Antigenotoxicity protection of Carapa guianensis oil against mitomycin C and cyclophosphamide in mouse bone marrow.

The aim of this study was to evaluate the possible protective of C. guianensis oil against MMC and CP, which are direct- and indirect-acting chemical mutagens, using the micronucleus test. Three experiments were performed. First the C. guianensis oil was co-administered to mice at doses of 250, 500 and 1000 mg/kg bw with 4 mg/kg bw MMC or 50 mg/kg bw CP. Second, the mutagenic drug (CP) was administered ip 50 mg/kg bw and after 6 and 12 hours 250 and 500 mg/kg bw of C. guianensis oil were administered. In the last, C. guianensis oil was administrated (250 and 500 mg/kg bw) during five days and after it was administered ip 50 mg/kg bw CP. The results obtained showed that the C. guianensis oil is not cytotoxic neither genotoxic to mouse bone marrow. Regarding the antimutagenic effect, all doses of C. guianensis oil were significantly (p < 0.05) effective in reducing the frequency of micronucleated polychromatic erythrocytes, when compared with MMC or CP alone. Based on these results, our results suggest that the C. guianensis oil shows medicinal potential as an antimutagenic agent, modulating the mutagenicity caused by both direct- and indirect-acting chemical mutagens, in a mammalian model.

Role of recombinant human erythropoietin in mitomycin C-induced genotoxicity: analysis of DNA fragmentation, chromosome aberrations and micronuclei in rat bone-marrow cells.

Mitomycin C (MMC) is one of the most effective chemotherapeutic agents. However, during clinical use several side effects may occur. Recombinant human erythropoietin (rhEPO), a glycoprotein that regulates haematopoiesis, has been shown to exert an important cyto-protective effect in many tissues. The aim of this study was to explore whether rhEPO protects against MMC-induced genotoxicity in rat bone-marrow cells. Adult male Wistar rats were divided into six groups of 18 animals each: a control group, a 'rhEPO alone' group, an 'MMC alone' group and three 'rhEPO+MMC' groups (pre-, co- and post-treatment conditions). Our results show that MMC induced a noticeable genotoxic effect in rat bone-marrow cells. rhEPO reduced the effects of MMC significantly in every type of experiment conducted, such as the frequency of micronuclei, the percentage of chromosome aberrations and the level of DNA damage measured with the comet assay. The protective effect of rhEPO was more efficient when it was given 24h prior to MMC treatment.

Assessment of the cytotoxic, genotoxic, and antigenotoxic activities of Celtis iguanaea (Jacq.) in mice.

Ethnobotanical surveys of Cerrado native plants show that leaves of Celtis iguanaea (Jacq.) Sargent (Cannabaceae), popularly known in Brazil as "esporão de galo", are used in folk medicine for body pain, asthma, cramps, poor digestion, urinary infection, kidney dysfunctions, as well as a stimulant and diuretic. This work aimed at evaluating possible C. iguanaea aqueous leaf extract (CALE) cytotoxicity, genotoxicity, and antigenotoxicity using the mouse bone marrow micronucleous test. To assess CALE genotoxicity, Swiss mice were orally treated with three different extract concentrations (100, 300, and 500 mgkg-1). To evaluate its antigenotoxicity, the same doses were used simultaneously with a single i.p. dose of mitomycin C (MMC, 4mg.kg-1). The frequencies of micronucleated polychromatic erythrocytes (MNPCE) were evaluated 24 h and 48 h after administration except for the negative control (24 h). Genotoxicity was evaluated using the frequency of micronucleated polychromatic erythrocytes (MNPCE), whereas cytotoxicity was assessed by the polychromatic and normochromatic erythrocytes ratio (PCE/NCE). The results showed that CALE did not exhibit a significant reduction in the PCE/NCE ratio, neither a considerable increase in the frequency of MNPCE. Nonetheless, CALE reduced bone marrow toxicity (increased PCE/NCE ratio) and decreased the micronuclei frequency induced by MMC. We can conclude that CALE presented no cytotoxic and genotoxic effects, but showed antigenotoxic and anticytotoxic actions under the experimental conditions applied in this study.

