Oxygen tolerance and coupling of mitochondrial electron transport

Oxygen is critical to aerobic metabolism, but excessive oxygen (hyperoxia) causes cell injury and death. An oxygen-tolerant strain of HeLa cells, which proliferates even under 80% O2, termed "HeLa-80," was derived from wild-type HeLa cells ("HeLa-20") by selection for resistance to stepwise increases of oxygen partial pressure. Surprisingly, antioxidant defenses and susceptibility to oxidant-mediated killing do not differ between these two strains of HeLa cells. However, under both 20 and 80% O2, intracellular reactive oxygen species (ROS) production is significantly (approximately 2-fold) less in HeLa-80 cells. In both cell lines the source of ROS is evidently mitochondrial. Although HeLa-80 cells consume oxygen at the same rate as HeLa-20 cells, they consume less glucose and produce less lactic acid. Most importantly, the oxygen-tolerant HeLa-80 cells have significantly higher cytochrome c oxidase activity (approximately 2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. Indeed, preferential inhibition of cytochrome c oxidase by treatment with n-methyl protoporphyrin (which selectively diminishes synthesis of heme a in cytochrome c oxidase) enhances ROS production and abrogates the oxygen tolerance of the HeLa-80 cells. Thus, it appears that the remarkable oxygen tolerance of these cells derives from tighter coupling of the electron transport chain.     

Cytochrome C oxidase activity and oxygen tolerance

Most cultured cells and intact animals die under hyperoxic conditions. However, a strain of HeLa cells that proliferates under 80% O(2), termed "HeLa-80," has been derived from wildtype HeLa cells ("HeLa-20") by selection for resistance to stepwise increases of oxygen partial pressure. The tolerance of HeLa-80 cells to hyperoxia is not associated with changes in antioxidant defenses or susceptibility to oxidant-mediated killing. Rather, under both 20 and 80% O(2), mitochondrial reactive oxygen species (ROS) production is approximately 2-fold less in HeLa-80 cells, likely related to a significantly higher cytochrome c oxidase (COX) activity ( approximately 2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. We now report that in HeLa-80 cells elevated COX activity is associated with a >2-fold increase in the regulatory subunit COX Vb, whereas expression levels of other subunits are very close to wild type. Small interfering RNA against Vb selectively lowers COX Vb expression in HeLa-80 cells, increases mitochondrial ROS generation, decreases COX activity 60-80%, and diminishes viability under 80% (but not 20%) O(2). In addition, overexpression of subunit Vb increases COX activity and decreases ROS production in wild-type HeLa-20 cells, along with some increase in tolerance to hyperoxia. Overall, our results indicate that it is possible to make cells tolerant of hyperoxia by manipulation of mitochondrial electron transport. These observations may suggest new pharmaceutical strategies to diminish oxygen-mediated cellular damage.     

三氧化二砷（As2O3）对蚕豆根尖毒性效应的细胞遗传学研究

本文以蚕豆根尖为材料,研究不同浓度As2O3在不同的处理时间内对蚕豆胚根根尖的细胞遗传学毒性效应。结果表明：不同浓度的As2O3在不同处理时间内均能诱发较高的微核率和染色体畸变率,并能有效地积累中期分裂相,阻止其进入后期与末期。结论是As2O3对蚕豆根尖细胞具有明显的细胞遗传学毒性效应,并具有积累有丝分裂中期细胞的效应。 Abstract:The cytogenetic toxic effects of different concentration of As2O3 within different time on the cell of Vicia Faba root tip were studied.The results indicate that the different concentrations of As2O3 can induce high frequency of micronucleus and chromosome aberration.Besides,it can accumulate the metaphases in mitosis efficiently and prevent the cells from continuing their cell cycle.It shows that As2O3 has marked cytogenetic toxic effect on the root tip cells of Vicia Faba,and effect of accumulating the metaphases in mitosis.     

