European Journal of Pharmacology 691 (2012) 268274

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Immunopharmacology and inammation

Ferulic acid, a dietary phenolic acid, modulates radiation effects in Swiss albino mice
Janakiraman Shanthakumar, Arumugam Karthikeyan, Venkata Reddy Bandugula, Nagarajan Rajendra Prasad n
Department of Biochemistry & Biotechnology, Annamalai University, Annamalainagar 608002, Tamilnadu, India

a r t i c l e i n f o
Article history: Received 12 February 2012 Received in revised form 19 June 2012 Accepted 20 June 2012 Available online 26 June 2012 Keywords: Ferulic acid Radioprotection Radiotherapy Lipid peroxidation Jejunum

a b s t r a c t
The radioprotective efcacy of Ferulic acid (FA) against whole body gamma radiation was studied in Swiss albino mice. To study the radiation protection, mice were administered with ferulic acid intraperitoneally (i.p) (50 mg/kg body weight.), once daily for ve consecutive days. One hour after the last administration of ferulic acid on the sixth day, animals were whole body exposed to 8 Gy gamma radiations. Effect of ferulic acid pretreatment on radiation-induced changes in antioxidant enzymes and lipid peroxidation status in spleen, liver and intestine was analyzed. A signicant increase in the antioxidant enzymatic status and decreased lipid peroxidation marker levels were observed in ferulic acid pretreated group, when compared to the irradiated animals. Our study also shows increased % tail DNA, tail length, tail moment and Olive tail moment in irradiated mice blood lymphocytes. Ferulic acid (50 mg/kg body weight) pretreatment signicantly decreased the % tail DNA, tail length, tail moment and Olive tail moment in irradiated mice lymphocytes. The histological observations indicated a decline in the villus height and crypt number with an increase in goblet and dead cell population in the irradiated group, which was normalized by ferulic acid pretreatment. In conclusion, present study indicated ferulic acid treatment prevents radiation-induced lipid peroxidation, DNA damage and restored antioxidant status and histopathological changes in experimental animals. & 2012 Elsevier B.V. All rights reserved.

1. Introduction Radiotherapy is an important modality for cancer treatment and more so when surgical removal of the cancer mass is not possible or when surgery might debilitate the patient (Hall, 2000). The amounts of ionizing radiation that can be given to treat malignant tumors are often limited by the toxicity to surrounding normal tissues and organs (Weiss, 1997). The only drug approved for clinical use in cancer therapy is amifostine, a synthetic phosphorothioate compound, which also produces dose limiting drug toxicity at the maximum effective dose (Kemp et al., 1996; Foster-Nora and Siden, 1997). Moreover, amifostine is very expensive. Therefore, there is a need for non-toxic and inexpensive drugs for clinical radiation protection. Radioprotectors might be useful in military as well as in clinical oncology, space travel, radiation site clean-up and radiological terrorism (Nair et al., 2001). During the last six decades a number of compounds of diverse structures have been considered as countermeasures for radiation (Weiss and Landauer, 2009). Recent studies have indicated that some

commonly used medicinal plants may be good sources of potent but non-toxic radioprotectors. Ferulic acid (FA) (Scheme 1) is a monophenolic phenylpropanoid occurring in plant products such as rice bran, green tea and coffee beans (Zhao and Moghadasian, 2008). Ferulic acid acts as a well-known antioxidant, effective in scavenging peroxyl radicals and other active oxygen species like superoxide and hydroxyl radical (Kikuzaki et al., 2003). It has an inhibitory effect on 4-nitroquinoline 1-oxide (4-QO)-induced rat tongue carcinogenesis (Tanaka et al., 1993). Our previous report showed ferulic acid decreased radiation induced micronuclei formation in cytokinesis blocked cells and dicentric aberration in human blood lymphocytes (Prasad et al., 2006). The present study was carried out to investigate whether ferulic acid had radioprotective activity in mice irradiated by 60Co g-rays. 2. Materials and methods 2.1. Chemicals Ferulic acid, thiobarbituric acid, phenazine methosulphate (PMS) nitroblue tetrazolium (NBT), 5, 5-dithiobis 2-nitrobenzoic acid (DTNB), 2, 2-diphenyl-1-picryl hydrazyl (DPPH) and nicotinamide

Corresponding author. Tel.: 91 954144 238343; fax: 91 954144 239141. E-mail address: drprasadnr1@gmail.com (N. Rajendra Prasad).

