termed oxidative stress (Kovacic and Jacintho, 2001; Valko et al., 2001; Ridnour et al., 2005). This occurs in biological systems when there is an

Transcription

1 Chapter 4: Protective effect of cultured mycelium of Volvariella volvacea against in vitro and in vivo oxidative damage

2 4.1. Introduction The harmful effect of free radicals causing potential biological damage is termed oxidative stress (Kovacic and Jacintho, 2001; Valko et al., 2001; Ridnour et al., 2005). This occurs in biological systems when there is an overproduction of ROS/RNS on one side and a deficiency of antioxidants on the other. In other words, oxidative stress results from the metabolic reactions that use O2 and represents a disturbance in the equilibrium status of prooxidant/antioxidant reactions in living organisms. The excess ROS can damage cellular lipids, proteins or DNA, inhibiting their normal function. Because of this, oxidative stress has been implicated in a number of human diseases as well as in the ageing process. Free radicals are being constantly generated in our body by a large number of reactions involving either endogenous systems, exogenous xenobiotics or during exposure to physicochemical agents and pathological conditions. There are many exogenous sources that generate ROS or RNS. These include redox cycling of xenobiotics, exposure to ionizing radiations such as X-rays and -rays, besides visible light or UV in the presence of oxygen and a photosensitizer, which can be in the form of cytochromes, porphyrins, riboflavins, tetracycline, phenothiazines, chemical toxicants or air pollutants (Devasagayam and Kesavan, 1996). Free radicals or reactive oxygen and nitrogen species such as superoxide, hydrogen peroxides, hydroxy radicals, nitric oxide as well as peroxynitrite radicals are involved in several diseases and in a number of pathological conditions. The existence of radical in free state is a relatively recent and their relevance in biology was discovered still recently. Overwhelming evidences indicate that oxidative stress leads to cell and tissue injury. They are involved in the regulation of signal transduction and gene expression, activation of receptors and nuclear transcription factor, antimicrobial, cytotoxic activity, 56

3 immune system cells, neutrophils and macrophages as well as in ageing and degenerative diseases. They also cause extensive DNA damage and mutation leading to carcinogenesis and hence are implicated in cancer and tumour promotion. Hence oxidative damage has been implicated in many major human health problems; antioxidants have attracted great attention in recent years. Antioxidants act as major defense against radical mediated toxicity by protecting the damages caused by free radicals or ROS. They have significant role in the prevention of free radical mediated pathological conditions. Antioxidants help to prevent oxidation and also help to increase immune protection. In recent years, the use of some synthetic antioxidants has been restricted because of their possible toxic and carcinogenic effects (Gazzani et al., 1998; Frankel et al., 1995). Several epidemiological findings demonstrate that increased uptake of dietary antioxidants might contribute to chemoprevention of some human cancer (Wasser and Weis, 1999a). This concern has resulted in an increased interest in the investigation of the effectiveness of naturally occurring compounds with antioxidant properties (Duh et al., 1992). Foods rich in antioxidants have been shown to play an essential role in the prevention of cardiovascular diseases (Dragsted et al., 1993), neurodegenerative diseases, Parkinson s and Ahlzeimer s diseases (Joseph et al., 1999), inflammation and problems caused by cell and cutaneous ageing (Ames et al., 1993; Gaulejac et al., 1999; Cao and Prior, 2000). Thus the natural antioxidants present in foods and other biological materials have attracted considerable interest because of their presumed safety and potential nutritional and therapeutic effects (Ames et al., 1993). Ionizing radiation and chemotherapeutic drugs are the most important source of oxidative stress. There has been extensive research on radio protective 57

4 compounds during the past 50 years because of the relevance of these compounds in military, clinical and industrial applications. They are capable to reduce the oxidative stress produced during exposure to physicochemical agents. The development of effective radioprotectors and radiorecovery drugs is of great importance in view of their potential application during both planned radiation exposure (e.g. radiotherapy) and unplanned radiation exposure (e.g. in the nuclear industry, space exploration, natural background radiation emanating from the earth or other sources) (Arora and Goel, 2000; Bump and Malaker, 1998; Coleman et al., 2003; Moulder, 2002; Nair et al., 2001). These drugs are also likely to be useful in nuclear warfare to provide protection to personnel (Giambaressi and Jacobs, 1987). However, no ideal, safe synthetic radioprotectors are available to date, so the search for alternative sources has been ongoing for several decades. Damage due to ionizing radiations are mainly occurs through free radical mediated mechanism. Several mushrooms have been used to treat free radical-mediated ailments for centuries and, therefore, it is logical to expect that such mushrooms may also render some protection against radiation damage. A systematic screening approach may helps to identify potential new candidate drugs from mushroom sources, for mitigation of radiation injury. Recent investigations have demonstrated that a number of mushrooms possess antioxidant and radioprotective activities (Chang, 1996; Lakshmi et al., 2004; Pillai et al., 2006). Mushroom mycelia are ideal source for developing novel pharmaceutical agents derived from mushrooms. Several novel medicinal substances have been developed from mushroom mycelium produced by biotechnological processes. Paddy Straw mushroom is an excellently edible mushroom extensively cultivated in South-East Asia. The genus Volvariella (paddy straw mushroom) 58

