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Protective effect of ferulic acid on 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis in Swiss albino mice
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Experimental and Toxicologic Pathology 61 (2009) 205–214 www.elsevier.de/etp
Protective effect of ferulic acid on 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis in Swiss albino mice Linsa Mary Aliasa, Shanmugam Manoharana,, Lakshmanan Vellaichamya, Subramanian Balakrishnana, Cinnamanoor Rajamani Ramachandranb a
Department of Biochemistry and Biotechnology, Faculty of science, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India b Department of Oral & Maxillofacial Pathology, Rajah Muthiah Dental College & Hospital, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India Received 7 May 2008; accepted 3 September 2008
Abstract Our aim was to evaluate and compare the chemopreventive potential of topically applied and orally administered ferulic acid in 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis. Estimating the status of phase I and phase II detoxication agents, lipid peroxidation byproducts and antioxidants during DMBA-induced skin carcinogenesis assessed the mechanistic pathway for its chemopreventive efficacy. Skin squamous cell carcinoma was induced in the shaved back of mice, by painting with DMBA (25 mg in 0.1 mL1 acetone) twice weekly for 8 weeks. We have observed 100% tumor formation in the 15th week of experimental period in mice treated with DMBA alone. Marked alterations in the status of phase I and phase II detoxication agents, lipid peroxidaton byproducts and antioxidants were observed in tumor bearing mice. Oral administration of ferulic acid completely prevented the formation of skin tumors, whereas topically applied ferulic acid did not show significant chemopreventive activity during DMBA-induced mouse skin carcinogenesis. Also, oral administration of ferulic acid reverted the status of phase I and phase II detoxication agents, lipid peroxidaton byproducts and antioxidants to near-normal range in DMBAtreated mice. Our results thus demonstrate that orally administered ferulic acid has potent suppressing effect on cell proliferation during DMBA-induced skin carcinogenesis. This is probably due to its modulating effect on the status of lipid peroxidation, antioxidants and detoxication agents during DMBA-induced skin carcinogenesis. r 2008 Elsevier GmbH. All rights reserved. Keywords: Skin cancer; Ferulic acid; Lipid peroxidation; Antioxidants; Detoxication agents
Introduction Skin, a major environmental interface for the body, is accidentally or occupationally exposed to a number of Corresponding author. Tel.: +91 4144 238343; fax: +91 4144 238080. E-mail address: [email protected] (S. Manoharan).
0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.09.001
chemical mutagens and carcinogens. Skin cancer accounts for 30% of all newly diagnosed cancers in the world. Epidemiological studies have reported that the incidence of skin cancer is significantly rising worldwide due to increased cumulative ultraviolet exposure. Epithelial tumors, basal cell carcinoma and squamous cell carcinoma are the most important skin tumors (Jemal et al., 2003; Kyriazi et al., 2006). 7,12-dimethylbenz[a]anthracene
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(DMBA), a potent organ-specific carcinogen, can act either as complete carcinogen or as an initiator of mouse skin carcinogenesis. Dihydrodiolepoxide, the ultimate carcinogen of DMBA, mediates skin carcinogenesis by inducing chronic inflammation, over production of reactive oxygen species (ROS) and oxidative DNA damage. DMBA-induced mouse skin carcinogenesis is widely employed to test the chemopreventive efficacy of medicinal plants and their constituents (Miyata et al., 2001; Rastogi et al., 2007). ROS such as superoxide radical, hydroxyl radical and hydrogen peroxides are frequently generated in the biological systems either by normal metabolic pathways or as a consequence of exposure to physical, chemical and biological agents. ROS attack biomembranes and lead to oxidative destruction of polyunsaturated fatty acids (PUFA) by a chain reaction known as lipid peroxidation. ROS interfere with the structure and function of the cells, making them weak and defenseless. Overproduction of ROS within tissues can damage DNA and contribute to mutagenesis and carcinogenesis. ROS-mediated oxidative stress has been implicated in the pathogenesis of several diseases including cancer (Ray and Husain, 2002). Human body has, however, an array of sophisticated antioxidant defense mechanism to combat the deleterious effects of ROS-mediated oxidative damage. This defense mechanism includes nonenzymatic antioxidants (vitamin E, vitamin C and glutathione) and enzymatic antioxidants [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] (Ray and Husain, 2002; McCord, 2000). Previous reports from our laboratory have documented the status of lipid peroxidation and antioxidants in experimental carcinogenesis (Manoharan et al., 2006; Kolanjiappan and Manoharan, 2005). Chemoprevention is a promising pharmacological approach to prevent, delay or reverse the multi-step process of carcinogenesis. A large number of plant phenolics act as potent inhibitors for mutagenesis and carcinogenesis induced by polycyclic aromatic hydrocarbons such as DMBA (Wood et al., 1982; Newmark, 1987). The anticancer effect of phytochemicals is based on their ability to quench ROS and thereby protecting critical cellular molecules from oxidative insult. Also, antiproliferative activity and induction of apoptosis in cancer cells are other important anticancer mechanisms of phytochemicals. Ferulic acid, 4-hydroxy-3-methoxycinnamic acid, (Fig. 1) arises from the metabolism of phenylalanine and tyrosine by Shikimate pathway in plants. Ferulic acid is rich in many staple food such as grain bran, whole grain foods, citrus fruits, banana, coffee, orange juice, eggplant, bamboo shoots, beet root, cabbage, spinach and broccoli. It exhibits beneficial effects against various diseases like cancer, diabetes, cardiovascular and neurodegenerative disorders. Ferulic acid has potent free radical scavenging activity and can
O
OH
OH OCH3
Fig. 1. Molecular structure of ferulic acid.
effectively scavenge superoxide anion radicals. In certain countries ferulic acid has been approved as food additive to prevent lipid peroxidation (Zhao and Moghadasian, 2008; Graf, 1992; Srinivasan et al., 2007). Asanoma et al. (1994) have demonstrated that topical application of ferulic acid showed weak chemopreventive activity in skin carcinogenesis. To the best of our knowledge, we have found no scientific evidence on the chemopreventive potential of orally administered ferulic acid in DMBA-induced skin carcinogenesis. The present study was therefore designed to compare the chemopreventive potential of topically applied and orally administered ferulic acid in DMBA-induced mouse skin carcinogenesis.
Materials and methods Chemicals DMBA and ferulic acid were purchased from Sigma Aldrich Chemical Pvt. Ltd., Bangalore, India. All other chemicals used were of analytical grade.
Animals Male Swiss albino mice 4–6 weeks old, weighing 15–20 g were purchased from the National Institute of Nutrition, Hyderabad, India, and maintained in the Central Animal House, Rajah Muthaiah Medical College and Hospital, Annamalai University. The animals were housed in groups of four or five in polypropylene cages and provided standard pellet diet and water ad libitum and maintained under controlled
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conditions of temperature and humidity, with a 12 h light/dark cycle. The animals were maintained as per the principles and guidelines of the ethical committee for animal care of Annamalai University in accordance with the Indian National Law on animal care and use.
Experimental design The Institutional animal ethical committee [Reg. no.: 160/1999/CPCSEA], Annamalai University, Annamalainagar, India, approved the experimental design. A total of 30 male Swiss albino mice were divided into five groups of six each. Skin carcinogenesis was developed in Swiss albino mice according to the method of Azuine and Bhide (1992). Depilatory cream was applied to remove hair from the back of each mouse and the mice were left untreated for 2 days. Mice having no hair growth after 2 days were selected for the experimental study. The depilated back of group I mice was painted with acetone (0.1 ml/mouse) twice weekly for 8 weeks (vehicle-treated control). The depilated back of groups II–IV mice were painted with DMBA (25 mg in 0.1 ml acetone/mouse) twice weekly for 8 weeks. Group II mice received no other treatment. Group III mice were administered ferulic acid (40 mg/kg b.w in 1 ml distilled water) orally starting 1 week before the exposure to the carcinogen and continued for 25 weeks (3 times/week on alternate days) thereafter. Group IV animals were topically painted with ferulic acid (1.5 mg/animal/day) starting 1 week before the exposure to the carcinogen and continued for 25 weeks (3 times/week on alternate days) thereafter. At the end of the experimental period all the animals were sacrificed by cervical dislocation.
