Involvement of calcium-dependent protein kinase C in arsenite-induced genotoxicity in chinese hamster ovary cells

Arsenic is the first metal to be identified as a human carcinogen. Arsenite, one inorganic form of arsenic, has been found to induce sister chromatid exchange, chromosome aberrations, and gene amplification in a variety of in vitro systems. In this study of arsenite-induced genotoxicity represented as micronuclei production in Chinese hamster ovary cells (CHO-K1), we found that the calcium channel blocker, verapamil, can potentiate arsenite-induced micronuclei. And after arsenite treatment, the elevation of intracellular calcium was observed. When extracellular calcium was depleted during arsenite treatment, the arsenite-induced micronuclei formation was significantly suppressed. These data indicated that a calcium ion plays an essential role in arsenite-induced genotoxicity. Further, it was found that the cotreatment of arsenite and a calcium ionophore, A23187, can increase the micronuclei induction. In contrast, pretreatment of the intracellular calcium chelator, quin 2, significantly inhibited micronuclei production of arsenite administration. In addition, we measured the activity of calcium-and phospholipid-dependent protein kinase C (PKC) and found that arsenite can activate PKC activity in a dose-dependent manner. Subsequently, some PKC activators and inhibitors were applied to investigate the involvement of PKC on arsenite-induced micronuclei formation. It was found that H7, a PKC inhibitor, can depress but TPA, a PKC activator, can enhance arsenite-induced micronuclei significantly. These data indicated that arsenite exposure perturbs intracellular calcium homeostasis and activates PKC activity. As a result, the activation of PKC activity may play an important role in arsenite-induced genotoxicity. J. Cell. Biochem. 64:423–433. © 1997 Wiley-Liss, Inc.     

环磷酰胺诱发蚕豆体细胞遗传损伤的研究

利用蚕豆根尖研究环磷酰胺的遗传毒性效应, 结果表明:环磷酰胺(0.1~5.0 mg/mL)能够降低蚕豆根尖细胞有丝分裂指数, 使根尖细胞中具有微核、核出芽及核固缩的细胞明显增多, 并诱发染色体结构和行为异常, 产生染色体断片、滞后和桥。环磷酰胺处理组根尖中具有核固缩和微核的细胞数呈剂量依赖性增加, 且与作用时间呈正相关, 而分裂指数的降低也具有剂量和时间效应关系。研究结果表明, 低浓度长时间接触或高浓度短时间接触环磷酰胺均可产生遗传毒害, 因此, 有关的作业人员应注意防护。     

Sewage sludge does not induce genotoxicity and carcinogenesis

Through a series of experiments, the genotoxic/mutagenic and carcinogenic potential of sewage sludge was assessed. Male Wistar rats were randomly assigned to four groups: Group 1 - negative control; Group 2 - liver carcinogenesis initiated by diethylnitrosamine (DEN; 200 mg/kg i.p.); Group 3 and G4-liver carcinogenesis initiated by DEN and fed 10,000 ppm or 50,000 ppm of sewage sludge. The animals were submitted to a 70% partial hepatectomy at the 3^rd week. Livers were processed for routine histological analysis and immunohistochemistry, in order to detect glutathione S-transferase positive altered hepatocyte foci (GST-P⁺ AHF). Peripheral blood samples for the comet assay were obtained from the periorbital plexus immediately prior to sacrificing. Polychromatic erythrocytes (PCEs) were analyzed in femoral bone-marrow smears, and the frequencies of those micronucleated (MNPCEs) registered. There was no sewage-sludge-induced increase in frequency of either DNA damage in peripheral blood leucocytes, or MNPCEs in the femoral bone marrow. Also, there was no increase in the levels of DNA damage, in the frequency of MNPCEs, and in the development of GST-P AHF when compared with the respective control group.     

Mitochondriotropic action and DNA protection: Interactions between phenolic acids and enzymes

The protective action of caffeic (CA) and syringic (SA) acids on the genotoxicity exercised by snake venoms was investigated in this study. Molecular interactions between phenolic acids and the enzyme succinate dehydrogenase were also explored. In the electrophoresis assay, SA did not inhibit the genotoxicity induced by the venom. However, CA partially inhibited DNA degradation. In the comet assay, SA and CA exerted an inhibitory effect on the venom‐induced fragmentation. Succinate dehydrogenase presented, in computational analyzes, favorable energies to the molecular bond to both the malonic acid and the phenolic compounds evaluated. In the enzymatic activity assays, SA inhibited succinate dehydrogenase and interfered in the interaction of malonic acid. Meanwhile, CA potentiated the inhibition exerted by the malonic acid. The results suggest transient interactions between toxins present in venoms and phenolic acids, mainly by hydrogen interactions, which corroborate with the data from previous works.     

