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Oxidative damage induced by the fullerene C 60 on photosensitization in rat liver microsomes

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We have examined the ability of a commonly used fullerene, C60, to induce oxidative damage on photosensitization using rat liver microsomes as model membranes. When C60 was incorporated into rat liver microsomes in the form of its cyclodextrin
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  Chemico-Biological Interactions 114 (1998) 145–159 Oxidative damage induced by the fullerene C 60  onphotosensitization in rat liver microsomes Jayashree P. Kamat  a , Thomas P.A. Devasagayam  a, *,K.I. Priyadarsini  b , Hari Mohan  b , Jai P. Mittal  b a Cell Biology Di   ision ,  Bhabha Atomic Research Centre ,  Mumbai  - 400085  ,  India b Chemistry Group ,  Bhabha Atomic Research Centre ,  Mumbai  - 400085  ,  India Received 15 December 1997; received in revised form 7 April 1998; accepted 8 April 1998 Abstract We have examined the ability of a commonly used fullerene, C 60 , to induce oxidativedamage on photosensitization using rat liver microsomes as model membranes. When C 60 was incorporated into rat liver microsomes in the form of its cyclodextrin complex andexposed to UV or visible light, it induced significant oxidative damage in terms of (1) lipidperoxidation as assayed by thiobarbituric acid reactive substances (TBARS), lipid hydroper-oxides and conjugated dienes, and (2) damage to proteins as assessed by protein carbonylsand loss of the membrane-bound enzymes. The oxidative damage induced was both time-and concentration-dependent. C 60  plus light-induced lipid peroxidation was significantlyinhibited by the quenchers of singlet oxygen ( 1 O 2 ),   -carotene and sodium azide, anddeuteration of the buffer-enhanced peroxidation. These observations indicate that C 60  is anefficient inducer of peroxidation and is predominantly due to  1 O 2 . Biological antioxidantssuch as glutathione, ascorbic acid and   -tocopherol significantly differ in their ability toinhibit peroxidation induced by C 60 . Our studies, hence, indicate that C 60 , on photosensitiza-tion, can induce significant lipid peroxidation and other forms of oxidative damage inbiological membranes and that this phenomenon can be greatly modulated by endogenousantioxidants and scavengers of reactive oxygen species. © 1998 Elsevier Science Ireland Ltd.All rights reserved. * Corresponding author. Fax:  + 91 22 5560750.0009-2797 / 98 / $19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII   S0009-2797(98)00047-7  J  . P .  Kamat et al  .  /   Chemico - Biological Interactions  114 (1998) 145–159  146 Keywords :   C 60 ; Lipid peroxidation; Membrane damage; Photoexcitation; Protein oxidation;Rat liver microsomes; Singlet oxygen 1. Introduction Fullerenes have attracted considerable attention in the last few years. Due totheir unique structures and properties fullerenes exhibit widely differing activities.There are many investigations on the physical and chemical characteristics of fullerenes, including photophysical and photochemical properties [1–5]. Only re-cently the studies of biological effects have been started due to the preparation andavailability of suitable derivatives [6–13]. Because fullerenes, as exemplified by C 60 ,are being produced in macroscopic amounts [14] there is a need to study theirbiological effects.Fullerenes have been shown to be present in sooty flames [15,16]. Topicalapplication of fullerenes in benzene to mice along with the tumor promoter phorbolester resulted in the formation of benign skin tumors but did not induce eithermalignant or benign tumors if administered along with polycyclic aromatic hydro-carbons [14]. C 60 , dissolved in polyvinyl pyrrolidone, was mutagenic for somestrains of   Salmonella  in the presence of rat liver microsomes and irradiated byvisible light. The observed mutagenicity was also shown to be due to oxidizedphospholipids in rat liver microsomes [9]. Free and water-insoluble C 60  as aphotosensitizer can be used to mediate the inactivation of enveloped viruses [6].Fullerenes are also capable of being incorporated into phosphatidylcholine lipo-somes [8] and in artificial lipid membranes [17]. Further studies have shown