Primary Human Hepatocytes, but Not HepG2 or Balb/c 3T3 Cells, Efficiently Metabolize Salinomycin and Are Resistant to Its Cytotoxicity.

Salinomycin is a polyether antibiotic showing anticancer activity. There are many reports of its toxicity to animals but little is known about the potential adverse effects in humans. The action of the drug may be connected to its metabolism. That is why we investigated the cytotoxicity of salinomycin and pathways of its biotransformation using human primary hepatocytes, human hepatoma cells (HepG2), and the mouse fibroblast cell line (Balb/c 3T3). The cytotoxicity of salinomycin was time-dependent, concentration-dependent, and cell-dependent with primary hepatocytes being the most resistant. Among the studied models, primary hepatocytes were the only ones to efficiently metabolize salinomycin but even they were saturated at higher concentrations. The main route of biotransformation was monooxygenation leading to the formation of monohydroxysalinomycin, dihydroxysalinomycin, and trihydroxysalinomycin. Tiamulin, which is a known inhibitor of CYP450 izoenzymes, synergistically induced cytotoxicity of salinomycin in all cell types, including non-metabolising fibroblasts. Therefore, the pharmacokinetic interaction cannot fully explain tiamulin impact on salinomycin toxicity.

Cellular distribution and degradation of cobalt ferrite nanoparticles in Balb/3T3 mouse fibroblasts.

The effect of the concentration of cobalt ferrite (CoFe(2)O(4)) nanoparticles (NPs) on their intracellular location and distribution has been explored by synchrotron radiation X-ray and fluorescence microscopy (SR-XRF) monitoring the evolution of NPs elemental composition as well. In cells exposed to low concentrations of CoFe(2)O(4) NPs, the NPs preferentially segregate in the perinuclear region preserving their initial chemical content. At concentrations exceeding 500 µM the XRF spectra indicate the presence of Co and Fe also in the nuclear region, accompanied by sensible changes in the cellular morphology. The increase of the Co/Fe ratio measured in the nuclear compartment indicates that above certain concentrations the CoFe(2)O(4) NPs intracellular distribution could be accompanied by biodegradation resulting in Co accumulation in the nucleus.

Cyclotron production of radioactive CeO(2) nanoparticles and their application for in vitro uptake studies.

Nowadays, a wide variety of nanoparticles (NPs) are applied in different fields such as medical science and industry. Due to their large commercial volume, the OECD Working Party on Manufactured Nanomaterials (NMs) has proposed to study a set of 14 nanomaterials, one of which being cerium oxide (CeO(2)). In particular, CeO(2) based NPs are widely used in automotive industry, healthcare, and cosmetics. In this paper, we propose a method for the production of radioactive CeO(2) NPs.We demonstrate that they maintain the same physicochemical characteristics as the "cold" ones in terms of size distribution and Zeta potential; we develop a new protocol to assess their cellular interaction in immortalized mouse fibroblast cell line Balb/3T3, a model for the study of basal cytotoxicity and carcinogenic potential induced by chemicals and in the present case by NPs. Experimental result of this work, which shows a quasi-linear concentration-uptake response of cells, can be useful as a reference dose-uptake curve for explaining effects following biological uptake after exposure to CeO(2) NPs.

Toxicogenomics applied to in vitro carcinogenicity testing with Balb/c 3T3 cells revealed a gene signature predictive of chemical carcinogens.

