Assessment of the genotoxic and cytotoxic potential of an anti-epileptic drug, phenobarbital, in mice: a time course study.

To examine if chronic oral administration of phenobarbital (PB), a widely used anti-epileptic drug, has any genotoxic and cytotoxic potential in mice, a mammalian model, cytogenetic assays through several endpoints such as chromosome aberrations, induction of micronuclei, mitotic index of bone marrow cells, sperm-head anomaly in testis and enzymatic assays of several toxicity marker enzymes have been conducted by use of standard techniques. Mice of both treated (chronically receiving an oral dose of PB at 1.2 mg/kg bw) and control (without receiving PB) groups were sacrificed at 7, 15, 30, 60, 90 and 120 days for the study with all the above-mentioned protocols. Further, total protein profiles in liver of both control and treated mice were analyzed through the SDS-PAGE technique at day 60. The results of all these studies, when compared with controls, showed that PB has both genotoxic and cytotoxic potential in apparently increasing intensity at longer periods of chronic feeding in mice, which would warrant due consideration in its long-term use on human subjects.

Effect of amaranth leaves on dimethylhydrazine-induced changes in multicomponent antioxidant system of rat liver.

Effect of prefeeding dehydrated amaranth (A. gangeticus) leaves at 10 and 20% levels on a chemical toxicant, dimethylhydrazine (DMH)-induced free radical stress in rat liver was evaluated. DMH-induced rise in hepatic malondialdehyde (MDA), was diminished by AL. AL intake resulted in a significant increase in hepatic glutathione (GSH). The feeding of AL at 10% level increased the hepatic glucose-6-phosphate dehydrogenase (G-6-PDH) activity, while that at 20% level increased the hepatic glutathione reductase (GSSGR) as well, in addition to G-6-PDH. Amaranth leaves at 10 and 20% levels of feeding diminished the hepatic superoxide dismutase and glutathione peroxidase (GSH-Px) activities. DMH influenced adversely the hepatic antioxidant enzyme activities. Simultaneous administration of DMH and feeding of AL enhanced the DMH-induced decrease in hepatic GSH-Px. DMH enhanced formation of micronuclei was reverted significantly by AL intake. Hence, it was concluded that the consumption of AL at 20% level reduced DMH-induced impaired antioxidant status in rat liver.

Role of micronucleus-limited DNA in programmed deletion of mse2.9 during macronuclear development of Tetrahymena thermophila.

Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important cis-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growth of Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision.

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.

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.

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.

Cytotoxic and genotoxic effects of beta-carotene breakdown products on primary rat hepatocytes.

According to Siems and colleagues, free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria. This finding may be an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators of the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo8'- carotenal (apo8') and beta-carotene utilizing primary cultures of rat hepatocytes. The end-points tested were: the mitotic index, the percentage of necrotic and apoptotic cells, micronucleated cells, chromosomal aberrations and sister chromatid exchanges (SCE). Our results indicate a genotoxic potential of both CP and apo8' already at the concentrations 100 nM and 1 microM, i.e. at pathophysiologically relevant levels of beta-carotene and beta-carotene breakdown products. A 3 h treatment with CP induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 10 microM and chromosomal aberrations at concentrations of 1, 5 and 10 microM. Apo8' induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 5 microM and chromosomal aberrations at concentrations of 0.1, 1 and 10 microM. Statistically significant increases in SCE induction were only observed at a concentration of 10 microM CP and apo8'. In contrast, no significant cytotoxic effects of these substances were observed. Since beta-carotene induced neither significant cytotoxic nor genotoxic effects at concentrations ranging from 0.01 up to 10 microM, these observations indicate that most likely beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study and the Beta-CArotene and RETinol Efficacy Trial (CARET).

Immunomodulatory and cytoprotective role of RP-1 in gamma-irradiated mice.

RP-1 has been reported to provide protection against lethal gamma-irradiation in mice. The present study was undertaken to understand its mechanism of action, especially with respect to modulation of radiation-induced changes in immune cell function, plasma antioxidant potential, cell cycle perturbations, apoptosis in mouse bone marrow cells, and micronuclei frequency in mice reticulocytes. 2 Gy reduced mitogenic response of splenic lymphocytes significantly at 48 h. Pre-irradiation RP-1 treatment significantly countered the radiation-induced loss of splenocyte proliferation. RP-1 treatment, with or without radiation, suppressed macrophage activation as compared to control. Irradiation decreased plasma antioxidant status significantly (p < 0.05) at 1 and 2 h (4.8 +/- 0.224 and 4.9 +/- 0.057 mM Fe2+) as compared to control (6.29 +/- 0.733 mM Fe2+) that was countered by RP-1 pre-treatment significantly (p < 0.05). RP-1 and irradiation individually caused G2 delay in bone marrow cells. RP-1 pre-treatment augmented radiation-induced G2 delay and elicited significant (p < 0.05) recovery in S-phase fraction at 48 h in comparison to irradiated group. Radiation-induced apoptosis (3%) was significantly higher than the control. RP-1 pre-treatment further enhanced apoptosis frequency (7.2%) in bone marrow cells. RP-1 pre-treatment significantly (p < 0.05) reduced (1.23%) the radiation-induced MN frequency (2.9%) observed at 48 h post-irradiation interval. Since the radioprotective manifestation of RP-1 is mediated through multiple mechanisms, needs further investigation.

