CARNOSIC ACID: A POTENT CHEMOPREVENTIVE AGENT AGAINST ORAL CARCINOGENESIS

Chemico-Biological Interactions 188 (2010) 616–622

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Carnosic acid: A potent chemopreventive agent against oral carcinogenesis Shanmugam Manoharan ∗ , MuthamizhSelvan VasanthaSelvan , Simon Silvan , Nagarethinam Baskaran , Arjun Kumar Singh, Veerasamy Vinoth Kumar Department of Biochemistry & Biotechnology, Annamalai University, Annamalainagar 608 002, Cuddalore, Tamil Nadu, India

a r t i c l e

i n f o

Article history: Received 6 August 2010 Received in revised form 26 August 2010 Accepted 27 August 2010 Available online 22 September 2010 Keywords: Carnosic acid Chemoprevention Oral cancer Hamsters DMBA

a b s t r a c t The present study was aimed to investigate the chemopreventive potential of carnosic acid in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. The chemopreventive potential was assessed by analyzing the tumor incidence, tumor volume and burden as well as by measuring the status of lipid peroxidation, non-enzymatic and enzymatic antioxidants and phase I and phase II detoxification enzymes. Oral squamous cell carcinoma was developed in the buccal pouch of golden Syrian hamsters by painting with 0.5% DMBA in liquid paraffin three times a week for 14 weeks. In the present study, 100% tumor formation was observed in hamsters treated with DMBA alone. Also, the status of lipid peroxidation, antioxidants and phase I and phase II detoxification enzymes were significantly altered during DMBA-induced oral carcinogenesis. Oral administration of carnosic acid at a dose of 10 mg/kg body weight/day to DMBA-treated animals completely prevented the tumor formation in the hamsters’ buccal pouches. Also, carnosic acid exerted potent anti-lipid peroxidative function and stimulated the detoxification cascade during DMBA-induced hamster buccal pouch carcinogenesis. The results of the present study suggest that the chemopreventive potential of carnosic acid is probably due to its anti-lipid peroxidative potential and modulating effect on carcinogen detoxification enzymes during DMBA-induced oral carcinogenesis. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Oral cancer is one of the most common malignant neoplasm and fifth most frequent cancer worldwide. Despite recent advancement in cancer treatment, the morbidity and mortality due to oral cancer is still higher in developing countries including India, where this form of cancer accounts for 40–50% of all cancers. Tobacco and alcohol consumption are the principal risk factors of oral carcinogenesis, and 80% of patients with oral cancer are habituated to tobacco and alcohol consumption [1,2]. Squamous cell carcinoma accounts for 90% of all oral cancers and 5-year survival rate of patients with oral cancer are still at 50%. Oral carcinogenesis proceeds through three distinct phases, initiation, promotion and progression. Golden Syrian hamsters have contributed significantly to the understanding of precancerous and cancerous lesions of oral carcinogenesis. 7,12Dimethylbenz(a)anthracene (DMBA), a potent site and organ specific carcinogen, is widely used to induce oral squamous cell carcinoma in hamsters’ buccal mucosa. DMBA is also used to induce carcinogenesis in skin and mammary tissues. DMBA manifests its carcinogenic effect in the target tissues through formation of DNA

∗ Corresponding author. Tel.: +91 4144 239141x230; fax: +91 4144 238080. E-mail address: [email protected] (S. Manoharan). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.08.009

adducts, induction of chronic inflammation, over production of reactive oxygen species (ROS) and oxidative DNA damage [3,4]. DMBA-induced tumors in the hamsters’ buccal mucosa closely mimics the human oral cavity tumors, morphologically, histologically, biochemically and at molecular level. Also, DMBA-induced pathological lesions and ultra-structural changes closely resemble precancerous and cancerous lesions of human oral cavity [5–7]. Since the buccal pouch of golden Syrian hamster is more accessible to complete examination, DMBA-induced hamster buccal pouch carcinogenesis is commonly employed and widely accepted model to investigate the chemopreventive potential of natural products. Lipid peroxidation, a free radical mediated chain reaction, causes oxidative deterioration of lipids, particularly polyunsaturated fatty acids. Lipid peroxides, at physiological concentrations, play an important role in the control of cell division. An inverse relationship has been reported between lipid peroxidation and the rate of cell proliferation [8]. Low levels of thiobarbituric acid reactive substances (TBARS) have been well documented in tumors of the oral cavity [9]. Enhanced lipid peroxidation due to over production of reactive oxygen species causes oxidative damage to DNA, proteins and lipids [10]. Living organisms are, however, endowed with several antioxidant mechanisms that counterbalance the potential deleterious effects of reactive oxygen species (ROS). The antioxidant defense system consists of enzymatic [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] and non-

