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.

Mitochondrial effects of the pleiotropic proteasomal mutation mpr1/rpn11: uncoupling from cell cycle defects in extragenic revertants.

We have previously characterized a Saccharomyces cerevisiae mutant which contains a mutation in the essential rpn11/mpr1 gene coding for the proteasomal regulatory subunit Rpn11. The mpr1-1 mutation shows the phenotypic characteristics generally associated with proteasomal mutations, such as cell cycle defects and accumulation of polyubiquitinated proteins. However, for the first time, mitochondrial defects have also been found to be a consequence of a mutation in a proteasomal gene (Mol. Biol. Cell 9 (1998) 2917-2931). Since the mutant strain is thermosensitive both on glucose and on glycerol, we searched for revertants in order to shed light on the Rpn11/Mpr1 functions. Spontaneous revertants able to grow on glucose but not on glycerol at 36 degrees C were isolated, and, only from them, revertants able to grow at 36 degrees C on glycerol were selected. Revertants of the two classes were found to be extragenic. The detailed characterization of these extragenic suppressors demonstrates that the phenotypes related to cell cycle defects can be dissociated from those concerned with mitochondrial organization.

G2-phase radiation response in lymphoblastoid cell lines from Nijmegen breakage syndrome.

The relationship between G2-phase checkpoint activation, cytoplasmic cyclin-B1 accumulation and nuclear phosphorylation of p34CDC2 was studied in Nijmegen breakage syndrome cells treated with DNA damaging agents. Experiments were performed on lymphoblastoid cell lines from four Nijmegen breakage syndrome patients with different mutations, as well as on cells from an ataxia telangiectasia patient. Lymphoblastoid cell lines were irradiated with 0.50-2 Gy X-rays and the percentage of G2-phase accumulated cells was evaluated by means of flow cytometry in samples that were harvested 24 h later. The G2-checkpoint activation was analysed by scoring the mitotic index at 2 and 4 h after treatment with 0.5 and 1 Gy X-rays and treatment with the DNA double-strand break inducer calicheamicin-gamma1. Cytoplasmic accumulation of cyclin-B1 was evaluated by means of fluorescence immunostaining or Western blotting, in cells harvested shortly after irradiation with 1 and 2 Gy. The extent of tyrosine 15-phosphorylated p34CDC2 was assessed in the nuclear fractions. Nijmegen breakage syndrome cells showed suboptimal G2-phase checkpoint activation respect to normal cells and were greatly different from ataxia telangiectasia cells. Increased cytoplasmic cyclin-B1 accumulation was detected by both immunofluorescence and immunoblot in normal as well as in Nijmegen breakage syndrome cells. Furthermore, nuclear p34CDC2. phosphorylation was detected at a higher level in Nijmegen breakage syndrome than in ataxia telangiectasia cells. In conclusion, our data do not suggest that failure to activate checkpoints plays a major role in the radiosensitivity of Nijmegen breakage syndrome cells.

Strong antioxidant activity of ellagic acid in mammalian cells in vitro revealed by the comet assay.

Oxidative stress due to oxygen and various radical species is associated with the induction of DNA single- and double-strand breaks and is considered to be a first step in several human degenerative diseases, cancer and ageing. Naturally occurring antioxidants are being extensively analysed for their ability to protect DNA against such injury. We studied three naturally occuring compounds, Ascorbic Acid, Melatonin and Ellagic acid, for their ability to modulate DNA damage produced by two strong radical oxygen inducers (H2O2 and Bleomycin) in cultured CHO cells. The alkaline Comet assay was used to measure DNA damage and a cytofluorimetric analysis was performed to reveal the intracellular oxidative species. The data showed a marked reduction of H2O2- and Bleomycin-induced DNA damage exerted by Ellagic Acid. On the contrary Ascorbic acid and Melatonin appeared to induce a slight increase in DNA damage per se. In combined treatments, they caused a slight reduction of H2O2-induced damage, but they did not efficiently modulate the Bleomycin-induced one. The Dichlorofluorescein diacetate (DCFH-DA) cytofluorimetric test confirmed the strong scavenging action exerted by Ellagic Acid.

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.

Inhibitory action of melatonin on H2O2- and cyclophosphamide-induced DNA damage.

