**5.6 Troxerutin**

490 Selected Topics in DNA Repair

doses of 5 or 15 mg/kg, administered either i.p. or in drinking water, reduced radiationinduced frequency of chromosomal aberrations. Farooqi and Kesavan, had shown that administration immediately after radiation exposure also significantly reduced chromosome aberrations (Farooqi and Kesavan 1992). In our other study, George et al showed that when caffeine (80 mg/kg) was administered i.p. 1 h before irradiation it increases the survival of the mice (George et al. 1999). Radioprotection in mice at the same dose of caffeine may be related to modulation of radiation-induced apoptotic genes, for example, the depression of bax mRNA (Kim et al. 2003). This same high dose of caffeine protected against local radiation (35 Gy) skin reactions of mice. However, caffeine injection into a mouse

Chlorophyllin (CHL) (scheme 2B), is a water-soluble mixture of sodium-copper salts of green plant pigment, chlorophyll. CHL is widely marketed for a variety of dietary and medicinal uses. Recently, it has also been shown to occur naturally in a constituent of traditional Chinese medicine (Chiu et al. 2003). It has chemopreventive, antimutagenic and anticarcinogenic properties (Egner et al. 2001; Guo et al. 1995). Our lab has explored it radioprotective property in various model systems. It exhibited protection against radiation and chemical induced cytogenetic damage (Kumar et al. 1999)). CHL inhibited lipid peroxidation induced by 2,2'-azobis(2-propionimidinedihydrochloride) (AAPH) in lymphocytes in vitro. It also partially prevented radiation-induced suppression of mitogenic

Ferulic acid (FA) (Scheme 2C) is a monophenolic phenylpropanoid occurring in plant materials such as rice, green tea and coffee beans. It is well known antioxidant and has ability to scavenge the free radicals. As for toxicity of this compound, no *in vivo* data is available, but *in vitro* cytotoxicity in rat hepatocytes showed an LD50 of 25 mM (Maurya et al. 2005b). We have explored the effect of ferulic acid on gamma-radiation-induced relaxation of plasmid pBR322 DNA and induction of DNA strand breaks in peripheral blood leukocytes and bone marrow cells of mice exposed to whole body gamma-radiation. Presence of 0.5 mM ferulic acid significantly inhibited the disappearance of supercoiled (ccc) plasmid pBR322 with a dose modifying factor (DMF) of 2.0. Intraperitoneal administration of different amounts (50, 75 and 100 mg/kg body weight) of FA 1 h prior to 4Gy gammaradiation exposure showed dose-dependent decrease in the yield of DNA strands breaks in murine peripheral blood leukocytes and bone marrow cells as evidenced from comet assay. The dose-dependent protection was more pronounced in bone marrow cells than in the blood leukocytes. It was observed that there was a time-dependent disappearance of radiation induced strand breaks in blood leukocytes (as evidenced from comet parameters) following whole body radiation exposure commensurate with DNA repair (Maurya et al. 2005b). Administration of 50 mg/kg body weight of FA after whole body irradiation of mice resulted disappearance of DNA strand breaks at a faster rate compared to irradiated controls, suggesting enhanced DNA repair in FA treated animals. When normal and tumor cells were treated with FA and the DNA damage measured after radiation exposure it was found that FA is having preferential protection to normal cells compared to tumor cells both under *ex vivo* and *in vivo* conditions (Maurya and Nair 2006). Recently, Ma et al have

fibrosarcoma did not affect the tumor response to irradiation (Hebbar et al. 2002).

stimulation of lymphocytes in vitro (Kumar et al. 2004).

