Molecular toxicology最新文献

An assay using established cell lines, human A431 epidermal cells and mouse 3T3 fibroblasts, has been developed to predict the phototoxic potential of compounds. The test determines the viability of the two cell lines in response to UV light in both the presence and absence of the test compound. The end point for cytotoxicity is determined from the mitochondrial dehydrogenase conversion of a tetrazolium salt (MTT) to a colored formazan product. The cytotoxicity of the test compound is established prior to UV exposure, and the highest no-effect concentration observed is then applied to cells that are subsequently exposed to different periods of UVA and UVA plus UVB light. A phototoxic effect is considered to have occurred when a biologically significant enhancement of toxicity is shown for the UV light with the compound present when compared to that of UV light alone. The test system has been validated with 30 compounds classified as strong, idiosyncratic, and negative based on the frequency of reported adverse reactions in humans. The in vitro phototoxicity assay was able to highlight the potential for phototoxicity in the strong category of phototoxic compounds and several of the idiosyncratic compounds. Only one of the negative compounds produced any activity in the assay in terms of enhancing UV toxicity. Some of the compounds were shown to protect the cells from the toxic effects of UV exposure.

The effects of 26 different cosmetic ingredients (e.g., permanent wave and hair dye compounds, emulsifiers, resins, and detergents such as quats) were assessed by four end points indicative for qualitatively and quantitatively different cytotoxicity: (1) neutral red uptake reduction after 24 h of treatment (NR-90 and NR-50); (2) cell detachment from culture dish after 4 h of treatment (CD-25); (3) growth inhibition after 48 h of treatment (GI-50); and (4) membrane permeability measured by fluorescent dye retention (fluorescence shift FS-25) and dye exclusion (viability ratio VR-25). The cytotoxicity potentials of the test agents were ranked for each in vitro test and compared with the in vivo eye irritation in guinea pigs (Draize test) after application of 5 or 2.5% (w/v) solutions of the same test batches. Strong irritants could be easily detected by most of the in vitro tests, but the neutral red uptake assay (especially NR-50) was the only one that was able to distinguish the minimally irritating test agents from strong irritants as well as from nonirritants. (I) All three extremely irritating quaternary ammonia compounds were identified as the strongest cytotoxic agents. (II) Nine out of 12 minimally irritating substances (mainly emulsifiers and resins) were ranked in the intermediate group. (III) Eight out of 11 non-or practically nonirritating chemicals (mainly permanent wave compounds) showed cytotoxic effects at very high concentrations only. The distinction of these three groups was better by means of NR-50 than by NR-90 data. At least two of the other cell tests (CD-25, GI-50, FS-25, and VR-25) had to be considered to allow an adequate interpretation of in vitro cytotoxicity.

The hepatotoxicity of N-acetyl-p-aminophenol (acetaminophen, paracetamol) was investigated in hepatocyte cultures obtained from eight different human liver biopsies. Incubation of hepatocytes with paracetamol resulted in a dose- and time-dependent glutathione depletion. Glutathione decreased linearly for 8 h, reaching a minimum after 12 h of exposure. Cytotoxicity, assessed as loss of cellular protein from plates, was observed only when glutathione decreased below 20% for more than 12 h. However, in one donor, cytotoxicity was observed with even a moderate glutathione decrease. Prestimulation of hepatocytes with 1 mM phenobarbital or 2 microM methylcholanthrene for 48 h did not lead to a significant increase of paracetamol toxicity, although the glutathione levels in 3-methylcholanthrene-treated cells were somewhat lower. Several metabolic precursors were examined in vitro for their ability to increase intracellular glutathione and the results showed the following sequence: N-acetylcysteine greater than thioproline greater than cysteine greater than 2-oxo-4-thiazolidine carboxylic acid greater than methionine. However, only N-acetylcysteine, thioproline, and cysteine substantially increased glutathione levels when 1 mM paracetamol was present in the incubation medium and thus prevented its toxicity. N-acetylcysteine elevated glutathione even after 24 h of preexposure to paracetamol. The fact that cell damage did not correlate with glutathione levels in all human cultures suggests that glutathione depletion may not be the only determinant of paracetamol toxicity in human hepatocytes.

