The effect of antioxidants on oxidative DNA damage induced by visible-light-irradiated camphorquinone/N,N-dimethyl-p-toluidine

Previous investigations have found that visible-light (VL)-irradiated camphorquinone (CQ), in the presence of a tertiary amine (e.g., N,N-dimethyl-p-toluidine, DMT), generates reactive oxygen species and causes oxidative DNA damage in vitro. In this study, oxidative DNA damage produced by VL-irradiated CQ/DMT, in the presence and absence of antioxidants (glutathione, N-acetyl-L-cysteine (NAC), mannitol, vitamin C, and vitamin E), was measured by the conversion of PhiX-174 RF I supercoiled (SC) double-stranded plasmid DNA into open and linear forms. VL-irradiated CQ/DMT, lacking antioxidant, damaged 99.4 +/- 1% of the PhiX-174 RF I SC double-stranded plasmid DNA. Our results revealed that glutathione (10.0, 5.0, 2.5, 1.0, and 0.5 mm) and NAC (10.0, 5.0, and 2.5 mm) significantly (p < 0.02) reduced oxidative DNA damage produced by VL-irradiated CQ/DMT. Vitamin E, vitamin C, and mannitol were ineffective at reducing oxidative DNA damage produced by VL-irradiated CQ/DMT. Furthermore, vitamin E (10.0 and 5.0 mm) and vitamin C (10.0, 5.0, 2.5, 1.0, 0.5 mm) treatment significantly (p < 0.02) enhanced VL-irradiated CQ/DMT-induced oxidative DNA damage and caused significant (p < 0.001) DNA damage following VL-irradiation in the absence of CQ/DMT. As a result, future studies should evaluate whether glutathione and NAC effectively reduce or prevent oxidative damage induced by VL-irradiated CQ/DMT in vivo.     

The effect of N-acetyl-l-cysteine and ascorbic acid on visible-light-irradiated camphorquinone/N,N-dimethyl-p-toluidine-induced oxidative stress in two immortalized cell lines

Recent studies have revealed that visible-light (VL)-irradiated camphorquinone (CQ), in the presence of a tertiary amine (e.g., N,N-dimethyl-p-toluidine, DMT), generates initiating radicals that may indiscriminately react with molecular oxygen forming reactive oxygen species (ROS). In this study, the ability of the antioxidants N-acetyl-l-cysteine (NAC) and ascorbic acid (AA) to reduce intracellular oxidative stress induced by VL-irradiated CQ/DMT or VL-irradiated hydrogen peroxide (H(2)O(2)) was assessed in an immortalized Murine cementoblast cell line (OCCM.30) and an immortalized Murine fibroblast cell line, 3T3-Swiss albino (3T3). Intracellular oxidative stress was measured with the membrane permeable dye, 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA). VL-irradiated CQ/DMT and VL-irradiated H(2)O(2) each produced significantly (p<0.001) elevated intracellular oxidative levels in both cell types compared to intracellular ROS levels in VL-irradiated untreated cells. OCCM.30 cementoblasts were found to be almost twice as sensitive to VL-irradiated CQ/DMT and VL-irradiated H(2)O(2) treatment compared to 3T3 fibroblasts. Furthermore, 10mm NAC and 10mm AA each eliminated oxidative stress induced by VL-irradiated CQ/DMT and VL-irradiated H(2)O(2) in both cell types. Our results suggest that NAC and AA may effectively reduce or eliminate oxidative stress in cells exposed to VL-irradiated CQ/DMT following polymerization.     

