Lipid Peroxidation, Superoxide Dismutase and Catalase Activities in ...

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activity in brain tumor tissue was 106.3% higher than that in controls. Keywords: Catalase, lipid peroxidation, oxidative stress, superoxide dismutase. Introduction.

Archives of Physiology and Biochemistry 2003, Vol. 111, No. 5, pp. 455–459

Lipid Peroxidation, Superoxide Dismutase and Catalase Activities in Brain Tumor Tissues B. Popov1, V. Gadjeva1, P. Valkanov2, S. Popova1 and A. Tolekova3 Department of Chemistry and Biochemistry; 2Department of Neurosurgery; and 3Department of Physiology, Medical Faculty, Thracian University, Stara Zagora, Bulgaria

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Abstract Free radical-mediated damages may play an important role in cancerogenesis. To investigate their relevance in the cancer process, malonyl dialdehyde (MDA) level, superoxide dismutase (SOD), and catalase (CAT) activities were determined in the normal brain tissue and brain tumor tissue. When compared with the normal brain tissue, we have detected: (i) significantly lower MDA concentration in brain tumor tissue (1.63 nmol/mg Pr vs 2.04 nmol/mg Pr; p = 0.03); (ii) SOD activity in brain tumor tissue was significantly lower (3.15 U/mg Pr vs 4.97 U/mg Pr; p = 0.0002); and (iii) CAT activity in brain tumor tissue was 106.3% higher than that in controls. Keywords: Catalase, lipid peroxidation, oxidative stress, superoxide dismutase.

Introduction Oxygen is critical for life, but when oxygen is utilized, reactive oxygen species (ROS) are also produced (Sahu, 1991). ROS include hydroxyl radicals, superoxide anions, hydrogen peroxide and nitric oxide. They are highly unstable molecules that interact quickly and aggressively with polyunsaturated fatty acids, DNA and proteins (Halliwell & Gutteridge, 1984). Free radicals and other reactive oxygen species have long been known to be mutagenic; furthermore, these agents have more recently emerged as mediators of the other phenotypic and genotypic changes that lead from mutation to neoplasia (Guyton & Kensler, 1993; Lawrence, 2000).

Under normal conditions free radicals and other reactive oxygen species are essential for life, because they are involved in cell signaling and are used by phagocytes for their bactericidal action. Cells are protected against the toxic effects of high concentrations of ROS by a balanced level of endogenous enzymatic and non-enzymatic antioxidants. When ROS generation is increased to an extent that overcomes the cellular antioxidants, the result is oxidative stress. It is now clear that several biological molecules, which are involved in cell signaling and gene regulation systems, are very sensitive to redox status of the cell (Mates, 1999). It ha been suggested that oxidative stress can act as a contributing factor in the pathogenesis of many diseases, including cancer (Oberley & Buettner, 1979; Slater, 1984; Farber et al., 1990; Oberley, 1990; Sangeetha et al., 1990; Sahu, 1991; Guyton & Kensler, 1993; Farber, 1994; Wiseman & Halliwell, 1996; Borek, 1987, 1997; Mates et al., 1999). The prevention of oxidation is an essential process in all the aerobic organisms, as decreased antioxidant protection may lead to cytotoxicity, mutagenicity and/or carcinogenicity (Mates, 2000). The human brain is especially vulnerable to free radical attack because of its high oxygen consumption and high concentrations of easily oxidizable polyunsaturated fatty acids. In addition, the brain antioxidant capacity is lowered compared with other organs and thus the brain may be more susceptible to oxidative damage (Halliwell & Gutteridge, 1989). The purpose of this report is to compare the level of lipid peroxidation and antioxidant enzyme superoxide dismutase

Accepted: 15 July, 2004 Address correspondence to: Assoc. Prof. Vesselina Gadjeva, Ph.D., Department of Chemistry and Biochemistry, Medical Faculty, 11 Armeiska Str., Stara Zagora, BG-6000, Bulgaria. Tel.: + 359 42 600 879; Fax: + 359 42 600 005; E-mail: [email protected] DOI: 10.1080/13813450312331342328

© 2003 Taylor & Francis Ltd.

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and catalase activities in brain tumor tissue and in normal brain tissue.

