In vitro & molecular toxicology最新文献

The cytotoxicity of MEIC chemicals Nos. 11-30 was evaluated by determination of neutral red uptake in Balb/c 3T3 mouse fibroblasts with and without the addition of a microsomal activation mixture. The use of microsomes significantly decreased the cytotoxicity of malathion, 2,4-dichlorophenoxyacetic acid, propranolol, thioridazine, lithium sulfate, copper sulfate and thallium sulfate, whereas the cytotoxicity of 1,1,1-trichloroethylene, phenol, nicotine, and paraquat was significantly increased by use of the microsomal activation mixture. These cytotoxicity data are in line with observations in other studies on microsomal modulation of the cytotoxicity of the test substances. Moderate to good correlations were found between the cytotoxicity data and rodent lethality data, and the addition of microsomes slightly improved the in vitro/in vivo concordance. The evidence to support the relevance of the in vitro/in vivo correlations obtained in the MEIC project is limited due to a high variability on the in vivo lethality data and a large interlaboratory variation on the in vitro data.

The emergence of engineered tissues and new cell strains has called for the need to explore solutions that can be used to store these cells and tissues in a state of near suspended animation without using cryopreservation. The ability of the hypothermic solutions ViaSpan (DuPont Merck Pharmaceutical Company, Wilmington, DE), HypoThermosol (Cryomedical Sciences, Rockville, MD), HypoThermosol supplemented with either ethylenediaminetetraacetic acid (EDTA) or Vitamin E and HypoThermosol supplemented with apoptosis protease inhibitors were tested for their abilities to cold-protect Madin Darby Canine Kidney (MDCK) cells at 4 degrees C. Alamar Blue, a nontoxic metabolic indicator was used to measure cell viability. The order of cold protection was HypoThermosol with Vitamin E and EDTA > HypoThermosol with Vitamin E > HypoThermosol with EDTA > HypoThermosol > ViaSpan > Dulbecco's Modified Eagle's Medium (DMEM). Membrane integrity tests supported the Alamar Blue data that EDTA and Vitamin E conferred a benefit to the cold-storage capabilities of HypoThermosol. MDCK cells that died subsequent to 1 to 6 days cold-storage detached from the substratum and their DNA was harvested after being placed at 37 degrees C. This DNA was compared to DNA retrieved from adherent cells in the same cultures that survived the cold-storage regime. Gel electrophoresis of cells dying due to 1 to 4 days of cold-storage showed a DNA ladder indicating that cells died through apoptosis, programmed cell death. Dead cells harvested at 5 to 6 days of cold storage, however, had randomly cleaved DNA indicative of necrotic cell death. HypoThermosol supplemented with apoptosis protease inhibitors was better able to cold-protect cells than the base HypoThermosol. These data suggest that the inhibition of apoptosis should be considered in the future cold-storage formulations.

Release of endogenous glutamate (GLU) evoked by an exogenous excitotoxin such as kainate can contribute to central nervous system (CNS) excitotoxicity. In this study, the possibility that this mechanism accounts for reported domoic acid (DOM) toxicity was investigated using the isolated chick retina. Exposing retinas to 0-100 µM DOM for 40 min caused an increased efflux of lactate dehydrogenase (LDH) and a dose-related release of GLU into the incubation medium. Neuronal damage following exposure to DOM was confirmed by histologic examination of the retina. DOM-induced GLU release occurred at 27 and 37 degrees C and was more pronounced in medium containing a low calcium concentration (0.1 mM). In contrast, K(+) evoked GLU release occurred only at 37 degrees C in 2 mM calcium, but not at 27 degrees C. DOM-induced GLU release was reduced in hyperosmolar medium (medium 1 100 mM sucrose). Similar to GLU release, the LDH release occurred at both 27 and 37 degrees C and was reduced in hyperosmolar medium. These results suggest that an increase in membrane permeability secondary to osmotic swelling and lysis rather than a calcium-dependent vesicular exocytosis mechanism is responsible for GLU and LDH release. GYKI 52466, a selective noncompetitive antagonist of the non-NMDA receptor, prevented neuronal degeneration and GLU/LDH release. MK-801, a highly potent noncompetitive NMDA receptor blocker, reduced neuronal injury (33% decrease in LDH release), but did not reduce DOM-evoked GLU release significantly. Typical excitotoxic lesions were produced at all concentrations tested, with amacrine cells in the inner nuclear layer being the most severely affected. Released GLU contributed to further excitotoxic injury, exacerbating the neurotoxic action of DOM in isolated embryonic chick retina.