0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.06.027

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treatment with ferulic acid. All mice were sacriced by cervical dislocation on day 2 after irradiation. 2.5. Preparation and mode of administration of drug Ferulic acid procured from Sigma Aldrich was dissolved in dimethylsulphoxide (DMSO) immediately before use and was administered intraperitoneally, at a dose of 50 mg/kg body weight for 5 consecutive days (Maurya et al., 2005; Maurya and Nair, 2006). 2.6. Biochemical estimations Animals were sacriced by cervical dislocation on day 2 after irradiation. Spleen, liver and intestine were dissected out after transcardial perfusion with ice-cold saline. In each group six samples (n 6) were processed. The level of lipid peroxidation was determined by analyzing TBA-reactive substance according to the protocol of Niehaus and Samuelsson (1968). The pink colored chromogen formed by the reaction of 2-TBA with breakdown products of lipid peroxidation was measured. Superoxide dismutase activity was assayed by the method of Kakkar et al. (1984), based on the inhibition of the formation of (NADHPMS NBT) complex. Catalase activity was assayed by the procedure of Sinha (1972) quantifying the hydrogen peroxide after reacting with dichromate in acetic acid. The activity of Glutathione peroxidase was assayed by method of Rotruck et al. (1973), a known amount of enzyme preparation was allowed to react with hydroperoxides (H2O2) and reduced glutathione for a specied time period. Then the reduced glutathione content remaining after the reaction was measured. The total reduced glutathione content was measured by method of Ellman (1959). 2.7. Single-cell gel electrophoresis (comet assay) The comet assay was carried out under alkaline conditions, as described by Singh et al. (1988). Peripheral blood was collected by tail bleeding and the lymphocytes were isolated from the whole blood by using histopaque 1077 (Sigma-Aldrich). Damage to DNA in peripheral blood lymphocytes, after whole-body exposure of mice to 8 Gy g-radiations, was studied at 24 h post-irradiation. Two slides per animal were prepared. Agarose gels were prepared on fully frosted slides coated with 1% normal melting point (NMP) agarose. 50 ml of mice peripheral blood lymphocytes was mixed with 0.5% low melting point agarose (LMP) placed on the slides and covered with layer of 0.5% LMP agarose. The slides were immersed for 2 h in freshly prepared ice-cold lysis solution. Denaturation and electrophoresis were carried out at 4 1C under dim light in freshly prepared electrophoresis buffer. After 20 min of denaturation, the slides were randomly placed in the horizontal gel-electrophoresis tank, facing the anode. Electrophoresis at 25 V and 300 mA lasted another 20 min. After electrophoresis, the slides were washed with a neutralization buffer. Slides were stained with ethidium bromide (20 mg/mL). For visualization of DNA damage, observations were made using a 20 objective on an epiuorescent microscope equipped with an excitation lter of 510560 nm and a barrier lter of 590 nm. One or two hundred comets on duplicated slides were analyzed (for each group we prepared two comet assay slides and in each slide we scored 100 comets for analysis). Images were captured with a digital camera with networking capability and analyzed by image analysis software, CASP (http://casp.sourceforge.net). DNA damage was quantied by the torsional moment of the tail (tail moment), tail length (Olive et al., 1990), whose distribution was adjusted by a two-parameter, Weibull model (Ejchart and Sadlej-Sosnowska, 2003).

