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.     

Chromosomal response of human lymphocytes to streptozotocin

The induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the methylating agent streptozotocin (STZ) and the effect of this compound on mitotic index (MI) and cell cycle progression in human lymphocytes were investigated. Unstimulated (G(0)) or cycling lymphocytes derived from whole blood or purified lymphocyte cultures were pulse-treated with increasing doses of STZ for 0.5-24h. Induction of CAs by STZ was only observed in cycling lymphocytes derived from whole blood cultures (WBC) (P<0.05). On the contrary, STZ produced a significant and dose-response increase in the yield of SCEs in unstimulated as well as cycling lymphocytes (P<0.05). In addition, STZ induced a dose-dependent decrease in the MI but had a slight effect on cell cycle progression. These results suggest that SCEs are the most sensitive endpoint for evaluating the chromosomal effects of STZ on these cells.     

Effect of recombinant interferon-alpha on streptonigrin-induced chromosome aberrations and sister-chromatid exchanges in hamster cells

The effect of recombinant interferon-alpha-2a (rIFN-alpha-2a) on the induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the radiomimetic antibiotic streptonigrin (SN, 250 ng/ml, 20 min, 37 degrees C) in Chinese hamster ovary (CHO) cells was investigated. Recombinant IFN-alpha-2a (4500-45,000 IU/ml) was added to the cell cultures 30 min before SN and left in the culture medium until the end of SN treatment or until cell harvesting. A statistically significant increase in the frequency of CAs and SCEs was observed following treatment with SN (P < 0.05), whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs and SCEs over control values. Low rIFN-alpha-2a doses produced a reduction in the frequency of CAs and an increase in the yield of SCEs induced by SN, while high doses of the cytokine caused an increase in the yield of CAs and a reduction in the frequency of SCEs induced by the antibiotic. In addition, rIFN-alpha-2a caused a marked inhibition (around 50%) on the yield of SN-induced chromatid-type aberrations in the G(2) phase of the cell cycle. It is suggested that the inhibitory effect of rIFN-alpha-2a on the SN-induced chromosome damage is due to the stimulation of DNA synthesis and repair by the cytokine. On the other hand, our results give further support to our previous hypothesis that the induction of CAs and SCEs by SN is based on different mechanisms.     

Spontaneous sister chromatid exchange and chromosome aberration frequency in humans: the familial effect.

The possible effects of environmental and genetic factors on spontaneous frequencies of sister chromatid exchanges (SCEs) and cells with chromosome aberrations (CAs) in human lymphocytes were investigated by analysing 177 completed families (mother, father and at least one child). After removing the effects of methodological, biological and life-style factors by the use of multifactor analysis of variance (MANOVA), SCEs and CAs residuals were analysed by simple correlation analysis and principal component analysis. SCEs and CAs inter-familiar variability was higher than that found within families. A significant correlation was found between the average SCE frequencies shared by parents (the so-called 'midpoint parents', or 'midparent') and offspring (linear slope b=0.26+/-0.07, p<0.05), but also between mother and father (b=0.23+/-0.11, p<0.05) suggesting the presence of an effective environmental factor. The midparent-offspring correlation was found to be sustained by the mother-offspring relationship (b=0.28+/-0.08, p<0.05), being the father-offspring correlation not significant (b=0.16+/-0.11, p0.05). Concerning CAs, no statistically significant correlation between parents was found, but the strong relationship between mother and offspring was confirmed (b=0.468+/-0.11, p<0.001). The SCEs correlation between mother vs. offspring disappeared for older offspring (over 23 years old). The obtained findings strongly showed that the genetic make-up is barely detectable in the presence of domestic environment factors which are shown to play the major role in determining the interfamilial variability of SCE and CA in a general population. These results strengthen the suitability of the use of SCEs and CAs analysis in human cytogenetic surveillance for the detection of effective environmental factors.     

