Protective Effect of Sanguinarine on Ultraviolet B-mediated Damages in SKH-1 Hairless Mouse Skin: Implications for Prevention of Skin Cancer

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Protective Effect of Sanguinarine on Ultraviolet B-mediated Damages inSKH-1 Hairless Mouse Skin: Implications for Prevention of Skin Cancer

Haseeb Ahsan, Shannon Reagan-Shaw, David M. Eggert, Thomas C. Tan, Farrukh Afaq, Hasan Mukhtarand Nihal Ahmad*

Department of Dermatology, University of Wisconsin, School of Medicine and Public Health, Madison, WI

Received 30 March 2007; accepted 27 April 2007; DOI: 10.1111 ⁄ j.1751-1097.2007.00156.x

ABSTRACT

Excessive exposure of solar ultraviolet (UV) radiation, partic-

ularly its UVB component (280–320 nm), to human skin is the

major cause of skin cancers. UV exposure also leads to the

development of precancerous conditions such as actinic kera-

tosis and elicits a variety of other adverse effects such as

sunburn, inflammation, hyperplasia, immunosuppression and

skin aging. Therefore, there is a need to intensify our efforts

towards the development of novel mechanism-based approa-

ches ⁄ agents for the protection of UVB-mediated damages.

Chemoprevention is being investigated as a potential approach

for the management of UV damages including skin cancer. We

have earlier shown that sanguinarine, a benzophenanthridine

alkaloid, inhibits UVB exposure-mediated damages in HaCaT

keratinocytes. In this study, to determine the relevance of our

in vitro findings to in vivo situations, we assessed the effects of

sanguinarine on UVB-mediated damages in SKH-1 hairless

mice. Our data demonstrated that a topical application of

sanguinarine (5 lmol 0.3 mL)1 ethanol per mouse), either as a

pretreatment (30 min prior to UVB) or posttreatment (5 min

after UVB), resulted in a significant decrease in UVB-mediated

increases in skin edema, skin hyperplasia and infiltration of

leukocytes. Further, sanguinarine treatments (pre and post)

also resulted in a significant decrease in UVB mediated (1)

generation of H2O2 and (2) increases in the protein levels of

markers of tumor promotion ⁄ proliferation viz. ornithine

decarboxylase (ODC), proliferating cell nuclear antigen

(PCNA) and Kiel antigen-67. Based on this data, we suggest

that sanguinarine could be developed as an agent for the

management of conditions elicited by UV exposure including

skin cancer. However, further detailed studies are needed to

support this suggestion.

INTRODUCTION

Ultraviolet (UV) radiation from sun, particularly its UVB

component (290–320 nm), is the major cause of skin cancerthat accounts for more than a million new cases each year inthe USA alone (1). Further, exposure to UVB may lead to

precancerous lesions such as actinic keratoses and may also

elicit a variety of other adverse effects including erythema,sunburn, inflammation, hyperplasia, skin aging, hyperpigmen-tation and immunosuppression (2–5). UVB radiation causestumor initiation through DNA damage and its promoting

activity includes transcriptional modulation of genes involvedin tumor promotion as well as activation of several signaltransduction pathways (2–5). Whereas low doses of UVB

cause DNA mutation leading to tumor initiation, high dosesresult in irreparable DNA damage causing apoptosis (sun-burn) and eventually cell death (2–5). UVB also indirectly

damages DNA by increasing levels of reactive oxygen species(ROS) which facilitate DNA oxidation (6). Cells in theepidermis with common UV mutations represent ‘‘initiatedcells’’ which can then undergo clonal expansion to become

tumors (7).The available prevention strategies have not been able to

effectively manage the increasing incidence of skin cancers

and other UV-related conditions. This warrants immediateattention towards developing novel mechanism-basedapproaches for the prevention as well as treatment of skin

cancer. Chemoprevention by naturally occurring plant-basedagents is being investigated as a potential approach forprevention as well as treatment of skin cancer as well as

other UV exposure related conditions. Sanguinarine, derivedfrom the root of Sanguinaria canadensis and other poppy-fumaria species, is a benzophenanthridine alkaloid and astructural homolog of chelerythrine and has been shown to

possess antimicrobial, antioxidant and anti-inflammatoryproperties (8,9). It is often used in toothpastes and mouth-washes for its anti-inflammatory effects (9–11). We have

shown previously that sanguinarine induces apoptosis inhuman squamous carcinoma A431 cells but not in normalhuman epidermal keratinocytes at micromolar doses (12). In

addition, sanguinarine was shown to induce apoptosis viaactivation of Bcl-2 family proteins and the mitochondrialpathway in immortalized human HaCaT keratinocytes (13).