Melatonin, the pineal gland hormone known for its ability to modulate circadian rhythm, has recently been studied in its several functions. It is believed to inhibit cancer growth, to stimulate the immune system and to act as an antioxidant. In particular, this latter activity is ascribed to two different mechanisms: stimulation of radical detoxifying enzymes and scavenging of free radicals. We used this compound in mammalian cells in vitro to investigate its mechanism of action in modulating DNA damage. Cytogenetic and cytofluorimetric analyses were performed. We show that melatonin is able to modulate chromosome damage (chromosomal aberrations and sister chromatid exchanges) induced by cyclophosphamide. Conversely, its involvement in modulating oxidative processes, thereby reducing DNA damage, is less clear. In particular, melatonin is able to decrease H2O2-induced chromosomal aberrations but not sister chromatid exchanges and has been found to induce oxygen species in a cytofluorimetric test (DCFH assay).

Impaired p53-mediated DNA damage response, cell-cycle disturbance and chromosome aberrations in Nijmegen breakage syndrome lymphoblastoid cell lines.

PURPOSE: To investigate the p53 ionizing radiation-induced response, G1/S cell-cycle block and cytogenetic damage in Nijmegen breakage syndrome (NBS) cells characterized by different haplotypes. MATERIAL AND METHODS: Lymphoblastoid cell lines derived from three normals, five NBS and two ataxia telangiectasia (AT) individuals were treated with moderate doses of X-rays and changes in the p53 response were studied by dose-response and time-course experiments. Multiparametric flow cytometry analysis of bromodesoxyuridine-incorporated cells was carried out to analyse G1/S checkpoint alterations. Cytogenetic damage induced by 2 Gy radiation was assessed in cells harvested 28 h later. RESULTS: Comparison of mean values of p53 accumulation in NBS, AT and control cells indicated that protein induction in NBS cells was between normal and AT cells. Cell-cycle experiments showed a markedly reduced S-phase fraction in irradiated samples of normal cell lines, while NBS, and particularly AT cells, showed less reduction in S-phase fraction. Irrespective of differences in p53 induction and G1/S block, chromatid-type aberrations were induced at a comparable level in both syndromes, while being almost absent in normal cells. CONCLUSIONS: The data suggested that failure of NBS cells to initiate cell-cycle delay cannot account alone for their extreme sensitivity to radiation.

Phyllanthus orbicularis aqueous extract: cytotoxic, genotoxic, and antimutagenic effects in the CHO cell line.

The present work evaluates the cytotoxic, genotoxic, and antimutagenic effects of Phyllanthus orbicularis (plant of genus Phyllantus) aqueous extract in Chinese hamster ovary (CHO) cells. P. orbicularis aqueous extracts are used in Cuban traditional medicine for their antiviral activity against Hepatitis B virus and A and B flu virus. The cytotoxicity of the extract was tested by means of colony-forming ability and growth-inhibition assays as well as by measuring the mitotic index. Apoptosis induction and cell-cycle kinetics were analyzed by cytofluorimetric methods. Chromosome aberration assays were performed to study the genotoxic and antimutagenic activity of the extract. Results show that doses of up to 100 microg/ml of the extract did not induce any cytotoxic effects. Cell survival and mitotic index decreased significantly at doses higher than 100 microg/ml as a function of dose as well as of treatment time. Moreover, continuous treatments of up to 18 h induced the appearance of a significant number of apoptotic cells. Following a 3-h exposure to a dose of 750 microg/ml, cells accumulated significantly in G(2)-M phase and remained blocked in G(1-) and G(2)-M phases after several posttreatments in fresh growth medium. The aqueous extract alone did not induce chromosome aberrations but, in combined treatment with H(2)O(2), significantly reduced H(2)O(2)-induced chromosome aberrations. Flow cytometric analysis of DCFH intracellular oxidation showed that the extract decreased the oxidizing power of H(2)O(2.) This ability could possibly explain the extract's antigenotoxic activity. Absence of cytotoxicity at the lower tested doses and the antimutagenic properties of the extract stimulate the interest in studying possible new pharmaceutical uses of P. orbicularis.

DNA damage and cytotoxicity induced by beta-lapachone: relation to poly(ADP-ribose) polymerase inhibition.

beta-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5, 6-dione) was previously shown to enhance the lethality of X-rays and radiomimetic agents and its radiosensitizing role in mammalian cells was attributed to a possible interference with topoisomerase I activity. Furthermore, beta-lapachone alone was found to induce chromosomal damage in Chinese hamster ovary (CHO) cells. The aim of the present study was to further elucidate the possible mechanisms by which beta-lapachone exerts its genotoxic action in cultured mammalian cells. Flow cytometry analysis of beta-lapachone-treated CHO cells indicated a selective cytotoxic effect upon S phase of the cell cycle. beta-lapachone produced DNA strand breaks as determined by alkaline elution assay; alkaline elution profiles from treated cells showed a bimodal dose-response pattern, with a threshold dose above which a massive dose-independent DNA degradation was observed. Furthermore, beta-lapachone increased the capacity of crude CHO cellular extracts to unwind supercoiled plasmid DNA, while significantly inhibiting in vitro poly(ADP-ribose) polymerase (PARP). These results suggest that damage induction is probably mediated by the interaction between beta-lapachone and cellular enzymatic function(s), rather than reflecting a direct action on the DNA. We suggest that the inhibition of PARP plays a central role in the complex biological effects induced by beta-lapachone in CHO cells.

