WIL2-NS Cells Research Articles

Bisphenol A-induced autophagy ameliorates human B cell death through Nrf2-mediated regulation of Atg7 and Beclin1 expression by Syk activation

The widely used plasticizer bisphenol A (BPA) is known as an endocrine-disrupting chemical (EDC). Many studies have shown that BPA contributes to diseases involving immune system alterations, but the underlying mechanisms have yet to be elucidated. We previously reported that BPA at concentration of 100 μM caused human B cell death in accordance with an increase in nuclear factor (erythroid-derived 2)-like 2(Nrf2) expression. Autophagy is a cellular process that degraded and recycles cytoplasmic constituents. Here, we investigated whether BPA induces autophagy through Nrf2, which is associated with regulation of B cell death using human WiL2-NS lymphoblast B cells. Then, cell viability was assessed by various assays using trypan blue, MTT or Celltiter glo luminescent substrate and DAPI. When WiL2-NS cells were treated with BPA, cell viability was decreased and LC3 autophagy cargo protein/puncta was increased. BPA-induced autophagy was confirmed by the modification of LC3 puncta formation or autophagy flux turnover with the treatment of hydroxychloroquine(HCQ), NH4Cl and PI3K inhibitors including 3-methyladenine(3-MA), LY294002 and wortmannin. BPA treatment increased the expression of autophagy-related gene(Atg)7 and Beclin1 as well as Nrf2 induced by the production of reactive oxygen species (ROS). The inhibition of autophagy with siAtg7 or siBeclin1 and Nrf2 depletion aggravated BPA-induced cell death. BPA enhanced the bound of Nrf2 to the specific region on Beclin1 and Atg7 promoter. Spleen tyrosine kinase(Syk) activity was enhanced in response to BPA treatment. Bay61–3606, Syk inhibitor, decreased LC3 and the expression of Atg7 and Beclin1, leading to the increase of BPA-induced B cell death. The results suggest that BPA-induced autophagy ameliorates human B cell death through Nrf2-mediated regulation of Atg7 and Beclin1 expression.

Genotoxicity of advanced glycation end products in vitro is influenced by their preparation temperature, purification and cell exposure time.

Advanced glycation end products (AGEs) are formed via non-enzymatic reactions between amino groups of proteins and the carbonyl groups of reducing sugars. Previous studies have shown that highly glycated albumin prepared using a glucose-bovine serum albumin (Glu-BSA) model system incubated at 60°C for 6 weeks induces genotoxicity in WIL2-NS cells at 9 days of exposure measured by the cytokinesis-block micronucleus cytome (CBMNcyt) assay. However, this AGE model system is not physiologically relevant as normal body temperature is 37°C and the degree of glycation may exceed the extent of albumin modification in vivo. We hypothesised that the incubation temperature and purification method used in these studies may cause changes to the chemical profile of the glycated albumin and may influence the extent of genotoxicity observed at 3, 6 and 9 days of exposure. We prepared AGEs generated using Glu-BSA model systems incubated at 60°C or 37°C purified using trichloroacetic acid (TCA) precipitation or ultrafiltration (UF) and compared their chemical profile (glycation, oxidation, and aggregation) and genotoxicity in WIL2-NS cells using the CBMNcyt assay after 3, 6 and 9 days of exposure. The number of micronuclei (MNi) was significantly higher for cells treated with Glu-BSA incubated at 60°C and purified via TCA (12 ± 1 MNi/1000 binucleated cells) compared to Glu-BSA incubated at 37°C and purified using UF (6 ± 1 MNi/1000 binucleated cells) after 9 days (P < 0.0001). The increase in genotoxicity observed could be explained by a higher level of protein glycation, oxidation, and aggregation of the Glu-BSA model system incubated at 60°C relative to 37°C. This study highlighted that the incubation temperature, purification method and cell exposure time are important variables to consider when generating AGEs in vitro and will enable future studies to better reflect in vivo situations of albumin glycation.

Methylglyoxal induces chromosomal instability and mitotic dysfunction in lymphocytes.

Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis.

