Suppressing effect of tannic acid on the frequencies of mutagen-induced sister-chromatid exchanges in mammalian cells

The effects of tannic acid (m-galloyl gallic acid) and 7 of its analogues on the frequencies of sister-chromatid exchanges (SCEs) were investigated in cultured Chinese hamster cells. SCEs induced by UV-light or mitomycin C (MMC) were suppressed by post-treatment with tannic acid and 5 of its analogues. These effects were independent of the extension of the cell cycle. The compounds which showed an SCE-suppressing effect have a common structure of 3 neighboring hydroxy or methoxy groups substituted on the phenyl group in benzoic acid or ester. These decreasing effects of tannic acid were observed in the G1 phase but not in the S or G2 phase of the cell cycle and a greater decline of the frequencies of UV-induced SCEs during liquid holding was seen in the presence of tannic acid. However, cells irradiated with X-rays were not influenced by tannic acid. In cells from a xeroderma pigmentosum (XP) patient, a Fanconi's anemia (FA) patient, and a normal human embryo, MMC-induced SCEs were also decreased by post-treatment with tannic acid. Tannic acid reduced the SCE frequencies in UV-irradiated FA and normal human cells but not in UV-irradiated XP cells. Our results suggest that tannic acid modifies DNA-excision repair and that the decrease in the amount of unrepaired DNA damage might cause the reduction of induced SCEs.     

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.     

Hypersensitive character of Bloom syndrome B-lymphoblastoid cell lines usable for sensitive carcinogen detection

Various carcinogens were tested with regard to the induction of sister-chromatid exchanges (SCEs) and chromosome aberrations using 3 types of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) (type I with normal frequency of SCEs and normal karyotype; type II with high frequency of SCEs and normal karyotype; type III with high frequency of SCEs and abnormal karyotypes) in the presence and absence of S9 mix. Three types of BS B-LCLs and normal cells showed different responses to the various carcinogens in the level of SCE induction. BS type I cells had the same SCE response as normal cells to carcinogens. Some carcinogens that require metabolic activation (S9 mix) had little effect on type II cells without S9 mix but had high SCE levels with S9 mix. BS type III cells were highly susceptible to both direct and indirect carcinogens with respect to high SCE increase without S9 mix (ca. 140 SCEs/cell), though some carcinogens produced SCEs rated in the medium (ca. 120 SCEs/cell) range, and had a high rate (more than 10%) of centromere spreading (CS), in addition to quadriradials. Therefore BS type III is a unique cell line which can be used to detect carcinogens.     

Enhancing effects of heterocyclic amines and beta-carbolines on the induction of chromosome aberrations in cultured mammalian cells.

The effects of post-treatment with heterocyclic amines and beta-carbolines on the induction of chromosome aberrations were studied in Chinese hamster CHO K-1 cells and SV40-transformed excision repair-deficient human XP2OSSV cells. The number of chromosome aberrations induced by UV and MMC were increased by post-treatment with Trp-P-1 and Trp-P-2, in both the presence and the absence of S9 mix. A alpha C, MeA alpha C, Glu-P-1, Glu-P-2, IQ, MeIQ, harman and harmine increased chromosome aberrations only in the presence of S9 mix. Glu-P-2, IQ, MeIQ, harman, and harmine did not induce chromosome aberrations by themselves at the concentrations used in this study. Trp-P-1, Trp-P-2, A alpha C, MeA alpha C and Glu-P-1 were weak clastogens by themselves, but at much higher concentrations than those at which they increased the induction of chromosome aberrations in cells pretreated with UV or MMC. Therefore, the increases in chromosome aberrations were not considered to be additive.     

Differential sensitivity of peripheral blood lymphocytes of untreated leprosy patients to mitomycin C

The effects of a bifunctional alkylating agent mitomycin C (MMC), an effective inducer of chromosome aberrations and sister-chromatid exchanges (SCEs), have been studied in untreated leprosy patients. This was done to study the mutagen sensitivity of the leprosy patients. The frequency of chromosomal aberrations induced by MMC (conc. 0.01 microgram/ml) was 2.5% in controls, 3.6% in paucibacillary (PB), and 6.8% in multibacillary (MB) patients. The difference in the frequency of MMC-induced chromosome aberrations between the 3 groups studied was highly significant (p less than 0.01). Cultures grown with MMC showed the frequency of SCEs/cell to be 12.70 +/- 1.19 in controls, 19.97 +/- 3.51 in PB, and 29.66 +/- 5.92 in MB patients. The differences in the frequency of MMC-induced SCEs between the 3 groups were found to be highly significant (p less than 0.01). The enhanced frequencies of spontaneous and MMC-induced chromosome aberrations and SCEs observed in PB and MB patients indicate a clear differential mutagen sensitivity between PB and MB patients who are known to have different immunological status and thereby differ in the severity of the disease.     

