A mixture of isothiocyanates induces cyclin B1- and p53-mediated cell-cycle arrest and apoptosis of human T lymphoblastoid cells.

As with other candidate chemopreventive agents, most of our knowledge on the biological effects of isothiocyanates (the many sulfur-containing metabolites found in cruciferous vegetables) comes from studies of single natural or synthetic compounds. To investigate whether the biological/chemopreventive effects of administration of single isothiocyanates can differ from those of a mixture of isothiocyanates, we tested the effects of a mixture of four different isothiocyanates on cell-cycle progression and apoptosis in human T leukemia Jurkat cells, and identified some of the molecular pathways triggered by the mixture. The mixture affected critical points of the cell cycle via modulation of the expression of cyclin B1. Moreover, it induced apoptosis, mediated by an increase in p53 and bax (expression of bcl-2 was unaffected). Comparison of the data with those previously obtained with the single isothiocyanates under identical experimental conditions provides evidence that the quantitative effects of a single, specific isothiocyanate can be significantly different from those of an isothiocyanate mixture at realistic doses.

Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in non-transformed human T lymphocytes.

Despite experimental evidence that sulforaphane can exert chemopreventive effects, whether these effects are specific for neoplastic cells is not known. Following our previous demonstration that sulforaphane induces cell cycle arrest and apoptosis in human T lymphoblastoid Jurkat leukemia cells and increases p53 and bax protein expression, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human lymphocytes. Here, we demonstrate that sulforaphane arrested cell cycle progression in G, phase, through a decrease in the protein expression of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53. These findings suggest that sulforaphane is a growth modulator for T cells. Our in vitro evidence that sulforaphane is active and even cytotoxic in normal as well as transformed lymphocytes raises important questions regarding its suitability for cancer chemoprevention.

Micronuclei induction, cell cycle delay and apoptosis as markers of cellular stress caused by ursodeoxycholic acid in human lymphocytes.

Ursodeoxycholic acid (UDCA) is a bile acid (BA) used for cholesterol gallstone dissolution. Since epidemiological evidence indicates that BAs can be involved in the etiology of colorectal cancer, we investigated the effects of UDCA and its physiologically produced taurine conjugate tauroursodeoxycholic acid (TUDCA) on human lymphocyte cultures in terms of genetic damage in the form of micronuclei (MN) production, cell cycle modifications and induction of apoptosis. With respect to controls, treatment with UDCA (from 10 microg/ml) caused a dose-related increase in MN, whereas TUDCA caused no significant increase (up to 1000 microg/ml). Fluorescence in situ hybridization (FISH) analysis using pancentromeric probes suggested that UDCA exerts aneugenic activity. Bromodeoxyuridine/Hoechst flow cytometry showed that both BA significantly inhibit cell cycle progression (UDCA at 100 microg/ml, and TUDCA, more markedly at 300-1000 microg/ml). Neither UDCA nor TUDCA affected induction of apoptosis, as evaluated by the Annexin-V-Fluos assay. We conclude that UDCA is potentially genotoxic. However, taking into account the characteristics of other physiological BA, our findings are in line with the concept that long-term UDCA treatment may be safely administered. The multi-assay approach reported here could be useful in the toxicological evaluation of newly developed BA analogs as candidates for pharmacological use.

Early functional apoptotic responses of thymocytes induced by Tri-n-butyltin.