Evaluation of the genotoxic and antigenotoxic effects of exopolysaccharide pullulan in human lymphocytes in vitro.

Pullulan is a biocompatible and water-soluble exo-polysaccharide produced by primary strains of the fungus Aureobasidium pullulans. It is frequently used in the pharmaceutical and food industries. In this study, possible cytotoxic effect of pullulan was assessed using the MTT assay in the human breast cancer (MCF-7) cell line. Micronucleus (MN), micronucleus-FISH (MN-FISH), random amplified polymorphic DNA (RAPD-PCR), and comet assays were used to investigate genotoxic and antigenotoxic effects of pullulan against mitomycin C (MMC) (at MN assay) and hydrogen peroxide (at comet assay) in human lymphocytes. Antigenotoxicity was determined using two different applications: 1 h pretreatment and simultaneous treatment. In the MTT assay, pullulan significantly reduced the cell viability at 15.6-2000 µg/mL compared to the control. No significant alterations in MN rates were found in human lymphocytes treated with different concentrations of pullulan compared to the control. In contrast, co-treatment of pullulan and MMC decreased the frequency of MN in almost all the treatment concentrations and durations compared to the MMC. No significant change was observed in the frequency of the centromere-positive C + or negative C- MNi compared to the positive control. In comet assay, pullulan did not affect comet tail intensity compared to the negative control. On the contrary, pullulan in combination with H2O2 significantly decreased tail intensity at almost all the concentrations compared to the positive control. The changes occurring in RAPD-PCR profiles following pullulan treatments included an increase or decrease in band intensity and gain or loss of bands. These results indicate that exopolysaccharide Pullulan is not genotoxic; moreover, it possesses a protective effect against MMC and H2O2 induced genotoxicity. In breast cancer cells, pullulan induced cytotoxic/anti-proliferative effect.

Protective effect of melatonin against mitomycin C-induced genotoxic damage in peripheral blood of rats.

Mitomycin C (MMC) generates free radicals when metabolized. We investigated the effect of melatonin against MMC-induced genotoxicity in polychromatic erythrocytes and MMC-induced lipid peroxidation in brain and liver homogenates. Rats (N = 36) were classified into 4 groups: control, melatonin, MMC, and MMC + melatonin. Melatonin and MMC doses of 10 mg/kg and 2 mg/kg, respectively, were injected intraperitoneally. Peripheral blood samples were collected at 0, 24, 48, 72, and 96 hours posttreatment and homogenates were obtained at 96 hours posttreatment. The number of micronucleated polychromatic erythrocytes (MN-PCE) per 1000 PCE was used as a genotoxic marker. Malondialdehyde (MDA) plus 4-hydroxyalkenal (4-HDA) levels were used as an index of lipid peroxidation. The MMC group showed a significant increase in MN-PCE at 24, 48, 72, and 96 hours that was significantly reduced with melatonin begin coadministrated. No significant differences were found in lipid peroxidation. Our results indicate that MMC-induced genotoxicity can be reduced by melatonin.

Antigenotoxic effect of apigenin against anti-cancerous drugs.

Mitomycin C and cyclophosphamide are well known anti-tumor drugs. Their genotoxic effects are well established in various test systems. The genotoxic effects in non-tumor cell are of special significance due to the possibility that they may induce secondary tumors in cancer patients. Apigenin is a well known anti-oxidant and possess number of properties that are beneficial in some way to humans. With this view, the present study deals with the effect of apigenin against the genotoxic doses of mitomycin C and cyclophosphamide using chromosomal aberrations, sister chromatid exchanges and cell cycle kinetics as a parameters. The treatment of apigenin results in a significant, dose dependent decrease in the genotoxic damage, induced by mitomycin C and cyclophosphamide. It is concluded that the apigenin is potent in reducing the genotoxic damage, induced by anti-cancerous drugs, thereby reducing the chances of developing secondary tumors during the therapy.

Lithium chloride attenuates mitomycin C induced necrotic cell death in MDA-MB-231 breast cancer cells via HMGB1 and Bax signaling.

