Sequestration of mammalian Rad51-recombination protein into micronuclei.

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after gamma irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.

Vaccinia virus DNA replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei.

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ~45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [(3)H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

Gamma-irradiation-induced micronuclei from mouse hepatoma cells accumulate high levels of the tumor suppressor protein p53.

The p53 tumor suppressor protein plays an important role in the G1 arrest of cells treated with DNA-damaging agents. Mouse hepatoma cells with wild-type or mutated p53 genotype were gamma-irradiated and the time course of p53 expression was determined by immunocytochemical staining. In p53 wild-type cells, gamma-irradiation led to a transient accumulation of the protein in the nuclei, whereas no such accumulation occurred in p53-mutated cells. Micronuclei were induced by gamma-irradiation in both wild-type and mutated cells in a dose- and time-dependent manner, but only micronuclei from p53 wild-type cells demonstrated a strongly positive staining reaction for p53 protein. This accumulation of p53 protein in micronuclei was not associated with a block in DNA synthesis as evidenced by bromodeoxyuridine labeling experiments.

Oxidative metabolism modulates signal transduction and micronucleus formation in bystander cells from alpha-particle-irradiated normal human fibroblast cultures.

The role of oxidative metabolism in the up-regulation/activation of stress-induciblesignaling pathways as well as induction of micronucleus formation in bystander cells was investigated. By immunoblotting and in situ immunofluorescence, active Cu-Zn superoxide dismutase (SOD) enzyme and active catalase enzyme were shown to inhibit the up-regulation of p21(Waf1) as well as the induction of micronucleus formation in bystander cells from confluent cultures of normal human diploid fibroblasts irradiated with 0.3-3 cGy of alpha-particles. Enzyme activity assays indicated that exogenous SOD became significantly associated with the cells. Reactive oxygen species apparently derived from a flavin-containing oxidase enzyme [presumably an NAD(P)H-oxidase] appeared to be major contributors to the bystander-induced up-regulation of p53 and p21(Waf1) as well as micronucleus formation, as evidenced by the inhibition of these effects with diphenyliodonium. Rapid activation of nuclear factor kappaB, Raf-1, extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase and their downstream effectors activator protein 1, ELK-1, p90RSK, and activating transcription factor 2 was also observed in cultures exposed to very low fluences of alpha-particles. Significant attenuation in the activation of these kinases and transcription factors occurred in irradiated cultures treated with either SOD or catalase. Overall, these results support the hypothesis that superoxide and hydrogen peroxide produced by flavin-containing oxidase enzymes mediate the activation of several stress-inducible signaling pathways as well as micronucleus formation in bystander cells from cultures of human cells exposed to low fluences of alpha-particles.

Role of wild type p53 in the G2 phase: regulation of the gamma-irradiation-induced delay and DNA repair.

Upregulation of the p53 protein was shown to induce cell cycle arrest at the G1/S border and in some cases at the G2/M border. Furthermore, it was suggested that p53 is associated with the induction of the various DNA repair pathways. Previously, we demonstrated that cells co-expressing endogenous wild type p53 protein, together with dominant negative mutant p53, exhibit deregulation of apoptosis, G1 arrest and delay in G2 following gamma-irradiation. In the present study, we investigated the role of p53 protein in the DNA damage response at the G2 phase. Using p53-null, wild type p53 and mutant p53-producer cell lines, we found that the two C-terminally spliced p53 forms could prevent gamma-irradiation induced mutagenesis prior to mitosis, at the G2/M checkpoint. We found that at the G2 phase, p53 may facilitate repair of DNA breaks giving rise to micronuclei, and regulate the exit from the G2 checkpoint. At the G1 phase, only the regularly spliced form of p53 caused growth arrest. In contrast, both the regularly and the alternatively spliced p53 forms directed postmitotic micronucleated cells towards apoptosis. These results provide a functional explanation for the cell cycle-independent expression of p53 in normal cycling cells, as well as in cells where p53 is up-regulated, following DNA damage.

