Comparative study of the cytotoxicity and genotoxicity of kaurenoic acid and its semi-synthetic derivatives methoxy kaurenoic acid and kaurenol in CHO-K1 cells.

The diterpene kaurenoic acid (KA) has vasorelaxant, antimicrobial, anti-tumoural and anti-leishmanial effects. Semi-synthetic derivatives were obtained to achieve more satisfactory responses. The assessment of genotoxicity is part of the toxicological evaluation of therapeutic compound candidates. The present study investigated the cytotoxicity and genotoxicity of KA and its semi-synthetic derivatives methoxy kaurenoic acid (MKA) and kaurenol (KRN) using the CHO-K1 cell line. The cytotoxicity evaluation demonstrated that treatments with 200 and 400 µM KA reduced cellular proliferation to 36.5 and 4.43%, respectively, and that 100 and 200 µM KA reduced the survival fraction (SF) to 48.1 and 5.5%, respectively. MKA and KRN at concentrations of 400 µM reduced proliferation to 81 and 86.8%, respectively, while 100 and 200 µM KRN reduced the SF to 50%, and 200 µM MKA reduced the SF to 74%. No genotoxicity was observed for KA or MKA. However, 100 µM KRN increased the DNA damage index, as detected by comet assay, although a micronucleus assay did not confirm these data. The results demonstrated that KA and its semi-synthetic derivative MKA were not genotoxic when tested at noncytotoxic concentrations, but KRN was genotoxic at the highest concentration that was tested, as demonstrated by the comet assay.

The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.

A combination of the cytokinesis-block micronucleus cytome assay and centromeric identification for evaluation of the genotoxicity of dicamba.

The purpose of this study was to further investigate the cytotoxic and genotoxic effects of dicamba and Banvel(®) employing the cytokinesis-block micronucleus cytome (CBMN-cyt) assay estimated by the analysis of the nuclear division index (NDI), the frequency of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs). Besides, for mechanism of MN induction CREST anti-kinetochore antibody analysis was performed. The activities of both compounds were tested within the range of 50-500 µg/ml on Chinese hamster ovary (CHO-K1) cells. Overall, dicamba and Banvel(®) produced a NDI dose-dependent decrease but the response was statistically significant only in cultures treated with Banvel(®) at a 100-500 µg/ml concentration range. A dose-dependent induction of MN was observed after dicamba- and Banvel(®)-treatments within the 50-400 µg/ml and 50-500 µg/ml concentration-ranges, respectively. Induction of NPBs and NBUDs was significantly enhanced by both test compounds. The NPBs/MN ratio values found for dicamba and Banvel(®) were 0.04-0.11 and 0.05-0.18, respectively. Results clearly demonstrated that dicamba and Banvel(®) exerted both cyto- and genotoxic damage on CHO-K1 cells. Furthermore, the CBMN-cyt assay employed confirmed our previous investigations concerning the cellular and DNA damaging capabilities of dicamba and highlights that both clastogenic and aneugenic mechanisms are implicated in the MN induction.

Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro.

INTRODUCTION: The purpose of this study was to evaluate whether corrosion eluates obtained from commercially available orthodontic brackets are able to induce genetic damage in vitro. MATERIAL AND METHODS: Genotoxicity was assessed by the single cell gel (comet) assay using Chinese hamster ovary (CHO) cells. The following orthodontic metallic brackets were used: Morelli (Sorocaba, Brazil); Abzil (São José do Rio Preto, Brazil); Dentaurum (Pforzheim, Germany); and 3M Unitek (Puchheim, Germany). Each dental bracket was submitted to a corrosion process in a solution containing equal amounts of acetic acid and sodium chloride at 0.1 M concentration for 1, 3, 7, 14, 21, 35, and 70 days. CHO cells were exposed to eluates for 30 minutes at 37°C. The negative control was treated with the same solution used for corrosion process for 30 minutes at 37°C. Independent positive control was performed with methyl methanesulfonate (MMS) (Sigma Aldrich, St. Louis, Mo) at 1 ug/mL for 1 hour. RESULTS: None of the eluates was found to exhibit genotoxicity, regardless of the different commercial brands of orthodontic appliance used. CONCLUSIONS: In summary, our results indicate corrosion eluates obtained from orthodontic brackets do not induce genetic damage as assessed by single cell gel (comet) assay.

