Neutral Red Uptake Cytotoxicity Assay Research Articles

Homo- and heterometallic complexes of Zn(II), {Zn(II)Au(I)}, and {Zn(II)Ag(I)} with pentadentate Schiff base ligands as promising anticancer agents.

Two families of homo- and heterometallic complexes, [Zn2L1(μ-OH)(H2O)2](ClO4)2, [Zn2L2(μ-OH)(H2O)2](ClO4)2, [Zn2L3(μ-OH)(H2O)2](ClO4)2, 1∞[{L1Zn2(μ-OH)}{μ-[Ag(CN)2]}](ClO4), [{L1Zn2(μ-OH)}2{μ-[Au(CN)2]}{[Au(CN)2]2}](ClO4)·H2O, 1∞[{L2Zn2(μ3-OH)}2(H2O){μ-[Ag(CN)2]}](ClO4)3·THF·0.5MeOH, 1∞[{L2Zn2(μ3-OH)}2(H2O){μ-[Au(CN)2]}](ClO4)3·THF·H2O, and 1∞[{L3Zn2(μ-OH)}{μ-[Ag(CN)2]}][Ag(CN)2]·H2O, respectively, have been synthesized and characterized. The Schiff bases used as ligands were obtained by condensation reactions of 2,6-diformyl-p-cresol with N,N-dimethyl-ethylenediamine (HL1), 2-aminomethyl-pyridine (HL2), and 2-aminoethyl-pyridine (HL3), respectively. The cytotoxic/cytostatic and genotoxic effects in cultured human MCF-7 (luminal type A breast cancer), MDA-MB-231 (triple negative breast cancer), HeLa (cervical carcinoma), and Lep-3 (non-tumor embryonal fibroblastoid cells) were studied. The investigations were performed by thiazolyl blue tetrazolium bromide test (MTT test), neutral red uptake cytotoxicity assay, crystal violet staining, hematoxylin and eosin staining, double staining with acridine orange and propidium iodide, AnnexinV/FITC, and Comet assay in short-term experiments (24-72 h, with monolayer cell cultures) as well as by 3D colony-forming method in long-term experiments (28 days, with 3D cancer cell colonies). The results obtained revealed that: (i) applied at a concentration range of 0.1-100 μg mL-1, the compounds investigated decrease in a time- and concentration-dependent manner the viability and/or proliferation of the treated cells; (ii) complexes of {Zn(II)Au(I)} show relatively higher cytotoxic/genotoxic activity and antitumor potential as compared to {Zn(II)Ag(I)}; (iii) some of the complexes demonstrate more pronounced cytotoxic potential than commercially available antitumor agents cisplatin, oxaliplatin, and epirubicin.

Preparation and in vitro biocompatibility of PBAT and chitosan composites for novel biodegradable cardiac occluders

Abstract The biodegradable composites were prepared by melt blending of chitosan (CS) and poly(butyleneadipate-co-terephthalate) (PBAT). By utilizing Fourier transformed infrared spectroscopy, scanning electron microscopy-energy dispersive spectroscopy, mechanical properties analysis, water contact angle measuring, differential scanning calorimetry, and thermogravimetric analysis, it was demonstrated that the CS of the PBAT-CS10 composite was relatively evenly dispersed in the PBAT matrix, the mechanical properties were significantly improved, the hydrophilicity was increased, the cold crystallization temperature was increased, and a good range of melt working temperature was obtained. The PBAT-CS10 composite was used to fabricate a cardiac occluder by fused deposition modeling of three-dimensional printing, and finite element analysis, and in vitro implantation testing proved the occluder’s mechanical support and sealing function under extreme boundary conditions. In vitro degradation experiments, neutral red uptake cytotoxicity assay, CCK-8 cell proliferation detection, immunofluorescence staining of the cytoskeleton, cell apoptosis detection, and reactive oxygen species assay were all performed on the composite, confirming that it and the occluder made of it could be hydrolyzed under physiological conditions and had no adverse effects on the cell membrane, lysosome membrane, cell proliferation, cell morphology, cell apoptosis, or ROS level, and had good biocompatibility.