**5.4 Chlorophyllin** 

**5.5 Ferulic acid** 

Troxerutin (Scheme 2D), a derivative of the natural flavonoid rutin extracted from *Sophora japonica* (Japanese pogoda tree) has been commonly used in the treatment of Chronic Venous Insufficiency (CVI) disease (Lefebvre and Lacombe 1991). In clinical trials, troxerutin has been given in doses up to 7 g per day orally for up to 6 months with no contraindications (Glacet-Bernard et al. 1994). It has been reported that during radiotherapy of head and neck cancer, administration of a mixture of troxerutin and coumarin offered protection to salivary glands and mucosa. We have shown that troxerutin inhibited lipid peroxidation in membrane of sub-cellular organelles as well as normal tissues of tumorbearing mice exposed to gamma-radiation. Further, it was found that administration of troxerutin resulted in differential protection of DNA in blood leucocytes and bone marrow cells and not in cells of tumor in whole body irradiated tumor-bearing mice. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under *ex vivo* condition of irradiation (2Gy)

Fig. 2. A. Baicalein, B. Chlorophyllin, C. Ferulic acid, D. Troxerutin, E. Vanillin.

Role of Radioprotectors in the Inhibition of DNA Damage

such as that of radiotherapy for cancer.

cells. *Blood* Vol.104, No.4, pp. 978-985

*Biochim Biophys Acta, Vol.* 1282, No 1, pp.63-70

*Mutation Research*, Vol. 269, No.2, pp.225-230

Vol.19, No.2, pp.171-176

1065. *Mutation Research*, Vol. 356, No.2, pp.147-154

*Research,* Vol.46, No.1 pp.37-41

**7. References** 

and Modulation of DNA Repair After Exposure to Gamma-Radiation 493

cytoprotective or immunomodulatory property or their combination have been evaluated extensively for their radioprotective potentials in both in vitro and in vivo models (Maurya et al. 2006; Weiss and Landauer 2009). So far no synthetic radioprotective molecule is available that is perfectly safe and effective. Herbal origin radioprotectors have a hope to overcome the toxicity issues seen with the synthetic compounds. There are many reports on the pre-irradiation radioprotectors, using synthetic or natural compounds. There are hardly a few post-irradiation radioprotectors. Such radioprotectors based on induction of DNA repair and related mechanisms will have great potential in reducing accidental exposure such as those that occur due to natural calamities like earthquakes and tsunami as in the case of Fukushima daiichi nuclear reactors. To overcome this kind of situation, there is a need to locate more effective post-irradiation effective radioprotector that can enhance the DNA repair. Since the DNA is the critical target for ionizing radiation, future, studies should focus on compounds that have potential to enhance the process of DNA repair which is essential after radiation exposure. These also can be used in other cases of exposure

Charrier, S., Michaud, A., Badaoui, S., Giroux, S., Ezan E, Sainteny, F., Corvol, P., &

Cherdyntseva, N., Shishkina, A., Butorin, I., Murase, H., Gervas, P., & Kagiya, T.V. (2005).

Chiu LC, Kong CK, and Ooi VE. (2003). Antiproliferative effect of chlorophyllin derived

MCF-7 cells. I*nternational Journal of Oncology*, Vol. 23, No.3, pp.729-735 Damron, T.A., Spadaro, J.A., Horton, J.A., Margulies, B.S., Strauss, J.A., & Farnum, C.E.

physis. *International Journal of Radiation Biology*, Vol. 80, No. 3, pp.217-228 Devasagayam, T.P., Kamat, J.P., Mohan, H., & Kesavan, P.C. (1996). Caffeine as an

Diamond, A.M., Dale, P., Murray, J.L., & Grdina, D.J. (1996). The inhibition of radiation-

Egner, P.A., Wang, J.B., Zhu, Y.R., Zhang, B.C., Wu, Y., Zhang, QN., Qian, G.S., Kuang, S.Y.,

Farooqi, Z., & Kesavan, P.C. (1992). Radioprotection by caffeine pre- and post-treatment in

George, K.C., Hebbar, S.A., Kale, S.P., & Kesavan, P.C. (1999). Caffeine protects mice against

*National Academy of Science*, U S A, Vol. 98, No.25, pp.14601-14606

Vainchenker, W. (2004). Inhibition of angiotensin I-converting enzyme induces radioprotection by preserving murine hematopoietic short-term reconstituting

