Sister chromatid exchanges of human peripheral blood lymphocytes induced by N,N-diethylaniline in vitro

N,N-Diethylaniline is a reagent used in organic synthesis and is an important intermediate in the manufacturing of dyes. To evaluate its genotoxicity, we examined whether it can induce sister chromatid exchanges (SCEs) in human lymphocytes. We found that N,N-diethylaniline significantly increased the frequency of SCEs both in the absence and presence of S-9 mix. The SCEs from cultures treated by N,N-diethylaniline in the presence of S-9 mix displayed a marked increase which was about 5-fold greater than the control. ANOVA analyses indicated that there is a dose–response relationship between doses of N,N-diethylaniline and the frequency of SCEs, especially in the presence of S-9 mix. The results suggested that N,N-diethylaniline has genotoxicity.     

Effects of auxin and cytokinin on induction of sister chromatid exchanges in cultured cells of wheat (Triticum aestivum L.)

Summary In order to know the mutagenic effects of synthetic auxins (NAA, 2,4-D, and 2,4,5-T) and a cytokinin (kinetin) in vitro, sister chromatid exchanges (SCEs) were analyzed in cultured cells of a hexaploid wheat (Triticum aestivum L.). In the MS medium supplemented with 2.0 mg/l 2,4-D, the mean number of SCEs per cell was 15.2, and per pg of DNA, 0.42. No significant effect was found in the treatments of NAA or 2,4-D at concentrations of 0.5–10.0 mg/l, whereas more than 2.0 mg/l of 2,4,5-T induced dramatic increases of SCEs. Kinetin itself had no significant effect on SCE induction, but there was a tendency that SCEs induced by 2,4,5-T were suppressed by kinetin.     

Influence of duration of exposure to styrene oxide on sister chromatid exchanges and cell-cycle kinetics in cultured human blood lymphocytes in vitro

Styrene-7,8-oxide, an intermediate of styrene, is a known alkylating mutagen. The present study was carried out to investigate the influence of duration of exposure to styrene-7,8-oxide (styrene oxide) on induction of sister chromatid exchanges (SCEs) and inhibition of cell-cycle kinetics using cultured human blood lymphocytes in vitro. Phytohemagglutinin-stimulated whole-blood lymphocyte cultures obtained from heparinized whole blood from healthy donors were exposed to 100 μM styrene oxide for 22, 36, 48 and 72 h. A reduction of SCEs induction with increase in duration of exposure to styrene oxide was observed, i.e. a clear significant inverse relationship between exposure time and frequencies of SCEs induction due to styrene oxide was obtained. Styrene oxide induces significant elevations in unscheduled DNA synthesis DNA repair as well as S-phase synthesis in human blood lymphocytes in vitro, depending on the duration of exposure. The decrease in the induction of SCEs due to styrene oxide with increasing duration of its exposure may be principally due to an increased DNA repair and partly due to an increasing metabolic transformation to styrene glycol with increasing duration of its exposure as well as to some extent due to cell death at the maximum period of exposure, i.e. 72 h. Although the proliferations of lymphocytes exposed to 100 μM styrene oxide were significantly inhibited at different durations of exposure, no linear relationship between the replication index and the duration of exposure was noticed (r=0.47, p>0.05). Similarly, there was no relationship between replication index and SCE frequency (r=−0.36, p>0.05), suggesting that these two parameters may reflect two different endpoints for the cytogenotoxic effects of styrene oxide.     

Induction of sister-chromatid exchanges in ICR 2A frog cells exposed to 265-313 nm monochromatic ultraviolet wavelengths and photoreactivating light

Exposure of ICR 2A frog cells to 265 nm, 289 nm, 302 nm or 313 nm monochromatic ultraviolet (UV) wavelengths induced the formation of sister-chromatid exchanges (SCEs). However, treatment of cells with photoreactivating light (PRL) following the UV irradiations resulted in a lower level of SCEs compared with cells incubated in the dark. Hence, it can be concluded that pyrimidine dimers are the principal photoproducts responsible for the induction of SCEs in cells exposed to 265-313 nm UV due to the specificity of DNA photolyase for the light-dependent monomerization of dimers in DNA. It was also found that the maximum yield of induced SCEs in 313 nm-irradiated cells was only about 7 SCEs per cell whereas the plateau values for the shorter wavelengths were approximately 15-20 SCEs per cell. In addition, treatment of cells with 313 nm plus 265 nm light resulted in a lower level of SCEs than in cells exposed to 265 nm UV alone. These results can be interpreted in the context of a replication model for SCE, in which the high level of non-dimer damages produced in the DNA of 313 nm-irradiated cells inhibits the induction of SCEs by the pyrimidine dimers that are also produced by this wavelength.     

