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The effect of selenium applied as sodium selenite (Na2SeO3) on the cytotoxicity of vanadyl sulphate (VOSO4) was examined using CHO-K1 cells. From the resazurin-based assay, it appears that Na2SeO3 at low doses (0.5 and 1 μM) can enhance 100 μM VOSO4-induced cell damage. The two-way ANOVA analysis revealed that the increased cell damage was a consequence of a synergistic interaction of 0.5 μM Na2SeO3 with VOSO4 and 1 μM Na2SeO3 with VOSO4. Observations performed with a phase-contrast microscope showed most cells to be rounded upon treatment with VOSO4 alone. In turn, a majority of cells co-treated with VOSO4 and 1 μM Na2SeO3 were elongated, and exhibited cytoplasmic vacuolization. These results warn of the potential contribution of inorganic selenium to vanadium-induced toxicity.

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. Fil: Tossolini, Ileana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina Fil: López Díaz, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Intellectics Inc.; Estados Unidos. Salk Institute for Biological Studies; Estados Unidos Fil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina Fil: Prieto, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Cellargen Biotech S.R.L.; Argentina

Made available in DSpace on 2018-11-26T17:39:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-07-25 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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 mu M) or ISA (0.5 to 50 mu M). 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 mu M) and HeLa cells (10 to 200 mu M), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10 mu M; HeLa: 5 and 10 mu M). 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. Univ Estadual Londrina, Ctr Biol Sci, Dept Gen Biol, Celso Garcia Cid Rd,PR 445 Km 380, BR-86057970 Londrina, Parana, Brazil Sao Paulo State Univ, Araraquara Inst Chem, Araraquara, Brazil Sao Paulo State Univ, Araraquara Fac Pharmaceut Sci, Dept Biol Sci, Araraquara, Brazil Sao Paulo State Univ, Expt Campus Paulista Coast, Sao Vicente, Brazil Yale Sch Med, Abraham Ribicoff Res Facil, New Haven, CT USA Sao Paulo State Univ, Araraquara Inst Chem, Araraquara, Brazil Sao Paulo State Univ, Araraquara Fac Pharmaceut Sci, Dept Biol Sci, Araraquara, Brazil Sao Paulo State Univ, Expt Campus Paulista Coast, Sao Vicente, Brazil

Yingxiao Li,1,2 Kai Chun Cheng,1 Chiang-Shan Niu,3 Shih-Hsiang Lo,3,4 Juei-Tang Cheng,2,5 Ho-Shan Niu31Department of Psychosomatic Internal Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan; 2Department of Medical Research, Chi Mei Medical Center, Yong Kang, Tainan City, 3Department of Nursing, Tzu Chi College of Technology, Hualien City, 4Division of Cardiology, Department of Internal Medicine, Chung Hsing Branch of Taipei City Hospital, 5Institute of Medical Sciences, College of Health Science, Chang-Jung Christian University, Guei-Ren, Tainan City, TaiwanBackground: G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) has been shown to participate in glucose homeostasis. In animal models, a TGR5 agonist increases incretin secretion to reduce hyperglycemia. Many agonists have been developed for clinical use. However, the effects of TGR5 blockade have not been studied extensively, with the exception of studies using TGR5 knockout mice. Therefore, we investigated the potential effect of triamterene on TGR5.Methods: We transfected the TGR5 gene into cultured Chinese hamster ovary cells (CHO-K1 cells) to express TGR5. Then, we applied a fluorescent indicator to examine the glucose uptake of these transfected cells. In addition, NCI-H716 cells that secrete incretin were also evaluated. Fura-2, a fluorescence indicator, was applied to determine the changes in calcium concentrations. The levels of cyclic adenosine monophosphate (cAMP) and glucagon-like peptide (GLP-1) were estimated using enzyme-linked immunosorbent assay kits. Moreover, rats with streptozotocin (STZ)-induced type 1-like diabetes were used to investigate the effects in vivo.Results: Triamterene dose dependently inhibits the increase in glucose uptake induced by TGR5 agonists in CHO-K1 cells expressing the TGR5 gene. In cultured NCI-H716 cells, TGR5 activation also increases GLP-1 secretion by increasing calcium levels. Triamterene inhibits the increased calcium levels by TGR5 activation through competitive antagonism. Moreover, the GLP-1 secretion and increased cAMP levels induced by TGR5 activation are both dose dependently reduced by triamterene. However, treatment with KB-R7943 at a dose sufficient to block the Na+/Ca2+ exchanger (NCX) failed to modify the responses to TGR5 activation in NCI-H716 cells or CHO-K1 cells expressing TGR5. Therefore, the inhibitory effects of triamterene on TGR5 activation do not appear to be related to NCX inhibition. Blockade of TGR5 activation by triamterene was further characterized in vivo using the STZ-induced diabetic rats.Conclusion: Based on the obtained data, we identified triamterene as a reliable inhibitor of TGR5. Therefore, triamterene can be developed as a clinical inhibitor of TGR5 activation in future studies.Keywords: triamterene, CHO-K1 cells, TGR5, transfection, sitagliptin&nbsp