In a recent study, we have shown that the plant alkaloidsanguinarine protects HaCaT skin keratinocytes from UVB-mediated damages via enhancing the apoptotic eliminationof UVB-damaged cells (14). This study was designed

to investigate the relevance of these findings to in vivosituations in SKH-1 hairless mouse skin that possesses

*Corresponding author email: [email protected] (Nihal Ahmad)� 2007TheAuthors. JournalCompilation.TheAmericanSociety ofPhotobiology 0031-8655/07

Photochemistry and Photobiology, 2007, 83: 986–993

986

relevance to human situations. Our data demonstrated that atopical application of sanguinarine resulted in a significantdecrease in UVB-mediated (1) increases in skin edema, skinhyperplasia and infiltration of leukocytes, (2) generation of

H2O2 and (3) increases in the protein levels of markers oftumor promotion ⁄proliferation viz. ornithine decarboxylase(ODC), proliferating cell nuclear antigen (PCNA) and Kiel

antigen-67 (Ki-67). To our knowledge, this is the first studyshowing prevention of UVB-mediated damages in skin bysanguinarine.

MATERIALS AND METHODS

Materials. Sanguinarine chloride (>99.0% pure) was purchased fromSigma Chemical Co. (St. Louis, MO). The following antibodies wereused: PCNA (Biosource International, Camarillo, CA), ODC (Neo-markers ⁄Lab Vision, Fremont, CA), Ki-67 (Santa Cruz Biotechno-logy, Santa Cruz, CA) and b-actin (Sigma-Aldrich, St. Louis, MO). Allother chemicals were of analytical grade.

Animals and UVB light source. Female SKH-1 hairless mice (6-weeks old) obtained from Charles River Laboratories (Wilmington,MA) were used in this study. The mice were allowed to acclimatizefor 2 weeks prior to the start of the experiments. The animals had anad libitum access to Purina Chow diet and tap water. Throughout theexperimental protocols, the mice were maintained at standardconditions (temperature 24 ± 2�C; relative humidity 50 ± 10%;12 ⁄ 12 h light ⁄dark cycle). A ‘‘Daavlin Research Irradiator’’ obtainedfrom Daavlin Company (Bryan, OH) was used for UVB exposure.This equipment consists of a fixture mounted on fixed legs andcontains four UVA and four UVB lamps. The exposure system iscontrolled using two Daavlin Flex Control Integrating Dosimeters.The dose units, in this equipment, could be entered as mJ cm)2 (forUVB) or Joules (for UVA). For accuracy, the machine is periodicallycalibrated using International Light IL 1400, digital light meter(Daavlin Company).

Treatments of animals. Female SKH-1 hairless mice were dividedinto six groups of eight animals each. The mice in the first group didnot receive any treatment and served as a control. The second groupreceived a topical application of 0.3 mL ethanol (vehicle control).The mice in the third group received a topical treatment ofsanguinarine (5 lmol in 0.3 mL ethanol per mouse) on their dorsalskin. The mice in the fourth group received UVB (180 mJ cm)2)treatment. The mice in the fifth group received a topical application

5

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UVB Sang

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n p

un

ch w

eig

ht

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iam

eter

)

*

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Figure 1. Inhibitory effect of sanguinarine on UVB-induced skinedema in SKH-1 hairless mice. Twenty-four hours after UVBtreatment, the skin edema was determined by weighing 4 mm-diameterpunch skin as described in text. At least four determinations weremade at different dorsal skin sites per mouse in each group. The datarepresent the mean ± SE of eight mice (*P < 0.05 compared withuntreated and vehicle control and #P < 0.05 compared with UVB).

