Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate.

Organolead compounds enter the environment primarily through the combustion of leaded gasoline and industrial discharge. Lead and lead-containing compounds have been shown to induce a broad spectrum of toxic effects, including hematopoietic, renal, neurologic, and carcinogenic effects. In this study, the mutagenic activity of triethyllead acetate (Et3PbAc) was determined by measuring the induction of chromosomal aberrations in Chinese hamster ovary cells. The results indicate that Et3PbAc is very cytotoxic and a potent clastogen. In preliminary cytotoxicity studies used to determine appropriate test concentrations for chromosomal aberration analysis, the LC50 of Et3PbAc was approximately 10 microM in the absence of metabolic activation, and 80 microM in the presence of metabolic activation. The maximal response was greater with metabolic activation than without. However, a much higher dose was required to elicit a significant response in the presence of metabolic activation than in its absence.     

Methyl mercury uptake and associations with the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells

In order to evaluate possible health effects of environmental exposure of humans towards methyl mercury species, relevant exposure experiments using methyl mercury chloride in aqueous solution and Chinese hamster ovary (CHO) cells were performed. The solution was monitored for the presence of monomethyl, dimethyl and elemental mercury by several analytical techniques including chromatographic as well as atomic absorption and mass spectrometric methods. Methyl mercury induces structural chromosomal aberrations (CA) and sister chromatid exchanges (SCE) in CHO cells. At a concentration of methyl mercury in the culture medium of 1.0 x 10(-6) M where the frequencies of CA and SCE are significantly elevated, the intracellular concentration was 1.99 x 10(-16) mol/cell. Possible biochemical processes leading to the cytogenetic effects are discussed together with toxicological consequences, when humans (e.g. workers at waste deposits) are exposed to environmental concentrations of methyl mercury.     

Restriction endonuclease Bam H I induces chromosomal aberrations in Chinese hamster ovary (CHO) cells

Treatment of Chinese hamster ovary (CHO) cells with the restriction endonuclease Bam H I (recognition site: G/GATCC) leads to high frequencies of chromosomal aberrations. Experiments with bromodeoxyuridine-labelled chromosomes show that the aberrations occur nearly exclusively in first post-treatment metaphases. The results are interpreted to mean that only some of the cells take up the enzyme and that these cells are the ones showing the aberrations. Cells which do not take up the enzyme show up as differentially stained metaphases and have no aberrations. Why some cells take up the restriction enzyme and others not is not known, possibly this is dependent on the physiological condition of the cells.     

Participation of active oxygen species in the induction of chromosomal aberrations by cadmium chloride in cultured Chinese hamster cells

The effect of various scavengers of active oxygen species on the induction of chromosomal aberrations by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Incidences of chromosomal aberrations by CdCl2 were partially or fully reduced by the presence of catalase, mannitol (a scavenger of hydroxyl radicals) and butylated hydroxytoluene (BHT, an antioxidant). These findings may indicate participation of the active oxygen species such as hydrogen peroxide (H2O2) or hydroxyl radicals in the clastogenicity of cadmium. In contrast, superoxide dismutase (SOD) and dimethylfuran (a scavenger of singlet oxygen) did not influence incidences of chromosomal aberrations by CdCl2. These results suggest that superoxide anion and singlet oxygen are not directly involved in the clastogenicity of the metal. The presence of aminotriazole (an inhibitor of catalase) increased incidences of chromosomal aberrations by CdCl2. This emphasizes participation of H2O2 in the clastogenicity of cadmium.     

Elevation of Alu I-induced frequencies of chromosomal aberrations in Chinese hamster ovary cells by Neurospora crassa endonuclease and by ammonium sulfate

The frequencies of chromosomal aberrations induced by the restriction endonuclease Alu I (recognition site AG/CT) can be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from Neurospora crassa (EC 3.1.30.1), or with ammonium sulfate in which the Neurospora endonuclease is suspended. These data indicate that Alu I does not produce DNA single-strand breaks in the chromatin of living cells, which can be recognized by the Neurospora endonuclease. The salt may induce conformational changes in the chromatin which make more recognition sites available for Alu I. Experiments with recovery times between the treatments with Alu I and the salt indicate that Alu I can act in the nucleus for at least 40 min.     