Materials and methods Materials Brain biopsies used in this study were taken from 32 patients with surgically resectable brain cancer (8 men, 24 women, age range: 31–78 years, mean age: 58 years). For controls we used spasements taken at autopsies from 19 postmorten nonnecrotic tumor-free brain tissues (6 men, 13 women, age range: 44–81 years, mean age: 70.2 years). The mean postmortem interval was 4.5 h for control subjects. Specimens for control were taken from three anatomic sites – gyrus precentrales, capsula externa and nuclei centrali (regions with most frequently located brain tumors). Immediately upon removal, specimens were placed in tissue cassettes with 0.9% NaCl. All specimens (from patients and controls) were analyzed on the same day. All assays were performed in duplicate. Brain tumor/control comparisons were carried out using Student’s t-test. The difference was considered statistically significant when p < 0.05. All chemicals and enzymes used in this study were purchased from Sigma Chemical Co (St Louis, Mo). Methods

fraction product was read at 532 nm. The lipid peroxidation was expressed as nmol MDA/mg of protein. Measurement of antioxidant enzymes activities Superoxide dismutase enzyme activity assay Total SOD activity was determined by the xanthine/xanthineoxidase/ nitroblue tetrazolium (NBT) method according to Sun et al. (1988) with minor modification. Xanthine/ xanthine-oxidase-produced O2- reduces NBT to formazan, which can be assessed spectrophotometrically at 560 nm. SOD competes with NBT for the dismutation of O2- and inhibits its reduction. The level of this reduction is used as a measure of SOD activity. The total SOD activity was expressed in units/mg of protein, where one unit is equivalent to the SOD activity that causes 50% inhibition of the reaction rate without SOD. Catalase enzyme activity assay The assay of CAT activity was that of Beers & Sizer (1952). Briefly, hydrogen peroxide (30 mM) was used as a substrate and the decrease in H2O2 concentration at 22°C in a phosphate buffer (50 mM, pH 7.0) was followed spectroscopically at 240 nm for 1 min. The activity of the enzyme was expressed in units per mg of protein and 1 unit equals the amount of an enzyme that degrades 1 mM H2O2 per minute.

Tissue extraction Extractions were prepared from approximately 0.25 g samples of wet tissue. The samples were weighed, and 5 ml of PBS buffer (pH 7.4) containing 2.68 mM KCL, 1.47 mM KH2PO4, 136 mM NaCl, 1.4 mM Na2HPO4 and 1 mM EDTA, was added. After addition of a buffer, the tissue was homogenized and stored on ice until all samples were processed. The tissue homogenates were centrifuged at 15,000 rpm for 10 min at 4°C, and the final supernatants were obtained. They were used for determination of protein concentration, lipid peroxidation, and superoxide dismutase and catalase activities. The protein concentration of the supernatants was determined using the method of Lowry et al. (1951).

Results The results of MDA level, SOD and CAT activity are presented in Figures 1 and 2.

Discussion Lipid peroxides resulted from hydroxyl radical-attack on polyunsaturated fatty acids are hydroxyl radical markers. Elevated level of lipid peroxides thus strongly suggest

Analysis of lipid peroxidation Basal levels of lipid peroxidation, as indicated by thiobarbituric acid-reactive substances (TBARS), were determined using the thiobarbituiric acid (TBA) method, which measures the malondialdehyde (MDA) reactive products (Plaser & Cushman, 1966). In the TBARS assay 1 ml of the supernatant, 1 ml of normal saline, and 1 ml of 25% trichloro-acetic acid were mixed and centrifuged at 2000 rpm for 20 min. 1 ml of protein free supernatant was taken, mixed with 0.25 ml of 1% thiobarbituric acid (TBA) and boiled for 1 h at 95°C. After cooling, the absorbance of the pink color of the obtained

Fig. 1. Comparison of MDA level in brain tumor tissue to normal brain tissue. The results are expressed as mean ± SD, p = 0.03.

MDA, SOD and CAT in Brain Tumors

Fig. 2. Comparison of SOD and CAT actities in brain tumor tissue to normal brain tissue. The results are expressed as mean ± SD, for SOD p < 0.05; CAT activity in brain tumor tissue was 106.3% to that in controls.