Untreated Vehicle UVB

Sanguinarine Sanguinarine + UVB UVB + Sanguinarine

Figure 2. Effect of sanguinarine on UVB-mediated hyperplasia and infiltration of leukocytes in SKH-1 hairless mice. Twenty-four hours after UVBirradiation, the animals were killed and skin biopsies were processed for hematoxylin and eosin staining. Representative pictures are shown. Thedetails are given under Materials and Methods.

Photochemistry and Photobiology, 2007, 83 987

of sanguinarine (5 lmol in 0.3 mL ethanol per mouse) followed byUVB (180 mJ cm)2, 30 min following sanguinarine treatment;pretreatment group). The animals in the sixth group received UVB(180 mJ cm)2) treatment first followed by sanguinarine treatment(5 lmol in 0.3 mL ethanol; 5 min following UVB treatment; post-treatment group).

Skin edema. To assess the effect of sanguinarine on UVB-inducedskin edema, we evaluated the increase in bifold skin punch weightafter 24 h following the last treatment (15). The increase in bifoldskin weight was measured using a 4 mm diameter biopsy punch.At least four determinations were made at different dorsalskin sites per mouse in each group. An increase in the weightfollowing UVB and ⁄ or sanguinarine treatments was obtained bysubtracting the values for the untreated controls from treatedanimals.

Histopathology. For histopathological analysis of skin, the skinsamples obtained from control and treated mice were fixed in 10%formalin and embedded in paraffin. Vertical section (6 lMM) were cut,mounted on a glass slide and stained with hematoxylin and eosin(H&E) followed by microscopic evaluation and digital photography ofthe slides.

Histochemical staining of hydrogen peroxide. Histochemical detec-tion of H2O2 was performed following the method described byDannenberg et al. (16). Briefly, frozen skin sections (6 lm thick) wereincubated with 0.1 MM Tris–HCl buffer (pH 7.5), containing 1 mg mL)1

glucose and 1 mg mL)1 diaminobenzidine (DAB) for 5–6 h at 37�C.Sections were then washed in distilled water and counterstained inmethyl green (2% for 60 min). The DAB-peroxidase gave a brownreaction product and methyl green provided a blue nuclear counter-stain. The slides were analyzed under microscope and pictures werecaptured using digital camera.

Epidermis isolation, preparation of cytosol and ODC activity. Forisolation of epidermis, skin was made free of the subcutaneoustissue and then into a solution of 50 U mL)1 diaspase overnight at4�C. Thereafter, the epidermal cell layer was separated from thedermal layer using forceps. The epidermis was washed with PBS,weighed and homogenized with a polytron homogenizer in PBScontaining KCl and centrifuged at 18 000 g for 15 min at 4�C toprepare cytosolic fractions (10% wt ⁄ vol) as described previously

(17). ODC enzyme activity was determined in cytosol by measuringthe release of 14CO2 from the DD,LL-[14C]ornithine as describedpreviously (18) and expressed as picomoles of CO2 released perhour per milligram protein.

Preparation of epidermal lysate and immunoblot analysis. From thewhole skin, the epidermis was separated as described above andhomogenized in a lysis buffer with freshly added protease inhibitorcocktail. The homogenate was centrifuged at 13 000 g for 25 min at4�C, and the supernatant (total cell lysate) was collected and theprotein content was measured by bicinchoninic acid (BCA) proteinassay kit (Pierce Biotechnology, Rockford, IL).

For immunoblot analysis, 30 lg protein was subjected toSDS–PAGE (10–15% Tris–HCl gel). The protein was transferredonto a nitrocellulose membrane and blocked with tris buffered saline(TBS)-Tween plus 5% dry milk. The membrane was probed with anappropriate primary antibody followed by a secondary horseradishperoxidase (HRP)-conjugated antibody. The protein was detected byfreshly prepared chemiluminescent solution.

Immunohistochemical analysis. Whole skin was frozen in optimalcutting temperature (OCT) medium and kept at )80�C until process-ing. Histological sections (6 lMM) were prepared from frozen tissues andfixed in acetone and stored at )80�C. The slides were incubated withappropriate primary antibodies and antiperoxidase secondary anti-body. The slides were stained with DAB reagent and counterstainedwith methyl green before mounting in permount medium.