Scheme 1. Structure of ferulic acid (FA).

adenine dinucleotide (NAD) were purchased from Sigma chemical Co., St. Louis, USA. Other chemicals, low melting agarose, normal melting agarose, and phosphate buffered saline (PBS) and reduced glutathione were purchased from Himedia, Mumbai. All other chemicals and solvents were of analytical grade. All other chemicals used were of analytical purity. 2.2. Experimental animals The experimental protocol was approved by the Institutional Animal Ethics Committee, Annamalai University (No. 611, Registration Number: 160/1999/CPCSEA) and animals were cared in accordance with the Guide for the care and use of laboratory animals (NIH, 1985) and Committee for the purpose of control and supervision on experimental animals. Swiss albino mice (5 to 6 weeks old, weighing 1822 g), obtained from Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College, Annamalai University, were used for the experiments. They were housed in plastic bottom cages and maintained under controlled conditions of temperature and light (light:dark 14:10 h). They were provided standard mice feed (supplied by Hindustan Lever Ltd., Bangalore, India) and water ad libitum. 2.3. Experimental design The mice were randomly divided into four groups (n 6) and were given intraperitoneal administration for 5 consecutive days. 1. Group I: Sham control 1 ml/kg body weight; i.p; DMSO. 2. Group II: Ferulic acid control 50 mg/kg body weight; i.p. 3. Group III: Radiation control 1 ml/kg body weight; i.p; DMSO 30 min prior to g-radiation. 4. Group IV: Irradiationferulic acid 50 mg/kg body weight; i.p.

2.4. Irradiation protocol Whole-body irradiation of experimental animals was performed using a cobalt-60-g-radiation source of the Cancer Treatment Centre, Dr. Kamakshi Memorial Hospital, Chennai. Animals were restrained in well-ventilated perpex boxes and exposed to whole-body g-irradiation at a dose rate of 1.66 Gy/min to deliver 8 Gy (Kim et al., 1998) as required by the study, 30 min after the

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2.8. Intestinal toxicity Based on the preliminary experiments, the ferulic acid dose of 50 mg/kg body weight was selected for this study. Jejunum was removed 24 h post-irradiation; sections of 5 mm were prepared and scored for the following parameters: villus height, number of goblet cells per crypt section, mucosal erosion, number of mitotic cells per crypt section, inammatory cells and basement membrane, etc. 2.9. Statistical analysis All the values were expressed as mean 7S.D. of six determinations. Statistical analysis of the data were carried out by one-way ANOVA on SPSS (Statistical package for social sciences) and the group mean compared by Duncans Multiple Range Test (DMRT). A value of Po0.05 was considered signicant.

Fig. 2. Ferulic acid modulates SOD activity in irradiated mice spleen, liver and intestine. Values are given as means 7S.D. of six experiments in each group. Values not sharing a common marking (a, b, c, etc.) differ signicantly at Po 0.05 (DMRT).

3. Results 3.1. Biochemical estimations Fig. 1 shows the levels of thiobarbituric acid in spleen, liver and intestine of sham control and experimental animals in each group. The levels of thiobarbituric acid were signicantly increased in radiation control compared to sham control and ferulic acid treated mice. Intraperitoneal administration of ferulic acid for 5 consecutive days reverted the levels of thiobarbituric acid to near normal range. Mice treated with ferulic acid alone showed no signicant difference in spleen, liver and intestine thiobarbituric acid as compared to sham control. Figs. 25 show the activities of superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione levels in spleen, liver and intestine tissue of sham control and experimental animals in each group, respectively. The activities of superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione levels were signicantly decreased in mice spleen, liver and intestine of irradiated control as compared to sham control. Administration of ferulic acid in treatment group reverted the activities of enzymatic and non-enzymatic antioxidants level to near normal range. Control mice treated with ferulic acid alone showed no signicant difference in spleen, liver and intestine tissue both in enzymatic and non-enzymatic antioxidant status as compared to control mice.

