In vitro RHE skin sensitisation assays: Applicability to challenging substances.

In the last 20 years, alternative approaches to the identification of skin sensitisation hazards have been at the forefront of the 3Rs and have helped refine the validation and acceptance processes. However, experience with the local lymph node assay showed that, post-validation, challenges still occurred, particularly when a wider diversity of chemical substances was addressed, a situation which will arise with validated in vitro alternatives. In the present work, a range of substances potentially challenging to assess in current nonanimal OECD test guidelines were evaluated in several of the emerging in vitro alternatives. Twelve such substances (of which just over half were known skin sensitisers) were assessed in 4 assays, all based on reconstructed human epidermis (RHE) models. For hazard identification, the overall predictive accuracy ranged around 70% for three assays, although for one (SensCeeTox), it fell below 50% when human data was used as the benchmark. In most cases, sensitivity was high, such that sensitisation was overpredicted. As the substances were challenging to assess in other nonanimal methods, the results indicate that the 3D RHE models may be a useful tool for assessing skin sensitisation potentials without needing to revert to animal use.

CON4EI: EpiOcular™ Eye Irritation Test (EpiOcular™ EIT) for hazard identification and labelling of eye irritating chemicals.

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. The objective of the CON4EI project was to develop tiered testing strategies for eye irritation assessment. A set of 80 reference chemicals (38 liquids and 42 solids) was tested with eight different methods. Here, the results obtained with the EpiOcular™ Eye Irritation Test (EIT), adopted as OECD TG 492, are shown. The primary aim of this study was to evaluate of the performance of the test method to discriminate between chemicals not requiring classification for serious eye damage/eye irritancy (No Category) and chemicals requiring classification and labelling. In addition, the predictive capacity in terms of in vivo drivers of classification (i.e. corneal opacity, conjunctival redness and persistence at day 21) was investigated. EpiOcular™ EIT achieved a sensitivity of 97%, a specificity of 87% and accuracy of 95% and also confirmed its excellent reproducibility (100%) from the original validation. The assay was applicable to all chemical categories tested in this project and its performance was not limited to the particular driver of the classification. In addition to the existing prediction model for dichotomous categorization, a new prediction model for Cat 1 is suggested.

Antiproliferative activity and apoptosis induced by 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline on cells of leukemia lines.

UNLABELLED: Quinazolines are known to be multitarget agents with broad spectrum of biological activity. AIM: To investigate anticancer activity of newly prepared 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline (BMAQ) towards L1210, HL-60 and U-937 leukemia cells. MATERIALS AND METHODS: Growth inhibition of BMAQ-treated cells was determined by cell counting using trypan blue staining technique. Apoptosis and cell cycle profile changes were analysed using internucleosomal DNA fragmentation assay, fluorescence microscopy and flow cytometry. Activity of caspase-3 was determined using colorimetric method. RESULTS: Cell proliferation assay showed that BMAQ caused significant decrease of cell number in a dose-dependent manner. BMAQ induced cell death by apoptosis, based on results from DNA fragmentation, fluorescence microscopy and caspase-3 assays. CONCLUSION: Presented results clearly demonstrate that BMAQ is a promising anticancer agent with significant antiproliferative and apoptotic activities towards leukemia cells in vitro.

4-Amino-3-acetylquinoline-induced apoptosis of murine L1210 leukemia cells involves ROS-mitochondrial-mediated death signaling and activation of p38 MAPK.

Quinolines are known to be multitarget agents with a broad spectrum of biological activity. In a previous study, we showed that newly prepared 4-amino-3-acetylquinoline (AAQ) possesses strong anticancer activities. In this study, we investigated whether AAQ has cytotoxicity in murine L1210 leukemia cells. Results from cell proliferation assays showed that AAQ caused significant decrease in cell number in a dose-dependent manner. The cell death induced by AAQ appeared to involve apoptosis, based on evidence from apoptotic DNA fragmentation, flow cytometry, fluorescence microscopy, and Western blot analyses. We found that AAQ-treated cells had activated p38 MAPK and that apoptosis was processed through a reactive oxygen species (ROS)-dependent mitochondrial pathway. In summary, our results suggest that AAQ can induce apoptosis, at least in part, through the activation of the p38 MAPK pathway in L1210 leukemia cells.

