Search

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. [ABSTRACT FROM AUTHOR]

The role of the FGER and GER short peptides containing a common tripeptide fragment in the regulation of the adhesive response of CHO-K1 cells was studied in this work. Both peptides increased cell adhesion both on untreated plastic and on gelatin-coated plastic, but did not affect the rate of cell attachment to poly-L-lysine-coated plastic. The GER peptide stimulated cell adhesion more on the untreated plastic. The FGER peptide increased the rate of cell attachment to gelatin over a wider range of concentrations compared to that to untreated plastic. The variativity of the process of cell spreading on different substrates under the influence of the studied peptides was noted. On untreated plastic, both peptides almost equally stimulated cell spreading. On gelatin, the FGER peptide retained the ability to stimulate cell spreading, and the GER peptide partially inhibited spreading compared to that on untreated plastic. It was found that the insertion of an additional N-terminal hydrophobic amino-acid residue Phe to the GER tripeptide fragment changes the regulatory activity of the peptide in the model of cell adhesion, depending on the stage of interaction of cells with the substrate and/or on the properties of the attachment surface. The structural and functional activity of the studied peptides with respect to various structural components of adhesive structures is discussed. [ABSTRACT FROM AUTHOR]

Introduction: Hybrid modeling combining First-Principles with machine learning is becoming a pivotal methodology for Biopharma 4.0 enactment. Chinese Hamster Ovary (CHO) cells, being the workhorse for industrial glycoproteins production, have been the object of several hybrid modeling studies. Most previous studies pursued a shallow hybrid modeling approach based on three-layered Feedforward Neural Networks (FFNNs) combined with macroscopic material balance equations. Only recently, the hybrid modeling field is incorporating deep learning into its framework with significant gains in descriptive and predictive power.Methods: This study compares, for the first time, deep and shallow hybrid modeling in a CHO process development context. Data of 24 fed-batch cultivations of a CHO-K1 cell line expressing a target glycoprotein, comprising 30 measured state variables over time, were used to compare both methodologies. Hybrid models with varying FFNN depths (3-5 layers) were systematically compared using two training methodologies. The classical training is based on the Levenberg-Marquardt algorithm, indirect sensitivity equations and cross-validation. The deep learning is based on the Adaptive Moment Estimation Method (ADAM), stochastic regularization and semidirect sensitivity equations.Results and conclusion: The results point to a systematic generalization improvement of deep hybrid models over shallow hybrid models. Overall, the training and testing errors decreased by 14.0% and 23.6% respectively when applying the deep methodology. The Central Processing Unit (CPU) time for training the deep hybrid model increased by 31.6% mainly due to the higher FFNN complexity. The final deep hybrid model is shown to predict the dynamics of the 30 state variables within the error bounds in every test experiment. Notably, the deep hybrid model could predict the metabolic shifts in key metabolites (e.g., lactate, ammonium, glutamine and glutamate) in the test experiments. We expect deep hybrid modeling to accelerate the deployment of high-fidelity digital twins in the biopharma sector in the near future.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. Key points: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing [ABSTRACT FROM AUTHOR]

In the last few years, quantum dots (QDs) have attracted research interest in different fields of science and technology. Despite their applications, it is essential to understand how nanomaterials (with different crystal sizes) are metabolized inside organisms. Thus, the focus of this study was on an evaluation of how crystal sizes of CdSTe QDs affect the viability and response of the cytochrome P450 system in CHO-K1 and HEP-G2 cells. CdSTe QDs were synthesized using a microwave-assisted system at different reaction temperatures (60, 120, 150, and 180 °C) to obtain different crystal sizes. The optical and structural characterization confirmed four crystal sizes from 3 to 8 nm. Fluorescence microscopy confirmed that CdSTe QDs are incorporated into both cell lines. Viability studies suggested that CHO-K1 cells are more sensitive than HEP-G2 cells to CdSTe QDs and Cd+2 ions. The responsible mechanisms for the toxicity of QDs and Cd+2 are apoptosis followed by necrosis. The activity of CYP 1A1, 1A2, and 3A4 isoenzymes suggests that the smallest CdSTe crystals are recognized in a manner similar to that of Cd+2. Furthermore, the largest CdSTe crystals can have different metabolic routes than Cd+2. [ABSTRACT FROM AUTHOR]

Flux balance analysis (FBA) is currently the standard method to compute metabolic fluxes in genome-scale networks. Several FBA extensions employing diverse objective functions and/or constraints have been published. Here we propose a hybrid semi-parametric FBA extension that combines mechanistic-level constraints (parametric) with empirical constraints (non-parametric) in the same linear program. A CHO dataset with 27 measured exchange fluxes obtained from 21 reactor experiments served to evaluate the method. The mechanistic constraints were deduced from a reduced CHO-K1 genome-scale network with 686 metabolites, 788 reactions and 210 degrees of freedom. The non-parametric constraints were obtained by principal component analysis of the flux dataset. The two types of constraints were integrated in the same linear program showing comparable computational cost to standard FBA. The hybrid FBA is shown to significantly improve the specific growth rate prediction under different constraints scenarios. A metabolically efficient cell growth feed targeting minimal byproducts accumulation was designed by hybrid FBA. It is concluded that integrating parametric and nonparametric constraints in the same linear program may be an efficient approach to reduce the solution space and to improve the predictive power of FBA methods when critical mechanistic information is missing. [ABSTRACT FROM AUTHOR]

Abstract 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.

