The clastogenic activity of 1,6-dinitropyrene in peripheral human lymphocytes

The ability of 1,6-dinitropyrene to induce chromosome damage in peripheral human lymphocyte cultures has been demonstrated. Low levels of clastogenic activity were detected following 3-h treatments with 1,6-dinitropyrene in the presence of a rat-liver cytosol fraction. The clastogenic activity reached a peak at a concentration of 1.25 micrograms/ml of 1,6-dinitropyrene after which the frequency of aberrations decreased. This unusual genotoxic dose response is similar to that found previously in yeast and rat-liver cells. The fact that a positive result was obtained using human lymphocytes shows that, in the presence of the appropriate activation system, dinitropyrene is genotoxic in human cells.     

Induction of DNA repair in human and rat hepatocytes by 1,6-dinitropyrene

1,6-Dinitropyrene (DNP) was found to be an extremely potent genotoxicant in metabolically competent primary cultures of human and rat hepatocytes. Dose-dependent increases in DNA repair as measured by unscheduled DNA synthesis (UDS) were observed in the range from 0.05 to 5 microM 1,6-DNP for both species, indicating that the rat-hepatocyte assay is an appropriate model for assessing genotoxic potential in human hepatocytes for this class of compound. Unlike some nitroaromatic compounds, 1,6-DNP did not require gut flora for metabolic activation. No DNA repair was observed in hepatocytes isolated from rats treated with 50 mg/kg 1,6-DNP in corn oil by gavage 2, 12 or 24 h previously. The reason for the lack of a response in vivo is not known, but may relate to detoxification or distribution of the compound in the animal.     

The effects of 5-bromodeoxyuridine on the growth and morphology of transformed rat liver cells

The effects of bromodeoxyuridine (BrdUrd) on the growth, morphology, and tumorigenicity of the spontaneously transformed rat liver cell line R72/3 were studied. These cells grow either in suspension or in a monolayer and are tumorigenic. In monolayer cultures, cells treated with low concentrations (2.5 μg/ml) of BrdUrd were larger, more spread out, and more firmly attached to the substratum than were untreated controls. Treated cells failed to grow in suspension or on confluent monolayers of 3T3 cells and did not form colonies in soft agar. Scanning electron microscopy revealed extensive flattening of treated cells and a dramatic reduction in the number of microvilli on the cell surface. Transmission electron microscopy showed an increase in polyribosomes and rough endoplasmic reticulum, as well as an enlargement of endoplasmic reticulum cisternae and a complete absence of the bundles of intermediate size filaments that were conspicuous in untreated cells. The persistence of these changes required the continuous presence of BrdUrd in the medium. The effects of BrdUrd were readily reversed by withdrawal of BrdUrd and were not expressed in the presence of excess thymidine.     

The mutagenic potency of 1,8-dinitropyrene in cultured mouse lymphoma cells

Although non-toxic, 1,8-dinitropyrene (1,8-DNP) was mutagenic for mouse lymphoma L5178Y cells when assayed for induced resistance to 6-thioguanine, methotrexate, ouabain and 1-β-D-arabinofuranosyl cytosine. In bacteria, nitropyrenes are potent inducers of frame-shift mutations, and the induction of ouabain-resistant mutants, believed to be due to base-pair substitutions, suggests that the mechanism of action may be different in mouse cells and bacteria. Long treatment times were required to detect 1,8-DNP-induced mutants in L5178Y cells, suggesting the possibility of an inducible activation system. 4-Nitroquinoline 1-oxide was both toxic and mutagenic to these same 4 mutation assays after short (2 h) treatment times. The dilemma that exists when comparing the mutagenic potential of test chemicals when concentration of mutagen, treatment times and toxicity are markedly different, is discussed.     

Effect of colchamine on the morphology of transformed fibroblast-like cells in culture]

Colcemid-induced changes in the morphology of cultured transformed fibroblast-like mouse cells were studied using scanning electron microscope. Colcemid interferes with the normal cell polarization so that all the cell edges became active. Colcemid-treated normal cells remained well spread over the substrate. Transformed cells lost their polarization after colcemid treatment--the long, narrow, poorly attached processes of different form appeared along all the cell periphery. As in the case of the absence of colcemid, transformed cells remained less spread and worse attached to the substrate than normal ones. It is suggests that a) the same colcemid-sensitive structures are responsible for the polarization of transformed and normal cells; b) morphological differences between transformed and normal cells are determined mainly by structures different from colcemide-sensitive ones.     