Cytotoxicity of hydroxyapatite, fluorapatite and fluor-hydroxyapatite: a comparative in vitro study.

The purpose of this study was to evaluate the cytotoxicity of two formulations of hydroxyapatite (HA), namely fluorapatite (FA) and fluor-hydroxyapatite (FHA). HA is used as carrier material for antibiotics or anticancer drugs during treatment of bone metastasis. Negative control, represented by HA, was included for comparative purposes. Leukemia cells were used as a model cell line, and the effect of eluates of tested biomaterials on cell proliferation/viability and mechanism of antiproliferative activity were assessed. Study design attempted to reveal the toxicity of tested biomaterials with an emphasis to decide if tested biomaterials have promise for further studies in vivo. Results showed that eluates of FA and FHA inhibit the growth of leukemia cells and induce programmed cell death through mitochondrial/caspase-9/caspase-3-dependent pathway. Due to these differences compare to HA, it is concluded that FA and FHA have promise for evaluation of their behaviour in vivo.

In vitro effects of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity.

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard-tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH(-) groups and F(-) ions. The aim of this experimental investigation was to evaluate cytotoxic, genotoxic and mutagenic effects of FHA and FA eluates on Chinese hamster V79 cells and to compare them with the effects of hydroxyapatite (HA) eluate. Cytotoxicity of the biomaterials tested was evaluated by use of the cell colony-formation assay and by direct counting of the cells in each colony. Genotoxicity was assessed by single-cell gel electrophoresis (comet assay) and mutagenicity was evaluated by the Hprt gene-mutation assay and in bacterial mutagenicity tests using Salmonella typhimurium TA100. The results show that the highest test concentrations of the biomaterials (100% and 75% eluates) induced very weak inhibition of colony growth (about 10%). On the other hand, the reduction of cell number per colony induced by these concentrations was in the range from 43% to 31%. The comet assay showed that biomaterials induced DNA breaks, which increased with increasing test concentrations in the order HA

New [1,2,4]triazolo[4,3-c]quinazoline enhances cisplatin- and temozolomide-induced growth inhibition and apoptosis in HL-60 cells.

The purpose of this study was to investigate the therapeutic potential of a newly synthesized [1,2,4]triazolo[4,3-c]quinazoline (NTCHMTQ) alone and in combination with two anticancer drugs (cisplatin and temozolomide) against HL-60 leukemia cell line. The IC50 value of NTCHMTQ toward HL-60 cells was 19.7 microM. No apoptosis and cell cycle changes were observed in cells treated with 5 microM NTCHMTQ alone. Combination of non-toxic concentrations of NTCHMTQ (1-5 microM) with cisplatin or temozolomide sensitized HL-60 cells to these two drugs and significantly enhanced their efficacies, that is illustrated by combination indexes, sub-G0 cell fraction, apoptotic DNA fragmentation and caspase-3 activity. The results suggest that combined therapy of non-toxic concentrations of NTCHMTQ with chemotherapeutics may provide synergistic regimen for treatment of leukemia. However, further in vitro and in vivo experimental drug-cell and drug-drug studies are warranted.

[Cytotoxicity screening and mode of action of natural and synthetically prepared compounds usable in tumour chemotherapy]. / Skríning cytotoxicity in vitro a mechanizmus pôsobenia prírodných a synteticky pripravených látok vyuzitel'ných v chemoterapii nádorov.

The paper reviews the current approaches to cytotoxic effect screening and mode of action of natural and synthetic compounds usable in tumour chemotherapy. Oncological diseases belong to the most frequently occurring and the most serious diseases that threaten millions of human lives. A serious complication of anticancer therapy is an inadequate therapeutic answer which is caused by the resistance of the human organism to the employed drugs, chemotherapeutic agents. Therefore the resistance is one of the driving forces which constantly force us to search for new anticancer effective drugs of natural or synthetic origin. Primary screening in vitro, in which by different methods the sensitivity of cancer cells growing in vitro to cytotoxic compounds is monitored, is commonly and routinely used for searching for new potential cytostatics today. The properties of a potential anticancer compound are characterized by different parameters, which include antiproliferative activity, monitoring of structural and functional changes in the cytoplasmic membrane, changes in cell proteins and nucleic acids content, cell metabolism changes, cell cycle changes, induction of apoptosis, enzymatic activity (dihydrofolate reductase, proteinases, proteinkinases, topoisomerases, tymidylatsynthetase), effect on mitochondria, cell cytoskeleton, telomerase activity, etc. By monitoring these parameters, the mode of action of a cytotoxically effective compound can be followed up.