Induction of chromosome damage by aniline and its C-hydroxylated metabolites in the root cells of Vicia faba

The induction of sister-chromatid exchanges (SCEs) and chromosome aberrations (CAs) by aniline hydrochloride (AH) and its C-hydroxylated metabolites, o-, m- and p-aminophenol, in the root cells of Vicia faba was examined. AH induced CAs, but not SCEs. All the C-hydroxylated metabolites of aniline induced both SCEs and CAs. However, the treatment of cells with these metabolites at concentrations that did not cause significant increases in CAs resulted in significant increases in SCEs. These results seem to suggest that the substance that induced CAs in root cells treated with AH was not the C-hydroxylated metabolites of aniline.     

Induction of chromosome damage by benzo[a]pyrene, 2-aminofluorene and cyclophosphamide in the root cells of Vicia faba

The induction of sister-chromatid exchanges (SCEs) and chromosome aberrations (CAs) by benzo[a]pyrene (BP), 2-aminofluorene (2-AF) and cyclophosphamide (CP) in the root cells of Vicia faba was examined. BP and 2-AF induced CAs, but not SCEs. CP induced both SCEs and CAs.     

Induction of sister-chromatid exchanges by pesticides in primary rat tracheal epithelial cells and Chinese hamster ovary cells

Possible induction of sister-chromatid exchanges by butachlor, paraquat, phorate and monocrotophos was examined in primary rat tracheal epithelial (RTE) and Chinese hamster ovary (CHO) cells. At dose levels that killed less than 50% of the cell population, monocrotophos induced SCEs positively in CHO and RTE cells, while paraquat was positive only in RTE cells. In two trials of the same experiment, paraquat and butachlor in CHO cells, and phorate in either RTE or CHO cells failed to induce a significant number of SCEs at any dose level within the ranges assayed. On the other hand, in RTE cells, butachlor induced a significant number of SCEs at a dose level of 5 micrograms/ml in one trial, but was insignificant in another. The inductions in these assays were, however, dose-dependent. The addition of S9 mixture did not alter the results of SCE induction by these 4 pesticides in CHO cells. RTE cells were more vulnerable to paraquat in cytotoxicity and SCE assays than CHO cells. Cytotoxicities were ranked as butachlor greater than phorate greater than paraquat greater than monocrotophos to CHO cells and paraquat greater than butachlor greater than phorate greater than monocrotophos to RTE cells. Significant cell cycle delays were only found in the treatments with the highest dose levels of butachlor, paraquat and phorate in CHO cells. In addition, this is the first report on SCE induction in RTE cells.     

Uncoupling of the induction of mutations and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA

The REP mutagenesis protocol, which involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of deoxyribonucleoside triphosphate (dNTP) pool imbalance, has been shown to induce both mutations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. However, when a Syrian hamster melanoma-derived cell line, called 2E, which was selected for its ability to replace all of the thymine residues in DNA with BrUra, was subjected to the REP mutagenesis protocol, the correlation between the induction of mutations and SCEs was no longer observed. The 2E cells were found to be much more sensitive to the induction of mutations by REP mutagenesis than were the CHO cells. This increased sensitivity to REP mutagenesis was found to correlate with increased perturbations of the dNTP pools that have been shown to be involved in the mutagenic mechanism of this protocol. In contrast, when the induction of SCEs by the REP protocol was measured, it was found that although a baseline level of SCEs was detected in 2E cells, no significant induction of SCEs due to dNTP pool perturbation was observed. It was shown that high levels of SCEs were readily induced in 2E cells by other agents, e.g. mitomycin C. A model, which discusses the fate of mismatched bases thought to be generated by the REP mutagenesis protocol as the determining factor for the induction of mutations of SCEs, is proposed to explain the uncoupling of mutagenesis and SCE induction in 2E cells.     

Cytogenetic monitoring of chemical workers in production of the drug 1-propoxy-2-acetamino-4-nitrobenzol with reference to their smoking habits

The induction of chromosome aberrations (CAs) and sister-chromatid exchanges (SCEs) was examined in cultured lymphocytes from 23 individuals employed in the production of the drug 1-propoxy-2-acetamino-4-nitrobenzol. Lymphocytes of workers were cultured before and 13 weeks after starting professional activity. The latter showed a significant increase in both CAs and SCEs. Smokers (11 individuals) and non-smokers (12 individuals) were indistinguishable with respect to the frequencies of CAs or SCEs before starting professional activity, 13 weeks later smokers exhibited significantly more SCEs than non-smokers. This strongly suggests a comutagenic interaction of cigarette smoke and exposure to chemicals during drug production. On the basis of the technology applied the compound inducing CAs and SCEs in lymphocytes of smoking workers seems to be 2-acetamino-4-nitrophenol.     

