Nano-Scaled Particles of Titanium Dioxide Convert Benign Mouse Fibrosarcoma Cells into Aggressive Tumor Cells
2009; Elsevier BV; Volume: 175; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.080900
ISSN1525-2191
AutoresKunishige Onuma, Yu Sato, Satomi Ogawara, Nobuyuki Shirasawa, Masanobu Kobayashi, Jun Yoshitake, Tetsuhiko Yoshimura, Masaaki Iigo, Junichi Fujii, Futoshi Okada,
Tópico(s)Nanoplatforms for cancer theranostics
ResumoNanoparticles are prevalent in both commercial and medicinal products; however, the contribution of nanomaterials to carcinogenesis remains unclear. We therefore examined the effects of nano-sized titanium dioxide (TiO2) on poorly tumorigenic and nonmetastatic QR-32 fibrosarcoma cells. We found that mice that were cotransplanted subcutaneously with QR-32 cells and nano-sized TiO2, either uncoated (TiO2−1, hydrophilic) or coated with stearic acid (TiO2−2, hydrophobic), did not form tumors. However, QR-32 cells became tumorigenic after injection into sites previously implanted with TiO2−1, but not TiO2−2, and these developing tumors acquired metastatic phenotypes. No differences were observed either histologically or in inflammatory cytokine mRNA expression between TiO2−1 and TiO2−2 treatments. However, TiO2−2, but not TiO2−1, generated high levels of reactive oxygen species (ROS) in cell-free conditions. Although both TiO2−1 and TiO2−2 resulted in intracellular ROS formation, TiO2−2 elicited a stronger response, resulting in cytotoxicity to the QR-32 cells. Moreover, TiO2−2, but not TiO2−1, led to the development of nuclear interstices and multinucleate cells. Cells that survived the TiO2 toxicity acquired a tumorigenic phenotype. TiO2-induced ROS formation and its related cell injury were inhibited by the addition of antioxidant N-acetyl-l-cysteine. These results indicate that nano-sized TiO2 has the potential to convert benign tumor cells into malignant ones through the generation of ROS in the target cells. Nanoparticles are prevalent in both commercial and medicinal products; however, the contribution of nanomaterials to carcinogenesis remains unclear. We therefore examined the effects of nano-sized titanium dioxide (TiO2) on poorly tumorigenic and nonmetastatic QR-32 fibrosarcoma cells. We found that mice that were cotransplanted subcutaneously with QR-32 cells and nano-sized TiO2, either uncoated (TiO2−1, hydrophilic) or coated with stearic acid (TiO2−2, hydrophobic), did not form tumors. However, QR-32 cells became tumorigenic after injection into sites previously implanted with TiO2−1, but not TiO2−2, and these developing tumors acquired metastatic phenotypes. No differences were observed either histologically or in inflammatory cytokine mRNA expression between TiO2−1 and TiO2−2 treatments. However, TiO2−2, but not TiO2−1, generated high levels of reactive oxygen species (ROS) in cell-free conditions. Although both TiO2−1 and TiO2−2 resulted in intracellular ROS formation, TiO2−2 elicited a stronger response, resulting in cytotoxicity to the QR-32 cells. Moreover, TiO2−2, but not TiO2−1, led to the development of nuclear interstices and multinucleate cells. Cells that survived the TiO2 toxicity acquired a tumorigenic phenotype. TiO2-induced ROS formation and its related cell injury were inhibited by the addition of antioxidant N-acetyl-l-cysteine. These results indicate that nano-sized TiO2 has the potential to convert benign tumor cells into malignant ones through the generation of ROS in the target cells. Nanomaterials are produced in tonnage quantities in the world every year, mainly as a result of the recent advances in nanotechnologies.1Veranth JM Kaser EG Veranth MM Koch M Yost GS Cytokine responses of human lung cells (BEAS-2B) treated with micron-sized and nanoparticles of metal oxides soil dusts.Part Fibre Toxicol. 