Nanosized titanium dioxide particles do not induce DNA damage in human peripheral blood lymphocytes

Industrial application of titanium dioxide nanoparticles (TiO₂‐NPs) as an additive in pharmaceutical and cosmetic products is increasing. However, the knowledge about the toxicity of this material is still incomplete and data concerning health and environmental safety and results of recent studies on TiO₂ nanotoxicology are inconsistent. The in vitro geno‐ and cytotoxicity of TiO₂‐NPs in the anatase crystal phase was evaluated in human peripheral blood lymphocytes from 10 male donors. Initially, transmission electron microscopy (TEM) was performed to describe particle morphology and size, the degree of particle aggregation, and the intracellular distribution. Cells were exposed to nanoparticles in increasing concentrations of 20, 50, 100, and 200 μg/ml for 24 hr. Cytotoxic effects were analyzed by trypan blue exclusion test and the single‐cell microgel electrophoresis (comet) assay was applied to detect DNA double‐strand breakage. TiO₂‐NPs were sphere shaped with a diameter of 15–30 nm. Despite dispersive pretreatment, a strong tendency to form aggregates was observed. Particles were detected in the cytoplasm of lymphocytes, but also a transfer into the nucleus was seen. The trypan blue exclusion test did not show any decrease in lymphocyte viability, and there was no evidence of genotoxicity in the comet assay for any of the tested concentrations. In conclusion, TiO₂‐NPs reached the cytoplasm as well as the nucleus and did not induce cyto‐ or genotoxic effects in human peripheral blood lymphocytes. Complement investigations on different human cell systems will be performed to estimate the biocompatibility of TiO₂‐NPs. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss, Inc.     

二氧化硫吸入对小鼠脾细胞凋亡的诱导作用

目的:探讨二氧化硫大剂量吸入对小鼠脾细胞凋亡和脾脏组织学结构的影响.方法:以不同浓度的二氧化硫分别对小鼠连续染毒7d,用透射电镜法、DNA琼脂糖凝胶电泳法和流式细胞技术观察小鼠脾脏组织学结构和细胞凋亡改变.结果:在二氧化硫染毒组小鼠脾脏的红髓区和白髓区均有典型的脾细胞凋亡发生,在边缘区可见大量核变形淋巴细胞,此外还可见巨噬细胞出现明显的凋亡改变和网状内皮细胞受损.168mg/m3二氧化硫染毒可引起小鼠脾细胞凋亡率增加.结论:大剂量二氧化硫吸入可引起小鼠脾脏超微结构改变,在一定剂量和时间范围内可引起脾细胞凋亡加速,从而对机体的免疫功能造成一定损伤.     

Sensitivity of peripheral blood lymphocytes from breast cancer patients to glucocorticoids

We measured DNA-dependent RNA polymerase activity, calculated hormone-binding sites and intracellular association constant under conditions of phytohemagglutinin stimulation and dexamethasone treatment, and studied the interaction of cells with the glucocorticoid covalently bound to an inert carrier not penetrating into cells. Reduced sensitivity of peripheral blood lymphocytes from breast cancer patients to glucocorticoids was demonstrated. The biological mechanisms for hormonal regulation are reviewed here. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 1, pp. 80–84, January, 2006     

Titanium dioxide nanoparticles trigger p53-mediated damage response in peripheral blood lymphocytes

Titanium dioxide nanoparticles (nano-TiO2) are widely used as a photocatalyst in air and water remediation. These nanoparticles are known to induce toxicity; however, their cytotoxic mechanism is not fully understood. In this study, we investigated the underlying mechanism of nano-TiO2-induced cytotoxicity in peripheral blood lymphocytes. We examined the genotoxic effects of nano-TiO2 in lymphocytes using alkaline single-cell gel electrophoresis (Comet) and cytokinesis-block micronucleus (CBMN) assays. Lymphocytes treated with nano-TiO2 showed significantly increased micronucleus formation and DNA breakage. Western-blot analysis to identify proteins involved in the p53-mediated response to DNA damage revealed the accumulation of p53 and activation of DNA damage checkpoint kinases in nano-TiO2-treated lymphocytes. However, p21 and bax, downstream targets of p53, were not affected, indicating that nano-TiO2 does not stimulate transactivational activity of p53. The generation of reactive oxygen species (ROS) in nano-TiO2-treated cells was also observed, andN-acetylcysteine (NAC) supplementation inhibited the level of nano-TiO2-induced DNA damage. Given that ROS-induced DNA damage leads to p53 activation in the DNA damage response, our results suggest that nano-TiO2 induces ROS generation in lymphocytes, thereby activating p53-mediated DNA damage checkpoint signals.     