Genotoxic effects of o-phenylphenol metabolites in CHO-K1 cells

The effects of microsomal activation and/or deactivation on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) in cultured Chinese hamster ovary cells (CHO-K1 cells) by o-phenylphenol (OPP) were studied, and concurrently the metabolites were determined. After a 3-h incubation in the presence of 15% S9 mix (45 microliters/ml of S9), OPP (25-150 micrograms/ml) dose-independent SCEs and chromosomal aberrations were induced, while the amount of phenylhydroquinone (PHQ) metabolite produced from OPP did not increase linearly in the higher doses. The maximum induction of chromosomal aberrations was 18% at the 150 micrograms/ml dose, and of SCEs 13.8/cell at 75 micrograms/ml. The corresponding control values were 3% and 5.8/cell. The lowest dose required to induce SCEs in the presence of S9 mix was 25 micrograms/ml. Changing the percent of S9 mix (0-50%) while holding the OPP dose constant (100 micrograms/ml) produced a correlation between SCEs and the production of PHQ. PHQ caused cytogenetic effects both with and without S9 mix, however, in the absence of S9 mix it was more lethal and was oxidized to phenylbenzoquinone (PBQ). These results suggest that the enhanced cytogenetic effects of OPP by the addition of S9 mix correlated with the amount of PHQ produced or with the further oxides of PHQ such as phenylsemiquinone and/or PBQ which are capable of being produced from PHQ spontaneously or by the mixed-function oxidase system.     

Cytogenetic studies on 1,1-dichloroethylene and its two isomers in mammalian cells in vitro and in vivo

Chromosomal aberration and sister-chromatid exchange (SCE) tests in vitro on 1,1-dichloroethylene (1,1-DCE), its two isomers, cis- and trans-1,2-DCE, and two possible metabolites of 1,1-DCE, chloroacetyl chloride and chloroacetic acid, were carried out using a Chinese hamster cell line, CHL. 1,1-DCE induced chromosomal aberrations in the presence of S9 mix prepared from the rat liver, but not in the absence of S9 mix. SCEs were also slightly induced by 1,1-DCE only in the presence of S9 mix. On the other hand, two isomers and two metabolites of 1,1-DCE induced neither chromosomal aberrations nor SCEs with and without S9 mix. 1,1-DCE, however, was negative even at a sublethal dose in the micronucleus test using mouse bone marrow, fetal liver and blood.     

Induction of sister chromatid exchanges and chromosome aberrations in cultured mammalian cells treated with aminophenylnorharman formed by norharman with aniline

Aminophenylnorharman (APNH) is a newly identified mutagenic heterocyclic amine formed by coupling of norharman with aniline in the presence of S9 mix. Furthermore, mutagenic amino-3'-methylphenylnorharman (AMPNH) and aminophenylharman (APH) have been identified from a reaction mixture of norharman and o-toluidine and that of harman and aniline, respectively, with S9 mix. Among these three heterocyclic amines, APNH shows most potent mutagenic activity towards Salmonella typhimurium TA98 and YG1024 with S9 mix. In the present study, the induction of sister chromatid exchanges (SCEs) by APNH was examined in Chinese hamster lung (CHL) cells in vitro, comparing it to those of AMPNH and APH. On incubation with rat S9 for 6h, followed by a recovery culture period of 18h, a dose-dependent effect was found at concentrations between 0.00125 and 0.01 microg/ml for APNH and between 0.3125 and 5 microg/ml for AMPNH and APH. The approximate chemical concentrations leading to a three-fold of control SCE levels calculated from slopes of the linear regressions of induced SCEs were 0.005 for APNH, 0.51 for AMPNH and 1.7 microg/ml for APH. Because of the very strong SCE-causing ability of APNH, we further explored its genotoxicity by examining the induction of chromosome aberrations in CHL cells. A dose-dependent effect was found for chromosome aberrations at concentrations between 0.00125 and 0.04 microg/ml of APNH. The aberrations observed were primarily chromatid exchanges (cte) and breaks (ctb). In conclusion, the potency of SCE induction and clastogenic activity induced by APNH is stronger than Actinomycin D, Mitomycin C (MMC) or 1,8-dinitropyrene which are considered to be the potent clastogens in the literature. Further studies are needed for elucidating mechanisms of the genotoxic actions of these compounds and for evaluating their potential hazards to human health.     