S. Manoharan et al. / Chemico-Biological Interactions 188 (2010) 616–622

enzymatic antioxidants [vitamins C, E, and reduced glutathione (GSH)] [11]. Over production of ROS and/or reduction in the activities of antioxidants results in oxidative stress, that has been implicated in the pathogenesis of several disorders including cancer [12]. Liver plays prominent and profound role in the metabolism and detoxification of mutagenic and carcinogenic substances. Glutathione-S-transferase (GST), glutathione reductase (GR) and reduced glutathione act together to detoxify carcinogen either by destroying their reactive centers or facilitating their excretion by conjugation process. Measurement of phase I and phase II detoxification enzymes in liver and buccal mucosa could help to assess the chemopreventive potential of the test compound [13,14]. Chemoprevention, a novel approach and recent trends in experimental oncology, is used to test the anti-tumor initiation and promoting potential of natural and synthetic agents. Agents that intervene early in the process of carcinogenesis to eliminate pre-malignant cells before they become malignant, or protect normal cells from undergoing malignant transformation are identified to have significant chemopreventive potential [15]. Chemopreventive agents that suppress promotion and/or progression of pre-malignant cells are found to have anti-mutagenic, anti-carcinogenic, inhibiting effects on cellular proliferation, modulating effect on phase I and II detoxification enzymes and free radical scavenging properties [16]. Rosemary (Rosmarinus officinalis) has many phytochemicals, which are potential sources of natural antioxidants. The antioxidant activity of rosemary is attributed mainly to its carnosic acid, carnosol and rosmarinic acid components. Carnosic acid, the primary phenolic diterpene, is abundantly present in the leaves of rosemary. It has been reported that carnosic acid protected biological membranes and prevented lipid peroxidation through its strong antioxidant potential. Carnosic acid scavenges number of reactive oxygen species including hydroxyl radical and lipid peroxy radicals [17]. Wijeratne and Cuppett [18] have reported that carnosic acid inhibited lipid peroxidation by 88–100% under oxidative stress conditions. Carnosic acid also exerted anti-proliferative, anti-inflammatory, anti-tumorigenic and neuroprotective effects [19,20]. It has been demonstrated that carnosic acid induced cell cycle arrest predominantly at G2 /M phase [21]. To the best of our knowledge, there were no scientific studies on chemopreventive potential of carnosic acid in DMBA-induced hamster buccal pouch carcinogenesis. The present study was therefore designed to evaluate the chemopreventive potential of carnosic acid in DMBAinduced oral carcinogenesis. The chemopreventive potential of carnosic acid was assessed by analyzing the tumor incidence, tumor burden and volume and by measuring the status of lipid peroxidation, antioxidants and detoxification agents in DMBA-treated hamsters. 2. Materials and methods 2.1. Chemicals DMBA and carnosic acid were obtained from Sigma–Aldrich Chemical Pvt. Ltd., Bangalore, India. All other chemicals used were of analytical grade, purchased from Hi-media Laboratories, Mumbai, India. 2.2. Animals and treatment Male golden Syrian hamsters, aged 8–10 weeks, weighing 80–120 g, were purchased from the National Institute of Nutrition, Hyderabad, India and were maintained in the Central Animal House, Rajah Muthaiah Medical College and Hospital, Annamalai

