RESUMO

Lambda-cyhalothrin (LCT) and its microformulation Karate® (25 % a.i.) were analysed for its genotoxicity and cytotoxicity on Chinese hamster ovary (CHO-K1) cells. Cytokinesis-block micronucleus cytome (CBMN-cyt) and alkaline single-cell gel electrophoresis (SCGE) bioassays were selected to test genotoxicity. Neutral red uptake (NRU), succinic dehydrogenase activity (MTT) and apoptogenic induction were employed for estimating cytotoxicity. Both compounds were analysed within a concentration range of 0.1-100 µg/mL. Only LCT produced a significant augment in the frequency of micronuclei (MNs) when the cultures were exposed to highest concentrations of 10 and 100 µg LCT/mL. A noticeable decrease in NDI was observed for cultures treated with LCT at 10 and 100 µg/mL. Karate® induced the inhibition of both the proportion of viable cells and succinic dehydrogenase activity and triggered apoptosis 24 h of exposition. Whilst an increased GDI in CHO-K1 cells was observed in the treatments with 1-100 µg Karate®/mL, the GDI was not modified in the treatments employing LCT at equivalent doses. SCGE showed that Karate® was more prone to induce genotoxic effects than LCT. Only 50 µg/mL of Karate® was able to increase apoptosis. Our results demonstrate the genomic instability and cytotoxic effects induced by this pyrethroid insecticide, confirming that LCT exposure can result in a severe drawback for the ecological equilibrium of the environment.

RESUMO

Myricitrin (MYR), a flavonol consumed in the leaves and fruits of plants of the Myrtaceae family, presents anti-proliferative, anti-inflammatory, anti-diabetic, and antioxidant properties in humans. However, there are few studies regarding the cyto-genotoxicity and the chemopreventive potential of MYR. Using the in vitro Micronucleus test, the cytostasis, mutagenicity, and modulatory effect of MYR in CHO-K1 cells were assessed. The concentrations of 39 and 78 µg/mL (p < 0.001.) of MYR decrease the cytokinesis-block proliferation index (CBPI) in the short exposure treatment (4 h), while in the extended treatment (24 h), concentrations of 4.8, 9.7, 19.5, 39 and 78 µg/mL (p < 0.001.) decreased the CBPI. MYR associated with oxaliplatin decreased CBPI at all tested concentrations in the pre-(p < 0.001) and post-treatments (p < 0.001), but there was no decrease when associated with bleomycin. As for chromosome instability, MYR did not increase the frequency of micronuclei (MNi), nucleoplasmic bridges (NPBs), or nuclear buds (NBUDs) in the 4 h exposure time, however, in the 24 h treatment, MYR increased the frequency of MNi and NPBs at concentration 19.5 µg/mL (p < 0.001). As for the modulatory effect, MYR associated with bleomycin decreased the frequency of MNi, NPBs, and NBUDs at all concentrations in the pretreatment (MNi and NPBs p < 0.001, NBUDs p < 0.05) and simultaneously (MNi, NPBs and NBUDs p < 0.001). When associated with oxaliplatin, the simultaneous treatment decreased the frequency of MNi (p < 0.001) and NBUDs (p < 0.01) at all concentrations, however, in the post-treatment, MYR increased MNi (p < 0.001) and NPBs p < 0.05) in CHO-K1 cells, when compared to oxaliplatin alone. The results demonstrated that MYR could modulate the mutagenic and cytostatic actions of bleomycin and oxaliplatin, demonstrating distinct behaviors, depending on the mechanism of action of the chemotherapeutic agent.

Assuntos

Citostáticos , Humanos , Oxaliplatina , Testes para Micronúcleos/métodos , Bleomicina/toxicidade , Instabilidade Cromossômica , Dano ao DNA

RESUMO

Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we describe a highly efficient process based on third-generation lentiviral vectors as delivery method for the production of reCG in suspension CHO-K1 cells, with productivities above 20 IU 106 cell-1.d-1 and 70% purification yields after one purification step. Importantly, reCG demonstrated biological activity in cattle, since around 30 µg of reCG were needed to exert the same biologic effect of 400 IU of PMSG in an ovulation synchronization protocol. The results obtained demonstrate that the developed strategy represents an attractive option for the production of reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.

