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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]

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]

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]

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]

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 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]

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. [ABSTRACT FROM AUTHOR]