Effect of cyanidin 3-O-beta-glucopyranoside on micronucleus induction in cultured human lymphocytes by four different mutagens

The anthocyanin cyanidin 3-O-beta-glucopyranoside (Cy-g) is reported to be one of the most effective antioxidants, but little is currently known regarding its potential chemopreventive properties. In this study, we evaluated the ability of Cy-g to protect cultured human lymphocytes from micronucleus (MN) induction by four different mutagens: ethyl methanesulfonate (EMS), colchicine (COL), H(2)O(2), and mitomycin C (MMC). To gain insight into the mechanisms of action of Cy-g, the cultures were treated with the compound before, during, and after treatment with the mutagens; in addition, the cultures were evaluated for the induction of apoptosis. When used by itself, up to 100 microg/ml of Cy-g was nongenotoxic, while 100 microg/ml Cy-g reduced the replicative index of the cells by nearly 50%. In addition, Cy-g was able to reduce the frequency of micronuclei induced by EMS, COL, and H(2)O(2) using all three treatment protocols, but it had no significant effect on MN induction by MMC in any of the protocols. Apoptosis was produced in the cultures treated with Cy-g alone and was increased under conditions in which Cy-g produced anti-genotoxic effects, suggesting that Cy-g mediated-apoptosis may remove highly damaged cells. However, increases in apoptosis were found under conditions in which Cy-g was not significantly anti-genotoxic, indicating that the increases in apoptosis were not sufficient to account for the anti-genotoxicity of Cy-g. Taken together, our findings indicate that Cy-g possesses anti-genotoxic activity in vitro, which suggests its potential use as a chemopreventive agent.     

Different effects of mutagens on sister chromatid exchange induction in three Chinese hamster cell lines

The sister chromatid exchange (SCE) induction of mutagens with different mechanisms of action was comparatively investigated on permanent cell lines of the Chinese hamster (CHO, V-79, and DON) with and without exogenous metabolic activation and with the use of various experimental protocols. CHO and V-79 cells were treated with ethylmethanesulfonate (EMS), a direct mutagen; with the two indirect mutagens cyclophosphamide (CP) and benzo[a]pyrene (BP); as well as with the radical former hydrogen peroxide (H2O2) and hydroxyurea (HU), an inhibitor of DNA synthesis. Aside from an increased basal SCE level and a higher bromodeoxyuridine (BrdUrd) sensitivity, there was no decisive difference between CHO and V-79 cells. However, there was a distinct relationship between SCE induction and the experimental protocol used, which was most pronounced after HU treatment. Neither cell line was able to metabolize the indirect mutagen BP. Only in CHO cells did CP lead to increased SCE frequencies. However, in all cases, the simultaneous application of S9 mix produced a distinct SCE induction. In contrast, BP caused SCE induction in DON cells, whereas CP was not metabolized. The reason for these findings must obviously be sought in the metabolization of CP and BP via different monooxygenase systems, whose activity can differ in these permanent cell lines. One notable finding was that the number of SCE induced by H2O2 could be distinctly reduced by the simultaneous application of S9 mix. This effect can be explained by the fact that S9 mix contains H2O2-degrading enzymes. The results indicate that closely related cell lines differ in their capability for inducing SCE and that investigations of SCE inductions performed on only one cell line do not necessarily produce a representative response.     

Syk is required for p38 activation and G2/M arrest in B cells exposed to oxidative stress

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. In this study, we have investigated the role of Syk in p38 activation and the regulation of cell-cycle progression upon oxidative stress. In B cells, p38 is activated by hydrogen peroxide (H(2)O(2)) stimulation. Syk is required for p38 activation following stimulation with 10-100 microM H(2)O(2), but not with 1 mM H(2)O(2). H(2)O(2)-induced p38 activation is abrogated in phospholipase C-gamma2 (PLC-gamma2)-deficient as well as Syk-deficient cells, suggesting that Syk activates p38 through PLC-gamma2 upon H(2)O(2) stimulation. Although stimulation with 20-100 microM H(2)O(2) induces cellular apoptosis in B cells, pretreatment with SB203580, a p38-specific inhibitor, has no effect on H(2)O(2)-induced apoptosis. Flow cytometric analysis reveals that B cells exposed to 10-20 microM H(2)O(2) exhibit cell-cycle profile of G2/M arrest, and pretreatment with SB203580 inhibits only a little H(2)O(2)-induced G2/M arrest. On the other hand, Syk-deficient cells show no induction of G2/M arrest following H(2)O(2) stimulation. These findings indicate that Syk plays a role in the regulation of cell-cycle progression in G2/M phase via p38-dependent and -independent pathways after oxidative stress.     