Assuntos

Gonadotropina Coriônica , Gonadotropinas Equinas , Animais , Células CHO , Bovinos , Gonadotropina Coriônica/farmacologia , Cricetinae , Cricetulus , Feminino , Gonadotropinas Equinas/farmacologia , Cavalos , Ovulação , Gravidez

RESUMO

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

RESUMO

Spatial memory performance declines in both normal aging and Alzheimer's disease. This cognitive deficit is related to hippocampus dysfunction. Gene therapy using neurotrophic factors like Glial cell line-derived neurotrophic factor (GDNF) emerges as a promising approach to ameliorate age-related cognitive deficits. We constructed a two vector regulatable system (2VRS) which consists of a recombinant adenoviral vector (RAd) harboring a Tet-Off bidirectional promoter flanked by GDNF and Green Fluorescent Protein (GFP) genes. A second adenovector, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are cotransduced by the 2VRS, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is silenced. We tested the 2VRS in CHO-K1 cells where we observed a dose-dependent GFP expression that was completely inhibited by DOX (1 mg/ml). The 2VRS injected in the hippocampal CA1 region transduced both neurons and astrocytes and was efficiently inhibited by DOX added to the drinking water. In order to assess GDNF biological activity we injected 2VRS and its Control (CTRL) vector in the hypothalamus and monitored body weight for one month. The results showed that GDNF retards weight recovery 6 days more than CTRL. In conclusion, our 2VRS demonstrated optimal GFP expression and showed a bioactive effect of transgenic GDNF in the brain.

Assuntos

Fator Neurotrófico Derivado de Linhagem de Célula Glial/administração & dosagem , Proteínas de Fluorescência Verde/administração & dosagem , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Adenoviridae , Animais , Células CHO , Cricetinae , Cricetulus , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Ratos

RESUMO

A combined approach employing alkaline single cell gel electrophoresis (SCGE) and cytokinesis-blocked micronucleus (MNs) cytome bioassays was adopted to assess the deleterious properties of the auxinic 2,4-dichlorophenoxyacetic acid (2,4-D) and its microparticulated low volatility product Dedalo Elite (30% a.i.) on Chinese hamster ovary (CHO-K1) cells. Cytotoxicity was estimated by neutral red uptake (NRU), succinic dehydrogenase activity (MTT) and apoptosis assessment. Both compounds were assayed at 0.1-10 µg/ml concentration range. Whereas exposed CHO-K1 cells revealed a statistically significant enhancement of MNs when 10 µg 2,4-D/ml was assayed, MNs were only achieved in cells treated with 2 µg Dedalo Elite/ml. A diminution in the nuclear division index was only achieved after exposure to Dedalo Elite within the 1-10 µg/ml concentration range. Whereas increased genetic damage index was achieved when 6 and 10 µg 2,4-D/ml were assayed, GDI induction was observed in treatments employing 4 µg Dedalo Elite/ml. Both compounds induced cytotoxicity by inhibition of both lysosomal and MTT activities by enhancing the frequencies of early and late apoptotic cells. Our results not only indicate the genotoxic and cytotoxic potential of 2,4-D and its microparticulated marketplace formulation, but also highlight the risk of these agrochemicals present towards the biota and human health.

Assuntos

Ácido 2,4-Diclorofenoxiacético/toxicidade , Herbicidas/toxicidade , Mutagênicos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Testes de Mutagenicidade

