mmol/l BHT, 0.67% TBA, 1%

hilled OPA and tissue homogenate (10%). The mixtureas incubated at 90 ◦C for 45 min. The absorbance of

upernatant was measured at 535 nm. The rate of LPOas determined as nmol of TBA reactive substances

TBARS) formed/h/g of tissue using a molar extinctionoefficient of 1.56 × 105 M−1cm−1.

.4.2. Hydrogen peroxideHydrogen peroxide was measured by the method of

ick and Keisari [24]. Briefly, a 0.5 ml solution of phe-ol red (0.1 mg/ml phosphate buffer, 0.1 M, pH 7.4) andRPO (50 �g/ml in phosphate buffer, 0.1 M, pH 7.4)ere mixed with 0.5 ml PMS and incubated at 37 ◦C for0 min. The reaction was terminated by the addition of.0 ml NaOH (1 M) and the optical density of the prod-ct was measured at 610 nm. Hydrogen peroxide wasalculated as nmol H2O2/h/g tissue.

.4.3. Xanthine oxidaseXanthine oxidase activity was measured by the

ethod of Stripe and della-Corte [25]. The reactionixture containing 0.2 ml PMS diluted to 1 ml with

hosphate buffer (0.1 M, pH 7.4) was first incubated formin at 37 ◦C. The reaction was started by adding 0.1 ml

nteractions 172 (2008) 195–205 197

xanthine and incubation at 37 ◦C for 20 min. Reactionwas terminated by the addition of 0.5 ml ice-cold per-chloric acid (10%). After 10 min, 2.5 ml distilled waterwas added and the mixture was centrifuged at 4000 × gfor 10 min and the absorbance of supernatant was mea-sured at 290 nm. The result was expressed as uric acidformed/mg protein.

2.4.4. Myeloperoxidase (MPO) activitymeasurement

Skin homogenate was prepared in 50 mM K2HPO4buffer (pH 6.0) containing 0.5% hexadecyl trimethylammonium bromide (Sigma) and enzyme activity wasmeasured by the method of Bradley et al. [26]. After threecycles of sonication and freezing–thawing, the sampleswere centrifuged at 2500 × g for 30 min at 4 ◦C. MPOactivity in supernatant (0.1 ml) was assayed by mixing itwith 2.9 ml phosphate buffer (50 mM, pH 6.0) contain-ing 0.167 mg/ml o-dianisidine dihydrochloride (Sigma)and 0.0005% hydrogen peroxide (Sigma). The changein absorbance resulting from decomposition in H2O2 inthe presence of o-dianisidine dihydrochloride was mea-sured at 460 nm for 5 min. The results are expressedas Units/min/mg protein. One unit of MPO activitywas defined as that degrading 1 �mol of peroxide permin.

2.4.5. GSHGSH was measured by the method of Haque et al.

[27]. PMS (1 ml) was precipitated with 1 ml of sulfosal-icylic acid (4.0%). The samples were kept at 4 ◦C for 1 hand then centrifuged at 1200 × g for 15 min at 4 ◦C. Theassay mixture contained 0.2 ml of filtered aliquot, 2.6 mlof sodium phosphate buffer (0.1 M, pH 7.4) and 0.2 mlDTNB (stock 100 mmol/l in sodium phosphate buffer) ina total volume of 3 ml. The absorbance of reaction prod-uct was read at 412 nm. The GSH content was expressedas nmol GSH/g tissue.

2.4.6. Catalase (CAT)CAT activity was assayed by the method of Clai-

borne [28]. The assay mixture consisted of 1.95 mlphosphate buffer (0.05 M, pH 7), 1 ml H2O2 (0.09 mol/l)and 0.05 ml of 10% PMS in final volume of 3 ml. Changein absorbance was recorded kinetically at 240 nm. Cata-lase activity was calculated in terms of nmol H2O2consumed/min/mg protein.

2.4.7. Glutathione S-transferaseGlutathione S-transferase (GST) activity in skin PMS

was measured by the method of Haque et al. [27]. The

ogical I

TPA-treated animals (Group II). TPA-treated animals(Group II) showed a significant increase in the activ-ity of MPO (p < 0.001) when compared with controlgroup (Group I) (Fig. 2). Group III and IV animals when

198 S. Rahman et al. / Chemico-Biol

reaction mixture consisted of 1.675 ml sodium phos-phate buffer, 0.2 ml GSH (1 mmol/l), 0.025 ml of CDNB(1 mmol/l) and 0.1 ml of PMS (10%) in a total volumeof 2 ml. The absorbance change was recorded at 340 nmand the enzyme activity calculated as nmol CDNB con-jugates formed/min/mg protein using a molar extinctioncoefficient of 6.22 × 103 M−1cm−1