CXCR4 promotes B cell viability by the cooperation of nuclear factor (erythroid-derived 2)-like 2 and hypoxia-inducible factor-1\u03b1 under hypoxic conditions

B cells that interact with T cells play a role in regulating the defense function by producing antibodies and inflammatory cytokines. C-X-C chemokine receptor type 4 (CXCR4) is a specific receptor for stromal cell-derived factor 1 (SDF-1) that controls various B cell functions. Here, we investigated whether CXCR4 regulates B cell viability by inducing hypoxia-inducible factor (HIF)-1α and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) under a hypoxic condition in WiL2-NS human B cells. Nrf2 and CXCR4 expressions increased significantly when WiL2-NS cells were incubated under a hypoxic condition. Interfering with CXCR4 expression using CXCR4-siRNA inhibited cell viability. CXCR4 expression also decreased after treatment with a HIF inhibitor under the hypoxic condition, leading to inhibited cell viability. Increased reactive oxygen species (ROS) levels and the expression of HIF-1α and Nrf2 decreased under the hypoxic condition following incubation with N-acetylcysteine, a ROS scavenger, which was associated with a decrease in CXCR4 expression. CXCR4 expression was augmented by overexpressing Nrf2 after transfecting the pcDNA3.1-Nrf2 plasmid. CXCR4 expression decreased and HIF-1α accumulation decreased when Nrf2 was inhibited by doxycycline in tet-shNrf2-expressed stable cells. Nrf2 or HIF-1α bound from −718 to −561 of the CXCR4 gene promoter as judged by a chromatin immunoprecipitation assay. Taken together, these data show that B cell viability under a hypoxic condition could be regulated by CXCR4 expression through binding of HIF-1α and Nrf2 to the CXCR4 gene promoter cooperatively. These results suggest that CXCR4 could be an additional therapeutic target to control B cells with roles at disease sites under hypoxic conditions.

BAFF attenuates oxidative stress-induced cell death by the regulation of mitochondria membrane potential via Syk activation in WiL2-NS B lymphoblasts

Cell survival is facilitated by the maintenance of mitochondrial membrane potential (MMP). B cell activating factor (BAFF) plays a role in survival, differentiation, and maturation of B cells. In the present study, we examined whether BAFF could attenuate oxidative stress-induced B cell death by the regulation of MMP collapse via spleen tyrosine kinase (Syk) activation using WiL2-NS human B lymphoblast cells. BAFF binds to receptors on WiL2-NS cells. When the cells were incubated in serum-deprived conditions with 1% fetal bovine serum (FBS), BAFF reduced the percentage of dead cells as determined through trypan blue staining and caspase 3 activity. BAFF also inhibited MMP collapse with 1% FBS, as indicated by a decrease in the number of cells with high-red fluorescence of MitoProbe™ JC-1 reagent or a decrease in the percentage of DiOC6-stained cells. Reactive oxygen species (ROS) production was reduced by incubation with BAFF in the presence of 10% or 1% FBS. BAFF inhibited MMP collapse, cell growth retardation, dead cell formation, and caspase 3 activation caused by treatment with H2O2. Syk phosphorylation on tyrosine (Y) 525/526 was increased in cells incubated with 1% FBS in the presence of BAFF than cells incubated with 1% FBS or BAFF alone. BAY61-3606, a Syk inhibitor reduced the effect of BAFF on MMP collapse, caspase 3 activation, cell growth retardation, and dead cell formation. Together, these data demonstrate that BAFF might attenuate oxidative stress-induced B cell death and growth retardation by the maintenance of MMP through Syk activation by Y525/526 phosphorylation. Therefore, BAFF and Syk might be therapeutic targets in the pathogenesis of B cell-associated diseases such as autoimmune disease.

Can a digital slide scanner and viewing technique assist the visual scoring for the cytokinesis-block micronucleus cytome assay?

