Impairment of cell cycle progression by aflatoxin B1 in human cell lines.

Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression.

Expression and DNA binding activity of the Ku heterodimer in bladder carcinoma.

BACKGROUND: The Ku protein is a tightly associated heterodimer, comprised of 70-kilodalton (kD) and 86-kD subunits, that forms the DNA-dependent protein kinase (DNA-PK) complex together with the 470-kD DNA-PKcs catalytic subunit, and is involved mainly in DNA double-strand breaks (DSBs) repair. The objective of the current study was to investigate the expression and DNA-binding activity of the Ku protein in fresh tissues from patients with bladder carcinoma and to compare it with that in nontumor tissues obtained from the same organ. Moreover, the DNA-binding activity of Ku was assessed after exposure of the tumor cells to 1 or 2 grays (Gy) of X-rays. Furthermore, the level of phosphorylated Ku was analyzed in both the nuclear and cytoplasmic compartment of normal tissue after exposure to 2 Gy of X-rays. METHODS: The expression and DNA-binding activity of Ku protein were assessed in tumor samples from patients who all were diagnosed with transitional cell carcinoma (TCC) of the bladder using Western blot analysis and the electrophoretic mobility shift assay, respectively. RESULTS: Enhanced Ku activity and expression were found in tumor tissue compared with normal tissue for each patient. Moreover, variations in Ku activity were found in a dose-dependent manner after the tumor cells were exposed to 1 or 2 Gy of X-rays. A decrease in phosphorylated Ku in the cytoplasm and a parallel increase in the nucleus of normal tissue cells were observed after exposure to X-rays. CONCLUSIONS: The results of the current study suggest a possible role of Ku in regulating the DNA-PK activity of DSBs repair in bladder tumors.

Calibration curves for biological dosimetry by fluorescence in situ hybridisation.

Dose-response curves were measured for the induction of chromosomal aberrations in peripheral blood lymphocytes after acute exposure in vitro to 60Co gamma rays. Blood was obtained from four different healthy donors, and chromosomes were either observed at metaphase, following colcemid accumulation, or prematurely condensed by calyculin A. Cells were analysed in three different Italian laboratories. Chromosomes 1, 2, and 4 were painted, and simple-type interchanges between painted and non-painted chromosomes were scored in cells exposed in the dose range 0.1-3.0 Gy. The chemical-induced premature chromosome condensation method was also used combined with chromosome painting (chromosome 4 only) to determine calibration curves for high dose exposures (up to 20 Gy X rays). Calibration curves described in this paper will be used in our laboratories for biological dosimetry by fluorescence in situ hybridisation.

Evaluation of genotoxic and cytotoxic properties of pesticides employed in Italian agricultural practices.

In a program coordinated by the Italian Ministry of Works, we tested in vitro four pesticides widely employed in a developed agricultural region of central Italy. The four commercial agents were chosen on the basis of their diffusion in agricultural practice, knowledge of their active principle(s), and scant availability of data concerning their toxic and genotoxic activity. The agents were Cirtoxin, Decis, Tramat Combi (TC), and Lasso Micromix (LM). All substances were tested in three in vitro systems: Chinese hamster ovary (CHO) cells, a metabolically competent hamster cell line (Chinese hamster epithelial liver; CHEL), and root tips of Vicia faba (VF). The cytotoxic and genotoxic end points challenged were micronuclei and root tip length (RTL) in VF and mitotic index (MI), proliferation index (PI), cell survival (CS), cell growth (CG), cell cycle length (CCL), sister chromatid exchanges, chromosomal aberrations, and single-cell gel electrophoresis, or comet assay, in CHEL and CHO cells. Tested doses ranged from the field dose up to 200x the field dose to take into account accumulation effects. On the whole, tested agents appear to induce genotoxic damage only at subtoxic or toxic doses, indicating a low clastogenic risk. MI, PI, CS, CG, RTL, and CCL appear to be the less sensitive end points, showing no effects in the presence of a clear positive response in some or all of the other tests. Using cytogenetic tests, we obtained positive results for TC and LM treatments in CHO but not in CHEL cells. These data could be accounted for by postulating a detoxifying activity exerted by this cell line. However, cytogenetic end points appear to be more sensitive than those referring to cytotoxicity.

Damage proneness induced by genomic DNA demethylation in mammalian cells cultivated in vitro.

Variations in the genomic DNA methylation level have been shown to be an epigenetic inheritable modification affecting, among other targets, the sister chromatid exchange (SCE) rate in mammalian cells in vitro. The inheritable increase in SCE rate in affected cell populations appears as a puzzling phenomenon in view of the well established relation between SCE and both mutagenesis and carcinogenesis. In the present work we demonstrate that, in a treated cell population, demethylation could be responsible for the inheritable induction of damage proneness affecting both damage induction and repair. Normal and ethionine or azacytidine treated Chinese hamster ovary cells, subclone K1 (CHO-K1), were challenged with UV light (UV) or mitomycin-C (MMC) at different times from the demethylating treatment. The SCE rate was measured with two main objects in view: (i) the induction of synergism or additivity in combined treatments, where mutagen (UV or MMC) pulse is supplied from 0 to 48 h after the end of the demethylating treatment; and (ii) the pattern of damage extinction, for the duration of up to six cell cycles after the end of the combined (demethylating agent + mutagen) treatment. Results indicate both a synergism in SCE induction by mutagens in demethylated cells even if supplied up to four cell cycles after the end of the demethylation treatment and a delay in recovery of induced damage, compared with normally methylated cells. These data are discussed in the light of the supposed mechanism of SCE increase and of the possible biological significance in terms of mutagenesis and carcinogenesis.

Specificity of the G1 block induced by ethionine in human lymphocytes in vitro: a flow cytometric analysis.

Ethionine is the ethyl analogue of the amino acid methionine. The agent is well known to have a weak demethylating activity. In addition, its capacity to reversibly block the cell cycle progression in G1 human lymphocytes (HL) without interfering with blastic transformation has been reported. In order to better understand the mechanism by which the agent is able to induce cell cycle block, experiments have been performed by using flow cytometry, in HL. In particular the hypothesis of the involvement of a specific target at the G0/G1 boundary was tested by treating HL at different times after blastic transformation. Starting from the 40th hour after PHA stimulation, ethionine loses its blocking capacity in such a way that cells challenged by the agent do not differ from controls in any one of the tested cell cycle-related parameters. We suggest the agent exerts its blocking activity at a specific stage of the transformation pathway.