Fig. 3. Ferulic acid increases CAT activity in irradiated mice spleen, liver and intestine. Values are given as means 7S.D. of six experiments in each group. Values not sharing a common marking (a, b, c, etc.) differ signicantly at Po 0.05 (DMRT).

Fig. 4. Ferulic acid increases GPx activity in irradiated spleen, liver and intestine. Values are given as means 7S.D. of six experiments in each group. Values not sharing a common marking (a, b, c, etc.) differ signicantly at P o0.05 (DMRT).

Fig. 1. Effect of ferulic acid on g-radiation-induced thiobarbituric acid levels in Swiss albino mice. Values are given as means7 S.D. of six experiments in each group. Values not sharing a common marking (a, b, c, etc.) differ signicantly at Po 0.05 (DMRT).

3.2. Effect of ferulic acid on gamma radiation-induced DNA damage in mice lymphocytes Studies were carried out to evaluate the oxidative damage to cellular DNA in mice with or without ferulic acid administration,

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following irradiation. For this purpose, mice blood lymphocytes from different groups were subjected to single cell gel electrophoresis and DNA damage was analyzed by using CASP software (http://casp.sourceforge.net). Cells were stained with ethidium bromide after conducting the comet assay. Gamma-irradiation caused signicant tail fraction (Fig. 6(6.3)) in mice lymphocyte DNA, whereas ferulic acid treated shows the decreased comet length (Fig. 6(6.4)). Fig. 7(7.1 and 7.2) shows the changes in the levels of DNA damage (% tail DNA and tail length) in the control, gamma plus ferulic acid pretreated blood. Gamma irradiation signicantly increased % tail DNA and tail length. Pretreatment with ferulic acid for 5 consecutive days signicantly decreased the levels of mice blood lymphocyte DNA. Fig. 7(7.3 and 7.4) shows the effect of ferulic acid on g-irradiation reduced tail

moment and Olive tail moment. We observed the increased tail moment and Olive tail moment in g-irradiated mice blood lymphocyte DNA. Pre-treatment with ferulic acid for 5 consecutive days signicantly decreased tail moment and Olive tail moment in mice blood lymphocyte DNA. 3.3. Histopathological study In our study, the histopathological examination of intestine of sham mice and ferulic acid treated alone showed normal villous architecture, submucosa and muscularies (Fig. 8(8.1 and 8.2)). Intestine of mice exposed to 8 Gy irradiation displayed a distortion of villi and crypts with evidence of cryptitis (Fig. 8(8.3)). Treatment with ferulic acid prior to 8 Gy-irradiation revealed normal villi and crypts (Fig. 8(8.4)). Overall improvement is evident from the gross anatomy of the sections (Fig. 8(8.3 and 8.4)).

4. Discussion Our study suggests that the administration of ferulic acid at 50 mg/kg body weight pretreatment signicantly reduced thiobarbituric acid levels in spleen, liver and intestine of mice after 24 h irradiation. Ferulic acid acts as an antioxidant against peroxyl radical induced oxidation in neuronal culture and synaptosomal membranes (Scott et al., 1993). Previous reports have shown that ferulic acid is an effective scavenger of free radicals and it has been approved in certain countries as food additive to prevent lipid peroxidation (Schafer and Buettner, 2001). It scavenges the reactive oxygen species such as hydroxyl radical (OH), hypochlorous acid (HOCl) and peroxyl radical [RO2] (Toda et al., 1991) and stable free radical 1, 1-diphenyl-2-picrylhydrazyl [DPPH] (Kikuzaki et al., 2003). We observed gamma radiation exposure resulted in a decline in superoxide dismutase, catalase and glutathione peroxidase activities after 24 h post irradiation.

Fig. 5. Ferulic acid increases reduced glutathione levels in irradiated mice spleen, liver and intestine. Values are given as means7 S.D. of six experiments in each group. Values not sharing a common marking (a, b, c, etc.) differ signicantly at P o0.05 (DMRT).

