Molecular toxicology最新文献

The possibility of digitization and processing of microscope images in "real time" (i.e., at video rates) has opened a variety of ways to dramatically improve the quality of microscopic images and to create new applications of light microscopy. One of the new techniques, video-enhanced contrast (VEC) microscopy, enables one to increase contrast and magnification to the extent that positions and movements of biological objects as small as 15-20 nm (e.g., small membrane-bounded vesicles and microtubules) can be analyzed in living cells. Organelle motion can be quantitatively described by a number of parameters such as velocity, straightness of path, and reversals of direction. The second group of videomicroscopic techniques is based on the measurement of fluorescence intensities of intracellular compounds by monochromatic light microscopy or using other low-light signals (video-intensified microscopy, VIM). The resulting images are two-dimensional arrays of fluorescence or absorption spectroscopy measurements and contain information on the amounts of intracellular metabolites or exogenous agents. Typical parameters for VIM measurements include Ca2+ concentration, pH value, metabolites and membrane potentials. Specific nontoxic dyes are also available to verify the identity of the organelles seen by the VEC techniques and to quantitate their abundance. The whole battery of new tests based on videomicroscopy can be applied at selected time intervals to a given set of cultured cells to obtain simultaneous measurements of multiple end points. However, since quantitative data for these parameters can be calculated from the live images in real time and encoded in the form of gray-shaded or pseudocolor images, they can also be continuously recorded and yield video films of the complete sequence of intracellular events during and after exposure to toxic or pharmacological agents. Videomicroscopy allows multiparametric studies to be performed with cultured cells, yielding a wealth of information that could be reached in the past only in animal experimentation. In addition, videomicroscopy enables us to observe directly and quantitate metabolic, physiological, and morphological parameters in the living cell that were not accessible by animal experimentation. It is therefore to be expected that videomicroscopy in the near future will catalyze a major shift from animal to in vitro experimentation in the fields of toxicology, pharmacology, and experimental pathology.

Prostaglandin-H-synthase (PHS) peroxidase has been suggested to mediate drug metabolism particularly in extrahepatic tissues low in monooxygenase (MFO) activity. PHS can oxidize various xenobiotics in vitro; its contribution in vivo is still uncertain and is currently assessed by differences in the MFO- and PHS-catalyzed product/adduct formation of a few suitable substrates. Cells in culture that are PHS competent but MFO deficient can provide an additional approach for further investigating the role of PHS in the metabolic activation of foreign compounds. To this end, a cell line has been derived from ram seminal vesicles (SEMV), a tissue known as a good source of PHS but shown to be devoid of MFO activity. SEMV cells can be cultured in IBR or in RPMI medium supplemented with fetal calf serum, and have been subcultured until passage 30. The arachidonic acid (AA) metabolism in these cells has been characterized: besides incorporation in the lipid pool, AA was mainly metabolized to prostaglandin (PG) E2; minor products were PGF2 alpha and the lipoxygenase products 12- and 15-HETE. The PGE2 production (17 nmol/10(6) cells.24 h) of SEMV cells (passage 10) exceeded at least 10-fold that of other cells cultured under similar conditions. These data, indicative of high PHS activity, suggest that the cells can be a useful tool for future studies on the objectives outlined above.

A differentiating keratinocyte cell line derived from explant cultures of rat sublingual epithelium has been used as a potential in vitro model for topical (skin) irritation of a range of detergents. The end points used to assess toxicity were acid phosphatase (AP) activity after 4 h of dosing and neutral red (NR) uptake and kenacid blue (KB) staining after 3 d to assess cell viability and number. The acid phosphatase activity increased to a sharp peak with increasing doses, then fell equally sharply for the anionic detergent sodium dodecyl sulfate (SDS) and cationic detergents TMABs (trimethylammonium bromides). With the Tweens (the least toxic group) there was no acid phosphatase peak or it appeared at the highest dose level used (1.0 mg/ml). The dose-response curves for NR uptake paralleled those for KB staining. With all of the three end points, it was apparent that the order of toxicity for the different groups was TMABs greater than SDS greater than Tweens, with a difference of one order of magnitude between consecutive groups when using NR and KB.

