Induction of chromosome aberrations and sister-chromatid exchanges in CHO-K1 cells by o-phenylphenol

o-Phenylphenol (OPP), is used in Japan as a fungicide in food additives for citrus fruits. The induction of chromosome aberrations and sister-chromatid exchanges (SCEs) by OPP in cultured Chinese hamster ovary (CHO-K1) cells was studied. Cells were exposed to various concentrations of OPP ranging from 50 to 175 micrograms/ml for 3 h, and further incubated for 27 and 42 h. These incubation periods are almost equal to 2 and 3 cell cycles. SCEs and chromosome aberrations were induced by OPP at concentrations of 100, 125 and 150 micrograms/ml after the incubation for 27 h. For chromosome aberrations, chromatid breaks and exchanges there was a dose-dependent increase. Diplochromosomes due to endoreduplication were also caused by the same concentrations of OPP in a dose-dependent manner. After incubation for 42 h, chromosome aberrations were also increased by OPP at concentrations of 100 and 125 micrograms/ml, but the frequencies of SCEs were not significantly different from those of the control. These results suggest that OPP has a cytogenetic toxicity, and that the DNA damage resulting in SCEs induced by OPP is relatively short-lived and can be repaired during the longer incubation time.     

In vitro chromosome damage due to PCB interactions

In order to study the possible mutagenic properties of polychlorinated biphenyls (PCBs), human lymphocyte cultures were examined for chromosome breakage, rearrangements, sister-chromatid exchange, and mitotic delay. The present study, which used cyclophosphamide as a positive control, shows that one planar PCB congener, 3,4,3',4'-tetrachlorobiphenyl, caused dose-related chromosome breakage in human lymphocytes exposed in vitro to 0.1-10(-4) micrograms/ml. In contrast, the non-planar PCB, 2,5,2',5', did not cause chromosome damage in comparable tests even at concentrations as high as 1 microgram/ml. However, when 3,4,3',4' at a concentration lower than that which causes chromosome breakage (10(-5) micrograms/ml) was combined with a non-clastogenic concentration of 2,5,2',5', the chromosomal damage observed was far in excess of what one would expect from higher doses of 3,4,3',4' alone. These results suggest that some PCB congeners may interact to cause synergistic genotoxic effects.     

Analysis of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed in vitro to Hydergine.

Hydergine (dihydroergotoxine mesylate, Sandoz) was examined for its capability to induce chromosome damage and sister-chromatid exchanges (SCEs) in human lymphocyte in vitro. For the chromosome-aberration study, cultures set up from 6 individuals were divided into 5 groups: negative control, positive control (caffeine, 0.5 mg/ml), and Hydergine (0.1, 0.25 and 0.5 micrograms/ml). For the SCE examination, which used 8 individuals, 4 cultures were made per person in the following way: negative control, positive control (mitomycin C, 0.1 microgram/ml), and Hydergine (0.1 and 0.5 micrograms/ml). Lymphocytes were cultivated for 72 h, being exposed to the respective treatments during the final 24 h. The results showed that Hydergine induced no chromosome damage in human lymphocytes in vitro.     

The role of foetal red blood cells in protecting cultured lymphocytes against diepoxybutane-induced chromosome breaks

Diepoxybutane (DEB) is an established mutagen that induces chromosome damage following in vitro treatment of peripheral blood lymphocytes. It is widely used to identify patients with Fanconi Anemia (FA), a clinical situation that is characterized, besides the hypersensitivity to DEB, by an elevated foetal haemoglobin (HbF) content in the peripheral blood. In a previous study, we showed that red blood cells (RBC) from normal individuals can protect cultured lymphocytes against chromosomal breaks induced by DEB and demonstrated the particular role of haemoglobin in the protective effect. In the present work, we studied the influence of RBC extracted from umbilical cord blood of neonates (F cells) on the frequency of DEB-induced chromosome breaks in lymphocyte cultures from normal individuals. Simultaneously, we determined individual GSTT1 and GSTM1 genotypes and the activity of Pi-class glutathione S-transferase (GSTP), catalase and superoxide dismutase (SOD) in adult and foetal RBC. Our results show that F cells, in comparison with adult RBC, elicit a better protection of cultured lymphocytes from normal individuals against chromosome breaks induced by DEB. Variability in the protective effect among RBC from different individuals was observed; we confirmed that the GSTT1 genotype modulates this inter-individual variability, but it is not sufficient to explain all of the protective effect of F cells. Our results suggest that the increased protective effect of F cells can be, at least in part, correlated with an increase in the activity of glutathione S-transferase, catalase and superoxide dismutase, in particular Cu/Zn SOD, in F cells compared with adult RBC.     