5 comprised a group of several species, which can be found growing on a variety of substrates in tropical and sub-tropical regions. Volvariella volvacea (Bull.) Singer is probably the best known species, as it has been traditionally cultivated in Southeast Asia since the 18th century (Chang, 1977). Through the present study we investigated in vitro antioxidant activities of cultured mycelium of paddy straw mushroom including its ABTS radical scavenging activity and oxygen radical scavenging capacity. The protective effect of V. volvacea against radiation and chemical induced membrane damage was analysed by means of lipid peroxidation. The ability of V. volvacea to defend the in vivo radiation induced oxidative stress was also analyased Materials and methods Preparation of the extract Aqueous-ethanolic extract of V. volvacea mycelium was prepared as described in section For in vitro antioxidant studies the extract was dissolved in distilled water and for pulse radiolysis studies was dissolved in Millipore water. All experiments were carried out with different concentrations of the extract in order to evaluate whether an increase in the concentration influences the antioxidant activity Animals Three months old male Wistar rats were used in the study of membrane damage and male Swiss albino mice (weighing 25 2gm) were used for the study of radiation induced oxidative stress in vivo Assay for in vitro antioxidant activity ABTS radical scavenging assay In the ABTS radical scavenging assay, the scavenging activities of extract was determined using Long and Halliwell (2001) protocol with some 59

6 modifications. The assay was carried out by interacting the extract with a model stable-free radical derived from 2, 2 - azinobis (3-ethylbenzothiazolin- 6-sulphonic acid) (ABTS). To a stock solution of ABTS (7mM) prepared in water, ammonium persulphate (2.45mM final concentration) was added and the solutions were allowed to react for duration of more than 16 hrs in the dark at room temperature. ABTS and per sulphate react with each other leading to the incomplete oxidation of ABTS to generate ABTS radical. The ABTS radical solution was diluted to an absorbance of 0.75 at 734nm in PBS and 10 l of different concentrations of the extract (1-50 μg/ml) were added to 1ml of ABTS radical solution. Absorbance was measured by a spectrophotometer at 6 minutes after initial mixing, using PBS as reference ABTS radical scavenging by pulse radiolysis ABTS radical scavenging ability of aqueous-ethanolic extract of V. volacea mycelium was also evaluated by pulse radiolysis technique using a linear accelerator. In pulse radiolysis, significant concentrations of transient species are generated in a very short interval of time by giving a high energy-intense electron pulse to the sample. The transient species are monitored by following the changes in optical absorption with respect to time. The pulse radiolysis systems with 7 MeV electrons used in the present study. The dosimetry was carried out using an air-saturated aqueous solution containing 5 x 10-2 mol dm -3 KSCN (G = 23,889 dm 3 mol -1 cm -1 per 100 ev at 500 nm). The width of the electron pulse was 500 ns and the dose was 20 Gy per pulse (Adhikari and Mukherjee, 2002). Pulse radiolysis study of ABTS radical involves scavenging of primary radicals (. OH) by azide (N3 - ) producing azidyl radical (N3. ), which in turn generates ABTS radical when ABTS is present in solution (Scotl et al., 1993; Lakshmi et al., 2004). The standard pattern of decay with ascorbic acid having three different concentrations of 5, 10 and 20 µg/ml showed typical 60

