Plant extracts induce chromosome aberrations and sister-chromatid exchanges in Chinese hamster ovary cells and human lymphocytes

Effects of extracts from Vicia faba were compared with those of Zea mays for the induction of sister-chromatid exchanges (SCEs) and of chromosome aberrations (CAs) in Chinese hamster ovary (CHO) cells. CA induction by the maize extract was also tested in human lymphocytes. The extracts from roots and leaves of Vicia faba induced CAs and SCEs in CHO cells. The extracts from maize leaves also induced SCEs and CAs in CHO cells, and CAs in human lymphocytes. Maize extracts were more potent in inducing SCEs than Vicia extracts and the SCE- and CA-inducing capacity of maize extracts decreased during preincubation before addition to cells.     

The effect of fluoride on chromosome aberration and sister-chromatid exchange frequencies in cultured human lymphocytes

Sodium fluoride, at concentrations of up to 60 times the level normally used in drinking water for the prevention of dental decay, was compared with 2 other inorganic salts for its ability to induce chromosome aberrations and sister-chromatid exchanges (SCE) in cultured human lymphocytes. No significant increases in the frequencies of aberrations of SCEs were found.     

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.     

Role of the Fancg gene in protecting cells from particulate chromate-induced chromosome instability

Particulate hexavalent chromium (Cr(VI)) is a known human lung carcinogen. Cr(VI)-induced tumors exhibit chromosome instability (CIN), but the mechanisms underlying these effects are unknown. We investigated a possible role for the Fanconi anemia (FA) pathway in particulate Cr(VI)-induced chromosomal damage by focusing on the Fancg gene, which plays an important role in cellular resistance to DNA interstrand crosslinks. We used the isogenic Chinese hamster ovary (CHO) KO40 fancg mutant compared with parental and gene-complemented cells. We found that fancg cells treated with lead chromate had lower intracellular Cr ion levels than control cell lines. Accounting for differences of Cr ion levels between cell lines, we discovered that fancg cells treated with lead chromate had increased cytotoxicity and chromosomal aberrations, which was not observed after restoring the Fancg gene. Chromosomal damage was manifest as increased total chromosome damage and percent metaphases with damage, specifically an increase in chromatid and isochromatid breaks. We conclude that Fancg protects cells from particulate Cr(VI)-induced cytotoxicity and chromosome damage, which is consistent with the known sensitivity of fancg cells to crosslinking damage and the ability of Cr(VI) to produce crosslinks.     

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.     

In vitro genotoxic effects of the anticancer drug gemcitabine in human lymphocytes

This research was carried out to investigate in vitro genotoxic effects of the anticancer agent gemcitabine on the induction of chromosomal aberrations and sister-chromatid exchange in human lymphocytes. Three doses of gemcitabine (0.001, 0.002 and 0.004 microg/ml) were applied to lymphocyte cultures from 15 donors. There was a significant increase in the induction of chromosome aberrations and in the occurrence of sister-chromatid exchange in these cells. In addition, gemcitabine significantly decreased the mitotic index and replicative index for all doses. Dose-response regression lines were used to compare the individual susceptibilities to gemcitabine with respect to the chromosome aberration and sister-chromatid exchange frequencies. Our results indicate that gemcitabine is able to induce both cytotoxic and genotoxic effects in human lymphocyte cultures in vitro in a dose-dependent manner.     

Cytogenetic effects of 2-methoxyethanol and its metabolite,methoxyacetaldehyde, in mammalian cells in vitro

Glycol ethers such as 2-methoxyethanol (2-ME) are reproductive toxins. The genotoxicity of 2-ME, especially its metabolites: methoxyacetaldehyde (MALD) and methoxyacetic acid (MAA), is not adequately investigated yet. We have shown previously that MALD induced mutation in the bacterial gpt gene which is inserted in an autosome of CHO-AS52 cell line but not in the hprt gene on the X chromosome of CHO-K1-BH4 cell line. These data suggest that MALD induces major deletion-type mutation. If this prediction is correct we would expect to observe that MALD is an efficient inducer of chromosome aberrations in both CHO cell lines. We have conducted a cytogenetic study using both CHO cell lines and human lymphocytes to investigate this phenomenon. Our results show that human lymphocytes treated with 10–30 mM MALD for 1 h or 0.05–0.5 mM MALD for 24 h induced significant dose-dependent increase of sister-chromatid exchanges (SCE) (p < 0.05). It also induced significant dose-dependent increase (p < 0.05) of chromosome aberrations in human lymphocytes (10–40 mM treated for 1 h, or 0.05–2.5 mM for 24 h) and in both CHO cell lines (1.25–20 mM for 3 h). Treatment of these cells with the parent compound, 2-ME did not induce chromosome aberrations nor SCE unless very high doses of the chemical were used. In conclusion, these results indicate that MALD is clastogenic to different cell types therefore it is potentially carcinogenic. The genotoxic effects of 2-ME in humans will be dependent upon the metabolic capability of individuals to bioactivate 2-ME to MALD.     