Cytotoxic/antiproliferative effects of new [1,2,4]triazolo[4,3-c] quinazolines in tumor cell lines HeLa and B16.

Quinazolines - 1,3-benzodiazines are biological active compounds, and some of them act as anticancer drugs. We evaluated cytotoxic/antiproliferative activity of new synthetically prepared [1,2,4]triazolo[4,3-c]quinazolines using tumor cell lines HeLa and B16. The in vitro cytotoxic studies of the most active derivative 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin- 4-yl[1,2,4]triazolo[4,3-c]quinazoline (NTCHMTQ) were complemented by cell cycle analysis, and determination of apoptotic DNA fragmentation. Possible direct interaction of NTCHMTQ with calf thymus DNA was tested by the DNA-modified screen-printed electrode. Five quinazoline derivatives tested acted cytotoxically on both tumor cell lines. The melanoma cells B16 were more sensitive to quinazolines treatment than HeLa cells. The most effective derivative was NTCHMTQ which manifested significant in vitro cytotoxic/antiproliferative effect. NTCHMTQ at micromolar concentrations induced morphological changes and necrosis of B16 cells. NTCHMTQ at concentrations tested did not cause changes in cell cycle, did not induce apoptotic cell death in the B16 cells and did not even behave as a typical intercalating agent.

The effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on cell growth, cell cycle, induction of DNA fragmentation, and activity of caspase 3 in murine leukemia L1210 cells and fibroblast NIH-3T3 cells.

Quinazolines are multitarget agents, which have broad spectrum of biological activity, and some of them are now in cancer clinical testing. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects on cancer cell lines HeLa and B16. Quinazoline, at micromolar concentrations, induced morphological changes and necrosis of B16 cells, and at nanomolar concentrations it produced changes of F-actin cytoskeleton. It did not cause changes in the cell cycle, did not induce apoptotic cell death in B16 cells, did not have a mutagenic effect, and did not even behave as a typical intercalating agent. Little significant reduction of tumor volume in intramuscular transplanted B16 cells was observed. The aim of the present study was to examine the cytotoxic effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on murine leukemia L1210 cells and fibroblast NIH-3T3 cells. Induction of cell morphology and cell cycle changes, induction of apoptosis and caspase 3 activity were studied. Quinazoline acted cytotoxically on both cell lines. The sensitivity of leukemia L1210 cells to the quinazoline was higher than that of fibroblast NIH-3T3. The IC(100) was 12 microM for L1210 cells and 24 microM for NIH-3T3 cells. No effect of quinazoline on the cell cycle profile of L1210 and NIH-3T3 was detected, however, quinazoline induced an increase of the sub-G(0) cell fraction, apoptotic DNA fragmentation, and apoptotic morphological changes at a concentration of 12 microM. This quinazoline concentration induced caspase 3 activity. Our results demonstrated that induction of apoptotic cell death via activation of caspase 3 contributed to the cytotoxic effects of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline in murine leukemia L1210 cells.

Antimicrobial activity of some substituted triazoloquinazolines.

Fifteen substituted 1,2,4-triazolo[4,3-c]quinazolines were tested for antibacterial and antifungal effects. The most effective derivatives had the triazoloquinazoline skeleton substituted with the pharmacologically active chromophores--morpholine, chlorine and nitro group. The broadest antimicrobial activity was found with 5-morpholin-4-yl-3-(5-nitrothien-2-yl)[1,2,4]triazolo[4,3-c]quinazoline in concentration of 10 mg/L for B. subtilis, 50 mg/L for S. aureus and 100 mg/L for C. tropicalis. The highest tested concentration of derivative caused 83% growth inhibition of R. nigricans.