7 concentrated-dependent curves. The ascorbic acid equivalent was computed by extrapolating the results with the standard graph Oxygen Radical Absorbance Capacity (ORAC) assay In ORAC assay, the chemical damage to -phycoerythrin (PE) through the decrease in its fluorescence emission was detected. The fluorescence of PE is highly sensitive to conformational and chemical integrity of protein. Loss of PE fluorescence in presence of RS (reactive species) is an index of oxidative damage of the protein. The inhibition of RS by antioxidant reflects in protection against loss of PE fluorescence in this assay as a measure of antioxidant capacity against RS. Reaction mixture (200 µl) contained 175 µl of 75 mm phosphate buffer (ph 7.0), 10 l of PE (0.5 mg in 7.3 ml buffer), 10 l of the extract and 5 l of 160 mm AAPH. For standard assay, 50 M Trolox was added in place of the extract. The fluorescence was recorded every 5 min till the last reading becomes less than 5% of first reading ( Exit- 540 nm and Emis- 565 nm) (Cao and Prior, 1999) Systems used to induce oxidative damage a. Ionizing radiation ( 60 Cobalt - gamma source) Oxidative damage was induced in biological systems by exposure to -rays from a 60 Co source. All in vivo and in vitro gamma irradiations were carried out using gamma irradiator, blood irradiator and junior theratron teletherapy machine. Mitochondria and microsomes from rat liver were exposed to 450Gy gamma radiation at a dose rate of 60Gy/min. For whole body irradiation, animals were placed in perspex covered boxes and were exposed to 2Gy at a dose rate of 3.1Gy/min. 61

8 b. AAPH for peroxyl radicals Peroxyl radical-induced damage was observed using 2, 2 -azobis (2- amidinopropane) dihydrochloride (AAPH). AAPH is an azo compound, which on thermal activation produces azo radicals. These then react with molecular oxygen and produce peroxyl radicals. In our system, membrane damage was induced by incubating the mitochondria and microsomes with AAPH (final concentration 10 mm) Protection against membrane damage by V. volvacea Isolation of mitochondria and microsomes Three months old male Wistar rats were killed by cervical dislocation and the livers were quickly removed, cleaned and washed with pre chilled isolation medium consisting of 0.25 mm sucrose and 10 mm tris HCl, (PH 7.4) (Shetty, 2002). A 10% liver homogenate was made in the isolation medium. The homogenate was centrifuged at 750 xg for 10 minutes to remove nuclear fraction and debris. The supernatant was centrifuged at 10,000 xg for 10 minutes to pellet out mitochondrial fraction. After separating the mitochondrial fraction, the supernatant was ultra centrifuged at 1, 00,000 xg for 45 minutes to obtain microsomal pellet. Both mitochondrial and microsomal fractions were washed twice with ice cold 5mM potassium phosphate buffer and suspended in the same buffer to give a mg protein /ml. Aliquots of mitochondria and microsomes were immediately frozen in liquid N2 and stored at C to be used within two months for the assays. The protein concentration was determined by Bradford s method (Bradford, 1976) (section 3.2.5) Induction of membrane damage Membrane damage was induced either by exposure to -rays from a 60 Co source or by reaction with AAPH. For radiation induced damage, 62

9 mitochondria and microsomes from rat liver treated with or without the extract (50, 100, 250, 500 μg/ml) in vitro were exposed to 450Gy gamma radiation at a dose rate of 60Gy/min. For AAPH induced damage, mitochondria and microsomes (final concentration 0.2 mg/ml) with or without the extract (50, 100, 250, 500 μg/ml) were incubated with AAPH (final concentration 10 mm) at 37 C for 30 min in a shaker water bath with continuous bubbling of oxygen (Devasagayam et al., 1996). The reaction mixture after the treatment with radiation or AAPH was used to assess the damage produced Parameters used to assess membrane damage Lipid peroxidation Lipid peroxidation (LP) is a free radical mediated chain reaction, which is a highly damaging process. It has been defined as an oxidative deterioration of polyunsaturated fatty acids (PUFA). Various products of LP include conjugated dienes, lipid hydroperoxides, aldehydes and ketones. These are fairly stable molecules, at physiological temperatures, and can be measured using different assays a. Lipid hydroperoxide Out of various techniques used for detection and quantitation of hydro peroxides, the xylenol orange assay or FOX II method (Nourooz- Zadeh, 1996) is found to be very successful to measure LOOH concentration in cell defined systems. This is a simple and sensitive spectrophotometric procedure, which detects hydroperoxides present in lipid phase such as in lipoproteins, membranes and fats. Hydroperoxides oxidize Fe 2+ to Fe 3+ under acidic conditions (Jiang et al., 1992). The resultant Fe 3+ can be determined using ferric-sensitive dyes as an indirect measure of hydroperoxide concentration. The dye, xylenol orange, complexes with an equal molar concentration of Fe 3+ 63