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University. The animals were housed five in a polypropylene cage and provided with a standard pellet diet (Agro Corporation Pvt. Ltd., Bangalore, India) and water ad libitum. The animals were maintained under controlled conditions of temperature (27 ± 2 ◦ C) and humidity (55 ± 5%) with a 12 h light/dark cycle. The Institutional Animal Ethics Committee (Reg. No. 160/1999/CPCSEA), Annamalai University, Annamalainagar, India, approved the experimental design. A total number of 40 hamsters were randomized into four groups and each group contained 10 hamsters. Group I animals served as the control and were treated with liquid paraffin (vehicle) alone three times a week for 14 weeks on their left buccal pouches. Group II animals were treated with 0.5% DMBA in liquid paraffin three times a week for 14 weeks on their left buccal pouches. Group II animals received no other treatment. Group III animals were treated with DMBA as in group II, received in addition oral administration of carnosic acid (10 mg/kg body weight/day), dissolved in 1 ml of 5% dimethyl sulphoxide (DMSO), starting 1 week before exposure to the carcinogen and continued on alternate days to DMBA painting until the animals were sacrificed. Group IV animals received oral administration of carnosic acid (10 mg/kg body weight/day) alone, as in group III, throughout the experimental period. The experiment was terminated at the end of 16 weeks and all animals were sacrificed by cervical dislocation. Biochemical studies were conducted on the plasma, liver and buccal mucosa tissues. For histopathological examination, buccal mucosa tissues were fixed in 10% formalin and routinely processed and embedded with paraffin, 2–3 ␮m sections were cut in a rotary microtome and stained with haematoxylin and eosin. 2.3. Induction of oral squamous cell carcinogenesis Tumors were induced in each hamster’s buccal pouch with topical application of 0.5% DMBA in liquid paraffin three times a week for 14 weeks. The total number of tumors in the hamster’s buccal pouch was determined macroscopically at the time of sacrifice of animals. 2.4. Samples (plasma and tissue preparation) Blood samples were collected into heparinized tubes. Plasma was separated by centrifugation at 1000 × g for 15 min. Tissue samples from the animals were washed with ice cold saline and homogenized using an appropriate buffer (GST: 0.3 M phosphate buffer, pH 6.5; GR: 0.1 M phosphate buffer, pH 7.4; TBARS: 0.025 M Tris–HCl buffer, pH 7.5; GSH and GPx: 0.4 M phosphate buffer, pH 7.0; SOD: 0.025 M sodium pyrophosphate buffer, pH 8.3; CAT: 0.01 M phosphate buffer, pH 7.0) in an all-glass homogenizer with a teflon pestle and used for biochemical estimations. 2.5. Biochemical analysis The activity of glutathione-S-transferase in liver and buccal mucosa tissue homogenate was assayed using the method employed by Habig et al. [22]. GST activity was measured by incubating the tissue homogenate with the substrate 1-chloro-2,4dinitrobenzene (CDNB). The absorbance was followed for 5 min at 540 nm after the reaction was started by the addition of reduced glutathione. Glutathione reductase activity in liver tissue homogenate was assayed using the method employed by Carlberg and Mannervik [23]. The enzyme activity was assayed by measuring the formation of reduced glutathione when the oxidised glutathione (GSSG) is reduced by reduced nicotinamide adenine dinucleotide phosphate (NADPH). The levels of cytochrome P450 and b5 in the liver and buccal mucosa were determined according to the method of Omura and Sato [24]. Cytochrome P450 was mea-