Epstein-Barr virus latent membrane protein 1 induces micronucleus formation, represses DNA repair and enhances sensitivity to DNA-damaging agents in human epithelial cells.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a viral oncogene and it is essential for the transformation of resting B cells by the virus. The protein acts as a ligand-less membrane receptor and triggers numerous cellular signaling pathways. Cellular transformation frequently has been associated with genomic instability. To investigate whether EBV LMP1 induces chromosomal aberrations, micronucleus (MN) formation was examined in LMP1-expressing epithelial cells. The expression of wild-type LMP1 enhanced both spontaneous and bleomycin-induced MN formation. MN formation may be induced by inactivation of DNA repair and, therefore, we investigated the effect of LMP1 on DNA repair, using a host cell reactivation (HCR) assay. In the HCR assay, LMP1 reduced the capacity for DNA repair of both NPC-TW01 (p53-wild-type) and H1299 (p53-deficient) cells. As reduction of DNA repair by LMP1 occurs in p53-wild-type and p53-deficient cells, it seems that LMP1 can repress DNA repair in a p53-independent manner. Inactivation of DNA repair may render cells sensitive to DNA-damaging agents. In this study, H1299 cells harboring LMP1 were shown to be more sensitive to UV and bleomycin than those with a vector control. Using various deletion mutants of EBV LMP1 to determine the regions of LMP1 required to enhance MN formation, inhibit DNA repair and sensitize cells to DNA-damaging agents, we found that the region a. a. 189-222 (located within the CTAR1 domain) was responsible for sensitizing cells to UV and bleomycin, as well as for enhancing MN formation and repressing DNA repair. Based on these results, we suggest that disruption of DNA repair by LMP-1 results in an accumulation of unrepaired DNA and consequent genomic instability, which may contribute to the oncogenesis of LMP1 in human epithelial cells.

Mitotic aberration coupled with centrosome amplification is induced by hepatitis B virus X oncoprotein via the Ras-mitogen-activated protein/extracellular signal-regulated kinase-mitogen-activated protein pathway.

Multinucleated cells have been noted in pathophysiological states of the liver including infection with hepatitis B virus (HBV), the status of which is also closely associated with genomic instability in liver cancer. Here, we showed that hepatitis B virus X oncoprotein (HBx) expression in Chang cells results in a multinuclear phenotype and an abnormal number of centrosomes (n >or=3). Regulation of centrosome duplication in HBx-expressing ChangX-34 cells was defective and uncoupled from the cell cycle. HBx induced amplification of centrosomes, multipolar spindle formation, and chromosomal missegregation during mitosis and subsequently increased the generation of multinucleated cells and micronuclei formation. Treatment with PD98059, a mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2 inhibitor, significantly reduced the number of cells with hyperamplified centrosomes and decreased the multinucleated cells and micronuclei formation. Consistently, the phospho-ERK level during cell progression was substantially higher in ChangX-34 cells than that of Chang cells. In contrast, neither wortmannin, an inhibitor of phosphoinositide-3 kinase, nor SB203589, an inhibitor of p38 mitogen-activated protein kinase (MAPK), showed any effects. Introduction of Ras dominant-negative (D/N) and MEK2 D/N genes into ChangX-34 cells significantly alleviated centrosome amplification, whereas introduction of the PKC D/N and PKB D/N genes did not. Thus, our results demonstrate that the HBx induced centrosome hyperamplification and mitotic aberration by activation of the Ras-MEK-MAPK. Intervention of this signaling pathway could suppress the centrosome amplification as well as mitotic aberration. These findings may provide a possible mechanism by which HBx promotes phenotypic progression by predisposing chromosomal alteration in HBV-infected liver.

Oxidative damage and cytogenetic analysis in leukocytes of Parkinson's disease patients.