2.4.8. Glutathione peroxidase (GPx)GPx activity was assayed according to the method of

Haque et al. [27]. The assay mixture consisted of 1.44 mlsodium phosphate buffer, 0.1 ml EDTA (1 mmol/l),0.1 ml sodium azide (1 mmol/l), 0.05 ml of GR (1 IU/ml),0.1 ml GSH (1 mmol/l), 0.1 ml NADPH (0.02 mmol/l),0.01 ml H2O2 (0.25 mmol/l) and 0.1 ml PMS (10%)in a total volume of 2 ml. Oxidation of NADPHwas recorded spectrophotometrically at 340 nm. Theenzyme activity was calculated as nmol NADPHoxidized/min/mg of protein, using molar extinction coef-ficient of 6.22 × 103 M−1cm−1.

2.4.9. Glutathione reductaseGR activity was assayed by the method of Mohandas

et al. [29]. The assay system consisted of 1.65 ml phos-phate buffer (0.1 M, pH 7.6), 0.1 ml EDTA (0.5 mM),0.05 ml oxidized glutathione (GSSG, 1 mM), 0.1 mlNADPH (0.1 mM) and 0.1 ml of PMS (10%, w/v)in a total volume of 2.0 ml. The enzyme activitywas measured at 25 ◦C as disappearance of NADPHat 340 nm, and was calculated as nmol NADPHoxidized/min/mg protein using a molar extinction coef-ficient of 6.223 × 103 M−1cm−1.

2.4.10. Superoxide dismutase (SOD) activitymeasurement

SOD activity was measured according to the methodof Marklund and Marklund [30]. The assay systemconsisted of 2.875 ml Tris–HCl buffer (50 mM, pH8.5), pyrogallol (24 mM in 10 mM HCl) and 100 �lPMS in a total volume of 3 ml. The enzyme activ-ity was measured at 420 nm and was expressed asunits/mg protein. One unit of enzyme is defined as theenzyme activity that inhibits autoxidation of pyrogallolby 50%.

2.5. Histological investigation

The skin samples were processed for histologi-

nteractions 172 (2008) 195–205

cells into the dermis when compared with that of thecontrols. Intercellular edema (accumulation of fluidbetween the epidermal cells) was scored as presentor absent. The number of nucleated cell layers inthe epidermis was determined by counting the aver-age numbers at five randomly selected locations perslide.

2.6. Statistical analysis

One-way analysis of variance (ANOVA) was appliedto determine significant differences in results of vari-ous groups. P values <0.05 were considered significant.Subsequently, Tukey’s t-test was applied for analyzingthe significance of changes between different treat-ment groups. The values are expressed as means± S.E.

3. Results

3.1. Inflammatory responses

Double TPA application, at a dose of 10 nmol eachwith a 24 h interval led to discernible edema for-mation (Fig. 1). TPA application caused significantedema formation when compared with Group I animals(p < 0.001). Group III and Group IV animals which weretreated with both the doses of vitamin E (20, 40 �mol)applications on TPA-applied skin reduced the edemaresponse and the changes in response was significant(p < 0.05 and p < 0.01, respectively) when compared with

Fig. 1. Effect of vitamin E and TPA on edema formation in skin ofmice. Values are expressed as means ± S.E. (n = 5) of wt/punch/in mg.Significant differences are indicated by ***p < 0.001 when comparedwith control animals (Group I) and #p < 0. 05 and ##p < 0.01 whencompared with TPA-treated animals (Group II).

S. Rahman et al. / Chemico-Biological Interactions 172 (2008) 195–205 199

Fig. 2. Effect of vitamin E and TPA on MPO activity in skin of mice.Values are expressed as means ± S.E. (n = 5) of units of MPO/min/mgpcp

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Fig. 4. H2O2 production in skin of mice from different treatmentgroups. Values are expressed as means ± S.E. (n = 5) measured as nmol

rotein. Significant differences are indicated by ***p < 0.001 whenompared with control animals (Group I) and ###p < 0.001 when com-ared with TPA-treated animals (Group II).

reated with vitamin E (20, 40 �mol) on TPA-appliedkin showed significant (p < 0.001) reduction in MPOctivity when compared with TPA only treatment groupGroup II).

