Artesunate does not affect oocyte maturation and early embryo development of bovine.

Despite the cytotoxicity and embryotoxicity previously reported artesunate is a recommended drug to treat malaria for adults, children, and women in the first trimester of pregnancy. To address the putative effects of artesunate on female fertility and preimplantation embryo development, when the pregnancy is not detectable yet, artesunate was added to the oocyte in vitro maturation and in vitro embryo development of bovine. Briefly, in experiment 1 the cumulus-oocyte complexes (COCs) were in vitro matured for 18 h with 0.5, 1, or 2 µg/mL of artesunate or not (negative control) and then checked for nuclear maturation and subsequent embryo development. In experiment 2, the COCs were in vitro matured and fertilized without artesunate, which was added (0.5, 1, or 2 µg/mL) from the 1st to the 7th day of embryo culture along with a negative and a positive control group with doxorubicin. As a result, the use of artesunate on oocyte in vitro maturation did not differ from the negative control (p > 0.05) regarding nuclear maturation, cleavage, and blastocyst formation. Also, artesunate on in vitro embryo culture did not differ from negative control (p > 0.05) regarding cleavage and blastocyst formation, except for positive control, with doxorubicin (p < 0.05). In conclusion, under the conditions investigated, there was no evidence of artesunate toxicity on oocyte competence and the preimplantation period of in vitro embryo development in the bovine model, however, artesunate use still should be taken carefully as the outcome of implantation after oocytes and blastocysts exposure to artesunate remains unknown.

Artesunate added to in vitro maturation did not impair oocyte competence in bovine.Artesunate added to in vitro culture did not affect cleavage and blastocyst formation.No evidence of artesunate toxicity in oocytes and embryos of bovine was found.

Study of Genotoxicity, Activities on Caspase 8 and on the Stabilization of the Topoisomerase Complex of Isoeleutherin and Analogues.

This study evaluated the genotoxicity of Ethanol Extract (EEEp), Dichloromethane Fraction (FDCMEp) and isoeleutherin isolated from Eleutherine plicata, using the micronucleus test and the impact of structural alterations on toxicity and molecular docking (topoisomerase II and DNA complex). The extract was obtained by maceration and fractionation in a chromatography column. The genotoxicity was evaluated by the micronucleus test in human hepatoma cells (HepG2). Isoeleutherin was the starting molecule in the search for analogues by structural similarity, using the ZINC and e-Molecules databases. Isoeleutherin and analogues were subjected to in silico toxicity prediction, and compounds free of toxicological risks (CP13, CP14, CP17 and isoeleutherin) were selected for molecular docking in Topoisomerase II (PDB: 1ZXM). In the micronucleus test, isoeleutherin was less genotoxic. Among the 22 isoeleutherin analogues there were variations in the toxicity profile. Molecular docking studies showed that the compounds have good complementarity in the active site with important hydrogens bonds. Therefore, the structural changes of isoeleutherin led to the obtaining of a molecule with a lower mutagenic potential, and the CP13 can be considered a prototype compound for the development of new molecules with pharmacological potential.

Antitumoral effect of novel synthetic 8-hydroxy-2-((4-nitrophenyl)thio)naphthalene-1,4-dione (CNN16) via ROS-mediated DNA damage, apoptosis and anti-migratory effect in colon cancer cell line.