Induction of chromosome damage by methylene chloride in CHO cells

The genotoxicity of methylene chloride was determined using sister-chromatid exchange (SCE) and chromosome aberration assays in cultured Chinese hamster ovary (CHO) cells. Methylene chloride caused extensive chromosome aberrations both with and without metabolic activation. However, the results of the SCE assay were negative for methylene chloride. These results agree with previously observed genotoxic effects of methylene chloride in Salmonella typhimurium and Saccharomyces cerevisiae. The fact that methylene chloride causes chromosome aberrations without increasing the SCE level indicates that complete reliance on the induction of SCE as a test system for assessing chromosomal effects is not valid.     

Sister chromatid exchanges induced in cultured mammalian cells by chromate

Chromate compounds induced sister chromatoid exchanges (SCEs) and chromosome aberrations in cultured mammalian cells. Similar increases in SCE frequency were observed in human fibroblasts exposed to the compounds K2Cr2O7 and K2CrO4. Marked increases in SCE frequency in cells exposed to chromate for a 48-h period were detected at concentrations between 10(-7) and 10(-6) M. Chromosome aberrations (primarily chromatid breaks) were also produced in human cells exposed to K2CrO4 at concentrations between 8 . 10(-7) and 3 . 10(-6) M. K2CrO4, but not the trivalent compound CrCl3, induced SCEs in Chinese hamster ovary (CHO) cells at low concentrations.     

Chromosome aberrations and sister-chromatid exchange frequencies in pathology staff occupationally exposed to formaldehyde

Past studies have shown that formaldehyde is mutagenic in microbial tests and Drosophila and causes chromosomal aberrations in cultured mammalian cells. Chromosomal analysis of bone marrow cells and spermatocytes from exposed laboratory animals has failed to show any genotoxic effect. Information on individuals occupationally exposed is limited and there is no evidence to date that formaldehyde can induce chromosome damage at occupational levels of exposure. This study examines the chromosome aberration and sister-chromatid exchange frequencies in lymphocytes from a group of 6 pathology workers and 5 unexposed controls. No detectable differences could be found between the groups in either chromosomal aberration induction or sister-chromatid exchange frequencies.     

Vitamin C is positive in the DNA synthesis inhibition and sister-chromatid exchange tests

Ascorbate caused a dose-dependent increase in sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells and in human lymphocytes. Moreover, in the DNA synthesis inhibition test with HeLa cells, ascorbate gave results typical of DNA-damaging chemicals. Catalase reduced SCE induction by ascorbate, prevented its cytotoxicity in CHO cells, and prevented its effect on HeLa DNA synthesis. Ascorbate reduced induction of SCE in CHO cells by N-methylN′-nitrosoguanidine (MNNG) by direct inactivation of MNNG.     

Effects of vanillin on sister-chromatid exchanges and chromosome aberrations induced by mitomycin C in cultured Chinese hamster ovary cells

Effects of vanillin on the induction of sister-chromatid exchanges (SCEs) and structural chromosome aberrations by mitomycin C (MMC) were investigated in cultured Chinese hamster ovary cells. Vanillin induced neither SCEs nor chromosome aberrations by itself. However, an obvious increase in the frequency of SCEs was observed when MMC-treated cells were cultured in the presence of vanillin. The effect of vanillin was S-phase-dependent. On the contrary, the frequency of cells with chromosome aberrations was significantly decreased by the post-treatment with vanillin at G2 phase.     