Effects of combinations of ROS scavengers on oxidative DNA damage caused by visible-light-activated camphorquinone/N,N-dimethyl-p-toluidine

The objective of this investigation was to analyze whether various combinations of the ROS scavengers glutathione (GSH), N-acetyl-cysteine (NAC), and vitamins C and E decrease DNA damage due to visible-light-irradiated (VL-irradiated) camphorquinone/N,N-dimethyl-p-toluidine (CQ/DMT) compared with individual vitamin C or E. PhiX-174 RF plasmid DNA was used to determine single and double strand breaks as parameters of DNA damage. Individual ROS scavengers and combinations of the antioxidants were added to plasmid DNA treated with VL-irradiated CQ/DMT/Cu (II). After incubation, DNA was loaded into a 1% agarose gel. Following electrophoresis, gels stained with 0.5 microg/mL ethidium bromide were photographed under ultraviolet illumination and analyzed with NIH ImageJ software. Results were evaluated between groups for statistical significance using Student's paired t-test (p < 0.05). Glutathione significantly reduced oxidative DNA damage at all test concentrations when combined with vitamin C or vitamin E. The concentration of damaged DNA observed in the presence of combinations of GSH with vitamin C or vitamin E was significantly lower compared with all other combinations of antioxidants investigated in our study (p < 0.05). In contrast to GSH, NAC was not able to compensate the pro-oxidative effects of vitamin C and vitamin E. Only at a concentration of 2 mM, NAC combined with vitamin C efficiently prevented CQ/DMT/Cu (II)-associated DNA damage. Our data indicate that solely the combinations of GSH with vitamin C or vitamin E significantly reduce the severity of oxidative DNA damage caused by CQ/DMT, whereas NAC may even increase the pro-oxidant activity of vitamin C and vitamin E.     

Protective effects of antioxidants on micronuclei induced by camphorquinone/N,N-dimethyl-p-toluidine employing in vitro mammalian test system

Camphorquinone (CQ) is widely used as an initiator in modern visible-light (VL) cured resin systems. CQ is also characterized as a potential allergenic compound. To date, there is growing concern that CQ may produce genetic damage by inducing mutation. In this study, CQ in the presence of reducing agent N,N-dimethyl-p-toluidine (DMT) with or without VL irradiation was analyzed for the induction of chromosomal aberrations indicated by micronuclei (MN) induced in CHO cells. Our data demonstrated that an increase in the numbers of MN was observed with CQ/DMT with or without VL irradiation (p < 0.05). Significant prolongation of cell cycles was observed by the treatment with CQ/DMT with or without VL irradiation (p < 0.05). In addition, VL irradiated CQ/DMT was found to exhibit significantly genotoxic and cytotoxic effects as compared with CQ/DMT alone (p < 0.05). Furthermore, to determine whether oxidative stress could modulate the MN induced by CQ/DMT with or without VL irradiation in CHO cells, cells were pre-treated with various antioxidants 10 mM N-acetyl-L-cysteine (NAC), 2 mM ascorbic acid, and 2 mM alpha-tocopherol. The pre-treatment with antioxidants could antagonize not only the increased MN cells but also the prolonged cell cycle induced by CQ/DMT with or without VL irradiation in CHO cells (p < 0.05). Our findings provide the evidences for the induction of MN by CQ/DMT employing mammalian test system, indicating clastogenic activity of CQ/DMT with or without VL irradiation in vitro. In addition, VL irradiated CQ/DMT exhibits higher genotoxic and cytotoxic effects than CQ/DMT alone. Moreover, NAC, ascorbic acid, and alpha-tocopherol act as the antagonists against the genotoxicity and cytotoxicity of CQ/DMT with or without VL irradiation.     

