Mutational and pseudomutational effects of 5-bromodeoxyuridine in human lymphoblasts

We have studied the effects of 5-bromodeoxyuridine (BrdUrd) at two genetic loci in diploid human lymphoblast cells. In thymidine kinase heterozygotes (tk +/-), a 2-h dose of BrdUrd induced a transient, non-heritable resistance to the thymidine analogue, trifluorothymidine (F3TdR). We have called this phenomenon pseudomutation and have shown that affected cells acquire the ability to survive in the presence of F3TdR and then, after degradation of F3TdR in the medium, return to an apparently normal wild-type state. Our data suggest that BrdUrd incorporation into DNA as a thymidine analogue is responsible for the effect, which we interpret as a temporary loss of thymidine kinase activity. This effect is not seen in tk +/+ homozygotes. In contrast, at the hypoxanthine-guanine phosphoribosyl transferase locus in tk +/- heterozygotes, BrdUrd did not induce a permanent, heritable resistance to 6-thioguanine (gene locus mutation). We detected such mutations only in the tk +/+ homozygote and only at external BrdUrd concentrations considerably higher than those which saturate the uptake of BrdUrd into DNA as a thymidine analogue. We postulate that the reduced TK enzyme levels (30%) in the heterozygote prevent the build-up of a sufficiently high intracellular BrdUrd triphosphate pool to promote the misincorporations as deoxycytidine triphosphate which may be responsible for gene locus mutation.     

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

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

Relative efficacy of short-term tests in detecting genotoxic effects of cadmium chloride in mice in vivo

The ability of intraperitoneally administered cadmium chloride (0.42-6.75 mg/kg) to induce genotoxic damage in somatic and germ cells of mice was evaluated using chromosomal aberrations, sister-chromatid exchanges (SCE), micronuclei and sperm-head abnormalities as end-points. A significant increase in the frequency of chromosomal aberrations and SCEs was observed in almost all treated series when compared to the negative control. Micronucleus formation in polychromatic erythrocytes was not affected significantly except at the highest concentration used (6.75 mg/kg). Significant differences were observed in the frequency of sperm with abnormal head morphology at all concentrations tested except the lowest one. The clastogenic effects of cadmium chloride in both somatic and germinal cells are found to depend directly on the concentrations used.     

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.     

In vitro genotoxic effects of ZnO nanomaterials in human peripheral lymphocytes

In this study, possible genotoxic effects of zinc oxide (ZnO) nanoparticles were investigated in cultured human peripheral lymphocytes by using chromosome aberrations and micronucleus assays (MN). For this purpose, the cells were treated with ZnO (1, 2, 5, 10, 15 and 20 μg/mL) for 24 and 48 h. In this research, four types of chromosome aberrations were observed as chromatid and chromosome breaks, fragment and dicentric chromosomes. ZnO induced significant increase of the ratio of chromosomal aberrations as well as percentage of abnormal cells at concentrations of 1, 5, 10 and 20 μg/mL in 24 h treatments. In 48 h treatments, while ZnO nanomaterials induced significant increase of the percentage of abnormal cells only at a concentration of 10 μg/mL, and of chromosome aberration per cell in comparison to the control at concentrations of 5 and 10 μg/mL. On the other hand, this material significantly increased the micronuclei frequency (MN) at concentrations of 10 and 15 μg/mL in comparison to the control. Cytokinesis-block proliferation index was not affected by ZnO treatments. It also decreased the mitotic index in all concentrations at 24 h but not at 48 h. The present results indicate that ZnO nanoparticles are clastogenic, mutagenic and cytotoxic to human lymphocytes in vitro at specific concentrations and time periods.     

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity.

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.     