Statistical analysis. A two-tailed Student’s t-test was used to assessthe statistical significance between the groups. A P-value of <0.05 wasconsidered statistically significant.

RESULTS

Inhibitory effect of sanguinarine on UVB exposure-mediated

cutaneous edema

Because studies have shown that UV exposure to the skin

results in cutaneous edema, which is regarded as a marker ofUV damage, we evaluated the protective effect of sanguinarine



Figure 3. Effect of sanguinarine on UVB-mediated production of H2O2 in SKH-1 hairless mice. Twenty-four hours after UVB irradiation, theanimals were killed and histochemical detection of H2O2-producing cells was performed using the DAB-peroxidase reaction. Representativepictures are shown. The details are described under Materials and Methods.

988 Haseeb Ahsan et al.

on UVB-mediated skin edema in SKH-1 hairless mice. Asdetermined by the weight of punch biopsy of the dorsal skin,UVB exposure to SKH-1 mouse skin resulted in a significantskin edema at 24 h post-UVB treatment compared with

control and vehicle-treated groups (Fig. 1). The topical appli-cation of sanguinarine (pre- as well as posttreatments) to skinwas found to result in a significant inhibition in UVB-mediated

skin edema in SKH-1 hairless mice. The topical application ofsanguinarine alone did not cause any increase in skin edema(Fig. 1).

Inhibitory effect of sanguinarine on UVB exposure-mediated

epidermal hyperplasia and infiltration of leukocytes

We next determined the effect of sanguinarine on epidermalhyperplasia, characterized by a thickening of the epidermis,

which is another marker of UV damage and is considered ahyperproliferative response. As shown in Fig. 3, our datademonstrated that UVB exposure to SKH-1 hairless mouse

skin resulted in a marked increase in epidermal hyperplasiaat 24 h following UVB, compared with control animals.



ODC

β-actin

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1200(a)

(b)

(c)

Unt Veh UVB Sang Sang +UVB

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Figure 4. Effect of sanguinarine on UVB-mediated induction of epidermal ODC in SKH-1 hairless mice at 24 h posttreatment. (a) Epidermalcytosolic fraction was prepared, and ODC activity was determined. Data is shown as ODC activity in picomoles per hour per milligram of protein.(b) Epidermal protein lysate was prepared, and ODC protein expression was determined by western blotting. Equal loading of protein wasconfirmed by stripping the immunoblot and reprobing it for b-actin. (c) From skin biopsies, 6 lm thick sections were cut and the effects oftreatments on ODC were assessed by immunohistochemical analysis. The representative pictures are from five to six skin biopsies captured by aSpot Insight QE Camera attached to a Nikon microscope. Details of experiments are provided under Materials and Methods.


Sanguinarine treatment (pre- and posttreatments) resulted in asignificant decrease in UVB-mediated epidermal hyperplasia(Fig. 2). Further, since the infiltration of leukocytes, which isbelieved to be initiated by locally generated chemotactic

stimuli and is often accompanied by simultaneous increasesin vascular permeability and blood flow, is an important eventduring many acute inflammatory reactions in skin (19),

we determined the effect of sanguinarine on UVB-mediatedleukocyte infiltration. The topical application of sanguinarine,however, prior to or after UVB exposure resulted in a marked

inhibition of UVB-mediated increase in infiltration of leuko-cyte into the dermis (Fig. 2). Sanguinarine alone did notinduce epidermal hyperplasia or leukocyte infiltration in SKH-

1 hairless mouse skin (Fig. 2).

Inhibitory effect of sanguinarine on UVB-mediated hydrogen

peroxide generation

Based on many studies, H2O2 generation is a well-accepted

marker of oxidative stress (20–22) and because sanguinarine hasbeen shown to possess antioxidant effects (23), we assessedwhether the preventive effects of sanguinarine are mediated via

modulating the levels of H2O2 in the cells. As shown in Fig. 3,compared with controls, the UVB exposure was found to causean increased generation of H2O2 in the skin of SKH-1 hairlessmice, and sanguinarine application (both pre- and posttreat-

ments) resulted in a marked decrease in this response of UVBsuggesting that sanguinarine protects UVB-mediated damagesto the skin, at least in part, via its antioxidative effects.