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.     

Sister chromatid exchanges in Drosophila melanogaster cell lines in vitro

The frequency of sister chromatid exchanges (SCEs) in two cell lines of Drosophila melanogaster with different karyotypes (XX and XY) was determined, considering (1) the distribution of SCEs within each chromosome, with reference to eu- and heterochromatin and (2) the distribution of SCEs in different chromosomes. A comparison was made between chromosome pairs within each karyotype and between the two different karyotypes. The following results were obtained. The SCEs are not randomly distributed along chromosomes, since exchanges were never observed in heterochromatin. SCEs are more frequent in XY than in XX cells; moreover, in both cell types there exists a significantly higher frequency of SCEs in the X chromosome than in the autosomes. These findings are discussed in relation to chromosome aberrations and mitotic recombination.     

Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes

Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.     

Increased sister-chromatid exchanges (SCEs) and chromosomal fragilities by BrdU in a human mutant B-lymphoblastoid cell line

From an X-irradiated human B-lymphoblastoid cell line (CCRF-SB), we have isolated a unique mutant clone (CCRF-SB-T1) which reveals high frequencies of sister-chromatid exchanges (SCEs) and chromosomal fragilities in the C-band regions of chromosomes Nos. 1, 9 and 16, when exposed to high concentrations of bromodeoxyuridine (BrdU). A clear BrdU dose-dependent increase of SCEs (9.6 SCEs/cell at 0.05 mM, 40 SCEs/cell at 0.37 mM on average) in this mutant was observed. Relative contributions of nucleoside and a thymidine (dT) analog of BrdU to high SCEs were studied, since an unusual SCE response to BrdU led us to suspect the significance of BrdU incorporation into DNA and dT pool disturbances. Addition of deoxycytidine (dC), dT or both dC and dT causes an increase of SCEs. On the other hand, deoxyadenosine (dA) and deoxyguanosine (dG) did not have significant effects on SCEs in SB-T1 cells. These results suggest that disturbances of pyrimidine-nucleotide synthesis, including gross imbalance of nucleotide pools, play a pivotal role in the high SCE induction of SB-T1 cells by BrdU.     

Lack of association between <Emphasis Type="Italic">GSTT1</Emphasis> polymorphism and endogenous or benzo[a]pyrene-induced sister chromatid exchanges as analyzed in metaphase or G2-phase lymphocytes

The objectives of the present work are (1) to verify whether genetic polymorphism in the detoxification gene GSTT1 influences the endogenous sensitivity in terms of sister chromatid exchanges (SCEs)/cell in healthy donors and (2) to test whether in vitro exposure to B[a]P in terms of SCEs/cell can be associated with polymorphism of GSTT1 gene. The presence or absence of the homozygous deletion in GSTT1 gene was determined in peripheral blood cells using multiplex-PCR. For SCEs quantitation, the cytogenetic method used thus far is based on the analysis in metaphase chromosomes. Consequently, G2-arrested cells are not included in the analysis. To overcome this shortcoming of the conventional method, we applied here SCE analysis in G2-phase prematurely condensed chromosomes (G2-PCCs) induced by calyculin-A, using a modified fluorescence-plus-Giemsa staining protocol. Compared to metaphase, a statistically significant increase in the yield of SCEs was notified in the G2-phase analysis after 48 h exposure of peripheral blood lymphocytes to 0.01–1 mM B[a]P, in both GSTT1-positive and -null donors. Therefore, the analysis of SCEs in the G2-phase using calyculin-A induced PCC methodology was shown to be more sensitive compared to the analysis at the metaphase level. Nevertheless, the results obtained do not show an association between the GSTT1 polymorphism with increased endogenous and/or B[a]P-induced SCE-frequencies in peripheral blood lymphocyte chromosomes in vitro. These results highlight not only the effect of B[a]P on cell cycle kinetics but also they demonstrate that conventional cytogenetic analysis at metaphase underestimates the cytogenetic effects of chemicals that delay cell cycle progression in G2-phase.     