Macroscopic evaluation Body weight of experimental animals in each group was recorded at weekly intervals, throughout the experimental period. During the sacrifice of animals, liver was dissected out and blotted dry before weighing. A part of the liver was used for the assay of phase I and phase II detoxication agents. The relative organ weight was calculated as the ratio between organ weight and body weight. The number of skin tumors was counted and the diameter of each tumor was measured with a caliper. The tumor volume was calculated by the formula n ¼ (4/3)p[D1/2][D2/2][D3/2], where D1, D2 and D3 are the three diameters (mm) of the tumors. Tumor burden was calculated by multiplying tumor volume and the number of tumors/mice.
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Histological investigation Skin tissues from control mice, DMBA alone treated mice, DMBA+ferulic acid treated mice and ferulic acid alone treated mice were removed, measured and weighed. The skin tissues were fixed overnight in buffered neutral formalin and processed for histological evaluation. The tissues were routinely processed and embedded with paraffin wax, sectioned at 2–3 mm in a rotary microtome and stained with hematoxylin for 10 min. Subsequently, they were washed in running tap water for 5 min and then rinsed in 0.5% acid alcohol (0.5% HCl in 95% alcohol). The slides were dried on a hot plate and stained with 0.5% eosin in 95% ethanol for 5 min. The sections were rapidly rinsed in 95% ethanol and dehydrated in two changes of absolute ethanol for 5 min each. The slides were then cleared in xylene and mounted in resinous medium. Single sections of each specimen were evaluated by light microscopy. The epidermal changes were categorized into (i) hyperkeratosis, when there is an increase in the superficial cornified layer, (ii) hyperplasia, when the epidermal thickness is more than the normal thickness without any dysplastic features, (iii) dysplasia, for which the criteria proposed by Pindborg et al. (1997) is considered and (iv) carcinoma, when there is a breach in the basement membrane and invasion of epithelial cells into the underlying stromal tissue.
Biochemical analysis Biochemical studies were conducted on plasma, erythrocytes and skin tissues of control and experimental animals in each group. Blood samples were collected in heparinized tubes. The plasma was separated by centrifugation at 3000 rpm for 15 min. After plasma separation, the buffy coat was removed and the packed cells were washed thrice with physiological saline. A known volume of erythrocytes was lysed with hypotonic buffer at pH 7.4. The hemolysate was separated by centrifugation at 10,000 rpm for 15 min at 20 1C. The erythrocyte membrane was prepared by the method of Dodge et al. (1963) modified by Quist (1980). The erythrocytes remaining after the removal of plasma were washed three times with 310 mM isotonic Tris–HCl buffer (pH 7.4). The washed erythrocyte suspension was treated with 20 mM hypotonic Tris–HCl buffer (pH 7.2) to perform hemolysis. The erythrocyte membranes were sediment in a high-speed cooling centrifuge at 20,000g for 40 min. The supernatant was decanted and the erythrocyte membrane pellet was made up to a known volume using 0.2 M isotonic Tris–HCl buffer (pH 7.4). Aliquots from these preparations were used for the biochemical estimations.
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In plasma, thiobarbituric acid reactive substances (TBARS) was assayed by the method of Yagi (1987). Plasma was deproteinised with phosphotungstic acid and the precipitate was treated with thiobarbituric acid at 90 1C for 1 h. The pink color formed gives a measure of the TBARS, which was read at 530 nm. TBARS in erythrocytes and erythrocyte membranes was estimated by the method of Donnan (1950). Absorbance of pink chromogen formed by the reaction of thiobarbituric acid with breakdown products of lipid peroxides was read at 535 nm. The reduced glutathione level in erythrocytes, liver and skin tissues was determined by the method of Beutler and Kelly (1963). 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 5,50 dithiobis-2-nitrobenzoic acid. Superoxide dismutase activity in erythrocytes and skin tissues was assayed by the method of Kakkar et al. (1984), based on the 50% inhibition of formation of NADH–phenazine methosulphate nitro blue tetrazolium (NBT). 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 NBT reduction. The activity of CAT in erythrocytes and skin tissues was assayed by the method of Sinha (1972), based on the utilization of H2O2 by the enzyme. The color developed was read at 620 nm. One unit of the enzyme is expressed as mmoles of H2O2 utilized per minute. The activity of GPx in erythrocytes and skin tissues was determined using the method of Rotruck et al. (1973), based on the utilization of reduced glutathione by the enzyme. One unit of the enzyme is expressed as mmoles of GSH utilized per minute. The activity of glutathione-S-transferase (GST) in skin tissues and liver tissue homogenate was assayed by the method of Habig et al. (1974). GST activity was measured by incubating the tissue homogenate with the substrate 1-chloro-2,4-dinitrobenzene (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 skin
Table 1.
Statistical analysis Values are expressed as mean7SD. Statistical analysis was performed by one-way analysis of variance, followed by Duncan’s multiple range test. The values were considered statistically significant if p value was less than 0.05.
Results Body weight and liver weight Table 1 shows the body and liver weight and relative organ weight of control and experimental animals in each group. The body weight and liver weight were significantly decreased in DMBA-treated animals as compared to control animals. Oral administration of ferulic acid three times per week for 25 weeks
Effect of ferulic acid on body weight, liver weight and relative organ weight of experimental animals in each group
Groups
1. 2. 3. 4. 5.
tissues and liver tissue homogenate was assayed by the method of Carlberg and Mannervik (1985). The enzyme activity was assayed by measuring the formation of reduced glutathione when the oxidized glutathione (GSSG) is reduced by reduced nicotinamide adenine dinucleotide phosphate (NADPH). The levels of cytochrome P450 and b5 in liver and skin tissues were determined according to the method of Omura and Sato (1964). Cytochrome P450 was measured 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 oxidized cytochrome was used as an index to measure the level of cytochrome b5. The activity of DTdiaphorase was estimated according to the method of Ernster (1967) based on the measurement of reduction at 550 nm using reduced NADPH as the electron donor and 2,6-dichlorophenol indophenol as the electron acceptor.
Body wt. (g)
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Initial
Final
22.8071.1a 23.1171.3a 23.1871.1a 23.2171.3a 22.8471.2a
28.1371.5a 22.1671.7b 27.3471.6a 23.4671.6b 28.8771.9a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05.
Liver wt. (g)
Relative organ weight (liver wt./body wt.)
1.1670.07a 0.8870.09b 1.0970.08a 0.9170.07b 1.1870.06a
0.04170.002a 0.04070.004a 0.04070.003a 0.03970.003a 0.04170.002a
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significantly increased the body and liver weight in DMBA-treated animals. However, topical application of ferulic acid to DMBA-painted animals slightly increased the body and liver weight in DMBA-treated animals. Oral administration of ferulic acid alone to mice (group V) showed no significant difference in body and liver weight as compared to control animals (group I). We also found no statistically significant difference in the relative organ weight of control and experimental animals in each group.
(Fig. 3C). Moderate preneoplastic lesions, hyperplasia, hyperkeratosis and dysplasia, and well-differentiated squamous cell carcinoma were noticed in DMBApainted animals that received topical application of ferulic acid (group IV) (Fig. 3D). Oral administration of ferulic acid to DMBA-treated mice showed normal skin and the presence of subcutaneous tissue (Fig. 3E).