• Establishment of a fast and reliable expression system in CHO-K1 cells that allows to develop cell lines in six weeks expressing dimeric IgAs and glyco-engineered human secretory component, hSC. • Establishment of a protocol to purify dimeric IgAs and the hSC. • Establishment of a protocol to reconstitute SIgA. • Validation of the functionality of two dimeric and secretory IgAs against toxins TcdA and TcdB produced with our system. Despite the essential role secretory IgAs play in the defense against pathogenic invasion and the proposed value of recombinant secretory IgAs as novel therapeutics, currently there are no IgA-based therapies in clinics. Secretory IgAs are complex molecules and the major bottleneck limiting their therapeutic potential is a reliable recombinant production system. In this report, we addressed this issue and established a fast and robust production method for secretory IgAs in CHO-K1 cells using BAC-based expression vectors. As a proof of principle, we produced IgAs against Clostridium difficile toxins TcdA and TcdB. Recombinant secretory IgAs produced using our expression system showed comparable titers to IgGs, widely used as therapeutic biologicals. Importantly, secretory IgAs produced using our method were functional and could efficiently neutralize Clostridium difficile toxins TcdA and TcdB. These results show that recombinant secretory IgAs can be efficiently produced, thus opening the possibility to use them as therapeutic agents in clinics. [ABSTRACT FROM AUTHOR]

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR., Competing Interests: JT, LH, GG, HH, AO No competing interests declared, DR Reviewing editor, eLife, (© 2024, Tung et al.)

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. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the study was to determine the concentration of elements using the two methods: total reflection X-ray fluorescence (TXRF) and wavelength dispersive X-ray fluorescence (WD-XRF) in two media, DMEM + and PBS+. Materials and methods: Tests were carried out at 37 and 0 °C, irradiated by gamma radiation doses of 0, 0.25, 0.5, 5 Gy, both with and without contact with CHO-K1 cells. The survival of non-irradiated CHO-K1 cells was determined after transmission of media from irradiated CHO-K1. Results: Normalized concentrations of elements as a percentage of control data (i.e. 0 Gy dose) for Al, P, S, Cl, K, Ca, Zn, Br, were determined using the TXRF method and for Na, P, S, Cl, K, Ca determined using the WD-XRF method in DMEM + and PBS + without and with contact with cells at two temperatures, 37 and 0 °C, and three absorbed doses of 0.25, 0.5 and 5 Gy. Concentration of elements, presented on the coordinates of the two principal components (PC) for media without contact with cells, determined by the TXRF method and in contact with cells, determined by the TXRF and WD-XRF methods were presented. Treatments to which the media were subjected, presented as co-ordinates determined by the first two PC when media were without and in contact with cells (TXRF method) and for media in contact with cells (WD-XRF method) were shown. Conclusions: The results showed that a statistically significant difference occurred in elemental concentrations for media in contact with the cells at the temperatures used. From principal component analysis (PCA), it was observed that the concentrations of elements such as Al, K, Ca, Zn, Br were similar to each other, in contrast to the concentrations of P, Cl, S, both with contact and without contact with cells. A high correlation between the treatment of media within the group at doses of 0.25 Gy and for the group with 0.5 and 5 Gy doses was confirmed. Numerous correlations were observed between the concentrations of elements for media that were in contact with cells, which were not observed in media without contact with cells. The survival of non-irradiated CHO-K1 cells, was determined after transmission of media from irradiated CHO-K1 cells showing no statistically significant differences. [ABSTRACT FROM AUTHOR]

Measurement of cell transmembrane potential (TMP) is a complex methodology involving patch-clamp methods or fluorescence-based potentiometric markers, which have limited to no applicability during ultrafast charging and relaxation phenomena. In such a case, analytical methods are applied for evaluation of the voltage potential changes in biological cells. In this work, the TMP-based electrotransfer mechanism during ultra-high frequency (≥1 MHz) electric fields is studied and the phenomenon of rapid membrane charge accumulation, which is non-occurrent during conventional low-frequency electroporation is simulated using finite element method (FEM). The influence of extracellular medium conductivity (0.1, 1.5 S/m) and pulse rise/fall times (10–50 ns) TMP generation are presented. It is shown that the medium conductivity has a dramatic influence on the electroporation process in the high-frequency range of applied pulsed electric fields (PEF). The applied model allowed to grasp the differences in polarization between 100 and 900 ns PEF and enabled successful prediction of the experimental outcome of propidium iodide electrotransfer into CHO-K1 cells and the conductivity-dependent patterns of MHz range PEF-triggered electroporation were determined. The results of this study form recommendations for development and pre-evaluation of future PEF protocols and generators based on ultra-high frequency electroporation for anticancer and gene therapies. [ABSTRACT FROM AUTHOR]

