Evaluation of the toxic activity of anorectic diethylpropion in Chinese hamster ovary cells.

Diethylpropion has been available in the market for treating obesity for over 50 years. Refined studies are lacking to fully elucidate its action spectrum. The aim of our study was to evaluate possible toxic effects of anorectic diethylpropion in Chinese hamster ovary (CHO) cells. Comet assay (detects breaks in the DNA strand), micronucleus test (detects clastogenic/aneugenic damage), and cell survival test (detects cytotoxic damage) were used to evaluate the toxic effects. In comet assay, we found that the damage scores with diethylpropion treatments at the concentrations of 20 and 40 µg/mL were more significant ( p < 0.05) than that of the negative control. When assessing the possible aneugenic and/or clastogenic damage caused by the drug in CHO cells, we found no difference ( p > 0.05) in the values of micronucleated cells when comparing different diethylpropion treatments and the negative control. Regarding the cell viability, for all the diethylpropion concentrations tested, higher values ( p < 0.05) of apoptosis were found compared with those of the negative control. In relation to the number of necrotic cells, no difference ( p > 0.05) was noted between the means of the three concentrations of diethylpropion evaluated and the negative control. In the experimental conditions, we conclude that diethylpropion has weak genotoxic and cytotoxic activities.

Genotoxic effect of Physalis angulata L. (Solanaceae) extract on human lymphocytes treated in vitro

Physalis angulata L (Solanaceae) is a medicinal plant from North of Brazil, whose different extracts and infusions are commonly used in the popular medicine for the treatment of malaria, asthma, hepatitis, dermatitis and rheumatism. However, the genotoxic effects of P. angulata on human cells is not well known. The main purpose of the present study was to evaluate the in vitro genotoxic effects of aqueous extract of P. angulata using the comet assay and the micronucleus assay in human lymphocytes provided from 6 healthy donors. Treatments with P. angulata extracts were performed in vitro in order to access the extent of DNA damage. The comet assay has shown that treatments with P. angulata at 0.5, 1.0, 2.0, 3.0 and 6.0 microg/mL in culture medium were genotoxic. Lymphocytes treated with P. angulata at the concentrations of 3.0 and 6.0 microg/mL in culture medium showed a statistically significant increase in the frequency of micronucleus (p<0.05), however, the cytokinesis blocked proliferation index (CBPI) was not decreased after P. angulata treatment. In conclusion, the present work demonstrated the genotoxic effects of P. angulata extract on human lymphocytes in vitro.

Genotoxic effect of Physalis angulata L. (Solanaceae) extract on human lymphocytes treated in vitro

Physalis angulata L (Solanaceae) is a medicinal plant from North of Brazil, whose different extracts and infusions are commonly used in the popular medicine for the treatment of malaria, asthma, hepatitis, dermatitis and rheumatism. However, the genotoxic effects of P. angulata on human cells is not well known. The main purpose of the present study was to evaluate the in vitro genotoxic effects of aqueous extract of P. angulata using the comet assay and the micronucleus assay in human lymphocytes provided from 6 healthy donors. Treatments with P. angulata extracts were performed in vitro in order to access the extent of DNA damage. The comet assay has shown that treatments with P. angulata at 0.5, 1.0, 2.0, 3.0 and 6.0 microg/mL in culture medium were genotoxic. Lymphocytes treated with P. angulata at the concentrations of 3.0 and 6.0 microg/mL in culture medium showed a statistically significant increase in the frequency of micronucleus (p<0.05), however, the cytokinesis blocked proliferation index (CBPI) was not decreased after P. angulata treatment. In conclusion, the present work demonstrated the genotoxic effects of P. angulata extract on human lymphocytes in vitro.(AU)

Formocresol mutagenicity following primary tooth pulp therapy: an in vivo study.