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sured by the formation of pigment on reaction between reduced cytochrome P450 and carbon monoxide. The pigment was read with an absorbance maximum at 450 nm. The difference spectrum between reduced and oxidised cytochrome was used as an index to measure the level of cytochrome b5 . The activity of DTdiaphorase in the liver was estimated according to the method of Ernster [25] based on the measurement of reduction at 550 nm using reduced nicotinamide adenine dinucleotide phosphate as the electron donor and 2,6-dichlorophenol indophenol as the electron acceptor. Lipid peroxidation was estimated as evidenced by the formation of thiobarbituric acid reactive substances. Thiobarbituric acid reactive substances in plasma were assayed by the method described by Yagi [26]. Plasma was deproteinised with phosphotungstic acid and the precipitate was treated with thiobarbituric acid at 90 ◦ C for 1 h. The pink color formed gives a measure of the thiobarbituric acid reactive substances (TBARS), which was read at 530 nm. Buccal mucosa lipid peroxidation was done using the method employed by Ohkawa et al. [27]. The color formed by the reaction of thiobarbituric acid with break down product of lipid peroxidation was measured colorimetrically at 532 nm. The reduced glutathione levels in the plasma and buccal mucosa were determined by the method described by Beutler and Kelly [28]. The technique involves protein precipitation by meta-phosphoric acid and spectrophotometric assay at 412 nm of the yellow derivative obtained by the reaction of the supernatant with 5,5 -dithiobis-2-nitrobenzoic acid. The oxidised glutathione level in the buccal mucosa was determined by the method of Tietze [29]. The oxidised glutathione content in the buccal mucosa was measured enzymically using glutathione reductase and NADPH. The vitamin E level in the plasma was determined colorimetrically using the method described by Desai [30]. Vitamin E presents in the lipid residue forms a pink colored complex with bathophenanthroline–phosphoric acid reagent, which was measured at 536 nm. Buccal mucosa vitamin E was measured using the fluorimetric method described by Palan et al. [31]. The lipid extracts were dried under nitrogen and the residues were suspended in 66% ethanol, followed by the addition of 4 ml of hexane and 0.6 ml of 60% sulphuric acid. The tubes were vortexed and centrifuged. The upper hexane phase was removed and its fluorescence intensity was measured at an excitation of 295 nm and emission of 320 nm, with ␣-tocopherol used to determine the standard curve. Superoxide dismutase activity was assayed in plasma and buccal mucosa using the method employed by Kakkar et al. [32] based on the 50% inhibition of formation of NADH-phenazine methosulphate nitroblue tetrazolium (NBT) formation. The color developed was read at 520 nm. One unit of enzyme is taken as the amount of enzyme required to give 50% inhibition of nitroblue tetrazolium (NBT) reduction. The activity of catalase in plasma and buccal mucosa was assayed using the method described by Sinha [33] based on the utilization of H2 O2 by the enzyme. The color developed was read at 620 nm. One unit of the enzyme is expressed as micromoles of H2 O2 utilized per minute. The activity of glutathione peroxidase in plasma and buccal mucosa was deter-

mined using the method employed by Rotruck et al. [34] based on the utilization of reduced glutathione by the enzyme. One unit of the enzyme is expressed as micromoles of GSH utilized per minute. 2.6. Protein determination The protein content was determined by the method of Lowry et al [35]. The peptide bonds (–CONH–) in polypeptide chain react with copper sulphate in an alkaline medium to give a blue colored complex. In addition, tyrosine and tryptophan residues of proteins cause reduction of the phosphomolybdate and phosphotungstate components of the Folin–Ciocalteau reagent to give bluish products read at 640 nm, which contribute towards enhancing the sensitivity of this method. 2.7. Statistical analysis The data is expressed as mean ± standard deviation (SD). Statistical comparisons for biochemical parameters were performed by one-way analysis of variance followed by Duncan’s multiple range test (DMRT). The tumor incidence was, however, statistically analyzed using Chi-Square (2 ) test. The results were considered statistically significant if the p-values were less than 0.05. 3. Results 3.1. Incidence of oral neoplasm The incidence of oral neoplasm in control and treated animals in each group are shown in Table 1. The tumor incidence was 100% in hamsters treated with DMBA alone and tumors were histopathologically confirmed as well-differentiated squamous cell carcinoma. The total number of oral tumors in the buccal pouches was counted and the diameter of each tumor was measured with a vernier caliper. Oral administration of carnosic acid (10 mg/kg body weight/day), on alternate days to DMBA painting, to DMBA-treated hamsters for 14 weeks completely prevented the formation of oral squamous cell carcinoma. 3.2. Histopathology The histopathological features observed in the buccal mucosa of the control and treated animals in each group are shown in Table 2 and Fig. 1. We observed severe hyperkeratosis, hyperplasia, dysplasia and well-differentiated squamous cell carcinoma in the buccal pouches of DMBA-alone treated hamsters (Fig. 1b). Although well-differentiated squamous cell carcinoma was not seen in the buccal pouches of DMBA + carnosic acid (group III) treated hamsters, hyperplasia, hyperkeratosis and dysplasia were noticed (Fig. 1c). Hamsters administered with carnosic acid alone showed well-defined and intact epithelial layers similar to that of the control hamsters (Fig. 1a and d).