The relative biological effectiveness (RBE) values were determined for single- and mixed-ion beams containing carbon and oxygen ions. The CHO-K1 cells were irradiated with beams with the linear energy transfer (LET) values of 236–300 and 461–470 keV/μm for 12C and 16O ions, respectively. The RBE was estimated as a function of dose, survival fraction (SF) and LET. The SF was not affected by varying contributions of the constituent ions to the total mixed dose. The RBE has the same value for single-ion exposures with ions with LET 300 (12C) and 470 keV/μm (16O). • The relative biological effectiveness (RBE) of low-energy ions was estimated. • RBE for the individual and mixed 12C and 16O beams was calculated. • Survival fractions of the CHO-K1 cells after such irradiations are presented. • At specific LET values, carbon and oxygen ions have equal RBEs. • Survival fractions do not depend on the mixed beam composition. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Low intensity (< 2 Vpp/cm (peak to peak voltage/cm)), high frequency (10–30 MHz), and 10 min alternating electric fields (sine wave with no DC component) induce non-contact and enzyme-free cell detachment of anchorage-dependent cells directly from commercially available cell culture flasks and stack plates. 0.25 Vpp/cm, 20 MHz alternating electric field for 10 min at room temperature (RT) induced maximum detachment and separated 99.5 ± 0.1% (mean ± SEM, n = 6) of CHO-K1 and 99.8 ± 0.2% of BALB/3T3 cells from the culture flasks. Both vertical and lateral alternating electric field applications for 10 min at RT detach the CHO-K1 cells from 25 cm2 culture flasks. The alternating electric field application induced cell detachment is almost noncytotoxic, and over 90% of the detached cells remained alive. The alternating electric field applied CHO-K1 cells for 90 min showed little or no lag phase and immediately enter exponential phase in cell growth. Combination of the 20 MHz alternating electric field and enzymatic treatment for 4 min at 37 °C showed synergetic effect and quickly detached human induced pluripotent stem cells from a laminin-coated culture flask compared with the only enzymatic treatment. These results indicate that the rapid cell detachment with both the electric field application and the enzymatic treatment could be applied to subcultures of cells that are susceptible to prolonged enzymatic digestion damage for mass culture of sustainable clinical use. [ABSTRACT FROM AUTHOR]

Brazilin and brazilein, the major compounds of Caesalpinia sappan L. (CS) have been reported to possess antioxidant and cytotoxic activities and could potentially be used as an antigenotoxic as well as an anticancer. This study was conducted to investigate the cytotoxic and antigenotoxic effects of CS ethanolic extract (CEE). In vivo mammalian micronucleus test of CEE at the dose of 500 mg/kg BW and 1000 mg/kg BW decreased the number of MNPCE and increased the ratio of PCE to NCE meaning that CEE performed antigenotoxic effect in an in vivo model. In contrast, CEE and doxorubicin (DOXO) performed cytotoxic effect on CHO-K1 cells under MTT assay with IC50 values of 67 μg/mL and 6 μM, respectively. Interestingly, treatment of CEE in combination with DOXO and H2O2 as genotoxic inducer decreased intracellular ROS levels. In addition, in vitro genotoxicity study by using cytokinesis-block micronucleus (CBMN) assay, both of Giemsa staining and flow cytometric analysis showed that the treatment of CEE increased the number of micronuclei and correlated with apoptotic induction results. Moreover, the combination of CEE and DOXO induced cells accumulation in Sub-G1 and G2/M phase. In conclusion, CEE performed antigenotoxic effect in an in vivo model and cytotoxic effect on CHO-K1 cells. [ABSTRACT FROM AUTHOR]

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.

A current trend within photo-dynamic therapy (PDT) is the development of molecular systems targeting hypoxic tumors. Thus, type I PDT sensitizers could here overcome traditional type II molecular systems that rely on the photo-initiated production of toxic singlet oxygen. Here, we investigate the cell localization properties and toxicity of two polymeric anthracene-based fluorescent probes (neutral Ant-PHEA and cationic Ant-PIm). The cell death and DNA damage of Chinese hamster ovary cancer cells (CHO-K1) were characterized as combining PDT, cell survival studies (MTT-assay), and comet assay. Confocal microscopy was utilized on samples incubated together with either DRAQ5, Lyso Tracker Red, or Mito Tracker Deep Red in order to map the localization of the sensitizer into the nucleus and other cell compartments. While Ant-PHEA did not cause significant damage to the cell, Ant-PIm showed increased cell death upon illumination, at the cost of a significant dark toxicity. Both anthracene chromophores localized in cell compartments of the cytosol. Ant-PIm showed a markedly improved selectivity toward lysosomes and mitochondria, two important biological compartments for the cell’s survival. None of the two anthracene chromophores showed singlet oxygen formation upon excitation in solvents such as deuterium oxide or methanol. Conclusively, the significant photo-induced cell death that could be observed with Ant-PIm suggests a possible type I PDT mechanism rather than the usual type II mechanism.