Genetic diversity and toxic activity of Aggregatibacter actinomycetemcomitans isolates.

INTRODUCTION: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes. METHODS: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB. RESULTS: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells. CONCLUSION: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.

Effects of copper ions released from metallic copper on CHO-K1 cells.

The aim of this study was to investigate the cytotoxic and genotoxic effect of copper extracts obtained from metallic copper in Chinese hamster ovary (CHO-K1) cell line using neutral red (NR), sister chromatid exchange (SCE), chromosomal aberrations (CA) and cell-cycle kinetics tests. Cells were cultured in Ham-F10 with different copper-containing extracts obtained after the immersion of copper disks for 1, 2, 3, 9, 12, 24, 48 and 72 h in culture medium. Results from cytotoxicity assay showed an inverted U-shape response evidenced in changes in lysosomal activity and mitotic index. The analysis of CA revealed an increase of abnormal metaphases for copper concentration (cCu) in the 5.67-7.42 mg/L dose-range (p<0.001). In addition, SCE frequencies were higher for treated cells when compared with controls in the 1.56-7.42 mg/L concentration range (p<0.001). The absence of metaphases indicated cytotoxicity for cCu≥10.85 mg/L. Results show that cells close to copper-containing materials releasing copper ions are susceptible to cytotoxic and genotoxic effects.

Genotoxicity of corrosion eluates obtained from endosseous implants.

PURPOSE: Commercially pure titanium alloys are currently used as metallic biomaterials in implantology. Corrosion phenomena appear to play a decisive role in metallic implant long-term behavior. Thus, the goal of this study was to examine the genotoxic potential of corrosion eluates obtained from dental implants using Chinese ovary hamster cells in vitro by the single-cell gel (comet) assay. This technique detects deoxyribonucleic acid strand breaks in individual cells in alkaline conditions. MATERIALS AND METHODS: The materials tested included 3 dental implants commercially available. Each of the tested materials was corroded in a solution consisting of equal amounts of acetic acid and sodium chloride (0.1 M) for 1, 3, 7, 14, and 21 days. The Chinese ovary hamster cultures were then exposed to all corrosion eluates obtained from endosseous dental implants for 30 minutes at 37 degrees C. RESULTS: None of the eluates was found to exhibit genotoxicity, regardless of the type of dental implant used. CONCLUSION: The results suggest that all dental implants tested in this study did not induce deoxyribonucleic acid breakage as depicted by the single-cell gel (comet) assay.

Biocompatibility of gutta-percha solvents using in vitro mammalian test-system.

OBJECTIVES: Taking into consideration that DNA damage and cellular death play important roles during carcinogenesis, the purpose of the present study was to evaluate in vitro genotoxic or cytotoxic effects of chloroform and eucalyptol by single cell gel (comet) assay and trypan blue exclusion test, respectively. STUDY DESIGN: Chloroform and eucalyptol were exposed to Chinese hamster ovary cells in culture directly for 3 hours at 37 degrees C at final concentrations ranging from 1.25 to 10 microL/mL. The negative control group was treated with vehicle control (phosphate-buffered solution), and the positive control group was treated with methyl metasulfonate (MMS, at 1 microg/mL concentration). All data were analyzed by the Kruskal-Wallis nonparametric test followed by the Dunn test. RESULTS: The results showed that both gutta-percha solvents were cytotoxic at concentrations of 2.5, 5, and 10 microL/mL (P < .05). On the other hand, both solvents did not induce DNA breakage at 1.25 microL/mL concentration. CONCLUSIONS: These results suggest that both chloroform or eucalyptol are strong cytotoxicants, but they may not be a factor that increases the level of DNA lesions in mammalian cells.

Butylated hydroxytoluene does not protect Chinese hamster ovary cells from chromosomal damage induced by high-dose rate 192Ir irradiation.