RESUMO

Ribavirin is an important component of the treatment for hepatitis C virus (HCV) infection and, in combination with the new direct-acting antiviral (DAA) agents, comprises the major current therapeutic regimens. This study evaluated the cytotoxicity and chromosomal instability induced by ribavirin using the in vitro cytokinesis-block micronucleus cytome (CBMN-Cyt) assay in two cell lines with different expression levels of drug-metabolizing enzymes: human hepatocellular carcinoma cells (HepG2) and Chinese hamster ovary (CHO-K1) cells. HepG2 cells were treated with nine concentrations (from 15.3 µg/ml to 3.9 mg/ml) and CHO-K1 cells were exposed to eight concentrations (from 15.3 µg/ml to 1.9 mg/ml) of ribavirin for 24 h. Ribavirin inhibited cell proliferation in both cell lines, but at different concentrations: 3.9 mg/ml in HepG2 and 244.2 µg/ml in CHO-K1 cells. No significant differences were observed regarding aspects of cell death in HepG2 and CHO-K1 cells, reflecting the absence of cytotoxic effects associated to ribavirin. Ribavirin did not increase the frequency of nucleoplasmic bridges (NPBs) and nuclear bud (NBUD). However, when compared to the negative control, a significant increase in micronuclei (MNi) frequency was observed in both cell lines. However, chromosomal instability was induced by higher concentrations of ribavirin in HepG2 cells (from 61.1 to 976.8 µg/ml), compared with CHO-K1 cells (15.3 and 30.5 µg/ml). These results demonstrate the potential of ribavirin to promote chromosomal instability, and suggest that cells with different expressions of drug-metabolizing enzymes show different susceptibility to ribavirin effects.

Assuntos

Antivirais/toxicidade , Proliferação de Células/efeitos dos fármacos , Instabilidade Cromossômica/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Ribavirina/toxicidade , Animais , Antivirais/metabolismo , Apoptose/efeitos dos fármacos , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Inativação Metabólica , Testes para Micronúcleos , Ribavirina/metabolismo

RESUMO

Chinese hamster ovary (CHO) derived cell lines are the preferred host system for the production of therapeutic proteins. The aim of this work was to explore the regulation of suspension-adapted CHO-K1 host cell line bioprocesses, especially under a temperature gradient from 37 °C to 31 °C. We analyzed cell cycle behavior through flow cytometry of propidium iodide stained cells and high throughput transcriptome dynamics by RNA sequencing. We found a cell culture state characterized by G0/G1 synchronization, mainly during the late exponential growth phase and towards the last days of the stationary phase. We successfully identified key genes and pathways connected with the particular culture states, such as response to low temperature, modulation of the cell cycle, regulation of DNA replication and repair, apoptosis, among others. The most important gene expression changes occurred throughout the stationary phase when gene up-regulation markedly prevailed. Our RNA-seq data analysis enabled the identification of target genes for mechanism-based cell line engineering and bioprocess modification, an essential step to translate gene expression data from CHO-K1 host cells into bioprocess-related knowledge. Further efforts aim at increasing desirable phenotypes of CHO cells, and promoting efficient production of high quality therapeutic proteins can highly benefit from this type of studies.

Assuntos

Células CHO/citologia , Técnicas de Cultura de Células/métodos , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Animais , Ciclo Celular , Cricetulus , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Temperatura

RESUMO

Histamine H3 receptors (H3Rs) signal through Gαi/o proteins and are found in neuronal cells as auto- and hetero-receptors. Alternative splicing of the human H3R (hH3R) originates 20 isoforms, and the mRNAs of two receptors of 445 and 365 amino acids (hH3R445 and hH3R365) are widely expressed in the human brain. We previously showed that the hH3R445 stably expressed in CHO-K1 cells experiences homologous desensitization. The hH3R365 lacks 80 residues in the third intracellular loop, and in this work we therefore studied whether this isoform also experiences homologous desensitization and the possible differences with the hH3R445. In clones of CHO-K1 cells stably expressing similar receptor levels (211 ± 12 and 199 ± 16 fmol/mg protein for hH3R445 and hH3R365, respectively), there were no differences in receptor affinity for selective H3R ligands or for agonist-induced [35S]-GTPγS binding to membranes and inhibition of forskolin-stimulated cAMP accumulation in intact cells. For both cell clones, pre-incubation with the H3R agonist RAMH (1 µM) resulted in functional receptor desensitization, as indicated by cAMP accumulation assays, and loss of receptors from the cell surface and reduced affinity for the agonist immepip in cell membranes, evaluated by radioligand binding. However, functional desensitization differed in the maximal extent (96 ± 15% and 58 ± 8% for hH3R445 and hH3R365, respectively) and the length of pre-exposure required to reach the maximum desensitization (60 and 30 min, respectively). Furthermore, the isoforms differed in their recovery from desensitization. These results indicate that the hH3R365 experiences homologous desensitization, but that the process differs between the isoforms in time-course, magnitude and re-sensitization.