Costus spiralis extract restores kidney function in cisplatin-induced nephrotoxicity model: Ethnopharmacological use, chemical and toxicological investigation

Ethnopharmacological relevanceCostus spiralis (Jacq.). Roscoe (Costaceae) is traditionally used in Brazil for the treatment of kidney diseases such as pyelonephritis, urethra inflammation, kidney stones, and inflammatory conditions. There are reports of its use by Brazilian Indians since the 17th century when it was known as “pacocatinga.” Currently, the use of the Costus species in Brazil is widespread, which was evidenced by the inclusion of the genus in the Brazilian National List of Medicinal Plants of Interest to the Unified Health System (RENISUS). Aim of the studyThis study aimed to confirm the ethnopharmacological use of Costus spiralis in the treatment of kidney diseases, toxicity study using animal models, and the phytochemistry of the species. Materials and methodsThe chemical profile of Costus spiralis leaves extract (CSLE) was obtained for the hydroethanolic extract by ultra-performance liquid chromatography coupled to a mass spectrometer and ultraviolet detector with diode array (UPLC-UV/DAD-ESI-MS). The acute oral toxicity of the extract was predicted using the neutral red uptake cytotoxicity assay. Wistar rats were used in a model in vivo for confirmation of acute oral toxicity (2000 mg/kg p.o. for 14 days.) and determination of the effect on a cisplatin-induced nephrotoxicity model. ResultsThe analysis by UPLC-UV/DAD-ESI-MS showed that the chemical composition of the extract is mostly di-glycosylated flavones of apigenin. In the extract were identified the flavones vicenin II and schaftoside. The quantification of total flavonoids by spectrometry showed 0.880%. CSLE proved to be safe for acute oral administration (2000 mg/kg) with an IC50 value of 222.9 μg/mL and predicted oral toxic dose of 523.82 μg/mL in a neutral red uptake cytotoxicity assay. The absence of death allows the classification of the extract in class 5 according to OECD 423 guidelines and therefore it can be considered as a high acute safety product, which is highly relevant, considering the wide popular use of the species. In the cisplatin-induced nephrotoxicity model, C. spiralis extract (5, 15, and 30 mg/kg) significantly improved renal function, reversing almost completely the effects on plasma creatinine levels and creatinine clearance (p < 0.001). ConclusionsThis study demonstrates that oral administration of Costus spiralis extract leaves is safe and effective in restoring the renal function in rats in a cisplatin-induced nephrotoxicity. It is suggested that the observed activity is related to the flavonoids present. This hypothesis should be confirmed, and the participation of other secondary metabolites should be investigated in the future.

In vitro evaluation of mutagenic, cytotoxic, genotoxic and oral irritation potential of nicotine pouch products

Non-clinical in vitro studies were conducted to investigate the characteristics of extracts from tobacco free nicotine pouches alongside a reference snus product and/or 1R6F reference cigarette. In vitro investigations were conducted in the Neutral Red Uptake (NRU) cytotoxicity assay, Bacterial Reverse Mutation (Ames) assay, and in vitro Mammalian Cell Micronucleus (ivMN) assay. These products were also investigated for their oral irritation potential in the EpiGingival™ 3D tissue model. Results from the Ames, in vitro Micronucleus and NRU assays indicated that the tested products were non-mutagenic, non-genotoxic and non-cytotoxic in contrast to results obtained for the 1R6F reference cigarette. Results from Complete Artificial Saliva (CAS) extracts from these products also failed to be classified as irritants (as measured using the MTT assay), in the EpiGingival™ 3D tissue model

ZN(II)/AU(I) AND ZN(II)/AG(I) COMPLEXES WITH SALEN SCHIFF BASE EXPRESS PROMISING CYTOTOXIC ACTIVITY IN HUMAN CANCER CELLS