The clinical use of potent anticancer drug mitomycin C (MMC) has limited due to side effects and resistance of cancer cells. The aim of this study was to investigate whether lithium chloride (LiCl), as a mood stabilizer, can affect the sensitivity of MDA-MB-231 breast cancer cells to mitomycin C. The cells were exposed to various concentrations of mitomycin C alone and combined with LiCl and the viability determined by trypan blue and MTT assays. Proteins were analyzed by western blot and mRNA expression of HMGB1 MMP9 and Bcl-2 were analyzed by RT-PCR. Flow cytometry was used to determine the cell cycle arrest and percent of apoptotic and necrotic cells. Concentration of Bax assessed by ELISA. Exposure of the cells to mitomycin C revealed IC50 value of 20 µM, whereas pretreatment of the cells with LiCl induced synergistic cytotoxicity and IC50 value declined to 5 µM. LiCl combined with mitomycin C significantly down-regulated HMGB1, MMP9 and Bcl-2 gene expression but significantly increased the level of Bax protein. In addition, the content of HMGB1 in the nuclei decreased and pretreatment with LiCl reduced the content of HMGB1 release induced by MMC. LiCl increased mitomycin C-induced cell shrinkage and PARP fragmentation suggesting induction of apoptosis in these cells. LiCl prevented mitomycin C-induced necrosis and changed the cell death arrest at G2/M-phase. Taking all together, it is suggested that LiCl efficiently enhances mitomycin C-induced apoptosis and HMGB1, Bax and Bcl-2 expression may play a major role in this process, the findings that provide a new therapeutic strategy for LiCl in combination with mitomycin C.

The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro.

Brevetoxins (PbTxs) are highly potent trans-syn polyether neurotoxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. These neurotoxins act on voltage-sensitive sodium channels prolonging the active state. During red tides, the commercial fishing and tourism industries experience millions of dollars of lost revenue. Human consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). Additionally, blooms of K. brevis are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in coastal residents. There is little information regarding the full range of potential toxic effects caused by PbTxs. Recent evidence suggests that PbTxs are genotoxic substances. The purpose of this study was to determine if PbTxs could induce chromosomal aberrations and inhibit cellular proliferation in CHO-K1-BH4 cells, and if so, could the damage be negated or reduced by the PbTx antagonist brevenal. Results from the chromosomal aberrations assay demonstrated that PbTxs are potent inducers of CHO-K1-BH4 chromosome damage. Results from the inhibition of cellular proliferation assays demonstrated that PbTxs inhibit the ability of CHO-K1-BH4 cells to proliferate, an effect which can be reduced with brevenal.

Cytotoxicity of mitomycin C and adriamycin in freshly isolated rat hepatocytes: the role of cytochrome P450.

The role of cytochrome P450 (P450) in the cytotoxicity of mitomycin C (MMC) and Adriamycin (ADR) was investigated in freshly isolated hepatocytes from phenobarbital-induced rats. The loss of cell viability [measured as lactate dehydrogenase (LDH) leakage] upon MMC exposure was accompanied by a rapid and extensive intracellular glutathione (GSH) depletion and followed by minor lipid peroxidation (LPO). Coincubation of the hepatocytes with the P450 inhibitors, metyrapone and SK&F 525-A, strongly protected against MMC-induced LDH leakage, GSH depletion, and LPO. Inasmuch as the depletion of intracellular GSH by MMC, which is considered as a critical event in the development of MMC cytotoxicity, was not accompanied by a stoichiometric oxidation to oxidized GSH (GSSG), the formation of a MMC-GSH conjugate after one-electron reductive bioactivation of MMC by P450 was anticipated. In contrast to MMC, ADR was only cytotoxic to the hepatocytes upon prior depletion of intracellular GSH with diethylmaleate. Addition of metyrapone and SK&F 525-A completely protected the hepatocytes against ADR-induced LDH leakage and LPO. Moreover, the ADR-induced LDH leakage and LPO were strongly inhibited by dimethyl sulfoxide and ethanol, indicating that hydroxyl radicals were involved in the cytotoxicity of ADR. In conclusion, the present investigations indicate that P450 plays a major role in the cytotoxicity of both MMC and ADR in freshly isolated hepatocytes from phenobarbital-induced rats. The present findings lead to a better understanding of the mechanism of the cytotoxic actions of both MMC and ADR.