Evaluation of vitamin B12 effects on DNA damage induced by pioglitazone

Pioglitazone is a prototype of thiazolidinediones, used for the treatment of type 2 diabetes mellitus. Previous studies suggest that pioglitazone might cause DNA damage by generation of oxidative species. In this study, we investigated the mutagenic effects of pioglitazone using sister chromatid exchanges (SCEs), and chromosomal aberrations (CAs) assays in cultured human lymphocytes. In addition, oxidative DNA damage was evaluated in cells culture by measuring 8-hydroxy-2'-deoxyguanosine (8-OH-dG) marker. We also investigated the possible protective effects of vitamin B12, which is associated with DNA repair, on DNA damage induced by pioglitazone. Treatment of the human lymphocytes with pioglitazone (100μM) significantly increases the frequency of SCEs and CAs (p<0.01). In addition, significant elevation in 8-OH-dG release from lymphocytes was observed after treatment with pioglitazone (p<0.01). On the other hand, pretreatment of cultures with vitamin B12 (13.5μg/ml) protected lymphocytes from the genotoxic effect of pioglitazone. Therefore, we conclude that pioglitazone is genotoxic, and it induces chromosomal and oxidative DNA damage in cultured lymphocytes and this toxicity is prevented by pretreatment with vitamin B12.     

Cytogenetic response to 1,2-dicarbonyls and hydrogen peroxide in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes

Mutagenic 1,2-dicarbonyls have been reported to occur in coffee and other beverages and in various foods. We have measured the induction of sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) to determine the genotoxicity of various 1,2-dicarbonyl compounds in Chinese hamster ovary (CHO) AUXB1 cells and human peripheral lymphocytes. The 1,2-dicarbonyls glyoxal, methylglyoxal and kethoxal each induced highly significant increases in both SCEs and ERCs in AUXB1 cells. Glyoxal and kethoxal induced SCEs but not ERCs in human peripheral lymphocytes. In addition, hydrogen peroxide induced highly significant levels of SCEs and ERCs in AUXB1 cells. Bisulfite, which reacts with carbonyl groups to form addition products, significantly reduced the frequency of SCEs and the proportion of ERCs when glyoxal, methylglyoxal, kethoxal and diacetyl were administered to AUXB1 cells. In addition, bisulfite blocked the formation of ERCs, but not SCEs, induced by hydrogen peroxide. These in vitro results suggest that 1,2-dicarbonyls may play an important role in the genotoxicity of some foods and beverages.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

Effect of sulphur dioxide exposure on human chromosomes

The genotoxic effects of an average concentration of 41.7 mg/m³ of SO₂ exposure on 42 workers of a fertilizer factory were investigated. Mitotic index (MI), chromosomal aberrations (CAs), sister-chromatid exchanges (SCEs) and satellite associations (SA) were observed. In SO₂-exposed workers, a higher mitotic index (7.09) was recorded in comparison to controls (4.34). The MI, however, declined with duration of exposure. Satellite associations showed a two-fold increase (17.1) as compared to controls (8.11). Among chromosomes, D-G group associations were the highest (7.43%), while 3D type associations were the lowest (0.4%). There was a significant difference (p < 0.05) in the mean frequency of CAs per cell in the exposed workers (3.262%) and the controls (0.833%). The mean frequency of SCEs per cell increased from 3.32 ± 0.1 in controls to 7.72 ± 0.19 in the exposed group. The difference was significant (p < 0.05). In smokers, alcoholics and smoker-alcoholics, the frequency of CAs and SCEs per cell was significantly higher than the non-smokers and non-alcoholics, both in the controls and the SO₂-exposed workers and showed a correlation with the duration of exposure. SO₂ is therefore a clastogenic and genotoxic agent for which necessary precautions must be taken.     