Genotoxicity assessment in fish peripheral blood: a method for a more efficient analysis of micronuclei

The authors describe a method for analysis of micronuclei using a nucleic acid-specific fluorochrome, acridine orange, and ultraviolet microscopy in order to establish a simple and reliable technique for routine genotoxicity assessment in fish peripheral erythrocytes.     

Micronuclei in the blood and bone marrow cells of mice exposed to specific complex time‐varying pulsed magnetic fields

For 8 weeks, adult CD‐1 male mice were continuously exposed to complex time‐varying pulsed magnetic fields (PMF) generated in the horizontal direction by a set of square Helmholtz coils. The PMF were <1000 Hz and delivered at a peak flux density of 1 mT. Sham‐exposed mice were kept in a similar exposure system without a PMF. Positive control animals exposed to 1 Gy gamma radiation were also included in the study. Blood samples were collected before (time 0) and at 2, 4, 6, and 8 weeks. All mice were euthanized at the end of 8 weeks and their bone marrow was collected. From each blood and bone marrow sample, smears were prepared on microscope slides, fixed in absolute methanol, air‐dried, and stained with acridine orange. All slides were coded and examined using a fluorescence microscope. The extent of genotoxicity and cytotoxicity was assessed from the incidence of micronuclei (MN) and percent polychromatic erythrocytes (PCE) in the blood and bone marrow, respectively. The data indicated that both indices in PMF‐exposed mice were not significantly different from those observed in sham‐exposed animals. In contrast, positive control mice exhibited significantly increased MN, and decreased percentages of PCE in both tissues. Thus, the overall data suggested that 8 weeks of continuous exposure to PMF did not induce significantly increased genotoxicity and cytotoxicity in experimental mice. Further investigations are underway using other genotoxicity assays (comet assay, γ‐H2AX foci, and chromosomal aberrations) to assess genotoxicity following PMF exposure. Bioelectromagnetics 31:445–453, 2010. © 2010 Wiley‐Liss, Inc.     

浊漳河水体污染物对蚕豆根尖细胞的遗传毒性研究

运用蚕豆根尖细胞微核试验技术对浊漳河南段6个代表性样点水体中有机污染物的遗传毒性进行了研究,为浊漳河流域水体污染状况的有效监测提供理论依据.结果显示:(1)浊漳河南段各监测断面的水体有机污染物对蚕豆根尖细胞均产生了不同程度的损伤,表现出细胞微核、断片、染色体桥等多种异常形态,严重时导致细胞坏死和细胞凋亡.(2)6个样点水样处理的蚕豆根尖细胞微核率在21.47‰～64.77‰,极显著高于对照组(P<0.01).(3)各样点水样对蚕豆根尖细胞的遗传损伤程度依次为:王桥>店上>漳泽水库>北寨>黄碾>五阳;在有丝分裂后期,蚕豆根尖中异常细胞最高达70%,且异常细胞比率有随着污染程度增大而升高的趋势.研究表明,蚕豆根尖细胞微核是水质毒理检测的有效指标,其细胞有丝分裂后期异常细胞比率也可作为水质毒理检测的观察指标.     

Naked Sendai virus vector lacking all of the envelope-related genes: reduced cytopathogenicity and immunogenicity