Beta1-integrin and IL-1alpha expression as bystander effect of medium from irradiated cells: the pilot study.

Bystander effects have been proposed as a third action pathway of ionising radiation besides direct and indirect effects. The purpose of the study was to investigate whether expression of interleukin-1alpha (IL-1alpha) and beta1-integrin is elevated in bystander cells as a marker for bystander effects in comparison with classical markers such as the clonogenic assay, apoptosis and the presence of micronuclei. The hybrid cell line E.A. hy.926 obtained by fusion of HUVEC cells with the epithelial cell line A 459 was irradiated with 0-5 Gy. Bystander effects were established via medium transfer at 45 min and 4 h after irradiation from irradiated to nonirradiated cell populations. In order to exclude effects of the irradiated medium itself, irradiated medium only was also used for transfer to nonirradiated cells. Then, cells were fixed at 1, 2, 6, and 24 h after irradiation or medium transport and IL-1alpha and beta1-integrin were detected and evaluated. A higher number of beta1-integrin-positive cells was observed in both irradiated and bystander cell populations than in the control group at 1 and 24 h after irradiation with 1 Gy or medium transfer. Significantly higher numbers of IL-1alpha-positive cells were found at 1, 2, and 6 h after irradiation with 1 Gy or medium transfer as well as at 2 and 6 h after irradiation with 5 Gy or medium transfer. Clonogenic survival decreased dependently on the dose in irradiated cells but did not show any significant difference between the bystander cell populations and sham-irradiated cells. The irradiated medium itself did not have any effect. It is concluded that beta1-integrin and IL-1alpha expression may serve as more sensitive markers of post-irradiation responses in bystander cell populations than the classical radiobiological markers. Moreover, overexpression of beta1-integrin and IL-1alpha may induce increased susceptibility to inflammation of bystander cells.

Does a dose-response relationship reduce sensitivity to hidden bias?

It is often said that an important consideration in judging whether an association between treatment and response is causal is the presence or absence of a dose-response relationship, that is, larger ostensible treatment effects when doses of treatment are larger. This criterion is widely discussed in textbooks and is often mentioned in empirical papers. At the same time, it is well known through both important examples and elementary theory that a treatment may cause dramatic effects with no dose-response relationship, and hidden biases may produce a dose-response relationship when the treatment is without effect. What does a dose-response relationship say about causality? It is observed here that a dose-response relationship may or may not reduce sensitivity to hidden bias, and whether it has or has not can be determined by a suitable analysis using the data at hand. Moreover, a study without a dose-response relationship may or may not be less sensitive to hidden bias than another study with such a relationship, and this, too, can be determined from the data at hand. An example concerning cytogenetic damage among professional painters is used to illustrate.

Genotoxicity of hydrated sulfur dioxide on root tips of Allium sativum and Vicia faba.

Genotoxicity of sulfur dioxide (SO(2)) and its hydrates (bisulfite and sulfite) in human lymphocytes and other mammalian cells have been found earlier in our laboratory. In the present studies, we used Allium stavium and Vicia faba cytogenetic tests, which are the highly sensitive and simple plant bioassays. A mixture of sodium bisulfite and sodium sulfite (1:3), at various concentrations from 1 x 10(-4) to 2 x 10(-3)M was used for the treatment. Genotoxicity was expressed in terms of anaphase aberration (AA) frequencies in the Vicia-AA test and in terms of micronuclei (MCN) frequencies in both Vicia-MCN test and Alllium-MCN test. On average, the results showed a 1.7-3.9-fold increase of AA frequencies and a 3.5-4.5-fold increase of MCN frequencies in Vicia root tips as compared with the negative control. Similarly, results of Allium-MCN test also showed a significant increase in MCN frequencies in the treated samples. In addition, pycnotic cells (PNC) appeared in Allium root tips of treated groups. The frequencies of MCN, AA and PNC increased dose-dependently and the cell cycle delayed at the same time in bisulfite treated samples. Results of the present study suggest that the Vicia and Allium cytogenetic bioassays are efficient, simple and reproducible in genotoxicity studies of bisulfite.

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.

What do human micronuclei contain?

As micronuclei (MN) derive from chromosomal fragments and whole chromosomes lagging behind in anaphase, the MN assay can be used to show both clastogenic and aneugenic effects. The distinction between these phenomena is important, since the exposure studied often induces only one type of MN. This particularly concerns the use of MN as a biomarker of genotoxic exposure and effects, where differences in MN frequencies between exposed subjects and referents are expected to be small. A specific analysis of the induced type of MN may considerably improve the sensitivity of detecting the exposure effect. MN harbouring chromosomes can be distinguished from those harbouring acentric fragments by the presence of a centromere. The proportion of centromere-positive MN in human lymphocytes increases with age, which primarily reflects an age-dependent micronucleation of the X and Y chromosomes. The X chromosome especially tends to lag behind in female lymphocyte anaphase, being micronucleated more efficiently than autosomes. There is some evidence for an enhanced prevalence of fragments from chromosome 9 in spontaneous human lymphocyte MN and from chromosomes 1, 9 or 16 in MN induced in vitro by some clastogens; the breakage appears to occur in the heterochromatic block of these chromosomes. Although there are indications that centromere identification can improve the detection of clastogenic effects in humans in vivo, smokers have not shown an increase in centromere-negative MN in their cultured lymphocytes, although smoking is known to produce chromosomal aberrations. This may suggest that fragment-containing MN and chromosomal aberrations cover partly different phenomena. Understanding the mechanistic origin and contents of MN is essential for the proper use of this cytogenetic end-point in biomarker studies, genotoxicity testing and risk assessment.