The cytokinesis-block micronucleus cytome (CBMNcyt) assay is a comprehensive method to measure DNA damage, cytostasis and cytotoxicity caused by nutritional, radiation and chemical factors. A slide imaging technique has been identified as a new method to assist with the visual scoring of cells for the CBMNcyt assay. A NanoZoomer S60 Digital Pathology slide scanner was used to view WIL2-NS cells treated with hydrogen peroxide (H2O2) and measure CBMNcyt assay biomarkers using a high-definition desktop computer screen. The H2O2-treated WIL2-NS cells were also scored visually using a standard light microscope, and the two visual scoring methods were compared. Good agreement was found between the scoring methods for all DNA damage indices (micronuclei, nucleoplasmic bridges and nuclear buds) and nuclear division index with correlation R values ranging from 0.438 to 0.789, P < 0.05. Apoptotic and necrotic cell frequency was lower for the NanoZoomer scoring method, but necrotic frequency correlated well with the direct visual microscope method (R = 0.703, P < 0.0001). Considerable advantages of the NanoZoomer scoring method compared to direct visual microscopy includes reduced scoring time, improved ergonomics and a reduction in scorer fatigue. This study indicates that a digital slide scanning and viewing technique may assist with visual scoring for the CBMNcyt assay and provides similar results to conventional direct visual scoring.

Advanced glycation end-products accelerate telomere attrition and increase pro-inflammatory mediators in human WIL2-NS cells.

This study investigated the effect of dietary sugars and advanced glycation end-products (AGE) on telomere dynamics in WIL2-NS cells. Dietary sugars [glucose (Glu) and fructose (Fru); 0.1 M each] were incubated with bovine serum albumin (BSA) (10 mg/ml) at 60 ± 1°C for 6 weeks to generate AGE-BSA. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed total AGE levels as 87.74 ± 4.46 nmol/mg and 84.94 ± 4.28 nmol/mg respectively in Glu-BSA and Fru-BSA model. Cell treatment studies using WIL2-NS cells were based on either glucose, fructose (each 2.5-40 mM) or AGE-BSA (200-600 µg/ml) in a dose-dependent manner for 9 days. Telomere length (TL) was measured using qPCR. Nitric oxide (NO) production and tumour necrosis factor-α (TNF-α) levels were measured in WIL2-NS culture medium. An increasing trend for TNF-α and NO production was observed with higher concentration of glucose (R2 = 0.358; P = 0.019; R2 = 0.307; P = 0.027) and fructose (R2 = 0.669; P = 0.001; R2 = 0.339; P = 0.006). A decreasing trend for TL (R2 = 0.828; P = 0.000), and an increasing trend for NO production (R2 = 0.352; P = 0.031) were observed with increasing Glu-BSA concentrations. Fru-BSA treatment did not show significant trend on TL (R2 = 0.135; P = 0.352) with increasing concentration, however, a significant reduction was observed at 600 µg/ml (P < 0.01) when compared to BSA treatment. No trends for TNF-α levels and a decreasing trend on NO production (R2 = 0.5201; P = 0.019) was observed with increasing Fru-BSA treatment. In conclusion, this study demonstrates a potential relationship between dietary sugars, AGEs and telomere attrition. AGEs may also exert telomere shortening through the production of pro-inflammatory metabolites, which ultimately increase the risk of diabetes complications and age-related disease throughout lifespan.

Dietary sugars and related endogenous advanced glycation end-products increase chromosomal DNA damage in WIL2-NS cells, measured using cytokinesis-block micronucleus cytome assay