Information on the carcinogenic potential of chemicals is primarily available for High Production Volume (HPV) products. Because of the limited knowledge gain from routine cancer bioassays and the fact that HPV chemicals are tested only, there is the need for more cost-effective and informative testing strategies. Here we report the application of advanced genomics to a cellular transformation assay to identify toxicity pathways and gene signatures predictive for carcinogenicity. Specifically, genome-wide gene expression analysis and quantitative real time polymerase chain reaction (qRT-PCR) were applied to untransformed and transformed mouse fibroblast Balb/c 3T3 cells that were exposed to either 2, 4-diaminotoluene, benzo(a)pyrene, 2-acetylaminoflourene, or 3-methycholanthrene at IC20 conditions for 24 and 120 h, respectively. Then, bioinformatics was applied to define toxicity pathways and a gene signature predictive of the carcinogenic risk of these chemicals. Although bioinformatics revealed distinct differences for individual chemicals at the gene-level pathway, analysis identified common perturbation that resulted in an identification of 14 genes whose regulation in cancer tissue had already been established. Strikingly, this gene signature was identified in short-term (24 and 120 h) untransformed and transformed cells (3 weeks), therefore demonstrating robustness for its predictive power. The developed testing strategy thus identified commonly regulated carcinogenic pathways and a gene signature that predicted the risk for carcinogenicity for three well-known carcinogens. Overall, the testing strategy warrants in-depth validation for the prediction of carcinogenic risk of industrial chemicals in in vitro carcinogenicity assay.

PKCdelta survival signaling in cells containing an activated p21Ras protein requires PDK1.

Protein kinase C delta (PKCdelta) modulates cell survival and apoptosis in diverse cellular systems. We recently reported that PKCdelta functions as a critical anti-apoptotic signal transducer in cells containing activated p21(Ras) and results in the activation of AKT, thereby promoting cell survival. How PKCdelta is regulated by p21(Ras), however, remains incompletely understood. In this study, we show that PKCdelta, as a transducer of anti-apoptotic signals, is activated by phosphotidylinositol 3' kinase/phosphoinositide-dependent kinase 1 (PI(3)K-PDK1) to deliver the survival signal to Akt in the environment of activated p21(Ras). PDK1 is upregulated in cells containing an activated p21Ras. Knock-down of PDK1, PKCdelta, or AKT forces cells containing activated p21(Ras) to undergo apoptosis. PDK1 regulates PKCdelta activity, and constitutive expression of PDK1 increases PKCdelta activity in different cell types. Conversely, expression of a kinase-dead (dominant-negative) PDK1 significantly suppresses PKCdelta activity. p21(Ras)-mediated survival signaling is therefore regulated by via a PI(3)K-AKT pathway, which is dependent upon both PDK1 and PKCdelta, and PDK1 activates and regulates PKCdelta to determine the fate of cells containing a mutated, activated p21(Ras).

Identification of tumor promotion marker genes for predicting tumor promoting potential of chemicals in BALB/c 3T3 cells.

Tumor promoters can cause development of tumors in initiated cells and the majority of them are non-genotoxic carcinogens. The detection of tumor promoters is important for the prevention of cancer. The in vitro two-stage transformation assay, using BALB/c 3T3 cells, is a useful system, and benefits from a convenient protocol and high predictability of mammalian carcinogenicity. But these assays are time-consuming and often require expertise for microscopic observation. To construct an in vitro tumor promoting activity test system, we performed large-scale gene expression analyses, using DNA microarrays, of BALB/c 3T3 cells following treatment with nine chemicals that are known to induce tumor promotion: TPA, zinc chloride, sodium orthovanadate, okadaic acid, insulin, lithocolic acid, phenobarbital sodium, sodium saccharide, sodium arsenite. As a result of DNA microarray and real time PCR analyses, 22 marker genes were identified. These consisted of genes related to cell cycle, regulation of transcription, anti-apoptosis, and positive regulation of cell proliferation. There was a correlation between these 22 marker genes and the cell transformation assay results in BALB/c 3T3 cells. These results suggest that this tumor promoting activity test system, based on 22 marker genes, can become a valuable tool for screening potential tumor promoters.

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element.

Arginine rich, mutated in early stage of tumors (ARMET) was first identified as a human gene highly mutated in a variety of cancers. However, little is known about the characteristics of the ARMET protein and its expression. We identified ARMET as a gene upregulated by endoplasmic reticulum (ER) stress. Here, we show that the mouse homologue of ARMET is an 18-kDa soluble ER protein that is mature after cleavage of a signal sequence and has four intramolecular disulfide bonds, including two in CXXC sequences. ER stress stimulated ARMET expression, and the expression patterns of ARMET mRNA and protein in mouse tissues were similar to those of Grp78, an Hsp70-family protein required for quality control of proteins in the ER. A reporter gene assay using a mouse ARMET promoter revealed that the unfolded protein response of the ARMET gene is regulated by an ERSE-II element whose sequence is identical to that of the HERP gene. ARMET is the second fully characterized ERSE-II-dependent gene and likely contributes to quality control of proteins in the ER.