Overexpression of thioredoxin in Fanconi anemia fibroblasts prevents the cytotoxic and DNA damaging effect of mitomycin C and diepoxybutane.

Adult T cell leukemia derived factor (ADF)/thioredoxin (Trx) is known to be an important intracellular antioxidant involved in a number of redox reactions such as ribonucleotide reductase (RNR) as well as of tyrosinase. Since RNR is a key enzyme of nucleotide metabolism and DNA synthesis, a reduced Trx level would result in reduced enzymatic activity and cause DNA damage. Furthermore, Trx is considered to be an effective regulator of redox sensitive gene expression. The role of Trx in nucleotide metabolism and gene expression may be an explanation for increased chromosomal instability as well as hypersensitivity towards oxygen, ROI and ROI generating agents. The activity of tyrosinase, the key enzyme of melanin biosynthesis, is influenced by the thioredoxin level and by superoxide radicals. Low thioredoxin levels and high superoxide concentrations activate tyrosinase causing hyperpigmentation of the skin. In addition to the observed high superoxide concentration in Fanconi anemia (FA) patients, a low thioredoxin level might be responsible for the hyperpigmentation (café-au-lait spots) in this disease. We observed that overexpression of the thioredoxin cDNA in FA fibroblasts completely abolished the DNA damaging effects of mitomycin C and diepoxybutane and inhibited the constitutive activity of the nuclear factor kappaB (NF-kappaB) in SV40 transformed FA fibroblasts. However, spontaneous chromosomal breakage was not affected.

The modulating effects of quercetin and rutin on the mitomycin C induced DNA damage.

The present study was carried out to investigate the modulating effects of the two flavonoids quercetin and rutin on the mutagenic anticancer drug mitomycin C by single cell gel electrophoresis (Comet assay) in human lymphocytes. Lymphocytes were incubated with different concentrations of quercetin and rutin, with or without mitomycin C, and DNA damage was evaluated. Concentrations of 0.03, 0.15, 0.3, 0.6, 1.5 and 3mM quercetin significantly reduced the DNA strand breakage induced by mitomycin C (P<0.001) but the highest concentration of 6mM quercetin did not show a protective effect. The frequency of damaged cells induced by mitomycin C was not changed at 0.02 mM, and also at the highest concentrations of 1.64 and 3.28 mM rutin. However, at concentrations of 0.08, 0.16, 0.33 and 0.82 mM rutin cells were protected from DNA damage. Thus, in human lymphocytes quercetin and rutin displayed protective effects on DNA damage induced by mitomycin C, in a concentration-dependent manner.

The anticlastogenicity of beta-carotene evaluated on human hepatoma cells.

The efficiency of beta-carotene as a modulatory agent against clastogenicity induced by cyclophosphamide (CPA), an indirect-acting mutagen, and mitomycin C (MMC), a direct-acting mutagen, was evaluated in human hepatoma cells (Hep G2) using three different treatment regimes. Six doses of beta-carotene, 0.25, 0.5, 1.0, 2.0, 4.0 and 6.0 microM, were tested as pre-treatment, simultaneous treatment and pre- + simultaneous treatment. Since these cells are able to activate mutagens without any exogenous metabolizing system (S9 mix), some problems related to the use of S9 mix were eliminated. The data obtained show a statistically significant decrease in the frequency of micronuclei (MN) induced by CPA when the cells were treated with beta-carotene, for all treatments, and no effect of this provitamin on the clastogenicity of MMC was found. These results reinforce the anticlastogenicity of beta-carotene showing that its action is independent of the treatment regime used. On the other hand, the fact that beta-carotene had a protective action only on CPA-induced MN suggests an effect on activation of the promutagen and emphasizes the important utility of cell lines capable of metabolizing chemical mutagens, in such basic studies.

Phenethyl isothiocyanate modulates clastogenicity of mitomycin C and cyclophosphamide in vivo.

