Automated detection of hepatotoxic compounds in human hepatocytes using HepaRG cells and image-based analysis of mitochondrial dysfunction with JC-1 dye.

In this study, our goal was to develop an efficient in situ test adapted to screen hepatotoxicity of various chemicals, a process which remains challenging during the early phase of drug development. The test was based on functional human hepatocytes using the HepaRG cell line, and automation of quantitative fluorescence microscopy coupled with automated imaging analysis. Differentiated HepaRG cells express most of the specific liver functions at levels close to those found in primary human hepatocytes, including detoxifying enzymes and drug transporters. A triparametric analysis was first used to evaluate hepatocyte purity and differentiation status, mainly detoxication capacity of cells before toxicity testing. We demonstrated that culturing HepaRG cells at high density maintained high hepatocyte purity and differentiation level. Moreover, evidence was found that isolating hepatocytes from 2-week-old confluent cultures limited variations associated with an ageing process occurring over time in confluent cells. Then, we designed a toxicity test based on detection of early mitochondrial depolarisation associated with permeability transition (MPT) pore opening, using JC-1 as a metachromatic fluorescent dye. Maximal dye dimerization that would have been strongly hampered by efficient efflux due to the active, multidrug-resistant (MDR) pump was overcome by coupling JC-1 with the MDR inhibitor verapamil. Specificity of this test was demonstrated and its usefulness appeared directly dependent on conditions supporting hepatic cell competence. This new hepatotoxicity test adapted to automated, image-based detection should be useful to evaluate the early MPT event common to cell apoptosis and necrosis and simultaneously to detect involvement of the multidrug resistant pump with target drugs in a human hepatocyte environment.

Streamlined beta-galactosidase assay for analysis of recombinant yeast response to estrogens.

Here, we describe a rapid, convenient, and quantitative beta-galactosidase assay in liquid culture of recombinant yeast that expresses the estrogen receptor. This assay allows large-scale screening of chemicals (more than 600 samples/day) for the evaluation of their direct estrogenic potency and accurate determination of their EC50 with minimal manipulations. The assay, which is based on digestion of the yeast cell wall by lyticase (zymolase), a beta-glucanase isolated from Arthrobacter luteus, followed by a hypoosmotic shock lysis, is performed completely in 96-well plates. This protocol for using recombinant yeast with the two-hybrid technology significantly advances recombinant yeast manipulation.

Assessment of oestrogenic potency of chemicals used as growth promoter by in-vitro methods.

Three in-vitro bioassays were used to compare the oestrogenic potency of chemicals used as growth promoter in beef cattle in certain non-European Union countries (17beta-oestradiol, alpha-zearalanol, testosterone, trenbolone, trenbolone acetate, melengestrol acetate) or found as food contaminant such as the mycotoxin zearalenone and some of their metabolites (17alpha-oestradiol, oestrone, 17alpha-epitestosterone, 19-nortestosterone, androstendione, zearalanone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol, beta-zearalenol). The strong oestrogens 17alpha-ethinyl oestradiol and diethylstilboestrol were used as standards. The first bioassay was based on the activation of a reporter gene by oestrogens in recombinant yeast expressing human or rainbow trout oestrogen receptor. In the second bioassay, the vitellogenin gene induction of rainbow trout hepatocyte cultures was used as a biomarker for the exposure to oestrogens. The third bioassay was based on the alkaline phosphatase gene induction by oestrogens in the human endometrial Ishikawa cell line. The assessment of oestrogenic potency of these chemicals clearly demonstrates the strong oestrogenicity of the mycotoxin zearalenone and its metabolites and particularly alpha-zearalenol which was as potent as ethinyl oestradiol and diethylstilboestrol in the human endometrial Ishikawa cell line.

Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals.

The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries. Twenty chemicals were selected to represent direct-acting estrogens, compounds with estrogenic metabolites, estrogenic antagonists, and a known cytotoxic agent. Also included in the test panel were 17beta++-estradiol as a positive control and ethanol as solvent control. The test compounds were coded before distribution. Test methods included direct binding to the estrogen receptor (ER), proliferation of MCF-7 cells, transient reporter gene expression in MCF-7 cells, reporter gene expression in yeast strains stably transfected with the human ER and an estrogen-responsive reporter gene, and vitellogenin production in juvenile rainbow trout. 17beta-Estradiol, 17alpha-ethynyl estradiol, and diethylstilbestrol induced a strong estrogenic response in all test systems. Colchicine caused cytotoxicity only. Bisphenol A induced an estrogenic response in all assays. The results obtained for the remaining test compounds--tamoxifen, ICI 182.780, testosterone, bisphenol A dimethacrylate, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol dodecylethoxylate, butylbenzylphthalate, dibutylphthalate, methoxychlor, o,p'-DDT, p,p'-DDE, endosulfan, chlomequat chloride, and ethanol--varied among the assays. The results demonstrate that careful standardization is necessary to obtain a reasonable degree of reproducibility. Also, similar methods vary in their sensitivity to estrogenic compounds. Thus, short-term tests are useful for screening purposes, but the methods must be further validated by additional interlaboratory and interassay comparisons to document the reliability of the methods.