For a possible detection of aneuploidy induction by chemicals and ionizing radiation, fluorescein-bound antikinetochore antibodies (CREST-scleroderma antibodies) were used to discriminate between micronuclei deriving from acentric fragments or from chromosome loss induced in Chinese hamster cells. The cells were treated with aphidicolin, adriamycin, Hoechst 33258, colcemid, the alkylating agent diethyl sulfate, and ionizing radiation. The frequency of micronucleated cells, the fraction of kinetochore-positive and -negative micronuclei per cell, and the fraction of kinetochore-positive micronuclei was measured using immunofluorescence staining of kinetochores in micronuclei. Of the micronuclei and fragmented nuclei induced by colcemid, 99% contained kinetochores, whereas ionizing radiation induced only 4% of kinetochore-positive micronuclei. The other drugs induced variable, in some cases also cell-cycle-dependent, fractions of kinetochore positive micronuclei. With this technique a discrimination between clastogenic effects and effects that occur at the level of spindle formation of the agent studied seems to be possible. Flow karyotyping was used to study the induction of stable homogeneous and numerical aberrations in diploid Chinese hamster cell clones that had survived a dose of 15 Gy gamma-radiation. All analyzed clones showed deviations in their flow karyotypes: the mean number was 9.2 deviations per clone, compared to 1.1 deviations per clone in unirradiated control clones.

The aim of this study is to qualify the application the new microscopic methods fluorescence and AVEC-DIC (Allen video-enhanced contrast differential interference contrast) microscopy for toxicity testing. The effects of 2-OH-ethyl methacrylate (HEMA), a toxic acrylic monomer, on human fibroblasts was tested. The HEMA concentrations used were 0.01-1% at incubation times of 1-24 h. The cells were observed with AVEC-DIC microscopy and fluorescent staining to evaluate the velocity of lysosomal movement, the number and morphology of the mitochondria, and the fine structure of the cell. In the samples treated with the toxic compound the lysosomal movement changed, as did the morphology of the mitochondria and of the whole cells. The results are compared and discussed with regard to the results of conventional cytotoxicity tests performed in parallel. The new methods proved to be more sensitive and yielded more specific information on the cellular changes caused by the compound.

Adult human hepatocytes in chemically defined culture conditions were incubated with morphine, heroin, meperidine, and methadone to investigate their potential hepatotoxicity to human liver. Cytotoxic effects were observed at about 100 times the plasma concentrations required to produce analgesia in human nonaddicts. Concentrations of 1 mM morphine, heroin, and meperidine reduced the glycogen content by 50%, while even 0.2 mM methadone produced a depletion of 70% after 24 h of treatment. Concentrations of 0.8 mM morphine and heroin, 0.4 mM meperidine, and 0.005 mM methadone inhibited the albumin synthesis by about 50% after 24 h of pretreatment. Intracellular glutathione was reduced to 50% of that of controls after 2-3 h of incubation with 2 mM morphine and 1 mM heroin, while 1 mM meperidine and 0.2 mM methadone produced a reduction of about 30% after 6 h incubation. The results show that therapeutic doses of the opioids is unlikely to produce irreversible damage to human hepatocytes, but opiate doses during tolerance or abuse may be a cause of liver dysfunction.

Different methods of measuring cytotoxicity have been investigated in order to establish a test system for assessment of the toxicity of water samples. Ideally this should be highly sensitive and rapid to perform. Four variations of the neutral red uptake test have been compared with the MTT test and ATP determination. Chinese hamster V79/4 cells were used as the test system with 10 model toxins. All tests gave essentially similar results, with linear regression analysis producing correlation coefficients in excess of .93. No single test was most sensitive to all 10 compounds. The neutral red uptake and MTT tests could be performed in a single working day (approximately 8 h) if test compound and cells were plated out simultaneously. These tests are preferable to ATP determination, which is a complex and lengthy procedure, requiring expensive reagents.