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 sister-chromatid exchange in human blood lymphocytes by aqueous extract of palmyrah (Borassus flabellifer) flour

pPalmyrah palm (Borassus flabellifer) is widely consumed by people in certain tropical countries. The incidence of human malignant lymphomas, mutagenicity and toxicity in rats and bacteria encouraged us to study the potency of palmyrah crude aqueous extracts in inducing sister-chromatid exchanges (SCEs) in human blood lymphocytes in vitro. The extracts induced SCEs in a dose-related manner in both females and males. These effects apparently showed no consistency between batches. This result may be due to the intrinsic variation of different donors in their response to the induction of SCEs by palmyrah extracts. SCE frequency was proportional to chromosome length and SCEs at the centromeric region showed no difficulty in being scored. Concerning methods of short-term cytogenetic testing for detecting mutagenic and carcinogenic chemicals, we found that the SCE test was not more sensitive than the classic chromosome-breakage test.     

Cytogenetic studies in leprosy patients before and after chemotherapy

Summary The frequencies of chromosome aberrations and sister chromatid exchanges (SCEs), cell proliferation kinetics and mitotic indices were studied in peripheral blood lymphocyte cultures of leprosy patients both before and after chemotherapy. The differences in the frequencies of chromosome aberrations and SCEs between controls, paucibacillary and multibacillary patients were found to be statistically highly significant (P < 0.001). The extent of cytogenetic damage seemed to depend on the severity of the disease. Lymphocytes of untreated leprosy patients showed a low mitotic index and a slow rate of cell proliferation. Following combined treatment with dapsone and rifampicin there was an increase, but to a lesser degree (P < 0.01), in the frequency of SCEs and chromosome aberrations while the drug combination of dapsone, rifampicin and clofazamine had a non-mutagenic effect on chromosomes of the patient. Furthermore, after drug treatment, the cell proliferation rate and mitotic indices in paucibacillary patients were comparable to that of controls. These results indicate the clastogenic potency of Mycobacterium leprae and the remedial effects that follow therapeutic drug treatment.     

The effects of 4-thujanol on chromosome aberrations,sister chromatid exchanges and micronucleus in human peripheral blood lymphocytes

4-Thujanol, a bicyclic monoterpene alcohol, is present in the essential oils of many medicinal and aromatic plants. It is commonly used as a fragrance and flavouring ingredient in a lot of different products. The potential genotoxic effects of 4-thujanol on human peripheral blood lymphocytes (PBLs) were investigated in vitro by the chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests. The cells were treated with 13, 26 and 52 μg/mL 4-thujanol in the presence and absence of a metabolic activator (S9 mix). 4-Thujanol induced CA (P < 0.001) and MN formation (P < 0.05) at all concentrations (13, 26 and 52 μg/mL) in the presence and absence of the S9 mix without a concentration-dependent manner. However, the treatment of peripheral lymphocytes with 4-thujanol did not produce a statistical difference in the frequency of SCEs when compared with control group. Furthermore, this monoterpene did not significantly decrease the mitotic index (MI), proliferation index (PI), and nuclear division index (NDI). In conclusion, 4-thujanol had a significant clastogenic effect at the tested concentrations (13, 26 and 52 μg/mL) for human PBLs. In addition, no cytotoxic and/or cytostatic effects were observed regardless of the concentrations used. This work presents the first report on genotoxic properties of 4-thujanol.     

Sister chromatid exchange distribution in various human tissues exposed to MMC and BrdU

The effect of two known mutagens on different human tissues was examined in an attempt to determine if tissue specific responses exist in SCE distribution on chromosome. The tissues included human lymphocytes, skin fibroblasts, ovarian and testicular cells. All cell types were exposed to varying concentrations of 5-bromodeoxyuridine (BrdU), and mitomycin C (MMC). The numbers of SCEs were recorded from each tissue. Results indicated that certain of the tissues tested appeared more sensitive to particular agents. Results also showed that in all the tissues tested the larger chromosomes in groups A to C had greater numbers of SCEs than did the smaller chromosomes in groups D to G. There were very few SCEs in F and G group chromosomes including Y.     

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.     

Sister-chromatid exchanges in human lymphocytes induced by dimethoate, omethoate, deltamethrin, benomyl and their mixture.