Genotoxicity assessment in patients with thalassemia minor

Thalassemia is an inherited blood disorder that affects both genders and results in reduced synthesis of hemoglobin, and thus causing anemia. Previous studies have shown that the severe form of this disease, thalassemia major, is associated with genotoxicity. This includes increases in the level of sister chromatid exchange (SCEs), chromosomal aberrations (CAs) and micronuclei. In this study, we assessed genotoxicity in the lymphocytes of thalassemia minor subjects using sister chromatid exchange (SCE) and chromosomal aberration (CA) assays. In addition, we investigated the level of oxidative DNA damage by measuring 8-hydroxy-2'-deoxyguanosine (8OHdG) biomarker in urine samples. Eighteen thalassemia minor subjects and eighteen matched normal healthy controls were volunteered in the study. In addition, seven thalassemia major patients were recruited as positive controls. The results showed increases in the frequency of SCEs (P<0.05) in thalassemia minor compared to healthy controls. However, no difference in CAs frequency was detected between thalassemia minor and controls (P>0.05). Both SECs and CAs in thalassemia major patients were significantly higher compared to other groups (P<0.05). Regarding urine 8OHdG levels, the result showed a slight increase in thalassemia minor compared to healthy controls but the difference was not significant (P>0.05). In conclusion, our results showed that thalassemia minor is associated with genotoxicity to blood lymphocytes as indicated by SCEs assay.     

Effect of recombinant interferon-alpha on bleomycin-induced chromosome damage in hamster cells

The effect of recombinant interferon-alpha-2a (rIFN-alpha-2a) on the induction of chromosomal aberrations (CAs) by the radiomimetic antibiotic bleomycin (BLM, 5 microg/ml, 30 min, 37 degrees C) in Chinese hamster ovary (CHO) cells was investigated. Recombinant IFN-alpha-2a (4500-180,000IU/ml) was added to the cell cultures 0.5 or 24h before BLM (and left in the culture medium until the end of treatments) or immediately after BLM treatment (and left in the culture medium until harvesting). Cells were sampled at 18 or 2.5h after the end of treatments, in order to determine, respectively, the effect of rIFN-alpha-2a on the total chromosome damage induced by BLM and on the chromosome damage induced by this antibiotic in the G(2) phase of the cell cycle. A statistically significant increase in the frequency of CAs was observed following treatment with BLM (P<0.05), whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs over control values (P>0.05). The yield of CAs by BLM was significantly inhibited by rIFN-alpha-2a (P<0.05, 65.3% maximum inhibition). A strong inhibitory effect (around 80%) of rIFN-alpha-2a on the yield of BLM-induced CAs in the G(2) phase of the cell cycle was also observed. It is suggested that the inhibitory effect of rIFN-alpha-2a on the induction of CAs by BLM is mainly due to the stimulation of DNA synthesis and repair by the cytokine.     

Effect of cigarette-smoke condensate and norharman on the induction of SCEs by direct and indirect mutagens in CHO cells

Cigarette-smoke condensate and norharman were investigated either alone or in combination with a number of direct or indirect mutagens for the induction of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Cigarette-smoke condensate and norharman induced SCEs in these cells, only in the presence of a metabolic activation system. The number of SCEs induced by the direct-acting mutagens mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine was decreased in the presence of cigarette-smoke condensate or norharman. However, cigarette-smoke condensate and norharman showed synergistic effects in combination with the indirect mutagens 2-acetylaminofluorene, 2-aminofluorene, N-hydroxy-acetylaminofluorene, 2-aminoanthracene and benzo[a]pyrene. No synergism was observed when CHO cells were treated simultaneously with cigarette-smoke condensate or norharman and the indirect mutagen cyclophosphamide.     

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.     