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.

Highlights • This study combines cell cycle and transcriptome analysis of CHO-K1 host cell line. • The temperature gradient applied (37 °C–31 °C) triggers a coordinated cell response. • The main gene expression changes occurred in the stationary phase (at 31 °C). • Cell cycle, metabolism, DNA replication and apoptosis were significantly regulated. • Key targets for CHO cell line engineering and bioprocess modification were found. Abstract 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. [ABSTRACT FROM AUTHOR]

Carbon and oxygen ions were accelerated simultaneously to estimate the effect of irradiation of living cells with the two different ions. This mixed ion beam was used to irradiate the CHO-K1 cells, and a survival test was performed. The type of the effect of the mixed ion beam on the cells was determined with the isobologram method, whereby survival curves for irradiations with individual ion beams were also used. An additive effect of irradiation with the two ions was found. [ABSTRACT FROM AUTHOR]

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. • Lambda-cyhalothrin (LCT) and its microparticulated product Karate® were evaluated on CHO-K1 cells. • LCT increased the frequencies of MNs and NPBs. • LCT-based Karate® microformulation induced DNA single-strand breaks. • LCT-based Karate® reduced the lysosomal and mitochondrial activities. • Only LCT-based Karate® was able to increase apoptosis after treatment for 24 h. [ABSTRACT FROM AUTHOR]

In the present study, we examined properties of poly(lactide-co-glycolide) (PLGA)-based nanocarriers (NCs) with various functional or “smart” properties, i.e., coated with PLGA, polyethylene glycolated PLGA (PEG-PLGA), or folic acid-functionalized PLGA (FA-PLGA). NCs were obtained by double emulsion (water-in-oil-in-water) evaporation process, which is one of the most suitable approaches in nanoemulsion structural design. Nanoemulsion surface engineering allowed us to co-encapsulate a hydrophobic porphyrin photosensitizing dye—verteporfin (VP) in combination with low-dose cisplatin (CisPt)—a hydrophilic cytostatic drug. The composition was tested as a multifunctional and synergistic hybrid agent for bioimaging and anticancer treatment assisted by electroporation on human ovarian cancer SKOV-3 and control hamster ovarian fibroblastoid CHO-K1 cell lines. The diameter of PLGA NCs with different coatings was on average 200 nm, as shown by dynamic light scattering, transmission electron microscopy, and atomic force microscopy. We analyzed the effect of the nanocarrier charge and the polymeric shield variation on the colloidal stability using microelectrophoretic and turbidimetric methods. The cellular internalization and anticancer activity following the electro-photodynamic treatment (EP-PDT) were assessed with confocal microscopy and flow cytometry. Our data show that functionalized PLGA NCs are biocompatible and enable efficient delivery of the hybrid cargo to cancer cells, followed by enhanced killing of cells when supported by EP-PDT.

Histamine H 3 receptors (H 3 Rs) signal through Gα i/o proteins and are found in neuronal cells as auto- and hetero-receptors. Alternative splicing of the human H 3 R (hH 3 R) originates 20 isoforms, and the mRNAs of two receptors of 445 and 365 amino acids (hH 3 R 445 and hH 3 R 365 ) are widely expressed in the human brain. We previously showed that the hH 3 R 445 stably expressed in CHO-K1 cells experiences homologous desensitization. The hH 3 R 365 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 hH 3 R 445 . In clones of CHO-K1 cells stably expressing similar receptor levels (211 ± 12 and 199 ± 16 fmol/mg protein for hH 3 R 445 and hH 3 R 365 , respectively), there were no differences in receptor affinity for selective H 3 R ligands or for agonist-induced [ 35 S]-GTPγS binding to membranes and inhibition of forskolin-stimulated cAMP accumulation in intact cells. For both cell clones, pre-incubation with the H 3 R 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 hH 3 R 445 and hH 3 R 365 , 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 hH 3 R 365 experiences homologous desensitization, but that the process differs between the isoforms in time-course, magnitude and re-sensitization. [ABSTRACT FROM AUTHOR]

Context:Indigofera suffruticosaMiller (Fabaceae) andI. truxillensisKunth 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 apoptosisin vitro, their genotoxic potentialsin vitroandin vivo, and the IRN- and ISA-induced expression ofERCC1orBAXgenes. 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 assayin 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 genotoxicityin vivo(4 h). Conclusion:IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations. [ABSTRACT FROM PUBLISHER]