Reactive oxygen species generation and photo-cytotoxicity of eugenol in solutions of various pH

In order to clarify the mechanism of photo-damage caused by eugenol (4-allyl-2-methoxyphenol), we measured cell survival in the presence of eugenol at concentrations of 10(-3) - 10(-7) M, with and without VL (visible light) irradiation by a VL dental lamp and at various pHs (7.2, 7.8 and 8.2) using two different cells (HSG, a human submandibular gland tumor cell line; HGF, a human gingival fibroblast in primary culture). Also, ROS (reactive oxygen species) generation in the above adherent single cells was measured by ACAS laser cytometry combined with CDFH-DA, a peroxide probe. The survival of both HSG and HGF cells treated with eugenol was significantly decreased as the VL irradiation time and/or the pH of the medium was increased. The amount of ROS generated from eugenol was also enhanced by increasing the VL irradiation time and elevating the pH of the medium. Cytotoxicity and ROS generation of HGF cells were significantly lower than that of HSG cells. Glutathione (1 mM) or cysteine (1 mM) protected the photo damages. We conclude that the cytotoxicity of VL-irradiated eugenol possibly was caused by the generation of eugenol radicals and additionally by ROS, both of which were produced dependent on the dose of eugenol, length of irradiation time, and pH of the medium.     

Protective effects of antioxidants on micronuclei induced by irradiated 9-fluorenone/N,N-dimethyl-p-toluidine in CHO cells

9-Fluorenone (9F), the aromatic photosensitizer, is widely used as an initiator in visible-light (VL) cured resin systems. There is growing concern that 9F may produce genetic damage by inducing mutation. In this study, 9F in the presence or absence of reducing agent N,N-dimethyl-p-toluidine (DMT) with or without VL irradiation was analyzed for the induction of chromosomal aberrations indicated by micronuclei (MN) induced in CHO cells. Our data demonstrated that a dose-related increase in the frequency of MN and prolonged cell cycles in 9F with or without DMT in the presence or absence of VL irradiation (p < 0.05). The rank orders with respect to genotoxicity and cytotoxicity were found to be as follows: 9F/DMT +VL > 9F/DMT = 9F + VL > 9F. To determine whether oxidative stress could modulate MN induced by 9F/DMT with or without VL irradiation in CHO cells, cells were pretreated with N-acetyl-L-cysteine (NAC), ascorbic acid, and alpha-tocopherol. The pretreatment with antioxidants could diminish not only the prolonged cell cycle but also the decreased frequency of MN which is induced by 9F with or without DMT in the presence or absence of VL irradiation in CHO cells (p < 0.05). Our findings provide the evidences for the induction of MN by 9F in the presence or absence of DMT with or without VL irradiation in CHO cells, indicating clastogenic activity of 9F/DMT in vitro. These antioxidants act as the antagonists against the genotoxicity and cytotoxicity of 9F/DMT. Thus, leaching photoinitiator and reducing agent might be contributing the sources of oxidative stress.     

ROS formation and glutathione levels in human oral fibroblasts exposed to TEGDMA and camphorquinone

Glutathione (GSH) is important for the self-protection of cells against oxidative stress and toxic xenobiotics, whereas reactive oxygen species (ROS) at elevated concentrations may cause detrimental alterations of cell membranes, DNA, and other cellular structures. The present investigation addressed the effects of triethylene-glycoldimethacrylate (TEGDMA) and camphorquinone (CQ) on glutathione metabolism and the formation of ROS in oral cells. Primary human pulp fibroblasts were exposed to various concentrations of TEGDMA and CQ (0.1-5 mM). Subsequently, GSH concentration and ROS formation were analyzed with the use of the monobromobimane assay (GSH) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) (ROS). The endogenous ROS hydrogen peroxide (H2O2) was used as a positive control (0.02-2 mM). TEGDMA significantly decreased GSH at concentrations between 0.5 and 5 mM (p<0.05), but did not elevate ROS levels. Contrary, CQ increased ROS formation at concentrations>or=1 mM, but had only a moderate effect on GSH at the highest test concentration. Hydrogen peroxide increased ROS and simultaneously decreased GSH at concentrations of >or=0.2 mM. These data show that the investigated substances may cause cell damage due to various mechanisms, GSH decrease and/or ROS increase. As a consequence, TEGDMA and CQ released into an aqueous environment from resinous materials might interact, thus generating significant cytotoxic effects even at low concentrations.     