Influence of sodium butyrate on the induction of radiation-induced chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary (CHO) cells.

CHO cells were pre-treated with sodium butyrate (SB) for 24 h and then X-irradiated in G1. Metaphases were scored for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs). The data were compared with those obtained after irradiation of cells not pre-treated with SB and showed that SB has different effects on the endpoints examined. The frequencies of dicentric chromosomes were elevated and of small acentric rings (double minutes, DMs) reduced. These results are discussed to be a consequence of conformational changes in hyperacetylated chromatin which could lead to more interchromosomal and to less intrachromosomal exchanges. SB itself induces a few SCEs but suppresses the induction of SCEs by X-rays. We assume that a minor part of radiation induced SCEs are 'false' resulting from structural chromosomal aberrations, such as inversions, induced in G1. Inversions are the symmetrical counterparts of DMs. If inversions are suppressed by SB treatment to a similar extent as DMs a small reduction of SCEs by SB can be expected.     

Costunolide induces micronuclei formation,chromosomal aberrations,cytostasis, and mitochondrial-mediated apoptosis in Chinese hamster ovary cells

Costunolide (CE) is a sesquiterpene lactone well-known for its antihepatotoxic, antiulcer, and anticancer activities. The present study focused on the evaluation of the cytogenetic toxicity and cellular death-inducing potential of CE in CHO cells, an epithelial cell line derived from normal ovary cells of Chinese hamster. The cytotoxic effect denoting MTT assay has shown an IC₅₀ value of 7.56 μM CE, where 50% proliferation inhibition occurs. The oxidative stress caused by CE was confirmed based on GSH depletion induced cell death, conspicuously absent in N-acetylcysteine (GSH precursor) pretreated cells. The evaluation of genotoxic effects of CE using cytokinesis block micronucleus assay and chromosomal aberration test has shown prominent induction of binucleated micronucleated cells and aberrant metaphases bearing chromatid and chromosomal breaks, indicating CE’s clastogenic and aneugenic potential. The apoptotic death in CE treated cells was confirmed by an increase in the number of cells in subG1 phase, exhibiting chromatin condensation and membranous phosphatidylserine translocation. The apoptosis induction follows mitochondrial mediation, evident from an increase in the BAX/Bcl-2 ratio, caspase-3/7 activity, and mitochondrial membrane permeability. CE also induces cytostasis in addition to apoptosis, substantiated by the reduced cytokinetic (replicative indices) and mitotic (mitotic indices and histone H3 Ser-10 phosphorylation) activities. Overall, the cellular GSH depletion and potential genotoxic effects by CE led the CHO cells to commit apoptosis and lowered cell division. The observed sensitivity of CHO cells doubts unintended adverse effects of CE on normal healthy cells, suggesting higher essentiality of further studies in order to establish its safety efficacy in therapeutic explorations.     

Structure-activity relationship in the induction of chromosomal aberrations and spindle disturbances in Chinese hamster epithelial liver cells by regioisomeric phenanthrene quinones

Four regioisomeric phenanthrene (PH) quinones (Q) were investigated for their ability to induce chromosomal damage and spindle disturbances. PH 1,4-Q and PH 1,2-Q induced structural as well as numerical chromosomal aberrations, whereas the isomers PH 9,10-Q and PH 3,4-Q were virtually inactive in this respect. However, all four compounds enhanced the frequency of c-mitoses.     