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, is an effective chemopreventive agent against N-nitrosamine-induced carcinogenesis. We have investigated the extent to which PEITC modulates the clastogenicity of standard genotoxicants, mitomycin C and cyclophosphamide, using bone marrow cells of Swiss albino mice. PEITC, 1 mumol/kg body weight in corn oil was administered by gavage for 7 consecutive days to prime the animals. 24 h later, mice received a single dose of cyclophosphamide (10 or 20 mg/kg body weight) or mitomycin C (1 or 2 mg/kg body weight) intraperitoneally. Clastogenicity of the chemicals was compared using PEITC-primed and non-primed animals 24 h after clastogen treatment. As a single agent, PEITC is not clastogenic even after 7 days of priming. Oral priming with PEITC decreased the aberrations per cell values by 22-67% in all cases. PEITC could only alleviate the clastogenicity of 1 mg/kg body weight mitomycin C to near-control values (p < or = 0.05). Although PEITC is reported to be effective against N-nitrosamine-induced tumorigenesis by preventing metabolic activation and by blocking the reactive species formed, it is virtually ineffective against the clastogenicity of cyclophosphamide. The results of inhibition by PEITC of the clastogenicity of mitomycin C suggest that the modulation of mitomycin C bio-activation contributes to, but may not be sufficient for, PEITC chemoprevention of clastogenicity by mitomycin C.

Suppressing effects of S phase post-treatment with carbolines on sister-chromatid exchanges induced by mitomycin C in Chinese hamster cells.

The effects of post-treatment with four types of carbolines-alpha-, beta-, gamma-, and delta-carbolines-and aminoimidazoazaarenes during S phase on the frequency of SCEs induced by MMC were studied in synchronized Chinese hamster CHO cells. Post-treatment with alpha-carbolines (A alpha C, MeA alpha C), beta-carbolines (harman, norharman, harmine, and harmaline), gamma-carbolines (Trp-P-1 and Trp-P-2) and delta-carbolines (Glu-P-1, and Glu-P-2) during S phase at non-clastogenic concentrations caused a statistically significant decrease in the frequency of SCEs induced by MMC. Aminoimidazoazaarenes (IQ, MeIQ, and diMeIQx) showed no effects on SCEs, suggesting that the SCE-suppressing activity resides in the carboline structure. The data suggest that the suppressing effect may be due to inhibition of DNA replication.

Desmutagenic activity of natural humic acids: inhibition of mitomycin C and maleic hydrazide mutagenicity.

Humic acids are natural components of organic matter widespread in the environment. In spite of the incomplete knowledge about their composition, increasing interest in humic acid activity is justified by their ubiquity. Four different humic acids have been tested in Chinese hamster ovary cells in vitro, both alone and in combination with two well-known mutagens (mitomycin C and maleic hydrazide). Data about sister-chromatid exchanges, mitotic and proliferation indices were collected. Our results, on the whole, indicate: (i) a slight mutagenicity and toxicity of tested humic acids, probably due to chlorination during sample preparation; (ii) a desmutagenic rather than antimutagenic activity of the tested humic acids.

In vitro effects of prostaglandin E1 and indomethacin on mitomycin C-induced sister-chromatid exchanges in mitogen-stimulated human lymphocytes.

In this study, the individual and combined effects of prostaglandin E1 (PGE1) and Indomethacin on mitomycin C (MMC)-induced SCEs in human lymphocytes was investigated in vitro. All MMC-treated cultures showed a great increase of SCEs (approximately two-fold), indicating its ability to induce mutations. SCE data showed that MMC-induced SCEs were reduced significantly in the presence of PGE1 in pooled analysis of six experiments (60.55% reduction of SCEs at 10(-6) M, 34.13% reduction of SCEs at 10(-9) M). In contrast the presence of indomethacin in the medium during MMC treatment of cells failed to show a significant reduction of SCEs in pooled analysis (21.17%). However, individual analyses revealed only two of six donors with a significant SCE response. Thus, the findings suggest that PGE1 can modify the DNA damaging effect of carcinogens and thereby may prevent the initiation of the carcinogenic process.

Todralazine influence upon the mutagenicity of some direct- and indirect-acting mutagens.