BACKGROUND: Postmortem studies suggest excessive free radical toxicity in the substantia nigra of patients with PD. Increased lipid peroxidation and oxidative DNA damage have been reported in the CNS. Markers of oxidative stress have been identified in the blood of patients with PD. OBJECTIVE: To assess the presence of spontaneous chromosome and primary or oxidative DNA damage in peripheral blood leukocytes of patients with untreated PD. METHODS: Patients with de novo PD (20) and control subjects (16), matched for age, sex, and smoking habits, underwent cytogenetic analysis using the human lymphocyte micronucleus assay coupled with the fluorescence in situ hybridization technique and the Comet assay. RESULTS: Compared with controls, patients with PD showed an increase in the incidence of spontaneous micronuclei (p < 0.001); single strand breaks (p < 0.001); and oxidized purine bases (p < 0.05). Fluorescence in situ hybridization analysis showed micronuclei harboring acentric fragments. CONCLUSIONS: There is chromosomal, primary DNA damage and oxidative DNA damage demonstrable in lymphocytes of patients with untreated PD.

Lipid peroxidation status, somatic mutations and micronuclei in peripheral lymphocytes: a case observation on a possible interrelationship.

A controlled dietary study was conducted in healthy female volunteers and reported elsewhere [1]. In a subset of samples four different biomarkers were analyzed: plasma malondialdehyde (MDA) levels and urinary 8-isoprostaglandin-F(2alpha) were measured as markers for lipid peroxidation. The frequency of hprt (hypoxanthine guanine phosphoribosyl transferase) mutants and micronuclei in peripheral blood lymphocytes were analyzed as indicators of genotoxic effects. One of the ten individuals showed extremely high background levels in all of the four endpoints measured. This case observation raises the possibility that life style factors and dietary habits affect the level of DNA reactive lipid peroxidation products, which in turn increase mutagenic and cytogenetic effects. A possible association between these biomarkers, particularly in relation to dietary fat intake and antioxidant status, should now be studied in a larger trial.

Apoptosis and appearance of Trp53-positive micronuclei in murine tumors with different radioresponses in vivo.

The purpose of this paper is to determine the relationship between the response to radiation and the appearance of apoptosis and micronuclei with Trp53 protein in murine tumors after irradiation. Two murine tumors, EL4, which was derived from a mouse lymphoma, and FM3A, which was derived from a mouse mammary carcinoma, were locally irradiated with 15 Gy and sections were stained with H&E and an anti-Trp53 antibody. The response to radiation was greater in EL4 tumors than in FM3A tumors. The frequency of apoptotic cells in EL4 tumors was 6.1 +/- 1.2% at time zero, reached a peak of 36.3 +/- 3. 8% at 6 h, and then decreased with time through 72 h to 2.5 +/- 1.5% after 15 Gy irradiation. In FM3A tumors, no apoptotic cells were detected at 0, 1, 3, 6 or 24 h after exposure. At 48 and 72 h, the frequency was only 3.0 +/- 0.6% and 1.3 +/- 0.3%. Apoptotic cells increased significantly at 3, 6 and 24 h after irradiation in EL4 tumors (P < 0.008) and at 48 and 72 h in FM3A tumors (P < 0.006). The frequency of Trp53-positive cells was 17.9 +/- 2.2 and 15.2 +/- 2.3% at time zero in EL4 and FM3A tumors, respectively, increased to 74.5 +/- 4.5% in EL4 cells (P = 0.001), and increased to 33.9 +/- 1. 1% in FM3A cells (P = 0.005) 1 h after irradiation. Trp53-positive micronuclei appeared in cells in both tumors from 24 to 72 h after irradiation. The frequency of Trp53-positive micronuclei was 3.8 +/- 0.5 and 13.5 +/- 1.3% at 24 h in EL4 and FM3A tumors, respectively, and gradually decreased by 72 h. After exposure to 15 Gy, Trp53-positive micronuclei increased significantly in FM3A tumors compared to EL4 tumors at both 24 and 48 h (P < 0.02). The frequency of these micronuclei increased with increasing dose in FM3A tumors, and the difference between these percentages after 3 Gy and after 5, 10 and 15 Gy was significant (P < 0.02). Many apoptotic cells were observed in the radiosensitive EL4 tumor after irradiation. Death by apoptosis may be related to an early response to radiation in these tumors. The appearance of micronuclei may be an important mechanism of cell death in FM3A tumors in which no apoptosis was induced.