Using proper signal peptide and codon optimization are important factors that must be considered when designing the vector to increase protein expression in Chinese Hamster Ovary (CHO) cells. The aim of the present study is to investigate how to enhance Pertuzumab production through heavy and light chain coding gene optimization and proper signal peptide selection. First, CHO-K1 cells were transiently transfected with whole-antibody-gene-optimized, variable-regions-optimized and non-optimized constructs and then we employed five different signal peptides to improve the secretion efficiency of Pertuzumab. Compared to the native antibody gene, a 3.8 fold increase in Pertuzumab production rate was achieved with the whole heavy and light chain sequence optimization. Although an overall two fold increase in monoclonal antibody production was achieved by human albumin signal peptide compared to the control signal peptide, this overproduction was not statistically significant. Selected signal peptides had no effect on the binding of Pertuzumab to the ErbB2 antigen. The combined data indicate that human albumin signal peptide along with whole antibody sequence optimization can be used to improve Pertuzumab production rates. This sequence was used to produce Pertuzumab producing CHO-K1 stably transfected cells. This result is useful for producing Pertuzumab as a biosimilar drug. [ABSTRACT FROM AUTHOR]

The extensive demand of large scale therapeutic protein production in Chinese Hamster Ovarian cell lines increased the need of developing efficient and cost effective transfection reagent. Polyethylenimine has recently emerged as an attractive alternative to currently employed conventional transfection methods. In the present report, we attempted to develop inexpensive and easy to prepare polyethylenimine-based transfection reagent and compared its transfection efficiency with calcium phosphate, electroporation and lipoplex mediated transfection methods. Our results indicated highest transfection efficiency with in-house prepared polyplexes as marked by maximum percentage of enhanced cyan fluorescence protein positive cells. These findings contribute towards the development of economical and effective solution for high protein expression in Chinese hamster ovarian system. [ABSTRACT FROM AUTHOR]

The most common proteins used for reporter protein is the green fluorescent protein (GFP). It is very convenient to detect the GFP fluorescent by fluorescent microscopy or flowcytometry to monitor the successful transfection. The gfp gene can be introduced into the cells by transfecting of two different plasmid vectors or one vector containing both gfp and the gene of our interest. In this current experiment, we used pEGFP-c1 plasmid to express gfp in CHO-K1 cells. We transfected the CHO-K1 cells by using cationic lipid Lipofectamin 2000. We used this study as a way for predicting our human erythropoietin gene expression study in the CHO-K1 cells. In this study, we showed that expression of GFP decreased after incubation of the cells in selection medium containing G418. Expression of GFP seemed to be stable after about three weeks incubation in selection medium. Recombinant erythropoietin was also detected in the day 20.

There is considerable concern about the substances present in the environment and their potential to interfere with the endocrine system of vertebrates. Among these, the so-called endocrine-disrupting compounds, which can modulate or disrupt developmental and reproductive processes, substances with estrogenic activity have attracted most attention. Concerns about the presence of these compounds in the environment have led to the development of screening and testing assays that are able to detect such substances and evaluate their potential to induce adverse effects. In vitro systems such as mammalian and fish cell lines have become of growing importance in toxicity testing of such compounds. The cytotoxic and apoptotic effects induced by 17α-ethynylestradiol and diethylstilbestrol were studied on CHO-K1 cell line. Trypan blue exclusion method was used to determine the cell viability. Cytotoxicity of 17α-ethynylestradiol (0.34–34 μM) and diethylstilbestrol (0.37–37 μM) was found to be concentration-dependent with IC50 values of 12.8 and 10.4 μM after 72 h of exposure, respectively. In treated CHO-K1 culture cell death was assessed by determining morphological changes by haematoxilyn and eosin staining, nuclear morphology by fluorescein diacetate/propidium iodide staining and fluorescence microscopy, DNA fragmentation by TUNEL method and translocation of phosphatidyl serine by flow cytometry. The obtained results showed that 17α-ethynylestradiol induced apoptosis, while diethylstilbestrol induced necrosis in the treated CHO-K1 cells.

High concentrations of xenobiotics from urban and industrial wastes have contributed to the contamination of many aquatic environments. We used the comet assay to evaluate the genotoxic potential of water collected from the River Paraná, which receives a great deal of waste, at three points (Puerto Piray, Eldorado and Montecarlo) in the Misiones Province of Argentina. The in vivo comet assay used 40 freshwater clams (Corbicula fluminea) while the in vitro comet assay used Chinese hamster (Cricetulus griseus) K1 cell (CHO-K1) cultures with the mutagen ethyl methanesulfonate (EMS) as the positive control and phosphate buffered saline (PBS) as the negative control. Both assays showed statistically significant differences between the three sampling sites in relation to the negative control, the results of this preliminary study indicating that at these three sites water from the Paraná River presents genotoxic potential.