The effect of aging on the results of the rat micronucleus assay.

We conducted the micronucleus assay in 3-, 5-, 7-, 9-, 11- and 13-week-old male rats to determine whether the results varied with age. We administered cyclophosphamide orally at 0 (vehicle), 5, 10 or 20 mg/kg, twice, 24 h apart, to five rats per dosage group and collected bone marrow and peripheral blood 24 h after the second treatment. We observed an age-related decrease in micronucleus-inducing activity in both polychromatic erythrocytes (bone marrow) and reticulocytes (peripheral blood), which we attributed to an age-related decrease in hematopoiesis. In spite of the age-related decrease in sensitivity to the model chemical studied here, all age groups tested showed positive responses. We concluded that the rat is suitable for the micronucleus assay regardless of age.

Aneugenic potential of okadaic acid revealed by the micronucleus assay combined with the FISH technique in CHO-K1 cells.

Okadaic acid (OA) is a major toxin involved in diarrhetic shellfish poisoning in humans and has been shown to be both a potent tumor promoter in rodent skin and stomach and an inhibitor of serine/threonine protein phosphatases, specifically PP1 and PP2A. The research on the genotoxic potential of OA amounts to only a few studies, which give conflicting results. In order to evaluate the ability of OA to induce DNA damage, the cytokinesis-block micronucleus assay was performed in the CHO-K1 cell line. A statistically significant induction of micronuclei without strong cytotoxicity was obtained after a 24 h treatment with 20 (approximately 5-fold) and 30 nM (approximately 10-fold) OA. Then, in order to discriminate between a clastogenic or aneugenic effect of OA, the micronucleus assay was carried out in combination with fluorescence in situ hybridization (FISH) using a (TTAGGG)(n) DNA probe for centromere detection. FISH analysis showed that OA mainly induced centromere-positive micronuclei (68.9% induction with 20 nM OA and 77.0% with 30 nM). Therefore, OA can be considered aneugenic. Using the same assay, biotransformation of OA was studied after a 4 h treatment with and without metabolic activation. The results show that reactive metabolites of OA were generated with a significant increase in genotoxic potential. The relationship between the different components involved in the mitotic process and OA inhibition of protein phosphatase is also discussed.

Biomass growth, micronucleus induction, and antioxidant stress enzyme responses in Vicia faba exposed to cadmium in solution.

Biomass growth, micronucleus induction, and antioxidative stress enzymes (superoxide dismutase, peroxidase, glutathione reductase, and catalase) were investigated simultaneously in the Vicia faba plant exposed to cadmium in solution. The biomass lowest-observed-effect concentration (LOEC) value was 2,000 microM Cd2+. In the shoots, enzymic activities increased without concentration-response relationships. In the roots, after an initial increase, activities of all enzymes showed negative concentration-response relationships. A significant increase in micronucleus induction was observed at 20 microM Cd2+. Regarding sensitivity, our results showed that biomass endpoint was less sensitive than micronucleus induction, which was less sensitive than antioxidative enzyme activities. The increase of antioxidant stress enzyme activities in response to cadmium exposure may be taken as evidence for an enhanced detoxification capacity of V. faba plants toward reactive oxygen species (and derivatives) that might be generated in the stressed plants. Concomitant micronucleus induction may be also interpreted as a consequence of oxidative stress, upholding the view that cadmium-induced DNA damage is, to some extent, via generation of reactive (intermediate) oxygen species.

Antigenotoxic potential of certain dietary constituents.

The human diet contains a variety of compounds that exhibit chemopreventive effects towards an array of xenobiotics. In the present study, the antigenotoxic potential of selected dietary constituents including Diallyl sulfide (DAS), Indole-3-carbinol (I3C), Curcumin (CUR), and Black tea polyphenols (BTP) has been evaluated in the Salmonella typhimurium reverse mutation and mammalian in vivo cytogenetic assays. In addition, the anticlastogenic effect of the above dietary constituents was identified towards Benzo(a)pyrene (BaP) and cyclophosphamide- (CP) induced cytogenetic damage in mouse bone marrow cells. The induction of BaP and CP induced chromosomal aberrations, micronuclei formation, and sister chromatid exchanges (SCEs) were found to be inhibited in a dose-dependent manner by DAS, I3C, CUR, and BTP. Thus the study reveals the antimutagenic potential of these dietary compounds towards BaP- and CP-induced genotoxicity in microbial and mammalian test systems.

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.