OBJECTIVES: To investigate whether formocresol, in Buckley's original formulation, is mutagenic in vivo to lymphocyte cultures obtained from the peripheral blood of children aged from 5 to 10 years old. These children were recruited from those attending the dental clinics of Recife City Council and the University of Pernambuco School of Dentistry, Brazil. METHODS: The sample comprised 20 children who had primary teeth with cariously exposed vital pulps. Two venous blood samples were collected (6-8 ml) from each child, the first prior to vital pulpotomy (control group) and the second 24 h after pulpotomy (treated group). This research is a case-control study. The peripheral lymphocytes were grown in a complete culture medium consisting of 78% RPMI 1640 medium (a), supplemented with streptomycin (0.01 mg/ml), penicillin (0.005 ml(-1)), 20% fetal bovine serum (b) and 2% phytohemagglutinin (c). The lymphocytes were assessed for chromosomal aberrations via a previously published method which was modified. The cytogenetic analysis was performed in a blind test, where the slides were codified by an annotator and the scorers did not know which group they were analyzing. For each sample, this envolved the analysis of 200 metaphases. The level of significance adopted in the statistical test was 5.0% (p<0.05). RESULTS: There was no statistically significant difference in clinical doses between the control and treated groups, using Wilcoxon's Signed Ranks test, for the chromosomal aberrations (P=0.251) and for the total chromosomal breaks (P=0.149). Although there were no statistically significant differences between the control and treated groups, Buckley's formocresol was mutagenic for one patient, raising doubt about the desirability of its use for pulpotomies in children. CONCLUSIONS: The results revealed that, from a statistical standpoint, formocresol is not mutagenic. However, further investigations are required, preferably with a larger sample, in patients needing more than one pulpotomy in order to observe whether an increase in the quantity of the drug would increase the quantity of chromosome aberrations and also to verify individual susceptibility to chromosome alterations with the use of formocresol.

Study of chromosome damage in patients with breast cancer treated by two antineoplastic treatments.

The frequencies of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) were determined in peripheral blood lymphocyte cultures from women with breast cancer treated by chemotherapy (CT) with FEC (5-fluorouracil, epirubicin, and cyclophosphamide) or CMF (cyclophosphamide, methotrexate, and 5-fluorouracil) cocktail in six CT cycles. The number of patients in each CT cycle were from 1 to 3 for SCE and 2 to 5 for CA. Samples were collected before and 48 h after CT. Although the size of the sample was limited and interindividual variability was wide, it appears that a 21-day interval between CT sessions is sufficient for cell recovery. This fact was demonstrated by the reduction in CA and SCE frequencies between cycles in parallel with the unchanged mitotic index and proliferative index values.

Cytogenetic study of chronic ethanol consumption in rats.

Ethanol was supplied in the drinking water of Wistar rats at a concentration of 20% v/v for up to 30 days. The animals treated with ethanol demonstrated a nonsignificant increase in chromosomal aberration frequency when compared with control animals. The mitotic index values obtained indicated no significant differences between ethanol treatment and control groups. The final weights of control rats were significantly greater than those of the ethanol-treated group. Chronic administration of ethanol showed no clastogenic or cytotoxic effect. After chronic ethanol consumption, the cytochromes P450 activity increases, thus possibly preventing the ethanol that has entered the circulation from reaching excessive levels, leading to metabolic adaptation and/or tolerance.

Genotoxic action of the sesquiterpene lactone centratherin on mammalian cells in vitro and in vivo.

Centratherin is a sesquiterpene lactone known for its antimicrobial, anti-inflammatory, and trypanocidal activities. The aim of this study was to determine the clastogenic and cytotoxic potential of centratherin in human lymphocytes and in mice. Human lymphocytes in culture were submitted to either continuous treatment or treatment during G(2) phase of the cell cycle. After continuous treatment the 0.2 microg/ml concentration induced a significant increase in total of chromosomal aberrations (CA) and sister chromatid exchange compared to control, and it reduced the mitotic index (MI). In the treatment during G(2) phase, centratherin induced a significant increase in the frequency of CA for all concentrations tested (0.1, 0.3, and 0.5 microg/ml). In the in vivo test system all three concentrations tested in mice (3.3, 6.7, and 13.3 mg/kg b.w.) induced a significant increase in CA compared to the negative control. On the basis of these results, centratherin showed clastogenic and cytotoxic activity on in vitro and in vivo mammalian systems.

Assessment of the cytotoxic and clastogenic activities of the sesquiterpene lactone lynchnopholide in mammalian cells in vitro and in vivo.