The contribution of the DNA damage response to neuronal viability

Neurons are extremely active cells and metabolize up to 20% of the oxygen that was consumed by the organism. Despite their highly oxygenic metabolism, neuronal cells have a lower capacity to neutralize the reactive oxygen species (ROS) that they generate or to which they are exposed. High levels of ROS can lead to accumulation of damage to various cellular macromolecules. One of the cellular macromolecules highly affected by intracellular as well as extracellular insults is DNA. Neurons are also highly differentiated, postmitotic cells that cannot be replenished after disease or trauma. Since neurons are irreplaceable and should survive as long as the organism does, they need elaborate defense mechanisms to ensure their longevity. This review article mainly focuses on certain mechanisms that contribute to neuronal longevity, and concentrates on the DNA damage response in neuronal cells. The various mechanisms of DNA repair are briefly described, and focus is on those mechanisms that are activated in neuronal cells following DNA damage. Evidence is presented to show that proper DNA damage response is critically important, not just for normal neuronal development but throughout the entire life of any organism. Defective DNA damage response in older human age can generate neurodegenerative disorders such as Alzheimer's or Parkinson diseases.     

Comparison of radiation-induced reactive oxygen species formation in adult articular chondrocytes and that in human peripheral T cells: possible implication in radiosensitivity

Previously, we examined the formation of reactive oxygen species (ROS) in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In this study, we examined radiation-induced ROS formation in adult articular chondrocytes, which were demonstrated to be highly resistant to apoptosis in our previous study. We found that ROS formation was actually scarcely seen after irradiation of up to 20 Gy in these cells. Therefore, the origin of the great difference of radiosensitivity between T lymphocytes and adult articular chondrocytes is considered to lie in the degree of ROS formation following irradiation, with this difference possibly resulting from the scavenging acuity of these two kinds of normal tissue cells for free radicals including hydroxyl radicals.     

Radioprotection against DNA damage by an extract of Indian green mussel, Perna viridis (L).

This study describes the radioprotective ability of a hydrolysate prepared using an enzyme-acid hydrolysis method from the green mussel Perna viridis in terms of its ability to prevent radiation-induced damage in plasmid DNA, cell death, reactive oxygen species (ROS) formation, and DNA damage in mice lymphocytes. The mussel hydrolysate (MH) present during irradiation showed significant protection from gamma-radiation-induced strand breaks in plasmid DNA as evaluated by gel electrophoresis. Viability studies by trypan blue dye exclusion and MTT assay showed that preincubation of mice splenic lymphocytes with MH protected them from gamma-radiation-mediated killing. Moreover, the presence of MH during irradiation of isolated mice lymphocytes significantly decreased the DNA damage, as measured by comet assay. Measurement of intracellular ROS by dichlorofluorescein fluorescence revealed that the presence of MH effectively reduced the ROS generated in lymphocytes by both chemical method and gamma-irradiation. Prevention of DNA damage both in plasmid and lymphocytes and cell death in lymphocytes appears correlated with reduction of oxidatively generated free radicals. It is concluded that protection against radiation-induced cell death and DNA damage by MH was attributable to reduction of reactive free radical species generated by gamma-radiation.     

Synthesis and properties of thermotropic polyesters modified with a non-mesogenic rigid group