Genetic Characteristics of the Human Hepatic Stellate Cell Line LX-2

The human hepatic cell line LX-2 has been described as tool to study mechanisms of hepatic fibrogenesis and the testing of antifibrotic compounds. It was originally generated by immortalisation with the Simian Vacuolating Virus 40 (SV40) transforming (T) antigen and subsequent propagation in low serum conditions. Although this immortalized line is used in an increasing number of studies, detailed genetic characterisation has been lacking. We here have performed genetic characterisation of the LX-2 cell line and established a single-locus short tandem repeat (STR) profile for the cell line and characterized the LX-2 karyotype by several cytogenetic and molecular cytogenetic techniques. Spectral karyotyping (SKY) revealed a complex karyotype with a set of aberrations consistently present in the metaphases analyses which might serve as cytogenetic markers. In addition, various subclonal and single cell aberrations were detected. Our study provides criteria for genetic authentication of LX-2 and offers insights into the genotype changes which might underlie part of its phenotypic features.     

Cell-cycle phases and genetic profile of bone marrow-derived mesenchymal stromal cells expanded in vitro from healthy donors

Human mesenchymal stromal cells (MSCs) expanded in vitro for cell therapy approaches need to be carefully investigated for genetic stability, by employing both molecular and conventional karyotyping. Reliability of cytogenetic analysis may be hampered in some MSC samples by the difficulty of obtaining an adequate number of metaphases. In an attempt to overcome this problem, a methodology apt to evaluate the cell‐cycle structure on synchronous MSCs was optimised. Results obtained in five independent experiments by comparing cell‐cycle analysis of synchronous and asynchronous MSC populations evaluated at early and late culture passages documented that in synchronous MSCs, 30% of cells entered G2/M phase after about 27–28 h of culture, while in asynchronous MSCs only 8% of cells in G2/M phase could be observed at the same time point. Cytogenetic analysis on synchronous MSCs allowed us to obtain 20–25 valuable metaphases/slide, whereas only 0–4 metaphases/slide were detectable in asynchronous preparations. J. Cell. Biochem. 112: 1817–1821, 2011. © 2011 Wiley‐Liss, Inc.     

The tumorigenicity and metastasis of low-malignancy STHE-strain cells undergoing in vitro selection with peritoneal exudate cells]

We have previously shown that in vitro cocultivation of the low-malignant spontaneously transformed in vitro cells of STHE strain with LPS-activated peritoneal exudate cells (PECa) of normal Syrian hamsters resulted (in contrast to resident PEC (PECr)) in selection of the STHE cell variants resistant to macrophage- and hydrogen peroxide (H2O2)-mediated cytotoxic activity (CTA). In the present study the malignant characteristics, i.e. tumorigenic and metastasizing activities (TGA, MA), of the STHE cell variants in vitro selected with PECa and PECr as well as the parental ones were compared. By the use of quantitative transplantation test the relatively increased level of TGA of the STHE cell variants selected with PECa, especially on the late stages of in vitro selection, has been demonstrated. The increase of the tumour size of transplanted cells was demonstrated after in vitro selection of parental STHE cells with either PECa of PECr. The mean number of spontaneous lung metastases at 60th day after s.c. transplantation of the cell variants selected with PECa and PECr was increased as compared to parental STHE cells, however, as a rule, did not exceed 20-25. The study of experimental MA revealed that only STHE cell variants selected with PECa were able to form significantly larger numbers of experimental lung metastases, what was more expressed after the 4th, 5th and 10th cycles of the selection procedure. Thus, activated REC (mainly macrophages and lymphocytes) are capable to select in vitro tumour cell variants characterized with not only increased resistance to CTA of the macrophages and H2O2, but also more tumorigenic and metastasizing, i.e. more malignant.     

Cytogenetic analysis of peripheral blood lymphocytes in workers exposed to vinyl chloride

In the present study, the method of cytogenetic analysis of peripheral blood lymphocytes was used to examine 43 workers exposed to vinyl chloride monomer (average exposure 11.2 years) and 22 subjects selected from the same locality (control group). A total number of 8650 metaphases were analysed. All cytogenetic parameters examined were increased in the exposed group as compared to the control group and 3 parameters, chromatid breaks, percentage of aberrant cells and breaks per cell, were significantly increased (P less than 0.001).     