1,6-Dinitropyrene causes spindle disturbances and chromosomal damage in V79 Chinese hamster cells

We have investigated the cytogenetic effect of 1,6-dinitropyrene (1,6-DNP) in Chinese hamster V79 cells. The chemical caused a dose-dependent increase in the incidence of initial and full C-mitoses, polyploid mitoses, ana-telophases with lagging chromosomes, non-disjunction and multipolar configurations, in a range of 0.05-5 microM. These findings indicate that 1,6-DNP interferes with the functioning of the spindle apparatus in V79 cells. Early signs of spindle disturbances were seen at 1,6-DNP concentrations which only moderately reduced cell growth and division. Analysis of structural chromosomal aberrations revealed the appearance of chromatid-type aberrations with open breaks and exchanges accompanied by gaps. The results indicate that 1,6-DNP is both a spindle-disturbing and a clastogenic agent in V79 cells.     

Mitochondrial calcium response in human transformed lymphoblastoid cells

Human lymphoblastoid cell line (LCL) transformed by Epstein-Barr Virus (EBV) is a unique cellular model for the study of human diseases. Although pathophysiological significance of mitochondrial calcium regulation is drawing attention, it is not known whether or not mitochondria in LCLs play a role in intracellular calcium signaling. In this study, role of mitochondria of the lymphoblastoid cell line in calcium signaling was examined. Intra-mitochondrial calcium concentration ([Ca2+]m) was successfully measured using dihydro-Rhod-2, revealed by the decrease of fluorescence after application of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and intracellular localization patterns imaged by fluorescent microscope. Platelet activating factor (PAF) concentration-dependently increased cytosolic calcium concentration ([Ca2+]i), while no increase of [Ca2+]m was observed. In contrast, 10 microM thapsigargin increased [Ca2+]i as well as [Ca2+]m. LCLs may be used for the study of possible pathophysiological role of mitochondrial calcium regulation in human diseases.     

Genome expression of the DNA-containing human papovavirus in transformed human cells

In the course of study of the transformed cells of line 63 the phenomenon of cyclically repeated transitory infection of the level of separate cells accompanied by the periodically isolated DNA-containing virus has been shown. Virus reproduction was judged by the determination of the infectious and hemagglutinating activities and radioactivity. The application of activation methods (co-culturing, somatic hybridization and cells treatment with mytomycin C) led to the increase of virus synthesis in the transformed cells of line 63 during spontaneous production of it. We failed to express viral genome in refractor phase.     

The effects of shear stress and metastatic phenotype on the detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal, transformed, and reverted rat fibroblasts from a confluent monolayer of normal fibroblasts. In this method, known shear stresses were applied to the adherent cells and the percent of cells detached from the monolayer was determined. Results indicate that the detachment of all cell types increased with increasing shear stress and detachment of highly metastatic ras-transformed cells was significantly higher than that of either nonmetastatic normal cells or transformed cells reverted with the Kirsten ras revertant (K-rev 1a) gene, which are lowly metastatic. From these results, it is concluded that a correlation exists between the metastatic phenotype of the cell and its ability to detach from normal cells.     

Genotoxicity of 1,3- and 1,6-dinitropyrene: induction of micronuclei in a panel of mammalian test cell lines

1,3-Dinitropyrene (1,3-DNP) and 1,6-dinitropyrene (1,6-DNP) were assessed for their potential to increase the frequencies of micronuclei in a panel of test cell lines consisting of H4IIEC3/G, 5L, 5L/r-1,3-DNP1, 208F, V79, V79/r-1,6-DNP1, HepG2 and BWI-J cells, which have been partially characterized for their expression of xenobiotic metabolising enzymes. The micronuclei were analyzed for the presence or absence of kinetochores indicating the occurrence of aneuploidy or chromosome breakage, respectively. 1,3-DNP caused a substantial increase in the frequency of micronuclei only in V79 cells. 1,6-DNP was strongly genotoxic in lines H4IIEC3/G⁻, 208F, V79 and, to a minor degree, in 5L/r-1,3-DNP1. It caused the formation of kinetochore-positive as well as kinetochore-negative micronuclei in V79 cells but only of kinetochore-negative micronuclei in H4IIEC3/G⁻ and 208F cells. 1,6-DNP-induced formation of micronuclei was paralleled by the appearance of multinucleated cells. Treatment of V79 cells with 1,3-DNP resulted in the same types of damage as treatment with 1,6-DNP, although considerably higher concentrations were required.The results show that 1,6-DNP can be highly genotoxic in mammalian cells, whereas, at least in the panel of test cell lines used, 1,3-DNP possesses only a low genotoxic activity. 1,3-DNP appears to be activated to genotoxic products in V79 cells by the same pathway(s) as 1,6-DNP.     