Table 1 Incidence of oral neoplasm in control and treated animals in each group (n = 10). Parameter

Group I control

Group II DMBA alone

Group III DMBA + carnosic acid

Group IV carnosic acid alone

Tumor incidence (oral squamous cell carcinoma) Total number of tumors/animals Tumor multiplicity Tumor frequency Tumor volume (mm3 )/animals Tumor burden (mm3 )/animals

0% 0 0 0 0 0

100% 32/(10) 3.2 32 374.86 ± 41.72 1199.55 ± 133.50

0%a 0 0 0 0 0

0% 0 0 0 0 0

Tumor volume was measured using the formula, v = (4/3)[D1 /2][D2 /2][D3 /2] where D1 , D2 and D3 are the three diameters (mm) of the tumor. Tumor burden was calculated by multiplying tumor volume and the number of tumors/animal. Tumor multiplicity = average number of tumors per animal. Tumor frequency = number of tumors per group. a Significantly different from group II by Chi-Square (2 ) test.

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Table 2 Histopathological changes in the buccal pouch of hamsters in control and treated animals in each group. Parameter

Group I control

Group II DMBA alone

Group III DMBA + carnosic acid

Group IV carnosic acid alone

Hyperkeratosis Hyperplasia Dysplasia Squamous cell carcinoma

Absent Absent Absent Absent

Severe Severe Severe Well differentiated

Mild Mild Mild Absent

Absent Absent Absent Absent

3.3. Status of phase I and phase II detoxification agents The status of phase I (cytochromes P450 and b5 ) and phase II (GSH, GST, GR and DT-diaphorase) detoxification agents in the liver of treated and control animals in each group are given in Table 3. The status of phase I detoxification agents was significantly increased whereas phase II agents were decreased in the liver of DMBAtreated animals (group II) as compared to control animals (group I). Oral administration of carnosic acid to DMBA-treated animals (group III) brought back the status of phase I and phase II detoxification agents to near normal range in the liver. Oral administration of carnosic acid alone (group IV) showed no significant difference as compared to control animals. 3.4. Status of buccal mucosa phase I and phase II detoxification agents Table 4 shows the status of phase I and phase II detoxification agents in the buccal mucosa of control and treated hamsters in each group. The status of phase I (cytochromes P450 and b5 ) and phase II detoxification agents (GST and GSH) were significantly increased

whereas GSSG content was decreased in tumor-bearing hamsters (group II) as compared to control hamsters. Oral administration of carnosic acid to DMBA-treated hamsters (group III) significantly brought back the status of GSH, GSSG and GST to near normal range. Hamsters treated with carnosic acid alone (group IV) showed no significant difference in the status of GSH, GSSG and GST as compared to control hamsters (group I). 3.5. Status of plasma TBARS and antioxidants The status of thiobarbituric acid reactive substances and antioxidants (vitamins E and C, GSH, SOD, CAT and GPx) in plasma of control and treated animals in each group are shown in Table 5. The concentration of TBARS was increased, whereas the status of antioxidants was significantly decreased in tumor-bearing animals (group II) as compared to control animals. Oral administration of carnosic acid to DMBA-treated animals (group III) significantly brought back the concentrations of TBARS and antioxidants to near normal status. Hamsters treated with carnosic acid alone (group IV) showed no significant difference in TBARS and antioxidants status as compared to control hamsters (group I).

Fig. 1. Histopathological features observed in the buccal mucosa of control and experimental animals in each group. (a and d) Photomicrographs showing well-defined buccal pouch epithelium from control and carnosic acid alone treated hamsters respectively (H&E, 40×). (b) Photomicrograph showing well-differentiated squamous cell carcinoma with keratin pearls in hamsters treated with DMBA alone (H&E, 40×). (c) Photomicrograph showing moderate dyplastic epithelium in hamsters treated with DMBA + carnosic acid (H&E, 40×).

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Table 3 Status of phase I and phase II detoxification agents in the liver of control and treated animals in each group. Parameter

Group I control

Cyt P450 (UX /mg protein) Cyt b5 (UY /mg protein) GSH (nmol/mg protein) GR (nmol of NADPH oxidised/min/mg protein) GST (nmol of CDNB-GSH conjugate formed/min/mg protein) DT-diaphorase (UC /mg protein)

0.52 0.98 2.37 16.48 32.49 0.47

± ± ± ± ± ±

0.04a 0.08a 0.21a 1.23a 2.42a 0.03a

Group II DMBA alone 1.54 2.18 1.23 10.71 19.64 0.39

± ± ± ± ± ±

Group III DMBA + carnosic acid

0.10b 0.18b 0.10b 0.81b 1.70b 0.01b

0.60 1.18 2.13 14.57 28.92 0.43

± ± ± ± ± ±

Group IV carnosic acid alone

0.05c 0.16c 0.12c 1.07c 1.69c 0.03c

0.54 0.96 2.35 16.17 31.89 0.48

± ± ± ± ± ±

0.04a 0.07a 0.20a 1.38a 2.76a 0.04a

Values are expressed as mean ± SD for 10 hamsters in each group. Values that do not share a common superscript in the same column differ significantly at p < 0.05 (DMRT). X—micromoles of cytochrome P450 ; Y—micromoles of cytochrome b5 . C—micromoles of 2,6-dichlorophenol reduced per minute.

