BACKGROUND:Little is currently known regarding the cancer-preventive potential of cyanidin-3-O-beta-glucopyranoside (Cy-g) apart from its antioxidant activity.METHODS:We tested Cy-g on Jurkat and HL-60 leukemia cell lines and, to help elucidate whether the effects of Cy-g are specific for cancer cells, also on normal T lymphocytes.RESULTS:Cy-g induced apoptosis in all three cell systems, and this indicated that Cy-g was not selective towards leukemia cells. Moreover, Cy-g caused HL-60 differentiation. The induction of apoptosis and cytodifferentiation involved different proteins, thus suggesting that Cy-g-induced apoptosis and cytodifferentiation are two distinct events.CONCLUSIONS:Although obtained in vitro, our findings indicate that Cy-g possesses some interesting biological properties that should encourage further investigation as regards its chemotherapeutic potential.

BACKGROUND Little is known regarding the potentially chemopreventive activity of cyanidin-3-O-beta-glucopyranoside (Cy-g), the main anthocyanin present in the juice of pigmented oranges, apart from its antioxidant activity. After excluding a potential genotoxicity of Cy-g, its ability to induce apoptosis on transformed and normal T cells was analysed. In order to delineate the events leading to apoptosis, the expression of different proteins, known to be involved in apoptosis, was also measured. MATERIALS AND METHODS The evaluation of genotoxicity was performed by the micronucleus test. Flow cytometry was used for the analysis of apoptotic cells and proteins involved in the modulation of apoptosis. RESULTS Cy-g was nongenotoxic. Moreover, it induced apoptosis in both cell systems, modulated by an increase of p53 and bax proteins. CONCLUSION These interesting biological properties should encourage further studies into the chemopreventive potential of Cy-g. Nevertheless, its activity in normal T cells underlines the need for extensive toxicological investigation.

Cancer chemoprevention is a new approach in the management of cancer. Traditional cytotoxic chemotherapeutic approaches cannot cure most advanced solid malignancies. Chemoprevention can be defined as the use of non-cytotoxic drugs and natural agents to block the progression to invasive cancer. Recently, isothiocyanates, natural products found in the diet of humans, has been shown to function as cancer chemopreventive agents. They are strong inhibitors of phase I enzymes and inducers of phase II enzymes. They can also induce apoptosis and modulate cell-cycle progression of highly proliferating cancer cells. This commentary will review the mechanism of apoptosis and growth inhibition mediated by different isothiocyanates. Particular attention will be given to the effects of the new isothiocyanate 4-(methylthio)butylisothiocyanate (MTBITC). Since selective targeting and low toxicity for normal host tissues are fundamental requisites for proposed chemopreventive agents, we will also review the effects of different isothiocyanates on non-transformed human 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.

Abstract: Isothiocyanates exert chemopreventive effects against chemically induced tumors in animals, modulating enzymes required for carcinogens' activation/detoxification and/or the induction of cell‐cycle arrest and apoptosis in tumor cell lines. To investigate the chemopreventive potential of isothiocyanates, we studied proliferation, apoptosis induction and p53, bcl‐2 and bax protein expression in Jurkat T‐leukemia cells by the isothiocyanate sulforaphane. Sulforaphane caused G2/M‐phase delay and increase of apoptotic cell fraction in a time‐ and dose‐dependent manner. Necrosis was observed after prolonged exposure to elevated sulforaphane doses. Moreover, it markedly increased p53 and bax protein expression, and slightly affected bcl‐2 expression. Since selective targeting and low toxicity for normal host tissues are fundamental requisites for proposed chemopreventive agents such as sulforaphane, we tested sulforaphane on non‐transformed phytohemagglutinin‐stimulated human T‐lymphocytes. We demonstrated that sulforaphane arrested cell‐cycle progression in G1 phase by a significant down‐modulation of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53, whereas it exerted little effect on bcl‐2 and bax levels. These findings indicate that sulforaphane can exert protective effects inhibiting leukemic cell growth. Moreover, sulforaphane is active not only in transformed lymphocytes but also in their normal counterpart. Although in vitro studies do not necessarily predict in vivo outcomes, our findings raise important questions regarding the suitability of sulforaphane for cancer chemoprevention.

Glucosinolates (GL) can inhibit, retard or reverse experimental multistage carcinogenesis. When brassica plant tissue is broken, GLs are hydrolyzed by the endogenous enzyme myrosinase (Myr), releasing many products including isothiocyanates (ITC). Synthetic ITCs like sulforaphane exert chemopreventive effects against chemically induced tumors in animals, modulating enzymes required for carcinogens' activation/detoxification and/or the induction of cell-cycle arrest and apoptosis in tumor cell lines. To investigate the chemopreventive potential of ITCs while reproducing the circumstances of dietary contact with sulforaphane, we studied proliferation, apoptosis induction and p53, bcl-2 and bax protein expression in Jurkat T-leukemia cells by sulforaphane, the ITC generated in situ in a quantitative manner by Myr starting from glucoraphanin (GRA). Jurkat cells were treated with different doses of GRA-Myr mixture. Effects on cell growth or survival were evaluated by counting trypan blue-excluding cells. Cell-cycle progression, apoptosis and expression of p53, bax and bcl-2 proteins were analyzed by flow cytometry. Results were analyzed by two-sided Fisher's exact test. Sulforaphane, but not GRA, caused G(2)/M-phase arrest (P = 0.028) and increase of apoptotic cell fraction (P < 0.0001) in a time- and dose-dependent manner. Necrosis was observed after prolonged exposure to elevated sulforaphane doses. Moreover, it markedly increased p53 and bax protein expression, and slightly affected bcl-2 expression. These findings indicate that sulforaphane but not the native GL GRA can exert both protective and toxic effects inhibiting leukemic cell growth. Sulforaphane therefore deserves study as a potential chemopreventive/chemotherapeutic antileukemic agent.