.2. LPO, H2O2 and XO activity

Group II animals (TPA-treated) showed significantnhancement in levels of LPO (Fig. 3), H2O2 generationFig. 4) and XOD activity (Fig. 5) in skin when com-ared with Group I (control group). Group III and IV on

ig. 3. Effect of vitamin E on TPA-induced LPO in skin of mice. Val-es are expressed as means ± S.E. (n = 5) of nmol TBARS formed/h/gissue. Significant differences are indicated by ***p < 0.001 when com-ared with control animals (Group I) and ###p < 0.001 when comparedith TPA-treated animals (Group II).

H2O2/h/g tissue. Significant differences are indicated by ***p < 0.001when compared with control animals (Group I) and #p < 0.05 and###p < 0.001 when compared with TPA-treated animals (Group II).

3.3. Cutaneous antioxidants

The double application of TPA (Group II) caused asignificant decrease in the activities of all the glutathionemetabolizing enzymes such as GST (p < 0.001), GPx(p < 0.001) and GR (p < 0.001) when compared with con-trol (Group I) animals (Table 1). No significant changewas observed in vitamin E-treated animals (Group Vand VI) compared to acetone-treated control group data.

Fig. 5. Effect of vitamin E and TPA on XOD activity in skin prepara-tion of mice. Values are expressed as means ± S.E. (n = 5) of �g uricacid/mg protein. Significant differences are indicated by ***p < 0.001when compared with control animals (Group I) and ###p < 0.001 whencompared with TPA-treated animals (Group II).

200 S. Rahman et al. / Chemico-Biological Interactions 172 (2008) 195–205

Table 1Effect of vitamin E and TPA on the activities cutaneous antioxidant enzymes (GST, GPx, GR)

Group Antioxidant enzyme

GST GPx GR

I (Ac/Ac) 173.60 ± 4.71 272.01 ± 15.72 156.35 ± 8.62II (Ac/TPA) 113.08 ± 4.37*** 147.81 ± 8.38*** 88.70 ± 6.44***III [VE(20)/TPA] 138.19 ± 5.26# 198.74 ± 11.44# 126.01 ± 6.28#IV [VE(40)/TPA] 142.24 ± 6.88## 215.12 ± 11.43## 134.02 ± 7.16##V [VE(20)/Ac] 174.16 ± 4.30 274.22 ± 9.38 158.26 ± 7.85VI [VE(40)/Ac] 176.26 ± 5.48 277.69 ± 9.50 160.08 ± 8.22

Values are means ± S.E. (n = 5). GST is expressed as nmol CDNB conjugates/min/mg protein, GPx as nmol NADPH oxidized/min/mg protein andGR as nmol NADPH oxidized/min/mg protein. Significant differences are indicated by ***p < 0.001 when compared with control animals (GroupI), and #p < 0.05, ##p < 0.01 when compared with TPA-treated animals (Group II).

Table 2Effect of vitamin E and TPA on cutaneous GSH level and activities of CAT and SOD

Group Antioxidant

GSH CAT SOD

I (Ac/Ac) 1.16 ± 0.06 376.78 ± 15.50 11.37 ± 0.72II (Ac/TPA) 0.58 ± 0.06*** 247.74 ± 6.80*** 02.69 ± 0.26***III [VE(20)/TPA] 0.88 ± 0.05## 278.45 ± 5.02 06.07 ± 0.37##IV [VE(40)/TPA] 0.95 ± 0.04### 296.52 ± 6.42## 06.72 ± 0.29###V [VE(20)/Ac] 1.18 ± 0.06 379.30 ± 7.01 11.52 ± 0.73VI [VE(40)/Ac] 1.20 ± 0.03 382.66 ± 6.22 12.19 ± 0.90

Values are means ± S.E. (n = 6). GSH is expressed as nmol GSH/gm tissue, catalase as nmol H2O2 consumed/min/mg protein and SOD as units/mgcompa

Double application of TPA caused marked histo-logical alteration in skin mainly showing inflammatoryresponses in the tissue (Fig. 6B). These changes corre-

Table 3Effect of topical application of vitamin E on TPA-induced histologicalfindings in mouse skin as measured by number of epidermal layers,leukocyte infiltration and intercellular edema

Group Observation

No. of epidermallayers

Leukocyteinfiltration

Intercellularedema

I (Ac/Ac) 2–3 No change No changeII (Ac/TPA) 4–6 Severe SevereIII [VE(20)/TPA] 4–5 Moderate SlightIV [VE(40)/TPA] 3–4 Slight No changeV [VE(20)/Ac] 2–3 No change No changeVI [VE(40)/Ac] 2–3 No change No change

protein. Significant differences are indicated by ***p < 0.001 when###p < 0.01 when compared with TPA-treated animals (Group II).

dose vitamin E (40 �mol) + TPA treatment (Group IV)also showed significant increase in the activities of GST(p < 0.01), GPx (p < 0.01) and GR (p < 0.01) as com-pared to TPA-treated animals (Group II).