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.

Ascorbic acid and beta-carotene as modulators of oxidative damage.

Naturally occurring antioxidants are extensively studied for their capacity to protect organisms and cells from damage induced by oxygen reactive species. In fact, oxidative stress is considered a cause of aging, degenerative disease and cancer. We have focused our attention on two agents, ascorbic acid and beta-carotene, commonly considered to be antioxidants, but whose protective activity against cancer is insufficiently known. This paper reports on the ability of these agents to act against damage induced by H2O2 and bleomycin, in Chinese hamster ovary cells cultivated in vitro. Cytogenetic and cytofluorimetric analyses were performed. Both vitamins proved effective in reducing H2O2-induced sister chromatid exchanges, but increased H2O2- and bleomycin-induced chromosomal aberrations. Cytofluorimetric data, in contrast, showed that ascorbic acid and beta-carotene act as scavengers of endogeneous and H2O2-induced oxygen species.

Chromosomal sensitivity to clastogenic agents and cell cycle perturbations in Nijmegen breakage syndrome lymphoblastoid cell lines.

The relationship between chromosomal breakage and perturbations of cell cycle progression was investigated in lymphoblastoid cell lines established from a healthy donor, two subjects affected by Nijmegen Breakage Syndrome (NBS) and an ataxia-telangiectasia (AT) patient. The cytogenetic analysis revealed a similar chromosomal hypersensitivity in both NBS and AT cells exposed in the G1 phase to 200 cGy X-rays or in G2 to 15-30 cGy. Similarly, no differences were observed in the frequency of chromatid-type aberrations induced in G2 by 1-2 pg/ml calicheamicin gamma 1I, a DNA double-strand break inducer. In addition, as observed in AT cells, the rate of G2 radiation-induced chromosomal damage was less enhanced in NBS than in control cells following 3-h incubation with inhibitors of DNA synthesis/repair (cytosine arabinoside, aphidicolin, DMSO, hydroxyurea, caffeine). This is suggestive of an altered DNA lesion-processing pathway common to both syndromes. Despite the close resemblance of cellular phenotypes in the two syndromes, the analysis of mitotic indices carried out at 2 and 4 h postirradiation indicated that NBS sustained a G2-delay greater than that observed in AT cells, Furthermore, the flow cytometric analysis of 50-300 cGy irradiated cells at 10 and 20 h before harvesting showed that NBS cells sustained a G2/M phase arrest markedly lower than AT cells. Our data indicate that NBS and AT gene products are involved in a common pathway of radiation-induced chromosomal damage, but in a different one for cell cycle control after irradiation.

Modulation of radiation-induced chromosomal damage by inhibitors of DNA repair and flow cytometric analysis in ataxia telangiectasia cells with 'intermediate radiosensitivity'.

The relationship between repair processes and chromosomal aberrations and X-ray-induced cell cycle perturbations were investigated in ataxia telangiectasia (AT) cells with 'intermediate' (AT-INT) and 'classical' radiosensitivity. In the cytogenetic experiments, three AT-INT lymphoblastoid cell lines were X-irradiated in G2-phase and incubated in the presence of inhibitors of DNA polymerases alpha/delta/epsilon (cytosine arabinoside, aphidicolin, 10% v/v DMSO), ribonucleotide reductase (hydroxyurea) and presumed inhibitors of protein kinases (caffeine). Flow cytometric analysis was performed in cells harvested 20 h after irradiation and stained with either propidium iodide or antibody against 5-bromodeoxiuridine in order to investigate cell cycle distribution focusing on G2/Mphase accumulation. From our data it appears that: (i) chromosomal sensitivity to radiation in AT does not always reflect clinical features; (ii) the effects of DNA repair inhibitors are inversely correlated with chromosomal radiosensitivity; and (iii) radiation-induced G2/M phase accumulation is a feature of AT cells and not necessarily correlated with cellular and chromosomal sensitivity to ionizing radiation.

Transcriptional control of the Htf9-A/RanBP-1 gene during the cell cycle.