Fig. 6. Effects of ferulic acid on g-radiation induced DNA damages were determined by the changes in the levels of DNA damage in (6.1) Sham control, (6.2) ferulic acid control (50 mg/kg body weight), (6.3) cells from whole body irradiated at 8 Gy, (6.4) cells from whole body irradiated mouse at 8 Gy, after administration of ferulic acid (50 mg/kg body weight).

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Fig. 7. (7.17.4) Effect of ferulic acid on DNA damage (% tail DNA, tail length, tail moment and Olive tail moment).

Fig. 8. Effect of pretreatment with different doses of ferulic acid on jejunum excised 24 h after 8 Gy whole-body g-irradiation in Swiss albino mice. Sham control mice (8.1) showing normal villous architecture and muscularies. Ferulic acid control (8.2) showing normal villi, crypts, muscularies. Radiation control mice (8.3) showing distortion of villi and crypts with evidence of cryptitis and irradiated mice with ferulic acid (8.4) showing regeneration in distortion of villi and cupts with evidence of cryptitis.

Superoxide dismutase protects against the free radical injury by converting O radical to H2O2 and prevents the formation of 2 OH radical and then H2O2 can be removed by catalase or

glutathione peroxidase. The decrease in the activities of superoxide dismutase, catalase and glutathione peroxidase in g-irradiated rat hepatocytes is due to the inhibition or oxidative

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inactivation of enzyme protein caused by reactive oxygen species generation, which in turn can impair the antioxidant defense mechanism (Jagetia and Reddy, 2005). Pretreatment with ferulic acid signicantly increased the superoxide dismutase, catalase and glutathione peroxidase activities at 24 h post irradiation. These antioxidant enzymes are important in providing protection from radiation exposure; the proper balance of these enzymes in specic cells and in the whole organism is required for maximum radioprotection (Kumar and Kuttan, 2004). In addition to its antioxidant activity, ferulic acid modulates phase II enzymes like glutathione S-transferase (GST) and the radioprotection observed in the present study may also be attributed to the upregulation of antioxidant enzymes. Induction of GST and quinine reductase by ethylferulate was suggested as the mechanism of inhibition of carcinogenesis (Han et al., 2001). Exposure of mice to gamma radiation reduced the reduced glutathione levels in spleen, liver and intestine at 24 h post irradiation. Depletion of reduced glutathione in mice is known to inhibit glutathione peroxidase activity and has been shown to increase lipid peroxidation (Lewin, 1976). A similar correlation between the depletion of reduced glutathione and increase of lipid peroxidation has been proved (Nair et al., 2001). Pretreatment of mice with ferulic acid (50 mg/kg body weight) signicantly reduced the depletion in reduced glutathione concentration in spleen, liver and intestine at 24 h post-irradiation. Reduced glutathione has potent electron donating capacity, as indicated by the high negative redox potential of reduced glutathione/GSSH redox couple (Lewin, 1976). Reduced glutathione is a versatile protector and executes radioprotective function through free-radical scavenging, restoration of the damaged molecule by hydrogen donation, reduction of peroxides and maintenance of protein thiols in the reduced state (Biaglow et al., 1987; Revesz, 1985). The comet assay has provided useful insights into the behavior of individual cells exposed to ionizing radiation. Ionizing radiation is an effective oxidizing agent, producing hundreds of different types of base damages and single or double-strand breaks (Wallace, 1998). Using the comet assay, we observed ferulic acid pretreatment signicantly decreased the percentage of tail DNA, tail length, tail moment and Olive tail moment in the peripheral blood of whole body irradiated mice. This decrease in tail DNA, tail length, tail moment, Olive tail moment seen in ferulic acid treated plus gammairradiated mice indicates the protective effect of ferulic acid on radiation induced DNA damage. Previous report adds credence to our ndings that ferulic acid protects cellular DNA in peripheral blood leukocytes and bone marrow cells from radiation induced damage (Maurya et al., 2005). Previous studies have shown that ferulic acid protects the cells from oxygen species mediated DNA damage (Stagos et al., 2004). Natural antioxidants scavenge the free radicals and protect the cellular DNA against indirect effects of ionizing radiation (Rajakumar and Rao, 1993; Prasad et al., 2006). The intestine is a highly radiosensitive and dose-limiting organ with rapidly multiplying cells (crypt cells). Acute radiation enteropathy results from death of rapidly proliferating crypt cells, disruption of the epithelial barrier, mucosal inammation and lipid peroxidation (Paris et al., 2001). The pathogenesis of radiation enteropathy involves alterations of intestinal wall compartments, and changes in the absorption behavior during radiation sickness (Carr, 2001). In our study, pretreatment with ferulic acid at 50 mg/kg body weight signicantly reduced the number of inammatory cells, mitotic cells and dead cells, and maintained villus height. Nuclear enlargement was less in ferulic acid treated mice. The above protection to the intestinal epithelium could play a role in preventing malabsorption. Thus, our study reveals that ferulic acid shows potent radioprotective effects in Swiss albino mice and the mechanism of radioprotection may be due to its antioxidant nature. It can be