Objective: The aim of our study was to evaluate the influence of two complexes of Zn(II)/Au(I) and Zn(II)/Ag(I) with Schiff base ligand (H2Salen) obtained from the condensation reaction between salicylaldehyde and ethylenediamine (abbreviated ZnSalenAu, ZnSalenAg) on viability and proliferation of cultured human cancer cells.Methods: The following cell lines were used as model systems: Human cervical carcinoma (cervical carcinoma), A549 (non-small cell lung cancer [NSCLC]), glioblastoma multiforme (8MGBA), and A431 (squamous cell carcinoma) and its multidrug-resistant (MDR) clones A431-MDR, A431-MRP, and A431-ABCG2 that express mdr1, mrp1, or abcg2 gene, respectively. The investigations were performed by thiazolyl blue tetrazolium bromide test, neutral red uptake cytotoxicity assay, crystal violet staining, hematoxylin and eosin staining, double staining with acridine orange, and propidium iodide in short-term experiments (12–72 h, with monolayer cell cultures) as well as colony-forming method in long-term experiments (25 days, with three dimensional cancer cell colonies).Results: The results obtained revealed that ZnSalenAu and ZnSalenAg decreased significantly viability and proliferation of the treated cells in a time- and concentration-dependent manner being more active as compared to the free ligand H2Salen.Conclusion: The present study demonstrates for the 1st time the ability of two heterometallic complexes ZnSalenAu and ZnSalenAg to decrease significantly viability and proliferation of cultured cell lines established from some of the most common and aggressive human cancers (NSCLC, carcinoma of uterine cancer, 8MGBA, and squamous cell carcinoma) as well as MDR cancer cells.

ZN(II)/AU(I) AND ZN(II)/AG(I) COMPLEXES WITH SALEN SCHIFF BASE EXPRESS PROMISING CYTOTOXIC ACTIVITY IN HUMAN CANCER CELLS

Objective: The aim of our study was to evaluate the influence of two complexes of Zn(II)/Au(I) and Zn(II)/Ag(I) with Schiff base ligand (H2Salen) obtained from the condensation reaction between salicylaldehyde and ethylenediamine (abbreviated ZnSalenAu, ZnSalenAg) on viability and proliferation of cultured human cancer cells.Methods: The following cell lines were used as model systems: Human cervical carcinoma (cervical carcinoma), A549 (non-small cell lung cancer [NSCLC]), glioblastoma multiforme (8MGBA), and A431 (squamous cell carcinoma) and its multidrug-resistant (MDR) clones A431-MDR, A431-MRP, and A431-ABCG2 that express mdr1, mrp1, or abcg2 gene, respectively. The investigations were performed by thiazolyl blue tetrazolium bromide test, neutral red uptake cytotoxicity assay, crystal violet staining, hematoxylin and eosin staining, double staining with acridine orange, and propidium iodide in short-term experiments (12–72 h, with monolayer cell cultures) as well as colony-forming method in long-term experiments (25 days, with three dimensional cancer cell colonies).Results: The results obtained revealed that ZnSalenAu and ZnSalenAg decreased significantly viability and proliferation of the treated cells in a time- and concentration-dependent manner being more active as compared to the free ligand H2Salen.Conclusion: The present study demonstrates for the 1st time the ability of two heterometallic complexes ZnSalenAu and ZnSalenAg to decrease significantly viability and proliferation of cultured cell lines established from some of the most common and aggressive human cancers (NSCLC, carcinoma of uterine cancer, 8MGBA, and squamous cell carcinoma) as well as MDR cancer cells.

Investigating cell type specific mechanisms contributing to acute oral toxicity_suppl2

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of integrated approaches to testing and assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evalu­ation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system, and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 97 were identified with target organ specific effects, of which 94% (91/97) were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% (6/97) as non-toxic. Although specific target organ mechanisms of toxicity could in some cases explain the false negative prediction obtained with the cytotoxicity assay, in general it is difficult to explain in vitro misclassifications only on the basis of mechanistic information. This analysis will help to prioritize the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.

Investigating cell type specific mechanisms contributing to acute oral toxicity_suppl1

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of integrated approaches to testing and assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evalu­ation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system, and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 97 were identified with target organ specific effects, of which 94% (91/97) were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% (6/97) as non-toxic. Although specific target organ mechanisms of toxicity could in some cases explain the false negative prediction obtained with the cytotoxicity assay, in general it is difficult to explain in vitro misclassifications only on the basis of mechanistic information. This analysis will help to prioritize the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.