Inhibition of cytochrome P450 2E1 decreases, but does not eliminate, genotoxicity mediated by 1,3-butadiene.

1,3-Butadiene (BD), a rodent carcinogen, is metabolized to mutagenic and DNA-reactive epoxides. In vitro data suggest that this oxidation is mediated by cytochrome P450 2E1 (CYP2E1). In this study, we tested the hypothesis that oxidation of BD by CYP2E1 is required for genotoxicity to occur. Inhalation exposures were conducted with B6C3F1 mice using a closed-chamber technique, and the maximum rate of butadiene oxidation was estimated. The total amount of butadiene metabolized was then correlated with the frequency of micronuclei (MN). Three treatment groups were used: (1) mice with no pretreatment; (2) mice pretreated with 1,2-trans-dichloroethylene (DCE), a specific CYP2E1 inhibitor; and (3) mice pretreated with 1-aminobenzotriazole (ABT), an irreversible inhibitor of cytochromes P450. Mice in all 3 groups were exposed to an initial BD concentration of 1100 ppm, and the decline in concentration of BD in the inhalation chamber with time, due to uptake and metabolism of BD, was monitored using gas chromatography. A physiologically based pharmacokinetic model was used to analyze the gas uptake data, estimate V(max) for BD oxidation, and compute the total amount of BD metabolized. Model simulations of the gas uptake data predicted the maximum rate of BD oxidation would be reduced by 60% and 100% for the DCE- and ABT-pretreated groups, respectively. Bone marrow was harvested 24 h after the onset of the inhalation exposure and analyzed for frequency of micronuclei in polychromatic erythrocytes (MN-PCE). The frequency of MN-PCE per 1000 PCE in mice exposed to BD was 28.2 +/- 3.1, 19.8 +/- 2.5, and 12.3 +/- 1.9, for the mice with no pretreatment, DCE-pretreated mice and ABT-pretreated mice, respectively. Although inhibition of CYP2E1 decreased BD-mediated genotoxicity, it did not completely eliminate genotoxic effects. These data suggest that other P450 isoforms may contribute significantly to the metabolic activation of BD and resultant genotoxicity.

No evidence of increased chromosomal aberrations and micronuclei in lymphocytes from nonfamilial thyroid cancer patients prior to radiotherapy.

The relationship between the presence of high frequencies of chromosomal aberrations in peripheral lymphocytes and predisposition to cancer has been suggested for some cancer diseases. In nonfamilial thyroid cancer, the few reports available are equivocal. The aim of this study was to assess the possible chromosomal instability in peripheral blood lymphocytes from 22 patients suffering from nonfamilial thyroid cancer. For this purpose, 2 classic cytogenetic assays, the chromosomal aberrations assay and cytokinesis-blocked micronucleus assay, were chosen. The frequency of chromosomal aberrations excluding gaps (%) was 1.68 +/- 1.39 (mean value +/- SD) for the patients group versus 2.20 +/- 1.87 for the control group. The frequency of binucleated lymphocytes with micronuclei ( per thousand) was 5.41 +/- 3.51 (mean value +/- SD) for the patients group versus 5.37 +/- 3.21 for the control group. The results obtained revealed no significant differences between both groups. The present study reinforces the idea that constitutional chromosomal instability in peripheral blood lymphocytes is not visible in nonfamilial thyroid carcinomas.