Tumor incidence, volume and burden
Table 3 shows the levels of TBARS in plasma, erythrocyte membrane and skin tissues of control and experimental animals in each group. The levels of
Table 2 shows the tumor incidence, volume and tumor burden of control and experimental animals in each group. In DMBA-painted mice (group II), a 100% tumor formation with mean tumor volume (580.2 mm3) and tumor burden (1836.1 mm3) was observed. The gross appearance of skin tumors is depicted in Fig. 2. Oral administration of ferulic acid (40 mg/kg b.w) completely prevented tumor incidence in DMBA-painted mice. However, we noticed 66.6% tumor formation with mean tumor volume (300.1 mm3) and tumor burden (750.2 mm3) in DMBA-painted animals that received topical application of ferulic acid (1.5 mg/animal/day). No tumors were observed in control (group I) as well as ferulic acid alone treated animals (group V).
Status of TBARS
Histopathological observations The histopathological features observed in the skin tissues of Swiss albino mice in control and experimental animals in each group are depicted in Fig. 3(A–E). The histopathological studies on control animals (group I) showed normal skin and the presence of subcutaneous tissue (Fig. 3A). The skin tissues from DMBA-treated mice (group II) revealed hyperkeratosis, hyperplasia, dysplasia and well-differentiated squamous cell carcinoma with keratin pearls (Fig. 3B). The skin tissues from DMBA-painted animals received oral administration of ferulic acid (group III) showed mild hyperplasia Table 2.
Effect of ferulic acid on tumor incidence, tumor volume and tumor burden of experimental animals in each group
Groups 1. 2. 3. 4. 5.
Fig. 2. Gross appearance of mouse skin tumors in DMBA alone painted animals.
Control (vehicle treated) DMBA 1836.17128.59 DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Tumor incidence
Total number of tumors
Tumor volume (mm3)
Tumor burden (mm3)
0 100% (6/6)
0 19/(6)
0 580.2751.83
0
0 66.6% (4/6) 0
0 10/(4) 0
0 300.1728.6 0
0 750.2760.3 0
Values are expressed as mean7SD (n ¼ 6 mice). Tumor volume was measured using the formula n ¼ (4/3)p[D1/2][D2/2][D3/2] where D1, D2 and D3 are the three diameters (mm) of the tumors. Tumor burden was calculated by multiplying tumor volume and the number of tumors/animal. Number in parenthesis indicated total number of animals bearing tumors.
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Fig. 3. (A) Microphotograph of skin tissues from control animals showing well-defined subcutaneous tissues and intact epithelial layer. (B) Microphotograph of skin tissues from DMBA alone painted animals showing well-differentiated squamous cell carcinoma with hyperplasia (arrow head), dysplasia (arrow) and keratin pearls (block arrow). (C) Microphotograph of skin tissues from DMBA+ferulic acid (oral administration; 40 mg/kg b.w)-treated animals showing hyperplastic epithelium (arrow head). (D) Microphotograph of skin tissues from DMBA+ferulic acid (topical application; 1.5 mg/animal/day) treated animals showing hyperplastic (arrow head) and severe dysplastic epithelium (arrow). (E) Microphotograph of skin tissues from ferulic acid alone treated animals showing well-defined subcutaneous tissues and intact epithelial layer.
Table 3.
Effect of ferulic acid on TBARS in plasma, erythrocytes and skin tissues of experimental animals in each group
Groups
Plasma TBARS (nmol/ml)
Erythrocytes membrane TBARS (nmol/mg protein)
Skin tissues TBARS (mmol/100 g tissue)
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
2.1370.14a 4.8970.56b 2.3670.21c 3.5670.41d 2.0770.11a
0.3070.02a 1.0170.13b 0.3570.05c 0.8070.07d 0.2970.02a
100.1878.5a 115.73712.3b 106.1379.7a 113.56712.4b 101.0777.9a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05.
TBARS were significantly increased in plasma and erythrocyte membranes, whereas decreased in skin tissues of tumor bearing animals (group II) as compared to control animals. Oral administration of ferulic acid three times per week for 25 weeks to DMBA-painted animals reverted the levels of TBARS to near normal range. However, topical application of ferulic acid to DMBA-painted animals did not show significant effect. Control mice treated with ferulic acid alone (group V) showed no significant difference in plasma, erythrocyte
membrane and skin tissue TBARS as compared to control mice (group I).
Enzymatic and non-enzymatic antioxidants status Tables 4 and 5 show the activities of enzymatic antioxidants (SOD, CAT, GPx) and non-enzymatic antioxidant (GSH) level in erythrocytes and skin tissues of control and experimental animals in each group,
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Table 4. Effect of ferulic acid on status of enyzmatic and non-enzymatic antioxidants in erythrocytes of experimental animals in each group Groups
SOD (UA/ mg Hb)
CAT (UB/ mg Hb)
GPx (UC/ g Hb)
GSH (mg/dl)
1. 2. 3. 4. 5.
5.9670.37a 3.7370.32b 5.5670.48c 4.3870.51d 6.0170.42a
2.5370.18a 1.5670.20b 2.3270.28c 1.9370.24d 2.5970.19a
24.1871.51a 16.4371.93b 22.3072.28c 19.172.03d 24.2671.58a
42.173.01a 27.2873.17b 38.8773.96c 33.1273.09d 42.572.6a
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05. A. The amount of enzyme required to inhibit 50% NBT reduction. B. Micromoles of H2O2 utilized per second. C. Micromoles of glutathione utilized per minute.
Table 5. group
Effect of ferulic acid on status of enyzmatic and non-enzymatic antioxidants in skin tissue of experimental animals in each
Groups 1. 2. 3. 4. 5.
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
SOD (UA/ mg protein)
CAT (UB/ mg protein)
GPx (UC/ mg protein)
GSH (mg/ 100 mg tissues)
6.4370.48a 3.5970.41b 6.0170.53c 5.3270.56d 6.4970.39a
42.1873.23a 21.6772.56b 39.1473.53c 29.673.7d 42.2473.04a
43.5973.01a 29.3273.41b 40.0473.33c 36.1273.93d 43.6673.11a
40.373.76a 22.1472.97b 37.1173.92c 30.1573.59d 40.473.82a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05.
A. The amount of enzyme required to inhibit 50% NBT reduction. B. Micromoles of H2O2 utilized/s. C. Micromoles of glutathione utilized/min.
respectively. The activities of SOD, CAT, GPx and GSH level were significantly decreased in erythrocytes and skin tissues of tumor-bearing animals (group II) as compared to control animals. Oral administration of ferulic acid three times per week for 25 weeks to DMBA-painted animals reverted the activities of enzymatic and non-enzymatic antioxidants level to nearnormal range. However, topical application of ferulic acid to DMBA-painted animals did not show significant effect. Control mice treated with ferulic acid alone (group V) showed no significant difference in erythrocytes and skin tissue enzymatic antioxidants and nonenzymatic antioxidant status as compared to control mice (group I).
control and experimental animals in each group. The status of GSH, GST, GR was significantly decreased, whereas the status of cytochrome P450 and cytochrome b5 was increased in liver of tumor-bearing animals (group II) as compared to control animals. Oral administration of ferulic acid three times per week for 25 weeks to DMBA-painted animals reverted the status of phase I and phase II detoxification agents to nearnormal range. However, topical application of ferulic acid to DMBA-painted animals did not show significant effect. Control mice treated with ferulic acid alone (group V) showed no significant difference in the activities of phase II detoxification enzymes and reduced glutathione level as compared to control mice (group I).
Status of phase I and phase II detoxication agents
Discussion Tables 6 and 7 show the status of phase I (cytochrome P450 and cytochrome b5) and phase II detoxication agents (GST, GR and GSH), respectively, in the liver of
DMBA can act as a complete carcinogen or as an initiator of mouse skin carcinogenesis. In the present
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Table 6.
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Effect of ferulic acid on status of phase I detoxication agents in liver of experimental animals in each group
Groups 1. 2. 3. 4. 5.
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Cytochrome P450 (UA/mg protein)
Cytochrome b5 (UB/mg protein)
0.5970.03a 1.3370.10b 0.6570.06c 1.0170.11d 0.6070.04a
1.0870.07a 1.9670.17b 1.2070.14c 1.6370.19d 1.1070.06a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05. A. Micromoles of cytochrome per gram of tissue. B. Micromoles of cytochrome per gram of tissue.