Induction of chromosomal aberrations in cultured Chinese hamster cells by short-term treatment with cadmium chloride

Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride (CdCl2) was investigated under 3 different treatment conditions: (i) 2-h treatment in MEM medium supplemented with 10% fetal bovine serum (MEM + 10% FBS) or (ii) in HEPES-buffered Hanks' solution (HEPES-Hanks), and (iii) continuous treatment for 24 h in MEM + 10% FBS. Two-h treatment with CdCl2 in HEPES-Hanks or continuous treatment for 24 h in MEM + 10% FBS was respectively 2 or 3 times more cytotoxic than 2-h treatment with the metal in MEM + 10% FBS. Continuous treatment for 24 h with a CdCl2 concentration in excess of 5 X 10(-6) M was too toxic to the cells to allow chromosomal analysis, and moreover, only a slight increase in incidence of chromosomal aberrations was observed at a concentration of 5 X 10(-6) M CdCl2. In contrast, a marked and concentration-dependent increase in incidence of chromosomal aberrations was observed after post-treatment culture for 22 h follows 2-h treatment with 1 X 10(-6) M to 5 X 10(-5) M of CdCl2 in both MEM + 10% FBS and HEPES-Hanks. Two-h treatment with cadmium in HEPES-Hanks was approximately 3 times more potent for the induction of chromosomal aberrations than that in MEM + 10% FBS. Types of aberrations induced by CdCl2 mainly consisted of chromatid gaps and breaks, although a few exchanges, dicentrics and fragmentations were observed at high concentrations of cadmium. Increase in incidence of tetraploidy was also observed with a concentration dependency after 2-h treatment with CdCl2. Potency of CdCl2 to induce chromosomal aberrations after 2-h exposure was comparable to that of benzo[a]pyrene activated with S9 at equitoxic concentrations. Two-h treatment with cadmium markedly inhibited incorporation of [3H]thymidine, even at concentrations at which incorporation of [3H]uridine or [3H]leucine was less inhibited. However, the inhibition of [3H]thymidine incorporation by cadmium was reversible and the incorporation restored to the control level during 2-6 h of post-treatment incubation. These findings suggest that restoration of DNA synthesis after cadmium exposure is required for the efficient detection of chromosomal aberrations induced by the metal.     

Induction of chromosomal aberrations in cultured Chinese hamster cells in a superoxide-generating system

The induction of chromosomal aberrations in a superoxide-generating system using xanthine oxidase and hypoxanthine was investigated in cultured Chinese hamster cells. The production of chromosomal aberations in this system was inhibited by the addition of cytochrome C. This finding indicates that the generation of superoxide was the primary requirement for induction of chromosomal aberrations. On the other hand, superoxide dismutase showed no effect on the frequency of chromosomal aberrations, whereas catalase was effective in preventing the aberrations. It is conceivable, therefore, that the induction of chromosomal aberrations in the superoxide-generating system may be directly or indirectly due to hydrogen peroxide formed in the cultured medium as a result of the spontaneous dismutation reaction of superoxide.     

Potentiation of Alu I-induced chromosome aberrations by high salt concentrations in Chinese hamster ovary cells

The restriction endonuclease Alu I induces chromosome-type aberrations in Chinese hamster ovary cells whose frequencies are considerably elevated in the presence of high concentrations of MgCl2, (NH4)2SO4, CaCl2 or NaCl. The most plausible explanation for these findings is that salt leads to partial dehistonization of the chromatin which makes more recognition sites available for Alu I.     

Chromosomal aberrations caused by sesquiterpene lactones in chinese hamster ovary cells

The cytotoxic behaviour of 20 sesquiterpene lactones toward Chinese hamster ovary cells was examined. The structural pre-requisite for cytotoxicity was the α-methylene γ-lactone moiety. Certain sesquiterpene lactones caused chromosomal aberrations suggesting that DNA was the cellular target. The cellular target for most of these compounds, however, is probably not the nucleus and the cytotoxicity may be accounted for by Michael-type additions with sulphydryl groups of enzymes and other proteins.     