A CHO cell clone (CHO-PoIFN-β) with stable porcine IFN-β expression under control of CMV promoter was selected under G418 pressure. In a 25cm(2) cell culture flask (5 ml culture medium), the cumulative protein yield of recombinant PoIFN-β reached 2.3×10(6) IU/ml. This cells clone maintained stable expression for at least 20 generations even in the absence of G418 selection pressure. The expressed recombinant PoIFN-β could induce the expression of porcine Mx protein in PK15 cells, and activate the chicken Mx promoter-controlled luciferase reporter gene expression, confirming that the recombinant PoIFN-β has the biological activity of natural porcine type-I interferon. In addition, the recombinant PoIFN-β fully protected PK15 cells against 1000 TCID(50) of porcine transmissible gastroenteritis virus and pseudo-rabies virus infection, demonstrating its high potential in therapeutic applications. This is the first report of establishing a mammalian cell line with stable expression of porcine IFN-β.

The aim of this study was to determine the toxic effect of atrazine at the ovarian cellular level. Chinese Hamster Ovary (CHO-K1) cell line was used to evaluate the degree of in vitro atrazine cytotoxicity and the morphological changes were followed during the cell death. Application of four bioassays confirmed that atrazine decreases ovarian cell proliferation and IC(50) were determined with each assay after 72 h of exposure. The level of apoptosis in atrazine treated cells was low.

In vitro effects of the carbamates pirimicarb and zineb and their formulations Aficida® (50% pirimicarb) and Azzurro® (70% zineb), respectively, were evaluated in Chinese hamster ovary (CHO-K1) cells. Whereas the cytokinesis-blocked micronucleus cytome assay was employed to test for genotoxicity, MTT, neutral red (NR), and apoptosis evaluation were used as tests for estimating cell viability and succinic dehydrogenase activity, respectively. Concentrations tested were 10–300 μg/ml for pirimicarb and Aficida®, and 1–50 μg/ml for zineb and Azzurro®. All compounds were able to increase the frequency of micronuclei. A marked reduction in the nuclear division index was observed after treatment with 5 μg/ml of zineb and Azzurro® and 10 μg/ml of Azzurro®. Alterations in the cellular morphology not allowing the recognition of binucleated cells exposed to 300 μg/ml pirimicarb and Aficida® as well as 10–50 μg/ml zineb and Azzurro®. All four compounds induced inhibition of both cell viability and succinic dehydrogenase activity and trigger apoptosis in CHO-K1 cells, at least when exposed for 24 h. The data herein demonstrate the genotoxic and cytotoxic effects exerted by these carbamates and reveal the potential risk factor of these pesticides, still extensively used worldwide, for both human health and the environment. Fil: Soloneski, Sonia Maria Elsa. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Catedra de Citologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Kujawski, Maciej. City of Hope Comprehensive Cancer Center; Estados Unidos Fil: Scuto, Anna. City of Hope Comprehensive Cancer Center; Estados Unidos Fil: Larramendy, Marcelo Luis. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Catedra de Citologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