Cytogenetic effects of penicillamine

Penicillamine (PA), a drug used for the treatment of rheumatoid arthritis induces sister-chromatid exchanges (SCEs) and chromosome aberrations in cultivated mammalian cells. PA in concentrations from 400 micrograms/ml upward induced SCEs and proliferative delay in human blood cultures when added for the last 24 h of the culture period. In V79 Chinese hamster cells SCE induction was found after acute exposure to PA before the addition of BrdUrd and after chronic exposure during one cell cycle in the presence of BrdUrd. The effect of PA on SCE frequencies occurred both after treatment in complete medium and in serum-free medium and was not influenced by the application of an S9 mix. The simultaneous addition of peroxidase reduced the PA-induced SCEs whereas catalase did not show any effect. Chromosome analysis in the first mitosis after PA treatment revealed a significant increase in the incidence of chromosome aberrations and endoreduplication. The results are discussed with respect to the cause and the significance of the observed effects in connection with mutagenicity testing.     

Recombination repair pathway in the maintenance of chromosomal integrity against DNA interstrand crosslinks

DNA interstrand crosslinks (ICL) present a major threat to cell viability and genome integrity. In eukaryotic cells, the ICLs have been suggested to be repaired by a complex process involving Xpf/Ercc1-mediated endonucleolytic incision and homologous recombination (HR). However, the entire feature of the ICL tolerating mechanism is still poorly understood. Here we studied chromosome aberrations (CA) and sister chromatid exchanges (SCE) by the use of the crosslinking agent mitomycin C (MMC), in chicken DT40 cells with the HR genes disrupted by targeted replacement. The disruption of the Rad54, Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3 genes resulted in a dramatic reduction of spontaneous and MMC-induced SCEs. Interestingly, while HR-deficient cells were hypersensitive to cell killing by MMC, MMC-induced CAs were also suppressed in the HR-deficient cells except for Rad51D-, Xrcc2- and Xrcc3-deficient cells. These observations indicate that DNA double strand breaks (DSB) at stalled replication forks and those arising as repair intermediates present strong signals to cell death but can be tolerated by the HR repair pathway, where Rad54, Rad51B and Rad51C have an initiative role and repair can be completed by their paralogs Rad51D, Xrcc2 and Xrcc3. The impairment of the HR pathway, which otherwise leads to cell death, may be somewhat substituted by an alternative mechanism such as the Mre11/Rad50/Nbs1 pathway, resulting in reduced frequencies of SCEs and CAs.     

Effects of repair inhibition in the G1 phase of clastogen-treated human lymphocytes on the frequencies of chromosome-type and chromatid-type aberrations and sister-chromatid exchanges

It has been shown that certain types of DNA lesions induced by an S-dependent clastogen are converted to chromosome-type aberrations when their repair is inhibited in the G1 phase of the cell cycle. The purpose of the present study was to investigate which kinds of repair inhibitors have the ability to induce chromosome-type aberrations in cells having DNA lesions and which kinds of DNA lesions will be converted to chromosome-type aberrations when their repair is inhibited. For this purpose, human peripheral blood lymphocytes, which were treated with a clastogen in their G0 phase, were post-treated with one of several kinds of repair inhibitors in the G1 phase, and resulting frequencies of both chromosome-type and chromatid-type aberrations as well as of sister-chromatid exchanges (SCEs) were compared with those of the control cultures: chromatid-type aberrations and SCEs were adopted as cytogenetic indicators of lesions remaining in S and G2 phases. Chemicals used for the induction of DNA lesions were 4-nitroquinoline 1-oxide (4NQO), methyl methanesulfonate (MMS) and mitomycin C (MMC); inhibitors used were excess thymidine (dThd), caffeine, hydroxyurea (HU), 5-fluoro-2'-deoxyuridine (FdUrd), 1-beta-D-arabinofuranosylcytosine (ara C), 9-beta-D-arabinofuranosyladenine (ara A), 1-beta-D-arabinofuranosylthymine (ara T) and aphidicolin (APC). Induction of chromosome-type aberrations was observed in cells pretreated with 4NQO or MMS followed by ara C, ara A, ara T or APC, whereas other combinations of a clastogen and an inhibitor did not induce them. Among the inhibitors, ara C alone induced chromosome-type aberrations in cells without pretreatment. Chromatid-type aberrations were increased only in cells pretreated with MMC and their frequency was enhanced further by post-treatment with ara C. All of the clastogens used in the present experiments induced SCEs. Most inhibitors did not modify the SCE frequencies except for ara C which synergistically increased the frequency in MMC-treated cells. The present study offers further evidence that the lesions responsible for chromosome-type aberrations are those which are repaired quickly, and that they are converted to chromosome-type aberrations when repair by polymerase alpha is inhibited. The effects of ara C on MMC-induced lesions are considered residual effects of ara C treatment in the S or G2 phases rather than repair inhibition in the G1 phase.     

