Inhibition of potassium dichromate mutagenicity by todralazine

Todralazine, an antihypertensive drug of the hydrazinophthalazinegroup, markedly decreased the mutagenic activity of potassiumdichromate in standard bacterial tests. At the highest todralazinedose tested inhibition of potassium dichromate mutagenic activityby {small tilde}90% in the Ames test and up to 100% (complete)inhibition in the Bacillus subtilis rec^– assay was observed.Spectrophotometric analyses proved that todralazine inducedreduction of Cr(VI) to Cr(III) and complexation of Cr(III) ions.These spectrophotometric results may be a presumptive explanationof the observed mutagenic activity decrease, as it is knownthat Cr(III) is poorly transported across cell membranes andtherefore is not mutagenic to bacterial cells. We perceive ourexperiments as an example of attempts which should lead to aneffective reduction in chromium genotoxic and carcinogenic activityin exposed individuals. ¹To whom correspondence should be addressed. Tel: +48 71 486024: Fax: +48 71 215729     

Comparison of the aneugenic effect of vinorelbine and vincristine in cultured human lymphocytes.

Vinca alkaloids are used clinically against a variety of hematological and solid tumors. These compounds interact with tubulin subunits to prevent microtubule assembly, inducing abnormal chromosome segregation in dividing cells and causing aneuploidy. The vinca alkaloid vincristine sulfate (VCR) and the semisynthetic analog vinorelbine (VRB) were studied by analysis of micronuclei (MN) in cultured human lymphocytes using the cytokinesis block method. Furthermore, fluorescence in situ hybridization with a human alphoid satellite pancentromeric DNA probe was used to detect centromeres in isolated MN of VRB- or VCR-treated lymphocytes. At all the doses tested, both chemicals induced a significant increase in MN frequencies in binucleated (BN) cells (P < 0.001). The maximal effect was reached at a dose of 0.50 microgram/ml. At this dose, VRB produced an approximately 5-fold increase with respect to the control frequency of MN, while with VCR, this frequency increased 10-fold. Both drugs produced a slowing of the cell cycle, causing a decrease in the percentage of BN cells. This effect was lower with VRB. The percentages of centromere-positive MN were 89.79 and 87.60% in VRB- and VCR-treated cultures, respectively (control 27.03%). The high percentage of positive-signals in treated cultures (P < 0.001) indicates that the MN contained whole chromosomes. Our results confirm the aneugenic mode of action of these chemicals, VRB having less genetic effect.     

体外培养淋巴细胞基因转移的实验探讨

以ＰＨＡ、ＩＬ－２体外诱导培养２～３ｗｋ的淋巴细胞作为受体细胞，在脂质体介导下进行腺苷脱氨酶及β－半乳糖苷酶基因转移。结果表明：外周血淋巴细胞在体外经ＰＨＡ、ＩＬ－２诱导可大量增殖，其中ＣＤ３＋细胞占９０％以上。脂质体介导外源性ＤＮＡ阳性转染率可高达３０％～４０％以上。转移后细胞表达有活性的基因表达产物。提示应用脂质体对体外扩增的淋巴细胞实施基因转移，其方法简便，转移率高，转移后基因在受体细胞中能较好地表达     

The effect of organochlorine compounds on the induction of sister chromatid exchanges in cultured human lymphocytes

In this study, first, we investigated the effect of 7,8-benzoflavone (ANF), mitomycin C (MMC), a well-known genotoxic compound, and ANF plus MMC on the induction of sister chromatid exchanges (SCEs) in human whole-blood cultures. Second, we examined the effect of mixture of organochlorine compounds, which very resembled their contamination of healthy people in its composition, on the induction of SCEs in the same blood culture system in order to clarify their genotoxicity as a whole. The following results were obtained. 1. ANF and MMC significantly enhanced the number of SCEs/cell at the concentrations of 4 x 10(-5) M and 10(-8)M, respectively. When both of the compounds were simultaneously added in the blood cultures, their effects on the induction of SCEs seemed to be additive. 2. Without ANF in the blood culture system, namely, an usual system of the SCEs experiment, we could not find a dose-response relationship between the concentration of the mixture of organochlorine compounds and the induction of SCEs/cell. With ANF, however, we observed a fairly good dose-response relationship between them. 3. In the whole-blood culture system with ANF, we found significantly great number of SCEs/cell at the level of twenty times higher concentration of the organochlorine compounds than the ordinary level. According to the results described above and of our other studies, 50% effective concentration (EC50, about 2 SCEs/cell higher than control SCEs/cell) of the mixture was considered to be about 5 times greater level over the general one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genotoxicity of hydrochlorothiazide in cultured human lymphocytes. I. Evaluation of chromosome delay and chromosome breakage