10 to produce a blue-purple colour complex with an apparent extinction coefficient of 1.5 x 10 4 M -1 cm -1 at 560 nm. Hence the method is named FOX (Ferrous Oxidation in Xylenol orange). In presence of ROOH, yield of Fe 3+ -XO complex is higher than 1:1 because of chain oxidation of Fe 2+ by ROOH. In this instance, alkoxyl radicals generated in the ferrous oxidation step react rapidly with native lipid, generating further hydroperoxide in a chain reaction. Undesirable chain oxidation is prevented by lipid-soluble chain breaking antioxidant, BHT (butylated-hydroxy toluene), which presumably repairs alkyl radicals produced by reaction of alkoxyl radicals with unsaturated lipids. FOX II reagent was further adapted by the addition of methanol (90% v/v) in order to solubilize the lipids and BHT. Concentration of LOOH is then calculated with the help of standard graph using H2O2. FOX II gives for hydroperoxides as 4.46 x 10 4 M -1 cm -1. FOX II reagent contains solution A made up of 98 mg ammonium ferrous sulfate, 100 ml 250 mm H2SO4 and 79 mg xylenol orange, and solution B comprising of 969 mg BHT in 900 ml methanol, mixed in 1:9 ratio. It can be stored in dark at 4 C for 1 month. 875 l of FOX II reagent was added to 125 l of reaction mixture and incubated at 37 C for 30 min. It was centrifuged at 10,000 g for 15 min at 20 C and the absorbance was measured at 560 nm. For calculations, a standard graph of OD against concentration of H2O2 was plotted b. TBARS assay TBARS assay was performed by standard method using malondialdehyde equivalents derived from tetraethoxypropane. Malondialdehyde and other aldehydes have been identified as products of lipid peroxidation that react with thiobarbituric acid (TBA) to give a pink colored species that absorbs at 532 nm. It is one of the oldest and most frequently used tests for measuring 64

11 the peroxidation of fatty acids, membranes and foods (Gutteridge and Quinlan, 1983; Hartman, 1983). One great advantage of the TBA test is its ease of use and it works on biological material. The method involved heating of biological samples with TBA reagent (TBA- TCA-HCl-EDTA) for 20 min in a boiling water bath. TBA reagent contains 20% TCA, 0.5% TBA, 2.5 N HCl and 6 mm EDTA. After cooling, the solution was centrifuged at 2,000 rpm for 10 min and the precipitate obtained was removed. The absorbance of the supernatant was determined at 532 nm against a blank that contained all the reagents except the biological sample. Concentration of TBARS was then calculated with the help of standard graph using 1,1,3,3-tetraethoxypropane, as malondialdehyde equivalents. For correction of endogenous TBARS, fresh samples were boiled without radiation exposure, and values were subtracted (Sinnhuber and Yu, 1958; Hunter et al., 1963; Devasagayam et al., 1983) Protection against in vivo radiation induced oxidative damage by V. volvacea Male Swiss albino mice (weighing 25 2 gm) were divided into four groups of six animals. Group I animals were kept as normal group without any drug treatment. Group II animals serves as positive control groups that receive whole body irradiation with γ- radiation (4Gy). Group III and IV animals were administered with aqueous-ethanolic extract of V. volvacea orally, at doses 250 and 500 mg/ kg b.wt. respectively, once daily for 14 days. The group II, III and IV animals were received 4Gy gamma radiations, 24 hrs before sacrifice, as single whole body exposure. The animals were killed by cervical dislocation. Liver was removed and homogenized. The homogenate is then used to study the level of antioxidant enzymes, lipid and protein 65

12 oxidation. The protein concentration was determined by Bradford s method (Bradford, 1976) (section 3.2.5) Protection against in vivo lipid peroxidation Lipid peroxidation levels in liver homogenate after radiation exposure was estimated using TBARS method (Sinnhuber and Yu, 1958; Hunter et al., 1963; Devasagayam et al., 1983) and FOX II method (Nourooz- Zadeh, 1996) as described in section a and b Protection against in vivo protein peroxidation a. Protein carbonyls This method is based on the reaction of carbonyl groups with 2, 4- dinitrophenylhydrazine (DNPH) to form a 2,4-dinitrophenylhydrazone, which can be measured spectrophotometrically at 366 nm. The experiment was carried out using a blank set of tubes for every corresponding experimental tube. Initially both the sets were treated with 20 % TCA followed by vortexing and chilling on ice. The tubes were then centrifuged at 2,000 rpm for 20 min. The pellets obtained were treated with 2 ml of 10 mm DNPH in 2N HCl in the case of experimental set while only with 2 N HCl for the blank set. Both the sets were incubated at room temperature (RT) for 1 h with vortexing every min, followed by addition of 20% TCA. The tubes were centrifuged at 2,000 rpm for 10 min. The pellets obtained were washed thrice with a mixture of ethanol and ethyl acetate (1:1) to wash off the excess DNPH. The pellets finally obtained were dried thoroughly and dissolved in 6 M guanidine hydrochloride at 37 C for min. The absorbance was read at 366 nm. The difference in O.D. between blank and corresponding experiment gives amount of carbonyls formed (Palamanda and Kehrer, 1992). 66