Previous reports showed the protective effect of the synthetic antioxidant butylated hydroxytoluene (BHT) against the chromosomal damage induced by bleomycin (BLM), cadmium chloride and potassium dichromate. To test the hypothesis that this effect was exerted by inhibition and/or scavenging of reactive oxygen species (ROS), the effect of BHT on the chromosomal damage induced by a high dose-rate gamma rays (HDR (192)Ir). Experiments were carried out by irradiating G(1) CHO cells with nominal doses of 1, 2 or 3 Gy. BHT (doses of 1.0, 2.5 or 5.0 microg/ml) was added to the culture immediately before or immediately after irradiation. Cells were then incubated in the presence of BHT for 13 h until harvesting and fixation. Results obtained showed that BHT did not decrease the chromosomal damage induced by radiation in any consistent fashion. On the contrary, in cells post-treated with 5.0 microg/ml of BHT the yield of chromosomal aberrations increased in several experimental points. These results with ionizing radiation suggest that the previous observed protective effects of BHT on the chromosomal damage induced by chemical genotoxicants may not be mediated solely through the scavenging or inactivating reactive oxidative species. The decrease of the yield of chromosomal damage induced by BLM could be due to the union of BHT with a metallic ion, in this case Fe (II), required for the activation of BLM. In the same way, the protective effect of BHT on the chromosomal damage induced by cadmium chloride and potassium dichromate could be due to the decrease of the effective dose of both salts in the cell through the chelation of the cations by BHT.

Study of DNA damage induced by dental bleaching agents in vitro.

Dental bleaching is a simple and conservative procedure for aesthetic restoration of vital and non-vital discolored teeth. Nevertheless, a number of studies have demonstrated the risk of tissue damage from the contact of these agents with the oral mucosa. In the current study, the genotoxic potential associated with exposure to dental bleaching agents was assessed by the single cell gel (comet) assay in vitro. Chinese hamster ovary (CHO) cells in vitro were exposed to six commercial dental bleaching agents (Clarigel Gold - Dentsply; Whitespeed - Discus Dental; Nite White - Discus Dental; Magic Bleaching - Vigodent; Whiteness HP - FGM and Lase Peroxide - DMC). The results pointed out that all dental bleaching agents tested contributed to DNA damage as depicted by the mean tail moment, being the strongest effect observed with the highest dose of hydrogen peroxide (Whiteness HP and Lase Peroxide, at a 35% concentration). On the other hand, Magic Bleaching (Vigodent) induced the lowest level of DNA breakage. Negative and positive controls displayed absence and presence of DNA-damaging, respectively. Taken together, these results suggest that dental bleaching agents may be a factor that increases the level of DNA damage. A higher concentration of hydrogen peroxide produced higher noxious activities in the genome as detected by single cell gel (comet) assay.

The antibody to GD3 ganglioside, R24, is rapidly endocytosed and recycled to the plasma membrane via the endocytic recycling compartment. Inhibitory effect of brefeldin A and monensin.

Gangliosides are sialic acid-containing glycosphingolipids present on mammalian plasma membranes, where they participate in cell-surface events such as modulation of growth factor receptors and cell-to-cell and cell-to-matrix interactions. Antibodies to gangliosides have been associated with a wide range of clinically identifiable acute and chronic neuropathy syndromes. In addition, antibodies to tumor-associated gangliosides are being used as therapeutic agents. Their binding to and release from cell membranes and intracellular destinations have not so far been extensively examined. In this study, we characterized in both GD3 ganglioside-expressing Chinese hamster ovary (CHO)-K1 and SK-Mel 28 melanoma cells the intracellular trafficking and subcellular localization of the mouse monoclonal antibody to GD3, R24. By biochemical techniques and detailed confocal microscopic analysis, we demonstrate that the GD3-R24 antibody complex is rapidly and specifically internalized by a dynamin 2-independent pathway and then accumulates in the endocytic recycling compartment. In addition, we show that the R24 antibody exits the recycling compartment en route to the plasma membrane by a dynamin 2-dependent pathway sensitive to brefeldin A and monensin. Taken together, our results indicate that the GD3-R24 complex is endocytosed in GD3-expressing cells, accumulates in the recycling endosome, and is transported back to the plasma membrane via a route that involves clathrin-coated vesicles.