This study investigated the effect of glucose and fructose, and advanced glycation end-products (AGEs) on genome damage in WIL2-NS cells, measured using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The effect of AGEs was investigated using the bovine serum albumin (AGE-BSA) model system induced either with glucose (Glu-BSA) or with fructose (Fru-BSA). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed higher Nε-carboxymethyllysine (CML; 26.76 ± 1.09 nmol/mg BSA) levels in the Glu-BSA model. Nε-Carboxyethyllysine (CEL; 7.87 ± 0.19 nmol/mg BSA) and methylglyoxal-derived hydroimidazolone-1 (MG-H1; 69.77 ± 3.74 nmol/mg BSA) levels were higher in the Fru-BSA model. Genotoxic effects were measured using CBMN-Cyt assay biomarkers [binucleated(BN) cells with micronuclei (MNi), BN with nucleoplasmic bridges (NPBs) and BN with nuclear buds (NBuds)] following 9 days of treatment with either glucose, fructose, Glu-BSA or Fru-BSA. Fructose treatment exerted a significant genotoxic dose-response effect including increases of BN with MNi (R2 = 0.7704; P = 0.0031), BN with NPBs (R2 = 0.9311; P < 0.0001) and BN with NBuds (R2 = 0.7118; P = 0.0091) on cells, whereas the DNA damaging effects of glucose were less evident. High concentrations of AGEs (400-600 µg/ml) induced DNA damage; however, there was no effect on cytotoxicity indices (necrosis and apoptosis). In conclusion, this study demonstrates a potential link between physiologically high concentrations of reducing sugars or AGEs with increased chromosomal damage which is an important emerging aspect of the pathology that may be induced by diabetes. Ultimately, loss of genome integrity could accelerate the rate of ageing and increase the risk of age-related diseases over the long term. These findings indicate the need for further research on the effects of glycation on chromosomal instability and to establish whether this effect is replicated in humans in vivo.

Bisphenol A and its substitutes regulate human B cell survival via Nrf2 expression

B cells contribute to produce inflammatory cytokines and antibodies, to present autoantigens, and to interact with T cells, which lead to body defense and disease control. Nuclear factor (erythroid-derived 2)-like 2(Nrf2) is responsible for gene expression of antioxidant enzymes to protect cells from oxidative stress by reactive oxygen species(ROS) production. Bisphenol A(BPA) may not be safe due to the effect on body’s physiological functions. The chemicals that substitute for BPA may still have similar effects in the body. Tritan™ copolyester is a novel plastic form using BPA substitutes, 1,4-cyclohexanedimethanol(CHDM), dimethyl terephthalate(DMT), and 2,2,4,4-tetramethyl-1,3-cyclobutanediol(TMCD). Isosorbide(ISO) was also used as a substitute for TMCD and DMT. Here, we investigated whether B cell viability is influenced by BPA and its substitutes via Nrf2 induction using WiL2-NS human B lymphoblast cells. When cytotoxicity was measured by using assays with MTT, CellTiter-Glo, trypan blue and propidium iodide, cytotoxicity by BPA was higher than that by substitutes. BPA and its substitutes showed significant cytotoxicity and ROS production, which were attenuated by the treatment with N-acetylcysteine(NAC), a ROS scavenger. In addition, BPA treatment enhanced gene expression of antioxidant enzymes, heme oxygenase(HO)-1, catalase, superoxide dismutase(SOD) 1 and 2. As H2O2 treatment induced cell death and Nrf2 amount in WiL2-NS cells, BPA treatment increased Nrf2. Cell death by H2O2 was increased in doxycycline-inducible Nrf2-knockdown(KD) cells. In Cytotoxicity by the treatment with BPA or its substitutes was also enhanced in Nrf2-KD cells but that was reduced by Nrf2 overexpression compared to control cells. Taken together, these results implicate that B cell cytotoxicity by substitutes should be lower than BPA and Nrf2 can prevent B cells from BPA- or BPA substitutes-induced cytotoxicity via ROS production. Data suggest that the comprehensive studies or evaluation could be necessary to replace BPA in manufacture by other substitutes.

Spleen tyrosine kinase-dependent Nrf2 activation regulates oxidative stress-induced cell death in WiL2-NS human B lymphoblasts