Influence of dietary carrot on cytostatic drug activity of cyclophosphamide and its main directly acting metabolite: induction of sister-chromatid exchanges in normal human lymphocytes, Chinese hamster ovary cells, and their DNA repair-deficient cell lines

We have utilized an in vivo drug metabolism technique (i.e. injecting the chemical into rat and isolating plasma with metabolites from blood) for detecting the genotoxicity of indirectly acting cyclophosphamide and its directly acting metabolite phosphoramide mustard in cultures of human peripheral blood lymphocytes of normal individuals, Fanconi's anaemia (FA) and aplastic anaemia (AA) patients, wild-type Chinese hamster ovary cells (CHO) and its DNA repair-deficient mutant 43-3B cells. In addition, the influence of dietary carrot on the clastogenic activity of these 2 chemicals in all the different cell types was studied. The genotoxicity was assessed by the ability of the metabolites of these agents to induce sister-chromatid exchanges in the treated cells. A dose-dependent increase in the frequencies of sister-chromatid exchanges was observed in all cell strains following treatment with activated metabolites of cyclophosphamide or phosphoramide mustard. The sensitivity of lymphocytes from normal donors, FA and AA patients to these 2 chemicals was similar. In CHO cell lines the induced frequency of sister-chromatid exchanges was slightly higher after treatment with the metabolites of cyclophosphamide than with phosphoramide mustard. The mutant 43-3B cells responded with higher frequencies of SCEs when compared to the wild-type CHO cells, about 1.5-2-fold, at low doses. Pretreating of rats with fresh carrot juice effectively inhibited the increase in the frequencies of sister-chromatid exchanges induced by cyclophosphamide in wild-type and mutant CHO cells (P less than 0.01), and to a lesser extent in human lymphocytes (p less than 0.05). In contrast, no inhibitory effect was observed in any of these cell types in combination of dietary carrot for direct acting phosphoramide mustard on the frequency of induced sister-chromatid exchanges. The possibility that dietary carrot exerts its antimutagenic effect by affecting the processes of enzymatic activation of cyclophosphamide is discussed.     

Clastogenicity of lead chromate particles in hamster and human cells.

Several insoluble compounds of chromium, such as lead chromate, are respiratory carcinogens in experimental animals and suspected to be so in humans. Lead chromate induces neoplastic transformation in cultured cells but the mechanism of genotoxicity is unknown. We examined the effect of lead chromate on the integrity of chromosomes of Chinese hamster ovary (CHO) and human foreskin fibroblasts (HFF) after a 24-h exposure. At 0.4 microgram/cm2, 0.8 microgram/cm2, 2 microgram/cm2 and 8 microgram/cm2 lead chromate particles reduced survival of CHO cells to 86%, 62%, 2% and less than 1% respectively. These concentrations induced a dose-dependent 4-19-fold increase in the percent metaphases with damage. The HFF cells exhibited higher sensitivity in both cytotoxicity and clastogenicity. The spectrum of damage observed for both cell types was primarily achromatic lesions affecting one or both chromatids. To test for particle dissolution effects, CHO cells were treated for 24 h with either clarified medium that had been incubated for 24 h with lead chromate particles, or clarified medium that had been pre-conditioned by CHO cells treated with lead chromate particles for 24 h. No damage was detected in these cells, indicating that extracellular dissolution into ionic lead and chromate did not contribute to the genotoxicity. This is consistent with a previous study in which scanning electron micrographs illustrated internalization of the particles. These results suggest that clastogenesis may be a mechanism for lead chromate induced carcinogenesis.     

The effect of aphidicolin on Fanconi's anemia lymphocyte chromosomes

The cytogenetic effect of the DNA polymerase alpha inhibitor aphidicolin (APC) at a dose which did not affect cell cycle progression was determined in normal and Fanconi's anemia (FA) lymphocytes. APC enhanced sister-chromatid exchange (SCE) levels by about twice both in control and FA cells, while the yields of chromosome breakage increased up to 20 times in normal lymphocytes and 4 times in FA cells. APC did not act synergistically with the bifunctional alkylating diepoxybutane in terms of SCE either in normal or in FA lymphocytes. However, chromosome aberrations in cultures from normal subjects were much more than expected by an additive mode of action.     

Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells

Cytotoxicity, chromosome aberrations, and mutations to 6-thioguanine resistance were synergistically increased by incubating the ultraviolet light (UV)-irradiated Chinese hamster ovary (CHO) cells in medium containing sodium arsenite. However, the frequencies of sister-chromatid exchanges and mutations to ouabain resistance induced by UV were not synergistically increased by sodium arsenite. The synergistic effect of sodium arsenite on UV-induced chromosome aberrations varied with cell-harvesting time and decreased with increasing time intervals between UV and sodium arsenite treatments.     