Cytokinesis-block micronucleus assay in primary human liver fibroblasts exposed to griseofulvin and mitomycin C

Primary liver fibroblasts were applied in a cytokinesis-block micronucleus assay in combination with fluorescence in situ hybridization (FISH) using two protocols. In protocol A (Prot. A), cytochalasin B (Cyt B) was added at the end of the treatment time directly to the medium containing the standard compounds, whereas in protocol B (Prot. B) the chemical-containing medium was removed and fresh medium with Cyt B was added. The study was performed using the aneugen griseofulvin (GF) and the clastogen mitomycin C (MMC) as standard compounds. With both protocols GF induced a significant increase in MN frequency over controls in a dose-related manner at the lower concentrations tested (7.5 and 15 microg/ml). At the highest dose (30 microg/ml) the aneugen effect was substantially reduced. MN induction obtained with Prot. A was significantly higher ( approximately 3-fold) than with Prot. B at the most effective concentration. The aneugen effect induced by GF did not change when different cell densities were used, but again with Prot. A we obtained the highest effect. MN induced by MMC showed a dose- and time-dependent increase in both protocols. In contrast to GF, the greater clastogenic response induced by MMC in human liver fibroblasts was obtained with Prot. B, approximately 3-fold higher than Prot. A at the most effective concentration and approximately 2-fold with 24 h treatment at 0.17 microg/ml MMC. With GF, the FISH data in human liver fibroblasts (80% C+MN) were fairly consistent with those obtained in the rodent cell lines. In human whole blood cultures, the same dose used in our experiment produced a relatively higher percentage of C+MN. FISH analysis showed that MMC induced mainly MN containing acentric fragments rather than whole chromosomes. In conclusion we have demostrated that chemically induced genetic effects are strongly dependent on the cell culture employed, treatment schedule and intra- and post-treatment experimental conditions.     

Ceramide path in human lung cell death

Lung epithelium plays a significant role in modulating the inflammatory response to lung injury. Airway epithelial cells are targeted by hydrogen peroxide (H(2)O(2)) and oxygen radicals, which are agents commonly produced during inflammatory processes. The mechanisms and molecular sites affected by H(2)O(2) are largely unknown but may involve the induction of sphingomyelin (SM) hydrolysis to generate ceramide, which serves as a second messenger in initiating an apoptotic response. Here we show that exposure of human airway epithelial (HAE) cells to 50 to 100 microM H(2)O(2) induces within 5 to 10 min a greater than 2-fold activation of neutral sphingomyelinase activity with concomitant SM hydrolysis, ceramide generation, and apoptosis. On the other hand, activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate inhibits both H(2)O(2)-induced ceramide production and apoptosis. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. These findings indicate that ceramide, the product of SM hydrolysis, plays an important role in H(2)O(2)-induced apoptosis in HAE cells, and that PKC counteracts ceramide-mediated apoptosis in these cells. We suggest that the mediation of epithelial cell apoptosis by ceramide and its inhibition by PKC constitute a central mechanism by which inflammatory processes are modulated in the epithelium of the lung.     

Conversion of excision-repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes.

The human lymphocyte micronucleus (MN) assay is relatively insensitive to genotoxic agents that predominantly induce excision-repairable lesions such as adducts and abasic sites. In this study we have explored the possibility of using cytosine arabinoside (ARA) to convert excision-repairable DNA lesions to micronuclei (MN) within one cell cycle. The system consisted of human lymphocytes as target cells, the cytokinesis-block (CB) method for identifying cells that had completed one nuclear division only, and X-rays, methylnitrosourea (MNU), and ultraviolet light (UV) as mutagens. With each mutagen we have observed significant increments in induced MN in the cultures that had also been treated with ARA during G1. The slope of the dose-response curves for induction of MN was increased by a factor of approximately 1.8 for X-rays and 10.3 for UV and significant MNU induction of MN was only achieved in the cultures treated with ARA. Furthermore, a 24-hr gap between mutagen exposure and the start of the assay did not abolish the increased sensitivity in the cultures treated with ARA. These observations suggested that the combined ARA and cytokinesis-block micronucleus (CBMN) method may enhance the detection of exposure to genotoxic agents that predominantly induce excision-repairable lesions.     