Table 4 Status of phase I and phase II detoxification agents in the buccal mucosa of control and treated animals in each group. Parameter

Group I control

Cyt P450 (UX /mg protein) Cyt b5 (UY /mg protein) GST (UA /mg protein) GSH (␮g/g tissue) GSSG (␮g/g tissue) GSH/GSSG (␮g/g tissue)

1.59 0.29 1.28 66.2 12.18 4.02

± ± ± ± ± ±

Group II DMBA alone

0.09a 0.03a 0.14a 5.2a 1.04a 0.32a

3.34 0.73 3.30 109.2 7.21 12.69

± ± ± ± ± ±

Group III DMBA + carnosic acid

0.21b 0.07b 0.21b 17.4b 0.75b 1.3b

1.73 0.37 1.53 74.4 10.38 5.53

± ± ± ± ± ±

0.08c 0.06c 0.18c 5.3c 0.90c 0.61c

Group IV carnosic acid alone 1.57 0.30 1.26 66.4 12.27 4.21

± ± ± ± ± ±

0.12a 0.04a 0.17a 6.8a 1.81a 0.38a

Values are expressed as mean ± SD for 10 hamsters in each group. Values that do not share a common superscript in the same column differ significantly at p < 0.05 (DMRT). X—micromoles of cytochrome P450 ; Y—micromoles of cytochrome b5 . A—micromoles of 1-chloro-2,4-dinitrobenzene (CDNB)/reduced glutathione conjugate formed per minute.

Table 5 Status of plasma TBARS and antioxidants in control and treated animals in each group. Parameter

Group I control

TBARS (nmol/ml) SOD (UA /ml) CAT (UB /ml) GPx (UC /L) Vitamin E (mg/dL) GSH (mg/dL)

2.66 2.75 0.53 126.70 1.40 27.94

± ± ± ± ± ±

Group II DMBA alone

0.17a 0.21a 0.04a 11.81a 0.12a 2.81a

4.52 1.69 0.29 84.62 0.85 16.38

± ± ± ± ± ±

Group III DMBA + carnosic acid

0.39b 0.18b 0.01b 8.97b 0.07b 1.47b

2.92 2.45 0.47 111.41 1.20 23.36

± ± ± ± ± ±

0.18c 0.22c 0.03c 10.72c 0.16c 2.79c

Group IV carnosic acid alone 2.65 2.73 0.51 128.57 1.42 26.51

± ± ± ± ± ±

0.15a 0.20a 0.04a 11.29a 0.17a 1.74a

Values are expressed as mean ± SD for 10 hamsters in each group. Values that do not share a common superscript in the same column differ significantly at p < 0.05 (DMRT). A—the amount of enzyme required to inhibit 50% NBT reduction. B—micromoles of hydrogen peroxide utilized/s. C—micromoles of glutathione utilized/min.

3.6. Status of buccal mucosa TBARS and antioxidants

4. Discussion

The status of TBARS and antioxidants in the buccal mucosa of control and treated animals in each group is shown in Table 6. Decrease in the TBARS levels and disturbances in antioxidants status (vitamin E, GSH and GPx were increased; SOD and CAT were decreased) were noticed in tumor-bearing animals (group II) as compared to control animals. Oral administration of carnosic acid to DMBA-treated animals (group III) brought back the concentration of TBARS and antioxidants to near normal range. Hamsters treated with carnosic acid alone (group V) showed no significant difference in TBARS and antioxidants status as compared to control hamsters (group I).