Assuntos

Aminoácidos/biossíntese , Aminoácidos/genética , Receptores Histamínicos H3/biossíntese , Receptores Histamínicos H3/genética , Sequência de Aminoácidos , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Expressão Gênica , Agonistas dos Receptores Histamínicos/metabolismo , Agonistas dos Receptores Histamínicos/farmacologia , Humanos , Ligação Proteica/fisiologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética

RESUMO

Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg cell-1 d-1 ). After two purification steps, the active enzyme was recovered (2.4 × 106 U mg-1 ) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1334-1345, 2017.

Assuntos

Reatores Biológicos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Lentivirus/genética , Proteínas Recombinantes , alfa-Galactosidase , Animais , Células CHO , Cricetinae , Cricetulus , Doença de Fabry , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , alfa-Galactosidase/genética , alfa-Galactosidase/isolamento & purificação , alfa-Galactosidase/metabolismo

RESUMO

CONTEXT: Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood. OBJECTIVE: We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes. MATERIALS AND METHODS: HeLa and/or CHO-K1 cell lines were tested (3 or 24 h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72 h) tests after treatment with IRN (0.1 to 200 µM) or ISA (0.5 to 50 µM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24 h) by gavage (50, 100 and 150 mg/kg determined from the LD50 - 1 g/kg b.w.) and submitted to comet assay in vivo. RESULTS: IRN reduced the viability of CHO-K1 (24 h; 5 to 200 µM) and HeLa cells (10 to 200 µM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10 µM; HeLa: 5 and 10 µM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24 h) at all doses tested. IRN (100 and 150 mg/kg) also induced genotoxicity in vivo (4 h). CONCLUSION: IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.

Assuntos

Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/biossíntese , Endonucleases/biossíntese , Isatina/farmacologia , Mutagênese/fisiologia , Proteína X Associada a bcl-2/biossíntese , Animais , Antibióticos Antineoplásicos/isolamento & purificação , Antibióticos Antineoplásicos/farmacologia , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cricetinae , Cricetulus , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Endonucleases/genética , Feminino , Expressão Gênica , Células HeLa , Humanos , Indóis/isolamento & purificação , Indóis/farmacologia , Isatina/isolamento & purificação , Masculino , Camundongos , Mutagênese/efeitos dos fármacos , Componentes Aéreos da Planta , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Proteína X Associada a bcl-2/genética

RESUMO

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.

Assuntos

Diterpenos/toxicidade , Animais , Células CHO/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Cricetulus , Diterpenos/química , Diterpenos do Tipo Caurano/toxicidade , Relação Dose-Resposta a Droga , Testes para Micronúcleos , Testes de Toxicidade/métodos

RESUMO

Micronucleus (MN) assay constitutes a valuable surrogate to the chromosome aberration technique for in vitro testing of the genotoxicity of substances. As test substances, two peptidic compounds (DOTATATE and Ubiquicidin29-41) used in nuclear medicine, were tested for in vitro cytotoxicity and genotoxicity in CHO-K1 cells. None of the compounds showed detectable cytotoxicity (0.5-7.3 ng/mL for DOTATATE and 0.3-4.5 ng/mL for UBI29-41), genotoxicity (0.72, 7.2 and 72.0 ng/ml for DOTATATE and 0.45, 4.5 and 45.0 ng/mL for UBI29-41) or cell cycle changes as compared to untreated controls at the concentrations tested. Statistical analysis showed good concordance between two independent analysts. The results corroborate the notion of the safety of the compounds and present improvements of the in vitro MN assay when performed in a pre-clinical trial context that increase the throughput of small-to-medium testing facilities as an alternative to high content screening systems.