Hypertension is often treated with diuretics, like hydrochlorothiazide (HCTZ). Previous results on the in vitro genotoxicity of HCTZ are equivocal. In the present study, we have evaluated the genotoxicity of HCTZ in cultured human lymphocytes using the Cytokinesis Blocked Micronucleus (CBMN) assay. In addition, micronucleus (MN) induction was analyzed by Fluorescence In Situ Hybridization (FISH) with an alpha-satellite DNA centromeric probe to distinguish between clastogenic and aneugenic effects. Lymphocyte cultures from 32 healthy adults were exposed to 5 and 40 microg/ml HCTZ. Age, gender, and smoking were evaluated as factors affecting the MN analysis. We found that HCTZ increased MN frequencies. FISH analysis revealed that HCTZ exerts its genotoxicity more strongly at the 40 microg/ml concentration, and principally through chromosome delay (aneugenicity). Multiregression analysis of our results confirmed the known effect of age and gender on MN induction in human lymphocytes. Smoking was also a confounding factor for MN induction, especially for centromere-negative MN frequencies. Under the experimental conditions used, only age had a clear positive effect on the response of lymphocytes to HCTZ. These data indicate that HCTZ produces micronuclei in cultured human lymphocytes by a mechanism that involves chromosome delay and to a lesser extent through chromosome breakage.     

Immunoglobulin production by cultured human lymphocytes

The synthesis and secretion of immunoglobulin induced in cultured B-lineage cells is of interest for several reasons: (i) analysing the B-cell repertoire, (ii) recall of immunological activity retained in the circulating lymphocyte population, and (iii) study of factors needed for clonal expansion, immunoglobulin class switching, IgV-region mutation and maturation of cells to Ig secretion. Methods available are outlined and alternative procedures for cell separation and purification, helper cell provision and Ab/Ig assay systems are discussed. The aim is to provide practical guidance for those who intend to begin work in what is a vitally important, but experimentally difficult, area. There are a bewildering number of methods described in innumerable publications, old and new. The review provides a personal assessment of the present state of knowledge and prospects for improvements when all the new observations relating to cell-cell interactions and cytokines are integrated into existing technologies. The survey is chiefly concerned with physiologically based procedures, but artificial auxiliary methods are also briefly mentioned.     

Liquid nitrogen storage of cultured T lymphocytes

The conditions required for storing and recovering IL-2-dependent T cell clones from liquid nitrogen were investigated. For maximum cell recovery, cultured T lymphocytes were precooled at 4 degrees C for 15 min in medium containing 10% DMSO and 20% FCS before storage in liquid nitrogen. This method allowed adequate time for DMSO to penetrate the cells before freezing.     

A comparison of the antimutagenic potential of green, black and decaffeinated teas: contribution of flavanols to the antimutagenic effect

The present study was undertaken to compare the antimutagenicactivity of aqueous extracts, at the concentrations used forhuman consumption, from green, black and decaffeinated blacktea. Antimutagenic potential was evaluated against three indirect-actingdietary carcinogens, Glu-P-1, benzo(a)pyrene and nitrosopyrroudine.All three types of tea gave rise to strong and concentration-dependentsuppression of the mutagenicity of the three premutagens inthe presence of an activation system. No major difference inthe antimutagenic potential of the three types of tea couldbe discerned. Black tea, decaffeinated black tea and, to a lesserextent, green tea also antagonized the mutagenicity of the direct-actingmutagen 9-aminoacridine. All three types of tea inhibited markedlythe NADPHdependent reduction of cytochrome c and the O-dealkylationsof ethoxy-, methoxy- and, to a much lesser extent, pentoxy-resorufin.When the microsomal metabolism was terminated, after the metabolicactivation of the premutagens, incorporation of the aqueoustea extracts into the activation system caused a concentration-dependentsuppression of mutagenic response. No significant differencein the antimutagenic activity of the three types of tea in thissystem was evident. Bearing in mind the much higher concentrationof flavanols in green tea compared with the black teas, it maybe concluded either that these compounds are unlikely to bethe major tea components responsible for the antimutagenic,and possibly anticarcinogenic, properties of tea or that theirfermentation products are similarly active. ³To whom correspondence should be addressed     

Cytogenetic effects of permethrin in cultured human lymphocytes.