BACKGROUND: Programmed cell death, also termed apoptosis, is the main focus of interest in a variety of scientific and clinical areas. For a better understanding of the mechanisms of apoptosis, from the onset of the cellular death program to the late stages of apoptosis or apoptotic necrosis, very early functional events have to be quantified because they might be involved in temporal and causal relationships between apoptosis-related key processes. METHODS: We have established a flow cytometric technique to quantify time-dependent signals simultaneously with high temporal resolution (Deltat = 1 s) in living cells. With this technique, the response of cells to apoptosis-stimulating agents can be analyzed over 15 min. For this purpose, a thermostatted sample tube holder for repeatable interruption-free injection of substances into the cell suspension was developed. Early detectable fluorescence and scatter parameters were related to intracellular free Ca2+ concentration, [Ca2+]i (Indo-1 fluorometry), membrane permeability (propidium iodide [PI] influx), and cell volume (forward scatter). RESULTS: A T-cell line (Jurkat) served as a model system. Apoptosis was induced by the biozid Tri-n-butyltin (TBT). Dependent on the TBT concentration (0.3-10 microM), the mean free [Ca2+]i increased by a factor of 1.2-6 during a short time interval of just 2 min. Especially after low TBT concentrations (< 0.5 microM), this [Ca2+]i increase was nearly transient during the observation time of 15 min. Higher TBT concentrations (0.5-10 microM), however, induced a transient increase of [Ca2+]i (Ca-TR) only in a fraction of the cells; in another subpopulation, a steady-state Ca2+ signal (Ca-SST) was observed. The analysis of the simultaneously registered PI signals of the Ca-SST cells showed a shift to increasing PI fluorescence (by a factor of about 4) with increasing Ca2+ concentrations. In Ca-TR cells, the PI fluorescence remained nearly unchanged. These apoptosis-related changes (increase in [Ca(2+)]i and membrane permeability) could be confirmed by the additional observation of a TBT concentration-dependent decrease in cell volume measured during the same early time period. CONCLUSIONS: The simultaneously analyzed parameters (i.e., [Ca2+]i, membrane permeability, and cell volume) suggested that, in our model system of Jurkat T-cells treated with TBT, an apoptotic cell fate was indicated very early (within 15 min) by the steady-state [Ca2+]i level.

Fluorescence enhancement of DNA-bound TO-PRO-3 by incorporation of bromodeoxyuridine to monitor cell cycle kinetics.

BACKGROUND: The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycle. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser flow cytometry has been investigated. METHODS: Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is registered in combination with the fluorescence emission of the intercalating dye propidium iodide (PI) as a total DNA stain to give bivariate DNA/BrdUrd histograms. By the low concentration of only 0.3 mircoM TO-PRO-3, BrdUrd detection is optimized, and undisturbed total DNA content by PI can be detected as well. TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser. RESULTS: In order to understand the binding of TO-PRO-3, energy transfer from PI to TO-PRO-3 has been measured as well as the influence of an external DNA binding dye such as Hoechst 33258 with Adenine-Thymine (AT) binding specificity. Cell cycle studies of human SCL-2 keratinocytes and mouse 3T3 cells prove the method to be as generally applicable as the classical BrdUrd/Hoechst quenching technique, but without need for expensive ultraviolet laser excitation. No BrdUrd sensitivity could be found for the similar dyes TO-PRO-1 and YO-PRO-3, whereas TO-PRO-5 and YOYO-3 showed only very little sensitivity to BrdUrd labeling as compared with TO-PRO-3. CONCLUSIONS: Cell cycle studies of mammalian cells can be done by dual-laser flow cytometry without the need for ultraviolet lasers by using the BrdUrd-dependent fluorescence enhancement of TO-PRO-3. Total DNA content can be measured simultaneously using PI.

Genetic damage in operating room personnel exposed to isoflurane and nitrous oxide.

OBJECTIVES: To evaluate genetic damage as the frequency of sister chromatid exchanges and micronuclei in lymphocytes of peripheral blood of operating room personnel exposed to waste anaesthetic gases. METHODS: Occupational exposure was measured with a direct reading instrument. Venous blood samples were drawn from 10 non-smokers working in the operating room and 10 non-smoking controls (matched by age, sex, and smoking habits). Lymphocytes were cultured separately over 72 hours for each assay with standard protocols. At the end of the culture time, the cells were harvested, stained, and coded for blind scoring. The exchanges of DNA material were evaluated by counting the number of sister chromatid exchanges in 30 metaphases per probe or by counting the frequency of micronuclei in 2000 binucleated cells. Also, the mitotic and proliferative indices were measured. RESULTS: The operating room personnel at the hospital were exposed to an 8 hour time weighted average of 12.8 ppm nitrous oxide and 5.3 ppm isoflurane. The mean (SD) frequency of sister chromatid exchanges was significantly higher (10.2 (1.9) v 7.4 (2.4)) in exposed workers than controls (p = 0.036) the proportion of micronuclei (micronuclei/500 binucleated cells) was also higher (8.7 (2.9) v 6.8 (2.5)), but was not significant (p = 0.10). CONCLUSION: Exposure even to trace concentrations of waste anaesthetic gases may cause dose-dependent genetic damage. Concerning the micronuclei test, no clastogenic potential could be detected after average chronic exposure to waste anaesthetic gas. However, an increased frequency of sister chromatid exchanges in human lymphocytes could be detected. Although the measured differences were low, they were comparable with smoking 11-20 cigarettes a day. Due to these findings, the increased proportion of micronuclei and rates of sister chromatid exchanges may be relevant long term and need further investigation.