Induction of chromosomal aberrations in Chinese hamster ovary cells by the restriction endonuclease Dra I: dose-effect relationships and effect of substitution of chromosomal DNA with bromodeoxyuridine

Treatment of G1-phase Chinese hamster ovary (CHO) cells with the restriction endonuclease Dra I (recognition site TTT/AAA) leads to the induction of chromosome-type aberrations. The dose-effect relationships or the frequencies of polycentric chromosomes have a strong linear component. Prelabelling of the cells with bromodeoxyuridine (B) leads to a strong suppression by the chromosome breaking activity of Dra I. This may be explained by assuming that substitution of T by B renders the recognition site of Dra I resistant to being cut by the enzyme.     

Induction by chemical clastogens of aberrations in prematurely condensed interphase chromatin of Chinese hamster ovary cells

The clastogenic activities of diepoxybutane and bleomycin were comparatively studied on prematurely condensed interphase chromatin and metaphase chromosomes of Chinese hamster ovary cells. The yield of chromosomal aberrations was distinctly higher in G2-premature chromosome condensation as compared to metaphase. Most notably, the clastogenic activity of bleomycin was visible in premature chromosome condensation after application of much lower final concentrations than necessary for induction of chromosome aberrations in metaphase. In addition, the different mechanisms of action of both clastogens were reflected by the aberration yield in GI and G2 immediately after exposure. While bleomycin induced aberrations throughout all stages of interphase, diepoxybutane did not induce aberrations in GI or G2. Though certainly not a routine system for genotoxicity testing, premature chromosome condensation analyses provide a powerful opportunity to demonstrate relationships between DNA damage and repair, and the production of chromosomal changes at the site of their formation.Abbreviations BM bleomycin - BrdUrd bromodeoxyuridine - CHO Chinese hamster ovary - DEB diepoxybutane - DMSO dimethylsulfoxide - FCS fetal calf serum - PCC premature chromosome condensation, prematurely condensed chromosomes - PEG polyethylene glycol     

Influence of Neurospora endonuclease on trenimon-induced structural chromosomal aberrations and sister-chromatid exchanges in human peripheral lymphocytes and Chinese hamster ovary cells

Human peripheral lymphocytes and Chinese hamster ovary cells were treated in the G1 phase of the cell cycle with the trifunctional alkylating agent trenimon (TRN) and post-treated with a single-strand specific endonuclease from Neurospora crassa (NE). TRN induces chromosomal aberrations of the chromatid type (CA) and sister-chromatid exchanges (SCE). NE post-treatment leads to an elevation of the frequencies of CA but not of SCEs. This indicates that TRN induced CA are the result of DNA double-strand breaks and that the SCEs originate from other types of lesions, most probably base damage.     

Effects of iron chelators and glutathione depletion on the induction and repair of chromosomal aberrations by tert-butyl hydroperoxide in cultured Chinese hamster cells

The effects of iron chelators and glutathione (GSH) depletion on the induction of chromosomal aberrations by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. t-BuOOH in a concentration range of 0.1-1.0 mM induced chromosomal structural aberrations, consisting mainly of chromatid gaps and breaks, in a dose-dependent fashion. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. GSH depletion did not affect the formation of chromosomal aberrations and DNA single-strand breaks (ssb) by t-BuOOH. DNA ssb by 0.5 mM t-BuOOH were repaired within 60 min of treatment in both GSH-depleted (GSH-) and non-depleted (GSH+) cells. In contrast, chromosomal aberrations increased a little during the 60 min after treatment in both GSH- and GSH+ cells. The aberrations were then repaired in GSH+ cells but those in GSH- cells were maintained to a great extent during 20 h of post-treatment incubation. These results indicate that divalent iron mediates the induction of chromosomal aberrations by t-BuOOH. That t-BuOOH-induced chromosomal aberrations remain even after DNA ssb were repaired suggests involvement of other lesions than DNA ssb in the formation of chromosomal aberrations by the hydroperoxide.