Chromosomal composition of micronuclei in mouse NIH 3T3 cells treated with acrylamide, extract of Tripterygium hypoglaucum (level) hutch, mitomycin C and colchicine, detected by multicolor FISH with centromeric and telomeric DNA probes

The chromosomal composition of micronuclei (MN) induced by the model mutagens mitomycin (MMC) and colchicine (COL) as well as by acrylamide (AA) and the traditional Chinese medicine Tripterygium hypoglaucum (level) hutch (THH) in NIH 3T3 cells was analyzed by multicolor fluorescence in situ hybridization (FISH) using DNA probes for the centromere repeated minor satellite DNA and the telomeric hexamer repeat (TTAGGG). The majority of MN (78.6%) from treatment with MMC (0.1 microg/ml) did not show centromeric signals, reflecting the clastogenic action of MMC. Following treatment with COL (0.1 microg/ml), 74.5% of the MN showed centromeric signals and several telomeric signals, indicating that MN induced by this well-known aneugen were mainly composed of whole chromosomes. After treatment with AA (100, 200 and 400 microg/ml) both MN containing whole chromosomes and MN containing acentric fragments were found to increase in a dose-dependent manner, demonstrating that AA is not only a clastogen but also an aneugen. THH induced a high frequency of MN harboring whole chromosomes at all concentrations tested (5, 10 and 20 microl/ml) and produced a dose-dependent increase in fragment-containing MN, indicating that THH has both aneugenic and clastogenic potential.     

Identification of kinetochores and DNA synthesis in micronuclei induced by mitomycin C and colchicine in Chinese hamster ovary cells.

The presence of kinetochore and DNA synthesis in micronuclei (MN) induced in Chinese hamster ovary (CHO) cells by clastogenic and aneuploidogenic substances such as mitomycin C (MMC) and colchicine was determined by immunofluorescence technique using CREST antikinetochore antibodies and anti-bromodeoxyuridine (BrdUrd) antibodies. A cytofluorimetric analysis was also performed. Colchicine significantly increased micronucleated cells at least up to 96 h from the end of treatment. As expected, among colchicine-induced micronucleated cells the majority contained at least one CREST + MN. MMC induced a significant increase in micronucleated cells up to 120 h from the end of treatment and the great majority of MN lacked kinetochore fluorescence, indicating that MMC-induced MN were derived from acentric fragments. However, colchicine and MMC at 48 and 72 h from the end of treatment, induced a significant increase of CREST- and CREST + MN, respectively, suggesting an induction of clastogenicity by colchicine and aneuploidy by MMC. The clastogenic effect of colchicine after 48 h was also confirmed by the presence of chromatid fragments in metaphase cells. A cytofluorimetric analysis indicated that, as expected, colchicine and MMC interfere with the G2/M and S phases, respectively; however, a slight interference of colchicine with the S phase was also observed. DNA synthesis was present in MN and it was in most cases synchronous with synthesis in the main nucleus. The frequency of cells with MN in S phase observed in untreated or MMC-treated cells is in agreement with the proportion of cells without MN showing DNA synthesis. On the contrary, the frequency of cells with MN in S phase observed in colchicine-treated cells was significantly lower than that observed in control and MMC-treated cells.     