Colorectal cancer (CRC) is estimated as the third most incident cancer and second in mortality worldwide. Moreover, CRC metastasis reduces patients' survival rates. Thus, the study and identification of new compounds with anticancer activity selectively to tumor cells are encouraged in the CRC treatment. Naphtoquinones are compounds with several pharmacologic activities, including antitumoral properties. Therefore, this study aimed to investigate the anticancer mechanism of synthetic 8-Hydroxy-2-(P-Nitrothiophenol)-1,4-Naphthoquinone (CNN16) in colon cancer cell line HCT-116. CNN16 showed an IC50 of 5.32 µM in HCT-116, and 9.36, 10.77, and 24.57 µM in the non-cancerous cells MRC-5, MNP-01, and PMBC, respectively, evaluated by the MTT assay. CNN16 showed an anticlonogenic effect in HCT-116 and induced cell fragmentation identified by flow cytometry analysis. Furthermore, we observed that CNN16 presented genotoxicity and induces reactive oxygen species (ROS) after 3 h of treatment visualized by alkaline comet assay and DCFH-DA dye fluorescence, respectively. Furthermore, CNN16 caused cellular membrane disruption, reduction in the mitochondrial membrane polarization, and the presence of apoptotic bodies and chromatin condensation was visualized by differential stained (HO/FD/PI) in fluorescent microscopy along with PARP1, TP53, BCL-2, and BAX analyzed by RT-qPCR. Results also evidenced inhibition in the migratory process analyzed by wound healing assay. Therefore, CNN16 can be considered as a potential new leader molecule for CRC treatment, although further studies are still necessary to comprehend the effects of CNN16 in in vivo models to evaluate the anti-migratory effect, and toxicology and assure compound safety and selectively.

In Vitro Assessment of Cytoprotective Effects of CANOVA against Cell Death Induced by the Anti-malarial Artesunate - A Preliminary Experiment.

BACKGROUND: Artesunate (ATS) is a semi-synthetic compound derived from artemisinin, which is widely accepted in the treatment of malaria. However, there is evidence that ATS, under certain in vitro conditions, induces several impairments to normal cell functions. Canova (CA) is a Brazilian homeopathic formulation indicated for patients with depressed immune system. CA shows both in vitro and in vivo protective effects against mutagenic/carcinogenic compounds. Therefore, we aimed to assess in vitro the cytoprotective effects of CA against the cytotoxicity of ATS in Vero cells. METHODS: Viability of Vero cells exposed to ATS was assessed by MTT assay, whereas the anti-cytotoxic effect of CA was evaluated by apoptosis and necrosis quantification with fluorescent dyes. RESULTS: After 24 hours of ATS treatment, a reduction in cell viability was observed at 32 and 64 µg/mL, the latter being statistically significant (p < 0.05) in relation to the negative control. The concentration of 64 µg/mL was chosen for the subsequent experiments. ATS significantly induced both apoptosis and necrosis in Vero cells in relation to controls (p < 0.01). We also observed a statistically significant decrease in the number of apoptotic cells observed in the CA 16% + ATS co-treatment compared with ATS treatment (p < 0.01). Treatment with CA alone also had no influence on either type of cell death. CONCLUSION: Our results demonstrated that ATS is cytotoxic in the assessed conditions. However, such cytotoxicity was attenuated when the cells were treated simultaneously with ATS and CA.

In vitro assessment of cytotoxic, genotoxic and mutagenic effects of antimalarial drugs artemisinin and artemether in human lymphocytes.

Artemisinin is a substance extracted from the Chinese plant Artemisia annua L. widely used in natural medicine for the treatment of various diseases. Artemether is a substance synthesized from artemisinin, and both drugs are commonly administered in the treatment of malaria. Although considered effective antimalarial drugs, very little is known about the genotoxic, cytotoxic and mutagenic effects of these drugs. Therefore, in the present study, we evaluated the genotoxic, mutagenic and cytotoxic effects of artemisinin (12.5, 25 and 50 µg/mL) and artemether (7.46; 14.92 and 29.84 µg/mL) in cultured human lymphocytes using the comet assay, the micronucleus test and the cytotoxicity assay for detection of necrosis and apoptosis by acridine orange/ethidium bromide staining. Our results showed a significant increase (p < 0.05) in the rate of DNA damage measured by comet assay and in the micronucleus frequency after treatment with both drugs. It was also observed that only artemisinin induced a statistically significant increase (p < 0.05) in the number of lymphocytes with death by necrosis 48 h after treatment. The results demonstrated that these two drugs induce mutagenic, genotoxic and cytotoxic effects in cultured human lymphocytes. Our data indicate the need for caution in the use of such drugs, since genotoxic/mutagenic effects may increase the risk of carcinogenesis.

In vitro assessment of anticytotoxic and antigenotoxic effects of CANOVA(®).