Stable alterations in HeLa cell mitochondria following ethidium bromide treatment

This study describes the isolation of three HeLa cell clones after exposure of HeLa cells to ethidium bromide (EB) in culture medium for either 14 days, or 14 days plusreexposure for 30 days. All three EB-induced clones differed from the parental HeLa cell in various physical properties of their mitochondria. The ratio of mitochondrial DNA component I to component II was altered in clone HeLa-2A. In addition, the cytochrome content of the respiratory chain a + a₃, b and C₁ decreased, while the cytochroms c content remained unchanged. The amount of cytochromes b and c₁; which were not reduced by KCN treatment, but were reduced by dithionite, increased in clone HeLa-2A. The ultrastructure of HeLa-2A cells revealed several alterations characteristic of EB treatment. Some mitochondria had enlarged profiles, a reduced number of cristae and a more lucent electron density of the matrix. Other mitochondria were tightly packed with cristae, which occasionally showed a whorled configuration. These changes were observed 4 months (20–25 passages) after the omission of EB from the medium.     

Suppression of nuclear factor-kappa B activity by nitric oxide and hyperoxia in oxygen-resistant cells

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO +/- O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-kappaB activity. Cells lacking functional NF-kappaB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-kappaB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-kappaB activity in cells exposed to NO +/- O2, IkappaBalpha was degraded, suggesting that pathways regulating the steady-state levels of IkappaB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-kappaB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-kappaB activity.     

Chromosomal abnormalities in Day-6, in vitro-produced pig embryos

A cytogenetic study was undertaken to quantify, by chromosomal karyotyping, the incidence and type of chromosomal abnormalities present in Day-6 in vitro-produced (IVP) porcine embryos. Morphologically normal Day-6 blastocysts (n=318) were fixed and grouped into six classes according to the number of total cells (from < or =20 to 61-70). Of 248 embryos suitable for analysis, 97 (39.1%) displayed chromosomal abnormalities. The abnormalities included haploidy (9.3%), polyploidy (71.1%) and mixoploidy (19.6%). Within polyploid embryos, triploidy and tetraploidy showed the highest incidence (56.5 and 27.5%, respectively); among mixoploid embryos, diploid-triploid embryos (2n/3n) were prevalent (36.8%). Overall, the mean cell number was 34.3 +/- 12.1 and the mitotic index was 8.6 +/- 6.1. Chromosomally abnormal embryos had fewer (P<0.01) total cells compared to normal (2n) embryos (31.8 +/- 1.3 versus 35.9 +/- 1.0). In addition, the incidence of polyploidy decreased as the number of cells increased, while that of mixoploidy did not differ. These data indicate that polyploidy affects a large percentage of IVP porcine embryos capable of developing to blastocysts and the incidence of chromosomal abnormalities is much higher than that reported previously in in vivo embryos in this species. Given the ability of morphologically normal embryos with an abnormal chromosome complement to undergo preimplantation development in vitro, and the inability to identify blastocysts with abnormal karyotype without cytogenetic analysis, careful consideration should be given to factors affecting ploidy of IVP embryos, especially the incidence of polyspermic fertilization, when evaluating criteria of a porcine in vitro embryo production scheme.     

Longitudinal distribution of pulmonary vascular pressures as a function of postnatal age in rabbits.

The major purpose of this study was to determine whether the longitudinal distribution of pulmonary vascular pressures changes with postnatal age in rabbits. Using the direct micropuncture technique, we measured pressures in 20- to 80-microns-diam arterioles and venules in isolated lungs of rabbits of different postnatal ages. To determine the contribution of vasomotor tone, we added the vasodilator papaverine to the perfusate of some lungs of each age group. We compared vascular pressures measured at blood flow rates chosen to approximate in vivo cardiac outputs. In untreated lungs, the resistance across 20- to 80-microns-diam microvessels decreased from 12- to 72-h-old (0.022 +/- 0.009 cmH2O.min.kg.ml-1) to 5- to 15-day-old rabbits (0.008 +/- 0.007 cmH2O.min.kg.ml-1) and remained at this lower level in adults (0.013 +/- 0.008 cmH2O.min.kg.ml-1). In contrast, in papaverine-treated lungs, the resistance across 20- to 80-microns-diam microvessels did not change between 12- to 72-h-old (0.007 +/- 0.005 cmH2O.min.kg.ml-1) and 5- to 15-day-old rabbits (0.005 +/- 0.002 cmH2O.min.kg.ml-1) but increased between 5- to 15-day-old and adult rabbits (0.014 +/- 0.007 cmH2O.min.kg.ml-1). Thus vasomotor tone contributed to the postnatal change in the distribution of vascular pressures across lungs of rabbits.     