13 b. Protein thiol (P-SH) Aliquots of 250 μl of tissue homogenate were mixed in 5 ml test tubes with 750 μl of 0.2 M tris buffer (ph 8.2) and 50 μl of 0.01 M 5,5 dithiobis (2- nitrobenzoic acid) (DTNB). The mixture was made up to 5 ml with 3950 μl of absolute methanol. A reagent blank (without sample) and a sample blank (without DTNB) were prepared in a similar manner. The test tubes were stoppered with rubber caps, colour was developed for 15min. The absorbance of the supernatant was read in a spectrophotometer at 412nm and the amount of total thiol (T-SH) was calculated. Aliquotes of 250 μl of the homogenates were mixed in 5ml test tubes with 200 μl distilled water and 50 μl of 50% tricholoroacetic acid. The tubes were shaken intermittently for min and centrifuged for 15min at approximately 3000 g. Supernatant (200 μl) was mixed with 400 μl of 0.4M Tris buffer (ph 8.9), 10μl DTNB was added, and the sample was shaken mechanically. The absorbance was read within 5 min of the addition of DTNB at 412nm against a reagent blank containing no homogenate, from which the amount of non protein thiol (P-SH) was estimated. The molar extinction coefficient at 412 nm was 13,100 l mol -1 c m -1 in both T-SH (total thiol) and Np- SH (non-protein thiol) procedures. The concentration of protein-bound thiol groups (P-SH) was calculated by substracting the Np-SH from T-SH (Sedlak and Lindsay, 1968) Assessment of antioxidant levels in tissue The activities of the antioxidant enzymes super oxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in liver were analysed by the method of Mc Cord and Fridovich (1969), Beers and Sizer (1952) and Hafemann et al. (1974) as described in the section , section and 67

14 section The level of reduced GSH was determined by the method of Moron et al. (1979) (section 3.2.6) Results ABTS + radical scavenging activity The extract efficiently scavenged ABTS + radicals generated by the reaction between 2, 2 - azinobis (3-ethyl benzothiazolin-6-sulphonic acid) (ABTS) and ammonium per sulphate (Fig. 4.1). The activity was found to be in a dose dependent manner and showed an IC 50 value of 18.5±0.92 µg/ml ABTS radical scavenging by pulse radiolysis The antioxidant capacities of the aqueous ethanolic extract of V. volvacea was determined by pulse radiolysis by measuring the decay of ABTS radical. Fig.4.2a represents the decay in absence and presence of standard antioxidant, ascorbic acid 5 g/4ml; 10 g/4ml and 20 g/4ml. Fig.4.2b shows the decay of the ABTS radical monitored at 734 nm after pulse radiolysis of N2O-saturated solutions containing NaN3 (50 mm), ABTS (20 mm) and aqueous ethanolic extract of V. volvacea (0.25, 0.5 and 1 mg/ml). The standard pattern of decay with ascorbic acid having three different concentrations (5, 10 and 20 g/ml) showed typical concentration-dependent curves (Fig.4.2a). Using this as standard, the relative radical scavenging abilities of the aqueous ethanolic extract of V. volvacea were estimated. The activities 0.25, 0.5 and 1 mg/ml of V. volvacea extract was equivalent to 6.85, 8.6 and 11 µg of ascorbic acid per ml respectively. (Fig.4.2b). The extract showed significant ABTS radical scavenging activity in the pulse radiolysis assay. 68