BACKGROUND: CANOVA(®) (CA) is a homeopathic immunomodulator. It contains several homeopathic medicines prepares according to the Brazilian Pharmacopoeia. CA is indicated in clinical conditions in which the immune system is impaired and against tumors. N-methyl-N-nitrosourea (NMU) is an N-nitroso compound, with genotoxic/mutagenic properties. Although several studies have shown promising results in the use of CA, there are no studies reporting possible antigenotoxic effects. METHOD: This study evaluated the in vitro antigenotoxic and anticytotoxic effects of CA in human lymphocytes exposed to NMU. Samples of human lymphocytes that were subjected to different concentrations of a mixture containing CA and NMU were used. The genotoxicity/antigenotoxicity of CA was evaluated by the comet assay, anticytotoxicity was assessed by quantification of apoptosis and necrosis using acridine orange/ethidium bromide. RESULTS: CA significantly reduced DNA damage induced by NMU and reduced significantly the frequency of NMU-induced apoptosis after 24 h of treatment. CONCLUSION: CA has an important cytoprotective effect significantly reducing the DNA damage and apoptosis induced by the carcinogen NMU.

Quantitative difference of oral pathogen between individuals with gastric cancer and individuals without cancer.

The loss of teeth and lack of oral hygiene have been associated with the risk of developing gastric cancer (GC) in several populations evidenced in epidemiological studies. In this study, we quantitatively compared the proportion of oral pathogens in individuals with gastric cancer and individuals without cancer in a referral hospital in the city of Belém, Brazil. This study evaluated 192 patients with GC and 192 patients without cancer. Periodontal clinical examination was performed, and all individuals were submitted to the collection of salivary and dental biofilms. When comparing the median periodontal indexes in the gastric and cancer-free groups, it was statistically significant (p < 0.001) in the gastric cancer group compared to the probing depth of the periodontal pocket. Levels of bacterial DNA were observed in saliva and dental plaque, with a statistically significant difference (p < 0.001) between individuals with cancer and without neoplasia in all the bacteria surveyed. Significant relationships (p < 0.001) between biological agents and GC have been found in bacterial species that cause high rates of periodontal pathology and caries. The results suggest a different quantitative association in the presence of oral pathogens between individuals without cancer and patients with GC. As noted, it cannot be said that the bacteria present in the oral cavity increase the risk of gastric cancer or are aggravating factors of the disease. However, it is worth mentioning that, as it is part of the digestive system, the lack of care for the oral cavity can negatively affect the treatment of patients with gastric cancer.

Cytogenetic biomonitoring of inhabitants of a large uranium mineralization area: the municipalities of Monte Alegre, Prainha, and Alenquer, in the State of Pará, Brazil.

Uranium is a natural radioactive metallic element; its effect on the organism is cumulative, and chronic exposure to this element can induce carcinogenesis. Three cities of the Amazon region-Monte Alegre, Prainha, and Alenquer-in North Brazil, are located in one of the largest uranium mineralization areas of the world. Radon is a radioactive gas, part of uranium decay series and readily diffuses through rock. In Monte Alegre, most of the houses are built of rocks removed from the Earth's crust in the forest, where the uranium reserves lie. The objective of the present work is to determine the presence or absence of genotoxicity and risk of carcinogenesis induced by natural exposure to uranium and radon in the populations of these three cities. The frequency of micronuclei (MN) and chromosomal aberrations (CA) showed no statistically significant differences between the control population and the three study populations (P > 0.05). MN was also analyzed using the fluorescence in situ hybridization (FISH) technique, with a centromere-specific probe. No clastogenic and/or aneugenic effects were found in the populations. Using FISH analysis, other carcinogenesis biomarkers were analyzed, but neither the presence of the IGH/BCL2 translocation nor an amplification of the MYC gene and 22q21 region was detected. Clastogenicity and DNA damage were also not found in the populations analyzed using the alkaline comet assay. The mitotic index showed no cytotoxicity in the analyzed individuals' lymphocytes. Once we do not have data concerning radiation doses from other sources, such as cosmic rays, potassium, thorium, or anthropogenic sources, it is hard to determine if uranium emissions in this geographic region where our study population lives are too low to cause significant DNA damage. Regardless, genetic analyses suggest that the radiation in our study area is not high enough to induce DNA alterations or to interfere with mitotic apparatus formation. It is also possible that damages caused by radiation doses undergo cellular repair.