Disparate effects of 5-bromodeoxyuridine on sister-chromatid exchanges and chromosomal aberrations in Bloom syndrome fibroblasts

The existence of a high frequency of spontaneous sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) has thus far been supported by data on a small number of BS cell lines. To examine the cause of baseline SCEs more broadly, the frequencies of SCEs, as well as chromosomal aberrations (CAs) in 4 additional BS fibroblast strains were compared, under different assay and cell culture conditions, with those of normal cells in the range of approximately 0.9-90% 5-bromodeoxyuridine (BrdUrd) substitution into template DNA. SCEs at low levels of BrdUrd substitution were detected by an extremely sensitive immunofluorescent technique. From approximately 0.9% to 4.5% BrdUrd substitution, the SCE frequency in BS cells remained constant, at a level (40/cell) 8 times higher than that of normal cells. As BrdUrd substitution increased further, the SCE frequency in BS cells increased almost linearly, reaching 70-100 per cell at approximately 90% substitution, while the SCE increment in control fibroblasts was less than 5 per cell. Analysis of SCEs in 3 successive replication cycles similarly revealed that the SCE increment in BS cells depended on BrdUrd only at a high BrdUrd substitution level. In contrast to data on SCEs, CA induction by incorporated BrdUrd in BS cells was only slightly higher than that in normal cells. Thus, BS cells are extremely sensitive to BrdUrd for SCE induction, but much less so for CA induction.     

Aphidicolin-Resistant Mutants of Mouse Lymphoma L5178y Cells with a High Incidence of Spontaneous Sister Chromatid Exchanges

Two aphidicolin-resistant cell mutants (AC 12 and AC 41) with a fourfold increase in spontaneous frequency of sister chromatid exchanges (SCEs) were obtained out of over 400 aphidicolin-resistant mutants isolated from mouse lymphoma L5178Y cells. They also exhibited three- to fourfold increases in spontaneous frequency of chromosome aberrations (CAs). To determine whether the high level of SCE frequency in AC 12 is caused by 5-bromodeoxyuridine (BrdUrd) used for visualizing SCEs, the effect of BrdUrd incorporated into DNA on SCE induction was analyzed. The SCE frequencies in AC 12 remained constant at BrdUrd incorporation levels corresponding to 2-90% substitution for thymidine in DNA. In addition, the small amount of BrdUrd incorporated into both daughter and parenteral DNA strands in AC 12 had minimal effect on SCE induction. Furthermore, AC 12 and AC 41 were slightly resistant to BrdUrd with respect to the induction of CAs, the inhibition of cell-cycle progression and the decrease in mitotic activity. These findings suggest that the high incidence of SCEs in AC 12 and AC 41 is formed by their intrinsic defects, not by the effects of BrdUrd used. The analysis of SCE frequencies in hybrid cells between these mutants and the parental L5178Y revealed that the genetic defects in AC 12 and AC 41 appear to be recessive, and that these two mutants belong to the same complementation group. Furthermore, AC 12 belonged to a different complementation group from ES 4, which was isolated previously from L5178Y as an SCE mutant with a twofold higher frequency of spontaneous SCEs. This finding indicates that at least two different genetic defects participate in the formation of the high incidence of spontaneous SCEs in mouse cells. These SCE mutants would provide valuable cell materials for studying the molecular mechanism of SCE formation.     

Exposure of nuclear medicine patients to ionizing radiation is associated with rises in HPRT- mutant frequency in peripheral T-lymphocytes

When Chinese hamster fibroblasts were exposed to hydrogen peroxide or to a system consisting of xanthine oxidase and hypoxanthine, which generates superoxide anion plus hydrogen peroxide, sister-chromatid exchanges (SCEs) were formed in a dose-dependent manner. When the iron-complexing agent o-phenanthroline was present in the medium, however, the production of these SCEs was completely inhibited. This fact indicates that the Fenton reaction: Fe2+ + H2O2----OH0 + OH- + Fe3+ is responsible for the production of SCEs. When O2- and H2O2 were generated inside the cell by incubation with menadione, the production of SCE was prevented by co-incubation with copper diisopropylsalicylate, a superoxide dismutase mimetic agent. The most likely role of O2- is as a reducing agent of Fe3+: O2- + Fe3+----Fe2+ + O2, so that the sum of this and the Fenton reaction, i.e., the iron-catalyzed Haber-Weiss reaction, provides an explanation for the active oxygen species-induced SCE: H2O2 + O2(-)----OH- + OH0 + O2. According to this view, the OH radical thus produced is the agent which ultimately causes SCE. These results are discussed in comparison with other mechanisms previously proposed for induction of SCE by active oxygen species.