Menadione biphasically controls JNK-linked cell death in leukemia Jurkat T cells

Signals for cell-death induction by menadione were studied in Jurkat T cells. Low concentrations of menadione (10-20 microM) and H(2)O(2) (10-50 microM) induced cell death accompanying low (menadione: <5%) or moderate (H(2)O(2): 10-15%) levels of DNA fragmentation in Jurkat cells. These concentrations of menadione (10 microM) and H(2)O(2) also caused membrane (necrotic) cell death at unproportionally high (80%) and proportional (10-30%) levels, respectively. Higher concentrations (100-5,000 microM) of H(2)O(2) exclusively induced membrane cell death. Unexpectedly, 30-300 microM menadione induced ever-decreasing levels of necrotic cell death in a concentration-dependent manner. An in vitro kinase assay showed that 20-50 microM, but not >100 microM, menadione induced activation of c-Jun NH(2)-terminal kinase (JNK), whereas a striking activation of JNK was induced by 500-5,000 microM H(2)O(2). Induction of cell death by a low concentration of menadione was partially inhibited in dominant negative JNK gene-transfected Jurkat/VPF cells. A high concentration (300 microM) of menadione was found to inhibit cell-death induction by high concentrations (200-5,000 microM) of H(2)O(2). The JNK inhibitory activity of menadione was also demonstrated in a cell-free system. However, menadione did not activate JNK in vitro. These results suggest that JNK is required for induction of not only apoptotic cell death, but also necrotic cell death in Jurkat T cells and that menadione biphasically controls this JNK-linked signal for inducing cell death.     

Genotoxic effects induced by formaldehyde in human blood and implications for the interpretation of biomonitoring studies

Formaldehyde (FA) was tested for its genotoxicity in human blood cultures. We treated blood samples at the start of the culture to follow FA-induced DNA damage (DNA-protein crosslinks, DPX), its repair and its genetic consequences in form of sister chromatid exchanges (SCE) and micronuclei (MN). Our results clearly indicate that DPX (determined by the comet assay) are induced at FA concentrations of > or =25 microM. DPX induced by FA concentrations up to 100 microM are completely removed before lymphocytes start to replicate. SCE are induced at concentrations >100 microM parallel to the induction of cytotoxicity (measured as reduction of the replication index). MN were not induced by FA concentrations up to 250 microM (the highest analyzable concentration) added at the start of the blood cultures in the cytokinesis-block micronucleus (CBMN) test. FA-induced cytotoxicity (measured as reduction of the nuclear division index) possibly prevented division of damaged cells. MN were only significantly induced in human blood when proliferating cells were exposed to FA during the last cell cycle before preparation. Several human biomonitoring studies reported increased frequencies of SCE and MN in lymphocytes of subjects exposed to FA. Our results characterize the genotoxic potential of FA in cultured lymphocytes and lead to the conclusion that cytogenetic effects of FA are very unlikely to occur in blood cultures of FA-exposed subjects.     

Analysis of micronuclei induced in mouse early spermatids by mitomycin C, vinblastine sulfate or etoposide using fluorescence in situ hybridization

Non-radioactive in situ hybridization with mouse centromerespecific (major) gamma satellite DNA probe was used to analyzethe mechanism of induction of spermatid micronuclei (MN) causedby the alkylating agent mitomycin C (MMC), the spindle poisonvinblastine sulfate (VBL) or the DNA topoisomerase II inhibitoretoposide (VP-16). Male mice were treated with a single i.p.injection of 25 mg/kg VP-16, 5 mg/kg MMC or 2 mg/kg VBL, respectively.After 24 h (VP-16, VBL) or 13 days (MMC) stage I spermatid slideswere prepared and in situ hybridization was performed usinga polymerase chain reaction amplified mouse (major) gamma satelliteDNA probe. The observed MN frequencies for VP-16 and MMC, 6.2/1000and 7.5/1000 round spermatids, respectively, show a strong mutageniceffect on mouse germ cells compared with controls (1.4/1000spermatids). VBL, on the contrary, induced a much lower totalfrequency of MN (2.8/1000 spermatids) compared with previousresults on mouse somatic cells. Of MN in controls, 24% carrieda FISH signal. After correcting for background, MMC induced38.6% signal-positive MN, consistent with a predominantly clastogenicmode of action, while VBL induced 67.9% signal-positive MN,consistent with a mainly aneugenic mechanism. VP-16 induced65.5% signal-positive MN, indicating that its MN-inducing capacityis mainly due to whole chromosome lagging.     

