In vitro & molecular toxicology最新文献

Since 1997 the National Center for Documentation and Evaluation of Alternative Methods to Animal Experiments, ZEBET, in Berlin, has been coordinating a validation study aimed at prevalidation and validation of three in vitro embryotoxicity tests, funded by the European Center for the Validation of Alternative Methods (ECVAM) at the Joint Research Center (JRC, Ispra, Italy). The tests use the cultivation of postimplantation rat whole embryos (WEC test), cultures of primary limb bud cells of rat embryos (micromass or, MM, test), and cultures of a pluripotent mouse embryonic stem cell line (embryonic stem cell test or EST). Each of the tests was performed in four laboratories under blind conditions. In the preliminary phase of the validation study 6 out of 20 test chemicals comprising different embryotoxic potential (non, weakly, and strongly embryotoxic) were tested. The results were used to define biostatistically based prediction models (PMs) to identify the embryotoxic potential of test chemicals for the WEC test and the MM test. The PMs developed with the results of the preliminary phase of the validation study (training set) will be evaluated with the results of the remaining 14 test chemicals (definitive phase) by the end of the study. In addition, the existing, improved PM (iPM) for the EST, which had been defined previously, was evaluated using the results of the preliminary phase of this study. Applying the iPM of the EST to the results of this study, in 79% of the experiments, chemicals were classified correctly according to the embryotoxic potential defined by in vivo testing. For the MM and the WEC test, the PMs developed during the preliminary phase of this validation study provided 81% (MM test) and 72% (WEC test) correct classifications. Because the PM of the WEC test took into account only parameters of growth and development, but not cytotoxicity data, a second PM (PM2) was developed for the WEC test by incorporating cytotoxicity data of the differentiated mouse fibroblast cell line 3T3, which was derived from the EST. This approach, which has previously never been used, resulted in an increase to 84% correct classifications in the WEC test.

The use of hematopoietic progenitors in toxicology often requires scoring colonies, micro- and macroclusters, and evaluation of their distribution while taking into account their shape. Unfortunately, this evaluation is long and tedious, and classification errors may occur. Image analysis can improve the method if there is an automatic way of performing the classification analysis. It has been previously demonstrated that cell conglomerates can be automatically detected. The present paper describes three image analysis algorithms and reports on their capabilities to analyze cell heterogeneity in culture. The comparative tests are made on computer-generated images representing different cell distributions. Combining two of these three algorithms gives fairly good results on complex images created by adding various simple distributions.

Zinc toxicity has been linked to decreased reduced glutathione (GSH) and increased oxidized glutathione (GSSG) contents, which might be caused by a GSSG reductase inhibition by zinc. In this study we investigated zinc effects on GSH synthesis rates in various lung cell lines by thin-layer chromatography after (35)S-cysteine incorporation. Two alveolar epithelial cell lines (A549 and L2) and two human fibroblast-like lung cell lines (11Lu and 16Lu) were used in this study. Equipotent protein synthesis inhibition for the different cell lines was reached after 2 h (L2, 11Lu), 3 h (16Lu), and 4 h (A549) zinc exposure (15-200 microM) to cells. Here GSH depletion and GSSG increase in A549 cells were markedly lower than in the other cell lines tested. Incorporation of cysteine (Cys) into GSH was not different in the cell lines tested, while 11Lu cells only demonstrated a decrease of newly synthesized GSH after 1 h of (35)S-Cys exposure when cells were exposed to zinc. Only 11Lu cells showed a markedly decreased Cys availability as compared with the other cell lines. In all cell lines the availability of Cys was not affected by exposure to zinc. No compensating increase in GSH synthesis rates was found after zinc-mediated cellular GSH depletion.

The phototoxic antidiabetes drug glipizide (1) is photolabile under aerobic conditions and UV-B light. Irradiation of a phosphate-buffered solution of 1 under oxygen atmosphere produces 4 photoproducts as well as singlet oxygen, which was detected by trapping it with 2,5-dimethylfuran and by the histidine test. The photochemistry of 1 involves cleavage of the sulfonamine and the sulfonamine-R bonds. Red blood cell lysis, photosensitized by glipizide and the products of its aerobic photolysis were demonstrated. The photohemolysis rate was lower for 1 than for its photoproducts. Inhibition of this process on addition of 1, 4-diazabicyclo[2.2.2]octane (DABCO), reduced glutathione (GSH), Vitamin C, sodium azide, superoxide dismutase, and a-tocopherol confirmed the possibility of singlet oxygen, superoxide ion and free radicals participation. Furthermore, in a lipid-photoperoxidation test with linoleic acid the in vitro phototoxicity of glipizide was also verified. A low decreasing cell viability of lymphocytes and neutrophils was observed.