Change in the structural organization of Mycobacterium rubrum cells upon the action of ethidium bromide

The action of ethidium bromide on Mycobacterium rubrum cells was studied. The culture growth was found to depend on ethidium bromide (EB) concentration in the medium. The reaction of EB with nucleoid DNA was shown to be specific and changes in the nucleoid structure were detected. Low EB doses (ca. 2 micrograms/ml) caused DNA despiralization in many cells. The process was reversible, which accounted for the elevated ability of reactivation at low EB doses. A higher EB dose (ca. 5-10 micrograms/ml and more) made the nucleoid structure coarser and denser in most cells and the nucleoid broke down to small fragments. As a result, due to the pool of enzymes present in the cells prior to EB addition, secondary changes developed. They involved all the cellular structures as well as the metabolism of lipids, polyphosphates, and glycogen. As a rule, these changes were incompatible with the cell viability.     

Effects of theophylline on chromosomal breakage and sister-chromatid exchange

Frequencies of both sister-chromatid exchange (SCE) and chromosomal breakage (CB) were studied in the lymphocytes of normal individuals (10 and 7 individuals respectively). The cells were exposed in vitro to 3 different concentrations of theophylline (1, 10 and 100 micrograms/ml). A significant concentration effect of the drug was demonstrated for both SCEs and CB. Utilizing a Dunnett's test for individual comparisons, the 10 and 100 micrograms/ml concentrations both demonstrated a significant elevation of SCEs and CB compared to the untreated control cultures. This study suggests that in vitro concentrations of theophylline equal to or greater than 10 micrograms/ml, corresponding to serum levels attained during therapy, increase the frequency of SCEs and chromosome breakage in human lymphocytes.     

The effect of valproic acid on SCE and chromosome aberrations in epileptic children

Sister-chromatid exchange (SCE) and chromosome aberrations have been studied in peripheral lymphocytes of 20 epileptic children treated in monotherapy with valproic acid (VPA) for 6-52 months and in 2 matched control groups. The frequencies of SCE in the VPA-treated epileptic children were significantly higher than in the 2 control groups (p less than 0.01); rates of chromosome aberrations were slightly higher but not significantly different from the 2 control groups. We also examined SCE in 10 epileptic children before and after they took sodium valproate for 6-7 months; there was a statistically significant change in SCE following VPA. 9 normal children whose lymphocytes were exposed in vitro to sodium valproate (5-20 micrograms/ml) showed a significant increase in SCE.     

Evaluations of cellular proliferation and chromosome breakages after in vitro exposure of human lymphocytes to calcium or zinc DTPA

The analysis of mitotic indices (MI) and chromosome breakages in metaphases of 50-hr lymphocyte cultures exposed to the calcium or zinc chelates of diethylenetriamine pentaacetic acid (DTPA) demonstrated: (1) an 80% reduction in MI in cultures from three women but no reduction in those from two men after in vitro exposure to CaDTPA in concentrations as low as 10 micrograms/ml culture medium, and complete suppression of mitoses in cultures from men and women after exposure to 40 micrograms/ml CaDTPA; (2) minor suppression in MI in cultures from women and none in those from men after exposure to 40 or 80 micrograms/ml ZnDTPA; (3) no ring or dicentric chromosomes in 1700 metaphases from DTPA-treated cultures. Likewise, in other experiments we observed no differences in the frequency or distributions of rings and dicentrics in lymphocyte cultures from two persons after in vitro exposure to 250-R 60Co gamma radiation in the presence or absence of 10 micrograms/ml CaDTPA or 10 or 80 micrograms/ml ZnDTPA. These data indicate that while accurate estimates of the frequencies of radiation-induced rings and dicentrics in lymphocytes can be made in actinide-contaminated persons undergoing DTPA chelation therapy, blood samples for cytogenetic cultures should not be obtained from chelated patients until the compound has been cleared from the blood plasma.     