Significant decrease in activity of CAT (p < 0.001)and SOD (p < 0.001) was observed in the TPA-treatedgroup (Group II) when compared with Group I animals(Table 2). Group III animals when treated with vita-min E (20 �mol) + TPA showed a significant increasein the activities of SOD (p < 0.01) while no signifi-cant difference in the activity of catalase was observedwhen compared with TPA-treated animals (Group II).Vitamin E (40 �mol) + TPA (Group IV)-treated animalsshowed a significant increase in the activities of cata-lase and SOD when compared with Group II animals(Table 2).

Significant (p < 0.001) decrease in GSH wasobserved in the skin from TPA-treated animals (GroupII) when compared with Group I animals. Vita-

red with acetone-treated control animals (Group I) and ##p < 0.01,

3.4. Histological findings

Leukocytes infiltration and intensity of intercellular edema in skin tis-sues from various groups were recorded as slight, moderate or severedepending on degree of change, if any. The skin tissue samples wereprocessed for histology with hematoxylin and eosin (H & E) staining.Observations were scored under light microscope (H & E × 100).

S. Rahman et al. / Chemico-Biological Interactions 172 (2008) 195–205 201

Fig. 6. Histological sections of skin of mice from different groups. (A) Skin of control animal showing well defined epidermal and dermal layers. (B)Section of skin from mouse with double TPA treatment. The epidermal layer is markedly thickened and the upper dermis is infiltrated by prominentleukocytes and dermal edema as compared with control group. (C) Section of mouse skin treated with vitamin E (20 �mol) before topical applicationo tes andv epidermS pectiveln also gi

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f TPA. Here comparatively lesser thickened epidermal layer, leukocyitamin E (40 �mol) before TPA application. Histological findings ofections of skin from mice treated with vitamin E 20 and 40 �mol, reso marked change over controls. Detail of histological observations is

ated with incidence of edema and biochemical finding of

n epidermal thickness, inflammatory cell infiltration andhe intercellular edema was observed in case of TPA-

edema are apparent. (D) Section of skin treated with higher dose ofal layer, leukocytes and edema are apparently improved. (E) and (F)

y followed by acetone application showing normal skin structure withven in Table 3. (H & E × 100).

treated Group II animals (Fig. 6B; Table 3). However, in

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acetone showing normal skin structure (Fig. 6E and F).In TPA + vitamin E treatment groups (Group III and IV),cutaneous tissue showed marked histological improve-ment (Table 3). Leukocyte infiltration and intercellularedema were not observed in vitamin E-treated animals(Group V and VI) (Table 3).

4. Discussion

Topical application of TPA on mouse skin resultedin discernible inflammatory response as measured byedema and increased hydrogen peroxide productionand LPO. Inflammation induced by biological pro-cesses, chemical exposure and physical stresses has beenassociated with increased risk of disease and cancer.Inflammation activates an array of cells which induceand activate various oxidant generating enzymes. Theseinclude NADPH oxidase, inducible nitric oxide syn-thase, MPO and eosinophil peroxidase [31]. We alsoobserved that TPA application resulted in a significantincrease of MPO. MPO has been identified as crit-ical marker of inflammatory response [32,33]. MPOalong with other enzymes produce high concentrations ofdiverse free radicals including superoxide anion, nitricoxide, nitrogen dioxide, hydrogen peroxide, peroxini-trite, etc. [31]. TPA application resulted in significantincrease in H2O2 production. Some previous studieshave shown increased H2O2 in case of TPA applicationon skin and advocated a close relationship between thegeneration of ROS, including O2− and tumor promo-tion [34]. Thus, there is increasing evidence to suggesta putative role for oxidative stress in tumor promo-tion. This offers an opportunity to control or regulatethe promotional stages of tumor development. Addition-ally, Kensler et al. [35] proposed requirement of twoapplications of TPA for massive ROS generation. Wealso used double TPA application to generate inflam-matory response and subsequently control the eventsinvolved therein using chemopreventive agent, vitaminE. Each application of TPA induces two distinct bio-chemical events, priming and activation [36,37]. The firsttreatment causes chemotactic actions, i.e. recruitmentof neutrophils and the second application is responsi-ble for ROS generations. It is also established that thepromotion stage of multistage carcinogenesis has twooperationally distinct stages known as stage I and stageII tumor promotion [9,36,38].