Lychnopholide (LNP), a sesquiterpene lactone with antitumor, trypanocidal, and antimicrobial activities, was isolated from Vanillosmopsis erythropappa. The present study was carried out to assess the cytotoxic and clastogenic potential of this new agent in human cultured lymphocytes and Swiss bone marrow cells before the agent was used in medicine. The mitotic index, chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and proliferation index were investigated. There was no alteration in the number of CAs and SCEs in the continuous in vitro treatment. However, the highest concentration (0.2 microg/ml) of LNP was cytotoxic. LNP (0.1, 0.2, and 0.4 microg/ml) induced a significant increase in CA frequency at the G2 phase in all treated cultures. Only the highest concentration (26.67 mg/kg) caused a significant increase in the total number of CAs in the in vivo investigation. On the basis of these results, LNP had a clastogenic effect on both test systems and a cytotoxic effect in vitro.

Protective effect of thiourea, a hydroxyl-radical scavenger, on curcumin-induced chromosomal aberrations in an in vitro mammalian cell system.

Natural dietary antioxidants are extensively studied for their ability to protect cells from damage to DNA, protein, and lipids induced by antitumor agents or radiation that leads to the generation of free radical in normal cells in vivo and in vitro. Curcumin is a natural antioxidant known to possess therapeutic properties and has been reported to scavenge free radicals and to inhibit clastogenesis in mammalian cells. However, curcumin has been reported to induce a significant increase in the frequency of chromosomal aberrations in Chinese hamster ovary (CHO) cells. To investigate whether the clastogenic activity of curcumin in CHO cells in culture can be ascribed to a pro-oxidant behavior, mediated by free radical generation, experiments were carried out with the combination of curcumin (15 microg/ml) and thiourea (10, 20, or 40 microg/ml), a potent hydroxyl radical scavenger. The results showed that the clastogenic action of curcumin was statistically decreased in a dose-dependent manner in the presence of thiourea. These data have shown that curcumin-induced chromosomal damage in CHO cells can be mediated by hydroxyl radical generation in the present experimental conditions. Teratogenesis Carcinog. Mutagen. 21:175-180, 2001.

B chromosomes in two fish species, genus Rhamdia (Siluriformes, Pimelodidae).

Specimens belonging to two fish species genus Rhamdia were cytogenetically analysed from seven localities in Brazil and Argentina. In addition to the 58 chromosomes of the basic karyotype, one to five metacentric B chromosomes were observed carrying conspicuous heterochromatic blocks on the distal regions of both chromosome arms. These B chromosomes are mitotically stable and, in the two best-sampled populations in R. hilarii (Lobo and 29 reservoirs), they showed frequency distributions fitting a binomial distribution, though Bs were more frequent in the latter. The presence of B chromosomes with the same appearance in R. quelen suggests an ancient origin for these B chromosomes, presumably prior to speciation from a common ancestor.

Potentiation by turmeric and curcumin of gamma-radiation-induced chromosome aberrations in Chinese hamster ovary cells.

The effect of turmeric and curcumin, two natural antioxidants, on the frequencies of chromosome aberrations induced in Chinese hamster ovary (CHO) cells by gamma-radiation was investigated. Cells were treated with three concentrations of each drug, turmeric (100, 250, and 500 microg/ml) and curcumin (2.5, 5, and 10 microg/ml), and then irradiated (2.5 Gy) during different phases of the cell cycle. Turmeric was not clastogenic by itself, whereas curcumin at 10 microg/ml enhanced the chromosomal damage frequency. Neither of the two antioxidants showed protective effect against the clastogenicity of gamma-radiation. Instead, an obvious increase in the frequencies of chromosome aberrations was observed when turmeric at 500 microg/ml was associated with gamma-radiation during G2/S phase, and curcumin at 10 microg/ml plus gamma-radiation during S and G2/S phases of the cell cycle. The results clearly indicate the exacerbated effect of turmeric and curcumin on radiation-induced clastogenicity, suggesting that these antioxidants are also potentiating agents depending on the experimental conditions.

Comparative study of the effects of vitamin C and bleomycin on smokers' and non-smokers' lymphocytes in clastogenicity assays.

Free radicals are products of metabolic reactions and of external factors that can injure different biological molecules. However, different antioxidant agents can prevent the action of these reactive species and the damage they cause. Vitamin C (VC) is an important micronutrient found in the diet, which presents defense mechanisms against the free radicals that challenge the cells of the organism. The objective of the present study was to investigate the effect of VC as a modulator of the damage induced in DNA by bleomycin (BLM) in lymphocytes from smokers and non-smokers. The difference in response to the mutagenic potential of BLM between smokers and non-smokers was also investigated. Peripheral blood lymphocyte cultures were treated simultaneously with BLM (20 microg/ml) and/or VC (100, 200, and 400 microg/ml) in the G2 phase of the cell cycle. The results obtained did not demonstrate a statistically significant difference in the response to the antitumor agent BLM between smokers and non-smokers. The data also showed that VC had no significant modulating effect on the frequency of chromosome aberrations induced by BLM in the cells of smokers and non-smokers under the experimental conditions used.