RESUMO

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 µM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

Assuntos

Proteína Quinase C/metabolismo , Receptores Histamínicos H3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Células CHO , Carbazóis/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetulus/metabolismo , Humanos , Indóis/farmacologia , Fosforilação/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia

RESUMO

Background Polycosanols derived from plant species have traditionally been used in medicine as antiproliferative agents for treating various viruses (primarily the herpes simplex virus). However, few studies have studied their effects on hyperproliferative cell lines. In this work, the antiproliferative capacity of polycosanols from tall-oil pitch, obtained from black liquor soaps in the kraft pulping process of cellulose (specifically from Pinus radiata, Pinus taede, and Eucalyptus globulus), was evaluated on CHO-K1 and CRL-1974 human melanoma cell lines. Results The proliferative capacities and cell viabilities were measured for 72 and 140 h, respectively. Treatment with docosanol produced differential effects on the CHO-K1 and human melanoma cells and significantly affected their proliferation rates, but not their cell viabilities. Tetracosanol produced a significant negative effect on the proliferation of human melanoma cells, and this effect was less than that caused by docosanol. However, it had no effect on the proliferation of CHO-K1 cells and did not induce any significant effect on the viability of the studied cell lines. Conclusion Docosanol and tetracosanol induced antiproliferative effects on the studied cell lines and exhibited significantly greater effects on the oncogenic cell lines. Prior to this study, the capacity of these polycosanols has never been investigated. Future studies will be necessary to determine their mechanisms of action on these cell systems.

Assuntos

Humanos , Óleos de Plantas , Proliferação de Células/efeitos dos fármacos , Álcoois Graxos/farmacologia , Álcoois Graxos/química , Melanoma , Células CHO , Pinus , Linhagem Celular Tumoral , Eucalyptus

RESUMO

Histamine H3 receptors (H3Rs) modulate the function of the nervous system at the pre- and post-synaptic levels. In this work we aimed to determine whether, as other G protein-coupled receptors (GPCRs), H3Rs desensitize in response to agonist exposure. By using CHO-K1 cells stably transfected with the human H3R (hH3R) we show that functional responses (inhibition of forskolin-induced cAMP accumulation in intact cells and stimulation of [(35)S]-GTPγS binding to cell membranes) were markedly reduced after agonist exposure. For cAMP accumulation assays the effect was significant at 60 min with a maximum at 90 min. Agonist exposure resulted in decreased binding sites for the radioligand [(3)H]-N-methyl-histamine ([(3)H]-NMHA) to intact cells and modified the sub-cellular distribution of H3Rs, as detected by sucrose density gradients and [(3)H]-NMHA binding to cell membranes, suggesting receptor internalization. The reduction in the inhibition of forskolin-stimulated cAMP formation observed after agonist pre-incubation was prevented by incubation in hypertonic medium or in ice-cold medium. Agonist-induced loss in binding sites was also prevented by hypertonic medium or incubation at 4 °C, but not by filipin III, indicating clathrin-dependent endocytosis. Immunodetection showed that CHO-K1 cells express GPCR kinases (GRKs) 2/3, and both the GRK general inhibitor ZnCl2 and a small interfering RNA against GRK-2 reduced receptor desensitization. Taken together these results indicate that hH3Rs experience homologous desensitization upon prolonged exposure to agonists, and that this process involves the action of GRK-2 and internalization via clathrin-coated vesicles.

Assuntos

Endocitose/efeitos dos fármacos , Histamina/farmacologia , Receptores Histamínicos H3/metabolismo , Animais , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Endocitose/fisiologia , Humanos

RESUMO

The in-vitro effects of flurochloridone and its formulations Twin Pack Gold® (25% a.i.) and Rainbow® (25% a.i.) were evaluated in Chinese Hamster Ovary K1 (CHO-K1) cells. The cytokinesis-block micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were used. The activities were tested within the range of final concentrations of 0.25-15 µg flurochloridone/mL. The results demonstrated that both the flurochloridone and Rainbow® were not able to induce micronuclei (MN). On the other hand, Twin Pack Gold® only increased the frequency of MN at 5 µg/mL. Furthermore, 10 and 15 µg/mL of both formulations resulted in a cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. SCGE assay appeared to be a more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of flurochloridone than MN did. A marked increase in the genetic damage index was observed when 5 and 15 µg/mL of both flurochloridone and Rainbow® but only when 15 µg/mL of Twin Pack Gold® were used. This is the first report demonstrating that flurochloridone and its two commercial formulations are able to induce single-strand DNA breaks in vitro on mammalian cells.