that C 60 can be oxidatively modified by the drug-metabolizing cytochrome P-450 systemforming epoxides, which may have other biological functions [18]. The above pointsillustrate the biological significance of free fullerenes.Photosensitization involving UV or visible light, sensitizer and oxygen is apotentially damaging reaction in biological systems. It generates a number of reactive oxygen species and excited triplets capable of damaging different crucialbiomolecules [19–22]. Polyunsaturated fatty acids, present in cellular membranes,are especially prone to damage by these reactive species generated during photosen-sitization, and the resulting lipid peroxidation can have serious consequences to thetissues and the organism [20,23–25]. Lipid peroxidation plays a major role inmediating oxidative damage in biological systems. Among the various speciesgenerated during photosensitization, the peroxyl radical (ROO  ), hydroxyl radical(  OH) and singlet oxygen ( 1 O 2 ) are capable of inducing lipid peroxidation [26].Recent studies show that, besides peroxidation of membrane lipids, oxidation of proteins also is a highly damaging event capable of altering the integrity of cellularcomponents [27–29].High yield of C 60  triplets on photoexcitation, its high reactivity with oxygen andinertness to photooxidative destruction suggests it to be a potential generator of singlet oxygen ( 1 O 2 ) [30]. Singlet oxygen is capable of damaging crucial biological  J  . P .  Kamat et al  .  /   Chemico - Biological Interactions  114 (1998) 145–159   147 molecules such as DNA, lipids and proteins [19–23,31]. Studies have shown thatfullerenes can (1) mediate electron transport across lipid bilayers [32], (2) inactivateviral envelopes [6], and (3) modulate immune effects [33]. Recently Sera et al. [9]have shown that fullerene-induced mutagenicity by rat liver microsomes is mediatedthrough formation of lipid peroxides. However, the mechanisms involved in lipidperoxidation and other aspects of membrane damage induced by C 60  have not beenexamined in detail. The ability of natural compounds to protect against damageinduced by fullerenes also has not been studied earlier. Hence, to further under-stand the mechanisms of damage and its modulation, as relevant to the biologicaleffects of fullerenes, we have assessed the oxidative damage to lipids and proteinsinduced by C 60  using rat liver microsomes as model systems. The role of   1 O 2  in suchdamage and its possible prevention by natural antioxidants were also assessed. 2. Materials and methods 2.1.  Chemicals Adenosine triphosphate, ascorbic acid, ethylenediaminetetraacetic acid (EDTA),glutathione, glucose 6-phosphate, mannitol, nicotinamide, superoxide dismutase,tetraethoxypropane, 2-thiobarbituric acid, tryptophan,   -tocopherol and   -cyclo-dextrin were obtained from Sigma (St. Louis, MO). Sodium azide was from BDH(UK). Catalase was purchased from Boehringer Mannheim (Germany).   -Carotenewas a gift from Hoffman LaRoche and lipoic acid from Asta Pharma. Deuteriumoxide ( 2 H 2 O; 99.8%) was obtained from the Heavy Water Division of our ResearchCentre. C 60  was obtained from SES Research Corporation (USA) and used withoutfurther purification. Other chemicals used in our studies were of analytical gradefrom reputed manufacturers. 2.2.  Preparation of microsomes and incorporation of C  60  Female Wistar rats 3 months old and weighing approximately 270  30 g wereused for our studies. Hepatic microsomes were prepared as described earlier [34].The microsomal pellet obtained was washed thrice with 50 mM sodium phosphatebuffer, pH 7.4 (buffer A). A portion of the resulting sediment was suspended inbuffer A. The remaining part was suspended in 50 mM sodium phosphate buffer in 2 H 2 O, pD 7.4 (buffer B). These buffers were treated with Chelex-100 (Bio-RadCorporation) for several hours to remove traces of metal ions. For incorporatingC 60 , a solution of C 60  in hexane or 100   g of cyclodextrin–C 60  complex mg − 1 protein, as prepared earlier [35], was added to the microsomal pellet, homogenised,diluted to 11 ml with buffer A and resedimented at 105000 ×  g   for 1 h. Protein wasestimated and microsomes were resuspended at a concentration of 5 mg proteinml − 1 in buffer A or B, distributed as aliquots frozen in liquid nitrogen and storedat  − 20°C. For studying the spectra of microsomes, 50   g protein and 12.5   gC 60  –cyclodextrin complex were used.  