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.     

2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells

The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 micrograms/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture (greater than 5 micrograms/ml).     

Interchangeability of mutagens during fractionated effects of unequal concentrations of alkylating agents

The ability of thiophosphamide and dipin to substitute for each other in "clastogenic adaptation" of human lymphocytes was investigated at Go phase. There were used 5 low concentrations of mutagens 2, 0.2, 2.10(-2), 2.10(-3), 2.10(-4) micrograms/ml and the high one of 20 micrograms/ml with which cells were treated 2 hr after the effect of low concentrations. The "protective" concentrations for both mutagens were 0.2, 2.10(-2), 2.10(-3) micrograms/ml. The pretreatment with thiophosphamide caused the decrease in chromatid aberrations in "challenge" treatment with dipin, the pretreatment with dipin caused the decrease in chromosome aberrations in "challenge" treatment with thiophosphamide.     

Butachlor is cytotoxic and clastogenic and induces apoptosis in mammalian cells

The ability of butachlor to induce cytotoxicity, clastogenicity and DNA damage was assessed using Chinese hamster ovary cells (CHO), Swiss mouse embryo fibroblasts (MEF) and human peripheral blood lymphocytes. A dose and time dependent loss of viability was evident upon treatment of CHO cells with butachlor. Cell killing to an extent of 50% was observed when cells were treated with 16.2 micrograms/ml of butachlor for 24 hr or with 11.5 micrograms/ml for 48 hr. The herbicide induced micronuclei significantly in cultured lymphocytes at 24 and 48 hr of treatment suggesting that it is clastogenic. To understand the mechanism of cell death caused by butachlor, its effect on DNA strand breaks was studied in MEF. A concomitant decrease in cell viability was observed with increase in DNA strand breaks. Agarose gel electrophoresis of DNA from herbicide treated CHO cells and cytochemical staining indicate the induction of apoptosis by butachlor.     

Sister-chromatid exchanges in human lymphocytes exposed to 1-p-(3-methyltriazeno)benzoic acid potassium salt

The 1-p-(3-methyltriazeno) benzoic acid potassium salt (MTBA) is a triazeno analogue of dacarbazine, an antineoplastic agent capable of mediating the appearance of new antigenic specificities on cancer cells in mice, a phenomenon described as 'chemical xenogenization' (CX). Recently we reported the clastogenic potential of MTBA on human lymphocytes. Since sister-chromatid exchange (SCE) assay is more sensitive than clastogenic tests, at least at low drug concentrations, we assessed SCE frequencies induced by MTBA on human lymphocytes stimulated by PHA. Drug treatment at 2-500 micrograms/ml was performed in vitro prior to or after PHA addition. SCE values increased significantly in a dose-dependent manner up to 200 micrograms/ml. However, SCE frequencies, as well as chromosome breaks, did not increase dramatically. These data indicate that MTBA concentrations used for CX do not cause severe cytogenetic damage to immune cells at least in vitro.     