Table 7. Effect of ferulic acid on status of phase II detoxication enzymes and reduced glutathione in liver of experimental animals in each group Groups
1. 2. 3. 4. 5.
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Reduced glutathione (mg/g tissue)
Glutathione Stransferase (UA/ mg protein)
Glutathione reductase (UB/ mg protein)
2.6370.20a 1.4270.18b 2.4470.29c 1.8670.21d 2.6970.17a
162.36711.2a 101.22713.3b 151.27714.31c 133.30715.29d 165.13712.4a
39.8473.12a 23.5172.06b 35.8773.01c 30.1872.92d 39.8972.85a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05. A. Micromoles of CDNB–GSH conjugate formed per hour. B. Micromoles of NADPH oxidized per hour.
study the chemopreventive potential of orally administered and topically painted ferulic acid was evaluated in DMBA-induced mouse skin carcinogenesis by monitoring the percentage of tumor-bearing animals, tumor volume and burden as well as by analyzing the status of phase I and phase II detoxication agents, lipid peroxidation and antioxidants. Oral administration of ferulic acid to DMBA-painted mice completely prevented the formation of skin tumors. However topically painted ferulic acid failed to show significant chemopreventive activity in DMBA-painted mice. Our results thus demonstrate that orally administered ferulic acid has more suppressing effect on cell proliferation than topically applied ferulic acid. Liver, the major metabolic organ, performs an important role in the detoxication process and thus measuring the status of detoxication agents help to assess the chemopreventive efficacy of the test compound. GST detoxifies carcinogens either by destroying their reactive centers or facilitating their excretion by conjugation process. Glutathione reductase maintains the status of reduced glutathione by catalyzing NADPH-dependent reduction of glutathione disulfide to reduced glutathione. Altered activities of detoxication
agents in the liver indicate the insult by toxic foreign agents (Senthil et al., 2007; Dorai and Aggarwal, 2004). Chemopreventive agents exert their beneficial effects by modulating the metabolic activity of cytochrome P450 system required for the metabolic biotransformation of procarcinogens to ultimate carcinogens and by stimulating phase II detoxication enzymes to remove the intermediate electrophilic metabolites (Dorai and Aggarwal, 2004; Arts and Hollman, 2005; Wattenberg, 1990). In the present study, orally administered ferulic acid reverted the status of phase I and phase II detoxication agents to near-normal range in the liver of DMBA-painted mice. Our results suggest that ferulic acid antagonizes DMBA actions and stimulated phase I and phase II detoxication agents to enhance the detoxication of the carcinogen, DMBA. Oxidative stress, an imbalance in oxidant and antioxidant status, has been well documented in several cancers including skin cancer (Kolanjiappan and Manoharan, 2005). DMBA on metabolic activation excessively generates ROS that cause severe damage to DNA contributing to carcinogenesis. The structural integrity and function of red blood cell membrane are severely affected in cancerous conditions (Kolanjiappan
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et al., 2002). Enhanced susceptibility of erythrocytes to ROS-mediated oxidative damage has been shown in DMBA-induced skin carcinogenesis (Kolanjiappan and Manoharan, 2005). Assessment of plasma lipid peroxidation by-products is considered as the most reliable marker of tissue damage in pathological conditions (Gutteridge, 1995). Increase in plasma TBARS in tumor-bearing mice is probably due to overproduction and diffusion from the erythrocytes or tumor tissues with subsequent leakage into plasma. A slight increase in tumor tissue TBARS is due to repeated DMBA painting (twice a week for eight weeks) on the skin. Oral administration of ferulic acid reverted the status of TBARS in tumor tissues and circulation, which indicates its potent antilipidperoxidative property during DMBA-induced skin carcinogenesis. The phenolic nucleus and an extended unsaturated side chain in the structure of ferulic acid readily forms a resonance stabilized phenoxy radical, which accounts for its free radical scavenging property. Non-enzymatic and enzymatic antioxidants play a crucial role against ROS-mediated oxidative damage. Lowered activities of enzymatic antioxidants in circulation and tumor tissues are due to exhaustion of these enzymes to combat the deleterious effects of excessively generated ROS during carcinogenic process. Decreased levels of glutathione, glutathione peroxidase, superoxide dismutase and CAT confirm the status of oxidative stress in DMBA-treated mice. Oral administration of ferulic acid to DMBA-treated mice reverted the activities of antioxidants in skin tissues and circulation, which indicates its prominent role in improving the antioxidant defense mechanism during DMBA-induced skin carcinogenesis. In the present study, an interesting finding is that oral administration of ferulic acid alone to mice (group V) has no effect on any biochemical variables examined (i.e. when used in the absence of DMBA). Our study suggests that ferulic acid might have antagonized the actions of DMBA without affecting the status of lipidperoxidation by-products, antioxidants and detoxication agents during DMBA-induced mouse skin carcinogenesis. Sudheer et al. (2008) have also demonstrated that oral administration of ferulic acid alone to Wistar rats did not alter the status of lipid peroxidation and antioxidants. Our results lend credence to these observations. Further studies are however warranted to demonstrate the antagonizing effect of ferulic acid in DMBA-induced mouse skin carcinogenesis. Our study thus concludes that ferulic acid provides protection against DMBA-induced skin carcinogenesis by exhibiting antiproliferative activity, by acting as an antioxidant, and by modulating the activities of phase I and phase II detoxication agents. Our study also demonstrated that orally administered ferulic acid has better effect than topically applied ferulic acid in
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DMBA-induced mouse skin carcinogenesis. The present study thus concludes that oral administration of ferulic acid could be beneficial for clinical and therapeutic studies.
References Arts IC, Hollman PC. Polyphenols and disease risk in epidemiologic studies. Am J Clin Nutr 2005;81:317S–25S. Asanoma M, Takahashi K, Miyabe M, Yamamoto K, Yoshimi N, Mori H, et al. Inhibitory effect of topical application of polymerized ferulic acid, a synthetic lignin, on tumor promotion in mouse skin two-stage tumorigenesis. Carcinogenesis 1994;15:2069–71. Azuine MA, Bhide SV. Chemopreventive effect of turmeric against stomach and skin tumors induced by chemical carcinogens in Swiss mice. Nutr Cancer 1992;17:77–83. Beutler E, Kelly BM. The effect of sodium nitrite on red cell GSH. Experientia 1963;19:96–7. Carlberg I, Mannervik B. Glutathione reductase. Methods Enzymol 1985;113:484–90. Dodge JT, Mitchell C, Hanahan DJ. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 1963;100: 119–30. Donnan SK. The thiobarbituric acid test applied to tissues from rats treated in various ways. J Biol Chem 1950;182: 415–9. Dorai T, Aggarwal BB. Role of chemopreventive agents in cancer therapy. Cancer Lett 2004;215:129–40. Ernster L. DT-diaphorase. Methods Enzymol 1967:309–17 [Academic press, New York]. Graf E. Antioxidant potential of ferulic acid. Free Radical Biol Med 1992;13:435–48. Gutteridge JM. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 1995;41:1819–28. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130–9. Jemal A, Murray T, Samules A, Ghafoor A, Ward E, Thun MJ. Cancer statistics, 2003. CA Cancer J Clin 2003;53: 5–26. Kakkar D, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984;21:130–2. Kolanjiappan K, Manoharan S. Chemopreventive efficacy and antilipidperoxidative potential of Jasminum grandiflorum Linn. on 7,12-dimethylbenz(a)anthracene induced rat mammary carcinogenesis. Fundam Clin Parmacol 2005; 19:687–93. Kolanjiappan K, Manoharan S, Kayalvizhi M. Measurement of erythrocyte lipids, lipid peroxidation, antioxidants and osmotic fragility in cervical cancer patients. Clin Chim Acta 2002;326:143–9. Kyriazi M, Yova D, Rallis M, Lima A. Cancer chemopreventive effects of Pinus martima bark extract on ultraviolet radiation and ultraviolet radiation–7,12-dimethylbenz(a)anthracene induced skin carcinogenesis of hairless mice. Cancer Lett 2006;237:234–41.