Flow cytometric measurement of micronuclei induced in a permanent fish cell line as a possible screening test for the genotoxicity of industrial waste waters.

An in vitro micronucleus assay using the permanent fish cell line RTG-2 (rainbow trout gonads) was developed to test industrial waste waters for their genotoxic potential. Comparison of flow cytometric measurement and microscopic scoring of micronucleus frequency with the reference chemicals 1,4-butane sultone (0.2-1 mM), ethylmethane sulphonate (2-10 mM), potassium dichromate (20-100 microM) and benzo[a]pyrene (5-25 microM) showed similar dose-effect relationships. Thirty-eight industrial waste waters from 11 different branches of industry obtained from the Bavarian state office for water research were tested using the flow cytometric method (18 from metal processing, 10 from combined waste water, two from synthetic fibre production, one sample each from settlement wastes, non-iron metal manufacturing, leather production, sulphuric acid production, ore processing, graphite film production, cellulose production and flue gas washing). Fourteen of them showed a significant increase in micronucleus frequency.

Flow cytometric analysis of genetic damage, effect on cell cycle progression, and apoptosis by thiophanate-methyl in human lymphocytes.

Flow cytometric technique was used to study the effects of the fungicide Thiophanate-methyl on cell proliferation, micronucleus induction, and apoptosis in human peripheral blood lymphocytes treated in vitro. In particular, a combined approach of flow cytometry and fluorescence in situ hybridization (FISH) with a pancentromeric alpha-satellite probe was used to evaluate the mechanism of micronucleus induction by Thiophanate-methyl. Flow sorted micronuclei (MN) induced in human lymphocytes by Thiophanate-methyl were analyzed by FISH and the results were compared with results from FISH analysis on MN in binucleated cells. It could be shown that most MN induced by Thiophanate-methyl did not reveal any centromeric signal, thus demonstrating clastogenic action of this fungicide. Moreover, it was found that as a function of the concentration of Thiophanate-methyl, cellular proliferation was delayed and the frequency of apoptotic cells was increased.

Kinetics of radiation- and cytochrome c-induced modifications in liposomes analysed by FT-Raman spectroscopy.

Fourier transform Raman spectroscopy on artificial lipid membranes was used to study radiation-induced peroxidation processes as a function of time after radiation exposure. The time dependent intensity changes of the Raman lines of various C=C bondings were compared to results obtained by measuring conjugated dienes and by the thiobarbituric acid test for malondialdehydes. The results show that mainly the cis C=C bonds of the lipid chains are involved and, therefore, indicate that gamma-radiation induces conformational changes in the lipid chain while the mobility of the lipid chains is reduced. New Raman bands can be assigned to aldehyde products induced at the end of the peroxidation process. The immediate decrease of the =CH vibration lines was directly correlated with the formation of conjugated C=C double bonds suggesting that these vibration lines are in contrast to the C=C lines solely Raman active, when isolated C=C bonds are present. Cytochrome c (ox.) incorporated into the bilayer of the artificial membranes induced autooxidation processes not influenced by gamma-radiation. It was observed that cytochrome c (ox.)-induced changes of the relative intensity of the C=C bonds differ from those induced by gamma-radiation. These results of cytochrome c together with the inhibitory effects of the antioxidant alpha-tocopherol suggest that the radical species involved in the cytochrome c induced process might be different from the free radicals involved in the gamma-radiation-induced process.