Genotoxic activity of methyl mercury chloride and dimethyl mercury in human lymphocytes

The genotoxicity of methyl mercury chloride (MMC, 0–25 × 10⁻⁶M) and dimethyl mercury (DMM, 0–434 × 10⁻⁶M) was evaluated by chromosome metaphase analysis in human lymphocytes treated in vitro for 24 h. Structural (CA) and numerical (AN) chromosomal aberrations were scored for the assessment of induced genotoxic effects, while the variation in mitotic index (MI) was considered a monitor for induced cellular toxicity. MMC induced CA and AN in a dose-related manner at doses exceeding 0.6 × 10⁻⁶M, and the proportion of cells with CA was constantly and significantly higher than that of cells with AN. DMM was able to induce both effects as well, although to a lesser extent than MMC, CA and AN being induced at doses exceeding 43.4 × 10⁻⁶M and 1.73 × 10⁻⁶M, respectively. MMC was 6-fold more effective in inducing CA than DMM at equivalent toxic doses. On the other hand, no significant difference was observed between the two compounds in inducing AN. Therefore MMC was much more c lastogenic than DMM, whereas mitotic spindle disturbances appeared to be almost equally induced by both compounds.     

A study on the genotoxic effects of 8-Cl-cAMP on human lymphocytes in vitro

8-chloro-cyclic adenosine 3′,5′-monophosphate (8-Cl-cAMP) is the most potent cAMP analog that selectively inhibits a variety of cancer cell lines in vitro and tumors in vivo. Its action toward a variety of tumors, especially when coupled with other antitumor agents, have lead to phase I clinical investigations and recently phase II clinical investigations. Until today, very little was done to evaluate its genotoxic potential. In order to evaluate its genotoxic potential we used the cytogenetic and cytokinesis block micronucleus assay in vitro on peripheral blood lymphocytes of healthy individuals. In three concentrations (1 μM, 5 μM and 15 μM), 8-Cl-cAMP in normal human peripheral blood lymphocytes did not induce any cytogenetic aberrations of the structural type (chromatid breakage, isochromatid breakage and gaps), but did induce premature centromere separation (PCS) at all respective doses and increased the frequency of micronuclei (p < 0.05) only at the highest dose (15 μM). Antiproliferative action of 8-Cl-cAMP was estimated by using the cytokinesis block nuclear division index (NDI). The results showed a decrease in NDI of cells exposed to all doses of 8-Cl-cAMP when compared to control. Therefore, the overall results show a genotoxic potential of 8-Cl-cAMP in peripheral blood lymphocytes in vitro. This article was submitted by the authors in English.     

In vitro genotoxic effects of four Helichrysum species in human lymphocytes cultures

Helichrysum sanguineum, Helichrysum pamphylicum, Helichrysum orientale, Helichrysum noeanum (Asteraceae) are medicinal plants. For centuries, they have been used as tea in Turkey because of their medicinal properties. So far no scientific evidence has been found in a literature survey regarding the genotoxic effects of these plants. This work evaluated the genotoxic effects on human lymphocyte cultures induced by methanol extracts of these plants, assayed in different concentrations (0.01, 0.05, 0.1, 0.5 and 1 mg/mL). According to the results, Helichrysum noeanum, Helichrysum pamphylicum and Helichrysum sanguineum induced the formation of micronuclei and decreased the mitotic and replication indexes. Helichrysum orientale did not affect these parameters, whereas Helichrysum noeanum, Helichrysum pamphylicum and Helichrysum sanguineum were clearly genotoxic. They should therefore not be used freely in alternative medicine, although their antiproliferative activity may suggest antimitotic and anticarcinogenic properties. Helichrysum orientale could be used in alternative medicine.     

A study on the genotoxic effects of 8-Cl-cAMP on human lymphocytes in vitro

8-chloro-cyclic adenosine 3',5'-monophosphate (8-Cl-cAMP) is the most potent cAMP analogue that selectively inhibits a variety of cancer cell lines in vitro and tumors in vivo. Its action toward a variety of tumors, especially when coupled with other antitumor agents, have lead to phase I clinical investigations and recently phase II clinical investigations. Until today very little was done to evaluate its genotoxic potential. In order to evaluate its genotoxic potential we used the cytogenetic and cytokinesis block micronucleus assay in vitro on peripheral blood lymphocytes of healthy individuals. Using three concentrations (1 microM, 5 microM and 15 microM), 8-Cl-cAMP in normal human peripheral blood lymphocytes did not induce any cytogenetic aberrations of the structural type [chromatid breakage, isochromatid breakage and gaps], but did induce premature centromere separation (PCS) in all respective doses and increased the frequency of micronuclei (p <0.05) only in the highest dose (15 microM). Antiproliferative action of 8-Cl-cAMP was estimated by using the cytokinesis block nuclear division index (NDI). The results showed a decrease in the NDI of cells exposed to all doses of 8-Cl-cAMP when compared to control. Therefore, the overall results show a genotoxic potential of 8-Cl-cAMP in peripheral blood lymphocytes in vitro.     