Study of DNA damage induced by dental bleaching agents in vitro

Clareamento dental é um procedimento simples e conservador para restaurar esteticamente a cor de dentes vitais e não-vitais. Entretanto, alguns estudos têm demonstrado o risco de dano tecidual a partir do contato desses agentes com a mucosa bucal. Neste presente estudo, o potencial genotóxico associado à exposição aos agentes clareadores dentais foi avaliado pelo teste de células individualizadas em gel (teste do cometa) in vitro. Células de ovário de hamster chinês (CHO) in vitro foram expostas a seis agentes clareadores dentais comercialmente disponíveis (Clarigel Gold – Dentsply; Whitespeed – Discus Dental; Nite White – Discus Dental; Magic Bleaching – Vigodent; Whiteness HP – FGM e Lase Peroxide – DMC). Os resultados mostraram que todos os agentes clareadores testados contribuíram para os danos no DNA, como demonstrado pela média do momento da cauda, sendo o efeito mais forte observado na mais alta dose de peróxido de hidrogênio (Whiteness HP e Lase Peroxide, na concentração de 35%). Por outro lado, Magic Bleaching (Vigodent) induziu o menor nível de quebras no DNA. Os controles negativo e positivo apresentaram ausência e presença de danos no DNA, respectivamente. Em suma, esses resultados sugerem que os agentes clareadores dentais podem ser um fator que aumenta o nível de danos no DNA. Uma concentração de peróxido de hidrogênio mais elevada produziu atividades nocivas mais severas no genoma como detectado pelo teste do cometa.

Genotoxicity and cytotoxicity of mineral trioxide aggregate and regular and white Portland cements on Chinese hamster ovary (CHO) cells in vitro.

OBJECTIVE: Recently, mineral trioxide aggregate (MTA) and Portland cement have been used in dentistry as root-end-filling materials. However, the reported results concerning the biocompatibility of these materials are inconsistent. The goal of this study was to examine the genotoxicity and cytotoxicity of MTA and Portland cements in vitro by the single-cell gel (comet) assay and trypan blue exclusion test. STUDY DESIGN: Chinese hamster ovary (CHO) cells were exposed to MTA and regular and white Portland cements at final concentration ranging from 1 to 1000 microg/mL for 1 h at 37 degrees C. RESULTS: All compounds tested did not show genotoxic effects in all concentrations evaluated. No significant differences (P > .05) in cytotoxicity were observed for all compounds tested. CONCLUSIONS: Taken together, our results suggest that MTA and Portland cements are not genotoxins and are not able to induce cellular death.

Fucan inhibits Chinese hamster ovary cell (CHO) adhesion to fibronectin by binding to the extracellular matrix.

In recent years, sulfated fucans have emerged as an important class of natural biopolymers. In this study, the anti-adhesive activity of a fucan from the brown seaweed Spatoglossum schröederi was analyzed using tumorigenic cells: wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). Fibronectin (FN) was used as substrate for cell attachment. For both cell types, this fucan has shown a dose-dependent anti-adhesive effect, reaching saturation at around 400 mug/mL. This effect was abolished by desulfation of the fucan. In addition, this polymer exhibited the highest inhibitory effect in comparison to other sulfated polysaccharides. The fucan was biotinylated and used as a probe to identify its action sites. Biotinylated fucan was detected in the extracellular matrix environment by confocal microscopy and flow cytometric analysis, but not at the cell surface. The results suggest that the fucan shows anti-adhesive activity by binding directly to FN, and blocking FN sites that are recognized by cell surface ligands, possibly the integrin family.

Genotoxicity of antimicrobial endodontic compounds by single cell gel (comet) assay in Chinese hamster ovary (CHO) cells.

OBJECTIVE: In the current study, the potential DNA damage associated with exposure to a number of antimicrobial endodontic compounds was assessed by the single cell gel (comet) assay in vitro. STUDY DESIGN: Chinese hamster ovary (CHO) cells were exposed to formocresol, paramonochlorophenol, calcium hydroxide, or chlorhexidine at final concentration ranging from 0.01% to 1%. RESULTS: Formocresol, paramonochlorophenol, and calcium hydroxide, as well as chlorhexidine in all concentrations tested did not contribute to the DNA damage. CONCLUSION: These findings are clinically relevant since they represent an important contribution to the correct evaluation of the potential health risk associated with exposure to dental agents.

Fluoride does not induce DNA breakage in Chinese hamster ovary cells in vitro.