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.     

Cytogenetic effects of a food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and its metabolite, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), on human and Chinese hamster cells in vitro

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced structural chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in human lymphocytes and human diploid fibroblasts (TIG-7) at concentrations above 12.5 μg/ml in the presence of rat S9 mix. PhIP also elevated the frequencies of SCEs in human lymphocytes in the presence of rat S9 at concentrations above 2.0 μg/ml with dose-dependency. A proximate form of metabolites of PhIP, 2-hydroxyamino-1-methyl -phenylimidazo[4,5-b]pyridine (N-OH-PhIP), caused CAs in human and Chinese hamster fi fibroblast cells in the absence of S9 mix at concentrations above 0.75 μg/ml and 1.25 μg/ml, respectively, which were 10 times lower than the effective concentration of PhIP. No marked differenceswere observed in the cytogenetic sensitivity to N-OH-PhIP between human and Chinese hamster cells, except between lymphocytes obtained from different donors.     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

Analysis of cytogenetic effect in human lymphocytes induced by metabolically activated 2-nitropropane

Chromosome analyses were carried out in human lymphocytes treated in vitro with 2-nitropropane (2-NP) in the presence and absence of the mammalian metabolic activation system, S9 mix. Without S9 mix, only the frequency of gaps was significantly increased at 80 mM 2-NP as compared to controls. With S9 mix, the incidences of gaps and chromatid-type aberrations were significantly increased at 60 mM and 80 mM. Sister-chromatid exchanges (SCE) have been induced at concentrations as low as 7.5 mM. The present findings demonstrate that in human lymphocytes, 2-NP requires metabolic activation to express clastogenicity and SCEs.     

Genotoxic potential of ethinylestradiol in cultured mammalian cells

Ethinylestradiol, a steroidal estrogen, is widely used with various progestogens in oral contraceptives formulations. There are sufficient evidences for the carcinogenicity of ethinylestradiol in experimental animals. The reports on the genotoxic potential of ethinylestradiol are contradictory. Here in the present study we have tested the genotoxicity of ethinylestradiol in human lymphocytes using chromosomal aberrations (CAs), mitotic index (MI) and sister chromatid exchanges (SCEs) as a parameter. The study was carried out in the absence, as well as in the presence, of rat liver microsomal fraction, with and without NADP. Ethinylestradiol was studied at three different concentrations (1, 5 and 10 microM) and was found non-genotoxic in the absence of metabolic activation (S9 mix) and in S9 mix without NADP. Ethinylestradiol was found to be genotoxic at 5 and 10 microM in the presence of S9 mix with NADP. To study the possible mechanism of the genotoxicity of ethinylestradiol, superoxide dismutase (SOD) and catalase (CAT) were used separately and in combination along with 10 microM of ethinylestradiol at different doses. SOD treatment increased CAs and SCEs and decreases MI as compared with treatment with 10 microM of ethinylestradiol alone in the presence of S9 mix with NADP at both of the tested doses. CAT treatment decreased the frequencies of CAs and SCEs and increased MI, as compared with treatment with 10 microM of ethinylestradiol alone in the presence of S9 mix with NADP. CAT treatment in combination with SOD also decreased the frequencies of CAs and SCEs and increased MI suggesting a possible role of reactive oxygen species for the genotoxic damage.     