The effects of ionophores on steroidogenesis and morphology of avian granulosa cells

Summary Granulosa cells, isolated by collagenase digestion from the mature ovarian follicle of laying hens, were incubated in the presence of two ionophores, lasalocid (X537A) and ionomycin, to determine their effects on basal and stimulated steroidogenesis, as well as their effects on various cell parameters including DNA, RNA, and protein synthesis. Both ionophores caused a dose-dependent inhibition of agonist-promoted progesterone production and, in the presence of calcium, a small but significant increase in basal output of progesterone. Whereas the conversion of pregnenolone to progesterone was unaffected by the ionophores, the activity of cholesterol side-chain cleavage enzyme was inhibited in a dose-related manner. Both ionophores decreased cellular levels of ATP and inhibited the incorporation of radioactively-labeled precursors into DNA, RNA, and proteins. Morphologically, ionophoretreated cells showed swelling of the rough endoplasmic reticulum. Similar morphological changes were also observed in cells treated with oligomycin, a known metabolic inhibitor. These results suggest that the ionophores lasalocid and ionomycin impair release of energy and thereby exert the principal cause of the inhibited steroidegenic response by granulosa cells to a variety of agonists.     

Differential effects of dioctanoylglycerol on fibronectin localization in normal, partially transformed, and malignant human endometrial stromal cells

In this study, we describe the effects of direct activation of PKC by dioctanoylglycerol (DiC8) on cellular morphology and the localization of fibronectin (Fn) in normal, oncogene-transfected, and malignant human endometrial stromal cells. We questioned whether DiC8, an endogenous specific activator of PKC, would function as a second oncogene in partially transformed human endometrial stromal cells (HESC). Cells utilized were (1) normal HESC, (2) HESC transfected with a plasmid containing an origin-defective temperature-sensitive SV40 large T antigen alone or (3) in combination with an EJ ras oncogene, and (4) an endometrial sarcoma cell line (S7). Cell cultures were treated for 1 h with sn-dioctanoylglycerol (DiC8) and stained with a monoclonal fluorescein-labeled anti-Fn antibody. In normal HESC, DiC8 induced cell rounding and caused Fn localization to revert from the perinuclear region to the cell periphery. All experiments in this investigation were performed when cells were maintained at the permissive temperature for SV40 large T antigen function. In HESC expressing the SV40 large T antigen alone, Fn was localized to the perinuclear region and also occurred as parallel strands between cells. When these cells were treated with DiC8, Fn localization changed to intense punctate regions at the cell periphery or to matrix-like patterns between cells. Also, in these cells, DiC8 induced greater detachment of cells from the substrate than from other cells, resulting in an apparent piling up of cells. Control and treated SV40/EJ ras cells and uterine sarcoma cells expressed Fn in a matrix-like pattern between cells. The rounded cellular morphology of treated HESC and treated cells expressing SV40 resembled the morphology of control or treated SV40/EJ ras cells and uterine sarcoma cells. Thus, treated cells expressing the SV40 large T antigen resembled the SV40/EJ ras cells and uterine sarcoma cells with respect to Fn localization and cellular morphology. DiC8 did not appear to further transform HESC expressing SV40 and EJ ras. However, with regard to cell shape and Fn localization, our results suggest that DiC8 may function as a second oncogene in the signal transduction pathway, in cells expressing SV40 alone. It appears that, with regard to Fn localization, DiC8 may alter signal transduction analogously to that caused by the activated Ha-ras oncogene in HESC expressing the SV40 large T antigen.     

在低氧损伤中牛磺酸对视网膜神经节转化细胞RGC-5抗氧化作用的研究

目的:观察低氧(Hypoxia,Hyp)对大鼠视网膜神经节转化细胞(retina ganglion cell-5,RGC-5)氧化应激损伤的影响及牛磺酸(Taurine,Tau)的防护效应.方法:将RGC-5置于低氧条件(5%O2,5%CO2,90%N2),加入不同浓度的牛磺酸(0.05mM、0.1mM、0.5mM、1mM)预处理后培养12h,24h和48h,使用MTT法检测细胞活力,并通过对NO、GSH、MDA等指标的检测,观察牛磺酸对RGC-5的保护效应.结果:低氧处理后RGC-5细胞活力明显降低(P＜0.05),牛磺酸处理组细胞活力明显高于低氧组,其中0.1mM牛磺酸组作用最为显著(P＜0.05);低氧组与常氧组比较,RGC-5的NO、GSH含量明显降低(P＜0.05),而MDA含量显著升高(P＜0.05);牛磺酸处理组与低氧组比较,RGC-5细胞GSH,NO的含量显著升高(P＜0.05),而MDA的含量显著降低(P＜0.05).结论:牛磺酸能有效增强低氧损伤中RGC-5细胞的活力,其机制可能与牛磺酸可以提高其抗氧化能力有关.