used as an ideal adjuvant in the radiotherapy to protect normal tissues from deleterious effects of gamma-radiation.

References
Biaglow, J.E., Varnes, M.E., Epp, E.R., Clark, E.P., 1987. In: Cerrutti, P.A., Nygaard, O.F., Simic, M.G. (Eds.), Anticarcinogenesis and Radiation Protection. Plenum Press, New York, pp. 387. Carr, K.E., 2001. Effects of radiation damage on intestinal morphology. Int. Rev. Cytol. 208, 1119. Ejchart, A., Sadlej-Sosnowska, N., 2003. Statistical evaluation and comparison of comet assay results. Mutat. Res. 534, 8592. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 7077. Foster-Nora, J.A., Siden, R., 1997. Amifostine for protection from antineoplastic drug toxicity. Am. J. Health Syst. Pharm. 54, 787. Hall, E.J., 2000. Radiation, the two-edged sword: cancer risks at high and low doses. Cancer J. 6, 343350. Han, B.S., Park, C.B., Takasuka, N., Naito, A., Sekine, K., Noumra, E., Taniguchi, H., Tsuno, T., Tsuda, H., 2001. A ferulic acid derivative ethyl 3-(40 -genranyloxy-3methoxy phenyl)-2 proleonyl as a new candidate in rat. Jpn. J. Cancer Res. 92, 404409. Jagetia, G.C., Reddy, T.K., 2005. Modulation of radiation-induced alteration in the antioxidant status of mice by naringin. Life Sci. 77, 780794. Kakkar, Z.Y.P., Das, B., Viswanathan, P.N., 1984. A modied spectrophotometeric assay of superoxide dismutase. Indian J. Biochem. Biophys. 21, 130132. Kemp, G., Rose, P., Lurain, J., 1996. Amifostine pre-treatment for protection against cyclophosphamide-induced and cisplatin-induced toxicities: results of a randomised control trail in patients with advanced ovarian cancer. J. Clin. Oncol. 14, 2101. Kikuzaki, H., Hisamoto, M., Hirose, K., Akiyama, K., Taniguchi, H., 2003. Antioxidant properties of ferulic acid and its related compounds. J. Agric. Food Chem. 50, 21612168. Kim, S.G., Nam, S.Y., Kim, C.W., 1998. In vivo radioprotective effects of oltipraz in g-irradiated mice. Biochem. Pharmacol. 15 (55), 15851590. Kumar, K.B., Kuttan, R., 2004. Protective effect of an extract of Phyllanthusamarus against radiation induced damage in mice. J. Radiat. Res. 45, 133139. Lewin, S., 1976. Vitamin C: Its Molecular Biology and Medical Potential. Academic Press, New York, NY. (pp. 42). Maurya, D.K., Salvi, V.P., Nair, C.K.K., 2005. Radiation protection of DNA by ferulic acid under in vitro and in vivo conditions. Mol. Cell. Biochem. 280, 209217. Maurya, D.K., Nair, C.K.K., 2006. Preferential radioprotection to DNA of normal tissues by ferulic acid under ex vivo and in vivo conditions in tumor bearing mice. Mol. Cell. Biochem. 285, 181190. Nair, C.K.K., Parida, D.K., Nomura, T., 2001. Radioprotectors in radiotherapy. J. Radiat. Res. 42, 2137. Niehaus, W.G., Samuelsson, B., 1968. Formation of malondialdehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur. J. Biochem. 