A number of chemopreventive agents have been

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tocol treatment and observed that a number of putativemarkers of inflammatory response were positively mod-ulated by the application of vitamin E, a well-knownantioxidant and chemopreventive agent. Many mecha-nistic studies examined the anti-inflammatory effect ofvitamin E at the molecular level which show that vitaminE reduces oxidative stress and inflammation by decreas-ing the expression of pro-inflammatory cytokines, andsubsequent LPO [40,41]. Mechanistically, vitamin E hasbeen shown to interfere with many molecular events thatlead to inflammation [42,43]. By inhibiting the ROSwith its antioxidant capabilities, vitamin E can poten-tially increase its anti-inflammatory effects as ROS areknown to play a dual role by participating in the NF-�B activation cascade and by directly modulating DNAbinding affinity [43,44].

We observed that vitamin E application was effectivein preventing many inflammatory response of doubleTPA application. This was demonstrated by decreasedMPO, XO and LPO besides modulation of otherbiochemical parameters supporting its strong chemo-preventive role in skin cancer. Histological examinationalso revealed preventive effect of vitamin E. Vitamin Etreatment at both the doses reduced leukocyte infiltrationand intercellular edema. This suggests that vitamin E notonly improved antioxidant profile of skin tissue but it alsoshowed its preventive effect at cellular level. In severalstudies vitamin E has been proved to be effective in mit-igating the skin tissue damage. For example, Kuriyamaet al. [45] showed that skin ointment containing vita-min E suppressed chemically induced contact dermatitisin rats by stabilizing keratinocytes. Additionally, Uddinet al. [46] demonstrated that vitamin E protected miceagainst arsenite-induced enhancement of UV radiation-induced skin carcinogenesis with significant histologicalimprovement. Recently Cho et al. [47] reported anti-wrinkling effects of mixture of vitamin C, vitamin E,pycnogenol and evening primrose oil in UV-irradiatedfemale SKH-1 hairless mice. These studies support ourobservations on marked histological improvement byvitamin E treatment in skin of TPA-treated animals andsupplements its chemopreventive properties.

Vitamin E has been shown to protect skin carcino-genesis and UV-induced DNA damage [48]. In somecases vitamin E has been used with other known nat-ural antioxidants such melatonin and vitamin C. Forexample, Dreher et al. [19] used melatonin (N-acetyl5-methoxytryptamine) and vitamin C and E individu-

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ined treatment of vitamin E with either GSH or andelenium (Na2SeO3) was effective in inhibiting com-lete stage II tumor promotion by TPA and mezerein.owever, these treatments failed to inhibit significantly

he 7,12-dimethylbenz [a]-anthracene (DMBA)-inducedarcinogenicity as enhanced formation of skin papil-omas and carcinomas was observed by repeatedpplications of DMBA. Glauert et al. [50] also reportedhat vitamin E supplementation did not protect rats fromolychlorinated biphenyl (PCB)-induced altered hepaticoci. This suggests that vitamin E is not a universalnhibitor of promotion events. Promotion events in casef two different agents may follow different pathways.

The antioxidant role of vitamin E is well established.itamin E is the body’s major lipid soluble antioxidant,hich protects membranes from free radical attack. It is

lso the major peroxyl radical scavenger and ends thehain reaction damage caused by free radicals in mem-ranes. Vitamin E is particularly abundant in the stratumorneum of skin, and is delivered there in and protectshe outer layers of skin against pollutants and UV light51].

The TPA-induced cutaneous promotional events andV-induced events have many commonalities such asPO, increased production of free radicals, etc. There-

ore, the reported photoprotective action of vitamin End the protection observed by us in TPA-treated mousekin are mainly attributed to antioxidant property of vita-in E. Besides its antioxidant action, vitamin E is also

eported to regulate certain signaling pathway compo-ents such as nuclear factor-�B (NF-�B) and proteininases [44]. Modulation of these factors is suggested toe one of its main anti-inflammatory mechanisms. Pre-iously, some studies have shown that vitamin E inhibitsctivation of hepatic NF-�B in rats treated with a tumorromoter, phenobarbital [43]. More recently, Cheepalat al. [52] used microarray technique to study chemo-reventive effect of all-trans retinoic acid (ATRA) inkin cancer. They observed that approximately half ofhe genes regulated by TPA are oppositely regulatedhen ATRA is co-administered with TPA. Mainly theenes of Raf/Mek/Erk branch of mitogen-activated pro-ein (MAP) kinase pathway were found to be oppositelyegulated. This finding sheds new light on the mechanismf tumor promotion by TPA.

All the above-mentioned findings suggest that actionf vitamin E is not a universal protector of oxidativetress, inflammation and subsequent alternations associ-

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other agents. However, we found that vitamin E has def-inite anti-inflammatory and modulatory role in doubleapplication of TPA-induced massive inflammatory andoxidative stress responses. Now, we are evaluating mod-ulatory role of vitamin E in TPA-induced promotionalevents in immunosuppressed mouse skin.