Effect of tocopherol-monoglucoside (TMG), a water-soluble glycosylated derivate of vitamin E, on hematopoietic recovery in irradiated mice. *Journal of Radiation* 

from a traditional Chinese medicine Bombyx mori excreta on human breast cancer

(2004). Novel radioprotectant drugs for sparing radiation-induced damage to the

antioxidant: inhibition of lipid peroxidation induced by reactive oxygen species.

induced mutagenesis by the combined effects of selenium and the aminothiol WR-

Gange, S.J, Jacobson, L.P., et al. (2001). Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer. *Proceeding In* 

the bone marrow chromosomes of mice given whole-body gamma-irradiation.

whole-body lethal dose of gamma-irradiation. *Journal of Radiological Protection*,

(Maurya et al. 2004). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy gamma*-*radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. Measurement of DNA repair potential of the troxerutin shows that it enhances the DNA repair (Maurya et al. 2005a). So the observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals.

#### **5.7 Vanillin**

Vanillin (4-hydroxy-3-methoxybenzaldehyde), scheme 2E, is a compound used as a flavoring agent and as a dietary component. It is the major component of natural vanilla, which is one of the most widely used and important flavoring materials throughout the world. The source of vanilla is the bean, or pod, of the tropical Vanilla orchid (principally *Vanilla planifolia* Andrews, syn. *V. fragrans* (Salisb. Ames)). Vanillin is an antioxidant capable of protecting membrane against lipid peroxidation and DNA against strand breaks induced by reactive oxygen species like singlet oxygen. Vanillin and its analogs were strongly antimutagenic or anti-genotoxic in most studies. Vanillin also suppresses the chromosomal aberrations induced by X-rays in V79 cells *in vitro* (Keshava et al. 1998*)* and in mice *in vivo*  (Sasaki et al. 1990). Imanishi et al. have shown anti-mutagenic effect of vanillin against UV and X-rays in Chinese hamster V79 cells (Imanishi et al. 1990). We have explored the effect of vanillin on radiation-induced DNA damage in plasmid pBR322 (*in vitro*), human peripheral blood leucocytes and mouse splenic lymphocytes (*ex vivo*) and in mouse (*in vivo*), besides the possible mechanisms involved in terms of scavenging radiation related free radicals by pulse radiolysis. Our finding shows that presence of 0.5mM vanillin has a dosemodifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (*ex vivo*) to radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of -radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, 'tail moment' and 'Olive tail moment'. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (*in vivo*) to radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2h post-irradiation. This was more than that observed at 1 h (Maurya et al. 2007). The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair (Maurya et al. 2007).

#### **6. Future prospects for radioprotectors in mitigation of radiation damage**

Research in the development of radioprotectors worldwide has focused on screening a variety of chemical and biological compounds (Maurya et al. 2006; Nair et al. 2001; Weiss and Landauer 2009). Various natural or synthetic compounds having either antioxidant or cytoprotective or immunomodulatory property or their combination have been evaluated extensively for their radioprotective potentials in both in vitro and in vivo models (Maurya et al. 2006; Weiss and Landauer 2009). So far no synthetic radioprotective molecule is available that is perfectly safe and effective. Herbal origin radioprotectors have a hope to overcome the toxicity issues seen with the synthetic compounds. There are many reports on the pre-irradiation radioprotectors, using synthetic or natural compounds. There are hardly a few post-irradiation radioprotectors. Such radioprotectors based on induction of DNA repair and related mechanisms will have great potential in reducing accidental exposure such as those that occur due to natural calamities like earthquakes and tsunami as in the case of Fukushima daiichi nuclear reactors. To overcome this kind of situation, there is a need to locate more effective post-irradiation effective radioprotector that can enhance the DNA repair. Since the DNA is the critical target for ionizing radiation, future, studies should focus on compounds that have potential to enhance the process of DNA repair which is essential after radiation exposure. These also can be used in other cases of exposure such as that of radiotherapy for cancer.