Genotoxic effects of white fluorescent light on human lymphocytes in vitro.

Sources of light beams such as white fluorescent light, are present in our daily life to meet the needs of life in the modern world. This study was conducted with the objective of determining the possible genotoxic, cytotoxic and aneugenic effects caused by this agent in different stages of the cell cycle (G0/early G1, S, and late G2), using different cytogenetic parameters (sister chromatid exchanges--SCE, chromosome aberrations--CA, and detection of aneugenic effects) in lymphocytes from temporary cultures of human peripheral blood. WFL showed a genotoxic effect in vitro, expressed by an increase in the frequency of SCE's, regardless of the cell cycle stage. However, no increase in the frequency of CAs was observed. In addition, disturbances in cell cycle kinetics and chromosomal segregation were also observed. Taken together, such data may contribute to a better understanding and a different management in the use of phototherapy for some pathological conditions.

Cytotoxic and Genotoxic Effects of Fluconazole on African Green Monkey Kidney (Vero) Cell Line.

Fluconazole is a broad-spectrum triazole antifungal that is well-established as the first-line treatment for Candida albicans infections. Despite its extensive use, reports on its genotoxic/mutagenic effects are controversial; therefore, further studies are needed to better clarify such effects. African green monkey kidney (Vero) cells were exposed in vitro to different concentrations of fluconazole and were then evaluated for different parameters, such as cytotoxicity (MTT/cell death by fluorescent dyes), genotoxicity/mutagenicity (comet assay/micronucleus test), and induction of oxidative stress (DCFH-DA assay). Fluconazole was used at concentrations of 81.6, 163.2, 326.5, 653, 1306, and 2612.1µM for the MTT assay and 81.6, 326.5, and 1306µM for the remaining assays. MTT results showed that cell viability reduced upon exposure to fluconazole concentration of 1306µM (85.93%), being statistically significant (P<0.05) at fluconazole concentration of 2612.1µM (35.25%), as compared with the control (100%). Fluconazole also induced necrosis (P<0.05) in Vero cell line when cells were exposed to all concentrations (81.6, 326.5, and 1306µM) for both tested harvest times (24 and 48 h) as compared with the negative control. Regarding genotoxicity/mutagenicity, results showed fluconazole to increase significantly (P<0.05) DNA damage index, as assessed by comet assay, at 1306µM versus the negative control (DI=1.17 vs DI=0.28, respectively). Micronucleus frequency also increased until reaching statistical significance (P<0.05) at 1306µM fluconazole (with 42MN/1000 binucleated cells) as compared to the negative control (13MN/1000 binucleated cells). Finally, significant formation of reactive oxygen species (P<0.05) was observed at 1306µM fluconazole vs the negative control (OD=40.9 vs OD=32.3, respectively). Our experiments showed that fluconazole is cytotoxic and genotoxic in the assessed conditions. It is likely that such effects may be due to the oxidative properties of fluconazole and/or the presence of FMO (flavin-containing monooxygenase) in Vero cells.

Effect of diterpenoid kaurenoic acid on genotoxicity and cell cycle progression in gastric cancer cell lines.