Biosynthesis of methyl chloride in the fungusPhellinus pomaceus

The biosynthetic origin of themethyl group in the methyl chloride produced by cultures ofPhellinus pomaceus (Pers.) Maire has been investigated using stable isotope labeled substrates. Feeding ofd-[6,6-²H₂] glucose,Dl-[3,3-²H₂] serine andl-[methyl-²H₃] methionine led to the production of deuterated methyl chloride in which the major labeled species contained 2, 2, and 3 deuterium atoms, respectively. The data are consistent with the methyl chloride produced by this organism being derived solely from methionine with retention of all of the methyl protons.Abbreviation SAM S-adenosylmethionine - FH₄ tetrahydrofolate - N⁵-CH₃FH₄ N⁵-methyltetrahydrofolate - B₁₂ cobalamin     

Assessment of the cyto- and genotoxic effects of a nanoferromagnetic and a static magnetic field in vivo

The cyto- and genotoxic effects of ferromagnetic nanoparticles (FNs), a static magnetic field (SMF), or their combination were comparatively studied in Ehrlich ascites carcinoma (EAC) cells, bone marrow (BM) erythroid series cells, and peripheral blood lymphocytes at definite time intervals, using the MN test and the DNA-comet assay. The MN test and the DNA-comet assay were used as markers of genotoxic exposure. It has been shown that the limit value of insignificantly expressed changes in the EAC cell architectonics (due to FNs) is an FN concentration of 3 mg/kg. The FN concentration increases in tumor cells, because the expressed blebbing in the cytoplasmic membrane increases the amount of micronuclei and the percentage of DNA in the comet tail in EAC cells, BM erythroid series cells, and peripheral blood lymphocytes. It has also been demonstrated that an SMF alone, as an independent factor, does not produce cytogenotoxic effects on the studied cells. When SMF and FN were used in combination, we could observe the phenomenon of SMF induction by FNs, which manifested itself as an increased total effect of these factors on cells. The MN-test and the DNA-comet assay can be used for assessing the genomic stability of both tumor cells and somatic nonmalignized cells when testing the genotoxic effect of nanomaterials used in the design of vector systems. As was established, the DNA-comet method demonstrated a higher sensitivity in the assessment of the genotoxicity produced by the studied factors.     

Mutation of human lymphoblasts by methylnitrosourea

The lag in phenotype expression of methylnitrosourea(MNU)-induced mutation to 6-thioguanine (6TG) resistance has been studied in a diploid human lymphoblastoid cell line. We find that a considerable period (8–12 days) elapses before new mutants appear in treated cultures; after 2 weeks, however, a stable maximum fraction is attained, as would be expected for a genetic mutation. We present preliminary data linking this phenotypic lag to the slow degradation rate of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and to an apparent requirement for very low (<0.2% normal) cellular HGPRT content in order for cells to be resistant to 10 μg 6TG/ml. A series of reconstruction experiments are presented, the results of which support the conclusion that selective pressures in the assay procedure do not bias the quantitative estimates of induced mutant fraction.     

DNA replication in methotrexate-treated human lymphoblasts.

The rate of DNA synthesis in cultures of human lymphoblasts decreased more than 80% within 30 min after the cells were exposed to methotrexate, a potent inhibitor of dihydrofolate reductase. Despite this rapid initial inhibition, DNA continued to be synthesized for at least an additional 6 h. The mode of this subsequent replication appeared to be semiconservative, as indicated by the buoyant density of 5-bromodeoxyuridine-substituted DNA in alkaline CsCl gradients. The growth rates of DNA chains in cells exposed to methotrexate were determined by sedimentation rate analysis in alkaline sucrose gradients. DNA synthesized during 2-min or 10-min pulses with labeled deoxycitidine in the presence of methotrexate had about the same sedimentation coefficient, 35 S, as controls. When methotrexate-treated cultures were pulse-labeled for 10 min and then chased for various times, DNA fragments of about 80 S accumulated. DNA synthesized in the presence of methotrexate was stable and elongated to bulk-size DNA after methotrexate inhibition of growth was removed by addition of thymidine and deoxycytidine. The data suggest that methotrexate reduces the rate of DNA replication by inhibiting chain initiation independently of chain elongation.     