Dimethoate and omethoate, two common organophosphorus insecticides, induced a dose-related increase in the frequency of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro (P of the regression lines less than 0.01). Two other common pesticides, the pyrethroid insecticide deltamethrin and the systemic fungicide benomyl, induced a modest increase in SCEs which bordered on statistical significance (P = 0.053 and 0.055, respectively). Mixtures of the four pesticides at total concentrations of 41.5 and 83 micrograms/ml (composed of 43% dimethoate, 43% omethoate, 12% deltamethrin and 1.2% benomyl) induced a dose-dependent increase in SCEs (P less than 0.01). The effects of these mixtures of pesticides were variable using lymphocytes from different individuals, although these differences did not attain statistical significance. Moreover, low concentrations of the four pesticides that did not increase SCEs significantly when tested alone, were positive for SCE induction when tested as a mixture. The experiments show that sub-threshold doses of pesticides may increase SCEs when present in a mixture.     

Presence of abnormally high incidences of sister chromatid exchanges in three successive cell cycles in Bloom's syndrome lymphocytes

The frequencies of sister chromatid exchanges (SCEs) were examined in phytohaemagglutinin-stimulated blood lymphocytes of a normal individual, a Bloom's syndrome heterozygote (bl/+), and two Bloom's syndrome homozygotes (bl/bl). To determine the baseline SCE frequencies, lymphocytes were cultured with various concentrations of 5-bromodeoxyuridine (BrdUrd) for two cell cycles. The incidence of SCEs per two cell cycles inbl/bl lymphocytes levelled off at BrdUrd concentrations below 10 g/ml while that in normal andbl/+ lymphocytes stayed constant below 7.5 g/ml. The baseline SCE frequency in bl/bl cells was ten times higher than that in normal andbl/+ cells. At BrdUrd concentrations above 15 g/ml, SCEs inbl/bl cells were induced more frequently than in normal andbl/+ cells. These results indicate that at low concentrations BrdUrd has a minimal effect on the induction of SCEs in all individuals, while at higher concentrations the BrdUrd incorporated inbl/bl cells has a larger effect than that in normal andbl/+ cells. To elucidate the effect of BrdUrd incorporated into the daughter and parental DNA strands on SCE induction, SCEs occurring during each cell cycle were examined separately in three-way or two-way differentially stained, third-cycle metaphases. The incidence of SCEs detected in each cell cycle at 5 g/ml BrdUrd was constant in all individuals and the rates of SCEs in each cell cycle inbl/bl cells were remarkably higher than those observed in normal andbl/+ cells. These findings strongly indicate that most of the abnormally increased SCEs in thebl/bl cells used in our study occurred independently of any effect of BrdUrd incorporated into both the daughter and parental DNA strands. In addition, an abnormal response ofbl/bl cells to BrdUrd was not found for cell cycle progression or chromosomal aberration induction. Thus, the bl/bl cells did not exhibit an abnormal hypersensitivity to BrdUrd. From these results, it seems quite probable that the abnormally increased SCEs in thebl/bl lymphocytes used here were spontaneous.     

Trenimon-induced SCEs and structural chromosomal aberrations in early- and late-dividing lymphocytes

In cultures of human peripheral lymphocytes the frequencies of Trenimon-induced SCEs in second post-treatment metaphases and of structural chromosomal aberrations in first, second and third post-treatment metaphases were clearly lower at late as compared with early fixation times. These results, which are discussed, indicate that T cells (early dividing) are more sensitive with respect to the induction of SCEs and structural chromosomal aberrations by Trenimon as compared with B cells (late dividing).     

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.     

Enhancement of cytogenetic damage by chlorpromazine in human lymphocytes treated with alkylating antineoplastics and caffeine

In cultured human lymphocytes chlorpromazine (CPZ) was found to induce cell division delays and to have no effect on sister-chromatid exchanges (SCEs) or on mitotic indices (MIs). CPZ induces cytotoxic effects in combination with caffeine (CAF) and alkylating agents. In combination with CAF it induced cell division delays and suppression of MIs. In combination with melphalan (MEL) and CAF, CPZ synergistically induced SCEs, caused cell division delay and suppressed MIs. In combination with chlorambucil (CBC) and CAF, CPZ produced synergism on induction of SCEs, enhanced cell division delays and reduced MIs.