The goal of our study was to evaluate the effect of kaurenoic acid, obtained from copaiba oil resin, in gastric cancer (GC) and a normal mucosa of stomach (MNP01) cell lines. The compound was tested at concentrations of 2.5, 5, 10, 30 and 60µg/mL. Comet and micronucleus assays were used to access its potential genotoxicity in vitro. Moreover, we evaluated the effect of kaurenoic acid in cell cycle progression and in the transcription of genes involved in the control of the cell cycle: MYC, CCND1, BCL2, CASP3, ATM, CHK2 and TP53. Kaurenoic acid induced an increase on cell DNA damage or micronucleus frequencies on GC cell lines in a dose-dependent manner. The GC and MNP01 cell lines entering DNA synthesis and mitosis decreased significantly with kaurenoic acid treatment, and had an increased growth phase compared with non-treated cells. The treatment induced apoptosis (or necrosis) even at a concentration of 2.5µg/mL in relation to non-treated cells. GC cell lines presented reduced MYC, CCND1, BCL2 and CASP3 transcription while ATM, CHK2 and TP53 increased in transcription in relation to non-treated cells, especially at a concentration above 10µg/mL. The gene transcription in the MNP01 (non-treated non-cancer cell line) was designated as a calibrator for all the GC cell lines. In conclusion, our results showed that kaurenoic acid obtained from Copaifera induces DNA damage and increases the micronuclei frequency in a dose-dependent manner in GC cells, with a significant genotoxicity observed above the concentration of 5µg/mL. Moreover, this compound seems to be able to induce cell cycle arrest and apoptosis in GC cells.

Genotoxic effects of rotenone on cultured lymphocytes.

Rotenone is a heterocyclic compound widely used as an insecticide, acaricide and piscicide. Its toxicity is mainly caused by the inhibition of mitochondrial respiratory processes and ATP production, resulting in the generation of reactive oxygen species. Reactive oxygen species can interact with DNA, RNA and proteins, leading to cell damage, followed by death. We used the Comet assay, and we analyzed chromosome aberrations, in order to evaluate the genotoxic and clastogenic effects of rotenone on the different phases of the cell cycle. Cultured human lymphocytes were treated with 1.0, 1.5 and 2.0 microg/mL rotenone during the G1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle. Rotenone induced DNA damage and was clastogenic, but the clastogenicity was detected only with treatments conducted during the G1/S and S phases of the cell cycle. Rotenone also induced endoreduplication and polyploidy in treatments made during G1, while it significantly reduced the mitotic index in all phases of the cell cycle.

Genotoxic effects of the antimalarial drug lumefantrine in human lymphocytes in vitro and computational prediction of the mechanism associated with its interaction with DNA.

Lumefantrine (LF) is an aryl-amino alcohol antimalarial drug used in artemisinin-based combination therapies against malaria worldwide. In this study, we investigated the genotoxic effects of LF in human lymphocytes in vitro, and the potential noncovalent interaction of LF with DNA using a 3D DNA-docking model. The number of DNA breaks and the frequency of nuclear buds (NBUDS) was significantly increased (P < 0.01 and P < 0. 05, respectively) at LF concentrations of 60, 80, and 100 µg/mL (LF60, LF80, and LF100, respectively). Frequency (‰) of micronuclei (MN) formation also increased after LF treatments. However, this was only significant for LF100 (P = 0.01) and LF80 (P = 0.001). LF did not affect the frequency of nucleoplasmic bridges (NPBs) (P = 0.12) or the nuclear division index (NDI) (P = 0.32). Computational analysis suggests that LF may interact noncovalently with DNA via the DNA minor groove surface with a predicted binding affinity energy of -7.2 kcal/mol and showing a favorable shape complementary to this groove. Our results suggest that LF has clastogenic effects in human lymphocytes in vitro due to noncovalent interaction with the minor groove of DNA.

Evaluation of the mutagenic activity of hydroxyurea on the G1-S-G2 phases of the cell cycle: an in vitro study.

Hydroxyurea is considered an antineoplastic drug, which also plays an important role in the treatment of sickle cell anemia patients. We evaluated and compared the clastogenic and cytotoxic effects of hydroxyurea, using chromosomal aberrations and mitotic index, respectively, as endpoints. In vitro short-term cultures of lymphocytes were exposed to several concentrations of this drug, at various cell cycle phases. There was a significant increase in the cytotoxicity of hydroxyurea at G1 and G1/S as well in the G2 phase of the cell cycle. Hydroxyurea did not significantly increase chromosome aberrations. There was an S-dependent cytotoxic effect of hydroxyurea, which is expected based on the known activity of hydroxyurea as an inhibitor of ribonucleotide reductase.