The pyrethroid insecticide permethrin was tested for its ability to induce sister chromatid exchanges (SCE), micronuclei (MN) and structural chromosome aberrations (CA) in cultured human peripheral blood lymphocytes. Permethrin was tested in the range of 5-500 micrograms/ml in the absence and in the presence of a rat liver activation system (S9 mix). Small elevations in the SCE frequencies were found and even though statistically significant may have no biological meaning, the more so since there was no dose-effect relationship. Permethrin induced both MN and CA when it was evaluated in the absence of a metabolic activation system. Nevertheless, it cannot be said that S9 mix suppressed the activity in itself. The effect of permethrin seemed to be time of exposure dependent. Permethrin could be characterized as a S-phase independent agent with greater potential for inducing chromosomal damage than sister chromatid exchanges.     

Antiradical activity of ternary copolymers from the diallyl series in mediating the antimutagenic effect

Ternary copolymers of the diallyl series were tested for antiradical activity against the stable radical 1,1-diphenyl-2-picrylhydrazyl, and the numbers of active antitradical fragments which are present in their macromolecules and which may participate in the free-radical reactions induced by gamma radiation in living organisms were estimated. A correlation was found between the antiradical activity of ternary copolymers and their ability to protect barley seedling roots from mutagenic effects of gamma radiation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 9, pp. 265–267, September, 1995 Presented by V. N. Smirnov, Member of the Russian Academy of Medical Sciences     

大剂量硫酸锌与小剂量青霉胺对体外培养淋巴细胞染色体损伤的影响

目的　探讨大剂量硫酸锌与小剂量青霉胺对体外培养淋巴细胞染色体的损伤程度。方法　采用染色体分析技术对对照组、硫酸锌组、青霉胺组、(硫酸锌 +青霉胺 )组的姐妹染色单体互换 (SCE)频率进行观察。结果　硫酸锌组与对照组之间差异无显著性 (t =1.2 6 4 ,P >0 .0 5 ) ,(硫酸锌 +青霉胺 )组、青霉胺组与对照组差异有显著性 (t =8.12 ;6 .13,P <0 .0 0 1)结论　硫酸锌对染色体损伤无影响。青霉胺对染色体损伤有一定作用。     

Evaluation of the radioprotective effect of Aegle marmelos (L.) Correa in cultured human peripheral blood lymphocytes exposed to different doses of gamma-radiation: a micronucleus study

The radioprotective effect of a hydroalcoholic extract of Aegle marmelos (AME) was evaluated in cultured human peripheral blood lymphocytes (HPBLs) by the micronucleus assay. The optimum protective dose of the extract was selected by treating HPBLs with 1.25, 2.5, 5, 6.25, 10, 20, 40, 60, 80 and 100 microg/ml AME before exposure to 3 Gy gamma-radiation and then evaluating the micronucleus frequency in cytokinesis blocked HPBLs. Treatment of HPBLs with different doses of AME reduced the frequency of radiation-induced micronuclei significantly, with the greatest reduction in micronucleus induction being observed for 5 microg/ml AME. Therefore, this dose of AME was considered as the optimum dose for radioprotection and further studies were carried out treating the HPBLs with 5 microg/ml AME before exposure to different doses (0, 0.5, 1, 2, 3 and 4 Gy) of gamma-radiation. The irradiation of HPBLs with different doses of gamma-radiation caused a dose-dependent increase in the frequency of lymphocytes bearing one, two and multiple micronuclei, while treatment of HPBLs with 5 microg/ml AME significantly reduced the frequency of lymphocytes bearing one, two and multiple micronuclei when compared with the irradiated control. The dose-response relationship for both groups was linear. To understand the mechanism of action of AME separate experiments were conducted to evaluate the free radical scavenging of OH, O2(-), DPPH, ABTS(+) and NO in vitro. AME was found to inhibit free radicals in a dose-dependent manner up to a dose of 200 microg/ml for the majority of radicals and plateaued thereafter. Our study demonstrates that AME at 5 microg/ml protected HPBLs against radiation-induced DNA damage and genomic instability and its radioprotective activity may be by scavenging of radiation-induced free radicals and increased oxidant status.     

Strong antimutagenic effect of caffeine on 9-aminoacridine-induced frameshift mutagenesis in Escherichia coli K12

Caffeine, present at a concentration of 3 mg/ml during treatment with 100 microM 9-aminoacridine (9AA) of wild-type Escherichia coli K12, caused a decrease in the yield of frameshift reversions of more than three orders of magnitude. Antimutagenesis of caffeine was shown to be due to partial inhibition of induction by 9AA of frameshift replication errors, resulting in an increased efficiency of mismatch repair of the pre-mutations produced under these conditions.     