Acknowledgments

The financial support from the University GrantsCommission (UGC), Government of India in the formof Special Assistance Programme (SAP) to the Depart-ment for research on “Chemoprevention of cancer” isacknowledged. We thank Dr. A.K. Mukherjee for histo-logical interpretation of findings and Dr. Ehsan A. Khan,Head of the Department for suggestion on statisticalanalysis of results.

References

[1] F. Marks, G. Furstenberger, K. Muller-Decker, Tumor promotionas a target of cancer prevention, Recent Results Cancer Res. 174(2007) 37–47.

[2] A.M. Bode, Z. Dong, Targeting signal transduction pathways bychemopreventive agents, Mutat. Res. 555 (2004) 33–51.

[3] G.D. Stoner, M.A. Morse, G.J. Kelloff, Perspectives in cancerchemoprevention, Environ. Health Perspect. 105 (1997) 945–954.

[4] F. Marks, G. Furstenberg, Cancer chemoprevention throughinterruption of multistage carcinogenesis: the lesson learnt bycomparing mouse skin carcinogenesis and human large bowelcancer, Eur. J. Cancer 36 (2000) 314–329.

[5] G.D. Stoner, H. Mukhtar, Polyphenols as cancer chemopreventiveagents, J. Cell Biochem. 22 (1995) 169–180.

[6] D.R. Bickers, M. Athar, Novel approaches to chemopreventionof skin cancer, J. Dermatol. 27 (2000) 691–695.

[7] T. Nakadate, The mechanism of skin tumor promotion causedby phorbol esters: possible involvement of arachidonic acidcascade/lipoxygenase, protein kinase C and calcium/calmodulinsystems, Jpn. J. Pharmacol. 49 (1989) 1–9.

[8] H.Y. Ha, Y. Kim, Z.Y. Ryoo, T.Y. Kim, Inhibition of the TPA-induced cutaneous inflammation and hyperplasia by EC-SOD,Biochem. Biophys. Res. Commun. 348 (2006) 450–458.

[9] M. Lahiri-Chatterjee, S.K. Katiyar, R.R. Mohan, R. Agarwal, Aflavonoid antioxidant, silymarin, affords exceptionally high pro-tection against tumor promotion in the SENCAR mouse skintumorigenesis model, Cancer Res. 59 (1999) 622–632.

10] S. Gupta, H. Mukhtar, Chemoprevention of skin cancer: currentstatus and future prospects, Cancer Metastasis Rev. 21 (2002)363–380.

11] H. Mukhtar, N. Ahmad, Cancer chemoprevention. Future holdsin multiple agents: contemporary issues in toxicology, Toxicol.Appl. Pharmacol. 158 (1999) 207–210.

12] M.A. Soobrattee, T. Bahorun, O.L. Aruoma, Chemopreventiveactions of polyphenolic compounds in cancer, Biofactors 27(2006) 19–35.

13] S.C. Thomasset, D.P. Berry, G. Garcea, T. Marczylo, W.P.Steward, A.J. Gescher, Dietary polyphenolic phytochemicals—

ogical I

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

204 S. Rahman et al. / Chemico-Biol

promising cancer chemopreventive agents in humans? A reviewof their clinical properties, Int. J. Cancer. 120 (2007) 451–458.

14] R. Brigelius-Flohe, M.G. Traber, Vitamin E: function andmetabolism, FASEB J. 13 (1999) 1145–1155.

15] X. Wang, P.J. Quinn, Vitamin E and its function in membranes,Prog. Lipid Res. 38 (1999) 309–336.

16] D.J. Mustacich, R.S. Bruno, M.G. Traber, Vitamin E, Vitam.Horm. 76 (2007) 1–21.

17] M. Lopez-Torres, J.J. Thiele, Y. Shindo, Y. Han, L.D. Packer,Topical application of alpha-tocopherol modulates the antioxidantnetwork and diminishes ultraviolet-induced oxidative damage inmurine skin, Br. J. Dermatol. 138 (1998) 207–215.

18] C.S. Sander, H. Chang, F. Hamm, P. Elsner, J.J. Thiele, Role ofoxidative stress and the antioxidant network in cutaneous carcino-genesis, Int. J. Dermatol. 43 (2004) 326–335.

19] F. Dreher, B. Gabard, D.A. Schwindt, H.I. Maibach, Topical mela-tonin in combination with vitamins E and C protects skin fromultraviolet-induced erythema: a human study in vivo, Br. J. Der-matol. 139 (1998) 332–339.