Effect of sanguinarine on UVB-mediated induction of epidermal

ODC

Ornithine decarboxylase is the first and the rate-limiting

enzyme in the polyamine biosynthetic pathway and plays animportant role in both normal cellular proliferation and thegrowth and development of cancer (15,18). Therefore, in this

study, we next determined the effect of sanguinarine on UVB-mediated modulation in the epidermal ODC enzyme activity.As shown by the data in Fig. 4a, UVB exposure was found toresult in a dramatic increase in epidermal ODC activity, at 24 h

posttreatment compared with control and sanguinarine-alonegroups. Further, the pre- and postapplication of sanguinarineresulted in an inhibition of UVB exposure-mediated increase in



Unt

Veh UVBSang

Sang + UVB

UVB + Sang

PCNA

(a)

(b)

β-actin

←

←

Figure 5. Inhibitory effect of sanguinarine on UVB-mediated induction of PCNA in SKH-1 hairless mice at 24 h posttreatment. (a) Epidermalprotein lysate was prepared, and PCNA protein expression was determined by western blotting. Equal loading of protein was confirmed bystripping the immunoblot and reprobing it for b-actin. (b) From skin biopsies, 6 lm thick sections were cut and the effects of treatments on PCNAwere assessed by immunohistochemical analysis. The representative pictures are from five to six skin biopsies captured by a Spot Insight QECamera attached to a Nikon microscope. Details of experiments are provided under Materials and Methods.


ODC enzyme activity. Similar effects of sanguinarine on ODC

protein expression (Fig. 4b) and immunohistochemical analy-sis (Fig. 4c) were also observed.

Inhibitory effect of sanguinarine on the protein levels of the

markers of tumor promotion viz. PCNA and Ki-67

In our next set of experiments, we determined the effect ofsanguinarine on UVB exposure-mediated modulations in theprotein levels of proliferation ⁄ tumor promotion markers

PCNA and Ki-67. PCNA is a widely accepted qualitative aswell as quantitative molecular marker of cellular proliferation.As expected, UVB exposure resulted in a marked increase in

the protein levels and immunohistochemical staining of PCNAin mouse skin epidermis (Fig. 5). Topical pre- and postappli-cation of sanguinarine were found to result in an appreciabledecrease in PCNA (Fig. 5). Immunohistochemical staining of

Ki-67 shows similar results with UVB exposure significantlyincreasing expression and sanguinarine treatments downmodulating this response (Fig. 6).

DISCUSSION

This study was designed to determine the protective effects ofplant alkaloid sanguinarine against UVB radiation-mediatedskin damages in SKH-1 hairless mouse, which is a well-acceptedmodel for photodamage and photocarcinogenesis studies. The

rationale for our choice of sanguinarine for this study is basedon several pieces of evidence. First, sanguinarine has been usedto treat many proinflammatory conditions including cancer in

ancient times and is currently used in several anti-gingivitis and

anti-inflammatory preparations (mouthwashes, toothpastes,cough medicines etc.) (8,9). Second, sanguinarine has beenshown to strongly inhibit the activation of proinflammatoryand antiapoptotic transcription factor nuclear factor (NF)-jB,which has been implicated in many inflammatory conditionsand in cancer (24). Third, studies have shown that sanguinarineinduces apoptosis in several cancer cell lines without any effect

on normal cells, at similar concentrations (12). Fourth, we haverecently shown that sanguinarine imparts photoprotectiveeffects in human HaCaT keratinocytes via an apoptotic

elimination of damaged cells (14). Finally, studies from ourlaboratory as well as by others have suggested that sanguinarinepossesses significant antioxidant properties (14,23) and UVBradiation causes a significant generation of the ROS.