Synthesis, Characterization and Cytotoxic Activity of Co(II), Ni(II), Cu(II), and Zn(II) Complexes with Nonsteroidal Antiinflamatory Drug Isoxicam as Ligand

Four complexes of Co(II), Ni(II), Cu(II), and Zn(II) with isoxicam (H2ISO) as ligand, namely [Co(HISO)2(H2O)2]⋅3H2O (Co–Iso), [Ni(HISO)2(H2O)2]⋅0.5H2O (Ni–Iso), [Cu(HISO)2(H2O)] (Cu–Iso), [Zn(HISO)2(H2O)2]⋅0.5H2O (Zn–Iso), have been synthesized and characterized by FT-IR, UV–Vis–NIR, 1H NMR, TG/DSC, elemental analysis, magnetic and conductivity measurements. The following permanent cell lines were used to evaluate the cytotoxic activity of the compounds: LSCC-SF-Mc29, strain E7 (transplantable chicken hepatoma induced by the myelocytomatosis virus Mc29), LSR-SF-SR (transplantable sarcoma in rat, induced by Rous sarcoma virus, strain Schmidt-Ruppin), MCF-7 (luminal A type breast cancer), HeLa (human carcinoma of the uterine cervix) and Lep-3 (human non-tumor embryonal fibroblastoid cells). Short term experiments (24–72 h) with monolayer cultures were performed by MTT test, neutral red uptake cytotoxicity assay and double staining with acridine orange and propidium iodide as well as long term experiments (20 days) with 3D cancer cell colonies in semi-solid medium. The results obtained reveal that: (i) applied at a concentration range of 5–500 µg mL−1 the compounds investigated decrease in a time- and concentration-dependent manner viability and/or proliferation of the treated cells; (ii) metal complexes are more pronounced cytotoxic agents as compared to isoxicam; (iii) cell-specific response was observed—Cu–Iso expresses the highest cytotoxic activity in LSCC-SF-Mc29 (strain E7) cells, whereas Zn–Iso is the most effective compound in LSR-SF-SR and Lep-3 cells, and Co–Iso—in HeLa cells; (iv) in general, human non-tumor Lep-3 embryonic fibroblastoid cells are relatively less sensitive to the cytotoxic activity of the complexes as compared to human cancer MCF-7 and HeLa cells.

Investigating cell type specific mechanisms contributing to acute oral toxicity.

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of Integrated Approaches to Testing and Assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evaluation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 98 were identified with target organ specific effects, of which 93% were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% as non-toxic. This analysis will help to prioritise the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.

CON4EI: EpiOcular eye irritation tests – OECD TG 492 and ET-50 (time-to-toxicity) protocols

Non-clinical in vitro studies were conducted to investigate the characteristics of extracts from tobacco free nicotine pouches alongside a reference snus product and/or 1R6F reference cigarette. In vitro investigations were conducted in the Neutral Red Uptake (NRU) cytotoxicity assay, Bacterial Reverse Mutation (Ames) assay, and in vitro Mammalian Cell Micronucleus (ivMN) assay. These products were also investigated for their oral irritation potential in the EpiGingival™ 3D tissue model. Results from the Ames, in vitro Micronucleus and NRU assays indicated that the tested products were non-mutagenic, non-genotoxic and non-cytotoxic in contrast to results obtained for the 1R6F reference cigarette. Results from Complete Artificial Saliva (CAS) extracts from these products also failed to be classified as irritants (as measured using the MTT assay), in the EpiGingival™ 3D tissue model

3d metal complexes with meloxicam as therapeutic agents in the fight against human glioblastoma multiforme and cervical carcinoma