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component of glutathione peroxidase. Science 1973;179: 588–90. Senthil N, Manoharan S, Balakrishnan S, Ramachandran CR, Murali Naidu R. Chemopreventive and antilipidperoxidative efficacy of Piper longum (Linn) on 7,12-dimethylbenz(a)anthracene (DMBA) induced hamster buccal pouch carcinogenesis. J Appl Sci 2007;7:1036–42. Sinha KA. Colorimetric assay of catalase. Anal Biochem 1972;47:389–94. Srinivasan M, Sudheer AR, Menon VP. Ferulic acid: therapeutic potential through its antioxidant property. J Clin Biochem Nutr 2007;40:92–100. Sudheer AR, Muthukumaran S, Devipriya N, Devaraj H, Menon VP. Influence of ferulic acid on nicotine-induced lipid peroxidation, DNA damage and inflammation in experimental rats as compared to N-acetylcysteine. Toxicology 2008;243:317–29. Wattenberg LW. Inhibition of carcinogenesis by naturally occurring and synthetic compounds. Basic Life Sci 1990;52:155–66. Wood AW, Huang MT, Chang RL, Newmark HL, Lehr RE, Yagi H, et al. Inhibition of the mutagenicity of bayregion diol epoxides of polycyclic aromatic hydrocarbons by naturally occurring plant phenols: exceptional activity of ellagic acid. Proc Natl Acad Sci USA 1982;79: 5513–7. Yagi K. Lipid peroxides and human diseases. Chem Phys Lipids 1987;45:337–51. Zhao Z, Moghadasian MH. Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: a review. Food chem 2008;109:1–12.
Experimental and Toxicologic Pathology 61 (2009) 205–214 www.elsevier.de/etp
Protective effect of ferulic acid on 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis in Swiss albino mice Linsa Mary Aliasa, Shanmugam Manoharana,, Lakshmanan Vellaichamya, Subramanian Balakrishnana, Cinnamanoor Rajamani Ramachandranb a
Department of Biochemistry and Biotechnology, Faculty of science, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India b Department of Oral & Maxillofacial Pathology, Rajah Muthiah Dental College & Hospital, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India Received 7 May 2008; accepted 3 September 2008
Abstract Our aim was to evaluate and compare the chemopreventive potential of topically applied and orally administered ferulic acid in 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis. Estimating the status of phase I and phase II detoxication agents, lipid peroxidation byproducts and antioxidants during DMBA-induced skin carcinogenesis assessed the mechanistic pathway for its chemopreventive efficacy. Skin squamous cell carcinoma was induced in the shaved back of mice, by painting with DMBA (25 mg in 0.1 mL1 acetone) twice weekly for 8 weeks. We have observed 100% tumor formation in the 15th week of experimental period in mice treated with DMBA alone. Marked alterations in the status of phase I and phase II detoxication agents, lipid peroxidaton byproducts and antioxidants were observed in tumor bearing mice. Oral administration of ferulic acid completely prevented the formation of skin tumors, whereas topically applied ferulic acid did not show significant chemopreventive activity during DMBA-induced mouse skin carcinogenesis. Also, oral administration of ferulic acid reverted the status of phase I and phase II detoxication agents, lipid peroxidaton byproducts and antioxidants to near-normal range in DMBAtreated mice. Our results thus demonstrate that orally administered ferulic acid has potent suppressing effect on cell proliferation during DMBA-induced skin carcinogenesis. This is probably due to its modulating effect on the status of lipid peroxidation, antioxidants and detoxication agents during DMBA-induced skin carcinogenesis. r 2008 Elsevier GmbH. All rights reserved. Keywords: Skin cancer; Ferulic acid; Lipid peroxidation; Antioxidants; Detoxication agents
Introduction Skin, a major environmental interface for the body, is accidentally or occupationally exposed to a number of Corresponding author. Tel.: +91 4144 238343; fax: +91 4144 238080. E-mail address: [email protected] (S. Manoharan).
0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.09.001
chemical mutagens and carcinogens. Skin cancer accounts for 30% of all newly diagnosed cancers in the world. Epidemiological studies have reported that the incidence of skin cancer is significantly rising worldwide due to increased cumulative ultraviolet exposure. Epithelial tumors, basal cell carcinoma and squamous cell carcinoma are the most important skin tumors (Jemal et al., 2003; Kyriazi et al., 2006). 7,12-dimethylbenz[a]anthracene
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(DMBA), a potent organ-specific carcinogen, can act either as complete carcinogen or as an initiator of mouse skin carcinogenesis. Dihydrodiolepoxide, the ultimate carcinogen of DMBA, mediates skin carcinogenesis by inducing chronic inflammation, over production of reactive oxygen species (ROS) and oxidative DNA damage. DMBA-induced mouse skin carcinogenesis is widely employed to test the chemopreventive efficacy of medicinal plants and their constituents (Miyata et al., 2001; Rastogi et al., 2007). ROS such as superoxide radical, hydroxyl radical and hydrogen peroxides are frequently generated in the biological systems either by normal metabolic pathways or as a consequence of exposure to physical, chemical and biological agents. ROS attack biomembranes and lead to oxidative destruction of polyunsaturated fatty acids (PUFA) by a chain reaction known as lipid peroxidation. ROS interfere with the structure and function of the cells, making them weak and defenseless. Overproduction of ROS within tissues can damage DNA and contribute to mutagenesis and carcinogenesis. ROS-mediated oxidative stress has been implicated in the pathogenesis of several diseases including cancer (Ray and Husain, 2002). Human body has, however, an array of sophisticated antioxidant defense mechanism to combat the deleterious effects of ROS-mediated oxidative damage. This defense mechanism includes nonenzymatic antioxidants (vitamin E, vitamin C and glutathione) and enzymatic antioxidants [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] (Ray and Husain, 2002; McCord, 2000). Previous reports from our laboratory have documented the status of lipid peroxidation and antioxidants in experimental carcinogenesis (Manoharan et al., 2006; Kolanjiappan and Manoharan, 2005). Chemoprevention is a promising pharmacological approach to prevent, delay or reverse the multi-step process of carcinogenesis. A large number of plant phenolics act as potent inhibitors for mutagenesis and carcinogenesis induced by polycyclic aromatic hydrocarbons such as DMBA (Wood et al., 1982; Newmark, 1987). The anticancer effect of phytochemicals is based on their ability to quench ROS and thereby protecting critical cellular molecules from oxidative insult. Also, antiproliferative activity and induction of apoptosis in cancer cells are other important anticancer mechanisms of phytochemicals. Ferulic acid, 4-hydroxy-3-methoxycinnamic acid, (Fig. 1) arises from the metabolism of phenylalanine and tyrosine by Shikimate pathway in plants. Ferulic acid is rich in many staple food such as grain bran, whole grain foods, citrus fruits, banana, coffee, orange juice, eggplant, bamboo shoots, beet root, cabbage, spinach and broccoli. It exhibits beneficial effects against various diseases like cancer, diabetes, cardiovascular and neurodegenerative disorders. Ferulic acid has potent free radical scavenging activity and can
O
OH
OH OCH3
Fig. 1. Molecular structure of ferulic acid.
effectively scavenge superoxide anion radicals. In certain countries ferulic acid has been approved as food additive to prevent lipid peroxidation (Zhao and Moghadasian, 2008; Graf, 1992; Srinivasan et al., 2007). Asanoma et al. (1994) have demonstrated that topical application of ferulic acid showed weak chemopreventive activity in skin carcinogenesis. To the best of our knowledge, we have found no scientific evidence on the chemopreventive potential of orally administered ferulic acid in DMBA-induced skin carcinogenesis. The present study was therefore designed to compare the chemopreventive potential of topically applied and orally administered ferulic acid in DMBA-induced mouse skin carcinogenesis.