Sheets of MDCK cells were severely damaged by the commonly used antiseptic, chlorhexidine. Damage was indicated by changes in the characteristic blister formation normally observed in sheets of these cells growing on the base of culture flasks. Results are given for reduction in blister number with increasing chlorhexidine concentration. Damage was indicated also by increasing transport of radiolabeled inulin across the sheets after treatment with increasing concentrations of chlorhexidine. The mechanism of chlorhexidine damage is discussed. The conclusion is that chlorhexidine could be an important contributory factor in the failure of ultrafiltration in peritoneal mesothelial cells, and its use should be avoided with magnesium-containing dialysis fluids in kidney-failure patients.

Established renal epithelial cell lines of human, pig, and dog origin (293, LLC-PK1, MDCK) were examined in terms of nephrotoxicity and ability to biotransform cyclosporine A (CsA). All three cell lines exhibited a comparable concentration dependent cytotoxicity to CsA treatment. Alterations in cell function included a decreased transport of lysine, an inhibition of growth, and an activation of lysosomal and mitochondrial activity as indicated by the increased uptake of neutral red (NR) and increased reduction of the tetrazolium dye MTT at 1-6 microM CsA. Increased leakage of lactic dehydrogenase and activities of gamma-glutamyl transpeptidase (GGT) and N-acetyl-beta-D-hexosaminidase were observed at 48 h and 12 microM CsA. A discrimination between CsA and the less nephrotoxic cyclosporine-(CsH) was shown for DNA synthesis and NR uptake. The contribution of extrarenal parameters on kidney cell function was studied by the addition of medium from hepatocytes exposed to CsA to the kidney cell lines. A more potent inhibition of DNA synthesis and enhanced reduction of MTT resulted than by addition of equimolar CsA directly to the kidney cells. These data indicate that hepatocyte constituents present in the medium due to CsA treatment affect kidney cell function; additionally, the presence of CsA metabolites may contribute to the CsA-induced nephrotoxicity. The vascular nephrotoxicity induced by CsA, an increased deposition of platelets in the renal arterioles, was mimicked by cocultures of endothelial cell monolayers and platelets. CsA increased the aggregability and adherence of platelets to the endothelial cell monolayers, whereas CsH had no effect.

Changes in cytochrome P-450 isoenzymes were studied in rat liver and in primary cultures of rat hepatocytes after treatment with compounds belonging to various classes of inducers, including phenobarbital (PB), beta-naphthoflavone (BNF), and clofibrate/clofibric acid (CLOF/CLOFA). The enzyme activity toward specific substrates was measured, and the presence of apoprotein of several P-450 isoenzymes was determined semiquantitatively by Western blotting. In untreated cultures the P-450 content and activities of 7-ethoxyresorufin O-deethylation (EROD) and aniline 4-hydroxylation (AH) declined with time at different rates. In cultures treated with BNF, the protein levels of isoenzyme P-450IA1 and P-450IA2 were elevated, as in vivo. This induction was reflected in a markedly increased EROD activity. CLOFA enhanced the AH and EROD activity in primary cultures at the same level as in vivo. The monooxygenase activity pentoxyresorufin O-depentylation (PROD) was stimulated by PB and CLOF in vivo, which correlated with the enhanced protein level of P-450IIB1/2. In contrast, the PROD activity was not induced when cultures were treated with PB or CLOFA, although we could detect apoprotein of P-450IIB1/2 by immunoblotting.

Human keratinocytes are the most appropriate target cells for evaluating mechanisms of skin cytotoxicity and pharmacology of chemical agents. After having formed a confluent stratified epithelium with proliferating basal cells and differentiated cell layers, human keratinocytes were harvested after enzymatic detachment as stable, three-dimensional cell aggregates or used as adherent multilayers in microtiter plates to study the local cytotoxicity of different toxic compounds. The advantage of this test is that it uses the adequate target cells and that it evaluates both the ability of the test chemical to penetrate several cellular layers as well as the ability to interfere with cellular function. The end points are cell viability and cell metabolism, which are determined by neutral red uptake and MTT reduction, respectively. For the 13 chemicals evaluated in this study we found good correlation (r = .819) between the potency rankings of keratinocyte NR 50 values and in vivo irritancy data. There was also good agreement (r = .945) between ranking of these chemicals according to midpoint toxicity of both the 3T3 assay and the keratinocyte assay. This test system might be at the present stage a supplementation of the current test battery, which shall replace in vivo irritation tests like the Draize test.