The mouse Htf9-a gene encodes a small hydrophilic protein, named Ran-binding protein 1 (RanBP-1), for its interaction with the Ras-related Ran protein; RanBP-1 binds the biologically active form of Ran. Ran has been implicated in a variety of nuclear events including DNA replication, RNA export and protein import, and monitoring completion of DNA synthesis before the onset of mitosis; it biological activity is thought to be regulated in S phase. We have investigated whether expression of the Htf9-a/RanBP-1 gene is linked to cell proliferation. Expression of the Htf9-a/RanBP-1 transcript appeared to be dependent on the proliferating state of the cells and peaked in S phase. We have identified a promoter region required for Htf9-a cell cycle-dependent expression and for responsiveness to cell cycle regulators. An E2F-binding site was identified within the cell cycle-regulated promoter region. Expression of the E1A oncoprotein prevented Htf9-a down-regulation in quiescent cells; in addition, both pRb and its relative p107 inhibited transcription from the Htf9-a promoter. These results link the control of Htf9-a/RanBP-1 expression to the cell cycle progression.

Low environmental radiation background impairs biological defence of the yeast Saccharomyces cerevisiae to chemical radiomimetic agents.

Background radiation is likely to constitute one of the factors involved in biological evolution since radiations are able to affect biological processes. Therefore, it is possible to hypothesize that organisms are adapted to environmental background radiation and that this adaptation could increase their ability to respond to the harmful effects of ionizing radiations. In fact, adaptive responses to alkylating agents and to low doses of ionizing radiation have been found in many organisms. In order to test for effects of adaptation, cell susceptibility to treatments with high doses of radiomimetic chemical agents has been studied by growing them in a reduced environmental radiation background. The experiment has been performed by culturing yeast cells (Saccharomyces cerevisiae D7) in parallel in a standard background environment and in the underground Gran Sasso National Laboratory, with reduced environmental background radiation. After a conditioning period, yeast cells were exposed to recombinogenic doses of methyl methanesulfonate. The yeast cells grown in the Gran Sasso Laboratory showed a higher frequency of radiomimetic induced recombination as compared to those grown in the standard environment. This suggests that environmental radiation may act as a conditioning agent.

Taurine and ellagic acid: two differently-acting natural antioxidants.

Naturally occurring antimutagenic compounds are extensively analyzed for their capacity to protect cells from induced damage. We selected two agents, taurine and ellagic acid, treated in the literature as antioxidants, but whose activity is insufficiently known. This paper reports on the ability of these agents to act against damage induced by mitomycin-C and hydrogen peroxide in Chinese hamster ovary cells cultivated in vitro. Cytogenetic and cytofluorimetric analyses were performed. Ellagic acid proved to have more than one mechanism of action, probably as a scavenger of oxygen species produced by H2O2 treatment, and as a protector of the DNA double helix from alkylating agent injury. In our experimental conditions, taurine seems able to scavenge oxygen species.

Sensitivity of lymphocytes from vulcanizers to the in vitro induction of sister chromatid exchanges.

Spontaneous frequencies of sister chromatid exchanges (SCEs) and SCEs induced in vitro by chemicals with different mechanisms of action such as mitomycin C, 4-nitroquinoline oxide, and 3-aminobenzamide were examined in phytohemagglutinin-stimulated peripheral blood lymphocytes from a group of workers in a rubber plant and a control group, both of which had been analyzed for levels of spontaneous SCEs 2 years earlier. An interindividual variability in the induction of SCEs was found after in vitro treatments with the different mutagens, which did not correlate with occupational exposure. This variability in the sensitivity to the induction of SCEs might be correlated to genetic differences among individuals, which have to be taken into account in environmental monitoring programs.

Biological monitoring of workers in the rubber industry. I. Chromosomal aberrations and sister-chromatid exchanges in lymphocytes of vulcanizers.

To evaluate the possible genetic consequences of the industrial exposure among the vulcanizers of a rubber plant we measured the in vivo levels of chromosomal aberrations and sister-chromatid exchanges in peripheral lymphocytes of 34 vulcanizers and in an adequate control population. The observed chromosomal aberration frequencies were 1.9 +/- 1.4 aberrations/100 cells in the exposed group and 2.1 +/- 1.5 aberrations/100 cells in the controls. No difference was found between the two groups for the mean value of sister-chromatid exchanges (5.2 +/- 1.3 in the exposed, 5.2 +/- 0.7 in the control group). Cigarette-smoking was clearly associated with increased sister-chromatid exchange frequencies both in the exposed and in the control groups, while chromosomal aberration frequencies were not correlated with smoking habits.