Autoimmune rheumatic lesions are often characterised by the immune cell recruitment including B lymphocytes and the presence of reactive oxygen species (ROS), which increase antioxidant gene transcription via nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Spleen tyrosine kinase (Syk) has a major role in the signal transmission of all haematopoietic lineage cells including B/T cells, mast cells, and macrophages. In this study, we investigated whether B cell survival is regulated by Nrf2 via ROS-mediated Syk activation in WiL2-NS human B lymphoblast cells. When WiL2-NS cells were incubated with 1% foetal bovine serum (FBS), the survival rate and mitochondrial membrane potential (MMP) were reduced. In addition, 1% FBS increased caspase 3 activity, cytochrome C release, nuclear localisation of Nrf2, and ROS production. N-acetylcysteine attenuated ROS production and nuclear translocation of Nrf2. It also inhibited cell death, caspase 3 activation, MMP collapse, and cytochrome C release. Results from the 1% FBS treatment were consistent with those of H2O2 treatment. Syk phosphorylation at tyrosine 525/526 was increased by incubation with 1% FBS or treatment with 100 µM H2O2. Nuclear translocation of Nrf2 by H2O2 was inhibited by treatment with BAY61-3606, a Syk inhibitor. BAY61-3606 also promoted MMP collapse, cytochrome C release, caspase 3 activation, and cell death. Taken together, these results implicate that Syk controls oxidative stress-induced human B cell death via nuclear translocation of Nrf2 and MMP collapse. These results suggest that Syk is a novel regulator of Nrf2 activation.

Genotoxicity and cytotoxicity of chromium, copper, manganese and lead, and their mixture in WIL2-NS human B lymphoblastoid cells is enhanced by folate depletion

Heavy metal exposure or dietary deficiency is associated with increased genetic damage, cancer and age-related diseases. Folate (vitamin B9) required for DNA repair and synthesis may increase cellular susceptibility to metal induced genotoxicity. This study investigated the interactive effects of folic acid deficiency and sufficiency on genome instability and cytotoxicity induced by chromium (VI), copper (II), manganese (II), lead (IV), and their mixture (CCMP) in WIL2-NS human B lymphoblastoid cells. WIL2-NS cells were cultured in folic acid deficient (20nM) and replete (2000nM) RPMI 1640 medium treated with different concentrations (0.00–1000μM) of the metals and CCMP for 48h. Chromosomal damage and cytotoxicity were measured using the Cytokinesis-block Micronucleus Cytome assay. CCMP, Cr, Pb, Cu and Mn induced concentration dependent, increases in cells with chromosome damage (micronuclei, nucleoplasmic bridges, nuclear buds) and necrotic cells and decreased nuclear division index. The metals exhibited different cytotoxic and genotoxic potentials (CCMP>Cr>Pb>Cu>Mn) in both folate deficient and sufficient cells, with the cytogenotoxic effects being greater in folate deficient cells. Significant interaction between the metals and folic acid suggests that folic acid deficiency exacerbated cell proliferation inhibition and genome instability induced by metals. Folate deficiency, increasing metal concentration, and their interactions explained 3–11%, 74–92% and 4–12% of the variance of DNA damage biomarkers. In conclusion, exposure to the tested metals (0.01–1000μM) increased chromosomal DNA damage in WIL2-NS cells and this was exacerbated by folate deficiency.

Flow-cytometric analysis of in vitro micronucleus formation: Comparative studies with WIL2-NS human lymphoblastoid and L5178Y mouse lymphoma cell lines

The development of a flow cytometry-based micronucleus (FCMMN) assay for measuring the micronucleus (MN) frequency in cells following exposure to test chemicals has potential for improving reproducibility and reducing turn-around time when compared with the traditional microscopy-based micronucleus method. A major drawback of the FCMMN assay is that a false-positive interpretation could result from the presence of large numbers of apoptotic or necrotic bodies in the measured sample. Although several studies have reported ways in which the FCMMN assay could be improved using different staining techniques or electronic gating strategies, to date none of these protocols are suitable for use as a screening assay. To reduce the interference from apoptosis, performing the FCMMN assay with an apoptosis-resistant cell line may be an alternative approach. This study reports the use of p53-mutated cell lines to minimise the interference found in the FCMMN assay. Two commonly used cell lines (WIL2-NS and L5178Y) were investigated by comparison of (1) cytotoxicity and micronucleus induction in the FCMMN assay following treatment with model genotoxicants and (2) apoptotic responses after exposure to inducers of apoptosis. Both cell lines were responsive to all genotoxicants, producing concentration-dependent results with respect to genotoxicity. WIL2-NS cells were found to be more tolerant to apoptosis induction than L5178Y cells. This characteristic could be beneficial to minimise the interference from apoptotic nuclei in the FCMMN genotoxicity-screening assay.