Micronuclei containing whole chromosomes harbouring the selectable gene do not lead to mutagenesis.

Loss of heterozygosity is one genetic change observed in many tumours. We do not know whether the loss of chromosomal material through micronucleus formation is a viable mechanism associated with, and possibly leading to, genetic disease. Previously, we treated L5178Y mouse lymphoma cells with four aneugens. Although these aneugens induced micronuclei containing predominantly whole chromosomes, they did not induce mutations at Tk1, the selectable gene, under the same non-toxic conditions in which they induced micronuclei. This suggested that the induction of micronuclei containing whole chromosomes was not an early event leading to phenotypically expressed mutations in these cells under the conditions used. However, it is possible that chromosome 11, on which Tk1 resides, may be under-represented in the micronucleus population. To find out the frequency of induction of micronuclei containing chromosome 11, we applied fluorescence in situ hybridization using a chromosome 11 paint to micronuclei induced by colcemid and vinblastine. We found that the numbers of micronuclei containing chromosome 11 are more than sufficient to be detectable as mutations if these micronuclei lead to viable mutants. We conclude that the formation of micronuclei containing whole chromosomes does not lead to viable, dividing mutants in this system.

Flow cytometric analysis of micronuclei in cell cultures and human lymphocytes: advantages and disadvantages.

Flow cytometric techniques are described to quantify micronucleus (MN) induction in cell cultures and human lymphocytes. The advantages and disadvantages of these techniques are discussed. Because a suspension of nuclei and MN has to be prepared for flow cytometric measurements, care has to be taken to avoid unspecific debris that can influence the results. Using additional flow cytometric parameters, most of the unspecific particles in the suspension can, however, be gated out. Apoptotic cells and apoptotic bodies can overlap the MN during measurement, it is, therefore, proposed not to use the technique if apoptosis is induced by the respective treatment. Advantages of the automated flow cytometric techniques are that results can be obtained in short time intervals, the frequency of MN and the DNA distribution of MN can be measured simultaneously and flow sorting can be used for a further analysis of MN using other techniques.

Analysis of radiation-induced micronuclei by FISH using a combination of painting and centromeric DNA probes.

In situ hybridization with whole chromosome painting probes (chromosome 1, 7, 11, 14, 17 and 21) in combination with a human pancentromeric alpha-satellite probe was used to analyse the presence of specific chromosomal material in micronuclei (MN) induced in human lymphocytes by ionizing radiation. The purpose was to investigate the nature of radiation-induced cytogenetic damage, especially to study whether the fraction of paint-positive MN is proportional to the relative DNA content of the respective chromosomes which might indicate a random breakage of chromosomes. Flow-sorted MN and MN in binucleated cells were analysed with the six chromosome specific painting probes. It was found that the fraction of paint-positive MN increased linearly with the DNA content of the respective chromosomes. About 13% radiation-induced MN in human lymphocytes were found to contain centromeric signals independent of the presence of specific chromosome painting signals. The data obtained on flow-sorted MN and MN in binucleated cells agreed well, indicating that flow-sorted MN can be used for studying their chromosomal content with the FISH technique. If it is assumed that the chromosomal content of MN reflects radiation-induced damage, then these results support a random model of radiation-induced cytogenetic damage in human lymphocytes for the six chromosomes studied.

Quantitative and qualitative studies of micronucleus induction in mouse erythrocytes using flow cytometry. I. Measurement of micronucleus induction in peripheral blood polychromatic erythrocytes by chemicals with known and suspected genotoxicity.