Content of T and B lymphocytes in healthy human peripheral blood

It was shown by the rosette-formation method that the number of T and B lymphocytes in human blood varies with the time of day and season of the year. The number of T cells reaches a maximum in the morning and the number of B cells in the evening. The relative percentage of B cells is higher in the fall than in winter.Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 872–874, July, 1976.     

Mechanisms of realization of the combined action of cortisol and adenosine on peripheral blood lymphocytes in bronchial asthma

Department of Molecular Pharmacology and Radiobiology, and Department of Clinical Pharmacology, N. I. Pirogev Second Moscow Medical Institute. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol 111, No. 1, pp. 44–46, January, 1991.     

Interaction between human peripheral blood lymphocytes and autologous erythrocytes

Interaction between lymphocytes and autologous erythrocytes was discovered in patients with autoimmune diseases. The interaction was manifested macroscopically as hemagglutination and microscopically as rosette formation. Interaction between lymphocytes and autologous erythrocytes was not found in a group of healthy subjects or in patients free from autoimmune disturbances.Laboratory of Immunology, Institute of Clinical and Experimental Medicine, Siberian Division, Academy of Medical Sciences of the USSR, Novosibirsk. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 12, pp. 1469–1471, December, 1976.     

二氧化硫对大鼠缺血再灌注心脏的损伤作用

目的观察二氧化硫(SO2)在心肌缺血再灌注损伤中的作用。方法大鼠心脏分为:I/R组,SO2组及天冬氨酸异羟肟酸(HDX)组。采用Langendorff离体心脏灌注模型。MacLab数据采集系统监测离体心脏功能。结果SO2组心功能恢复率明显低于I/R组(P<0.05,P<0.01);HDX组显著高于I/R组(P<0.05,P<0.01)。SO2组冠脉流液中乳酸脱氢酶(LDH)、肌酸激酶(CK)、谷氨酸-草酰乙酸转移酶(GOT)活性、肌红蛋白(Mb)含量及心肌丙二醛(MDA)、共轭双烯键(CD)含量及GOT活性显著高于I/R组(P<0.05,P<0.01);HDX组冠脉流液中上述指标明显低于I/R组(P<0.05,P<0.01)。SO2组心肌还原型谷胱甘肽(GSH)含量明显低于I/R组(P<0.05);HDX组心肌GSH含量显著高于I/R组(P<0.01)。结论SO2参与了大鼠心肌缺血再灌注损伤。增加心肌脂质过氧化及降低心肌还原型谷胱甘肽含量可能与SO2心肌损伤有关。     

Subpopulation composition of peripheral blood lymphocytes in children with infectious mononucleosis

In patients with viral and bacterial infectious mononucleosis during the acute period of disease and clinical convalescence blood content of CD72⁺ and CD16⁺ lymphocytes increased compared to normal. The count of CD8⁺ cells increased in viral mononucleosis during convalescence and these changes persist in delayed periods after convalescence. In bacterial mononucleosis the content of CD72⁺ lymphocytes return normal 18 months after convalescence.     

Effect of dexamethasone on RNA synthesis in human peripheral blood lymphocytes

The action of dexamethasone on lymphoid tissue in a culture of partially purified peripheral blood lymphocytes was biphasic in character. After incubation of lymphocytes in vitro with the hormone for 6 h stimulation of RNA synthesis was found. Sedimentation analysis of labeled RNA fractionated on a column containing poly-V-sepharose indicated an increase in the mRNA content and enrichment of cytoplasmic RNA with polyA sequences. Meanwhile Mn⁺⁺-dependent RNA-polymerase, sensitive to -amanitine, was activated. After cultivation of the lymphocytes with the hormone for 24 h, RNA synthesis was inhibited. The biphasic character of the action of the steroid also was observed in the rosette-formation test.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 811–814, July, 1976.     

DNA damage in mice treated with sulfur dioxide by inhalation

Sulfur dioxide (SO2) is a ubiquitous air pollutant produced by the burning of fossil fuels. In this study, single-cell gel electrophoresis (the Comet assay) was used to evaluate the DNA damage produced by inhalation exposure of mice to SO2. Male and female mice were housed in exposure chambers and treated with 14.00 +/- 1.25, 28.00 +/- 1.98, 56.00 +/- 3.11, and 112.00 +/- 3.69 mg/m3 SO2 for 6 hr/day for 7 days, while control groups were exposed to filtered air. Comet assays were performed on blood lymphocytes and cells from the brain, lung, liver, spleen, kidney, intestine, and testicles of the animals. SO2 caused significant, dose-dependent increases in DNA damage, as measured by Olive tail moment, in all the cell types analyzed from both sexes of mice. The results indicate that inhalation exposure to SO2 damages the DNA of multiple organs in addition to the lung, and suggests that this damage could result in mutation, cancer, and other diseases related to DNA damage. Further work will be required to understand the ultimate toxicological significance of this damage. These data also suggest that detecting DNA damage in blood lymphocytes, using the Comet assay, may serve as a useful tool for evaluating the impact of pulmonary SO2 exposure in human biomonitoring studies.     