61, 126130. Olive, P.L., Banath, J.P., Durand, R.E., 1990. Detection of etoposide resistance by measuring DNA damage in individual Chinese hamster cells. J. Natl. Cancer Inst. 82, 779783. Paris, F., Fuks, Z., Kang, A., Capodieci, P., Juan, G., Ehleiter, D., Friedman, A.H., Cordon Cardo, C., Kolesnick, R., 2001. Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science 293, 293297. Prasad, N.R., Srinivasan, M., Pugalendi, K.V., Menon, V.P., 2006. Protective effect of ferulic acid on gamma-radiation induced micronuclei, dicentric aberration and lipid peroxidation in human lymphocytes. Mutat. Res. 603, 129134. Rajakumar, D.V., Rao, M.N., 1993. Dehydrozingerone and isoeugenol as inhibitors of lipid peroxidation and as free radical scavengers. Biochem. Pharmacol. 46, 20672072. Revesz, L., 1985. The role of endogenous thiols in intrinsic radioprotection. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 47, 361368. Rotruck, J.T., Pope, A., Ganther, H.E., Swanson, A.B., 1973. Selenium, biochemical roles as components of glutathione peroxidase. Science 179, 588590. Schafer, F.Q., Buettner, G.R., 2001. Redox environment of the cell as viewed through the redox state of the glutathione disulde/glutathione couple. Free Radic. Biol. Med. 30, 11911212. Scott, B.C., Butler, J., Halliwell, B., Aruoma, O.I., 1993. Evaluation of the antioxidant action of ferulic acid and catechins. Free Radic. Res. Commun. 19, 241253. Singh, N.P., Mc-Coy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184191. Sinha, K.A., 1972. Colorimetric assay of catalase. Anal. Biochem. 47, 389394. Stagos, D., Kouris, S., Kouretas, D., 2004. Plant phenolics protect from bleomycininduced oxidative stress and mutagenicity in Salomonella typhimurium TA 102. Anticancer Res. 24, 743745. Tanaka, T., Kojima, T., Kawamori, T., Suzui, M., Okamoto, K., Mori, H., 1993. Inhibition of 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis by the naturally occurring plant phenolics caffeic acid, ellagic acid, chlorogenic acid ferulic acid. Carcinogenesis 14, 3211325. Toda, S., Kumura, M., Ohnishi, M., 1991. Effects of phenolic carboxylic acids on superoxide anion and lipid peroxidation induced by superoxide anion. Plant Med. 57, 810.

274

J. Shanthakumar et al. / European Journal of Pharmacology 691 (2012) 268274

Wallace, S.S., 1998. Enzymatic processing of radiation-induced free radical damage in DNA. Radiat. Res. 150, S60S79. Weiss, J.F., Landauer, M.R., 2009. History and development of radiation-protective agents. Int. J. Radiat. Biol. 85, 539573.

Weiss, J.F., 1997. Pharmacologic approaches to protection against radiation-induced lethality and other damage. Environ. Health Perspect. 105, 14731478. Zhao, Z., Moghadasian, M.H., 2008. Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: a review. Food Chem. 109, 691702.