A Potential Role for Cx43-Hemichannels in Staurosporin-Induced Apoptosis

To address the role of gap junction hemichannels in apoptosis, the cell death induced by staurosporine (ST) was evaluated in wild type HeLa cells (HeLa-WT) and transfectants expressing either full-length connexin43 (HeLa-Cx43) or a C-terminal truncation of Cx43 (HeLa-ΔCT). Cell death was measured with fluorescence-activated cell sorting (FACS), both DNA and nuclear fragmentation methods and assays for PARP and caspase 3. The ST-mediated cell death was accelerated in HeLa-Cx43 cells compared to HeLa-WT and HeLa-ΔCT. To determine why HeLa-Cx43 cells were more susceptible to ST, the phosphorylation state and the localization of Cx43 protein within cells were examined using specific Cx43 antibodies. The phosphorylated forms of Cx43 were sharply reduced in HeLa-Cx43 cells treated with ST. Moreover, in ST-treated HeLa-Cx43 cells, Cx43 was mainly observed at the cell surface. In contrast, the truncated form of Cx43 found in HeLa-ΔCT cells, which lacks many of the normal phosphorylation sites, was observed in the cytosol with ST treatment. To examine the hemichannels in the plasma membranes of ST-treated HeLa-Cx43 cells, several dye uptake methods using carboxyfluorescein and propidium iodide were employed. While the number of fluorescent cells did not change in HeLa-WT and HeLa-ΔCT cells with ST treatment, the number of fluorescent HeLa-Cx43 cells increased more than ten-fold. These results indicate that the increases in cell surface Cx43 seen with immunofluorescence and the elevated hemichannel activities detected with dye uptake could help explain the accelerated cell death observed in ST-treated HeLa-Cx43 cells.     

Cytogenetic analysis of CpG-oligonucleotide DSP30 plus Interleukin-2-Stimulated canine B-Cell lymphoma cells reveals the loss of one X Chromosome as the sole abnormality

Human and canine lymphoid neoplasms are characterized by non-random cytogenetic abnormalities. However, due to the low mitotic activity of the B cells, cytogenetic analyses of B-cell lymphoid proliferations are difficult to perform. In the present study we stimulated canine B-cell lymphoma cells with the immunostimulatory CpG-oligonucleotide DSP30 in combination with interleukin-2 (IL-2) and obtained an adequate number of metaphases. Cytogenetic analyses revealed the loss of one X chromosome as the sole cytogenetic aberration. Chromosome analysis of the corresponding blood showed a normal female karyotype. Monosomy X as the sole clonal chromosomal abnormality is found in human hematopoietic malignancies as well, thus the dog may serve as a promising animal model.     

Cellular reactions induced by chronic exposure to 14C beta-rays

In V-79 cells the Effects of chronic exposure on induction of chromosome aberrations and abnormal metaphases as well as on efficiency of subsequent exposure to 2 Gy gamma-rays were investigated. It was found that chronic exposure increased the yield of chromosome aberrations as well as abnormal metaphases (spread-metaphases and apoptotic metaphases). In spite of the level of damages in cells, the chronic beta-exposure protected cells against the additional induction of chromosomal aberrations by subsequent exposure to higher acute dose of gamma-irradiation. Cytogenetic adaptive response was retained in the surviving progeny of irradiated cells which were cultured in free medium during 40, 70 days or one year after chronic exposure. At this time the level of residual chromosome aberrations, colony forming ability and distribution of the cells by the number of chromosomes were almost the same as in unirradiated cells. However, the high level of abnormal metaphases and half as much of cells in colony in the surviving progeny of irradiated cells in comparison with unirradiated cell, allow us to suggest that the chronic exposure induced the selection of adaptive forms in condition of the higher level of radiation.     