鼻咽癌患者外周血淋巴细胞对有丝分裂原的反应

本文探讨了PHA和Con A体外诱导NPC病人和VCA-IgA抗体阳性及阴性者外周血T_4、T_8细胞的变化。结果表明,NPC病人的PBMC对上述二种有丝分裂原诱导T_4和T_8细胞的能力最低,VCA-IgA抗体阳性组次之,VCA-IgA抗体阴性组最高。同时还证实了PHA主要是诱导T_4细胞,而Con A则主要是诱导T_8细胞。     

A mitochondria-targeted ratiometric fluorescent probe to monitor endogenously generated sulfur dioxide derivatives in living cells

Sulfur dioxide (SO₂) can be endogenously produced by enzymes in mitochondria during oxidation of H₂S or sulphur-containing amino acids, and plays important roles in several physiological processes. However, the design and synthesis of fluorescent probes which can detect mitochondrial SO₂ and its derivatives in living cells still remain unresolved. Herein, we report the preparation of a lipophilic cationic dye 1 (Mito-Ratio-SO₂), which targets the mitochondria in living cells and is sensitive to the presence of SO₂ derivatives. The ratiometric probe Mito-Ratio-SO₂ displays a 170 nm blue-shift in emission with two well-resolved emission bands upon addition of sulfite. Mechanistic studies indicate that three probe-SO₂ adducts coexist after reaction, as supported by liquid chromatography and density function theory investigations. Importantly, the ratiometric probe is highly selective for sulfite over other bio-species including H₂S. Fluorescence co-localization studies indicate that the probe localizes solely in the mitochondria of HeLa cells. Last but not least, fluorescent imaging of HeLa cells successfully demonstrates the detection of intrinsically generated intracellular SO₂ derivatives in living cells.     

Adhesion of peripheral blood lymphocytes of children with arthritis to human umbilical vein endothelial cells.

To determine whether adhesion of peripheral blood lymphocytes (PBL) of patients with juvenile rheumatoid arthritis (JRA) may be enhanced, adhesion of PBL of children with JRA, children with seronegative spondyloarthropathies (SSA), age-appropriate and adult controls, to human umbilical vein endothelial cells (HUVEC) was assessed in vitro. B and CD4 T lymphocytes in initial, adherent, and non-adherent cell fraction were identified by flow cytometry. B lymphocytes of all the younger subjects combined had a higher adherence to activated HUVEC compared with B lymphocytes of the adult donors. Except for greater adherence of HLA-DR+ CD4 T cells, lymphocytes of children with JRA showed no enhanced adhesion to either unactivated or activated HUVEC. The percentage of B cells adherent to activated HUVEC in each of the subject groups was 1.5-3.6-fold higher than adherent CD4 T lymphocytes. Surface analyses indicated higher percentages of CD49d (alpha 4)+ and CD29 (beta 1)+ CD4 T lymphocytes in adherent cells, but less of a differential in CD49 (alpha 4)+ and no difference in CD29 (beta 1)+ B lymphocytes. There were fewer Leu-8 (L-selectin)+ B and Leu-8+ CD4 T cells among adherent cells. The data suggest a greater adhesive capacity of B lymphocytes compared with CD4 T lymphocytes which is unrelated to disease, and the possibility that B lymphocytes may utilize adhesion molecules distinct from those of CD4 T lymphocytes. Only a small subset of T cells of patients with JRA may have an enhanced capacity for adhesion to endothelium.     

Role of peripheral blood lymphocytes in experimental allergic (Pertussis) encephalomyelitis

Peripheral blood lymphocytes of guinea pigs with experimental allergic encephalomyelitis (EAE) induced by a single injection of an oily suspension ofBordetella pertussis cells, when cultured with heterologous brain antigen undergo blast transformation, which is most marked on the 14th–21st day after sensitization. With the development of clinical symptoms of EAE the level of blast transformation of the lymphocytes fell and reached its minimum on the 40th day. It is postulated that this may be connected with the development of transient hypersensitivity and dissemination of sensitized lymphocytes into the CNS, leading to its injury.Laboratory of Allergy, I. I. Mechnikov Moscow Scientific-Research Institute of Vaccines and Sera, Ministry of Health of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR, A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 78, No. 9, pp. 75–78, September, 1974.     

Modulation of perforin expression in the decidual and peripheral blood cytotoxic lymphocytes in culture.