The tyrosine kinase Abl is required for Src-transforming activity in mouse fibroblasts and human breast cancer cells.

The cytoplasmic tyrosine kinase Src has been implicated in signal transduction induced by growth factors and integrins. Src also shows oncogenic activity when deregulated. Accumulating evidence indicates that the tyrosine kinase Abl is an important substrate for Src signalling in normal cells. Here we show that Abl is also required for Src-induced transformation of mouse fibroblasts. Abl does not mediate tyrosine phosphorylation of Stat3 and Shc, two important regulators of Src oncogenic activity. In contrast, Abl controls the activation of the small GTPase Rac for oncogenic signalling and active Rac partly rescued Src transformation in cells with inactive Abl. Moreover, Abl mediates Src-induced extracellular regulated kinase 5 (ERK5) activation to drive cell transformation. Finally, we find that Abl/Rac and Abl/ERK5 pathways also operate in human MCF7 and BT549 breast cancer cells, where neoplastic transformation depends on Src-like activities. Therefore, Abl is an important regulator of Src oncogenic activity both in mouse fibroblasts and in human cancer cells. Targeting these Abl-dependent signalling cascades may be of therapeutic value in breast cancers where Src-like function is important.

Enhanced protection of modified human acidic fibroblast growth factor with polyethylene glycol against ischemia/reperfusion-induced retinal damage in rats.

Molecular modification with polyethylene glycol (PEGylation) is an effective approach to improve protein biostability and decrease protein immunogenic activity. To create a PEGylated recombinant human acid fibroblast growth factor (rhaFGF) and improve its bio-stability, we have produced a rhaFGF mutant (rhaFGF(ser98,132)) by replacing the 98th and the 132nd cysteine residues with serine residues. The rhaFGF(ser98,132) that retains the bioactivity of rhaFGF was then site-specifically conjugated with PEG-maleimide at the 31st cysteine residue. PEGylated rhaFGF(ser98,132) has less effect than the native rhaFGF(ser98,132) on stimulating 3T3 cell proliferation in vitro; however, its biostability at a prolonged incubation under various temperatures and resistance to trypsinization were significantly enhanced, and half-life time in vivo was elongated while its immunogenicity was significantly decreased. The physiological function of PEGylated rhaFGF(ser98,132) was evaluated in a rat model of retinal ischemia/reperfusion injury, showing that in vivo supplementation of PEGylated rhaFGF(ser98,132) provided a significantly better protection than the native rhaFGF(ser98,132) against ischemia/reperfusion-induced retinal morphological changes and lipid peroxidation. The protection is probably mediated by antioxidant protective mechanisms.

Assessment of the phototoxic hazard of some essential oils using modified 3T3 neutral red uptake assay.

When substances are developed in the aim to be a constituent of personal care products, and to be applied on the skin, it is necessary to carry out an assessment of potential phototoxic hazard. Phototoxicity is skin reaction caused by concurrent topical or systemic exposure to specific molecule and ultraviolet radiation. Most phototoxic compounds absorb energy particularly from UVA light leading to the generation of activated derivatives which can induce cellular damage. This type of adverse cutaneous response can be reproduced in vitro using different models of phototoxicity such as the validated 3T3 Neutral Red Uptake (NRU) phototoxicity assay. In the present study we utilised two different cell lines (the murine fibroblastic cell line 3T3 and the rabbit cornea derived cell line SIRC) to compare the photo-irritation potential of a strong phototoxic compound, chlorpromazine, to a weaker composite, such as 8-methoxypsoralen and Bergamot oil. After comparison of the different systems, five other essential oils were tested with both cell lines. Cellular damage was evaluated by the NRU cytotoxicity test or by MTT conversion test.