BACKGROUND: Sendai virus (SeV) is a new class of cytoplasmic RNA vector that is free from genotoxicity that infects and multiplies in most mammalian cells, and directs high-level transgene expression. We improved the vector by deleting all of the envelope-related genes from the SeV genome and thus reducing its immunogenicity. METHODS: The matrix (M), fusion (F) and hemagglutinin-neuraminidase (HN) genes-deleted SeV vector (SeV/DeltaMDeltaFDeltaHN) was recovered in a newly established packaging cell line. Then, the generated SeV/DeltaMDeltaFDeltaHN vector was characterised by comparing with single gene-deleted type SeV vectors. RESULTS: This SeV/DeltaMDeltaFDeltaHN vector carrying the green fluorescent protein gene in place of the envelope-related genes could be propagated to a titer of more than 10(8) cell infectious units/ml. This vector showed an efficient transduction capability in vitro and in vivo, and the cytopathic effect and induction of neutralizing antibody in vivo were greatly reduced compared with those of single gene-deleted type SeV vectors. No activity of neutralizing antibody or anti-HN antibody was seen when SeV/DeltaMDeltaFDeltaHN was transduced ex vivo. Additional introduction of amino acid mutations that had been identified from SeV strains causing persistent infections was also effective for the reduction of cytopathic effects. CONCLUSIONS: The deletion of genes from the SeV genome and the additional mutation are very effective for reducing both the immunogenic and cytopathic reactions to the SeV vector. These modifications are expected to improve the safety and broaden the range of clinical applications of this new class of cytoplasmic RNA vector.     

Modulation of ochratoxin A induced DNA-damage in urothelial cell cultures

Despite good evidence for a genotoxic potential of ochratoxin A (OTA), the mechanism of OTA-induced genotoxicity (direct or indirect?) is still unclear. This calls for a further characterization of OTA-related DNA damage, and investigations of factors that may modulate dose-effect relationships in cells. Since bladder epithelium is a target tissue for the toxicity of OTA, its effects were studied in cultures of human bladder carcinoma (H5637) cells. Cytotoxicity of OTA, assessed by Neutral red (NR) uptake or Alamar-Blue assay, is concentration- and time-dependent: Upon 24 h treatment of 5637 cells, NR uptake is reduced by 50% with OTA concentrations of ≥0.2 microM, but not with 3 h treatment of the cells. Since cytotoxicity of OTA was not affected by addition of xenobiotic metabolizing enzymes (S-9 mix), it appears to be unrelated to biotransformation of the mycotoxin. Also, addition of S-9 mix did not significantly affect the genotoxicity of OTA as studied by alkaline single cell gel electrophoresis (Comet assay). DNA damage was detectable after 3 h treatment of cells at OTA concentrations between 0.1 and 1 microM, and increased further at higher concentrations. The magnitude of OTA-induced DNA damage did not increase with longer treatment times (18, 24 h), probably due to repair processes in the cells. Repair of OTA-induced lesions is quite efficient in kidney (Arch Toxicol 2002, 75, 734–741) and in porcine bladder cells (Föllmann and Lebrun, 2005, Mycotoxin Research, this volume). Interestingly, the genotoxicity of OTA is modulated by the pH of the culture medium, with higher damage at pH 5 compared to pH 7.5. In line with this, uptake studies with tritiated OTA show a higher cellular accumulation of the mycotoxin at pH 5 than in buffer of pH 7.5. Thus, bladder cells exposed to OTA in slightly acidic urine (which facilitates reabsorption) may be at higher risk.     

Genotoxicity of chloroquine in rat liver cells: Protective role of free radical scavengers

The genotoxic effect of chloroquine (CQ), a 4-aminoquinoline antimalarial drug was investigated in rat liver cells using the alkaline comet assay. Chloroquine (0–1000 μmol/L) significantly increased DNA strand breaks of rat liver cells dose-dependently. Rat liver cells exposed to CQ (100–500 μmol/L) and treated with endonuclease III and formamidopyrimidine-DNA glycosylase, the bacterial DNA repair enzymes that recognize oxidized pyrimidine and purine, respectively, showed greater DNA damage than those not treated with the enzymes, providing evidence that CQ induced oxidation of purines and pyrimidines. Treatment of cells with 5 mmol/L N-acetylcysteine, an intracellular reactive oxygen species (ROS) scavenger, and 100 μmol/L and 250 μmol/L deferoxamine, an established iron chelator, significantly decreased the CQ-induced strand breaks and base oxidation, respectively. Similarly, the formation of DNA strand breaks and oxidized bases was prevented by vitamin C (10 μmol/L) (a water-soluble antioxidant), quercetin (50 μmol/L) (an antioxidant flavonoid), and kolaviron (30 μmol/L and 90 μmol/L) (an antioxidant and a liver hepatoprotective phytochemical). The results indicate that the genotoxicity of CQ in rat liver cells might involve ROS and that free radical scavengers may elicit protective effects in these cells.