PROBLEM: Perforin (P) is a cytolytic molecule located in intracellular granules of cytotoxic lymphocytes both in the peripheral blood and decidua of pregnancy. The aim was to analyze the kinetics of P expression during in vitro culture and modulation of P expression by adherent cells, their supernatants and mitogen (PHA) stimulation. METHOD OF STUDY: P (intracellular antigen) was detected by flow cytometry in the suspension of first trimester pregnancy peripheral blood lymphocytes (PBL) and decidual lymphocytes (DL). RESULTS: A decrease of the percentage of P+ cells was obtained after 1 hr incubation and was prevented by addition of 30% of decidual adherent cells (DAC) or their supernatants. Upregulation of P expression was obtained when, in addition to adherent cells, DL and PBL were stimulated by PHA. DAC present in the culture in physiological concentrations prevent downregulation of P expression. CONCLUSION: DAC located in the vicinity of decidual cytotoxic lymphocytes, owing to their unique ability to produce a wide range of substances on demand, contribute to the high level of P expression in the decidua of pregnancy.     

Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes

Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4⁺ and CD8⁺ T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10⁻³-10⁻¹²M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G₁ phase of cell cycle and inhibits both IL-2 receptor (IL-2R) expression and IL-2 secretion. Apoptosis is clearly increased by PDN in PHA-activated human PBL, and the apoptotic effect of PDN is stronger on CD8⁺ than on CD4⁺ T lymphocytes. All these effects are dose- and time-dependent. The addition of exogenous IL-2 did not rescue lymphocytes from PDN-increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post-thymic level.     

An experimental model for the exposure of human ciliated cells to sulfur dioxide at different concentrations

Mucociliary transport is an important nonimmunological defense mechanism of the respiratory tract. The aim of this study was to investigate the effect of sulfur dioxide (S02) at different concentrations on ciliary beat frequency (CBF). Ciliated cells were obtained from 12 volunteers by nose brush. CBF was quantified using video-interference microscopy. The cells were placed on a polycarbonate membrane in contact with the surface of a reservoir filled with RPMI 1640 (bicarbonate buffered) or Ringer's (electrolyte) solution, allowing the cells to be supplied by capillarity. In an exposure chamber the cells were exposed for 30 min to SO₂ 2.5–12.5 ppm at 37°C and 100% air humidity. SO₂ induced a dose-dependent decrease in CBF of the cells cultured in Ringer's solution. SO₂ at 2.5 ppm caused a 42.8 % decrease and at 12.5 ppm a 96.5% decrease (8.1 ± 0.24 versus 0.28 ± 0.20 Hz). CBF of cells cultured in RPMI 1640 was reduced only moderately after 12.5 ppm SO₂ exposure (7.9 ± 0.26 versus 6.70 ± 0.30 Hz). In Ringer's solution a decrease in pH was observed after 30 min of S02 exposure to 12.5 ppm to a minimum value of 3.6. By contrast, the pH of RPMI 1640 remained constant at 7.5 under identical conditions. After adding RPMI 1640 to Ringer's solution, CBF increased in parallel to the pH to control values (5.0 ppm: 4.64 ± 0.45 to 8.51 ± 0.60 Hz). These data suggest that the highly water-soluble SO₂ reversibly eliminates CBF in correlation with a decrease in pH.Abbreviations CBF ciliary beat frequency Correspondence to: K. Kienast     

The peripheral blood compartment in patients with Graves' disease: activated T lymphocytes and increased transitional and pre‐naive mature B lymphocytes

Graves'' disease (GD) is an autoimmune disease that involves aberrant B and T lymphocyte responses. Detailed knowledge about lymphocyte subpopulation composition will therefore enhance our understanding of the pathogenesis of GD and might support the development of new immunomodulatory treatment approaches. The aim of this study was to gain detailed insight into the composition of the peripheral blood lymphocyte compartment in GD before and during anti-thyroid drug therapy. Major B and T lymphocyte subpopulations were investigated by flow cytometry in peripheral blood from newly diagnosed GD patients (n = 5), GD patients treated with anti-thyroid drugs (n = 4), patients with recurrent GD (n = 7) and healthy controls (HC; n = 10). In GD patients, numbers of activated T lymphocytes [human leucocyte antigen D-related (HLA-DR)⁺ and CD25⁺] were increased. The B lymphocyte compartment in GD was characterized by significantly higher numbers of transitional (CD38^highCD27⁻, P < 0·03) and pre-naive mature (CD38^lowCD27⁻IgD⁺CD5⁺, P < 0·04) B lymphocytes, while memory populations were slightly decreased. The increased numbers of CD5⁺, transitional and pre-naive mature B lymphocytes correlated positively with fT4 plasma levels. GD is associated with increased numbers of activated T lymphocytes and transitional and pre-naive mature CD5⁺ B lymphocytes within the peripheral blood. The increase in CD5⁺ B lymphocytes was due mainly to an increase in transitional and pre-naive mature B lymphocytes. Increased fT4 plasma levels might be associated with this increase in transitional and pre-naive mature CD5⁺ B lymphocytes.