Alteration of human breast tumor cell membrane functions by chromosome-mediated gene transfer

BOT-2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome-mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa-65 cells to the BOT-2 cells, correcting the permease deficiency. Poly-L-ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 X 10^?5 and 1 X 10^?5, respectively. Transferants MGP-1 and MGL-1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone-induced alkaline phosphatase activity. MGP-1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL-1 remains thymidine transport-deficient and survives on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL-1 is similar to HeLa-65, 2% of that in BOT-2, and in addition, is inducible 25-30-fold by 3 μM hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.     

Role of glutathione in the adaptive tolerance to H2O2

Endogenous antioxidant defense systems are enhanced by various physiological stimuli including sublethal oxidative challenges, which induce tolerance to subsequent lethal oxidative injuries. We sought to evaluate the contributions of catalase and the glutathione system to the adaptive tolerance to H2O2. For this purpose, H9c2 cells were stimulated with 100 microM H2O2, which was the maximal dose at which no significant acute cell damage was observed. Twenty-four hours after stimulation, control and pretreated cells were challenged with a lethal concentration of H2O2 (300 microM). Compared with the control cells, pretreated cells were significantly tolerant of H2O2, with reduced cell lysis and improved survival rate. In pretreated cells, glutathione content increased to 48.20 +/- 6.38 nmol/mg protein versus 27.59 +/- 2.55 nmol/mg protein in control cells, and catalase activity also increased to 30.82 +/- 2.64 versus 15.46 +/- 1.29 units/mg protein in control cells, whereas glutathione peroxidase activity was not affected. Increased glutathione content was attributed to increased gamma-glutamylcysteine synthetase activity, which is known as the rate-limiting enzyme of glutathione synthesis. To elucidate the relative contribution of the glutathione system and catalase to tolerance of H2O2, control and pretreated cells were incubated with specific inhibitors of gamma-glutamyl cysteine synthetase (L-buthionine sulfoximine) or catalase (3-amino-1,2,4-triazole), and challenged with H2O2. Cytoprotection by the low-dose H2O2 pretreatment was almost completely abolished by L-buthionine sulfoximine, while it was preserved after 3-amino-1,2,4-triazole treatment. From these results, it is concluded that both the glutathione system and catalase can be enhanced by H2O2 stimulation, but increased glutathione content rather than catalase activity was operative in the tolerance of lethal oxidative stress.     

Estimation of the contribution of thiophosphamide-induced chromosomal aberrations to perimplantation embryonic mortality in mice]

Thiotepa-induced perimplantation mortality in mice was analysed cytologically. The contribution of induced chromosomal aberrations to preimplantation embryonic death was studied. Under the influence of 1.25 mg/kg of thiotepa on late spermatids the stop of cleavage division was found at 2--16 cell stage in 27% of the cases and at 17--22 cell stage in 11.9% (totally in 38.9%) as compared to 6% in the controls. The cytogenetic analysis of embryos consisting of 2--16 cells revealed gross structural chromosomal aberrations in 75% metaphases suitable for analysis.     

Automated metaphase finding: an assessment of the efficiency of the METAFER2 system in a routine mutagenicity assay

The efficiency of the automated metaphase finding system METAFER2 is assessed in a routine mutagenicity assay using an aneuploid rat liver cell line treated with various promutagens. Data sets generated by automated and manual selection of metaphases are compared. It is demonstrated that METAFER2 routinely allows an efficient automatic identification of metaphases not only in lymphocyte preparations, but also in preparation from mammalian cell lines with varying chromosome numbers. Although larger slide areas are required for automated compared to manual metaphase scanning, the automatic system is faster by a factor of about 5. The interactive visual elimination of metaphases of insufficient quality is an easy and fast procedure.METAFER2 allows an unbiased selection of metaphases irrespective of their appearance as homogeneously stained first or harlequin-staines second division cells. Random selection of metaphases is neither influenced by various structural chromosome changes nor by increased frequencies of sister-chromatid exchanges.