Age-related increase of mitomycin C-induced micronuclei in lymphocytes from Down's syndrome subjects

Peripheral blood lymphocytes from 7 patients with Down's syndrome (DS; trisomy 21) and 14 healthy age-matched controls were studied for the induction of micronuclei (MN) by the cytokinesis-block method. The spontaneous incidence of MN in lymphocytes from DS subjects was lower than that of control cultures. When lymphocytes were treated with mitomycin C (MMC) at the beginning of the culture period, an increase in MN formation was found in cells from both DS and control subjects. In DS subjects this increase was much more marked than in control donors. This effect had to be ascribed to cells from older DS subjects (37–55 years old), which showed an MMC-induced MN formation that was markedly and significantly higher than that observed in cells from younger (9–16 years old) DS subjects.These data indicate that age has to be considered a major variable when studies on the genetic instability of DS subjects are performed.     

Different effectiveness of 4-nitroquinoline-1-oxide, mitomycin C and ethyl methanesulfonate to induce lesions in DNA leading to sister chromatid exchange throughout successive cell cycles in Chinese hamster ovary cells.

The present study was carried out in Chinese hamster ovary cells in order to determine whether lesions induced by three different mutagens, namely 4-nitroquinoline-1-oxide (4-NQO), Mitomycin C (MMC) and Ethyl methanesulfonate (EMS), can persist for more than one cell generation leading to sister chromatid exchanges (SCE) or, alternatively, they are efficiently repaired during the next replicative period after treatment. In order to accurately score the number of SCEs arising during the first (S1), second (S2) and third (S3) DNA synthetic periods, third-cycle (M3) metaphases showing three-way differential (TWD) staining were analyzed. Our results show that, even though the three compounds tested were efficient in increasing the yield of SCE, the frequency of SCE was more dramatically increased after MMC treatment. Differences were also observed among the three mutagens with regard to the persistence of the lesions leading to SCE throughout successive cell generations. EMS-induced lesions appeared as more persistent than those induced by MMC. However, most of the damage induced by the UV mimetic agent 4-NQO seems to be efficiently repaired after the first round of DNA replication following treatment with the drug.     

Inhibition of rat PC12 cell calpain activity by glutathione, oxidized glutathione and nitric oxide

The expression of catalase in cells of astroglia-rich primary cultures derived from the brains of newborn rats was investigated by double-labelling immunocytochemical staining. Strong catalase immunoreactivity was found in cells positive for glial fibrillary acidic protein and galactocerebroside, cellular markers for astroglial and oligodendroglial cells, respectively. The cells of these cultures dispose of exogenously applied hydrogen peroxide (initial concentration 200 microM) quickly with first order kinetics. In contrast, after inhibition of glutathione peroxidases by mercaptosuccinate the rate of the catalase-dependent disposal of H(2)O(2) declined with time and after about 10 min the extracellular concentration of H(2)O(2) remained almost constant at a concentration of about 100 microM. Catalase activity after 10 min of incubation under these conditions was no longer detectable. In contrast, in the absence of mercaptosuccinate catalase activity was maintained during H(2)O(2) disposal. These results demonstrate that in astroglia-rich cultures catalase is strongly expressed in the predominant astroglial cells and in the minor population of oligodendroglial cells and that the enzyme is rapidly inactivated during the disposal of H(2)O(2), if the glutathione system of the cells is compromised.     

In vitro exposure of acute promyelocytic leukemia cells to arsenic trioxide (As2O3) induces the solitary expression of CD66c (NCA-50/90), a member of the CEA family

Arsenic trioxide (As2O3) is a useful drug for the treatment of acute promyelocytic leukemia (APL), acting through a complex mechanism involving the induction of apoptosis. We investigated by flow cytometry whether in vitro treatment of APL leukemic cells with As2O3 determined specific surface membrane changes. Twelve APL bone marrow aspirates were analyzed following 7 days of in vitro treatment with As2O3 (0.25, 0.5 and 2.5 microM) with regard to the expression of a series of differentiation antigens. Twelve acute myeloid leukemia (AML) samples of non-APL morphotype were analyzed as controls. Exposure of APL as well as non-APL samples to any concentration of As2O3 did not affect the expression of beta2 integrins (CD11a and CD11b), CD45 isoforms (RA, RB and R0), CD44/H-CAM, CD33 and the CEA-related antigen family members CD66ade and CD66b, thus failing to disclose any maturating effect. Of interest, in all APL samples (but not in AML) every tested dose of As2O3 determined a dramatic upregulation of CD66c display; intermediate concentration (0.5 microM) of As2O3 increased the median percentage of CD66c+ cells from 5% in control cultures (25th-75th percentile 2-12%) to 80% in drug-exposed cultures (25th-75th percentile 58-90%) (P<0.001). The induction of solitary expression of CD66c is a new finding which demonstrates As2O3 capability of generating phenotypic changes absolutely restricted to APL cells Moreover, these results provide experimental basis for considering the involvement of the newly described CD66 signalling pathway in As2O3-driven programmed cell death.     