J  . P .  Kamat et al  .  /   Chemico - Biological Interactions  114 (1998) 145–159  148 2.3.  Exposing microsomes to photoexcitation Microsomes (final concentration 0.5 mg protein ml − 1 ) were suspended in bufferB (or buffer A for experiments to see the enhancing effect of deuteration) and wereexposed to a Hg lamp coated with phosphorus, emitting in the wavelength regionof 330–370 nm (Rayonet Photochemical Reactor; The Southern New EnglandUltraviolet Company, USA) or 400–700 nm (300 W, tungsten lamp) and a constantbubbling of oxygen or nitrogen. As determined by potassium ferrioxalate actinome-ter, the photon flux of the UV lamp was 1.0 × 10 17 photons ml − 1 min − 1 , and withtungsten lamp it was 7.3 × 10 15 photons ml − 1 min − 1 [36]. Vitamin E and lipoicacid were added in low volumes of alcohol (10   l; final concentration 0.25%) and  -carotene in tetrahydrofuran (10   l; final concentration 0.25%). The other antiox-idants, such as glutathione, nicotinamide and vitamin C, were water-soluble andused in buffer at a final concentration of 10 mM. 2.4.  Addition of ROS inhibitors The inhibitors of reactive oxygen species (ROS) used in our experiments werecatalase (inhibitor of H 2 O 2 ; 400 units per assay), mannitol (scavenger of    OH; 10mM), superoxide dismutase (scavenger of O −  2  ; 1200 units per assay), sodium azide(inhibitor of   1 O 2 ; 10 mM) and   -carotene (inhibitor of   1 O 2 ; 100   M). 2.5.  Assay of lipid peroxidation ,  protein oxidation and enzymes After photosensitization, the products of oxidative damage were estimated asthiobarbituric acid reactive substances (TBARS) using tetraethoxypropane as stan-dard [34], lipid hydroperoxide (LOOH) and conjugated dienes [37]. Lipid hydroper-oxide produced upon peroxidative damage was estimated by microiodometric assay[34,38]. To the lipid residue obtained by drying 0.5 mg of peroxidized lipid samplewas added 1 ml acetic acid–chloroform mixture (3:2, v / v) and 50   l of potassiumiodide (1.2 g ml − 1 deaerated water). After 5 min in the dark, the solution wasmixed with 3 ml of 1% cadmium acetate solution (to minimize autotoxidation of unreacted iodine) and centrifuged. The aqueous layer was removed and theabsorbance was recorded at 353 nm against a blank containing the completemixture except the lipid. As cited in our earlier reports standard assays were usedfor the estimations of glucose-6-phosphatase, total adenosine triphosphatase [39],and protein carbonyls [27]. 2.6.  Determination of singlet oxygen by histidine destruction assay The singlet oxygen generated in the photosensitizing system was measured byoxidation of   L -histidine followed by a spectrophotometric asssay. Destruction of histidine, as a measure of specific reaction with  1 O 2 , was determined by using aconcentration of 32.2 mM as a function of sensitizer concentration. This assay candetect the amount of histidine remaining after exposure to singlet oxygen, in therange of 10–1000   g [40,41].  J  . P .  Kamat et al  .  /   Chemico - Biological Interactions  114 (1998) 145–159   149 The experiments were carried out in quadruplicate and statistical significance wasdetermined by Student’s  t -test. 3. Results 3.1.  Spectral studies C 60 , either as a solid or as a solution in hexane, could not be incorporated intomicrosomes efficiently. But cyclodextrin–C 60  complex could be effectively intro-duced. The optical absorption spectrum of microsomes treated with cyclodextrin– C 60  exhibited broad absorption bands at 270 and 350 nm (Fig. 1), which suggestsits incorporation into the microsomes. However, the exact nature of the bondingwith the microsomes is not clear. Fourier transform infrared studies, carried out tounderstand the nature of the bonding between C 60  and microsomes, were notsuccessful as the characteristic bands of C 60  were masked by absorption bands of the microsomes. Fig. 1. Differential spectrum of rat liver microsomes containing cyclodextrin–C 60  complex. For thisexperiment microsomes (50   g protein equivalent) and 12.5   g C 60  –cyclodextrin complex (equivalent to5   g C 60 ) were used.
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