2007; 4: 2Crossref PubMed Scopus (217) Google Scholar Nanoparticles are defined as ultra-fine particles of a diameter less than 100 nm,2 and are basically fabricated from metal and ceramic oxides. 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4: 2Crossref PubMed Scopus (217) Google Scholar The objective of the present study was to determine the potential effects of nano-sized TiO2 particles on the acceleration of the carcinogenic process by using regressive tumor cells (QR clones).21Ishikawa M Okada F Hamada J Hosokawa M Kobayashi H Changes in the tumorigenic and metastatic properties of tumor cells treated with quercetin or 5-azacytidine.Int J Cancer. 1987; 39: 338-342Crossref PubMed Scopus (54) Google Scholar The regressive phenotype of QR clones is mediated by host immunity in normal mice,22Okada F Hosokawa M Hasegawa J Ishikawa M Chiba I Nakamura Y Kobayashi H Regression mechanisms of mouse fibrosarcoma cells after in vitro exposure to quercetin: diminution of tumorigenicity with a corresponding decrease in the production of prostaglandin E2.Cancer Immunol Immunother. 1990; 31: 358-364Crossref PubMed Scopus (38) Google Scholar, 23Kobayashi T Okada F Fujii N Tomita N Ito S Tazawa H Aoyama T Choi SK Shibata T Fujita H Hosokawa M Thymosin ß4 regulates motility and metastasis of malignant mouse fibrosarcoma cells.Am J Pathol. 2002; 160: 869-882Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar and tumorigenic QR clones24Okada F Hosokawa M Hamada J Hasegawa J Kato M Mizutani M Ren J Takeichi N, Kobayashi H: Malignant progression of a mouse fibrosarcoma by host cells reactive to a foreign body (gelatin sponge).Br J Cancer. 1992; 66: 635-639Crossref PubMed Scopus (62) Google Scholar, 25Okada F Hosokawa M Hasegawa J Kuramitsu Y Nakai K Yuan L Lao H Kobayashi H Takeichi N Enhancement of in vitro prostaglandin E2 production by mouse fibrosarcoma cells after co-culture with various anti-tumour effector cells.Br J Cancer. 1994; 70: 233-238Crossref PubMed Scopus (17) Google Scholar inhibit host anti-tumor immunity through the production of high levels of prostaglandin E2 (PGE2), which suppresses the induction of cytotoxic T lymphocytes.22Okada F Hosokawa M Hasegawa J Ishikawa M Chiba I Nakamura Y Kobayashi H Regression mechanisms of mouse fibrosarcoma cells after in vitro exposure to quercetin: diminution of tumorigenicity with a corresponding decrease in the production of prostaglandin E2.Cancer Immunol Immunother. 1990; 31: 358-364Crossref PubMed Scopus (38) Google Scholar Therefore, the tumorigenic potential of QR clones in mice can be estimated from measuring PGE2 secreted by cells converted from QR cells in vitro. We chose to use this model because we could monitor the conversion of a regressive phenotype to a tumorigenic/metastatic one in the presence of inflammation in mice.24Okada F Hosokawa M Hamada J Hasegawa J Kato M Mizutani M Ren J Takeichi N, Kobayashi H: Malignant progression of a mouse fibrosarcoma by host cells reactive to a foreign body (gelatin sponge).Br J Cancer. 1992; 66: 635-639Crossref PubMed Scopus (62) Google Scholar, 25Okada F Hosokawa M Hasegawa J Kuramitsu Y Nakai K Yuan L Lao H Kobayashi H Takeichi N Enhancement of in vitro prostaglandin E2 production by mouse fibrosarcoma cells after co-culture with various anti-tumour effector cells.Br J Cancer. 1994; 70: 233-238Crossref PubMed Scopus (17) Google Scholar, 26Tazawa H Okada F Kobayashi T Tada M Mori Y Une Y Sendo F Kobayashi M Hosokawa M Infiltration of neutrophils is required for acquisition of metastatic phenotype of benign murine fibrosarcoma cells: implication of inflammation-associated carcinogenesis and tumor progression.Am J Pathol. 2003; 163: 2221-2232Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 27Okada F Shionoya H Kobayashi M Kobayashi T Tazawa H Onuma K Iuchi Y Matsubara N Ijichi T Dugas B Hosokawa M Prevention of inflammation-mediated acquisition of metastatic properties of benign mouse fibrosarcoma cells by administration of an orally available superoxide dismutase.Br J Cancer. 