Cytotoxicity and genotoxicity of ultrafine crystalline SiO2 particulate in cultured human lymphoblastoid cells

Respirable crystalline silica has been classified as a human lung carcinogen. Ultrafine (diameter < 100 nm) silica particles may be important in carcinogenesis, although the mechanisms remain unclear. In the present study, WIL2-NS cells were incubated for 6, 24, and 48 hr with 0, 30, 60, and 120 microg/ml ultrafine crystalline SiO(2) (UF-SiO(2)). The cytotoxic and genotoxic effects caused by UF-SiO(2) in cultured human cells were investigated via a set of bioassays. Significant dose- dependent decreases in percent cell viability were seen with increasing dose of UF-SiO(2) in the methyl tetrazolium assay. Significant decreases were seen at 120 microg/ml (58, 38, and 57% for 6, 24, and 48-hr exposure, respectively). During 4 days growth in the flasks, there was a slight recovery observed after washing off UF-SiO(2) as measured by the population growth assay. Significant dose-dependent reduction in the cytokinesis block proliferation index was observed by the cytokinesis block micronucleus assay. Treatment with 120 microg/ml UF-SiO(2) for 24 hr produced a fourfold increase in the frequency of micronucleated binucleated cells (MNBNC). The increase in MNBNC was dose-dependent. The lowest dose that gave a statistically significant increase in MNBNC was 30 microg/ml (24-hr treatment), which had cytotoxicity of less than 10%. There was no significant difference in DNA strand breakage as measured by the Comet assay. A significant increase in induced mutant frequency was found at 120 microg/ml as detected by the hypoxanthine guanine phosphoribosyltransferase mutation assay. The results indicate that UF-SiO(2) is cytotoxic and genotoxic in cultured human cells.

Ultrafine Quartz-Induced Damage in Human Lymphoblastoid Cells in vitro Using Three Genetic Damage End-Points

ABSTRACTRespirable quartz is a potential human lung carcinogen. The mechanisms involved in this carcinogenesis, however, remain unclear, especially for the ultrafine particles (diameter <100 nm). The aim of the present study was to investigate the effects caused by ultrafine quartz (UF-quartz) in a human cell culture model. Genotoxicity and cytotoxicity induced by UF-quartz were investigated through the cytokinesis block micronucleus assay (CBMN), the Comet assay, the HPRT assay, the population growth assay, and the 3-(4, 5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. WIL2-NS cells were incubated for 10h with 0, 60, and 120 μg/mL UF-quartz. Significant decreases in percent of cell survival in the MTT assay were seen at higher doses, for example, 83%, and 64% relative survival at 60 μg/mL and 120 μg/mL, respectively. Only slight population regrowth was observed, with the population sizes recovering slightly by day 4 after quartz particles were removed. A significant increase in the frequency of micronucleated binucleated cells (MNed BNCs) was seen with 120 μg/mL quartz, from approximately 5 in 1000 BNCs in controls to 12 in 1000 BNCs. A significant reduction in the nuclear division index was observed by the CBMN assay, indicating inhibition of cell division by high-dose UF-quartz. A dose-dependent increase in induced HPRT-gene locus mutant frequency with increasing dose of UF-quartz was observed by the HPRT assay. No significant difference was found in DNA strand breakage as detected by the Comet assay. Collective findings suggest that UF-quartz can cause cytotoxicity and genotoxicity to human lymphoblasts in this model system.