C6 Rat glioma cells acquire an astrocyte-like morphology (cytoplasmic shrinking) after exposure to beta-adrenergic compounds (e.g., isoproterenol) in serum-free medium. This morphological response is mediated by elevation of the intracellular cAMP level and possibly by activation of a kinase phosphorylating and inactivating the myosin-L-kinase in analogy to observations with smooth muscle cells. The result is a disturbance of the stress fiber function, which depends on a cooperation between actin and myosin. Diethylstilbestrol (DES) induces an identical morphological response in these cells under serum-free conditions. However, under the influence of DES no increase of the cAMP level was observed. In addition, at concentrations not inducing these morphological responses, DES inhibits the isoproterenol-induced effect when administered simultaneously or prior to the beta-receptor agonist. DES does not inhibit the isoproterenol-mediated morphological response when added after isoproterenol treatment. Furthermore, if serum is added to cells showing the isoproterenol- or DES-mediated astrocyte-like morphology (DES or isoproterenol present all the time) the cells regain their normal fibroblast-like morphology within 30 min. In view of these results the question arises whether DES interferes with myosin-L-chain phosphorylation or whether it directly interacts with the cytoskeleton stress fiber components, as cytochalasin B1 does. Thus it remains to be established whether the observed effects are related to the estrogenic activity of DES. Similar effects were observed with Syrian hamster embryo fibroblasts under the influence of DES and the naturally occurring steroid estrogen 17-beta-estradiol.

The metabolism of the tropine indole-3-carboxylate ICS 205-930 (ICS), a highly potent and selective antagonist of 5-HT3 receptors, was investigated in continuous cell lines derived from rat or human liver and compared to the in vivo metabolism in rat and human. The well-differentiated rat hepatoma line 2sFou extensively metabolized ICS by hydroxylation of the indole moiety and subsequent conjugation to form the corresponding glucuronides and sulfates. The 2sFou cells also oxidized ICS at the tropinyl moiety to form both N-demethyl and N-oxide derivatives. The relative amount of the various metabolites was dependent on the substrate concentration. Pretreatment of the cells with dexamethasone increased the rate of metabolism for all pathways, while benz[a]anthracene caused an increase in hydroxylation at the indole moiety at the expense of N-oxidation. Phenobarbital pretreatment had no effect on ICS metabolism. The pattern of metabolites formed in 2sFou cells was qualitatively similar to that formed in rat urine. The human hepatoma line HepG2 metabolized ICS only to a small extent. The HepG2 cells failed to form detectable amounts of ICS conjugates found in human urine. The N-oxide-ICS was not found in HepG2 cells or in human urine. Virtually no ICS metabolites were found in human lung adenocarcinoma lines NCI-H358 or NCI-H322. The results suggest that continuous cell lines such as the differentiated rat hepatoma cells 2sFou might be used to mimic the metabolism of xenobiotics in rat and to clarify their complex metabolic pathways.

The Federal Health Office (BGA) has started in June 1988 a national interlaboratory study to validate alternatives to the Draize test. It is supported by grants of the Department of Research and Technology (BMFT) of West Germany. The aim of this collaborative study is to validate the classification of chemicals with regard to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg test-chorioallantoic membrane (HET-CAM) assay, according to Lüpke. Under the current research scheme, which is coordinated by the West German Public Health Office (BGA), 14 toxicology laboratories in the chemical industry, universities, the BGA, and other research institutions are to study 44 substances with a variety of chemical, biochemical, and toxicological characteristics. The validation test is intended to provide comparative data for the development of an alternative routine test scheme. The collaborative study is performed under routine laboratory conditions. The results should allow a decision on whether and to what extent the results of the NR/KB and the HET-CAM assay can replace the Draize test.