This article has been made available through the Brunel Open Access Publishing Fund. This article is distributed under the terms of the creative commons Attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s)and the source are credited. Combinations of genotoxic agents have frequently been assessed without clear assumptions regarding their expected (additive) mixture effects, often leading to claims of synergisms that might in fact be compatible with additivity. We have shown earlier that the combined effects of chemicals, which induce micronuclei (MN) in the cytokinesis-block micronucleus assay in Chinese hamster ovary-K1 cells by a similar mechanism, were additive according to the concept of concentration addition (CA). Here, we extended these studies and investigated for the first time whether valid additivity expectations can be formulated for MN-inducing chemicals that operate through a variety of mechanisms, including aneugens and clastogens (DNA cross-linkers, topoisomerase II inhibitors, minor groove binders). We expected that their effects should follow the additivity principles of independent action (IA). With two mixtures, one composed of various aneugens (colchicine, flubendazole, vinblastine sulphate, griseofulvin, paclitaxel), and another composed of aneugens and clastogens (flubendazole, doxorubicin, etoposide, melphalan and mitomycin C), we observed mixture effects that fell between the additivity predictions derived from CA and IA. We achieved better agreement between observation and prediction by grouping the chemicals into common assessment groups and using hybrid CA/IA prediction models. The combined effects of four dissimilarly acting compounds (flubendazole, paclitaxel, doxorubicin and melphalan) also fell within CA and IA. Two binary mixtures (flubendazole/paclitaxel and flubendazole/doxorubicin) showed effects in reasonable agreement with IA additivity. Our studies provide a systematic basis for the investigation of mixtures that affect endpoints of relevance to genotoxicity and show that their effects are largely additive. UK Food Standards Agency

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes. The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide. This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

The effects of synthetic estrogens 17α-ethynylestradiol (EE2) and diethylstilbestrol (DES) were compared on cell proliferation and morphology in Channel Catfish Ovary (CCO) and Chinese Hamster Ovary (CHO-K1) cells. EE2 exposure (0.1 and 0.5 μg/mL) induced stimulatory effect on CCO and CHO-K1 cell proliferation, while higher concentrations (1-10 μg/mL) showed cytotoxic effects. Increase in DES concentrations mainly resulted in dose-depended increase in cytotoxicity in both cell lines. Morphological changes induced by EE2 and DES exposure (5 μg/mL) showed disrupted cell monolayer and increased number and size of lysosomes. Comparison of IC(50) values showed almost equal sensitivity towards cytotoxicity of tested compounds in CCO and CHO-K1 cells.

Lindane, a toxic insecticide from the persistent organic pollutants (POP's) group, may act as an endocrine disrupter affecting crucial tissues of reproductive system. In this study a Chinese Hamster Ovary cell line (CHO-K1) was applied to assess the potential of lindane cytotoxicity at the cellular level. The methods of Trypan blue exclusion, MTT and Kenacid blue assays were used to assess cytotoxicity and confirmed a decrease in the number of viable CHO-K1 cells at 34.4-344 microM lindane during 24, 48 and 72 hours of exposure. The cell proliferation tests showed significant inhibition (p < 0.025-0.001 vs control) and a progressive increase in toxicity with increasing lindane concentrations. Corresponding IC(50) values were determined with each applied method. After 72 h of lindane exposure, IC(50) values were 184 microM according to the Trypan blue method and 272 and 256 microM with the Kenacid blue and MTT assays, respectively. Morphological changes induced by the cytotoxicity of lindane were followed by the fluorescence microscopy and only necrotic cells were detected. Vitamin E (25 and 50 microg/mL) was used for protection of ovarian cells against lidane-induced oxidative stress damage, and lipid peroxidation was postulated as a possible mechanism of lindane toxicity. The viability of cells pre-incubated with vitamin E was significantly enhanced (up to p < 0.025) compared to the results observed in cells exposed to lindane only, but vitamin E treatment could not prevent complete lindane-induced cytotoxicity. Results suggest that vitamin E may exert a slightly protective role in cell defense against lipophilic pro-oxidant xenobiotics such as lindane.

Cylindrospermopsin (CYN) is a cyanobacterial alkaloid that has been implicated in outbreaks of human morbidity and animal mortality. The principal mode of action for CYN is inhibition of protein and glutathione synthesis, and its toxicity seems to be mediated by cytochrome P-450-generated metabolites. It was also shown that CYN might be responsible for tumor initiation in animals; nevertheless, mechanisms leading to CYN-induced carcinogenesis are scarce and equivocal. The aim of the present study was to investigate the impact of metabolic activation on CYN-induced DNA damage. The effect of different doses of CYN (0.05–2 μg/ml) on DNA damage was determined in CHO-K1 cells after 3, 16 and 21 h of the treatment. The chromosome aberration assay with and without metabolic activation was applied to evaluate the clastogenic activity of CYN and its metabolite(s). In addition, the occurrence of apoptosis and necrosis was estimated by the annexin method using flow cytometry. The results revealed that CYN is not clastogenic in CHO-K1 cells irrespective of S9 fraction-induced metabolic activation. However, CYN significantly decreases the frequencies of mitotic indices and decreases proliferation irrespective of metabolic activation system. CYN increases the frequency of necrotic cells in a dose- and time-dependent manner, whereas it has a very slight impact on apoptosis. Moreover, the presence of metabolic activation influences a susceptibility to necrotic cell death but not an apoptotic one.