Cyto- and genotoxicity of ultrafine TiO 2 particles in cultured human lymphoblastoid cells

Titanium dioxide is frequently used in the production of paints, paper, plastics, welding rod-coating material, and cosmetics, because of its low toxicity. However, recent studies have shown that nano-sized or ultrafine TiO 2 (UF-TiO 2) (<100 nm in diameter) can generate pulmonary fibrosis and lung tumor in rats. Cytotoxicity induced by UF-TiO 2 in rat lung alveolar macrophages was also observed. This generates great concern about the possible adverse effects of UF-TiO 2 for humans. The cytotoxicity and genotoxicity of UF-TiO 2 were investigated using the methyl tetrazolium cytotoxicity (MTT) assay, the population growth assay, the apoptosis assay by flow cytometry, the cytokinesis block micronucleus (CBMN) assay, the comet assay, and the hypoxanthine-guanine phosphoribosyltransferase ( HPRT) gene mutation assay. WIL2-NS cells were incubated for 6, 24 and 48 h with 0, 26, 65 and 130 μg/ml UF-TiO 2. Significant decreases in viability were seen in the MTT assay at higher doses; for example, 61, 7 and 2% relative viability at 130 μg/ml for 6, 24 and 48-h exposure ( P < 0.01). A dose-dependent relationship was observed, while a time-dependent relationship was seen only at the highest dose (130 μg/ml) after exposure for 24 and 48 h. Treatment with 130 μg/ml UF-TiO 2 induced approximately 2.5-fold increases in the frequency of micronucleated binucleated cells ( P < 0.01). In addition, a significant reduction in the cytokinesis block proliferation index was observed by the CBMN assay ( P < 0.05). In the comet assay, treatment with 65 μg/ml UF-TiO 2 induced approximately 5-fold increases in olive tail moment ( P < 0.05). In the HPRT mutation assay, treatment with 130 μg/ml UF-TiO 2 induced approximately 2.5-fold increases in the mutation frequency ( P < 0.05). The results of this study indicate that UF-TiO 2 can cause genotoxicity and cytotoxicity in cultured human cells.

Anti-genotoxic effects of tea catechins against reactive oxygen species in human lymphoblastoid cells

Using the cytokinesis-block micronucleus assay in WIL2-NS cells, we investigated the effects of six tea constituents, (−)-epigallocatechin-3-O-gallate (EGCg), (−)-epicatechin-3-O-gallate (ECg), (−)-epigallocatechin (EGC), (−)-epicatechin (EC), (+)-catechin (+C) and gallic acid (GA), on chromosomal damage in two ways; induction by each component on its own and prevention against treatment of reactive oxygen species (ROS). None of the tea constituents induced chromosomal damage at <10μM. On the other hand, EGCg, EGC, ECg, +C and GA prevented H2O2-induced chromosomal damage in a dose-dependent manner with a significant effect detected at 1μM. Chromosomal damage induced by tert-butylhydroperoxide was apparently prevented by EGCg and ECg at 0.3μM, but not by EGC and GA even at 10μM, suggesting that the galloyl group linked to flavan-3-ol is needed for the observed protective effect. These results suggest that physiological concentration of tea constituents are not genotoxic but rather anti-genotoxic against ROS, although their preventive effects are slightly different depending on their chemical structure.

Grape seed extract prevents H(2)O(2)-induced chromosomal damage in human lymphoblastoid cells.

We examined the effects of grape seed extract (GSE) on chromosomal damage in two ways; induction on its own and prevention against treatment of reactive oxygen species (ROS). Chromosomal damage was evaluated by cytokinesis-block micronucleus assay (CBMN) in a human lymphoblastoid cell line, WIL2-NS cells. The GSE was composed of 89% proanthocyanidin with a degree of polymerization ranging from 2 to 15. GSE did not induce chromosomal damage in WIL2-NS cells at GSE concentrations up to 5 mg/l. In contrast, pretreatment with GSE dose-dependently prevented H(2)O(2)-induced chromosomal damage at an effective dose of 0.3 to 1 mg/l. A similar preventive effect of GSE was not detected in tert-butyl hydroperoxide-induced damage even at 5 mg/l. In a cell free system, GSE (<5 mg/l) directly scavenged H(2)O(2), but produced slight amounts of H(2)O(2) at higher concentrations (>50 mg/l). These results suggest that GSE is not genotoxic, but rather has an antigenotoxic effect against H(2)O(2) via direct scavenging action of H(2)O(2).