There is evidence for genetic polymorphism within the human histamine H 3 receptor (hH 3 R), and a Tyr to Cys exchange at position 197 (Y197C), located in the amino terminus of the fifth transmembrane domain, has been reported. In this work we compared the expression and the pharmacological and signaling properties of wild-type (hH 3 R WT ) and mutant (hH 3 R Y197C ) receptors transiently expressed in CHO-K1 cells. The hH 3 R Y197C cDNA was created by overlap extension PCR amplification. Receptor expression and affinity were assessed by N -α-[methyl- 3 H]-histamine binding to cell membranes and intact cells. Receptor function was evaluated by stimulation of [ 35 S]-GTPγS binding to cell membranes and by inhibition of forskolin-induced cAMP accumulation in intact cells. The hH 3 R WT and hH 3 R Y197C were expressed at similar levels (761 ± 68 and 663 ± 66 fmol/mg protein for membranes, and 13,434 ± 1533 and 15,894 ± 1884 receptors per cell, respectively). There were no significant differences in the affinities for H 3 R agonists or antagonists/inverse agonists between the hH 3 R WT and hH 3 R Y197C , and the H 3 R agonist RAMH was similarly efficacious and potent to stimulate [ 35 S]-GTPγS binding and to inhibit forskolin-induced cAMP accumulation. These results indicate that the Y197C mutation does not affect the expression, ligand affinity or signaling of the human H 3 receptor. [ABSTRACT FROM AUTHOR]

Electrocoagulation has recently attracted attention as a potential technique for treating toxic effluents due to its versatility and environmental compatibility, generating a residue chemically suitable to be used as a soil additive. In the present study, landfill leachate sludge hazardous effects were investigated prior and after electrocoagulation process using in vitro assays with the mammalian cells CHO-k1. An integrated strategy for risk assessment was used to correctly estimate the possible adverse landfill leachate sludge effects on human health and ecosystem. Electrocoagulation process proved to be an effective treatment due to possibility to improve effluent adverse characteristics and produce sludge with potential to be used as soil additive. Despite low cytoxicity, the residue presented genotoxic and mutagenic effects, indicating a capacity to induce genetic damages, probably due to induction of polyploidization process in cells. The observed effects demand an improvement of waste management methods for reduce negative risks of landfill leachate sludge application. [ABSTRACT FROM AUTHOR]

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H receptor of 445 amino acids (hHR) 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 hHR. In CHO-K1 cells stably transfected with the hHR direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished HR-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] i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished HR-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 HR-mediated stimulation of [S]-GTPγS binding to membranes from CHO-K1-hHR 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 HRs (−30.4 and −45.1 %) as evaluated by [H]-NMHA binding to intact cells. These results indicate that the hHR undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation. [ABSTRACT FROM AUTHOR]

Single-use bioprocessing bags are gaining popularity due to ease of use, lower risk of contamination, and ease of process scale-up. Bis(2,4-di- tert-butylphenyl)phosphate (bDtBPP), a degradant of tris(2,4-di- tert-butylphenyl)phosphite, marketed as Irgafos 168®, which is an antioxidant stabilizer added to resins, has been identified as a potentially toxic leachate which may impact the performance of single-use, multilayer bioprocessing bags. In this study, the toxicity of bDtBPP was tested on CHO-K1 cells grown as adherent or suspended cells. The EC50 (effective concentration to cause 50% cell death) for adherent cells was found to be one order of magnitude higher than that for suspended CHO-K1 cells. While CHO-K1 cells had good cell viability when exposed to moderate concentrations of bDtBPP, the degradant was shown to impact the viable cell density (VCD) at much lower concentrations. Hence, in developing an industry-standard assay for testing the cytotoxicity of leachates, suspended cells (as commonly used in the bioprocessing industry) would likely be most sensitive, particularly when reporting EC50 values based on VCD. The effects of mixing, cell culture volume, and exposure duration were also evaluated for suspended CHO-K1 cells. It was found that the sensitivity of cell culture to leachates from single-use plastic bags was enhanced for suspended cells cultured for longer exposure times and when the cells were subjected to continuous agitation, both of which are important considerations in the production of biopharmaceuticals. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1318-1323, 2016 [ABSTRACT FROM AUTHOR]