2006; 94: 854-862Crossref PubMed Scopus (36) Google Scholar, 28Okada F Nakai K Kobayashi T Shibata T Tagami S Kawakami Y Kitazawa T Kominami R Yoshimura S Suzuki K Taniguchi N Inanami O Kuwabara M Kishida H Nakae D Konishi Y Moriuchi T Hosokawa M Inflammatory-cell-mediated tumour progression and minisatellite mutation correlate with the decrease of antioxidative enzymes in murine fibrosarcoma cells.Br J Cancer. 1999; 79: 377-385Crossref PubMed Scopus (27) Google Scholar, 29Okada F Kobayashi M Tanaka H Kobayashi T Tazawa H Iuchi Y Onuma K Hosokawa M Dinauer MC Hunt NH The role of nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species in the acquisition of metastatic ability of tumor cells.Am J Pathol. 2006; 169: 294-302Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 30Okada F Tazawa H Kobayashi T Kobayashi M Hosokawa M Involvement of reactive nitrogen oxides for acquisition of metastatic properties of benign tumors in a model of inflammation-based tumor progression.Nitric Oxide. 2006; 14: 122-129Crossref PubMed Scopus (17) Google Scholar In this study, we demonstrate that nano-TiO2 alters the morphology of regressive QR-32 cells and enhances their PGE2-mediated tumorigenic properties through formation of intracellular reactive oxygen species (ROS), and that the associated biological and immunological changes differ according to the surface treatment of nano-TiO2. Nano-TiO2 (rutile crystal phase) was a generous gift, aimed for scientific investigation, from Ishihara Sangyo Kaisha LTD (Osaka, Japan). We used two types of nano-TiO2 with different surface modifications: TiO2−1 treated with ZrO2Al(OH)3, which is hydrophilic; and TiO2−2, treated with ZrO2Al(OH)3 and stearic acid, which is hydrophobic. Both of these forms were generated by the alkaline and acidic leaching method. The precise composition and the chemical properties of these nanoparticles are summarized in Table 1. As these nanoparticles are highly dispersed, we suspended them into 1.5% carboxymethylcellulose (217277, Wako Pure Chemical Industries, Osaka, Japan), and since nano-TiO2 aggregates spontaneously in carboxymethylcellulose solution, the suspension was vortexed and sonicated before it was used for experiments. N-acetyl-l-cysteine (017-05131) and aminoguanidine (014-02542) were obtained from Wako Pure Chemical Industries.Table 1Characteristics of Titanium Dioxide NanoparticlesNominal size (nm)NanoparticlesCrystalTiO2 contentsSurface treatmentSurface area (m2/g)Minor axisMajor axisOil absorption (g/100 g)pHHydropathyTiO2−1rutile84%–92%ZrO2Al(OH)370–9040–70200–30040–556.5–8.0HydrophilicTiO2−2rutile77%–86%ZrO2Al(OH)3 and stearic acid25–4540–70200–30029–45-Hydrophobic Open table in a new tab BMT-11, a transplantable fibrosarcoma, was induced in a C57BL/6 mouse with 3-methylcholanthrene, and its tumorigenic clone, BMT-11 cl-9, was subsequently isolated by limiting dilution. BMT-11 cl-9 cells were exposed to quercetin in vitro, which gave rise to a number of random subclones (QR cells).21Ishikawa M Okada F Hamada J Hosokawa M Kobayashi H Changes in the tumorigenic and metastatic properties of tumor cells treated with quercetin or 5-azacytidine.Int J Cancer. 1987; 39: 338-342Crossref PubMed Scopus (54) Google Scholar These cell spontaneously regressed when injected into normal syngeneic mice and the regressive phenotype was due to DNA hypermethylation induced by quercetin.21Ishikawa M Okada F Hamada J Hosokawa M Kobayashi H Changes in the tumorigenic and metastatic properties of tumor cells treated with quercetin or 5-azacytidine.Int J Cancer. 