Elsevier

Toxicology

Volume 209, Issue 3, 5 May 2005, Pages 225-235
Toxicology

Radioprotective effect of sesamol on γ-radiation induced DNA damage, lipid peroxidation and antioxidants levels in cultured human lymphocytes

https://doi.org/10.1016/j.tox.2004.12.009Get rights and content

Abstract

Sesamol pretreated (1, 5 and 10 μg/ml) lymphocytes were exposed to different doses of γ-radiation, i.e., 1, 2 and 4 Gray (Gy) and the cellular changes were estimated by using cytokinesis blocked micronucleus assay (MN), dicentric aberration (DC), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Radiation significantly increased MN, DC frequencies, TBARS levels and decreased GSH and antioxidant enzyme levels in a dose dependent manner. The highest damage to lymphocytes was observed at 4 Gy irradiation. On the other hand, sesamol pretreatment significantly decreased MN, DC frequencies, TBARS levels and increased GSH levels and SOD, CAT and GPx activities in a concentration dependent manner. At 1 Gy irradiation all concentrations of sesamol (1, 5 and 10 μg/ml) significantly protects the lymphocytes from radiation damage. At 2 Gy irradiation 5 and 10 μg/ml of sesamol shows significant radioprotection. Since the highest damage was observed at 4 Gy irradiation both 1 and 5 μg/ml of sesamol pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10 μg/ml of sesamol significantly (p < 0.05) protects the lymphocytes from radiation effect. Thus, sesamol pretreatment gives significant protection to cultured human lymphocytes against γ-radiation induced cellular damage. The possible mechanism involved in the radioprotective influence of sesamol is discussed.

Introduction

Deleterious impact of ionizing radiation, especially with low LET on biological systems is well documented. Molecular oxygen due to its biradical nature is the most important electron acceptor in the biosphere. It plays an important role in accepting unpaired electrons giving rise to a series of partially reduced species. In addition to this radiolytic product of water, eaq, OH and H also react with oxygen and generate various ROS (Von Sonnlag, 1987). These reactive species can induce damages to cellular macromolecules. Both direct and indirect effects of ionizing radiation damage cellular DNA. In the indirect effect, DNA damage is mainly induced by abstraction of the H atom from the C′-4 position of the deoxyribose or by attack of the bases via the hydroxyl radicals produced by the radiolysis of water (Spotheim-Maurizot et al., 1992). DNA lesions induced by ionizing radiations have already been suggested and it is believed that chromosomal aberrations such as dicentrics, translocations, etc., appear as a result of double strand breaks and misrepaired damage (Olive, 1998). Natarajan et al. (1980) and Bryant (1988) also suggested that double strand breaks are mainly responsible for the formation of chromosomal aberrations. Damage to chromosomes is also manifested as breaks and fragments, which appears as micronuclei in the rapidly proliferating cells (Hofer et al., 2000). Micronuclei are produced during mitosis due to various mechanisms, i.e., acentric fragments, multicentric chromosomes, damaged kinetochores and spindle fiber defects and it can be detected in the cytoplasm besides the cell nucleus as small nucleus-like particles (Muller and Strefter, 1994). Enhancement of the frequency of micronuclei and dicentric aberrations during γ-irradiation has been reported earlier in the human blood lymphocytes by several researchers (Konopacka Maria and Wolny, 2001, Schmid et al., 1995). Hence to quantify the level of DNA damage, the cytokinesis blocked micronucleus assay and dicentric aberration was used. These assays have been shown to be effective tool to measure cytogenetic damage of chemical agents as well as ionizing radiation in human lymphocytes in vitro (Fenech and Morely, 1985, Schmid et al., 1995).