Quantitative and qualitative aspects of the in vivo micronucleus-inducing potential of five chemicals were studied using flow cytometric enumeration of micronucleated polychromatic peripheral blood erythrocytes in mice. The chemicals were hydroquinone, vinblastine sulphate, chloral hydrate (tested in two different mouse strains), 5-bromo-2-deoxyuridine and 2-chlorobenzylidene malonitrile. Repeat samplings of peripheral blood were made at 0, 24, 40, 48 and 72 h and for low doses of 5-bromo-2-deoxyuridine 96 h after i.p. treatment. The agents hydroquinone (lowest effective dose 25 mg/kg), vinblastine sulphate (lowest effective dose 0.05 mg/kg) and 5-bromo-2-deoxyuridine (lowest effective dose 200 mg/kg) gave rise to significant increases in the frequencies of micronucleated polychromatic erythrocytes. No significant induction of micronucleated polychromatic erythrocytes by 2-chlorobenzylidene malonitrile or chloral hydrate was found. The frequencies of induced micronucleated polychromatic erythrocytes peaked at 40 h after hydroquinone treatment, at 48 h after vinblastine treatment and at 72 h after 5-bromo-2-deoxyuridine treatment with evident dose-dependent differences in the kinetics of the induction of micronucleated polychromatic erythrocytes. The mean relative Hoechst 33342 fluorescence of the populations of induced micronucleated polychromatic erythrocytes was used as an indicator of the DNA content of induced micronuclei. These values were found to be in agreement with the presumed mechanisms of micronucleus induction for hydroquinone, vinblastine sulphate and 5-bromo-2-deoxyuridine. Flow cytometric enumeration of micronucleated polychromatic erythrocytes in peripheral blood is an efficient method for the study of in vivo micronucleus induction, combining rapid analysis and high sensitivity with information on possible mechanisms of micronucleus induction. The method also allows a substantial reduction in the number of animals needed.

Quantitative and qualitative studies of micronucleus induction in mouse erythrocytes using flow cytometry. II. Analysis of micronuclei of aneugenic and clastogenic origin by dual-colour FISH on populations of bone marrow PCEs flow sorted on the basis of their relative DNA content.

Simultaneous dual-colour fluorescent in situ hybridization (FISH) with the major and minor mouse satellite probes was performed on flow-sorted fractions of micronuclei with defined Hoeschst 33342 fluorescence intensities, reflecting their relative DNA content. The purpose of the study was to investigate the relationship between probe binding and the size of micronuclei induced by a set of chemicals with different mechanisms of micronucleus induction. The agents studied were vinblastine, cyclophosphamide, hydroquinone and 5-bromo-2-deoxyuridine. The Hoechst 33342 fluorescence distributions of induced micronuclei differ markedly between cyclophosphamide, vinblastine and hydroquinone. While the distributions for cyclophosphamide and vinblastine agree well with those obtained for other model aneugens and clastogens, hydroquinone shows a distribution with characteristics similar to both clastogens and aneugens. A comparison between the Hoechst 33342 fluorescence distributions of hydroquinone-induced micronuclei positive for both major and minor probes and those positive for the major probe only indicates that the former consist of whole chromosomes, while the latter originate from chromosome fragments. The absolute frequency and Hoechst 33342 fluorescence distribution of major-only-positive micronuclei induced by vinblastine indicates that this model aneugen also shows a certain in vivo clastogenic potential. Micronuclei induced by 5-bromo-2-deoxyuridine indicate that this agent is a clastogen, preferentially inducing pericentric breaks, but may also to some extent be aneugenic.

Chromosome painting of radiation-induced micronuclei.

In this report, in situ hybridization with whole chromosome painting probes was used to paint radiation-induced micronuclei (MN) in three lymphoblastoid cells lines to investigate the frequency of radiation-induced MN. The results obtained for four different chromosomes showed that there was a significant deviation of the numbers of signal-positive MN from that expected on the basis of DNA proportionality. Restriction of the analysis to three chromosomes showed that the deviations arose primarily from chromosome 7, which was underrepresented in the numbers of signal-positive MN in the group of chromosomes studied.

Analysis of the DNA content distribution of micronuclei using flow sorting and fluorescent in situ hybridization with a centromeric DNA probe.