Todralazine decreased the mutagenic activity of tested direct- and indirect-acting mutagens. Despite the marked differences between efficient todralazine doses (ED50) it was observed that, in the case of tested indirect mutagens as well as in some of the direct mutagens, the decrease of mutagenicity by todralazine was very strong, exceeding 80% in some cases.

Inhibitory effects of coffee on transplacental genotoxicity in mice.

Experiments were carried out to ascertain whether or not coffee can modulate the genotoxicity of transplacentally active genotoxins/carcinogens. Coffee was orally administered to Swiss albino mice (gestation, 15-16 days), 90 min before exposure to cyclophosphamide (CPH), N-nitrosodiethylamine (DEN), N-nitroso-N-ethylurea (ENU) and mitomycin C (MMC). At the end of the treatment, the induction of micronucleated polychromatic erythrocytes (MnPCEs) was evaluated in the fetal liver (FL), fetal blood (FB) and maternal bone marrow (MBM). The results of this transplacental micronucleus test showed a consistent trend which suggests that the administration of coffee instead of water (control) can significantly inhibit the genotoxic effects of CPH, DEN, ENU and MMC in the FL and FB. When the fetal cells were evaluated either 22 and 28 h after CPH treatment, or 24 and 48 h after MMC treatment, there was no evidence for a significant interaction between the sampling time and the inhibitory effect of coffee (two-factor ANOVA). However, a significant interaction was observed between sampling time and the inhibitory effects of coffee when the fetal cells were sampled 24 and 40 h after DEN treatment (two-factor ANOVA). Coffee was also effective in significantly inhibiting the genotoxicity of CPH, ENU and MMC in the MBM. The differential response of fetal and maternal target cells was evident from this study.

Investigation of the antimutagenic effects of selected South African medicinal plant extracts.

Dichloromethane extracts from different parts of Rhamnus prinoides, Ornithogalum longibracteatum, Gardenia volkensii, Spirostachys africana, Diospyros whyteana, Syzigium cordatum and Prunus africana were investigated for mutagenic and antimutagenic effects in Salmonella/microsome and micronucleus tests. None of the extracts tested in the Ames test were found to induce mutations or to modify the effect of the mutagen 4-nitroquinoline-oxide (4NQO). In the micronucleus test, extracts from twigs/bark of R. prinoides, twigs of D. whyteana, P. africana and S. cordatum significantly lowered the effect of the mutagen mitomycin C (MMC). Extracts from twigs/bark of G. volkensii and S. africana were genotoxic in the micronucleus test, while extracts of O. longibracteatum leaves potentiated the genotoxicity of MMC. This preliminary investigation shows that plant extracts used in traditional medicine may have particular effects with regard to mutagenicity and antimutagenicity indicating careful use in some instances and the need to isolate their active principles for further research.

Studies on the anticlastogenic effect of turmeric and curcumin on cyclophosphamide and mitomycin C in vivo.

Turmeric and its main constituent curcumin were assessed in vivo for their anticlastogenic potential. In one experimental set, Swiss albino male mice were given turmeric (8, 12 and 16 mg/kg body weight) or curcumin (2, 4 and 8 mg/kg body weight) as a single intraperitoneal injection. In another set, the mice were given 8 mg/kg body weight of turmeric or one of three concentrations of curcumin (2, 4 and 8 mg/kg body weight) as a dietary supplement by gavage for 7 consecutive days. 30 min after the last dose the mice were administered a single acute dose of two known clastogens, cyclophosphamide (CP) (20 mg/kg body weight) or mitomycin C (MMC) (1.5 mg/kg body weight). After 18 hr, chromosome preparations were made from bone marrow cells. The endpoints studied were chromosome aberrations and damaged cells. Clastogenicity of the chemicals was compared using turmeric- or curcumin-primed and non-primed animals. As single agents turmeric and curcumin were not clastogenic even after 7 days of priming. Turmeric/curcumin could not inhibit CP- or MMC-induced clastogenicity. Although curcumin is reported to be the active chemopreventive principle in turmeric effective against a number of potential carcinogens in several experimental systems, it was virtually ineffective against the clastogenicity of CP or MMC at the doses tested.