A new hepatoma cell line for toxicity testing at repeated doses.

Many cell models that are used to assess basic cytotoxicity show a good correlation with acute toxicity. However, their correlation with the toxicity seen following chronic in vivo exposure is less evident. The new human hepatoma cell line HBG BC2 possesses the capacity of being reversibly differentiated in vitro and of maintaining a relatively higher metabolic rate when in the differentiated state (3 weeks) as compared to HepG2 cells, and thus may allow the conduct of repeated toxicity testing on cells in culture. In order to evaluate the genetic background of HBG BC2 cells, the expression of selected genes was analyzed in untreated cultures and, in addition, the behavior of HBG BC2 cultures under conditions of repeated treatment was studied with acetaminophen as a test substance and coupled with the use of standard staining techniques to demonstrate toxicity. Results showed that cultures of HBG BC2 cells retained a capacity to undergo apoptosis and proliferation, allowing probable replacement of damaged cells in the culture monolayer. MTT reduction was used to evaluate the toxicity of acetaminophen, acetylsalicylic acid, perhexiline, and propranolol, after both single and repeated (3 times/week for 2 weeks) administration. Under the conditions of repeated treatment, cytotoxicity was observed at lower doses as compared to single administration. In addition, the lowest nontoxic doses were in the same range as plasma concentrations measured in humans under therapeutic use. Our results suggest that the new human hepatoma HBG BC2 cell line is of interest for the evaluation of cell toxicity under conditions of repeated administration.

Evaluation of hydroquinone and chloral hydrate on the in vitro micronucleus test on isolated lymphocytes.

The cytochalasin B micronucleus test was performed in human peripheral lymphocyte cultures to assay the ability of hydroquinone and chloral hydrate to induce micronuclei, in the presence or absence of an exogenous metabolic activation system. Cultures and readings were performed in duplicate. No significant damage was found after treatment with chloral hydrate in this test system, whereas hydroquinone induced a clear positive response in one culture in the presence of metabolic activation during the G1 phase. Isolated lymphocytes used as a test system provide information about the test compound itself without interference by blood components. Comparison of the two readers' data showed few marked discrepancies in the number of micronuclei recorded in binucleated cells. Strict criteria for data analysis are therefore necessary to avoid intra-assay or operator variability.

The in vitro micronucleus test on isolated human lymphocytes.

The in vitro micronucleus test was performed on isolated human lymphocytes using the cytokinesis-block technique with and without a rat liver metabolizing system. Positive control substances were used to evaluate this test: a direct agent (vincristine) requiring no metabolic activation, and three promutagens (cyclophosphamide, benzo[a]pyrene and dimethylbenz[a]anthracene). All of them, when compared with controls, caused a significant increase in micronucleus frequency, with a clear dose response. Five compounds were then tested in this in vitro micronucleus test: safrole, azathioprine, procarbazine, diethylstilbestrol and o-toluidine. The chemicals were examined with and without exogenous metabolic activation. Of these five compound, o-toluidine was found to be a marked direct genotoxic agent and azathioprine gave positive results with or without metabolic activation (a better response was noted without the addition of S9 mix). Diethylstilbestrol gave conflicting results and was considered inconclusive. Two chemicals, safrole and procarbazine, were found to be non-genotoxic in this test system, whatever the protocol used.

A multicentre study of acute in vitro cytotoxicity in rat liver cells.

A multicentre validation study of the acute in vitro cytotoxicity of drugs involving six French laboratories from INSERM or pharmaceutical companies has been carried out. Thirty liquid or solid chemicals such as antibiotics, anticancer drugs and solvents were selected and incubated for 20 hr with normal rat hepatocytes and FaO hepatoma cells. Miniaturized and automated methods were defined for the evaluation of cytotoxic effects. Four endpoints were evaluated: the ratio of extracellular lactate dehydrogenase to total lactate dehydrogenase, total cellular protein content, reduction of a tetrazolium salt, and neutral red uptake. For each test IC(50) values were calculated. A good interlaboratory reproducibility was demonstrated. The neutral red assay was found to be the most sensitive and the least reproducible endpoint. More compounds were shown to be cytotoxic to hepatocytes than to hepatoma cells (18 v. 12). On the basis of the IC(50) values a few compounds were found to be much less cytotoxic than predicted from in vivo data, suggesting that a simple experimental protocol and non-specific cytotoxicity parameters are not sufficient to test certain drug families. However, such methods appear to provide a useful means of defining the concentration range of the drug that will be selected for further analysis using more specific tests.

Toxicological studies on a benzofuran derivative. III. Comparison of peroxisome proliferation in rat and human hepatocytes in primary culture.

Primary cultures of rat and human hepatocytes were used in our in vitro studies for investigating species differences in the response to a peroxisome proliferating benzofuran derivative, benzbromarone. Cyanide-insensitive palmitoyl coenzyme A oxidation (a marker of peroxisome fatty acid beta-oxidation) and electron microscopy were used to assess peroxisome proliferation. Hepatocytes were cultured essentially as described by Mitchell et al. (1984, Arch. Toxicol. 55, 239-246); clofibric acid and mono(2-ethylhexyl) phthalate (MEHP) were used as reference compounds, as they are well known to cause peroxisome proliferation in rat hepatocytes in primary culture. The benzofuran derivative, tested at drug concentrations ranging from 2.37 to 59.20 microM in rat hepatocyte primary cultures, induced, after 96 hr, a dose-related increase of the peroxisomal beta-oxidase activity correlated with an increased number of peroxisomes; this increase was much less marked than that obtained with clofibric acid or MEHP. By contrast, using the same range of concentrations, human hepatocytes in primary culture treated with benzbromarone revealed no enhancement of enzymatic activity and no concomitant statistically significant increase in the number of peroxisomes; the same observations were reported with clofibric acid and MEHP. These results demonstrate clearly that species differences in sensitivity to peroxisome proliferation with the benzofuran derivative do exist.

Variations of D (-)-beta-hydroxybutyrate dehydrogenase activity of rat liver mitochondria during post-natal development.

1. D(-)-beta-hydroxybutyrate dehydrogenase specific activity of rat liver mitochondria changes during ontogenesis: at birth, the activity is low, then increases to a maximum at 12 days, decreases until 50 days to keep constant thereafter. At the same time, mitochondrial protein amount increases regularly while succinatecytochrome c reductase specific activity slightly increases after birth to keep constant afterwards. 2. The observed changes in activity of D(-)-beta-hydroxybutyrate dehydrogenase are not related to possible interactions between the enzyme and phospholids since addition of lecithin to mitochondria does not change the activity. 3. Electrophoresis of mitochondrial proteins isolated from rats at different development stages demonstrates the presence of a protein band characterized by the same electrophoretic mobility as beta-hydroxybutyrate dehydrogenase and by significative changes of its proportion during maturation: the relative amount of this protein increases from the new-born to the 10-12 days old rat, to decrease afterwards. 4. These findings may signify that the increased activity of the enzyme with a maximum at 10-12 days followed by a decrease is related to the rate of the enzymes biosynthesis.