Many toxic effects are not caused by the administered compound itself, but are due to metabolites. All cell types express some xenobiotic-metabolizing enzymes, but levels and patterns are very variable. Critical metabolic steps may occur within the target cell and/or at other sites. This complex situation is difficult to mimic in vitro. The further problem is that cells that are taken into culture tend to rapidly cease the expression of important xenobiotic-metabolizing enzymes. Part of the problem may be solved by the addition of exogenous metabolizing systems, for example, in the form of freshly isolated hepatocytes, crude subcellular preparations, or purified enzymes. In these systems, the plasma membrane of the target cell may act as a barrier for the active metabolite and thereby lead to false negative results. The alternative is the use of metabolically active target cells. We therefore screened 18 cell lines for monooxygenase, cytochrome P-450 reductase, epoxide hydrolase, glutathione transferase, and UDP-glucuronosyl transferase activities. In further studies, IEC-17, IEC-18, and HuFoe-15 cells showed their capabilities of activating a broad spectrum of structurally heterogenous promutagens, as indicated by the induction of micronuclei. These cells, however, were not suited for the study of a more relevant genetic end point, the induction of hereditary functional changes (gene mutations), implying that a compromise had to be made on the level of the toxicodynamics. In the second approach, cDNAs encoding the rat cytochromes P-450IA1 and P-450IIB1, set under the control of a constitutive promoter, were transfected into V79 Chinese hamster cells, which do not express cytochromes P-450 but are ideal target cells for gene mutation assays. The resulting substrains (XEM1, XEM2, XEM3; SD1) stably expressed cytochromes P-450IA1 and P-450IIB1, respectively, and showed the corresponding monooxygenase activities. Aflatoxin B1, cyclophosphamide, dibutylnitrosamine, and benzo[a]pyrene mutated SD1 and/or XEM1 and XEM2 cells, but were inactive in parental V79 cells. The mutagenicity of benzo[a]pyrene 7,8-trans-dihydrodiol was about 1000 times more potent in XEM1 and XEM2 cells than in SD1 and V79 cells. Other promutagens were inactive in V79 as well as in the genetically engineered daughter lines. This system therefore is not yet optimal in general screening for the detection of new mutagens, but appears ideal in the identification of critical xenobiotic-metabolizing enzymes for a given mutagen.

Enzymatic activation of polycyclic aromatic hydrocarbons (PAHs) and its effect on cytotoxicity were studied using the neutral red viability assay as the end point. Benzo[a]pyrene was progressively cytotoxic to human hepatoma (HepG2) cells over a 1- to 3-d period, and after induction of monooxygenase activity with a polychlorobiphenyl (PCB) mixture (Arochlor 1254), cytotoxicity was increased about threefold. Concomitant with Arochlor exposure was an increase in the activity of 7-ethoxycoumarin odeethylase, which could be inhibited by exposure to alpha-naphthoflavone. Human keratinocytes (NHEK), but not fibroblasts (HFF), were sensitive to the cytotoxicity of benzo[a]pyrene. However, preexposure of the keratinocytes to Arochlor did not increase their sensitivity to benzo[a]pyrene. Neither the keratinocytes, fibroblasts, nor HepG2 cells were sensitive to acenaphthene. Addition of hamster or rat hepatic S9 mix, however, resulted in toxicity from benzo[a]pyrene and acenaphthene. 7,12-Dimethylbenz[a]anthracene was only mildly cytotoxic to the fibroblasts, and its cytotoxicity was not enhanced in the presence of rat or hamster S9 mix. Exposure of the HepG2 cells to 7,12-dimethylbenz[a]anthracene showed progressive toxicity over a 1- to 3-d period. Prior exposure of the HepG2 cells to Arochlor did not enhance their sensitivity to 7,12-dimethylbenz[a]anthracene. Human keratinocytes were sensitive to 7,12-dimethylbenz[a]anthracene, with cytotoxicity markedly increasing over a 1- to 3-d period.

In vitro neural systems can be predictive for CNS neurotoxicity, except where xenobiotics primarily affect the blood-brain barrier. The wide range of systems now used in neurobiological studies is available for mechanistic neurotoxicological investigations although the choice of system is generally arbitrary. A more rational approach may now be justified. There are many culture systems available including neural cell lines, organotypic explant or reaggregation cultures, and primary monolayer cultures of individual neural cell types: neurons, astrocytes, and oligodendrocytes. Of these models much success has recently been achieved using the organotypic explant culture type. Similarly in our laboratories, using rat whole-brain reaggregate cultures, we have demonstrated good in vitro/in vivo correlations for the cholinergic neurotoxicant ethylcholine mustard aziridinium (ECMA) where specific cholinergic lesions are produced using low concentrations of ECMA (12.5 microM). Higher concentrations (25-50 microM) were more cytotoxic, as shown, for example, by nonspecific effects on cerebellar glutamatergic granule neurons. Treatment of reaggregates lesioned with the cholinotoxin with a neurotrophic factor, nerve growth factor (NGF), did not reverse the lesion but treatment of control cells with NGF (50 ng/ml) elevated both choline acetyltransferase (ChAT) activity and muscarinic receptor binding. The "lesioned" reaggregate culture system may thus be of future value in evaluating potential therapeutic agents that could reverse such lesions in the CNS. By supplementing the information gained in the reaggregate system with tests using primary monolayer cultures of neurons or astrocytes we can propose a stepwise screening system for potential neurotoxicants in vitro. In its simplest form this is (1) screen initially using tumor-derived neural cell line, (2) test selected compounds in whole-brain reaggregates, and (3) supplement information with primary monolayer cultures of individual neural cell types.