Detection of micronuclei formation and nuclear anomalies in regenerative nodules of human cirrhotic livers and relationship to hepatocellular carcinoma.

Human cirrhosis is considered an important factor in hepatocarcinogenesis. The lack of substantial genetics and cytogenetics data in human cirrhosis led us to investigate spontaneous micronuclei formation, as an indicator of chromosomal damage. The analysis was performed in hepatocytes of regenerative, macroregenerative, and tumoral nodules from 30 cases of cirrhosis (paraffin-embedded archival material), retrospectively selected: cryptogenic, hepatitis C virus, and hepatitis C virus associated with hepatocellular carcinoma (HCC). Thirteen control liver samples of healthy organ donors were included. Micronucleated hepatocytes were analyzed with Feulgen-fast-green dyeing techniques. The spontaneous frequency of micronucleated hepatocytes in both regenerative and macroregenerative nodules of all cirrhotic patients was significantly higher than for the normal control group. There was no significant difference in frequency of micronucleated hepatocytes in regenerative nodules compared with macroregenerative nodules for all cases analyzed, whereas a significantly higher frequency of micronucleated hepatocytes was detected in tumoral nodules, compared with cirrhotic regenerative nodules and normal parenchyma. A higher frequency of the nuclear anomalies termed broken-eggs was observed in hepatitis C virus-related samples. Chromatinic losses and genotoxicity already existed in the cirrhotic regenerative nodules, which might predispose to development of HCC.

Induction of micronuclei and sister chromatid exchanges by polycyclic and N-heterocyclic aromatic hydrocarbons in cultured human lymphocytes.

Many natural environments are contaminated with carcinogenic polycyclic aromatic hydrocarbons (PAHs) and N-heterocyclic aromatic hydrocarbons (NHAs) as complex mixtures of coal tar, petroleum, and shale oil. These potentially hazardous substances are prevalent at many former tar production and coal gasification sites. Three polycyclic [benzo(a)pyrene (BaP), benz(a)anthracene (BAA), and 7,12-dimethylbenz(a)anthracene (DMBA)] and two N-heterocyclic [7H-dibenzo(c,g)carbazole (DBC), and dibenz(a,j)acridine (DBA)] aromatic hydrocarbons were analyzed for cytotoxic and genotoxic effects on human lymphocytes. All of these polyaromatic compounds are normally present in the environment, except for DMBA. Lymphocytes from healthy donors were isolated from whole blood. The 5-ring polycyclic aromatic BaP consistently induced micronuclei in a linear dose-dependent manner with doses from 0.1-10.0 micrograms/ml, whereas the 4-ring compounds (BAA and DMBA) had no effect on the induction of micronuclei above controls except at 5 and 10 micrograms/ml. Of the two N-heterocyclic compounds, DBC produced a significant increase in micronuclei in lymphocytes, but the dose response tended to plateau above 0.1 microgram/ml. DBA showed an effect on the frequency of micronuclei above controls only at high doses of 5 and 10 micrograms/ml. The average background frequency of micronuclei for 7 lymphocyte donors averaged 3.1 per 1,000 stimulated cells, whereas the average frequency of micronuclei at 10 micrograms/ml BaP was 36.8 per 1,000 stimulated cells. The lowest effective dose in 2 donors for BaP occurred at 0.1 microgram/ml. At a challenge dose of 1 microgram/ml (4 microM) of BaP, considerable variation in micronuclei induction between 7 individuals was observed, ranging from 2-6-fold increases above spontaneous frequency. Over a dose range of 1-10.0 micrograms/ml (4-40 microM), BaP also induced sister chromatid exchanges (SCEs) in lymphocytes, whereas BAA had no effect above controls. Parallel studies of both cytogenetic endpoints showed that the micronucleus assay is a more sensitive indicator of BaP exposure at equivalent doses. Mitotic and replication indices of BaP-exposed lymphocytes showed that cell proliferation is only moderately inhibited even at the highest dose; this shows that bulky DNA-adducts are generally compatible with cell survival. The cytogenetic data are consistent, first-off, with reports that individuals in the population vary widely with respect to the inducibility of the CYP1A1 gene, which is known to be involved in polycyclic aromatic hydrocarbon metabolism, in particular, in BaP. Secondly, the data support the fact that polyaromatic compounds differ with regard to micronucleus induction within the same sample(s) of human lymphocytes, indicating selective metabolism of polyaromatic compounds that may reflect carcinogen sensitivity of the individual.(ABSTRACT TRUNCATED AT 400 WORDS)