Fluoride has been widely used in dentistry because it is a specific and effective caries prophylactic agent. However, excess fluoride may represent a hazard to human health, especially by causing injury to genetic material. Genotoxicity tests represent an important part of cancer research to assess the risk of potential carcinogens. In the current study, the potential DNA damage associated with exposure to fluoride was assessed by the single cell gel (comet) assay in vitro. Chinese hamster ovary cells were exposed to sodium fluoride (NaF) at final concentration ranging from 7 to 100 micro/ml for 3 h, at 37 dgrees C. The results pointed out that NaF in all concentrations tested did not contribute to DNA damage as depicted by the mean tail moment and tail intensity. These findings are clinically important since they represent an important contribution to a correct evaluation of the potential health risk associated with the exposure to dental agents.

In vitro activity of two phenyl-carbamate derivatives, singly and in combination with albendazole against albendazole-resistant Giardia intestinalis.

Giardia intestinalis is one of the most prevalent parasites in adults and children in Mexico. Benzimidazoles have been proposed as a therapeutic alternative in the treatment of giardiasis. However, high-dose related toxicity and the development of resistance have emerged in clinical trials using this therapy. In the search of alternative drugs, we found that benzimidazole-resistant strains of fungi have shown increased sensitivity to phenyl-carbamates, hence, we developed several substituted phenyl-carbamates, two of which were tested against the protozoan parasite G. intestinalis in susceptible and albendazole-resistant Giardia strains. 4-R-ethyl-phenyl-carbamates IRE-6A and IRE-7B demonstrated antigiardial, albeit modest, activity when compared with albendazole, against susceptible and albendazole-induced resistant Giardia. However, when albendazole 0.38 microg/mL (MIC(50)) was combined with each IRE compound, a significant antigiardial synergism (fractional inhibitory concentration index (FICI < 0.5)) was obtained not only with sensitive cultures but also with resistant Giardia parasites. The results described here suggest a potential role for a combined therapy with phenyl-carbamates and sub-doses of benzimidazoles in the treatment of giardiasis.

Fluoride does not induce DNA breakage in Chinese hamster ovary cells in vitro

O flúor tem sido amplamente usado na Odontologia, pois é um agente profilático efetivo e específico contra a cárie dentária. Entretanto, o flúor em excesso pode representar perigos à saúde humana, especialmente por causar agressão ao material genético. Testes de genotoxicidade representam uma importante parte da pesquisa do câncer para a avaliação de risco de possíveis carcinógenos. Neste presente estudo, danos ao DNA associados à exposição ao flúor foram avaliados pelo teste de células individualizadas em gel de agarose (teste do cometa) in vitro. Células de ovário de hamster chinês foram expostas ao fluoreto de sódio (NaF) nas concentrações finais de 7 a 100 µg/ml, durante 3 h, a 37ºC. Os resultados mostraram que o NaF não contribuiu para os danos no DNA em todas as concentrações testadas, conforme demonstrado pelas médias do momento da cauda e da intensidade da cauda dos cometas. Esses achados são clinicamente importantes, uma vez que representam uma importante contribuição para a correta avaliação do potencial risco à saúde associada à exposição aos agentes odontológicos.

Distribution of breakpoints induced by etoposide and X-rays along the CHO X chromosome.

SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.

A mitogenic protein fraction in latex from Carica candamarcensis.

Latex from Caricaceae contains a number of proteins believed to be part of a defense mechanism that protects these plants from wounding. Prior evidence suggests that some components in Carica papaya improve healing of ulcerous wounds in mammals. This study shows the chromatographic isolation of a protein fraction from C. candamarcensis that stimulates cell proliferation of mammalian cells by measuring MTT reduction and thymidine incorporation. The effect appears to be cell specific as L929, MDA-MB231 and BHK-21 cells are stimulated while no effect is seen on CHO cells. The maximal stimulatory effect reaches 2.2-fold 72 h after addition of the active fraction to L929, 1.8-fold in MDA-MB231 cells and 1.6-fold in BHK cells. Proteolytic inactivation of the active fraction suggests that a protein is responsible for the proliferative activity and its size is estimated between 10 and 25 kDa. A potential candidate for this function is a 23 kDa protein found in the fraction that reacts with human EGF antibody.