Four series of copolyesters, namely BB6-DMT, BB5-DMT, BB6-DMI and BB5-DMI series, were prepared by melt polycondensation of dimethyl 4,4′-bibenzoate (BB) with a dimethyl phthalate (DMT: dimethyl terephthalate or DMI: dimethyl isophthalate) and an alkanediol (1,6-hexanediol or 1,5-pentanediol). The homopolyesters poly(hexamethylene 4,4′-bibenzoate) (BB6) and poly(pentamethylene 4,4′-bibenzoate) (BB5) exhibit a smectic phase. The thermotropic liquid crystalline and crystalline properties of the copolyesters are significantly influenced by the presence of the non-mesogenic rigid phthalate unit. All BB6-DMT copolyesters remain crystalline. As x, the molar fraction of the phthalate units in the diacid units, ≧ 0.7 the mesophase of the BB6-DMT copolyesters is destroyed completely. For BB5-DMT copolyesters, the mesophase disappears as x ≧ 0.4, and the copolyesters become amorphous as 0.5 ≦ x ≦ 0.8. The mesophase and the crystallinity of the BB6-DMI copolyesters are destroyed completely as x > 0.5. The BB5-DMI copolyesters lose the mesophase as x ≧ 0.3, and become amorphous as x ≧ 0.4. The results indicate that the non-linear isophthalate unit destroys mesophase and crystallinity of the copolyesters to a greater extent than the para-linked terephthalate unit.     

Polimorphonuclear Cell-Mediated Oxidative Stress: Sink for Reactive Oxygen Species and Cell Various Type Damage

Reactive oxygen species (ROS) are produced in animals and humans under physiologic and pathologic conditions. Polymorphonuclear cells (PMNs) and other professional phagocytes are able to generate large amounts of ROS that have not only antimicrobial capacity but are also deleterious to mammalian cells and responsible for many chronic diseases.

In particular, ROS produced in large amounts by the massively infiltrating leukocytes in inflammed tissues are believed to constitute a major tissue-destructive force and may contribute significantly to the pathogenesis of several inflammatory diseases.

Inflammation can accelerate the development of cancer: in fact, it seems that a part of the predisposition to cancer may be attributed to the oxidants released by the phagocytes at inflammatory site and then to the effects of continuous damage over a life span by ROS. The focus of this study was to investigate the differential capacity of ROS capture and the relative cellular damage degree in gastric, intestinal and fibroblastic cell lines. These various cell types were in vitro used as sink for ROS released by co-cultured fMLP-stimulated human polymorphonuclear cells.

Our data demonstrated that cell lines showed a differential capacity of ROS capture correlated to cellular damage, probably due to a different cell susceptibilty to the oxidative challenge produced by stimulated PMNs.     

Effects of sub-toxic concentrations of camphorquinone on cell lipid metabolism

The biological effects of camphorquinone (CQ), an initiator for light-polymerized resins, have been reported to relate to its ability to generate free radicals and cause radical-induced membrane damage via lipid peroxidation. However, the effects of CQ on lipids other than peroxidation may result in unfavorable tissue responses especially at concentrations that are not overtly toxic to cells. The purpose of the current study was to examine the effects of CQ on cell lipid metabolism at sub-toxic concentrations, with or without visible light irradiation. HCP and THP-1 cells were exposed to CQ with or without light irradiation under clinically relevant conditions and lipid metabolism was analyzed using ¹⁴C-labeling and thin-layer chromatography. We found that CQ increased synthesis of neutral lipids, such as triglycerides, from 7 to nearly 15% of the total and diglycerides from 2% to about 3% of the total in HCP cells, while synthesis of phospholipids, such as sphingomyelin, was decreased by 1–1.5%. In THP-1 cells cholesterol synthesis increased more than 2-fold and cholesterol ester synthesis increased more than 5-fold. Light-activated CQ did not differ significantly in terms of its bioactivity compared to no-light conditions. We conclude that CQ significantly altered the metabolism of several important structural lipids in two cell types at sub-toxic concentrations that are clinically relevant. These changes in lipid metabolism may in turn affect membrane integrity and permeability and possibly lead to significant changes in cell responses.     