hydroxyl radical activity, and reflect oxidative damage (Kenneth, 2000). Our results showed that lipid peroxidation in brain tumor cells was significantly lower compared with that in the controls. The lack of an increase in MDA level is consistent with previous studies in which decreases in MDA were observed. Cancer cells have highly evolved protective mechanisms to prevent lipid peroxidation so that rapid cell proliferation can occur (Kenneth, 2000). Several studies have demonstrated that lipid peroxidation is decreased significantly in tumor cells and tissues compared with that in corresponding normal cells and tissues (Lash, 1966; Gravela et al., 1975; Bartoli & Galeotti, 1979; Cheeseman et al., 1986). The primary mechanism by which cancer cells prevent lipid peroxidation consists of a marked increase, compared with normal cells, in vitamin E compared with the peroxidizable moieties (methylene groups) of the polyunsaturated fatty acids in their biological membranes (Cheeseman et al., 1986; Cheeseman et al., 1988). Compared with normal cells, tumor cells also have relatively low levels of the components of the NADPH-cytochrome P-450 electron transport chain, which results in fewer favorable conditions for the initiation and propagation of lipid peroxidation (Gravela et al., 1975; Player et al., 1979; Cheeseman et al., 1986, 1988). According to our results, total SOD activity showed significant decrease in brain tumor tissues. Mn-SOD is an essential primary antioxidant enzyme that converts superoxide radical to hydrogen peroxide and molecular oxygen within the mitochondrial matrix. Cu-Zn-SOD is found predominantly in the cytoplasm. Tumor cells have abnormal activities of the antioxidant enzymes when compared with an appropriate normal cell control. It has been found that tumor cells are nearly always low in Mn-SOD and usually low in Cu-Zn-SOD (Shijun et al., 2000). Levels of immunoreactive Mn-SOD are lowered in tumor cells when compared with normal cell control. It appears that at least part of the reason Mn-SOD activity is low in tumor cells is because the translatable mRNA is low and thus less Mn-SOD protein is made.

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Loss of Mn-SOD activity was found characteristic of tumor tissues and not of normal tissues (Sun, 1990). Several studies (Vladimirov, 1986; Borg & Schaich, 1988) have shown that neoplastic cell capability to produce superoxide radicals is low. This suggests that superoxide production coupled with diminished amounts of Mn-SOD may be a general characteristic of tumor cells. It has also been suggested that low SOD activity in tumors is a result of lowered oxygen concentration. However, there is good evidence that the lack of oxygen or oxygen radicals is not the only reason for the low SOD activity in neoplastic cells. Tumor cells are low in SOD activity because they have lost most of the capacity to undergo induction of the enzyme. In tumor cells activities of SOD correlate with the degree of differentiation of the cells. The more differentiated tumors indicate the higher SOD activity. Low SOD activity is consistent with the loss of mitochondria, while the increase in SOD activity precedes a morphological different ion. In contrast to what is seen in normal cells the SOD activity in tumor cells is inversely related to a growth rate and directly related to the degree of differentiation. The degree of SOD activity loss is inversely related to the growth rate of the tumors (Southorn & Powis 1988). In this study CAT activity showed little difference between normal and tumor brain tissues. Sun (1990), Sato et al. (1992), and Jaruga & Olinski (1994), reported that more of the tumor tissues were low in catalase activity. It is possible CAT activity to be sufficient for inactivation of ROS in brain tumor tissue. We propose that most of the superoxide obtained in a brain tumor cell dismutates into hydrogen peroxide, which is decomposed from a catalase.

Conclusion Cancer in humans and animals is a multi-step disease process. The complex series of cellular and molecular changes that occur through the development of cancer can be mediated by a diversity of endogenous and environmental stimuli. Free radicals may contribute widely to cancer development. The oxidant-antioxidant balance is thought to be important in the initiation, promotion, and therapy resistance of cancer. The SOD activity should be a very important factor influencing the choice of cytotoxic treatment according to Yoshii et al. (1999), since tumor cells in human gliomas with low SOD activity are effective for radiotherapy and anticancer drugs associated with oxidative stress. Other authors (Yoshikawa et al., 1995; Sawa et al., 2000; Fang et al., 2002) reported experimental models for a novel cancer chemotherapy, based on enzymatically produced oxygen radicals on tumor cells with low SOD and CAT activity. Huang et al. (2000) reported that superoxide dismutase may be a target for the selective killing of cancer cells. The active O2- production and low SOD activity in cancer cells may render the malignant cells highly dependent on SOD for survival and sensitive to inhibition of SOD. Their results indicate that tar-

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geting SOD may be a promising approach to the selective killing of cancer cells, and that mechanism-based combinations of SOD inhibitors with free-radical-producing agents may have clinical applications.

Acknowledgements We are most grateful to Dr Shomov, Dr Najdenov and Dr Stalev for their contributions.

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