Microcystin-LR (MC-LR), a potent inhibitor of PP1 and PP2A protein phosphatases, is related to tumor promotion and initiation. Although the genotoxic properties of this toxin have been extensively investigated with a variety of non-mammalian and mammalian test systems, the existing results are contradictory. Based on our previous results regarding the impact of MC-LR on the processes of DNA repair we decided to examine in greater detail its effect on the capacity of nucleotide excision repair (NER). CHO-K1 cells were pre-treated with increasing doses of MC-LR (1, 10 and 20 μg/ml) and then exposed to UV radiation (25 J/m2). Apoptosis was analyzed to exclude the possibility of false positive results in the comet assay. The results suggest that MC-LR targets the nucleotide excision repair mechanisms by interference with the incision/excision phase as well as the rejoining phase of NER and leads to an increased level of UV-induced cytogenetic DNA damage in CHO-K1 cells.

Background Monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) is a catabolic enzyme responsible for converting progesterone into biologically inactive 20α-hydroxyprogesterone, thereby playing a key role in the estrous cycle or pregnancy and allowing ovulation and parturition to occur in most mammalian animals. Monkey 20α-HSD was highly abundant in ovarian and placental tissues during the pre-ovulation and pre-parturition phase and was primarily localized in the syncytiotrophoblast of the placenta. In this study, we focused on the molecular characterization of the monkey 20α-HSD promoter region by conducting reporter assays in Chinese hamster ovary (CHO) K1 cells. Results A reporter assay using constructs of various lengths of the 5′-flanking region (-890-Luc, -513-Luc, -306-Luc, -273-Luc, and -70-Luc) revealed that a region corresponding to the activator protein 1 (Ap-1) located between -281 and -274 bp was essential for the transcriptional activity. Absence of the Ap-1 site in -273-Luc dramatically decreased the transcription levels to the control levels. When the reporter constructs were co-transfected with Ap-1 (Jun) and specificity protein (Sp-1) genes, the transcription activities of the constructs increased with the exception of -273 and -70, while that of the double construct was reduced compared to that of Ap-1 alone. Furthermore, mutational analysis demonstrated that a putative Ap-1 site played an important role in the expression of the reporter gene. These findings were confirmed by EMSA examining the interactions of the protein Ap-1 in a nuclear extract from CHO-K1 cells and the expression levels of the Ap-1 transcription factor in pre-parturition placenta and CHO-K1 cells. Although mut-1 and mut-2 of Ap-1 bound with nuclear extracts from CHO-K1 cells, the transcriptional activity of mut-3 was almost completely suppressed. Conclusions Our results indicate that the Ap-1 site (-281 → -274) (5′-TGTCTCAT-3′) plays a crucial role in the activation of the monkey 20α-HSD gene. Thus, we demonstrated that monkey 20α-HSD promoter activity is regulated by the transcription factor Ap-1 in CHO-K1 cells.

PD 128907 [4a R, 10 b R-(+)-trans-3, 4, 4a, 10 b-tetrahydro-4-n-propyl2 H,5H-[1]benzop-yrano[4,3-b] 1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (K-i, 1 nM) versus human D-2 receptors (K-i, 1183 nM) and a 10000-fold selectivity versus human D-4 receptors (K-i, 7000 nM) using [H-3]spiperone as the radioligand in CHO-K1-cells. Studies with [H-3]PD 128907, showed saturable, high affinity binding to human D-4 receptors expressed in CHO-K1 cells (CHO-K1-D-3) with an equilibrium dissociation constant (K-d) of 0.99 nM and a binding density (B-max) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D-2 receptors (CHO-K1-D-2). The rank order of potency for inhibition of [H-3]PD 128907 binding with reference DA agents was consistent with reported values for D-3 receptors. These results indicate that [H-3]PD 128907 is a new, highly selective D-3 receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DAD(3) receptors.

The m2 and m3 muscarinic acetylcholine receptors were expressed in CHO cells and were shown to couple to the release of calcium from intracellular stores. The m3 receptor, but not the m2 receptor, also coupled to calcium influx. Chimeric m2/m3 receptors were used to determine the structural domain of the m3 receptor linked to the regulation of calcium influx. It was found that the third intracellular loop of m3 receptor plays a fundamental role in regulating Ca2+ influx predicted to occur through Ca2+ channels located in the plasma membrane in CHO cells.