In this study, we examined the protective effect of sanguin-arine against UV exposure by following the known markers ofUV damages. Our data demonstrated that sanguinarine

treatment to mouse skin significantly inhibits the edema andhyperplasia caused by UVB exposure. Edema and hyperplasiaof skin are considered the classical markers of UV exposure

mediated damages to the skin (25,26). As in humans, hairlessmice develop elastic fiber hyperplasia followed by elastosis andultrastructural degradation after exposure to UV radiation;

and therefore, hairless mouse is believed to be a relevantmodel for photodamage and photoaging studies (27). We havealso found that sanguinarine (pre- as well as postapplications)strongly inhibited the UVB-mediated increases in the markers

of cellular proliferation viz. PCNA and Ki-67 in mouse skin.PCNA is a subunit of DNA polymerase that has been shown



Figure 6. Effect of sanguinarine on UVB exposure-mediated modulation in Ki-67 immunostaining in SKH-1 hairless mouse skin. The mice weresubjected to different treatments and 24 h after UVB irradiation, the animals were killed and skin biopsies were collected and frozen for furtherstudy. From the skin biopsies, 6 lm thick sections were cut and the effects of treatments on Ki-67 were assessed by immunohistochemical analysis.The representative pictures are from five to six skin biopsies captured by a Spot Insight QE Camera attached to a Nikon microscope. Details ofexperiments are provided under Materials and Methods.


to play a role in DNA replication and repair and serves as abiomarker of proliferation (28). Studies have shown that UVradiation induces Ki-67 and epidermal hyperplasia (29) andaberrant accumulation of p53 has been associated with Ki-67

in benign skin lesions (30). Similarly, UVB radiation has alsobeen shown to induce PCNA and p53 in the skin (31).Further, PCNA promoter has been shown to be transcrip-

tionally activated via wild-type p53 as a mechanism of cellularresponse to DNA damage (32). Furthermore, PCNA plays acritical role in cell cycle progression where the cyclin kinase

inhibitor (cki) WAF1 ⁄ p21 can inhibit cell cycle progression byforming a quaternary complex with cyclin, cyclin-dependentkinase (cdk) and PCNA (33). Thus, the observed inhibition

of UVB-mediated increase in PCNA by sanguinarine isimportant and it also suggests the involvement of p53signaling and a cki-cyclin-cdk network in the protective effectsof sanguinarine.

It is now established that UV exposure causes an oxidativestress in the skin that may be involved in a wide range ofconditions including immunosuppression, inflammation, can-

cer and skin aging (34–36). Studies have suggested thatsanguinarine possesses strong antioxidant activity(14,23,37,38). In this study, we have found that sanguinarine

markedly inhibits UVB exposure-mediated generation of H2O2

suggesting that the photoprotective effects of sanguinarinemay be mediated via its antioxidant effects. Also, studies haveshown that UVB exposure may cause an additional outburst of

ROS in the skin cells via infiltration of leukocytes, therebyinflicting further cellular damages (39). In this study, we havefound that both pre- and post-UVB treatment of sanguinarine

causes a decrease in the infiltration of leukocytes that furthersubstantiate our suggestion that the observed effect of san-guinarine could be through its antioxidant activity that reduces

leukocyte-mediated oxidative stress. This data is consistentwith our previous in vitro findings that sanguinarine imparts aphotoprotective effect via modulation of oxidative stress

pathways in vitro (14). Our data have also demonstrated thatthe topical application of sanguinarine dramatically inhibitedUVB-mediated induction of ODC activity as well as proteinexpression. This is an interesting observation because ODC

has been shown to be associated with cellular transformationthat is regarded as a marker of tumor promotion (40–43). Thisdata suggests that sanguinarine may be able to inhibit tumor

promotion which makes it an interesting and potential agentfor the management of UVB-induced skin cancers.

Thus, taken together, our results suggest that sanguina-

rine may provide protection against the damages caused byUVB exposure and these protective effects may be mediatedvia its antioxidant properties. Interestingly, the posttreat-ment of sanguinarine was found to impart equal protection

compared with the pretreatment suggesting that sanguina-rine-mediated responses are not sunscreen effects. Based onour study, we suggest that sanguinarine could be developed

as an agent for the management of skin cancer and otherhyperproliferative skin conditions. However, detailed in vivostudies in relevant animal models are needed to support this

suggestion.

Acknowledgements—This work was supported by funding from the

National Cancer Institute (CA114060 and CA099076) and from the

Department of Defense (W81XWH-04-1-0220).

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Protective Effect of Sanguinarine on Ultraviolet B-mediated Damages in SKH-1 Hairless Mouse Skin: Implications for Prevention of Skin Cancer

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