20] M. Placzek, S. Gaube, U. Kerkmann, K.P. Gilbertz, T. Herzinger,E. Haen, B. Przybilla, Ultraviolet B-induced DNA damage inhuman epidermis is modified by the antioxidants ascorbic acidand d-alpha-tocopherol, J. Invest. Dermatol. 124 (2005) 304–307.

21] D. Rocksen, B. Ekstrand-Hammarstrom, L. Johansson, A. Bucht,Vitamin E reduces transendothelial migration of neutrophils andprevents lung injury in endotoxin-induced airway inflammation,Am. J. Respir. Cell Mol. Biol. 28 (2003) 199–207.

22] M.T. Huang, C.T. Ho, Z.Y. Wang, T. Ferraro, T. Finnegen-Olive,Inhibitory effect of topical application of a green tea polyphe-nol fraction on tumor initiation and promotion in mouse skin,Carcinogenesis 13 (1992) 947–954.

23] M. Uchiyama, M. Mihara, Determination of malonaldehyde pre-cursor in tissues by thiobarbituric acid test, Anal. Biochem. 86(1978) 271–278.

24] E. Pick, Y. Keisari, A simple colorimetric method for the mea-surement of hydrogen peroxide produced by cells in culture, J.Immunol. Methods 38 (1980) 161–170.

25] F. Stripe, E. della-Corte, The regulation of rat liver xanthineoxidase. Conversion in vitro of enzyme activity from dehydro-genase (type D) to oxidase (type O), J. Biol. Chem. 244 (1969)3855–3863.

26] P.P. Bradley, D.A. Priebat, R.D. Christensen, G. Rothstein, Mea-surement of cutaneous inflammation: estimation of neutrophilcontent with an enzyme marker, J. Invest. Dermatol. 78 (1982)206–209.

27] R. Haque, B. Bin-Hafeez, S. Parvez, S. Pandey, I. Sayeed, M.Ali, S. Raisuddin, Aqueous extract of walnut (Juglans regiaL.) protects mice against cyclophosphamide-induced biochemicaltoxicity, Hum. Exp. Toxicol. 22 (2003) 473–480.

28] A. Claiborne, Catalase activity, in: R.A. Greenwald (Ed.), Hand-book of Methods for Oxygen Radical Research, CRC press, BocaRaton, FL, 1985, pp. 283–284.

29] J. Mohandas, J.J. Marshall, G.G. Duggin, J.S. Horvath, D.L.Tiller, Low activities of glutathione-related enzymes as factorsin the genesis of urinary bladder cancer, Cancer Res. 44 (1984)5086–5091.

30] S. Marklund, G. Marklund, Involvement of the superoxide anion

31] H. Ohshima, M. Tatemichi, T. Sawa, Chemical basis ofinflammation-induced carcinogenesis, Arch. Biochem. Biophys.417 (2003) 3–11.

[

nteractions 172 (2008) 195–205

32] I. Dahlen, C. Janson, E. Bjornsson, G. Stalenheim, C.G. Peterson,P. Venge, Changes in inflammatory markers following treatmentof acute exacerbations of obstructive pulmonary disease, Respir.Med. 95 (2001) 891–897.

33] K. Andelid, B. Bake, S. Rak, L.A. Linden, A. Rosengren, A.Ekberg-Jansson, Myeloperoxidase as a marker of increasing sys-temic inflammation in smokers without severe airway symptoms,Respir. Med. 101 (2007) 888–895.

34] H.L. Yoon, B. Marcus, R.W. Pfeifer, Induction of superoxide by12-O-tetradecanoylphorbol-13-acetate and thapsigargin, a non-phorbol type tumor promoter, in peritoneal macrophages elicitedfrom SENCAR and B6C3F1 mice: a permissive role for thearachidonic acid cascade in signal transduction, Mol. Carcinog.7 (1993) 116–125.

35] T.W. Kensler, P.A. Egner, B.G. Taffe, M.A. Trush, Role of freeradicals in tumor promotion and progression, in: T.J. Salaga,A.J.P. Klein-Szanto, R.K. Boutwell, D.E. Stevenson, H.L. Spitzer,B. D’Motto (Eds.), Skin Carcinogenesis, Progress in Clinical andBiological Research, Alan R. Liss Inc., New York, 1989, pp.233–248.

36] T.J. Slaga, Overview of tumor promotion in animals, Environ.Health Perspect. 50 (1983) 3–14.

37] J. DiGiovanni, Multistage carcinogenesis in mouse skin, Pharma-col. Ther. 54 (1992) 63–128.