Murine intestinal intraepithelial lymphocytes II. Comparison of freshly isolated and cultured intraepithelial lymphocytes

Highly enriched preparations of intraepithelial lymphocytes (IEL) containing a large subpopulation of granulated cells were isolated from the murine small intestinal mucosa. We cultured IEL in media containing interleukin 2 (growth media conditioned with 20% concanavalin A supernatant; Con A CM) or mast cell growth factor(s) (growth media conditioned with 40% WEHI-3 supernatant; WEHI CM) and compared the physical and functional properties of the cultured cells to freshly isolated IEL. IEL cultured in Con A CM developed enhanced cytotoxicity against YAC-1, compared to freshly isolated IEL, and spontaneous cytotoxicity for P815 targets. Most of these cultured cells were Thy-1+ Lyt-1- Lyt-2+, and contained cytoplasmic granules similar to those seen in electron photomicrographs of other cytotoxic cell populations. IEL cultured in WEHI CM gave rise to cells that morphologically resembled mast cells. Unlike freshly isolated IEL, the cells stained metachromatically, contained 200-450 ng of histamine/10(6) cells and expressed high-affinity receptors for IgE. Our data clearly show that, although IEL do not themselves have physical characteristics of mast cells, they do contain mast cell precursors. In addition, IEL grown in the presence of T cell growth factors give rise to an activated cytotoxic cell population which is mostly granulated and Thy-1+ Lyt-1- Lyt-2+.     

The effect of aging on cell-cycle kinetics and X-ray-induced chromosome aberrations in cultured lymphocytes from patients with Down syndrome

To evaluate the effect of aging on cytogenetic characteristics of lymphocytes from Down syndrome (DS), cell-cycle kinetics after PHA stimulation and chromosome-type aberration frequencies after X-ray exposure were investigated in vitro in the lymphocytes derived from 4 (or 3 for X-ray treatment) age groups of DS patients and age-matched controls. The results clearly showed higher mitotic and proliferation index levels in younger groups compared to older groups at the various culture intervals, whether the lymphocytes were from the DS patients or controls. The age-related changes of the proliferation index were mainly attributed to a delayed response to PHA as age increased. The changes of PHA responses seemed to be particularly marked during adolescence. Nonetheless, no significant differences were observed between the DS patients and age-matched controls for each age group. In all age groups, frequencies of both chromosome-type exchanges and deletions were elevated in the DS patients by about 1.3 times in comparison with the controls. The magnitude of radiosensitivity, however, seemed to decrease slightly in the 40–49 year group. To our knowledge, the present study is the first report in the literature to deal with the effect of aging on the greater radiosensitivity of DS lymphocytes.     

X irradiation and sister chromatid exchange in cultured human lymphocytes

Sister chromatid exchange (SCE) frequency was not increased in G₀ lymphocytes following irradiation up to 400 rads. Lymphocytes irradiated after 42 or 60 hours of culture showed a dose-dependent increase in exchange frequency at 100 and 200 rads. SCE was not increased in cells irradiated in G₂ (68.5 hours). Lymphocytes maintained in a 5-Bromodeoxyuridine (BrdUrd) free medium and irradiated 42 hours after culture initiation showed an increase in SCE if BrdUrd was added immediately after irradiation, but no increase was found if there was a 5 hour holding period prior to the addition of BrdUrd. The effect on induced SCE frequency of heightened radiosensitivity due to increased amounts of BrdUrd was also investigated. When the BrdUrd concentration was increased from 10 μg/ml to 50 μg/ml, the percent increase in X-ray-induced SCE was lower at 50 μg/ml. In addition, increased BrdUrd concentration only slightly increased the sensitivity of the SCE technique to irradiation doses of 50 rads.     

Functional properties of continuously cultured human T lymphocytes.

Human T lymphocytes (CCTC) have been maintained in continuous culture by a new method. The ability of CCTC to produce three types of T cell response has been determined and compared to the published responses of T cells grown in 'T cell growth factor' (TCGF) medium. Unlike TCGF cells, CCTC will not give proliferative responses when stimulated with PHA, Con A, or allogeneic lymphocytes even when supplemented with autologous PBL as a source of accessory cells. Instead, CCTC are potent inhibitors of the proliferative response of normal cells in MLC. This suppressive activity is not specific for histocompatible cells. Finally, CCTC express cytotoxic activity to a number of human lymphoid cell lines and this activity appears to be different from the polyclonal cytotoxicity reported for TCGF-maintained cells. Our method of long-term T cell culture therefore appears to grow cells with different properties from TCGF cells.