15 ORAC assay ORAC assay measures antioxidant activity against peroxyl radical induced by 2, 2 azobis (2 amidinopropane) dihydrochloride (AAPH). The inhibition of peroxyl radicals induced β-phycoerythrin oxidation and the protection by the mushroom extracts is estimated. ORAC assay has high specificity. It measures the capacity of an antioxidant to directly quench free radicals. The area under curve technique combines both inhibition percentage and the length of inhibition time of free radical action by an antioxidant into a single quantity, which makes it superior to similar methods that use either an inhibition percentage at a fixed time or a length of inhibition time at a fixed inhibition percentage (Cao et al., 1993). ORAC value for the extract was found to be 343 ± 5 micromoles of Trolox per gram of extract Protection against membrane damage by V. volvacea Phospholipids are major components in cell membranes, and their unsaturated fatty acids, because of their conjugated double bonds, are the first target of free radicals, particularly reactive oxygen species. The organic peroxides and hydroperoxides are common first products of the reaction between cellular constituents and free radicals or other reactive oxygen derivatives (Von Sonntag, 1987). Lipid peroxidation was assessed as formation of lipid hydroperoxides (LOOH) and thiobarbituric acid reactive substances (TBARS). Measurement of free MDA levels using thiobarbituric acid (TBA) test and LOOH level using xylenol orange assay are very good markers of comparative studies of oxidative stress induced by radiation and chemicals especially with membrane preparations. Data on the effect of V. volvacea on TBARS formation induced by radiation and AAPH in liver mitochondria and microsomes are given in Fig. 4.3, 4.5, 4.7 and 4.9 respectively. Exposure to radiation and AAPH cause a rise in TBARS 69

16 levels indicating an increase in lipid peroxidation. The treatment with the extract decreases the TBARS formation indicating its protection against the damage. The decrease in TBARS level was found to be in a concentration dependent manner. The higher concentration of the extract (100 µg/ml) was able to reduce TBARS formation as well as lipid peroxidation to normal levels. Exposure of rat liver mitochondria and microsome to gamma radiation and AAPH in the presence of the extract also showed a concentration dependent reduction in LOOH production. Results are expressed in Fig. 4.4, 4.6, 4.8 and 4.10 respectively. Here also the higher concentration of the drug (100 µg/ml) could effectively reduce the hydroperoxide formation to the normal levels. The results of both TBARS and LOOH assays indicate the high potential of V. volvacea extract to prevent lipid peroxidation in oxidative stress conditions Protection against in vivo radiation induced oxidative stress by V. volvacea 4.3.5a. Protection against in vivo lipid peroxidation One of the main forms of damage resulting from oxidative stress is lipo peroxidation, which is its own amplifier for the initial free radicals. Lipid hydroperoxides are the first by products of this process. The LH derived from unsaturated fatty acids and degrades in to different cytotoxic aldehydes such as 4-hydroxynonenal (4-HNE), various 2-alkenals and malondialdehyde. All these compounds are considered to play a huge role in cell injury or ageing and to trigger or to be involved in the initiation of numerous diseases such as atherosclerosis or cancer. The xylenol orange assay and TBARS assays are the simple, reliable methods that can be used successfully to measure the hydroperoxide and MDA concentrations in well defined systems for the assessment of oxidative stress in humans. 70

17 Lipids are easily peroxidised by ROS produced due to ionising radiation causing structural and functional impairment. Radiolysis of aqueous solution which produces OH. is well known stimulator of peroxidation of any lipids present in biological system. The free radical mediated lipid peroxidation is assessed in terms of TBARS and LOOH formation. The results are showed in the figure 4.11 and V. volvacea extract effectively prevented the formation of TBARS and lipid hydroperoxides induced by radiation b. Protection against in vivo protein peroxidation Radiation induces the formation of free radicals that can oxidize proteins increasing their hydrophobicity and sensitivity to proteolysis. Free radicals may react with amino acids or sulphur groups, leading to cross-linking and aggregation in proteins. Oxidation of proteins by ROS/RNS can generate a range of stable as well as reactive products such as protein hydroperoxides that can generate additional radicals particularly upon interaction with transition metal ions (Dean et al., 1997). Protection against protein oxidation was assessed as formation of protein carbonyls and depletion of protein thiols. The results are given in the figure 4.13 and It is clear from the results that V. volvacea significantly inhibit -radiation-induced in vivo protein oxidation. The amount of protein carbonyls in the homogenate was increased and there was a depletion of protein thiols after the irradiation. But the treatment with the extract decreased the formation of protein carbonyls and restored the protein thiol levels to normal suggesting the protection against radiation induced peroxidation of proteins in vivo c. Assessment of antioxidant levels in tissue Irradiation induced a significant reduction in the level of antioxidant enzymes GPx, SOD, Catalase and non enzymatic antioxidant GSH. But the administration of the extract increases the activity of these antioxidant 71