1987; 39: 338-342Crossref PubMed Scopus (54) Google Scholar The phenotype was stable since the cells did not acquire tumorigenicity or metastatic ability spontaneously in culture for several years (data not shown). The QR clones are not revertant from tumorigenic to normal cells, since they grow lethally in immunodeficient mice and show tumor cell properties as they conserve in vitro anchorage-independent and infinite growth22Okada F Hosokawa M Hasegawa J Ishikawa M Chiba I Nakamura Y Kobayashi H Regression mechanisms of mouse fibrosarcoma cells after in vitro exposure to quercetin: diminution of tumorigenicity with a corresponding decrease in the production of prostaglandin E2.Cancer Immunol Immunother. 1990; 31: 358-364Crossref PubMed Scopus (38) Google Scholar, 23Kobayashi T Okada F Fujii N Tomita N Ito S Tazawa H Aoyama T Choi SK Shibata T Fujita H Hosokawa M Thymosin ß4 regulates motility and metastasis of malignant mouse fibrosarcoma cells.Am J Pathol. 2002; 160: 869-882Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar One of these regressive cell clones, QR-32, was used in this study. QR-32 cells and derived cell lines were maintained in Eagle's minimum essential medium (Nissui Pharm., Tokyo, Japan) supplemented with 8% fetal bovine serum (1370978, GIBCO), sodium pyruvate, non-essential amino acids, and l-glutamine, at 37°C, in a humidified 5% CO2/95% air mixture. For in vitro cell growth analysis, cells were seeded into a 6-well plate (1 × 105 cells per well; 3506, Corning, NY) and the medium was changed every other day. The cells were harvested and counted every day from day 1 to day 7 using the trypan blue exclusion test; doubling time was calculated from the logarithmic phase of the growth curve. For evaluation of plating efficiency, 1 × 103 cells suspended in medium were plated into 60-mm dishes (430166, Corning) in triplicate. The dishes were incubated for 7 days, colonies were then fixed in Carnoy's fixative, stained with 0.1% crystal violet and scored. For determination of growth in soft agar, 2 × 102 cells were suspended in 1 ml medium containing 0.3% agar (0710-500G, MS technosystems, Japan) and 2× volume of fetal bovine serum, and applied onto the pre-solidified 0.6% agar (1 ml) in 6-well plates. Triplicate plates were prepared for each cell line and after 3 weeks of incubation, colonies larger than 0.1 mm in diameter were scored. Five-week-old female C57BL/6 mice were obtained from Nippon SLC (Hamamatsu, Japan). All of the mice were maintained in specific pathogen-free conditions, lit from 6:00 AM to 6:00 PM, at 23 ± 2°C and 45 ± 15% humidity, and fed with mouse diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and UV-irradiated water in the Institute for Animal Experimentation, Yamagata University Graduate School of Medicine. Diet and water were available ad libitum throughout the experiment. The experimental protocol was approved by the Committee of the Institute for Animal Experimentation, Yamagata University School of Medicine (#06-074). The mice (at 6 weeks of age) were used after 1 week of acclimatization. They were divided randomly into four groups. Group 1: QR-32 cells (1 × 105 cells/0.1 ml) were subcutaneously injected into mice. Group 2: Either TiO2−1 or TiO2−2 (5 mg/0.1 ml) were mixed at an equal volume with QR-32 cells (1 × 105 cells/0.1 ml), then 0.2 ml of the mixture were injected into the mice subcutaneously. Groups 3 and 4: Either TiO2−1 or TiO2−2 (5 mg/0.1 ml) was injected alone into the mice subcutaneously, and 30 days (Group 3) and 70 days (Group 4) later, QR-32 cells (1 × 105 cells/0.1 ml) were injected into the nano-TiO2 implantation sites. Tumor diameters were measured twice a week during the experiment. To assess whether the developing tumors had acquired metastatic properties, we removed the subcutaneously growing tumors aseptically, disaggregated them mechanically with scissors and used them for establishing individual cell lines. The detailed procedure has been described elsewhere.24Okada F Hosokawa M Hamada J Hasegawa J Kato M Mizutani M Ren J Takeichi N, Kobayashi H: Malignant progression of a mouse fibrosarcoma by host cells reactive to a foreign body (gelatin sponge).Br J Cancer. 