Photodynamic effects of two water soluble porphyrins evaluated on human malignant melanoma cells in vitro.

Two water soluble porphyrins: meso-tetra-4-N-methylpyridyl-porphyrin iodide (P1) and 5,10-di-(4-acetamidophenyl)-15,20-di-(4-N-methylpyridyl) porphyrin (P2) were synthesised and evaluated in respect to their photochemical and photophysical properties as well as biological activity. Cytotoxic and phototoxic effects were evaluated in human malignant melanoma Me45 line using clonogenic assay, cytological study of micronuclei, apoptosis and necrosis frequency and inhibition of growth of megacolonies. Both porphyrins were characterised by high UV and low visible light absorptions. Dark toxicity measured on the basis of the clonogenic assay and inhibition of megacolony growth area indicated that P1 was non-toxic at concentrations up to 50 microg/ml (42.14 microM) and P2 at concentrations up to 20 microg/ml (16.86 microM). The photodynamic effect induced by red light above 630 nm indicated that both porphyrins were able to inhibit growth of melanoma megacolonies at non-toxic concentrations. Cytologic examination showed that the predominant mode of cell death was necrosis.

Exfoliative cytology of normal buccal mucosa to predict the relative risk of cancer in the upper aerodigestive tract using the MN-assay.

The high frequency of second or third primary tumors was first explained by Slaughter et al. with the concept of field cancerisation. Another theory postulates micrometastatic lesions as a reason for this phenomenon. The micronuclei (MN)-assay was evaluated to provide evidence for the concept of field cancerisation and to quantify the premalignant field change of normal mucosa in order to predict the individual cancer risk. MN-assay was carried out in 55 patients with squamous cell carcinoma of the head and neck, in 16 patients with a leucoplakia and in 99 healthy controls. A detailed questionnaire for population monitoring was completed. Buccal cytosmears of healthy mucosa of the study participants were examined for the MN count per 1000 cells. There was a direct correlation between tobacco abuse and increasing MN count as a sign of a cytogenetic damage of buccal mucosa cells. Alcohol did not influence the formation of MN. Both buccal sites were damaged in the same degree as proof of field cancerisation. The relative cancer risk (odds ratio) for smoking healthy controls with a definite MN frequency was estimated. Our study underscores the importance of the MN-assay as a biomarker to predict the relative cancer risk in the upper aerodigestive tract under suspicion of the individual susceptibility and the exposition to known carcinogenic agents such as tobacco and alcohol. The concept of field cancerisation was confirmed.

Induction of micronucleated erythrocytes in mouse peripheral blood after cutaneous application of 5-fluorouracil.

BACKGROUND: For topically applied drugs such as 5-fluorouracil (5-FU), dosage is not as precise as for other drug administration pathways. Consequently, quantity of drug delivered may differ among individuals and applications. 5-FU is used in treatment of different diseases and has been reported as a clastogenic compound by micronucleus assay. METHODS: To determine whether 5-FU cream (5% 5-FU) absorbed through skin can produce genotoxic or cytotoxic effect in mouse bone marrow, induction of micronucleated erythrocytes (MNE) in mouse peripheral blood was examined after cutaneous application of 5-FU daily for 5 days. RESULTS: 5-FU cream induced significant micronuclei at doses of 37.5 mg (total weight of cream)/2 cm(2) and 75.0 mg/2 cm(2), as well as cytotoxic effects at doses of 150.0 and 300.0 mg/2 cm(2). CONCLUSIONS: Cutaneous application of 5-FU increased number of MNE in mouse peripheral blood. These data emphasize the importance of using correct dose when applying drugs topically.