Previously, evaluation of sodium metavanadate (NaVO3) cytotoxicity after 24 h exposure of Chinese hamster ovary K1 (CHO-K1) cells revealed different sensitivity of the in vitro assays used starting from the neutral red (NR, 3-amino-7-dimethylamino-2-methylphenazine hydrochloride) test (detecting lysosomal and possibly the Golgi apparatus damage) as the most sensitive followed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) and resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) tests (mitochondrial disruption). The trypan blue (TB) staining (plasma membrane permeability) showed cytotoxicity of NaVO3 at a much higher NaVO3 concentration than the above-mentioned assays. In the current study, using the same experimental approach, we have assessed the toxicity of vanadyl sulphate (VOSO4) and compared the obtained results with NaVO3 action. Unlike metavanadate, VOSO4 treatment at 24 h resulted in similar sensitivity of the NR and resazurin tests. Nevertheless, following the 48-h incubation with VOSO4, the NR test showed markedly higher sensitivity than the resazurin test when comparing the half maximal inhibitory concentration values (61 and 110 µM for the NR and resazurin test, respectively, p < 0.05). The TB staining method was the least susceptible for detecting vanadyl cytotoxicity at each exposure time point. In summary, both the NR and resazurin tests can be advocated as similarly sensitive in detection of VOSO4-induced cytotoxicity in the CHO-K1 cell line at 24 h. However, the longer incubation time with VOSO4 showed that the NR test is more sensitive than the resazurin assay. The differences in the results between the cytotoxicity tests employed probably arise from dissimilar susceptibility of the endpoints (targets) measured with these tests to the damage by vanadium. Considering this, the current and the previous studies highlight the role of lysosomes (and possibly the Golgi apparatus) apart from mitochondria in the toxicity mechanism induced by inorganic vanadium in mammalian cells. [ABSTRACT FROM AUTHOR]

Mycotoxins are produced by different genera of fungi; mainly Aspergillus , Penicillium and Fusarium . The natural co-occurrence of beauvericin (BEA), patulin (PAT) and sterigmatocystin (STE) has been proved in feed and food commodities. This study investigates the cytotoxicity of individual and combined mycotoxins BEA, PAT and STE. The cytotoxicity on immortalized ovarian cells (CHO–K1) was evaluated using the MTT assay. After 24, 48 and 72 h, the IC 50 values were 2.9 μM for PAT and ranged from 10.7 to 2.2 μM and from 25.0 to 12.5 μM for BEA and STE, respectively. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index (CI) value as a quantitative measure of the three mycotoxin interaction's degree. Binary and tertiary combinations showed a dose dependent effect. At low fraction affected, mycotoxin combinations were synergetic; whereas, at higher fraction affected, the combinations showed additive effect. Our results indicate that the co-occurrence of low concentrations of mycotoxin in food may increase their toxic effects. [ABSTRACT FROM AUTHOR]

The aim of this study was to verify various dosimetry methods in the irradiation of biological materials with a 12 C ion beam at the Heavy Ion Laboratory of the University of Warsaw. To this end the number of ions hitting the cell nucleus, calculated on the basis of the Si-detector system used in the set-up, was compared with the number of ion tracks counted in irradiated Solid State Nuclear Track Detectors and with the number of ion tracks detected in irradiated Chinese Hamster Ovary cells processed for the γ-H2AX assay. Tests results were self-consistent and confirmed that the system serves its dosimetric purpose. [ABSTRACT FROM AUTHOR]

The effect of selenium applied as sodium selenite (NaSeO) on the cytotoxicity of vanadyl sulphate (VOSO) was examined using CHO-K1 cells. From the resazurin-based assay, it appears that NaSeO at low doses (0.5 and 1 μM) can enhance 100 μM VOSO-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 NaSeO with VOSO and 1 μM NaSeO with VOSO. Observations performed with a phase-contrast microscope showed most cells to be rounded upon treatment with VOSO alone. In turn, a majority of cells co-treated with VOSO and 1 μM NaSeO were elongated, and exhibited cytoplasmic vacuolization. These results warn of the potential contribution of inorganic selenium to vanadium-induced toxicity. [ABSTRACT FROM AUTHOR]

We report the adaptation of the new CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system to disrupt the gene encoding fucosyltransferase 8 (FUT8), an α1,6-fucosyltransferase that directs fucose addition to derived antibody Fc region asparagine 297, in Chinese hamster ovary (CHO) cells. Compared to previously reported homologous recombination or zinc-finger nucleases (ZFNs) applications in CHO cells, CRISPR/Cas9 demonstrated higher targeting efficiency and easier customization. FUT8 disruptive clones (FUT8−/−) were obtained within 3 weeks at indel frequencies ranging from 9 to 25%, which could be enhanced to 52% with Lens culinaris agglutinin (LCA) selection. Based on the lectin blot method, the derived FUT8−/− clone had the ability to produce defucosylated therapeutic mAb with no detrimental effects on cell growth, viability, or product quality. The clone had the potential of industrial application for therapeutic antibodies manufacturing. We have demonstrated functionally that a gene related to product synthesis could be mutated using CRISPR/Cas9 technology, and consequently the glycan profile of expressed mAb was alternated. We believe that with its robustness and effectiveness, CRISPR/Cas9 can be widely applicable in cell line development leading to higher productivity and better quality of mAbs and other biological therapeutics. [ABSTRACT FROM AUTHOR]