18 systems in liver. This increase in the antioxidant level may give protection against the hazardous effect of radiation induced free radicals. The results are expressed in the table Discussion During the past 30 years, the field of free radical chemistry and biology have risen from relative obscurity to become mainstream elements of biomedical investigation and pharmaceutical development. Living organisms continually battle to maintain an appropriate balance of pro oxidants and antioxidants. If the balance is always in the direction of pro oxidants, oxidative stress can arise which, under normal circumstances, is controlled by a broad range of antioxidant enzymes, proteins and antioxidants provided by the diet. Antioxidants that can scavenge ROS or neutralize damage caused by them can be beneficial in reducing this oxidative stress. The formation of ROS is prevented by preventive antioxidants like superoxide dismutase (SOD) and catalase. Interception of free radicals or other reactive species is mainly by radical scavenging and is caused by various antioxidants like vitamins C and E, glutathione, other thiol compounds, carotenoids, flavonoids, etc. While at the repair and reconstitution level, mainly repair enzymes are involved (Halliwell and Aruoma, 1993; Sies, 1996). Breakdown or deficiency of this defence against ROS can lead to damage which has been strongly associated with a wide variety of chronic diseases including Alzheimers, autoimmune disease, cancer, cardio vascular disease, diabetics, multiple sclerosis, arthritis, neurodegenerative disorders and in the process of ageing. Presently the focus is on developing natural compounds as antioxidants that act at different levels and that can be possibly used to reduce damage caused by oxidative stress. Due to this ability, natural antioxidants have potential beneficial roles in various aspects of human health. Antioxidants are 72

19 abundant in fruits and vegetables as well as on other foods including nuts, grains and some meats, poultry and fish. The major food derived antioxidants include beta- carotene, leutin, lycopene, selenium, vitamin A, vitamin C and vitamin E. Antioxidants are widely used ingredients in dietary supplements for maintaining health, prevention and cure of various diseases. Edible mushrooms are excellent source of dietary antioxidants. Since human beings daily consume many of these mushrooms with no known side effects, they may be more easily and safely implemented for human use than other synthetic antioxidant chemicals. The present investigation clearly indicated the ability of V. volvacea extract to scavenge ABTS + radical and its possible mechanism through pulse radiolysis studies. The large ORAC value of the extract is the direct measure of its high antioxidant power. The ORAC methodology is arguably the most accepted and accurate indicator of antioxidant status, mainly because it is based on measurement of fluorescence rather than absorbance. This increases sensitivity and so permits a much lower molar ratio of antioxidant sample: reagents, thus minimising the likelihood of cross reactions between sample and reagent. In addition, the ORAC methodology measures total radical scavenging ability, since it is unique in that it takes reaction to completion, permitting a calculation of total area under the curve. The mitochondrion lies at the heart of cell life and cell death. Studies on mitochondria, the power houses of the cell has achieved great importance now a day because of its central role in energy production and programmed cell death. The enzyme complexes for respiratory chain and receptors for proper functioning of mitochondria lies in its membrane. Mitochondria are important source of ROS generation. Being an important source of ROS generation, mitochondria also form major targets of oxidative damage. Such 73

20 damage can lead to reduced cellular functions resulting from loss of energy generation and in cell death. Damage to mitochondria inevitably leads to disease. Mitochondrial dysfunction has been implicated in all the major neurodegenerative diseases Parkinson s, Alzheimer s, motor neuron disease, possibly in multiple sclerosis, cardiomyopathy, multi organ system failure in sepsis, the process of ageing and age related disease. The mechanisms involved in many human diseases such as hepatotoxicities, hepatocarcinogenesis, diabetes, malaria, acute myocardial infarction and skin cancer include lipid peroxidation as a main source of membrane damage (Yoshikawa et al., 2000). Lipid peroxidation can be a major contributor to the loss of cell function under oxidative stress situations. For example, peroxidation in microsomal membranes has been shown to lead to calcium release and uncontrolled activation of calcium dependent proteases and lipases and peroxidation and permeabilization of mitochondrial membranes can induce disruption of cellular energetics. The overall effects of lipid peroxidation are to decrease membrane fluidity, make it easier for phospholipids to exchange between the two monolayers, increase the leakiness of the membrane bilayer to substances that do not normally cross it other than through specific channels and inactivate membrane bound enzymes. Cross linking of membrane proteins decreases their lateral and rotational mobility. Lipid peroxidation is particularly harmful in mitochondria, that contain cardiolipin as a major component of inner mitochondrial membrane, and which required for the activity of cytochrome oxidase. Peroxidation products in mitochondrial membranes were significantly greater than in microsomal membrane. The major damages caused by radiation induced oxidative stress include tissue protein and lipid peroxidation. V. volvacea showed significant in vivo 74