Micronuclei, CREST-positive micronuclei and cell inactivation induced in Chinese hamster cells by radiation with different quality.

PURPOSE: To study the relative biological effectiveness-linear energy transfer (RBE-LET) relationship for micronuclei (MN) and cell inactivation, in Chinese hamster cells irradiated with low-energy protons (0.88 and 5.04 MeV, at the cell entrance surface). Chromosome loss was also investigated by means of antikinetochore CREST staining. MATERIALS AND METHODS: Cl-1 cells were exposed to different doses of X-rays, gamma-rays, 7.7 keV/microm and 27.6 keV/microm protons. The induction of MN, the distribution of MN per cell and the frequency of CREST-positive MN were evaluated in cytokinesis-blocked binucleated cells (BN cells) in the dose range 0.125-3 Gy. In parallel, cell survival experiments were carried out in samples irradiated with 0.5 to 4 Gy. RESULTS: MN yield and the frequency of BN cells carrying multiple MN (> or =2) were significantly higher after exposure to 27.6 keV/microm protons, compared with the other radiation types. In contrast, MN induction and MN distribution per BN cell were similar among 7.7 keV/microm protons, X- and gamma-rays up to 1 Gy. Cell survival experiments gave RBE values very close to those obtained with the MN assay. Both X-rays and 27.6 keV/microm protons yielded a significant proportion of CREST-positive MN at the highest doses investigated (0.75-3 Gy). CONCLUSIONS: Good correlations between MN induction and cell inactivation were observed for both low- and high-LET radiation, indicating that the MN assay can be a useful tool to predict cell sensitivity to densely ionizing radiation with implications for tumour therapy with protons.

Iron, alpha-tocopherol, oxidative damage and micronucleus formation in rat splenocytes.

The influence of low and high alpha-tocopherol diets in concert with a high polyunsaturated fat content and a modest increase in dietary iron has been studied. Iron supplementation at 5 times the recommended dietary level was not associated with any increased sensitivity of splenocytes to any of several oxidative challenges ex vivo. Despite the significantly higher alpha-tocopherol concentrations in plasma and liver in animals supplemented with this vitamin, there was no apparent protection against oxidative genotoxicity as judged by the formation of micronuclei in splenocytes subjected to oxidative stress ex vivo. These results add to the accumulating evidence that vitamin E supplementation has little effect against oxidative genomic damage, at least as demonstrated by an increase in micronucleus frequency.

Effects of cetirizine dihydrochloride on human lymphocytes in vitro: micronucleus induction. Evaluation of clastogenic and aneugenic potential using CREST and FISH assays.

Cetirizine dihydrocloride, a widely administered antiallergic drug with the amine piperazine in its molecule, was studied as to its ability to cause micronucleus formation in human lymphocyte cultures treated in vitro. Peripheral lymphocytes from four different donors were cultured and treated with different concentrations of the compound. Cetirizine dihydrocloride was shown to induce enhanced micronucleus frequency in a dose-dependent manner, although lymphocytes from the different donors showed different susceptibilities to the compound. The content of induced micronuclei was investigated in one of the four donors by two independent assays, CREST (the application of antikinetochore antibodies) and FISH (fluorescence in situ hybridization) on cytochalasin B-formed binucleated cells. It was shown that the induced micronuclei resulted from breakage events as well as chromosome loss, thus characterizing cetirizine dihydrocloride as both clastogen and aneugen. Since our results were derived only from in vitro experiments, we believe that an extensive in vivo study is necessary before drawing conclusions as to the effects of cetirizine dihydrochloride in patients.