Malignant tumors exhibit abnormal biochemical and molecular events as compared to their normal counterpart. DMBA induced oral tumor closely mimics the human oral tumor both histologically and morphologically. In the present study, 100% tumor formation was noticed in hamsters treated with DMBA alone at the end of experimental period. The tumors were histopathologically confirmed as well-differentiated squamous cell carcinoma. The tumor cells have pleomorphic, hyperchromatic nuclei with epithelial formation. Also, hamsters treated with DMBA alone revealed severe hyperplasia, hyperkeratosis and dysplasia. Though the tumor formation was not seen in hamsters treated with DMBA + carnosic

Table 6 Buccal mucosa TBARS and antioxidants in control and treated animals in each group. Parameter TBARS (nmoles/100 mg protein) SOD (UA /mg protein) CAT (UB /mg protein) GPx (UC /g protein) Vitamin E (mg/100 mg tissues) GSH (mg/100 g tissues)

Group I control 81.57 5.18 42.86 7.56 1.83 6.62

± ± ± ± ± ±

a

7.31 0.43a 3.71a 0.62a 0.16a 0.52a

Group II DMBA alone 45.16 3.47 25.91 14.68 3.12 10.92

± ± ± ± ± ±

b

3.98 0.38b 1.80b 1.27b 0.27b 1.74b

Group III DMBA + carnosic acid 72.19 4.59 37.79 8.80 2.02 7.44

± ± ± ± ± ±

c

6.82 0.36c 2.39c 1.19c 0.12c 0.53c

Group IV carnosic acid alone 83.67 5.15 41.71 7.55 1.85 6.64

± ± ± ± ± ±

7.43a 0.47a 3.11a 0.64a 0.10a 0.68a

Values are expressed as mean ± SD for 10 hamsters in each group. Values that do not share a common superscript in the same column differ significantly at p < 0.05 (DMRT). A—the amount of enzyme required to inhibit 50% NBT reduction. B—micromoles of hydrogen peroxide utilized/s. C—micromoles of glutathione utilized/min.

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acid, the buccal mucosa of these animals’ revealed severe hyperkeratosis, hyperplasia and mild dysplasia. The present study thus suggests that carnosic acid exerted pronounced chemopreventive potential by significantly preventing the tumor formation during DMBA-induced hamster buccal pouch carcinogenesis. In the present study, liver phase I detoxification enzymes were increased whereas phase II detoxification enzymes were decreased in hamsters treated with DMBA alone. Several reports suggested that in cancerous conditions including oral cancer, toxic metabolites are accumulated in liver due to increased activities of phase I detoxification enzymes and decreased activities of phase II detoxification enzymes [36,37]. Phase I and phase II detoxification cascade are stimulated when cells are exposed to carcinogenic agents including DMBA [38]. Our results lend credence to these observations. Oral administration of carnosic acid at a dose of 10 mg/kg body weight/day restored the activities of phase I and phase II detoxification agents in DMBA-treated hamsters. The results of the present study thus suggest that carnosic acid might have inhibited the metabolic activation of DMBA as evidenced by decreased activities of phase I enzymes and stimulated the activities of phase II detoxification enzymes to excrete the ultimate carcinogenic metabolite of DMBA, dihydrodiol epoxide. Chemical carcinogens mediate carcinogenesis in cells and tissues through reactive oxygen species mediated oxidative damage. DMBA, a pro-carcinogen, mediates carcinogenesis through its active metabolite dihydrodiol epoxide, which is generated upon metabolic activation of DMBA [39]. The possible mechanism of DMBA induced carcinogenesis includes induction of chronic inflammation, over production of reactive oxygen species and oxidative DNA damage [40]. Inverse association between lipid peroxidation and abnormal cell proliferation has been well documented [41]. The low availability of polyunsaturated fatty acids (PUFA) has been suggested to be a limiting factor for peroxidation in tumors tissues [9,42]. Profound studies demonstrated that oral cancer tissues have lower TBARS levels as compared to adjacent and normal tissues [43–45]. Lowered TBARS levels in the buccal mucosa of hamsters treated with DMBA alone are probably due to decreased PUFA content in tumor tissues or abnormal cell proliferation occurring in oral carcinogenesis. Tumor cells sequester nutrients from circulation and other host tissues to compromise its nutritional demand as well as for rapid growth [46]. Increase in vitamin E and glutathione in tumor tissues might be due to the above phenomena. Glutathione peroxidase and its co-substrate reduced glutathione are over-expressed in tumor tissues due to their regulatory effects on cell proliferation [47]. Lowered activities of superoxide dismutase and catalase were reported in several malignancies including oral cancer [48]. Our results lend credence to these observations. The decrease in TBARS levels and functional compromise of antioxidant defense mechanisms observed in the tumor tissues of DMBA-treated hamsters reflects a decreased susceptibility of oral tumor tissue to lipid peroxidation. Several experimental studies supported our findings [49,50]. Measurements of TBARS and antioxidants could be used to assess the extent of tissue damage. Elevated levels of TBARS and lowered activities of enzymatic antioxidants and non-enzymatic antioxidants levels were observed in the plasma of hamsters treated with DMBA. The reduced glutathione is the most effective intracellular antioxidant and the molar ratio of reduced glutathione to oxidised glutathione serves as an important marker of the antioxidant capacity of the cell [9]. Lowered levels of vitamin E and glutathione in plasma are probably due to their utilization by tumor tissues to meet the nutritional demand of growing tumor. Profound studies on experimental oral carcinogenesis demonstrated lowered activities of enzymatic antioxidants in plasma of tumorbearing animals [51,52]. Lowered activities of these enzymes are