Constitutive heterochromatin polymorphism and chromosome damage in viral hepatitis

The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) were scored in relation to constitutive heterochromatin in 100 patients with viral hepatitis B, 100 patients with viral hepatitis A and 100 age- and sex-matched normal controls. 23.4%, 15% and 4% of the cells showed chromosomal aberrations in patients with hepatitis B, hepatitis A and normal controls respectively. Non-random involvement of chromosomal aberrations were also noted in chromosome 1 of patients with hepatitis B and A as compared to normal controls. The frequencies of SCEs (mean +/- S.D.) were found to be 10.40 +/- 2.83 in hepatitis B and 8.70 +/- 2.34 in hepatitis A. These values were significantly higher than the SCE frequency (mean +/- S.D.) of 5.88 +/- 2.25 observed in normal controls (P less than 0.001). The intra-chromosomal distribution of SCEs revealed a relatively increased incidence of SCEs in chromosome 1 of patients with hepatitis B and A as compared to normal controls. Analysis of constitutive heterochromatin polymorphism showed chromosome 1 qh+ to be the most frequent variant in patients with hepatitis B and A as compared to normal controls. The increased involvement of C-band variant 1 qh+ in patients with hepatitis B and A as compared to normal controls may indicate that extra heterochromatin offers additional sites for viral integration.     

Chromosome aberrations, micronuclei and sister-chromatid exchanges (SCEs) in rat liver induced in vivo by hepatocarcinogens including heterocyclic amines

The induction of chromosome aberrations, micronuclei and SCEs was studied in hepatocytes of F344 rats exposed in vivo to hepatocarcinogens. Hepatocytes were isolated and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 h. Oral administration of dimethylnitrosamine at doses of 2.5-20 mg/kg body weight (bw) induced (1) chromosome aberrations in up to 27% of the metaphase cells 2-48 h after its administration, (2) SCEs with a frequency of up to 0.9 per chromosome 2-48 h after its administration, and (3) micronuclei in up to 2.9% of the cells 16-48 h after its administration. Oral administration of 2-acetylaminofluorene at doses of 6.25-200 mg/kg bw induced (1) chromosome aberrations in up to 35% of the metaphase cells after 2-48 h, (2) SCEs at up to 0.9 per chromosome and (3) micronuclei in up to 2.5% of the cells with a maximum after 4 h. Oral administration of CCl4, a non-genotoxic hepatocarcinogen, at a dose of 1600 mg/kg bw did not induce chromosome aberrations, SCEs or micronuclei within 4-72 h. Intraperitoneal injections of Trp-P-1, Glu-P-1, MeIQx, IQ and nitro-IQ resulted in chromosome aberrations in up to 16% of the metaphase cells and SCEs at up to 0.9 per chromosome, while injections of Trp-P-2 and Glu-P-2 produced SCEs at up to 0.7 and 1.1 per chromosome, respectively. The present method of in vivo cytogenetic assay using rats without partial hepatectomy or mitogen treatment in vivo should be useful for evaluating the tumor-initiating activities of hepatocarcinogens.     

Sister-chromatid exchanges induced by 1.3-butadiene and its epoxides in CHO cells

Sister-chromatid exchanges (SCEs) were analyzed in CHO cells after pulse treatment with 1,3-butadiene, 3,4-epoxy-1-butene (monoepoxybutene) and 1,2:3,4-diepoxybutane (diepoxybutane). A weak dose effect was observed after exposure to 1,3-butadiene but only in the presence of S9 mix. Monoepoxybutene and diepoxybutane were highly effective in inducing SCEs at concentrations of 0.1-1 microM both in the presence and in the absence of S9 mix. At higher concentrations the response was more pronounced without S9 mix.     

Chlorination of harman and norharman with sodium hypochlorite and co-mutagenicity of the chlorinated products

Harman and norharman are widely distributed in the environment and consequently contaminate in domestic waste-water. It has been reported that they have co-mutagenic activity in the presence of non- mutagenic aromatic amines such as aniline and o-toluidine with S9 mix. When these beta-carbolines were treated with sodium hypochiorite under mild conditions, chlorinated derivatives were produced. Among them, 6-chloroharman and 6-chloronorharman showed much more potent co-mutagenic activities than harman and norharman in the presence of o-toluidine toward Salmonella typhimurium TA98 with S9 mix. These results suggest that the chlorination of harman and norharman occurs during disinfection at the sewage plant to produce potent co-mutagens that contaminate river water.