Surface antigens of human embryonic stem cells: changes upon differentiation in culture*

We have analysed the surface antigen phenotype of a human embryonic stem (hES) cell line (H7) and the changes that occur upon differentiation induced by retinoic acid, hexamethylene bisacetamide and dimethylsulphoxide. The undifferentiated stem cells expressed Stage Specific Embryonic Antigen‐3 (SSEA3), SSEA4, TRA‐1‐60, and TRA‐1‐8 but not SSEA1. In these characteristics they closely resemble human embryonal carcinoma (EC) cells derived from testicular teratocarcinomas, and are distinct from murine EC and ES cells. The undifferentiated cells also expressed the liver/bone/kidney isozyme of alkaline phosphatase detected by antibody TRA‐2‐54, the class 1 major histocompatability antigens, HLA‐ABC, and the human Thy1 antigen. Differentiation of hES cells was induced by retinoic acid, HMBA and DMSO with the appearance of various cell types including neurons and muscle cells. The surface antigens characteristically expressed by hES cells were down‐regulated following induction of differentiation and other antigens appeared, notably several ganglioside glycolipids detected by antibodies VIN‐IS‐56 (GD3 and GD2), VIN‐2PB‐22 (GD2), A2B5 (GT3) and ME311 (9‐O‐acetyl‐GD3). Whereas the expression of HLA was slightly down‐regulated upon differentiation, its expression was strongly induced by interferon‐γ in both the undifferentiated and the differentiated cells, although the induction in the differentiated cultures was considerably stronger than in the stem cells. In all of these features the human ES cells, and their pattern of differentiation, resembled the pluripotent human EC cell line NTERA‐2 although clearly the range of cells generated by the hES cells was considerably greater.     

Three structurally homologous isothiocyanates exert “Janus” characteristics in human HepG2 cells

In this study, we used the single cell gel electrophoresis (SCGE) assay and the micronucleus (MN) test to investigate the DNA damaging effects and the antigenotoxic potencies of three structurally related ITCs in human HepG2 cells. The results show that all three ITCs possess the characteristic of a “Janus” compound, i.e., they exert both significant genotoxicity and antigenotoxicity, depending on the concentrations used in the test systems applied. Regression line analysis of the results derived by SCGE analysis showed genotoxic potency of the ITCs in the following order: 3‐methylthiopropyl ITC (MTPITC) > 4‐methylthiobutyl ITC (MTBITC) > 5‐methylthiopentyl ITC (MTPeITC); however, this order in genotoxic potency was not confirmed by MN analysis. Additionally, the MN test showed significant mutagenicity of the test substances at higher concentrations when compared with the SCGE assay. Twenty‐four hour‐treatment of the cells with the ITCs, followed by a 1‐hr recovery period, showed significant DNA repair in the SCGE assay at a concentration ≥10 μM MTPITC, ≥3 μM MTBITC, and ≥0.1 μM MTPeITC, respectively. In antigenotoxicity studies, the most effective concentration of MTPITC and MTPeITC toward B(a)P‐induced DNA damage was 0.1 μM in both test systems. MTBITC suppressed MN formation in B(a)P‐treated cells to the background level at a concentration of 1 μM. The ambivalent character of the ITCs under studymust be further clarified, especially in the possiblecontext of high dose therapeutic applications. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

The response of cells from patients with Huntington's chorea to mutagen-induced chromosome damage

