**5.2 Baicalein**

488 Selected Topics in DNA Repair

chemotherapy, with or without irradiation, followed by bone

DTPA is a FDA-approved agents and includes the calcium and zinc salts, used for the chelation of the transuranic radionuclides

SeM is a naturally occurring derivative of selenium, found in soya, grains, legumes, and selenium-enriched yeast (Whanger 2002). When administered i.p., SeM (4 mg/kg Se) significantly increased the survival of mice irradiated at 10-Gy 60Co at a low

plutonium, americium, and curium; Prussian blue for decorporation of 137Cs; and KI to block uptake of radioactive

DRF for 30-days survival in mice treated 30 min before

Genistein A DRF of 1.16 was obtained at a genistein dose (200 mg/kg SC) that

Table 1. A list of well known radioprotectors, mitigators and therapeutics.

irradiation with low doses (50 mg/kg) i.p. were 1.30 for orientin and 1.37 for vicenin. The same low doses of orientin or vicenin provided efficient protection against bone marrow damage, as measured by chromosomal aberrations and stem cell survival using the exogenous colony-forming unit spleen (CFU-S) assay.

did not result in any adverse pathology or behavioural toxicity.

**5. Role of radioprotectors in inhibition of DNA damages and enhancement of**

In order to restore the cellular function DNA repair is our important parameter that can be modified by radioprotective compounds to improve the radioprotection. Most of the cellular alterations induced by ionizing radiation is indirect and is mediated by the generation of free radicals and related reactive species, mainly derived from oxygen. Hence natural compounds with antioxidant activity have potential as good radioprotectors. Though a large variety of compounds have shown promise as radioprotector in laboratory studies, most of

Protection of cellular molecules including DNA from radiation can be achieved by various mechanisms. Protection to cellular DNA can be achieved by reducing the quantity of damage (by radical scavenging and chemical repair) followed by enhancement of biochemical repair of DNA to improved protection and recovery. Thus DNA repair is one of the important parameters that can be modified to attain improved protection. Many natural and related compounds are shown to be effective radioprotectors. They protect cellular molecules including DNA by various mechanisms. These include their antioxidant capacity and induction of repair mechanisms. Our studies have identified many compounds with radioprotective ability using *in vitro, ex vivo* and *in vivo* model systems. The systems used are rat liver and brain sub-cellular organelles, plasmid DNA, human lymphocytes, mammalian cells in culture and mouse. Different parameters also have been used to estimate damage and protection. Our studies have identified caffeine, chlorophyllin,

them failed even before reaching the preclinical stage due to toxicity and side effects.

Filgrastim patients with hematologic cancers undergoing high-dose

marrow transplantation.

iodine by the thyroid.

dose rate (0.2 Gy/min).

**DNA repair after radiation exposure**

**Protective property Ref.**

(Damron et al. 2004; Diamond et al. 1996)

(Nayak and Devi 2005; Uma Devi et al. 2000)

(Landauer et al.

2003)

**Radioprotectors/ mitigators and therapeutics**

Chelators such as diethylenetriamene pentaacetate (DTPA)

compounds such as Selenomethionine (SeM) and Sodium

Selenium

selenite

Orientin and vicenin from *Ocimum sanctum*

> Baicalein (scheme 2A), has been reported to possess lipid peroxidation inhibitory activity. Our results indicate that baicalein is a potent radioprotector at micromolar (5-50) levels. The protective effect, at 5 μM, was 80% against formation of thiobarbituric acid reactive substances (TBARS) and 50% against lipid hydroperoxide. The protective ability against protein carbonyl formation was 50% and protein hydroperoxide formation 85% at the same concentration. Similar protective effects were also observed against damage to glutathione peroxidase and superoxide dismutase. A concentration dependent effect also was seen with most of the parameters examined. Single-strand break formation induced by radiation also was accentuated with baicalein. It also inhibited the DNA binding caused by radiation. (Tilak and Devasagayam 2003). Recently it was found that baicalein has a radioprotective effect against NF-κB-mediated inflammatory response through MAPKs and the Akt pathway (Lee et al. 2011).

#### **5.3 Caffeine**

Caffeine is a bitter, white crystalline xanthine alkaloid that is a psychoactive stimulant present in coffee and cola-based soft drinks. In humans, caffeine acts as a central nervous system (CNS) stimulant, temporarily warding off drowsiness and restoring alertness. We have shown that caffeine protected against DNA strand breaks in plasmid pBR322, a system devoid of repair and replication machinery (Kumar et al. 2001). This protective effect was related to the demonstrated antioxidant properties of caffeine in vitro, including scavenging of primary and secondary ROS (Devasagayam et al. 1996). Pretreatment with caffeine at

Role of Radioprotectors in the Inhibition of DNA Damage

**5.6 Troxerutin**

pathway to inhibit apoptosis and oxidation (Ma et al. 2011b).

and Modulation of DNA Repair After Exposure to Gamma-Radiation 491

suggested that the radioprotective efficacy by FA may be a result of early recovery of hematopoietic cells due to enhanced production of G-CSF and erythropoietin (Ma et al. 2011a). They have also reported that FA had a radioprotective effect through the ERK

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.

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

#### **5.4 Chlorophyllin**

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

#### **5.5 Ferulic acid**

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 suggested that the radioprotective efficacy by FA may be a result of early recovery of hematopoietic cells due to enhanced production of G-CSF and erythropoietin (Ma et al. 2011a). They have also reported that FA had a radioprotective effect through the ERK pathway to inhibit apoptosis and oxidation (Ma et al. 2011b).