Protection and induction of chromosomal damage by vitamin C in human lymphocyte cultures.

Some chemotherapeutic approaches have proposed the use of antioxidants such as vitamin C (VC) to minimize the cytotoxicity and damage induced in normal tissue by antitumor agents that produce free radicals. Nevertheless, VC can also be cytotoxic, genotoxic, and harmful when combined with antitumor agents in human cells in vitro. The present study was undertaken to investigate the effects of VC (100, 200, 500, and 1,000 microg/ml) on human peripheral blood lymphocytes in vitro and its anticlastogenic effect on chromosomal aberrations induced by doxorubicin (DXR). VC did not show a clastogenic effect by itself, except at 1,000 microg/ml. At the concentration of 100 or 200 microg/ml of VC, administered in pre-, post-, or simultaneous treatment, there was a significant reduction in both chromosome aberrations and number of abnormal metaphases induced by DXR. At the doses of 500 or 1,000 microg/ml, VC did not present the same protective effect and was cytotoxic. Under the present experimental conditions, the efficiency of VC in protecting against chromosome damage was dependent on the doses used.

Modulatory effects of curcumin on the chromosomal damage induced by doxorubicin in Chinese hamster ovary cells.

Curcumin, a natural phenolic compound, is gaining importance as a free radical scavenger. This study was undertaken to investigate the modulatory effects of curcumin on the chromosomal damage induced by the antitumoral doxorubicin (DXR), a known free radical generator, in Chinese hamster ovary cells in culture. Cells were treated with three concentrations of curcumin (2.5, 5, or 10 microg/ml) and then treated with DXR (1.0 microg/ml) during different phases of the cell cycle. The results show that curcumin induces chromosomal damage in CHO at the highest concentration when compared to the untreated control. Neither treatment with curcumin shows a reduction in the clastogenicity of DXR. Instead, a statistically significant increase in the frequency of chromosomal damage was observed when the middle and the highest concentrations of curcumin were associated with DXR during the G1/S, S, and S/G2 phases of the cell cycle. The results clearly indicate the potentiating effect of curcumin on DXR-induced chromosomal damage.

Evaluation of the genotoxic potential of the isocoumarin paepalantine in in vivo and in vitro mammalian systems.

Paepalantine is an isocoumarin isolated from Paepalanthus vellozioides which showed antimicrobial activity in in vitro experiments. In the present study, paepalantine was tested for possible clastogenic and cytotoxic action. Cultures from different individuals were treated with paepalantine at concentrations of 20, 40 and 80 microg/ml. The effect of isocoumarin was also tested in an in vivo assay using Wistar rat bone marrow cells. Paepalantine was administered intraperitoneally at concentrations of 6.25, 12.5 and 25 mg/kg body weight. Under these conditions paepalantine did not have a clastogenic effect, but was significantly cytotoxic in the in vitro and in vivo mammalian cell systems tested in the present work.

Interaction effects of 5-azacytidine with topoisomerase II inhibitors on CHO cells, as detected by cytogenetic analysis.

Different cell treatment protocols with the hypomethylating agent 5 azacytidine (5-aza C) were used in exponentially growing Chinese hamster ovary (CHO) cells in order to test its influence on the induction of chromosomal aberrations (CAs) induced by topoisomerase II inhibitors, ellipticine (EPC) and teniposide (VM-26). Cells pre-treated with 1 microg/ml 5-aza C for 1 h during the S-phase and post-treated in the last 2 h of incubation with 0.6 microg/ml EPC or 0.04 microg/ml VM-26 showed a reduction of 48% and 45%, respectively, in the frequencies of CAs as compared to the sum value of the frequencies obtained for each drug alone. 5-aza C added to the cultures for the last 2 h before cell fixation after a 30-min pulse treatment with EPC or VM-26 caused a 38% and 28% reduction, respectively. Simultaneous treatments with 5-aza C plus EPC, or 5-aza C plus VM-26 during the last 2 h of incubation (G2-phase), showed a significant effect of CA reduction (24%) only for the combination of 5-aza C + EPC. Preliminary assays with 5-aza C alone added to the cultures at different times demonstrated its effectiveness in inducing chromosome damage during the S-phase. Since S-phase-treated CHO cells showed a higher degree of reduction in the frequencies of CAs induced by EPC and VM-26, we suggest that 5-aza C incorporation into DNA may change the topo II cleavage sites, protecting the DNA from the induction of damage, or that the hypomethylation induced by incorporation of 5-aza C into DNA may change the chromatin structure facilitating the access to DNA repair enzymes. An alternative possibility is that 5-azaC can reactivate methylated genes involved in the repair of DNA double-strand breaks induced by topo II inhibitors.