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probably due to exhaustion of these enzymes to scavenge excessively generated reactive oxygen species in the system. Increased levels of plasma TBARS in DMBA-treated animals could probably be due to insufficient antioxidant potential. Measurement of TBARS has limitations relative to assessing lipid peroxidation and therefore other markers of oxidative damage such as reactive oxygen species or carbonyl protein would also be useful for assessment of lipid peroxidation products. The status of reactive oxygen species and carbonyl protein might yield different results than what we reported and the measurement of these markers would be useful in the future experiments. Carnosic acid, an important bioactive principle of Rosemary, has been received considerable attention in recent years because of its diverse biological and pharmacological properties. Carnosic acid has been shown to have potent radioprotective, photoprotective anti-mutagenic, anti-genotoxic and antioxidant properties [17–20,53]. In the present study, oral administration of carnosic acid at a dose of 10 mg/kg body weight/day restored the status of lipid peroxidation and antioxidants in the plasma and buccal mucosa of DMBA-treated hamsters. The antioxidant potential of carnosic acid relies on the presence of two O-phenolic hydroxyl groups present at C11 and C12 of the molecule. The results of the present study thus suggest that carnosic acid might have maintained the balance of oxidant and antioxidant status; probably by scavenging excessively generated reactive oxygen species during DMBA-induced hamster buccal pouch carcinogenesis. The present study thus demonstrated the chemopreventive potential of carnosic acid in DMBA-induced hamsters buccal pouch carcinogenesis. The present study concludes that carnosic acid might have suppressed cell proliferation by inhibiting the metabolic activation of DMBA during DMBA-induced oral carcinogenesis. Also, its antilipid peroxidative (antioxidant) potential and modulating effect on detoxification cascade could play a possible role. Altered levels of the biomarkers (phase I and phase II detoxification enzymes, TBARS and antioxidants) in the tumor tissues are simply the reflection of profound alterations of cellular mechanisms in the tumor tissues that arise as a consequence of the process of carcinogenesis. A clear distinction between the cause and effect of carnosic acid could be achieved with time-course follow-up of these biomarkers by carrying out interim sacrifices of animals during DMBA-induced oral carcinogenesis. The effect of carnosic acid on molecular markers related to cell cycle and apoptotic pathway during DMBA-induced oral carcinogenesis is also under investigation. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgement Financial assistance from Indian Council of Medical Research (ICMR), New Delhi, is gratefully acknowledged. References [1] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, M.J. Thun, Cancer statistics 2008, CA Cancer J. Clin. 58 (2008) 71–96. [2] B. Rodu, C. Jansson, Smokeless tobacco and oral cancer: a review of the risks and determinants, Crit. Rev. Oral. Biol. Med. 15 (2004) 252–263. [3] N. Baskaran, S. Manoharan, S. Balakrishnan, P. Pugalendhi, Chemopreventive potential of ferulic acid in 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis in Sprague–Dawley rats, Eur. J. Pharmacol. 637 (2010) 22–29. [4] L.M. Alias, S. Manoharan, L. Vellaichamy, S. Balakrishnan, C.R. Ramachandran, Protective effect of ferulic acid on 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis in Swiss albino mice, Exp. Toxicol. Pathol. 61 (2009) 205–214.

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