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 G1 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.

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 μg/ml) caused a dose-related increase in MN, whereas TUDCA caused no significant increase (up to 1000 μg/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 μg/ml, and TUDCA, more markedly at 300–1000 μg/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.

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 (Delta = 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) sen ed as a model system. Apoptosis R as induced by the biozid Tri-n-butyltin (TBT). Dependent on the TBT concentration (0.3-10 muM), 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 muM), this [Ca2+](i) increase was nearly transient during the observation time of 15 min. Higher TBT concentrations (0.5-10 muM), 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 [Ca2+](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+], level. Cytometry 44:45-56, 2001. (C) 2001 Wiley-Liss, Inc.

Babesia ovata, which is transmitted by Haemaphysalis longicornis, is an intraerythrocytic protozoan parasite of cattle. Based on its morphology, B. ovata is classified as a large-type Babesia. The developmental stages of B. ovata have been described both in cattle and the tick vector. In infected adult female ticks, the parasite is transovarially transmitted to the tick eggs. The sexual reproduction of B. ovata has been demonstrated in the tick midgut. The diagnostic tools that are currently available for the specific detection of B. ovata in cattle include microscopy and polymerase chain reaction assays. The development of improved molecular and serological diagnostic tools has been constrained by the limited availability of genetic data. B. ovata has been reported in cattle populations in Japan, Korea, China, Mongolia and Thailand. B. ovata was thought to be a benign parasite; however, infections in immuno compromised or Theileria orientalis-infected animals are clinically significant. Thus, control strategies aimed at minimizing the prevalence of B. ovata are vital. The taxonomy of B. ovata is unclear, and the phylogenetic position has not been well defined. Consequently, non–B. ovata species have sometimes been classified as B. ovata. In this review, we provide an outline of the lifecycle, geographical distribution, and control of B. ovata, and critically discuss the taxonomy and phylogeny of this bovine Babesia.

Carcinogenesis is thought to require an initiating event that results in an irreversible genetic change in a subpopulation of cells. Apoptosis may attenuate this process by causing the deletion of genetically damaged cells. Surviving cells can contain sublethal levels of DNA damage and proliferate. This process facilitates the acquisition of mutations in the genome, possibly resulting in further insensitivity to apoptosis. For neoplastic growth to occur, an imbalance between proliferation and apoptosis must be established, such that cell growth predominates. Therefore, the analysis of genetic damage, cell proliferation and apoptosis can be used together as markers of the carcinogenic potential of xenobiotics. Cytogenetic techniques, such as the micronucleus assay, combined with flow cytometry, permit the analysis of genetic damage, cell cycle progression and apoptosis to be carried out in the same cell culture. This is possible, because only a small number of cells are required to perform such assays. Moreover, Annexin-V-Fluos/propidium iodide flow cytometry permits apoptotic cells to be distinguished from necrotic cells. This kind of approach was applied to study bile acids that have been positively connected with colon cancer incidence in humans. The results showed that ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) have genotoxic potential. Taurine conjugation in TUDCA and TDCA led to non-genotoxic compounds. All bile acids cause a delay of cell proliferation, while only DCA and TDCA increased the fraction of apoptotic cells after treatment for 4 hours. Longer exposure led to the occurrence of necrosis. Bile acids are an important group of compounds with structural and biological heterogenicity, and their study could reveal cellular and biochemical events that are important in the initiation and post-initiation stages of carcinogenesis.

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.

The common usage of flow cytometry (FCM) in research and clinical diagnostic is limited by the lack of visualising the fluorescence labelled cells. The Laser Scanning Cytometer (LSC) enables multicolour cytometric measurements on a slide featuring relocation of single cells for further investigation via brightfield and fluorescence microscopy. Additionally, it is possible to capture these images for documentation. In a FISH application, the LSC was successfully used for automated scoring techniques for evaluating the frequency of aneuploid sperm in humans and mice. In just 30 minutes, we were able to acquire more than 15,000 sperms, a task which normally takes more than a day. After relocation, genetic defects were identified and confirmed via fluorescence microscopy. In an ongoing study, we investigate via the LSC the remain of a new radiopaque material for high resolution echocardiography in the blood circulation. At first the results exhibited that the radiopaque material is endocysed by leukocytes just after application but is still detectable via echocardiography for up to 40 minutes. In conclusion, with the additional data acquisition by the LSC, it is possible to perform further detailed information from very small samples. Therefore, we are working up to now on developing new methods to introduce the LSC in our clinical diagnostic of neonates undergoing cardiac surgery.

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.

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.

Syh-Mo-Yiin (SMY), Guizhi-Fuling-Wan (GFW), Shieh-Qing-Wan (SQW) and Syh-Nih-Sann (SNS) are four prescriptions of Traditional Chinese Medicine (TCM) used in the remedy of liver trouble in various types. The hepatoprotective effects of water extracts of these four recipes against d-galactosamine (d-GalN) and carbon tetrachloride (CCl4)-induced acute hepatic damage were determined in rats. The results indicated that the serum glutamate-oxalate-transaminase (sGOT) and the serum glutamate-pyruvate-transaminase (sGPT) levels provoked by d-GalN and CCl4 decreased after treatment with these prescriptions of TCM. Histological changes around portal area (d-GalN-induced hepatotoxicity) and central vein (CCl4-induced hepatotoxicity) were simultaneously improved by the treatment with TCM mentioned above.

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.