This work evaluates the cytotoxicity of acephate, ethoprophos, and monocrotophos in the Chinese hamster ovary cell line. The neutral red incorporation (NRI), total cellular protein (TCP) content, and methyl tetrazolium (MTT) assays were followed to estimate the midpoint cytotoxicity values, NRI50, TCP50,and MTT50, respectively. The targeted cells were exposed to the tested pesticides in the presence and absence of fetal calf serum. The effects of the sublethal concentration (NRI25) on glutathione S-transferase, glutathione reductase, glutathione peroxidase, and total glutathione content have been evaluated. In addition, the ameliorative effects of extracellular reduced glutathione (GSH, 1 mM), vitamin C (70 µM), and vitamin E (30 µM) were investigated. The exposure period and protein content in the culture medium affected the cytotoxicity of the tested pesticides. Unexpectedly, acephate showed the least MTT50value, 56.6 µM. The tested antioxidants caused different and significant effects on the components of the glutathione redox cycle. [ABSTRACT FROM AUTHOR]

This investigation was undertaken to compare five different in vitro cytotoxicity assays for their power in revealing vanadium-mediated toxicity in Chinese hamster ovary (CHO)-K1 cells. The cells were exposed to sodium metavanadate (NaVO3) in the range of 10–1000 µM for 24 h and thereafter the cytotoxic effects of NaVO3 were measured by colorimetric in vitro assays: the neutral red (NR) test, the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay, the resazurin assay, the sulforhodamine B (SR-B) assay, and by microscopic assessment of cell viability using the trypan blue (TB) staining method. Among the assays used, the NR test was the most sensitive, since it revealed metavanadate cytotoxicity at the lowest NaVO3 dose (=50 µM). Also, NaVO3 cytotoxicity expressed as inhibitory concentration (IC) showed the lowest values for the NR test. Three other tests XTT, resazurin, and SR-B assays showed intermediate sensitivity revealing the cytotoxicity of NaVO3 at 100 µM. The corresponding IC10 and IC50 values calculated for the XTT, resazurin, and SR-B tests were similar. The TB staining method was the least sensitive, since it recorded metavanadate cytotoxicity at the highest NaVO3 concentration tested (=600 µM). Based on the cytotoxicity end points measured with the above assays, it can be concluded that lysosomal/Golgi apparatus damage (measured by NR assay) may be the primary effect of NaVO3 on CHO-K1 cells. The disintegration of mitochondria (assessed with the XTT and resazurin assays) probably follows lysosomal impairment. Plasma membrane permeability (staining with TB) occurs at a late stage of NaVO3-induced cytotoxicity on CHO-K1 cells. The results obtained in this research work show that the NR test can be recommended as a very sensitive assay for the assessment of NaVO3 cytotoxicity in the CHO-K1 cell culture model. Considering the convenience of assay performance along with adequate sensitivity, the XTT and resazurin assays can also be advocated for NaVO3 cytotoxicity assessment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Beauvericin (BEA) causes cytotoxicity, lipid peroxidation and reactive oxygen species in CHO-K1 cells. Resveratrol (RSV) is a polyphenol with multiple biological properties, including antioxidant effects. RSV has two forms: trans and cis . The aims of this study were to determine the cytoprotective effect of trans -RSV and diastereomers mixtures (50:50 trans/cis -RSV and 70:30 trans/cis -RSV) incubated alone and in combination with BEA in ovarian (CHO-K1) cells. The results demonstrated that cell viability increases (from 9% to 77%) when they were exposed to low concentration of RSV. Moreover, when the cells were pre-treated with RSV and then exposed to BEA, a cytoprotective effect (from 25% to 76%) and a ROS production diminution (from 27% to 92%) were observed, with respect to cells exposed to BEA without previous RSV exposure. RSV pre-treatment decreased the MDA levels (from 15% to 37%) when it is compared with cells exposed only to BEA. Therefore, it can be concluded that RSV could reduce the toxicological risk produced by BEA when they are in combination. [ABSTRACT FROM AUTHOR]

The cytotoxicity of the fungicides benalaxyl, metalaxyl, and triadimefon was evaluatedin vitrousing the Chinese hamster ovary (CHO-K1) cell line. The midpoint cytotoxicity values of neutral red (NR) incorporation (NRI50), total cellular protein content (TCP50), and the methyl tetrazolium assay (MTT50) were estimated. Benalaxyl was the most cytotoxic fungicide, followed by metalaxyl and triadimefon. Fetal calf serum (10%) caused a reduction in benalaxyl, metalaxyl, and triadimefon cytotoxicity by factors of 1.8, 1.3, and 1.3. The effects of sublethal concentrations (NRI25) of the three fungicides on the glutathione redox cycle components glutathioneS-transferase, glutathione reductase, glutathione peroxidase, and total glutathione content were studied. The ameliorative effects of extracellular glutathione (1 mmol L−1), vitamin C (70µmol L−1), and vitamin E (30µmol L−1) were also investigated. The three antioxidants led to significant effects on the glutathione redox cycle components. [ABSTRACT FROM PUBLISHER]

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. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 884-892, 2014. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]