Enhancing effects of cinoxate and methyl sinapate on the frequencies of sister-chromatid exchanges and chromosome aberrations in cultured mammalian cells

Sister-chromatid exchanges (SCEs) induced by mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) or UV-light in cultured Chinese hamster ovary cells (CHO K-1 cells) were enhanced by cinoxate (2-ethoxyethyl p-methoxycinnamate) or methyl sinapate (methyl 3,5-dimethoxy 4-hydroxycinnamate). Both substances are cinnamate derivatives and cinoxate is commonly used as a cosmetic UV absorber. Methyl sinapate also increased the frequency of cells with chromosome aberrations in the CHO K-1 cells treated with MMC, 4NQO or UV. These increasing effects of methyl sinapate were critical in the G1 phase of the cell cycle and the decline of the frequencies of UV-induced SCEs and chromosome aberrations during liquid holding was not seen in the presence of methyl sinapate. Both compounds were, however, ineffective in cells treated with X-rays. In cells from a normal human embryo and from a xeroderma pigmentosum (XP) patient, MMC-induced SCEs were also increased by the post-treatment with methyl sinapate. The SCE frequencies in UV-irradiated normal human cells were elevated by methyl sinapate, but no SCE-enhancing effects were observed in UV-irradiated XP cells. Our results suggest that the test substances inhibit DNA excision repair and that the increase in the amount of unrepaired DNA damage might cause the enhancement of induced SCEs and chromosome aberrations.     

Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19

The Chinese hamster ovary (CHO) cell mutant, EM9, is defective in rejoining strand breaks, hypersensitive to chlorodeoxyuridine (CldUrd), and has a high frequency of sister-chromatid exchange (SCE). Somatic cell hybrids constructed from fusion of EM9 cells with normal human lymphocytes and fibroblasts, and selected in CldUrd, extensively segregate human chromosomes but preferentially retain markers of human chromosome 19. The SCE frequency in the hybrid clones is low as in normal CHO cells, but in CldUrd-sensitive subclones, which lose the human chromosome 19 markers, SCE frequencies return to mutant levels. We therefore assign a human gene designated repair complementing defective repair in Chinese-hamster (RCC) to chromosome 19. Since this is the second (of two) human genes complementing repair-deficiency mutations in CHO cells assigned to the 19, the assignment and organization of DNA-repair genes is discussed in the light of hemizygosity in CHO cells and the evolutionary conservation of mammalian linkage groups.     

The relation between chemically induced sister-chromatid exchanges and chromatid breakage.

Studies of classical chromosome aberrations and sister-chromatid exchanges (SCES) suggest independent mechanisms for the two events despite some common features. Examination of chromosome breakage caused by X-rays, visible light, and viruses has shown that few chromatid breaks are accompanied by SCEs at the sites of breaks. No similar observations were available for chemically induced breaks, but it has been reported that rat chromosomes exposed to dimethylbenzanthracene (DMBA) contained a preponderance of both aberrations and SCEs in certain specific regions, implicating a common process in their formation. These conclusions were drawn from a comparison of breaks induced in vivo with SCEs induced in vitro. However, we used 7 chemical mutagens to induce both chromatid breaks and SCEs in "harlequin" chromosomes of cultured rat and Chinese hamster ovary (CHO) cells and found that 25% of the 914 breaks scored were associated with SCEs. The proportion of breaks accompanied by SCEs is related to the overall SCE frequency and falls into the range predicted on the basis that breaks and SCEs occur independently. The reported association between sites for SCEs and aberrations also reflects secondary factors, such as induction of SCEs and aberrations during DNA synthesis in late replicating regions of the chromosomes.     

Chromosome aberration test of Pigment Yellow 34 (lead chromate) in Chinese hamster ovary cells

Lead chromate pigment in the form of the commercial pigment, Pigment Yellow 34, CAS No. 1344-37-2, used in the plastics and coatings industries, did not induce chromosome aberrations in Chinese hamster ovary (CHO) cell line WB(L). Lead chromate pigment is essentially insoluble in water, and in an effort to test the material under realistic conditions, no attempt to solubilize the pigment was made. These results are significant because others have reported lead chromate to cause genotoxicity in various assays, but only under conditions in which its aqueous solubility was artificially enhanced.