Materials and methods Chemicals DMBA and ferulic acid were purchased from Sigma Aldrich Chemical Pvt. Ltd., Bangalore, India. All other chemicals used were of analytical grade.
Animals Male Swiss albino mice 4–6 weeks old, weighing 15–20 g were purchased from the National Institute of Nutrition, Hyderabad, India, and maintained in the Central Animal House, Rajah Muthaiah Medical College and Hospital, Annamalai University. The animals were housed in groups of four or five in polypropylene cages and provided standard pellet diet and water ad libitum and maintained under controlled
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conditions of temperature and humidity, with a 12 h light/dark cycle. The animals were maintained as per the principles and guidelines of the ethical committee for animal care of Annamalai University in accordance with the Indian National Law on animal care and use.
Experimental design The Institutional animal ethical committee [Reg. no.: 160/1999/CPCSEA], Annamalai University, Annamalainagar, India, approved the experimental design. A total of 30 male Swiss albino mice were divided into five groups of six each. Skin carcinogenesis was developed in Swiss albino mice according to the method of Azuine and Bhide (1992). Depilatory cream was applied to remove hair from the back of each mouse and the mice were left untreated for 2 days. Mice having no hair growth after 2 days were selected for the experimental study. The depilated back of group I mice was painted with acetone (0.1 ml/mouse) twice weekly for 8 weeks (vehicle-treated control). The depilated back of groups II–IV mice were painted with DMBA (25 mg in 0.1 ml acetone/mouse) twice weekly for 8 weeks. Group II mice received no other treatment. Group III mice were administered ferulic acid (40 mg/kg b.w in 1 ml distilled water) orally starting 1 week before the exposure to the carcinogen and continued for 25 weeks (3 times/week on alternate days) thereafter. Group IV animals were topically painted with ferulic acid (1.5 mg/animal/day) starting 1 week before the exposure to the carcinogen and continued for 25 weeks (3 times/week on alternate days) thereafter. At the end of the experimental period all the animals were sacrificed by cervical dislocation.
Macroscopic evaluation Body weight of experimental animals in each group was recorded at weekly intervals, throughout the experimental period. During the sacrifice of animals, liver was dissected out and blotted dry before weighing. A part of the liver was used for the assay of phase I and phase II detoxication agents. The relative organ weight was calculated as the ratio between organ weight and body weight. The number of skin tumors was counted and the diameter of each tumor was measured with a caliper. The tumor volume was calculated by the formula n ¼ (4/3)p[D1/2][D2/2][D3/2], where D1, D2 and D3 are the three diameters (mm) of the tumors. Tumor burden was calculated by multiplying tumor volume and the number of tumors/mice.
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Histological investigation Skin tissues from control mice, DMBA alone treated mice, DMBA+ferulic acid treated mice and ferulic acid alone treated mice were removed, measured and weighed. The skin tissues were fixed overnight in buffered neutral formalin and processed for histological evaluation. The tissues were routinely processed and embedded with paraffin wax, sectioned at 2–3 mm in a rotary microtome and stained with hematoxylin for 10 min. Subsequently, they were washed in running tap water for 5 min and then rinsed in 0.5% acid alcohol (0.5% HCl in 95% alcohol). The slides were dried on a hot plate and stained with 0.5% eosin in 95% ethanol for 5 min. The sections were rapidly rinsed in 95% ethanol and dehydrated in two changes of absolute ethanol for 5 min each. The slides were then cleared in xylene and mounted in resinous medium. Single sections of each specimen were evaluated by light microscopy. The epidermal changes were categorized into (i) hyperkeratosis, when there is an increase in the superficial cornified layer, (ii) hyperplasia, when the epidermal thickness is more than the normal thickness without any dysplastic features, (iii) dysplasia, for which the criteria proposed by Pindborg et al. (1997) is considered and (iv) carcinoma, when there is a breach in the basement membrane and invasion of epithelial cells into the underlying stromal tissue.
Biochemical analysis Biochemical studies were conducted on plasma, erythrocytes and skin tissues of control and experimental animals in each group. Blood samples were collected in heparinized tubes. The plasma was separated by centrifugation at 3000 rpm for 15 min. After plasma separation, the buffy coat was removed and the packed cells were washed thrice with physiological saline. A known volume of erythrocytes was lysed with hypotonic buffer at pH 7.4. The hemolysate was separated by centrifugation at 10,000 rpm for 15 min at 20 1C. The erythrocyte membrane was prepared by the method of Dodge et al. (1963) modified by Quist (1980). The erythrocytes remaining after the removal of plasma were washed three times with 310 mM isotonic Tris–HCl buffer (pH 7.4). The washed erythrocyte suspension was treated with 20 mM hypotonic Tris–HCl buffer (pH 7.2) to perform hemolysis. The erythrocyte membranes were sediment in a high-speed cooling centrifuge at 20,000g for 40 min. The supernatant was decanted and the erythrocyte membrane pellet was made up to a known volume using 0.2 M isotonic Tris–HCl buffer (pH 7.4). Aliquots from these preparations were used for the biochemical estimations.
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In plasma, thiobarbituric acid reactive substances (TBARS) was assayed by the method of Yagi (1987). Plasma was deproteinised with phosphotungstic acid and the precipitate was treated with thiobarbituric acid at 90 1C for 1 h. The pink color formed gives a measure of the TBARS, which was read at 530 nm. TBARS in erythrocytes and erythrocyte membranes was estimated by the method of Donnan (1950). Absorbance of pink chromogen formed by the reaction of thiobarbituric acid with breakdown products of lipid peroxides was read at 535 nm. The reduced glutathione level in erythrocytes, liver and skin tissues was determined by the method of Beutler and Kelly (1963). 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 5,50 dithiobis-2-nitrobenzoic acid. Superoxide dismutase activity in erythrocytes and skin tissues was assayed by the method of Kakkar et al. (1984), based on the 50% inhibition of formation of NADH–phenazine methosulphate nitro blue tetrazolium (NBT). 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 NBT reduction. The activity of CAT in erythrocytes and skin tissues was assayed by the method of Sinha (1972), based on the utilization of H2O2 by the enzyme. The color developed was read at 620 nm. One unit of the enzyme is expressed as mmoles of H2O2 utilized per minute. The activity of GPx in erythrocytes and skin tissues was determined using the method of Rotruck et al. (1973), based on the utilization of reduced glutathione by the enzyme. One unit of the enzyme is expressed as mmoles of GSH utilized per minute. The activity of glutathione-S-transferase (GST) in skin tissues and liver tissue homogenate was assayed by the method of Habig et al. (1974). GST activity was measured by incubating the tissue homogenate with the substrate 1-chloro-2,4-dinitrobenzene (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 skin
Table 1.
Statistical analysis Values are expressed as mean7SD. Statistical analysis was performed by one-way analysis of variance, followed by Duncan’s multiple range test. The values were considered statistically significant if p value was less than 0.05.
Results Body weight and liver weight Table 1 shows the body and liver weight and relative organ weight of control and experimental animals in each group. The body weight and liver weight were significantly decreased in DMBA-treated animals as compared to control animals. Oral administration of ferulic acid three times per week for 25 weeks
Effect of ferulic acid on body weight, liver weight and relative organ weight of experimental animals in each group
Groups
1. 2. 3. 4. 5.
tissues and liver tissue homogenate was assayed by the method of Carlberg and Mannervik (1985). The enzyme activity was assayed by measuring the formation of reduced glutathione when the oxidized glutathione (GSSG) is reduced by reduced nicotinamide adenine dinucleotide phosphate (NADPH). The levels of cytochrome P450 and b5 in liver and skin tissues were determined according to the method of Omura and Sato (1964). Cytochrome P450 was measured 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 oxidized cytochrome was used as an index to measure the level of cytochrome b5. The activity of DTdiaphorase was estimated according to the method of Ernster (1967) based on the measurement of reduction at 550 nm using reduced NADPH as the electron donor and 2,6-dichlorophenol indophenol as the electron acceptor.