Assuntos

Poluentes Ambientais/toxicidade , Herbicidas/toxicidade , Pirrolidinonas/toxicidade , Animais , Células CHO , Ensaio Cometa , Cricetinae , Cricetulus , Citocinese/efeitos dos fármacos , Quebras de DNA , Testes para Micronúcleos

RESUMO

A comet assay was used to analyze DNA damage kinetics in Chinese hamster ovary (CHO-K1) cells induced by antiparasitic ivermectin (IVM) and the IVM-containing technical formulation Ivomec® (IVO; 1% IVM). Cells were treated with 50 µg ml(-1) IVM and IVO for 80 min, washed and re-incubated in antiparasiticide-free medium for 0-24 h until assayed using the single-cell gel electrophoresis assay (SCGE). Cell viability remained unchanged up to 3 h of incubation. After 6 h of treatment, cell survival decreased up to 75% and 79% in IVM- and IVO-treated cultures, respectively, remaining unchanged within 12-24 h after treatment. For both anthelmintics, biphasic behavior in DNA damage occurred during the incubation time. A time-dependent increase of IVM- and IVO-induced DNA damage was observed within 0 to 3 h after pulse treatment, revealed by a progressive decrease of undamaged cells and an increase in slightly damaged and damaged cells. Finally, a time-dependent decrease in IVM- and IVO-induced DNA damage was revealed by a progressive decrease of slightly damaged cells and the absence of damaged cells simultaneously with an increase in the frequency of undamaged cells during the final 18 h of incubation. Flow cytometry analysis revealed that both compounds are able to induce a marked increase in early and late apoptosis. Based on our observations, we could conclude that the decrease in DNA lesions is mostly related to IVM-induced cytotoxicity rather than attributable to a repair process.

Assuntos

Anti-Helmínticos/toxicidade , Apoptose/efeitos dos fármacos , Dano ao DNA , Reparo do DNA , Ivermectina/toxicidade , Animais , Células CHO , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Cricetulus , Citometria de Fluxo , Cinética

RESUMO

An aqueous extract of Rhizophora mangle L. bark is used as raw material in pottery making in the State of Espirito Santo, Brazil. This extract presents large quantities of tannins, compounds possessing antioxidant properties. Tannin antioxidant activity, as a plant chemical defense mechanism in the process of stabilizing free radicals, has been an incentive to studies on anti-mutagenicity. The present work aimed to evaluate possible antimutagenic activity of a R. mangle aqueous extract, using the Allium cepa test-system and micronuclear (MN) assay with blockage of cytokinesis in Chinese hamster ovary cells (CHO-K1). The Allium cepa test-system indicated antimutagenic activity against the damage induced by the mutagenic agent methyl methanesulfonate. A reduction in both MN cell frequency and chromosome breaks occurred in both the pre and post-treatment protocols. The MN testing of CHO-K1 cells revealed anti-mutagenic activity of the R. mangle extract against methyl methanesulfonate and doxorubicin in pre, simultaneous and post-treatment protocols. These results suggest the presence of phyto-constituents in the extract presenting demutagenic and bio-antimutagenic activities. Since the chemical constitution of Rhizophora mangle species presents elevated tannin content, it is highly probable that these compounds are the antimutagenic promoters themselves.

RESUMO

An aqueous extract of Rhizophora mangle L. bark is used as raw material in pottery making in the State of Espirito Santo, Brazil. This extract presents large quantities of tannins, compounds possessing antioxidant properties. Tannin antioxidant activity, as a plant chemical defense mechanism in the process of stabilizing free radicals, has been an incentive to studies on anti-mutagenicity. The present work aimed to evaluate possible antimutagenic activity of a R. mangle aqueous extract, using the Allium cepa test-system and micronuclear (MN) assay with blockage of cytokinesis in Chinese hamster ovary cells (CHO-K1). The Allium cepa test-system indicated antimutagenic activity against the damage induced by the mutagenic agent methyl methanesulfonate. A reduction in both MN cell frequency and chromosome breaks occurred in both the pre and post-treatment protocols. The MN testing of CHO-K1 cells revealed anti-mutagenic activity of the R. mangle extract against methyl methanesulfonate and doxorubicin in pre, simultaneous and post-treatment protocols. These results suggest the presence of phyto-constituents in the extract presenting demutagenic and bio-antimutagenic activities. Since the chemical constitution of Rhizophora mangle species presents elevated tannin content, it is highly probable that these compounds are the antimutagenic promoters themselves.