Photodynamic inactivation of viruses using upconversion nanoparticles

Photodynamic therapy (PDT) is a promising treatment modality that utilizes light of an appropriate wavelength to excite photosensitive materials called photosensitizers, which upon excitation, generate reactive oxygen species (ROS) that are cytocidal and virucidal. However, problems such as hydrophobicity of photosensitizers and limited tissue penetration ability of the current light sources impeded its promotion as a mainstay in medical technology. Here, by using near-infrared (NIR)-to-visible upconversion nanoparticles (UCNs), we demonstrate UCN-based photodynamic inactivation as a potential antiviral strategy. These UCNs are nanotransducers which not only act as carriers of photosensitizers but also active participants in PDT by transducing NIR radiation to visible emissions appropriate for excitation of the attached photosensitizers. The UCNs effectively reduced the infectious virus titers in vitro with no clear pathogenicity in murine model and increased target specificity to virus-infected cells. Hence, this is a promising antiviral approach with feasible applications in the treatments of virus-associated infections, lesions and cancers.     

DNA damage in lymphocytes of Indian rickshaw pullers as measured by the alkaline Comet assay

Rickshaw pullers (RPs) engage in strenuous physical activity and are exposed to the air pollutants found in urban environments. Air pollutants and the reactive oxygen species generated by the physical activity both potentially can damage DNA. In the present study, the Comet assay, a sensitive tool for measuring DNA damage in single cells, was used to study genomic DNA damage in lymphocytes of Indian RPs. The study evaluated DNA damage in 118 healthy male volunteers, including 63 RPs whose work demanded high levels of physical activity for 7-9 hr/day, and 55 controls matched for age, habits, socio-economic status, and exposure to air pollution. A significant increase was found for the mean Olive tail moment (arbitrary units) among the RPs (4.13 +/- 0.11; P < 0.001) in comparison with the controls (3.21 +/- 0.10). Likewise, comet tail length (microm) (RPs: 58.98 +/- 1.01 vs. controls: 52.38 +/- 1.24) and tail DNA (%) (RPs: 13.52 +/- 0.31 vs. controls: 10.04 +/- 0.24) were also significantly higher for RPs compared with those of their matched controls (both, P < 0.001). To our knowledge, this is the first demonstration that physical activity due to occupation can produce DNA damage in peripheral lymphocytes.     

Mechanism of apoptotic resistance of human osteosarcoma cell line,HS-Os-1, against irradiation

In our previous study, we examined reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of Dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In this study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1, which was established from an osteoblastic tumor that arose in the left humerus of an 11-year-old girl and was already morphologically characterized in vitro and in vivo. We found that ROS formation and oxidative DNA damage were actually scarcely seen after irradiation of up to 30 Gy in these cells; that mitochondrial membrane potential was preserved; and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. Therefore, the origin of the close similarity of radiosensitivity between adult articular chondrocytes and the human osteosarcoma cell line HS-Os-1, is considered to involve the low degree of ROS formation following irradiation; the similarity possibly results from the strong scavenging ability of these two kinds of cells for free radicals including hydroxyl radicals.     

Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa

The objective of this study was to evaluate the effect of the generation of reactive oxygen species (ROS) on the integrity of the DNA of human spermatozoa, and to determine if pretreatment with antioxidants can reduce DNA damage. Samples were obtained from 47 men undergoing infertility investigation. ROS were generated in the samples by the addition of xanthine/xanthine oxidase (X/XO) with or without antioxidants. After incubation at timed intervals (0-2 h) with X/XO, the percentage of spermatozoa with DNA fragmentation was determined using the method of TdT-mediated DNA end-labelling (TUNEL). Time intervals were selected to mimic the clinical situation in which spermatozoa are held for a period of time after swim-up while the oocytes are prepared for ICSI. A significant increase in sperm DNA damage was evident when samples were incubated in the presence of ROS for intervals of 1 and 2 h, but not when incubated with ROS for <1 h (P = 0.0001). The addition of antioxidants significantly decreased the amount of DNA damage induced by ROS generation (P < 0.04). ROS can cause an increase in DNA fragmentation and pretreatment with antioxidants can reduce DNA damage.      