Apart from DNA damage lipid peroxidation also considered to be critical event of ionizing radiation effect (Agrawal and Kale, 2001). Lipid peroxidation has been found to increase with increase in radiation dose in rat liver mitochondria, microsomes and splenic lymphocytes (Bloor. et al., 2000, Santhosh Kumar et al., 2004, Priyadarsini et al., 2003). Lipid radicals (L) are believed to be formed by the reaction of OH radicals generated by ionizing radiation with polyunsaturated fatty acids (LH) which subsequently reacts with oxygen to form lipid peroxyl radical (LOO′) after undergoing molecular rearrangement of conjugation in double bonds and eventually a chain reaction is initiated on irradiation in oxygenated condition (Pandey and Mishra, 2000). Further lipid peroxidation products such as malondialdehyde forms adduct with cellular DNA. To maintain the redox balance in order to protect themselves from these free radicals action the living cells have evolved an endogenous antioxidant defense mechanism which include nonenzymatic entities like glutathione, ascorbic acid and also enzymes like catalase, superoxide dismutase, glutathione peroxidase, etc. (Mittal et al., 2001). Radiation exposure can alter the balance of endogenous protective systems such as glutathione and antioxidant enzyme systems (Fridovich, 1978, Dubner. et al., 1995, Samarth and Kumar, 2003). Therefore, to understand the mode of action of a antioxidant compound as a radioprotector it was necessary to investigate its effects on radiation induced DNA damage, lipid peroxidation with reference to alterations in the endogenous antioxidant defense mechanism.

Study on plant extracts and phytochemicals as modifiers of radiation effects is a new area of research. Indeed, human beings are consuming a variety of phytochemicals and are thus protected from radiation exposure. Therefore, it is necessary to assess the protective action of such commonly used phytochemicals and exploit their possible application in radiation therapy of cancer as an alternative source of non-toxic radioprotectors. Some reports are available regarding plant extracts and phytochemicals on radiation induced cellular damage in model as well as in animal systems (Choudhary et al., 1998, Shimoi et al., 1996, Goel et al., 2004) but the protective effects of plant extracts and phytochemicals on radiation induced DNA damage, lipid peroxidation and antioxidant status are not thoroughly investigated.

Sesamol is a potent antioxidant contained only in processed sesame oil (Ando et al., 2000). It is a phenolic derivative with a methylenedioxy group and like vitamin E is known to be an antioxidant (Uchida et al., 1996). Recent studies have shown that sesamol can act as a potent antioxidant (Suja et al., 2004) and possess chemopreventive, antimutagenic and antihepatotoxic properties (Kaur and Saini, 2000, Ohta et al., 1994, Kapadia et al., 2002). Very little work has been carried out on its role in the defense against radiation induced cellular damage. Thus, the present study attempted to delineate the effect of sesamol as a protector against γ-radiation mediated cellular damage in human blood lymphocytes in vitro.

Section snippets

Isolation of lymphocytes

Blood samples were aseptically collected in haparinized sterile glass tubes from median cubital vein of nonsmoking healthy individuals (22–25 years). Lymphocytes were isolated from blood using Ficoll-Histopague (Sigma, USA) and cultured as described previously (Boyum, 1968). Blood was diluted 1:1 with phosphate buffered saline (PBS) and layered onto Histopague with ratio of blood + PBS: Histopague maintained at 4:3. The blood was centrifuged at 1340 rpm for 35 min at room temperature. The

Effect of sesamol on MN and DC levels in cultured lymphocytes exposed to γ-radiation

The frequencies of dicentric aberrations (DC) during different doses of γ-irradiation in cultured human blood lymphocytes were presented in Fig. 1. Significant increases in the frequencies of DC were observed in a dose dependent manner. Maximum number of DC (63 ± 1.47) was observed at 4 Gy. All concentration of sesamol (1, 5 and 10 μg/ml) significantly decreased the DC frequencies when the lymphocytes were exposed to 1 Gy. At 2 Gy irradiation 5 and 10 μg/ml of sesamol brought the DC to near normal.

Discussion

The use of ionizing radiation has become an integral part of modern medicine. It is used for diagnostic as well as therapeutic purposes. In some cases, radiation may be the single best treatment of cancer. The radiation therapy of cancer depends on achieving a therapeutic differentiation between the cancer cell cytotoxicity and normal tissue toxicity. The therapeutic differentiation may be achieved with chemical radiation sensitizers or protectors. The development of radiation protectors is

Conclusion

Our results show that γ-irradiation induces increasing frequencies of micronuclei, dicentric aberration, elevated levels of TBARS, decreased levels of GSH and the decreased activities of antioxidant enzymes due to oxidative damage in cultured human blood lymphocytes. But sesamol probably through its free radical scavenging property protects lymphocytes from γ-irradiation. These results are particularly interesting since the effects of the sesamol were observed at very low concentrations and

Acknowledgement

We thank Tamilnadu State Council for Science and Technology for supporting the work in the form of Young Scientist Fellowship to the first author.

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