Comparative studies of embryotoxic action of ethylenethiourea in rat whole embryo and embryonic cell culture

The effects of ethylenethiourea (ETU) were investigated using rat (Wistar-imamichi) embryos cultured from days 11 to 13 of gestation or cultured rat embryonic cells extracted on day 11. Malformations in cultured embryos at the concentration of 30 micrograms/ml of ETU were found in the head and tail, which were severely affected, as well as the limb and face. All embryos exposed to 150 and 300 micrograms/ml of ETU had malformed heads, tails, limbs, and facial configurations. Protein contents of the cultured embryos were decreased dose-dependently at the concentrations ranging from 30 to 300 micrograms/ml. In the histological studies of the cultured embryos with ETU, thinner neuroepithelium in head was observed. In the embryonic cells extracted on day 11 of gestation, ETU dose-dependently inhibited the differentiation of midbrain (MB) cells into neurons and that of limb bud (LB) cells into chondrocytes at the concentrations ranging from 30 to 600 micrograms/ml of ETU. The concentrations of ETU that inhibited the production of differentiated foci by 50% (IC50) were 170 micrograms/ml in LB cells of day 11, and greater than 600 micrograms/ml in LB cells on day 12 of development. Therefore, differentiation of MB cells was more sensitive to ETU than the differentiation of LB cells. These results indicated that there was a reasonable correlation of ETU induced changes in cultured whole embryos and embryonic cells.     

Chromosomal changes induced by chrysotile fibres or benzo-3,4-pyrene in rat pleural mesothelial cells

The induction of chromosomal aberrations in rat pleural mesothelial cells (RPMC) following in vitro treatment with chrysotile fibres has been demonstrated. The production of chromosomal aberrations was also observed after treatment of the cells with benzo-3,4-pyrene (BP). The yield of abnormal metaphases was dose-dependent and reached 58% at a BP dose of 2 micrograms/ml. Chrysotile fibres at 7 micrograms/ml induced 21% abnormal metaphases and the frequency decreased with further increases in fibre concentration. Their decline is possibly related to a lethal effect. Chrysotile-induced chromosomal aberrations were primarily of the chromatid type and included breaks and fragments. BP induced chromosome exchanges which were not seen following chrysotile treatment. Minutes and double minutes were detected in BP-treated RPMC and occasionally found after chrysotile application. These results confirm that chrysotile fibres are clastogenic for some cultured cells and demonstrate that the fibres induce chromosome damage in target RPMC.     

Induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene

The induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene was investigated. 4 groups of 5 Swiss (ICR) male mice were given orally a solution of benzene every day for 14 days except days 5 and 10. The daily doses were 0, 36.6, 73.2 and 146.4 mg/kg. Mice were sacrificed on day 15, lymphocytes were obtained by perfusion of the spleen and the cells were cultured in RPMI 1640 medium. After 48 h of culture, cells were harvested for cytogenetic analysis. A significant dose-dependent increase in the frequency of cells with chromatid aberrations were found (p less than 0.001). A significant increase in polyploid cells were also observed (p less than or equal to 0.05). This study represents the first report on the induction of chromosome aberrations and polyploid cells in lymphocytes of mice after subchronic exposure to benzene. Such dual activity of benzene suggests that benzene may be responsible for more human health problems than currently estimated.     

Cellular cholesterol metabolism in mitogen-stimulated lymphocytes--requirement for de novo synthesis

The relationship between cholesterol synthesis and uptake in proliferating lymphocytes has been examined. [14C]Acetate incorporation into lymphocytes cultured under lipoprotein-deficient conditions increased initially in response to mitogen, decreased after 24 h, and increased rapidly between 72 and 96 h. Addition of LDL (10 micrograms/ml) to the culture during the 'trough' period caused [14C]acetate incorporation to return rapidly to baseline, while at peak periods LDL suppression of cholesterol synthesis was minimal. Lymphocytes cultured in the presence of the HMG-CoA reductase inhibitor, mevinolin, exhibited a time-dependent increase in their capacity to incorporate [14C]acetate into cholesterol, evident when mevinolin was removed by washing prior to assay. PHA enhanced 125I-labelled LDL receptor-mediated binding by lymphocytes cultured in lipoprotein-deficient medium over a 4 day period and mevinolin augmented the effect. [3H]Thymidine incorporation into mitogen-stimulated lipoprotein-deficient cultures was inhibited up to 75% by mevinolin (1 mumol/l). LDL (2.5-10 micrograms/ml) substantially reversed this inhibition in 72 h cultures, but only partially overcame inhibition in cells cultured for 96 h. Results suggest that endogenous cholesterol synthesis may be obligatory for lymphocyte proliferation after the initial round of cell division.     