Cultured blood lymphocytes from 15 patients with Huntington's chorea (HC) and matched controls were exposed to a series of graded doses of mitomycin C and ethyl methane sulphonate and examined for the incidence of sister chromatid exchange (SCE). The spontaneous SCE levels did not differ between HC patients and controls and although cells from the majority of HC patients showed a slightly enhanced response to SCE induction by the mutagens, the enhancement was small and significant only on the pooled data. Cultures from 4 HC patients and controls were exposed to a graded series of X-ray exposures and no difference was observed in the spontaneous aberration frequencies between HC cells and controls, or in their response to aberration induction by X-rays. Skin fibroblast cultures derived from three HC patients, two xeroderma pigmentosum patients and two healthy controls were exposed to MMC and the levels of unscheduled DNA synthesis determined. There was no difference between the response of HC cells and normal controls, although such synthesis in the xeroderma cells was severely depressed. It is concluded that: (i) fibroblasts and lymphocytes from HC patients show a normal response to the three mutagens studied; (ii) there is no evidence for any defect in processes involved in repairing the lesions induced; (iii) the slightly elevated response of HC lymphocytes to SCE induction may reflect the presence of a different proportion of a slightly more sensitive T cell sub-set in HC patients, and (iv) HC cells do not show a hypersensitivity to mutagens that could be used as a basis for diagnosis.     

Overexpression of regucalcin suppresses apoptotic cell death in the cloned rat hepatoma H4-II-E cells induced by a naturally occurring isothiocyanate sulforaphane

The effect of regucalcin, a regulatory protein in intracellular signaling system, on cell death and apoptosis was investigated. Sulforaphane, a naturally occurring isothiocyanate, is known to induce cell cycle arrest and apoptosis in cancer cells, although its effect has not been clarified in the cloned rat hepatoma H4-II-E cells. Hepatoma H4-II-E cells (wild-type) and stable regucalcin/pCXN2-transfected cells were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS). Cells with subconfluency were changed to a medium containing either vehicle or sulforaphane (10(-7) or 10(-6) M) in the absence of FBS. After medium change, cells were cultured for 24, 48, or 72 h. The number of wild-type cells was significantly decreased in the presence of sulforaphane (10(-7) or 10(-6) M). Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with sulforaphane (10(-7) or 10(-6) M) for 24 h. Sulforaphane (10(-7) or 10(-6) M)-induced cell number and DNA fragmentation was significantly suppressed in transfectants. The effect of sulforaphane (10(-6) M) in decreasing the number of wild-type cells was significantly prevented in the presence of caspase-3 inhibitor (10(-9) M), while the presence of Nomega-nitro-L-arginine methylester (NAME), an inhibitor of nitric oxide synthase, did not prevent sulforaphane-induced death of wild-type cells. Sulforaphane (10(-6) M) did not have a significant effect on cell number of transfectants in the presence of caspase-3 inhibitor or NAME. This study demonstrates that sulforaphane induces cell death and apoptosis in the cloned rat hepatoma H4-II-E cells, and that overexpression of regucalcin suppresses sulforaphane-induced apoptotic cell death which is partly mediated through caspase-3..     

Micronuclei and chromatid buds are the result of related genotoxic events

Chromatin buds (CHB), broken eggs, or budding cell nuclei are structures similar to micronuclei (MN) in shape, structure, and size, which are linked to the main nuclei of cells by a thread or stalks of chromatin. They have been observed in numerous cell types and there are reports of their existence relating them with MN or with genotoxic events. However, there is no systematic study reporting their frequency and no experiment has been done to ascertain whether they are really induced by genotoxins. Furthermore, they have been discarded as genotoxic events with the argument that they are not formed in dividing cells. Studies are presented here that indicate that CHB can be considered as genotoxic events and that their origin is comparable to that of MN. Bromodeoxyuridine (BrdU) was used to label proliferating lymphocytes, which were later identified by means of an immunohistochemical method, using the H2O2-DAB stain. The results show that CHB are consistently formed where MN are seen. CHB were induced by the clastogen mitomycin C (MMC) as well as by the aneuploidogen colcemid, with frequencies similar to MN in both cases, and to multinucleated cells in the case of colcemid. CHB occur in lymphocytes of smokers with frequencies similar to those of MN, and we found that the infection with Taenia solium metacestodes induced a comparable increase of both MN and CHB frequency in lymphocytes from pigs.