Physiological Concentrations of (−)-Epigallocatechin-3-O-Gallate (EGCg) Prevent Chromosomal Damage Induced by Reactive Oxygen Species in WIL2-NS Cells

We investigated the effects of (-)-epigallocatechin-3-O-gallate (EGCg) on chromosomal damage, which was evaluated by a cytokinesis-block micronucleus (CBMN) assay using WIL2-NS cells. EGCg itself induced chromosomal damage at 100 micromol/L. This damage was due to the production of H(2)O(2) by EGCg. In contrast, EGCg at < 10 micromol/L did not induce chromosomal damage and did not produce H(2)O(2). In addition, EGCg at < 10 micromol/L dose-dependently prevented chromosomal damage induced by H(2)O(2), tert-butyl hydroperoxide (tert-BuOOH) and superoxide, all of which are reactive oxygen species (ROS). A large amount of EGCg was present in cells after they were incubated with 0.3 micromol/L EGCg. When extracellular EGCg was removed and EGCg was present only inside of cells, the preventive effect of EGCg against tert-BuOOH-induced chromosomal damage was diminished but not that against the other two ROS tested. Direct interactions of EGCg with tert-BuOOH and superoxide but not with H(2)O(2) were detected. These findings suggest that physiological concentrations of EGCg (< 1 micromol/L) are not genotoxic but rather, can prevent ROS-induced chromosomal damage.

Detection of oxidant-induced slight chromosomal damage in cells by subsequent exposure to X-rays.

We examined whether slight oxidative stress and/or damage in cells could be amplified by subsequent ionizing irradiation and thus become detectable as obvious chromosomal damage. WIL2-NS cells, a human B lymphoblastoid cell line, were pretreated with an oxidant and then exposed to X-rays at 0.25 or 0.5 Gy. The chromosomal damage in the cells was evaluated by cytokinesis-block micronucleus (CBMN) assay. Pretreatment with a superoxide-generating system (hypoxanthine (HX)/xanthine oxidase (XO), 1 and 2 mU/ml of XO), tert-butyl hydroperoxide (t-BuOOH, 10 and 100 microM) or H2O2 (5 microM) alone did not induce significant chromosomal damage, but the oxidant-induced damage increased significantly with subsequent irradiation. The tested dose of these oxidants did not induce significant changes in cell viability, the nuclear division index, and the concentration of antioxidants, indicating that only weak oxidative stress was introduced into the cells. These results suggest that low-dose oxidant-induced chromosomal damage becomes detectable as obvious chromosomal damage with subsequent ionizing irradiation in vitro.

RPA foci are associated with cell death after irradiation.

MacPhail, S. H. and Olive, P. L. RPA Foci are Associated with Cell Death after Irradiation. Radiat. Res. 155, 672-679 (2001). Complexes containing replication protein A (RPA) were observed in human TK6 and WIL-2NS lymphoblast cells and SiHa cervical carcinoma cells exposed to 250 kV X rays. Image analysis of individual cells with fluorescence-tagged anti-RPA antibodies was used to measure numbers of discrete foci per cell. RPA foci formed in S-phase cells in response to radiation doses as low as 0.5 Gy, and the number of foci/nucleus was linearly related to dose up to 50 Gy. The maximum number of cells with foci occurred 4-8 h after exposure to 4 Gy, and subsequently declined. However, the number of RPA foci per nucleus (in those cells with foci) reached a maximum after 2-4 h. Apoptotic nuclei from irradiated TK6 and WIL-2NS cells initially contained foci, but these were lost as degradation continued. Radiation-induced micronuclei in SiHa cells were greatly enriched for RPA foci, and cells with nuclei without foci often contained micronuclei with multiple RPA foci. In SiHa cells examined up to 7 days after 4 Gy, RPA foci reappeared in one or more cells in up to 90% of the surviving colonies, and some cells contained 150 or more distinct foci. Reappearance of these complexes could be indicative of radiation-induced genomic instability. These results are consistent with the idea that RPA foci observed several hours after irradiation represent irreparable lesions and as such might be useful in identifying radiosensitive cells.