38] T.J. Slaga, S.M. Fischer, K. Nelson, G.L. Gleason, Studies on themechanism of skin tumor promotion: evidence for several stagesin promotion, Proc. Natl. Acad. Sci. U.S.A. 77 (1980) 3659–3663.

39] H. Inano, M. Onoda, N. Inafuku, M. Kubota, Y. Kamada, T.Osawa, H. Kobayashi, K. Wakabayashi, Chemoprevention bycurcumin during the promotion stage of tumorigenesis of mam-mary gland in rats irradiated with gamma-rays, Carcinogenesis20 (1999) 1011–1018.

40] I. Jialal, S. Devaraj, S.K. Venugopal, Oxidative stress, inflamma-tion, and diabetic vasculopathies: the role of � tocopherol therapy,Free Radic. Res. 36 (2002) 1331–1336.

41] E. Aghdassi, B.E. Wendland, A.H. Steinhart, S.L. Wolman, K.Jeejeebhoy, J.P. Allard, Antioxidant vitamin supplementation incrohn’s disease decreases oxidative stress, a randomized con-trolled trial, J. Gastroenterol. 98 (2003) 348–353.

42] R. Gonzalez, F. Sanchez de Medina, J. Galvez, M.E.Rodriguez-Cabezas, J. Duarte, A. Zarauelo, Dietry vitamin E sup-plementation protects the rat large intestine from experimentalinflammation, Int. J. Vitam. Nutr. Res. 71 (2001) 243–250.

43] K.G. Calfee-Mason, B.T. Spear, H.P. Glauret, Vitamin E inhibitshepatic NF�B activation in rats administered the hepatic tumorpromoter, Phenobarbital. J. Nutr. 132 (2002) 3178–3185.

44] M. Morante, J. Sandoval, M.C. Gomez-Cabrera, J.L. Rodrıguez,F.V. Pallardo, J.R. Vina, L. Torres, T. Barber, Vitamin E defi-ciency induces liver nuclear factor-kappaB DNA-binding activityand changes in related genes, Free Radic. Res. 39 (2005) 1127–1138.

45] K. Kuriyama, T. Shimizu, T. Horiguchi, M. Watabe, Y. Abe, Vita-min E ointment at high dose levels suppresses contact dermatitisin rats by stabilizing keratinocytes, Inflamm. Res. 51 (2002)483–489.

46] A.N. Uddin, F.J. Burns, T.G. Rossman, Vitamin E and organose-lenium prevent the cocarcinogenic activity of arsenite with

47] H.S. Cho, M.H. Lee, J.W. Lee, K.O. No, S.K. Park, H.S. Lee, S.Kang, W.G. Cho, H.J. Park, K.W. Oh, J.T. Hong, Anti-wrinklingeffects of the mixture of vitamin C, vitamin E, pycnogenol and

gical I

[

[

[

[

S. Rahman et al. / Chemico-Biolo

evening primrose oil, and molecular mechanisms on hairlessmouse skin caused by chronic ultraviolet B irradiation, Photo-dermatol. Photoimmunol. Photomed. 23 (2007) 155–162.

48] C. Weber, M. Podda, M. Rallis, J.J. Thiele, M.G. Traber, L.Packer, Efficacy of topically applied tocopherols and tocotrienolsin protection of murine skin from oxidative damage induced byUV-irradiation, Free Radic. Biol. Med. 22 (1997) 761–769.

49] J.P. Perchellet, N.L. Abney, R.M. Thomas, Y.L. Guislain, E.M.Perchellet, Effects of combined treatments with selenium, glu-tathione, and vitamin E on glutathione peroxidase activity,ornithine decarboxylase induction, and complete and multistagecarcinogenesis in mouse skin, Cancer Res. 47 (1987) 477–485.

[

nteractions 172 (2008) 195–205 205

50] H.P. Glauert, Z. Lu, A. Kumar, R.P. Bunaciu, S. Patel, J.C. Tharap-pel, D.N. Stemm, H.J. Lehmler, E.Y. Lee, L.W. Robertson, B.T.Spear, Dietary vitamin E does not inhibit the promotion of livercarcinogenesis by polychlorinated biphenyls in rats, J. Nutr. 135(2005) 283–286.

51] J.J. Thiele, Oxidative targets in the stratum corneum: a new basisfor antioxidative strategies, Skin Pharmacol. Appl. Skin Physiol.

52] S.B. Cheepala, Z. Syed, M. Trutschl, U. Cvek, J.L. Clifford,Retinoids and skin: microarrays shed new light on chemopre-ventive action of all-trans retinoic acid, Mol. Carcinog. 46 (2007)634–639.