Protective effects of the amino acid glutamine and of ascorbic acid against chromosomal damage induced by doxorubicin in mammalian cells.

The interaction of antioxidants can provide an essential protection against the damaging effects of free radicals. Beneficial interactions include radioprotection, protection against acute toxicity of chemicals, and antimutagenic and anticarcinogenic activity. The present study was undertaken to evaluate the protective effect of the amino acid glutamine (GLN) and ascorbic acid (AA) on the frequency of chromosomal aberrations induced by the antineoplastic agent doxorubicin (DXR). These micronutrients were tested separately and simultaneously in Wistar rat bone marrow and Chinese hamster ovary (CHO) cells. The treatments with GLN and/or AA significantly decreased the frequency of DXR-induced clastogenic damage in both test systems.

Effects of high doses of vitamins C and E against doxorubicin-induced chromosomal damage in Wistar rat bone marrow cells.

Doxorubicin (DXR) is one of the major antitumoral agents available for clinical use. In addition to intercalating into the DNA molecule, this drug generates free radicals. Vitamins C (VC) and E (VE) can protect normal cells from the damage caused by radicals without interfering with the cytotoxicity of DXR against tumors. The objective of the present study was to investigate the possible protective effect of VC and/or VE on mammalian cells treated with DXR in vivo. Animals treated with the lowest doses of VC and/or VE, alone or in combination, plus a single dose of DXR presented a statistically significant reduction in total number of chromosome aberrations and in number of abnormal metaphases. The highest vitamin doses tested caused no changes in the parameters analyzed when compared with control. Under the present experimental conditions, the efficiency of VC and/or VE in protecting against chromosome damage was dependent on the dose used.

In vitro and in vivo study of the clastogenicity of the flavone cirsitakaoside extracted from Scoparia dulcis L. (Scrophulariaceae).

The mutagenic effect of the flavone cirsitakaoside extracted from the medicinal herb Scoparia dulcis was evaluated in vitro by using human peripheral blood cultures treated with doses of 5, 10, and 15 microg of the flavone/ml culture medium for 48 h. The compound proved to be mutagenic at the highest concentration tested (15 microg/ml). Furthermore, the proliferative index was significantly reduced in all cultures treated with the flavone, although the mitotic index was not reduced. However, the clastogenic activity of the flavone cirsitakaoside was not observed when Swiss mice were treated orally with doses of 10, 20, and 30 mg/animal for 24 h.

Mutagenic and cytotoxic activity of an isocoumarin (Paepalantine) isolated from Paepalanthus vellozioides.

A new isocoumarin with antimicrobial activity was isolated from Paepalanthus vellozioides (a native Brazilian plant) and called paepalantine. This study was carried out to assess the mutagenic activity of this new agent in assays with Salmonella typhimurium TA100, TA98, and TA102 and in Chinese hamster ovary (CHO) cell cultures, as well as cytotoxicity to McCoy cells. paepalantine caused a significant dose-dependent increase in the frequency of revertants in the three strains used in the assay, both with and without S9 mix, in concentrations varying from 2 to 128 micrograms/plate. The mutagenicity was confirmed in assays with CHO cells treated in the G1, S, and G2 phases of the cell cycle. There was an increase in the chromosomal aberration frequency, mainly in the G2 phase. Furthermore, the mitotic index of the treated cultures (40,80, and 160 micrograms/ml) was significantly lower, indicating cytotoxicity. The midpoint cytotoxicity values of McCoy cells by the neutral red (NR) and microculture tetrazolium (MTT) techniques resulted in a NR50 and MTT50 of 30 and 38 micrograms/ml, respectively. Alterations to the paepalantine structure are suggested to reduce its mutagenic and cytotoxic activity in investigations for its antineoplastic potential.