The DNA content distributions of micronuclei induced in mouse 3T3 cells by ionizing radiation and chemicals was measured by flow cytometry. For a quantitative understanding of these distributions, micronuclei with increasing DNA contents were sorted and analysed for the presence of centromeric signals using fluorescent in situ hybridization (FISH) with a mouse centromeric gamma satellite probe. Radiation-induced micronuclei were found to be produced mainly by chromosome fragments, whereas micronuclei induced by the tear gas chlorobenzylidene malonitrile (CS) were found to be produced mainly by whole chromatids. In contrast, micronuclei induced by vinblastine (VBL) were, according to the shape of their DNA content distributions, produced mainly by whole chromosomes and by combinations of two or more whole chromosomes. With increasing DNA content, micronuclei induced by ionizing radiation also contained one or more whole chromosomes, whereas micronuclei induced by CS or VBL were found to contain several whole chromatids or chromosomes respectively. Computerized random breakage of chromosomes and random combination of chromosome fragments, whole chromatids and whole chromosomes were used according to the FISH results to simulate the measured DNA content distributions of micronuclei. A good agreement was obtained between measured and simulated distributions of micronuclei as well as between results of the measured frequency of micronuclei showing centromeric signals as a function of their DNA content and those predicted by the simulations. These results demonstrate the usefulness of flow cytometry and sorting combined with the FISH technique and computer simulations for producing a more detailed analysis of mechanisms of micronucleus induction.

Radiation-induced structural modifications in dsDNA analysed by FT-Raman spectroscopy.

Structural changes of double stranded DNA in aqueous solution induced by ionizing radiation were studied by Fourier-Transform-Raman spectroscopy. In addition to base damage, strand breaks, structural changes, i.e. unstacking of the bases, premelting effects and disordering of the B-form backbone could be observed. The amount of these different kinds of damage depended on the concentration of the DNA solution. Specifically, the following modifications were found depending on the gamma-ray dose and DNA concentration. (1) Intensity increase of the lines of dT (1240 cm-1) and dA (729, cm-1) indicating unstacking of these bases. (2) Intensity and frequency changes of the marker bands of all four bases indicating structural modifications. (3) Intensity decrease of the sugar marker lines showing change of the bonds in the deoxyribose and of bonds between the sugar moiety and the phosphodiester. (4) Intensity decrease of the lines of the phosphodiester groups (1094 and 790 cm-1) with simultaneous appearance of a difference peak at 1080 cm-1 and a new peak at 980 cm-1 suggesting the presence of strand breaks. (5) Intensity decrease of the B-form marker band (approximately 835 cm-1) and new lines at 876 cm-1), at approximately 660 cm-1 (C3'-endo/anti of dG) and at 1312 cm-1 (C3'endo/syn of dA) indicating decrease of the B-form conformation and the developing of partly new secondary forms of the DNA representing a helix-to-coil transition.

UV-B-induced cell cycle perturbations, micronucleus induction, and modulation by caffeine in human keratinocytes.

UV-B-induced perturbations of cell cycle progression in asynchronous human keratinocytes were analysed during two cell cycles with respect to their cell cycle stage at the time of irradiation using BrdUrd/Hoechst flow cytometry. Exponentially growing SCL-2-keratinocytes exposed to UV-B radiation showed a short delay in G1-phase exit and were blocked in the S and G2/M phases of the first cell cycle. UV-A wavelengths did not show any detectable effect on cell cycle progression. In contrast, 137Cs-irradiation of these cells induced a temporary G2 block only. Micronucleus frequency increased in gamma-irradiated cells as soon as the cells started to divide and reached a plateau when most of the cells had divided. Continuous treatment with caffeine starting immediately after 137Cs gamma-irradiation prevented accumulation of cells in G2 phase, but did not influence the frequency of micronuclei. In UV-B-irradiated keratinocytes, however, the damage-induced cell cycle perturbations were merely reduced by caffeine, but not eliminated. Compared with gamma-irradiation a moderate induction of micronuclei was observed in UV-B-irradiated cells. Caffeine, however, potentiated the induction of micronuclei by UV-B. These different effects on cell cycle kinetics and micronucleus induction indicate different mechanisms of DNA damage caused by UV-B- and gamma-irradiation that may be repaired through different pathways.