The aim of our study was to evaluate the influence of selective non-steroidal anti-inflammatory drug meloxicam and its metal (Cu(II), Zn(II), Co(II), Ni(II)) complexes on the viability and proliferation of cultured humancarcinoma of the uterine cervix (HeLa) and glioblastoma multiforme (8MGBA) cells. The investigations were performed by short-term (24 h–96 h, with monolayer cultures) and long-term (16 d, with three-dimensional colonies of cancer cells) experiments by using methods with different molecular/cellular targets and mechanisms of action, such as thiazolyl blue tetrazolium bromide (MTT) test, neutral red uptake cytotoxicity assay, crystal violet staining, double staining with acridine orange and propidium iodide, alkaline version of single cell gel electrophoresis (comet assay) and colony-forming technique. The obtained results revealed that the application of the examined compounds at concentrations ranging from 5 µg/mL to 500 µg/mL induced cytopathological changes, including DNA damages in the treated cells and a significant decrease of their viability and proliferation in a time- and concentration-dependent manner. Metal complexes were found to have a more pronounced cytotoxic/cytostatic effect, when compared to their ligand meloxicam.

P7.04 - Influence of Deoxycholic acid and its metal complexes on viability and proliferation of human tumor cell lines

ABSTRACT Introduction: The biological role of bile acids is well known and a number of derivatives of cholic acid are regarded as suitable candidates for the establishment of conjugated agents in carrying out site-directed anti-cancer therapy. At the same time there are data about carcinogenicity of some of these acids, which requires in-depth study of their relationship with cancer cells. The aim of our study was to evaluate the influence of deoxycholic acid (DCA) and its metal (CuII, ZnII, NiII) complexes on viability and proliferation of cultured human tumor cells. Materials and Methods: Human cell lines established from cancers of the breast (MCF-7), uterine cervix (HeLa), lung (A549), liver (HepG2), brain (8MGBA) and colon (HT29) were used as experimental models. A complex approach involving methods with different cellular / molecular targets was applied to evaluate the putative cytotoxic/cytostatic activities of the compounds: MTT test, neutral red uptake cytotoxicity assay, crystal violet staining, double staining with acridine orange and propidium iodide, Comet assay, colony-forming method. Data were processed by statistical analyses. Results: Administered at a concentration range of 10-200 µg/ml for 24–120 h the compounds examined decrease in various degree the viability and proliferation of the treated cells in a time- and concentration dependant manner and suppress their ability to form 3D colonies in semi-solid medium. Cytopathological changes including double stranded DNA damages have also been observed. Conclusions: The compounds investigated reduce significantly the growth of cell lines established from some of the most common and aggressive human cancers. Cu(II) and Zn(II) complexes of DCA are found to express the most promising antitumor activity. Applied independently, DCA is less effective as compared to its metal complexes. Among the cell lines involved as model systems in our study, A549 non-small cell lung cancer cells show the highest sensitivity to the cytotoxic/cytostatic properties of the tested compounds. Acknowledgements: Supported by Grant DFNI-Б02/30 from 12.12.2014 from National Scientific Fund in Bulgaria and a bilateral project between Bulgarian Academy of Sciences and Romanian Academy.

P7.05 - Metal complexes of Meloxicam and Isoxicam decrease viability and proliferation of virus-transformed cancer cells

ABSTRACT Introduction: There is growing evidence supporting the potential antitumor properties of non-steroidal anti-inflammatory drugs (NSAIDs). They are among the most consumed drugs over the world, including patients with cancer - a fact which further enhances the interest in the influence of these preparations on the survival and proliferative activity of tumor cells. It is assumed that for the realization of their biological activity is important linking to metal ions in the body. The aim of our study was to evaluate the effect of metal (CuII, ZnII, CoII, NiII) complexes of selective (Meloxicam) and non-selective (Isoxicam) NSAIDs on viability and proliferation of virus-transformed cancer cells. Materials and Methods: Permanent cell lines established from transplantable tumors in chicken (LSCC-SF-Mc29 –E7 - liver cancer, induced by the myelocytomatosis retrovirus Mc29) and rat (LSR-SF-SR–sarcoma, induced by RSV strain Schmidt-Rupin) were used as model systems. The investigations were performed by MTT test, neutral red uptake cytotoxicity assay, crystal violet staining, double-staining with acridine orange and propidium iodide, colony forming method and FACS analysis. The compounds were applied at a concentration range of 10-500 µg/ml for 24-120 h (short-term assays with monolayer cultures) and 18 days (long-term assay with 3D colonies). Results: The compounds examined decrease significantly viability and proliferation of treated cells as well as their ability to grow in semisolid medium in a time- and concentration-dependant manner. Cytopathological changes (including chromatin fragmentation and apoptotic bodies) and lower percentage of cells in G2 / M phase were also found. Conclusions: Cu(II) complexes with Meloxicam and Isoxicam are found to be the most active cytotoxic and cytostatic agents. They are highly effective against LSCC-SF-Mc29 cells that contain v-myc gene – disregulation of its cellular analogues is involved in pathogenesis of > 80% of human cancers. Applied independently, the ligands express lower cytotoxic/cytostatic activity as compared to metal complexes. Acknowledgements: Supported by Grant DFNI - Б 02/30 from 12.12.2014, Bulgaria, and a bilateral project between Bulgarian Academy of Sciences and Romanian Academy