1992; 66: 635-639Crossref PubMed Scopus (62) Google Scholar Each tumor cell line was then injected intravenously (1 × 106 cells) into mice. On day 35, the mice were sacrificed with excess inhalation of ether and metastatic nodules on the surface of the lungs and other organs were counted macroscopically. Nano-TiO2-implanted subcutaneous tissues were excised at the times indicated and fixed with Bouin's solution overnight, immersed sequentially in 50%, 75%, and 99% ethanol every 24 hours to remove picric acid, dehydrated, embedded in paraffin, sectioned at 4 μm, and mounted on glass slides either for Azan staining or immunohistochemistry. For immunostaining, after deparaffinization and rehydration, the tissue sections were incubated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase. After rinsing, the sections were incubated with mouse 8-hydroxy-2′-deoxyguanosine (8-OHdG) monoclonal antibody (MOG-100 at a concentration of 10 μg/ml) or mouse anti-4-hydroxy-2-nonenal (HNE) monoclonal antibody (MHN-100 at a concentration of 25 μg/ml; Nikken Foods, Fukuroi, Japan), respectively, in a humidified chamber at 4°C overnight. Then, the sections were incubated for 10 minutes with MAX-PO complex, which is a conjugation of amino acid polymer with the Fab′ portion of the secondary antibody and peroxidase (414321, Histofine mouse stain kit; Nichirei, Tokyo, Japan). After a final rinse, specific immunolabeling was examined with the use of 3,3′-diaminobenzidine (415171, Nichirei, Japan), as the chromogen, which was placed on the tissues for a few minutes. Development of 3,3′-diaminobenzidine was stopped by washing the tissues in distilled water. The sections were dehydrated, mounted, and photographed. The detailed procedure for isolating natural killer (NK) cells from mouse spleen and the NK cell-mediated cytolysis assay were described previously.22Okada F Hosokawa M Hasegawa J Ishikawa M Chiba I Nakamura Y Kobayashi H Regression mechanisms of mouse fibrosarcoma cells after in vitro exposure to quercetin: diminution of tumorigenicity with a corresponding decrease in the production of prostaglandin E2.Cancer Immunol Immunother. 1990; 31: 358-364Crossref PubMed Scopus (38) Google Scholar Briefly, the isolated NK cells were used as effector cells, and each tumor cell line was labeled with a fluorescent dye, PKH67, at a final concentration of 3.5 μmol/L (PKH67GL-1KT, Sigma, Tokyo, Japan). After 3 washes, the labeled tumor cells were suspended in RPMI medium and incubated with the NK cells at an effector-to-target cell ratio of 200:1 in a 96-well round-bottomed plate (3360, Corning) for 6 hours. The plate was centrifuged and the fluorescence intensity in the supernatants was measured at an excitation wavelength of 551 nm and an emission wavelength of 567 nm in a SpectraMax Gemini EM (01436, Molecular Devices, Tokyo, Japan). Spontaneous release and maximum release were determined by incubating target tumor cells without effectors in medium alone or in 1N hydrochloric acid, respectively. The specific percentage cytotoxicity was calculated as described below: Specific NK cytotoxicity (%) = (fluorescence intensity release due to the cytotoxicity of the NK cells − value of spontaneous release from target cells)/(fluorescence intensity caused by treatment with 1N hydrochloric acid − value of spontaneous release from target cells) × 100. Commercially available enzyme-linked immunosorbent assay kits for mouse vascular endothelial growth factor (MMV00, R & D Systems, Minneapolis, MN), mouse transforming growth factor (TGF)-ß (MB100, R & D Systems), and PGE2 (EA 02, Oxford Biomedical Research, Metamora, MI) were used to quantify the level of each mediator in conditioned media according to the manufacturer's instructions. For PGE2 samples, 1 × 105 cells were