The detection of differentiation-inducing chemicals by using green fluorescent protein expression in genetically engineered teratocarcinoma cells.

The murine embryonal teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the murineTert (mTert) promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. mTert expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert_GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert_GFP expression. In addition, it could be demonstrated that the mTert_GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert_GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells for ethanol, but there was a 2-fold increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested. This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.

Aurora-B/AIM-1 kinase activity is involved in Ras-mediated cell transformation.

Aurora-B, previously known as AIM-1, is a conserved eukaryotic mitotic protein kinase. In mammals, this kinase plays an essential role in chromosomal segregation processes, including chromosome condensation, alignment, control of spindle checkpoints, chromosome segregation, and cytokinesis. Aurora-B is overexpressed in various cancer cells, suggesting that the kinase activity perturbs chromosomal segregation processes. Its forced overexpression induces chromosomal number instability and progressive tumorigenicity in rodent cells in vitro and in vivo. Nevertheless, based on focus formation in BALB/c 3T3 A31-1-1 cells, Aurora-B is not oncogenic. Here, we show that Aurora-B kinase activity augments Ras-mediated cell transformation. RNA interference with short hairpin RNA inhibits transformation by Ras and its upstream oncogene Src, but not by the downstream oncogene Raf. In addition, the inner centromere protein, which is a passenger protein associated with Aurora-B, has a similar ability to potentiate the activity of oncogenic Ras. These data indicate that elevated Aurora-B activity promotes transformation by oncogenic Ras by enhancing oncogenic signaling and by converting chromosome number-stable cells to aneuploid cells.

In-house assessment of a modified in vitro cytotoxicity assay for higher throughput estimation of acute toxicity.

The main objective of this study was to assess an in-house 3T3 NRU cytotoxicity assay for compatibility with a prediction model for acute rodent oral toxicity endorsed by an NIEHS-ICCVAM workshop. The aim is to use the NRU assay as one test component of HTS strategies for both acute oral toxicity and acute skin irritation, enabling the rejection of the most toxic materials and prioritisation of other materials for further testing. Groups of model cytotoxins and irritants were tested using the NRU assay and their EC50 values obtained from dose-response curves. These values were compared with those estimated from a limited (three)-dose protocol, deemed more suitable for HTS. A good correlation was observed between the EC50 values from both dose-response curves (R2=0.94). The relationships between EC50 values and acute rodent oral toxicity were compared by application of the prediction model to the model cytotoxins. The results from both full and limited dose-responses fitted within the acceptance limits of the prediction model, with regression lines similar to that of the model. Results indicated that the performance of the currently used 3T3 NRU cytotoxicity assay was similar to that of the assays used to generate the data employed in developing the prediction model. This prediction model can be applied with both the standard and HT assays to estimate acute rodent oral toxicity.

Study of the potential cytotoxicity of dental impression materials.

The aim of this study was to assess the cytotoxicity of tow types of impression dental materials: polyethers (Impregum Penta, Permadyne Penta Heavy and Light) and vinyl polysiloxanes (Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty). Their cytotoxic effects were studied by indirect and direct tests. The indirect tests were performed by incubating impression materials in serum free cell culture medium to prepare the soluble extracts. Balb/c 3T3 cells were incubated with extract dilutions (25, 50, 75 and 100%) for 24 h. The extracts of polyether materials caused a decrease of cellular viability, evaluated by light microscopy, by cell counting and by MTT test. The extracts of vinyl polysiloxanes materials induced a slight effect on cellular number and viability. The direct tests were performed by placing the impression materials in the centre of Petri dishes while Balb/c 3T3 were settling. The cellular proliferation was drastically reduced by polyethers and it was unaffected by the presence of vinyl polysiloxanes. These results show that: (a) the polyether materials are more toxic than vinyl polysiloxanes in our experimental conditions, (b) the impression materials are cytotoxic to the same degree in all assay methods.