Metal Zn(II), Cu(II), Ni (II) complexes of ursodeoxycholic acid as putative anticancer agents

The aim of the study was to evaluate the influence of metal [Zn(II), Cu(II), Ni(II)] complexes with ursodeoxycholic acid (UDCA) on the viability and proliferation of tumour and non-tumour cells. Cell lines established from retrovirus-transformed chicken hepatoma (LSCC-SF-Mc29) and rat sarcoma (LSR-SF-SR) as well as from human cancers of the breast (MCF-7), uterine cervix (HeLa), lung (A549) and liver (HepG2) were used as model systems. Non-tumour human embryo (Lep-3) cells were also included in some of the experiments. The investigations were carried out by the thiazolyl blue tetrazolium bromide (MTT) test, neutral red uptake cytotoxicity assay, crystal violet staining, double staining with acridine orange and propidium iodide and the colony-forming method. The results obtained revealed that: (1) UDCA and its metal complexes in the tested concentrations decreased (to a varying degree) the viability and proliferation of the treated cells in a time- and concentration-dependent manner; (2) chicken hepatoma (LSCC-SF-Mc29) cells were most sensitive to the cytotoxic and antiproliferative action of the compounds tested, followed by rat sarcoma (LSR-SF-SR) cells; (3) Cu‒UDCA and Ni‒UDCA were more effective against animal LSCC-SF-Mc29 and LSR-SF-SR cells, while Zn‒UDCA significantly decreased the viability and proliferation of human tumour cell lines; (4) applied independently, UDCA expressed lower cytotoxic/cytostatic activity as compared to metal complexes; and (5) the sensitivity of the non-tumour embryonic Lep-3 cells to the effects of UDCA and its metal complexes was comparable or even higher than those of the human tumour cells.

A tutorial for analysing the cost-effectiveness of alternative methods for assessing chemical toxicity: the case of acute oral toxicity prediction.

Compared with traditional animal methods for toxicity testing, in vitro and in silico methods are widely considered to permit a more cost-effective assessment of chemicals. However, how to assess the cost-effectiveness of alternative methods has remained unclear. This paper offers a user-oriented tutorial for applying cost-effectiveness analysis (CEA) to alternative (non-animal) methods. The purpose is to illustrate how CEA facilitates the identification of the alternative method, or the combination of methods, that offers the highest information gain per unit of cost. We illustrate how information gains and costs of single methods and method combinations can be assessed. By using acute oral toxicity as an example, we apply CEA to a set of four in silico methods (ToxSuite, TOPKAT, TEST, ADMET Predictor), one in vitro method (the 3T3 Neutral Red Uptake cytotoxicity assay), and various combinations of these methods. Our results underline that in silico tools are more cost-effective than the in vitro test. Battery combinations of alternative methods, however, do not necessarily outperform single methods, because additional information gains from the battery are easily outweighed by additional costs.