cultured in 24-well plates (3526, Corning) in 2 ml medium. After 24 hours, the supernatants were obtained. For TGF-ß and vascular endothelial growth factor samples, 4 × 105 cells were cultured in 24-well plates in 0.5 ml medium for 24 hours. TGF-ß samples were acid-activated and neutralized, and the resultant immunoreactive forms were generated just before enzyme-linked immunosorbent assay analysis. Frozen tissues were crushed to a powder in a mortar with liquid nitrogen. Total RNA was extracted using TRIzol (15596-018, Invitrogen, Tokyo, Japan) according to the manufacturer's instructions. For real-time PCR, 1 μg of total RNA was subjected to cDNA synthesis in 10 μl of reaction mixture containing PrimeScript buffer consisting of dNTP mixture, MgCl2, 25 pM/L oligo dT primer and 50 pM/L random 6-mers, and PrimeScript reverse transcription enzyme mix I (RR037A, PrimeScript RT reagent kit, Takara, Otsu, Japan). The reverse transcription reaction was performed sequentially for 15 minutes at 37°C, for 5 seconds at 85°C, and thereafter at 4°C. Oligonucleotide primers were designed with the use of Primer 3 software version 0.4.0 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The primers were designed to produce an approximately 150-bp amplicon using the cDNA of interleukin (IL)-1ß (P10749), IL-2 (P04351), IL-4 (P07750), IL-6 (Q0PMN1), IL-10 (Q0VBJ1), interferon (IFN)-γ (P01580), TGF-ß (P04202), and tumor necrosis factor-α (Q3U593). To avoid amplification of genomic DNA, primers were placed within different exons close to an intron-exon boundary, with the probe spanning two neighboring exons whenever possible. The ß-actin (Q6IWE2) gene was used as an endogenous control to normalize for differences in the amount of total RNA present in each sample. Primer sequences for the amplification of above genes by real-time PCR were as follows: IL-1ß, sense, 5′-CCTCACAAGCAGAGCACAAg-3′; antisense, 5′-TGGGGAAGGCATTAGAAACA-3′; IL-2, sense, 5′-CCCACTTCAAGCTCCACTTC-3′; antisense, 5′-GGAGCTCCTGTAGGTCCATC-3′; IL-4, sense, 5′-TCAACCCCCAGCTAGTTGTC-3′; antisense, 5′-TGTGACCTCGTTCAAAATGC-3′; IL-6, sense, 5′-AAGCGAGAGTCCTTCAGAGAGA-3′; antisense, 5′-GAGCATTGGAAATTGGGGTA-3′; IL-10, sense, 5′-CTGTTTCCATTGGGGACACT-3′; antisense, 5′-AAGTGTGGCCAGCCTTAGAA-3′; IFN-γ, sense, 5′-GAGGAACTGGCAAAAGGATG-3′; antisense, 5′-GCTGATGGCCTGATTGTCTT-3′; TGF-ß1, sense, 5′-ATTCCTGGCGTTACCTTGG-3′; antisense, 5′-AGCCCTGTATTCCGTCTCCT-3′; Tumor necrosis factor-α, sense, 5′-ACGGCATGGATCTCAAAGAC-3′; antisense, 5′-AGATAGCAAATCGGCTGACG-3′; and, ß-actin, sense, 5′-TGAGGAGCACCCTGTGCT-3′; antisense, 5′-ACATGGCTGGGGTGTTGAAG-3′. Real-time PCR experiments were performed using a commercial kit (QPK-201, SYBR Green Realtime PCR master mix, Toyobo, Osika, Japan). Each reaction tube contained template DNA (200 ng) and 0.4 μmol/L each of forward and reverse primers. A negative control was assembled by using the same concentrations of reagents but omitting the template DNA (data not shown). Samples were amplified in a thermocycler (LightCycler 2.0 Instrument, Roche Applied Science, Tokyo, Japan) for 40 cycles: 1 minute at 95°C, 5 seconds at 60°C, and 10 seconds at 72°C. Comparative Ct (Fit Points method) was used to calculate the expression level of individual genes using LightCycler, Software (Ver. 3.5, Roche Diagnostics, Tokyo, Japan). A detailed description of reverse transcription (RT)-PCR for thymosin ß4 or glyceraldehyde-3-phosphate dehydrogenase gene amplification has been described previously.23Kobayashi T Okada F Fujii N Tomita N Ito S Tazawa H Aoyama T Choi SK Shibata T Fujita H Hosokawa M Thymosin ß4 regulates motility and metastasis of malignant mouse fibrosarcoma cells.Am J Pathol. 2002; 160: 869-882Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar The generation of reactive oxygen species in cell-free conditions was deter
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