Molecular toxicology最新文献

The proteins cross-linked to the DNA of cultured Chinese hamster ovary cells after exposure to cis-diamminedichloroplatinum(II) (cis-Pt), chromate, and formaldehyde were compared by two-dimensional (2D) gel electrophoresis, immunoblotting, and centrifugal assays that measured cross-link stability. Chromate and cis-Pt cross-linked seven of the same nonhistone proteins, such as actin, to DNA. In contrast, formaldehyde selectively formed histone-DNA cross-links. Immunoblotting experiments showed that all three chemicals cross-linked a 97-kDa nuclear protein to the DNA despite their different chemical reactivity with DNA and proteins. The chromate- and cis-Pt-induced cross-links were disrupted by thiourea, 2-mercaptoethanol, and EDTA, indicating that the metal could be chemically displaced from the cross-links. The formaldehyde-induced complexes required degradation with DNase 1 for the resolution of histones on 2D gels and were not chemically labile like the metal-induced cross-links. The agents and methodology used here could be applied to the study of additional nuclear proteins that bind or reside near the DNA.

We provide evidence for two new classes of halogenated aromatic hydrocarbon ligands for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or Ah) receptor: brominated naphthalenes and iodobenzenes. Polybrominated naphthalenes with four or more bromine atoms concentrated in lateral positions were shown to bind specifically and with high affinity (Kd approximately 10(-8) M) to the Ah receptor in rat liver cytosol preparations. The hexabrominated naphthalene isomers bind with high and nearly equal affinities but have been previously shown to have different toxicological properties. Possible explanations for these differences include differences in metabolism, antagonist versus agonist Ah receptor binding of some isomers, and the involvement of other binding sites in vivo that require different structural requirements. The moderate binding activity of the diiodobenzenes suggests that thyroid hormones should receive further study as possible endogenous ligands for the Ah receptor. It is difficult to explain the binding results with these two classes of compounds using previously developed molecular concepts for Ah receptor interactions based primarily on molecular size considerations.

Among the indicators of membrane damage, the leakage of intracellular components into the medium is the most directly related to the perturbations of the membrane molecular organization. The extent of the damage can be evaluated from the size of the released components. We have designed a protocol for the detection of membrane leakage based on the preincubation of cells with tritiated adenine for 24 h, followed by a 24-h chase in nonradioactive medium. The treatment takes place when the distribution of the precursor among its end products has reached the plateau, and thus the differences of radioactivity in the fractions obtained from the control and treated cultures (medium, nucleotide pool, RNA, DNA) correspond to actual quantitative variations induced by the test chemical. Aliquots of the medium are processed to determine which percentage of the released material is macromolecular, in order to distinguish between mild and severe membrane damage. The origin of the extracellular radioactivity can be recognized from the variations of RNA counts in the treated cells. DNA radioactivity is used to evaluate the number of cells that remain attached to the plates in the different conditions of treatment. By this means, generalized permeabilization of membranes to macromolecules is distinguished from complete solubilization of only a subpopulation of cells. We present some examples of application of the protocol with detergents (LAS, SDS, Triton X-100) and with Cr(VI), which damages cell membranes by a different mechanism of action.

The current strategy for the design of cytotoxicity tests is briefly reviewed in light of goals that need to be reached, and in the capacity of in vitro tests to fulfill the high public expectations for alternative methods to animal testing. Various cytotoxicity tests and parameters used for the assessment of topical toxicity and of neurotoxicity are chosen as examples and their relevance is discussed. Past experience with in vitro and short-term tests for mutagenicity shows not to look for one single supertest. A proposition for reasonable safety testing implies that a combined approach must be developed that integrates the results from a battery of cell tests and from structure-activity analyses as well as from kinetic and metabolic studies. The relevance of such an integrated approach must be aimed directly at the organisms that may be exposed.

Human keratinocytes in culture were able to take up hematoporphyrin derivatives (HPDs) used during photodynamic chemotherapy of tumors. In the absence of light, HPDs showed no cytotoxic effects to keratinocytes. However, after irradiation with visible light, HPDs induced immediate cytotoxicity as measured by the neutral red uptake assay. On the other hand, cell attachment as measured by protein estimation was not affected. When the cells treated with HPDs and irradiated with light were cultured for a further 72 h, they partially lost their ability to attach to the collagen surface. Most of the cells remaining attached after 72 h were no longer viable following treatment with HPDs and light. All parameters measured depended on the intracellular concentration of HPDs used (7-50 ng/10(5) cells) and the time of irradiation (0-30 min). These results suggest that human keratinocytes are a good model to study cytotoxic effects of photodynamically active drugs. Further, keratinocytes were unable to recover after damage caused by HPDs and light.

Previously, we have established that some peroxisome proliferators, a class of nongenotoxic hepatocarcinogens, are able to induce replicative DNA synthesis (RDS) in cultured hepatocytes. Hepatomegaly observed after short-term in vivo treatment correlated better with the ability to induce RDS than with the potency as peroxisome proliferator assessed in vitro. To clarify the challenging question of the limited sensitivity of primates to peroxisome proliferators, primary cultures of marmoset hepatocytes have been treated with nafenopin for some days. As expected from in vivo observations, no evidence for peroxisome proliferation could be observed. However, nafenopin induced a dose-dependent increase in the amount of RDS, but this induction was measurable only when the serum was absent from the culture medium. These results confirm that peroxisome proliferation and mitogenicity might be independent properties of peroxisome proliferators. Since in vivo the ability of compounds to induce RDS in liver cells is relevant to at least one key parameter of the hepatocarcinogenic response, it is suggested that measurement of RDS inducibility in cultured hepatocytes from different species might be relevant and useful to assess species differences in the liver tumor potency of nondirectly genotoxic compounds.

The in vitro cytotoxicities of 59 chemicals, expressed as ID50 values (i.e., concentrations of test chemicals that reduced the final cellular protein content of test cultures by 50% in comparison with appropriate solvent control cultures) and obtained using murine 3T3-L1 cells and the FRAME kenacid blue method, have been compared with rat oral and mouse intraperitoneal (ip) LD50 values. A better in vivo/in vitro correlation was obtained for the 59 chemicals with mouse ip LD50 values (r = .80) than with rat oral LD50 values (r = .76), but the best in vivo/in vitro correlation was found when the most toxic of the rat and mouse values were used in the comparison (r = .81).

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.