Radiation-induced reactive oxygen species formation prior to oxidative DNA damage in human peripheral T cells

Previously, we demonstrated that human peripheral T lymphocytes revealed early apoptotic changes (annexin V-positive) and late apoptotic changes (propidium iodide-positive), at 13 and 24 h, respectively, after irradiation of 5 Gy. Changes in mitochondrial membrane potential were observed at 10 h after irradiation of 5 Gy. Subsequently, mitochondrial cytochrome c-release was confirmed. In order to elucidate the mechanism which acts prior to the mitochondrial membrane potential changes, we examined in the previous study the radiation dose and the timing of oxidative DNA damage induced in human peripheral T lymphocytes following 10 MV X-ray irradiation. As a result, the production of 8-oxoguanine, i.e., the product of oxidative DNA damage, was clearly identified starting at 10, 6, and 3 h, after 2, 5, and 20 Gy of irradiation, respectively. Therefore, we examined in the present study reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation.     

Immunocytochemical characteristics of human osteosarcoma cell line HS-Os-1: possible implication in apoptotic resistance against irradiation

In our previous study, we examined reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy irradiation. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In the succeeding study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1. We found that ROS formation and oxidative DNA damage were actually scarcely seen after irradiation of up to 30 Gy in these cells, that mitochondrial membrane potential was preserved, and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. In the present study, we examined the immunocytochemical characteristics of the apoptotic-resistance of the HS-Os-1 cell line against irradiation in order to clarify its possible implications regarding radiosensitivity. The results showed that these cells lack P53 and Bax protein expression, and strong peroxidase activity was confirmed in the nuclei of the cells. Moreover, SODII (manganese superoxide dismutase II) protein expression was gradually increased in spite of irradiation of up to 30 Gy. Therefore, it is concluded that HS-Os-1 cells are originally apoptotic-resistant and that the cells possess a strong ability to scavenge for free radicals. To convert these cells to a state of apoptotic-susceptibility, a powerful oxidant such as hydrogen peroxide might exert such an effect in terms of the production of hydroxyl radicals in lysosomes in the cells as shown in our previous studies. The origin of the radioresistance of the human osteosarcoma cell line HS-Os-1 is considered to to be low degree of ROS formation following irradiation, reflecting the strong scavenging ability of these cells for free radicals including hydroxyl radicals.     

Development of an assay for quantification of linkage-specific O-acetylated sialoglycans on erythrocytes; its application in Indian visceral leishmaniasis

We have developed a noninvasive approach for the quantification of linkage-specific 9-O-acetylated sialoglycans on mammalian erythrocytes using a lectin, Achatinin-H, whose lectinogenic epitope has previously been defined as 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) alpha 2-->6 linked to subterminal GalNAc. Titration and checkerboard analysis were performed to optimize the assay using rabbit, rat and human erythrocytes that contain differing amounts of this glycotope. Assay specificity was established by decreased binding of erythrocytes to immobilised Achatinin-H when pre-incubated with excess lectin. The intra-assay coefficient of variation (CV) for rat and human erythrocytes was 8.6-9.2% and 11.1-13.0%, respectively. The inter-assay CV for rat and human erythrocytes was 9.9-10.1% and 15.2-16.6%, respectively. In previous studies, we have identified an enhanced presence of cell surface 9-O-AcSGs on the erythrocytes of patients with visceral leishmaniasis (VL) [Am. J. Trop. Med. Hyg. 58 (1998) 551]. Our assay when evaluated on erythrocytes from VL patients (n=30) showed a fourfold increase in lectin binding as compared to endemic controls. The mean +/- S.E.M. of the A(405) nm value was 1.14 +/- 0.04 vs. 0.23 +/- 0.03, respectively (p<0.0001). Following effective chemotherapy, a significant reduction of this glycotope on the erythrocytes of VL patients indicates that this assay has both a diagnostic and prognostic potential. Taken together, we conclude that this antigen-based assay is a specific and reproducible method for monitoring the disease status of VL patients and could be used in retrospective and prospective trials.