Cytotoxic and genotoxic effects of sodium hypochlorite on human peripheral lymphocytes in vitro

Chlorination is widely used method in the disinfection of drinking and utility water worldwide. In this study, cytotoxic and genotoxic effects of sodium hypochlorite were investigated by the cytokinesis-block micronucleus assay and chromosomal aberration analysis on human peripheral lymphocytes in vitro. A significant increase in chromosomal aberration frequency was observed in all treatments of NaOCl (0.030, 0.065, 0.100, 0.25, 0.5, 1, 2, 4 μg/mL) at 24 and 48 h compared with the negative control and mitomycin C (MMC, 0.3 μg/mL), which was used as a positive control. NaOCl significantly increased the frequency of micronuclei in a dose dependent manner. The results showed that there was a significant correlation between NaOCl concentration and chromosomal aberration, micronuclei frequency, necrotic cells, apoptotic cells and binucleated cells.     

Genotoxic activity of methyl mercury chloride and dimethyl mercury in human lymphocytes.

The genotoxicity of methyl mercury chloride (MMC, 0-25 x 10(-6) M) and dimethyl mercury (DMM, 0-434 x 10(-6) M) was evaluated by chromosome metaphase analysis in human lymphocytes treated in vitro for 24 h. Structural (CA) and numerical (AN) chromosomal aberrations were scored for the assessment of induced genotoxic effects, while the variation in mitotic index (MI) was considered a monitor for induced cellular toxicity. MMC induced CA and AN in a dose-related manner at doses exceeding 0.6 x 10(-6) M, and the proportion of cells with CA was constantly and significantly higher than that of cells with AN. DMM was able to induce both effects as well, although to a lesser extent than MMC, CA and AN being induced at doses exceeding 43.4 x 10(-6) M and 1.73 x 10(-6) M, respectively. MMC was 6-fold more effective in inducing CA than DMM at equivalent toxic doses. On the other hand, no significant difference was observed between the two compounds in inducing AN. Therefore MMC was much more clastogenic than DMM, whereas mitotic spindle disturbances appeared to be almost equally induced by both compounds.     

Signal transduction of somatostatin in human B lymphoblasts

Somatostatin (SST) and somatostatin receptors (SSTR) are widely distributed in lymphoid tissues. Here, we report on the stimulatory effects of SST in Epstein-Barr virus-immortalized B lymphoblasts. By RT-PCR, we demonstrated the exclusive expression of the somatostatin receptor isoform 2A (SSTR2A) in B lymphoblasts. Addition of SST rapidly increased the cytosolic free calcium concentration [Ca(2+)](i) maximally by about 200 nM, with an EC(50) of 1.3 nM, and stimulated the formation of inositol phosphates. Furthermore, SST increased binding of guanosine 5'-O-(3-thiotriphosphate) by 50% above basal. These effects were partly inhibited by pertussis toxin (PTX), which indicates the involvement of PTX-sensitive G proteins. We provide further evidence that Galpha(16,) a PTX-insensitive G protein confined to lymphohematopoietic cells, is involved in the otherwise unusual coupling of SSTR2A to phospholipase C activation. In addition, SST activated extracellular regulated kinases and induced a 3.5-fold stimulation of DNA synthesis and a 4.4-fold stimulation of B lymphoblast proliferation, which was accompanied by an enhanced immunoglobulin formation. Thus SST exerts a growth factor-like activity on human B lymphoblasts.     

Cytotoxic and genotoxic effects of extracellular generated singlet oxygen in human lymphocytes in vitro

The induction of sister-chromatid exchanges (SCE) together with the proliferation rate index (PRI) were studied in human lymphocytes in vitro after treatment with singlet oxygen. When produced outside the cells, singlet oxygen can increase the duration of the cellular cycle as measured by an enhancement of the differences between the proliferation rate indexes of the control and the treated cells. A dose-dependent increase in the SCE rate per chromosome was also detected after contact between the singlet oxygen and lymphocytes.