Body wt. (g)
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Initial
Final
22.8071.1a 23.1171.3a 23.1871.1a 23.2171.3a 22.8471.2a
28.1371.5a 22.1671.7b 27.3471.6a 23.4671.6b 28.8771.9a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05.
Liver wt. (g)
Relative organ weight (liver wt./body wt.)
1.1670.07a 0.8870.09b 1.0970.08a 0.9170.07b 1.1870.06a
0.04170.002a 0.04070.004a 0.04070.003a 0.03970.003a 0.04170.002a
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significantly increased the body and liver weight in DMBA-treated animals. However, topical application of ferulic acid to DMBA-painted animals slightly increased the body and liver weight in DMBA-treated animals. Oral administration of ferulic acid alone to mice (group V) showed no significant difference in body and liver weight as compared to control animals (group I). We also found no statistically significant difference in the relative organ weight of control and experimental animals in each group.
(Fig. 3C). Moderate preneoplastic lesions, hyperplasia, hyperkeratosis and dysplasia, and well-differentiated squamous cell carcinoma were noticed in DMBApainted animals that received topical application of ferulic acid (group IV) (Fig. 3D). Oral administration of ferulic acid to DMBA-treated mice showed normal skin and the presence of subcutaneous tissue (Fig. 3E).
Tumor incidence, volume and burden
Table 3 shows the levels of TBARS in plasma, erythrocyte membrane and skin tissues of control and experimental animals in each group. The levels of
Table 2 shows the tumor incidence, volume and tumor burden of control and experimental animals in each group. In DMBA-painted mice (group II), a 100% tumor formation with mean tumor volume (580.2 mm3) and tumor burden (1836.1 mm3) was observed. The gross appearance of skin tumors is depicted in Fig. 2. Oral administration of ferulic acid (40 mg/kg b.w) completely prevented tumor incidence in DMBA-painted mice. However, we noticed 66.6% tumor formation with mean tumor volume (300.1 mm3) and tumor burden (750.2 mm3) in DMBA-painted animals that received topical application of ferulic acid (1.5 mg/animal/day). No tumors were observed in control (group I) as well as ferulic acid alone treated animals (group V).
Status of TBARS
Histopathological observations The histopathological features observed in the skin tissues of Swiss albino mice in control and experimental animals in each group are depicted in Fig. 3(A–E). The histopathological studies on control animals (group I) showed normal skin and the presence of subcutaneous tissue (Fig. 3A). The skin tissues from DMBA-treated mice (group II) revealed hyperkeratosis, hyperplasia, dysplasia and well-differentiated squamous cell carcinoma with keratin pearls (Fig. 3B). The skin tissues from DMBA-painted animals received oral administration of ferulic acid (group III) showed mild hyperplasia Table 2.
Effect of ferulic acid on tumor incidence, tumor volume and tumor burden of experimental animals in each group
Groups 1. 2. 3. 4. 5.
Fig. 2. Gross appearance of mouse skin tumors in DMBA alone painted animals.
Control (vehicle treated) DMBA 1836.17128.59 DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Tumor incidence
Total number of tumors
Tumor volume (mm3)
Tumor burden (mm3)
0 100% (6/6)
0 19/(6)
0 580.2751.83
0
0 66.6% (4/6) 0
0 10/(4) 0
0 300.1728.6 0
0 750.2760.3 0
Values are expressed as mean7SD (n ¼ 6 mice). Tumor volume was measured using the formula n ¼ (4/3)p[D1/2][D2/2][D3/2] where D1, D2 and D3 are the three diameters (mm) of the tumors. Tumor burden was calculated by multiplying tumor volume and the number of tumors/animal. Number in parenthesis indicated total number of animals bearing tumors.
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Fig. 3. (A) Microphotograph of skin tissues from control animals showing well-defined subcutaneous tissues and intact epithelial layer. (B) Microphotograph of skin tissues from DMBA alone painted animals showing well-differentiated squamous cell carcinoma with hyperplasia (arrow head), dysplasia (arrow) and keratin pearls (block arrow). (C) Microphotograph of skin tissues from DMBA+ferulic acid (oral administration; 40 mg/kg b.w)-treated animals showing hyperplastic epithelium (arrow head). (D) Microphotograph of skin tissues from DMBA+ferulic acid (topical application; 1.5 mg/animal/day) treated animals showing hyperplastic (arrow head) and severe dysplastic epithelium (arrow). (E) Microphotograph of skin tissues from ferulic acid alone treated animals showing well-defined subcutaneous tissues and intact epithelial layer.
Table 3.
Effect of ferulic acid on TBARS in plasma, erythrocytes and skin tissues of experimental animals in each group
Groups
Plasma TBARS (nmol/ml)
Erythrocytes membrane TBARS (nmol/mg protein)
Skin tissues TBARS (mmol/100 g tissue)
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
2.1370.14a 4.8970.56b 2.3670.21c 3.5670.41d 2.0770.11a
0.3070.02a 1.0170.13b 0.3570.05c 0.8070.07d 0.2970.02a
100.1878.5a 115.73712.3b 106.1379.7a 113.56712.4b 101.0777.9a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05.
TBARS were significantly increased in plasma and erythrocyte membranes, whereas decreased in skin tissues of tumor bearing animals (group II) as compared to control animals. Oral administration of ferulic acid three times per week for 25 weeks to DMBA-painted animals reverted the levels of TBARS to near normal range. However, topical application of ferulic acid to DMBA-painted animals did not show significant effect. Control mice treated with ferulic acid alone (group V) showed no significant difference in plasma, erythrocyte
membrane and skin tissue TBARS as compared to control mice (group I).
Enzymatic and non-enzymatic antioxidants status Tables 4 and 5 show the activities of enzymatic antioxidants (SOD, CAT, GPx) and non-enzymatic antioxidant (GSH) level in erythrocytes and skin tissues of control and experimental animals in each group,
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Table 4. Effect of ferulic acid on status of enyzmatic and non-enzymatic antioxidants in erythrocytes of experimental animals in each group Groups
SOD (UA/ mg Hb)
CAT (UB/ mg Hb)
GPx (UC/ g Hb)
GSH (mg/dl)
1. 2. 3. 4. 5.
5.9670.37a 3.7370.32b 5.5670.48c 4.3870.51d 6.0170.42a
2.5370.18a 1.5670.20b 2.3270.28c 1.9370.24d 2.5970.19a
24.1871.51a 16.4371.93b 22.3072.28c 19.172.03d 24.2671.58a
42.173.01a 27.2873.17b 38.8773.96c 33.1273.09d 42.572.6a
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05. A. The amount of enzyme required to inhibit 50% NBT reduction. B. Micromoles of H2O2 utilized per second. C. Micromoles of glutathione utilized per minute.
Table 5. group
Effect of ferulic acid on status of enyzmatic and non-enzymatic antioxidants in skin tissue of experimental animals in each
Groups 1. 2. 3. 4. 5.
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
SOD (UA/ mg protein)
CAT (UB/ mg protein)
GPx (UC/ mg protein)
GSH (mg/ 100 mg tissues)
6.4370.48a 3.5970.41b 6.0170.53c 5.3270.56d 6.4970.39a
42.1873.23a 21.6772.56b 39.1473.53c 29.673.7d 42.2473.04a
43.5973.01a 29.3273.41b 40.0473.33c 36.1273.93d 43.6673.11a
40.373.76a 22.1472.97b 37.1173.92c 30.1573.59d 40.473.82a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05.