Studies of micronuclei and other nuclear abnormalities in red blood cells of Colossoma macropomum exposed to methylmercury

The frequencies of micronuclei (MN) and morphological nuclear abnormalities (NA) in erythrocytes in the peripheral blood of tambaqui (Colossoma macropomum), treated with 2 mg.L-1 methylmercury (MeHg), were analyzed. Two groups (nine specimens in each) were exposed to MeHg for different periods (group A - 24 h; group B - 120 h). A third group served as negative control (group C, untreated; n = 9). Although, when compared to the control group there were no significant differences in MN frequency in the treated groups, for NA, the differences between the frequencies of group B (treated for 120 h) and the control group were extremely significant (p < 0.02), thus demonstrating the potentially adverse effects of MeHg on C. macropomum erythrocytes after prolonged exposure.

Review article: chromosomal diagnosis in rhabdomyosarcoma

Os rabdomiossarcomas (RMS) são considerados tumores clinicamente agressivos com origem a partir de células mesenquimais imaturas e que se caracterizam pela presença de células com diferenciação pouco definida. O emprego das técnicas citogenéticas convencionais em RMS vem contribuindo consideravelmente para a diferenciação entre os rabdomiossarcomas alveolares e os outros tumores de células pequenas e redondas, além de fornecer informações prognósticas importantes referente ao rabdomiossarcoma do tipo alveolar. Assim, este trabalho visa a realizar uma revisão das alterações citogenéticas observadas nos diferentes subtipos histológicos de RMS, enfocando não só os trabalhos de citogenética convencional, mas também novas abordagens utilizadas para o estudo de neoplasias tais como FISH, CGH, SKY e M-FISH. Tais metodologias vêm contribuindo de maneira significativa para a melhor compreensão da heterogeneidade cariotípica observada nos RMS.

Mutagenicity of hydroxyurea in lymphocytes from patients with sickle cell disease

Hydroxyurea is commonly used in the treatment of myeloproliferative diseases and in patients with sickle cell disease (SCD). The use of this antineoplastic agent in patients with SCD is justified because of the drug's ability to increase fetal hemoglobin levels, thereby decreasing the severity of SCD. However, high doses or prolonged treatment with hydroxyurea can be cytotoxic or genotoxic for these patients, with an increased risk of developing acute leukemia. This danger can be avoided by monitoring the lymphocytes of patients treated with hydroxyurea. Cytogenetic tests are important endpoints for monitoring the physiological effects of physical and chemical agents, including drugs. In this work, we assessed the genotoxicity of hydroxyurea in short-term cultures of lymphocytes from SCD patients. Hydroxyurea was not cytotoxic or genotoxic at the concentrations tested in the G2 phase of the cell cycle. These results support the use of hydroxyurea in the treatment of SCD, although further work is necessary to understand the effects of this drug in vivo.

Cytogenetics in pediatric medulloblastomas

A meduloblastoma é um tipo de tumor frequentemente encontrada na infância e adolescência. É geralmente encontrada na fossa posterior (também conhecida como o compartilhamento infratentorial) e afeta, principalmente, o cerebelo. Embora um progresso significativo tenha sido alcançado no tratamento destes pacientes, muitos aspectos do comportamento biológico deste tumor ainda estão incertos. Portanto o estudo dos eventos genéticos envolvidos nestas neoplasias poderia ser considerado uma ferramenta útil na compreensão destes tumores, desde que, o comportamento biológico de um tumor passou a ser determinado pelas suas alterações genéticas. Em meduloblastoma, a alteração observada com maior frequência é um isocromossomo 17, mas sozinha esta aberração não constitui um fator prognóstico; apenas reflete uma proliferação celular desenfreada. Vários genes (por exemplo, TP53, ABR e HIC-1) parecem estar relacionados à gênese destes tumores, mas estudos mais aprofundados são necessários para esclarecer o assunto. Os avanços no campo de citogenética molecular tem permitido a gênese de processos de proliferação. Portanto, o objetivo desta revisão é apresentar uma revisão atualizada da literatura sobre meduloblastoma.