Evaluation of the cytotoxic and genotoxic potential of lecithin/chitosan nanoparticles

Nanoparticles-based drug targeting deliv- ery systems have been introduced in the treatment for various diseases because of their effective properties, although there have been conflicting results on the toxicity of nanoparticles. In the present study, the aim was to evaluate the cytotoxicity and the genotoxicity of different concentrations of lecithin/chitosan nano- particles with and without clobetasol-17-propionate (CP) by neutral red uptake (NRU) cytotoxicity assay and single cell gel electrophoresis (Comet) and cytokinesis-blocked micronucleus assays. The IC50 values of lecithin/chitosan nanoparticles with/without CP were found as 1.9 and 1.8 %, respectively, in the NRU cytotoxicity test. High concentrations of leci- thin/chitosan nanoparticles induced DNA damage in human lymphocytes as evaluated by comet assay. The micronucleus frequency was increased by the lecithin/ chitosan treatment in a dose-dependent manner. Also at the two highest concentrations, a significant increase in micronucleus formation was observed. Lecithin/chitosan nanoparticles with CP did not increase the frequency of micronucleus and also did not induce additional DNA damage when compared with lecithin/chitosan nanoparticles without CP; therefore, CP itself has not found to be genotoxic at the studied concentration.

Evaluation method for the cytotoxicity of cigarette smoke by in vitro whole smoke exposure

An in vitro whole smoke (WS) exposure method was established to evaluate the toxicological effects of fresh cigarette smoke using the VITROCELL® system associated with the neutral red uptake (NRU) cytotoxicity assay. The VITROCELL® system is a newly representative culture and exposure system for in vitro studies of gases or complex mixtures. The impacts of two factors on cytotoxicity measurements of cigarette smoke were investigated using this WS exposure system. The factors include synthetic air exposure and optimal time to perform the NRU assay after smoke exposure. Results showed that synthetic air exposure used in the system did not significantly alter cell survival; 24h after smoke exposure appeared to be an optimal time-point to assess the cytotoxicity of cigarette smoke. A clear dose–response relationship between smoke exposure and cell viability was demonstrated using this system, and the evaluation method was sensitive to distinguish the differences in smoke-induced cytotoxic effects from different cigarettes. In addition, we tried converting the values of EC50 from WS exposure testing into the values in unit used in total particulate matter (TPM) testing for a purpose of comparison, and the data indicate that the cytotoxicity of smoke measured by WS exposure is greater than that measured by TPM exposure.

In Vitro Activity of Biometal(II) Complexes of Monensin Against Virus-Induced Transplantable Animal Tumors

ABSTRACTThe anticancer properties of monensic acid (MonH) and its complexes with ions of Mg(II), Ca(II), Mn(II) and Co(II) were studied in cultured permanent cell lines originated from virus-induced transplantable tumors in chicken (hepatoma, LSCC-SF-Mc29) and in rat (sarcoma, LSR-SF-SR). The short-term experiments in monolayer adherent cells were performed by thiazolyl blue tetrazolium bromide (MTT) test (hepatoma and sarcoma cell lines) and neutral red (NR) uptake cytotoxicity assay (sarcoma cell line). The effect of monensin and corresponding complexes on the colony-forming ability of tumor cells was also evaluated. From the results obtained it can be concluded that all compounds significantly decrease the viability and proliferation of the treated cells in a time- and concentration-dependent manner, with metal(II) complexes being more effective than the non-coordinated ligand.

Assessment of the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity test method to identify substances not classified for acute oral toxicity (LD50 > 2000 mg/kg): Results of an ECVAM validation study

Assessing chemicals for acute oral toxicity is a standard information requirement of regulatory testing. However, animal testing is now prohibited in the cosmetics sector in Europe, and strongly discouraged for industrial chemicals. Building on the results of a previous international validation study, a follow up study was organised to assess if the 3T3 Neutral Red Uptake cytotoxicity assay could identify substances not requiring classification as acute oral toxicants under the EU regulations. Fifty-six coded industrial chemicals were tested in three laboratories, each using one of the following protocols: the previously validated protocol, an abbreviated version of the protocol and the protocol adapted for an automation platform. Predictions were very similar among the three laboratories. The assay exhibited high sensitivity (92–96%) but relatively low specificity (40–44%). Three chemicals were under predicted. Assuming that most industrial chemicals are not likely to be acutely toxic, this test method could prove a valuable component of an integrated testing strategy, a read-across argument, or weight-of-evidence approach to identify non toxic chemicals (LD50>2000mg/kg). However, it is likely to under predict chemicals acting via specific mechanisms of action not captured by the 3T3 test system, or which first require biotransformation in vivo.