A. The amount of enzyme required to inhibit 50% NBT reduction. B. Micromoles of H2O2 utilized/s. C. Micromoles of glutathione utilized/min.
respectively. The activities of SOD, CAT, GPx and GSH level were significantly decreased in erythrocytes and skin tissues of tumor-bearing animals (group II) as compared to control animals. Oral administration of ferulic acid three times per week for 25 weeks to DMBA-painted animals reverted the activities of enzymatic and non-enzymatic antioxidants level to nearnormal range. However, topical application of ferulic acid to DMBA-painted animals did not show significant effect. Control mice treated with ferulic acid alone (group V) showed no significant difference in erythrocytes and skin tissue enzymatic antioxidants and nonenzymatic antioxidant status as compared to control mice (group I).
control and experimental animals in each group. The status of GSH, GST, GR was significantly decreased, whereas the status of cytochrome P450 and cytochrome b5 was increased in liver of tumor-bearing animals (group II) as compared to control animals. Oral administration of ferulic acid three times per week for 25 weeks to DMBA-painted animals reverted the status of phase I and phase II detoxification agents to nearnormal range. However, topical application of ferulic acid to DMBA-painted animals did not show significant effect. Control mice treated with ferulic acid alone (group V) showed no significant difference in the activities of phase II detoxification enzymes and reduced glutathione level as compared to control mice (group I).
Status of phase I and phase II detoxication agents
Discussion Tables 6 and 7 show the status of phase I (cytochrome P450 and cytochrome b5) and phase II detoxication agents (GST, GR and GSH), respectively, in the liver of
DMBA can act as a complete carcinogen or as an initiator of mouse skin carcinogenesis. In the present
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Table 6.
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Effect of ferulic acid on status of phase I detoxication agents in liver of experimental animals in each group
Groups 1. 2. 3. 4. 5.
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Cytochrome P450 (UA/mg protein)
Cytochrome b5 (UB/mg protein)
0.5970.03a 1.3370.10b 0.6570.06c 1.0170.11d 0.6070.04a
1.0870.07a 1.9670.17b 1.2070.14c 1.6370.19d 1.1070.06a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05. A. Micromoles of cytochrome per gram of tissue. B. Micromoles of cytochrome per gram of tissue.
Table 7. Effect of ferulic acid on status of phase II detoxication enzymes and reduced glutathione in liver of experimental animals in each group Groups
1. 2. 3. 4. 5.
Control (vehicle treated) DMBA DMBA+ferulic acid (oral treatment) DMBA+ferulic acid (topical application) Ferulic acid alone (oral treatment)
Reduced glutathione (mg/g tissue)
Glutathione Stransferase (UA/ mg protein)
Glutathione reductase (UB/ mg protein)
2.6370.20a 1.4270.18b 2.4470.29c 1.8670.21d 2.6970.17a
162.36711.2a 101.22713.3b 151.27714.31c 133.30715.29d 165.13712.4a
39.8473.12a 23.5172.06b 35.8773.01c 30.1872.92d 39.8972.85a
Values are expressed as mean7SD (n ¼ 6 mice). Values that are not sharing common superscript in the same column differ significantly at po0.05. A. Micromoles of CDNB–GSH conjugate formed per hour. B. Micromoles of NADPH oxidized per hour.
study the chemopreventive potential of orally administered and topically painted ferulic acid was evaluated in DMBA-induced mouse skin carcinogenesis by monitoring the percentage of tumor-bearing animals, tumor volume and burden as well as by analyzing the status of phase I and phase II detoxication agents, lipid peroxidation and antioxidants. Oral administration of ferulic acid to DMBA-painted mice completely prevented the formation of skin tumors. However topically painted ferulic acid failed to show significant chemopreventive activity in DMBA-painted mice. Our results thus demonstrate that orally administered ferulic acid has more suppressing effect on cell proliferation than topically applied ferulic acid. Liver, the major metabolic organ, performs an important role in the detoxication process and thus measuring the status of detoxication agents help to assess the chemopreventive efficacy of the test compound. GST detoxifies carcinogens either by destroying their reactive centers or facilitating their excretion by conjugation process. Glutathione reductase maintains the status of reduced glutathione by catalyzing NADPH-dependent reduction of glutathione disulfide to reduced glutathione. Altered activities of detoxication
agents in the liver indicate the insult by toxic foreign agents (Senthil et al., 2007; Dorai and Aggarwal, 2004). Chemopreventive agents exert their beneficial effects by modulating the metabolic activity of cytochrome P450 system required for the metabolic biotransformation of procarcinogens to ultimate carcinogens and by stimulating phase II detoxication enzymes to remove the intermediate electrophilic metabolites (Dorai and Aggarwal, 2004; Arts and Hollman, 2005; Wattenberg, 1990). In the present study, orally administered ferulic acid reverted the status of phase I and phase II detoxication agents to near-normal range in the liver of DMBA-painted mice. Our results suggest that ferulic acid antagonizes DMBA actions and stimulated phase I and phase II detoxication agents to enhance the detoxication of the carcinogen, DMBA. Oxidative stress, an imbalance in oxidant and antioxidant status, has been well documented in several cancers including skin cancer (Kolanjiappan and Manoharan, 2005). DMBA on metabolic activation excessively generates ROS that cause severe damage to DNA contributing to carcinogenesis. The structural integrity and function of red blood cell membrane are severely affected in cancerous conditions (Kolanjiappan
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et al., 2002). Enhanced susceptibility of erythrocytes to ROS-mediated oxidative damage has been shown in DMBA-induced skin carcinogenesis (Kolanjiappan and Manoharan, 2005). Assessment of plasma lipid peroxidation by-products is considered as the most reliable marker of tissue damage in pathological conditions (Gutteridge, 1995). Increase in plasma TBARS in tumor-bearing mice is probably due to overproduction and diffusion from the erythrocytes or tumor tissues with subsequent leakage into plasma. A slight increase in tumor tissue TBARS is due to repeated DMBA painting (twice a week for eight weeks) on the skin. Oral administration of ferulic acid reverted the status of TBARS in tumor tissues and circulation, which indicates its potent antilipidperoxidative property during DMBA-induced skin carcinogenesis. The phenolic nucleus and an extended unsaturated side chain in the structure of ferulic acid readily forms a resonance stabilized phenoxy radical, which accounts for its free radical scavenging property. Non-enzymatic and enzymatic antioxidants play a crucial role against ROS-mediated oxidative damage. Lowered activities of enzymatic antioxidants in circulation and tumor tissues are due to exhaustion of these enzymes to combat the deleterious effects of excessively generated ROS during carcinogenic process. Decreased levels of glutathione, glutathione peroxidase, superoxide dismutase and CAT confirm the status of oxidative stress in DMBA-treated mice. Oral administration of ferulic acid to DMBA-treated mice reverted the activities of antioxidants in skin tissues and circulation, which indicates its prominent role in improving the antioxidant defense mechanism during DMBA-induced skin carcinogenesis. In the present study, an interesting finding is that oral administration of ferulic acid alone to mice (group V) has no effect on any biochemical variables examined (i.e. when used in the absence of DMBA). Our study suggests that ferulic acid might have antagonized the actions of DMBA without affecting the status of lipidperoxidation by-products, antioxidants and detoxication agents during DMBA-induced mouse skin carcinogenesis. Sudheer et al. (2008) have also demonstrated that oral administration of ferulic acid alone to Wistar rats did not alter the status of lipid peroxidation and antioxidants. Our results lend credence to these observations. Further studies are however warranted to demonstrate the antagonizing effect of ferulic acid in DMBA-induced mouse skin carcinogenesis. Our study thus concludes that ferulic acid provides protection against DMBA-induced skin carcinogenesis by exhibiting antiproliferative activity, by acting as an antioxidant, and by modulating the activities of phase I and phase II detoxication agents. Our study also demonstrated that orally administered ferulic acid has better effect than topically applied ferulic acid in
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DMBA-induced mouse skin carcinogenesis. The present study thus concludes that oral administration of ferulic acid could be beneficial for clinical and therapeutic studies.
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