21 radioprotection as analysed by measuring lipid and protein peroxidation after the administration of the extract for 14 days and a single whole body radiation treatment of 4 Gy to the Swiss albino mice. The extract was also found to maintain the antioxidant status of tissues which is considered to be the major mechanism against the radiation induced oxidative stress. In conclusion, the present study suggests that aqueous ethanolic extract of V. volvacea can scavenge most of the radicals of biological interest and prevents damage caused from radiation and chemical induced oxidative stress in vitro and in vivo and hence can be a valuable antioxidant with possible potential applications in health. 75

22 % of Activity Figure 4.1. ABTS radical scavenging activity of V. volvacea Concentration of the extract (µg/ml) Data represented as mean S.D., (n=3).

23 Figure 4.2. ABTS radical scavenging activity by pulse radiolysis a. Standard curve for the reaction of ascorbic acid with ABTS radical ABTS + Asc 734 nm only ABTS Absorbance ABTS + 5 g/ml Asc ABTS + 10 g/ml Asc ABTS + 20 g/ml Asc time, s b. Decay of ABTS radical in presence of V. volvacea extract nm a 0.15 Absorbance 0.10 b c 0.05 a: Only ABTS radical b: ABTS radical mg/ml v v c: ABTS radical + 0.5mg/ml v v d: ABTS radical + 1mg/ml v v d Time, s

24 μm LOOH/mg protein nmoles TBARS/mg protein Figure 4.3. Protection against radiation-induced lipid peroxidation in mitochondria in terms of TBARS Control Damage VV 50 VV 100 VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to damage. Figure 4.4. Protection against radiation-induced lipid peroxidation in mitochondria in terms of LOOH Control Damage VV 50 VV 100 VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to damage.

25 μm LOOH/mg protein nmoles TBARS/mg protein Figure 4.5. Protection against radiation-induced lipid peroxidation in microsomes in terms of TBARS Control Damage VV 50 VV 100 VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to damage. Figure 4.6. Protection against radiation-induced lipid peroxidation in microsomes in terms of LOOH Control Damage VV 50 VV 100 VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to damage.

26 μm LOOH/mg protein nmoles TBARS/mg protein Fig.4.7. Protection against AAPH-induced lipid peroxidation in mitochondria in terms of TBARS Control Damage VV 50 VV 100 VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to damage. Fig.4.8. Protection against AAPH-induced lipid peroxidation in mitochondria in terms of LOOH Control Damage VV 50 VV 100 VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to damage.

27 μm LOOH/mg protein nmoles TBARS/mg protein Fig.4.9. Protection against AAPH-induced lipid peroxidation in microsomes in terms of TBARS Control Damage VV 50 VV 100 VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to damage. Fig Protection against AAPH-induced lipid peroxidation in microsomes in terms of LOOH Control Damage VV 50 VV 100 VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to damage.

28 μm LOOH / mg protein nmoles TBARS / mg protein Figure Effect of V. volvacea on in vivo lipid peroxidation in terms of TBARS Normal Irradiated Control VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to irradiated control. Figure Effect of V. volvacea on in vivo lipid peroxidation in terms of LOOH Normal Irradiated Control VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to irradiated control.

29 nmol mg-1 protein nmol mg-1 protein Figure Effect of V. volvacea on protein carbonyl levels after irradiation Normal Irradiated Control VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to irradiated control. Figure Effect of V. volvacea on total thiol and protein thiol levels after irradiation T-SH P-SH 10 0 Normal Irradiated Control VV 250 VV 500 Values are mean ± S.D, n=3., P<0.01 with respect to irradiated control.

30 Table 4.1. Effect of V. volvacea on tissue antioxidant system Group Gpx (U/mg protein) SOD (U/mg protein) CAT (U/mg Protein) GSH (nmol/mg protein) Normal 4.20 ± ± ± ± 0.65 Irradiated control (4Gy) V. volcacea (250mg/kg b.wt.) V. volvacea (500mg/kg b.wt.) 2.29 ± ± ± ± ± 0.30 * 2.28 ± ± 0.43 * ± 0.81 * 4.72 ± 0.42 * 2.93 ± 0.17 * ± 0.76 * ± 0.43 * Values are mean ± S.D, (n=6)., *P < P<0.01 with respect to irradiated control.