Various structurally unrelated chemicals [2,5 hexandione, acrylamide, organophosphates like mipafox, beta,beta iminodipropionnitrile (IDPN), 3-nitropropionic acid (3-NP), potassium cyanide (KCN), paraquat, and NMDA (N-methyl-D-apartic acid)] are known to cause degenerative damage of the peripheral or central nervous system. Differentiated neuronal cell cultures obtained from fetal rats have been used to differentiate the mechanisms underlying this type of neurotoxicity. Cytotoxicity as measured by a viability assay was not sensitive enough and had to be supplemented by further endpoints covering effects on cytoskeleton and on the energy state of the cells [glucose consumption, mitochondrial membrane potential and adenosine 5'-triphosphate (ATP) concentration]. Compounds like the delayed neurotoxic organophosphates, exert a selective direct effect on cytoskeleton elements in this model at concentrations distinctly below cytotoxic concentrations. Other compounds, like KCN, paraquat, and 3-NP selectively disrupt the balance between energy supply and demand of the neurons either by interacting with mitochondrial respiration or glycolysis. For these compounds cytoskeletal damage seemed to be secondary to the energy depletion. For NMDA, 2,5 hexandione and acrylamide, both mechanisms may contribute to the neuronal damage. In conclusion, primary cortical neuronal cultures of the rat are well suited to detect a neurotoxic potential and to differentiate its underlying mechanisms. Damage of the cytoskeleton may be considered as an endpoint mechanistically related to degenerative neuropathic effects.

Previous studies from this laboratory have shown that L-tryptophan, after oxidation by either ultraviolet (UV) irradiation or ozone, causes induction of cytochrome P450 (CYP)1A1 mRNA, protein, and the corresponding 7-ethoxyresorufin O-deethylase (EROD) activity in wild type mouse hepatoma cells, Hepa lclc7 (Hepa-1), through the aryl hydrocarbon receptor (AhR). In the present study, we have examined the effect of temporary inhibition of protein synthesis by cycloheximide on oxidized tryptophan inducible CYP1A1 mRNA, protein, and EROD activity in Hepa-1 cells. The results demonstrate that combined exposure of wild-type Hepa-1 cells to either UV- or ozone-oxidized tryptophan and cycloheximide causes an increase in CYP1A1 mRNA, protein, and EROD activity, which is greater than the sum of the increases that were observed by exposure to each compound alone. The increase in EROD activity is dependent upon the dose and duration of cycloheximide treatment and is prolonged by actinomycin D when the latter compound was administered after removal of cycloheximide. Studies carried out to investigate the mechanism of this superinduction using various mutants of Hepa-1 cells, which are defective in either the AhR or AhR nuclear translocator protein indicated that the superinduction of oxidized tryptophan inducible EROD activity by cycloheximide occurs through the AhR. This is the first demonstration that oxidized tryptophan, in the presence of cycloheximide, causes superinduction of transcription of the Cyp1a1 gene with concomitant increase of CYP1A1 protein and EROD activity in Hepa-1 cells.

The mitogenic growth and differentiation factor (GDFs) oncostatin M (OM), epidermal growth factor (EGF), fibroblast growth factor 4 (FGF-4), platelet-derived growth factor AA (PDGF AA), PDGF AB, and PDGF BB and the anti-mitogenic GDF, transforming growth factor beta one (TGF-beta1), were tested in the 7-day continuous exposure and 24-h transient exposure Syrian hamster embryo (SHE) cell transformation assay to determine their reversible and irreversible transforming potential. OM was negative while EGF, FGF-4, and PDGF AA were positive for statistically significant morphological transformation (MT) in the 7-day exposure SHE cell transformation assay. PDGF AB and PDGF BB (but not EGF, FGF-4, and PDGF AA) were positive for statistically significant MT in the 24-h transient exposure SHE cell transformation assays. TGF-beta1 was not only negative for the induction of MT in the 7-day and 24-h exposure SHE cell transformation assays, but suppressed the spontaneous background transformation response. Investigation of the transformation suppression potential of TGF-beta1 demonstrated that TGF-beta1 was able to irreversibly suppress the transformation potential of a variety of transforming agents including growth factors, Ames assay positive carcinogens, and Ames assay negative carcinogens. PDGF AA and PDGF BB were investigated to better understand the reversible and irreversible transformation response. Differences in the receptors activated, the proteins phosphorylated by the receptors, and immediate early gene expressed were observed in SHE cells treated with either PDGF AA or PDGF BB. Importantly, SHE cells treated with TGF-beta1 and PDGF BB, two GDFs, which modulate SHE cell transformation irreversibly, altered DNA methylation; PDGF AA did not demonstrate this effect. Together these data demonstrate that the SHE cell transformation assay can be utilized to evaluate the transformation potential and mechanism of activation of GDFs.