Isolation and subregional mapping of a human cDNA clone detecting a common RELP on chromosome 12

Summary Peripheral blood lymphocytes from three patients with Down syndrome (DS; trisomy 21; aged 5–6 years) and three age-matched control children were studied for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs).Cells in G₀ were exposed to bleomycin (20–100 g/ml) for 3 h, and then cultured in medium containing 5-bromodeoxyuridine and phytohemagglutinin for 66 h. By the sister chromatid differential staining method, chromosome analyses were performed on metaphase cells that had divided one, two, or three or more times after treatment. The results indicate that DS cells exposed to bleomycin are hypersensitive to the production of dicentric and ring chromosomes compared to normal cells. Bleomycin also led to a dose-related increase in the frequency of SCEs, but no difference was found between the SCE frequencies in DS or normal lymphocytes exposed to bleomycin.     

Cytogenetic effects of pesticides. IV. Cytogenetic effects of the insecticides Gardona and Dursban.

The cytogenetic effects of the insecticides Gardona and Dursban were investigated. The toxicity and ability of both insecticides to induce chromosome aberrations and sister-chromatid exchange in vitro was tested in a primary culture of mouse spleen cells, in order to assess the potential mutagenicity of both insecticides. The concentrations 10(-7)-10(-3) M were used for testing the toxic effects of the insecticides. Both Gardona and Dursban were toxic to spleen cell cultures and the percentage of viable cells decreased as the concentration of the insecticide was increased. It reached 76.8% and 77.8% of control after treatment with the highest concentration tested (10(-3) M) of Gardona and Dursban respectively. Gardona at 0.25, 0.50, 1.0 and 2.0 micrograms/ml, and Dursban at 0.50, 1.0, 2.0 and 4.0 micrograms/ml were tested for the induction of chromosome aberrations and sister-chromatid exchanges. All of the tested concentrations of both insecticides induced a high percentage of metaphases with chromosomal aberrations in cultured mouse spleen cells after 4-h treatment. The frequency of SCEs/cell increased with increasing concentration of the insecticides. It reached 11.92 +/- 0.14/cell and 13.40 +/- 0.20/cell after treatment with Gardona (2 micrograms/ml) and Dursban (4 micrograms/ml), respectively, compared with 8.2 +/- 0.19/cell and 7.6 +/- 0.15/cell in the solvent control. The presented results indicate that both Gardona and Dursban in the tested concentrations are mutagenic in mouse spleen cell cultures.     

Aphidicolin and bleomycin induced chromosome damage as biomarker of mutagen sensitivity: a twin study

The induction of chromosome damage in cultured human lymphocytes by in vitro treatments with aphidicolin (APC) and bleomycin (BLM) has been proposed as test of sensitivity to mutagens. To assess their validity, we have investigated whether the individual expression of induced chromosome damage has a genetic rather than an environmental basis. Metaphase analysis for chromosomal aberrations (CA) and micronucleus (MN) assay in cytokinesis-blocked cells have been performed in peripheral blood lymphocytes from 19 healthy male twins (9 monozygotic and 10 dizygotic pairs), aged 70-78 years, after APC, BLM and APC+BLM treatments.Concordance between twins revealed a high genetic component in the sensitivity towards clastogenic action of APC both as percentages of CA and MN. The micronucleus assay demonstrated a genetic basis also in the expression of chromosome damage induced by BLM and APC+BLM treatments. Since twins were elderly people, to investigate the possible role of age, CA and MN frequencies were compared with those found in lymphocytes from 11 young male donors. Basal and APC-induced chromosome damage were clearly increased in the former. Following BLM and APC+BLM treatments, age significantly increased mitotic delay, as shown by the mitotic indexes (MI) and by the ratios between binucleated and mononucleated (B/M) cells.