William D. Pennie

Partial and weak oestrogenicity of the red wine constituent resveratrol: consideration of its superagonist activity in MCF-7 cells and its suggested cardiovascular protective effects

It was recently reported that the red wine phytoestrogen resveratrol (RES) acts as a superagonist to oestrogen‐responsive MCF‐7 cells. This activity of RES was speculated to be relevant to the ‘French paradox’ in which moderate red wine consumption is reported to yield cardiovascular health benefits to humans. We report here that RES binds to oestrogen receptors (ER) isolated from rat uterus with an affinity 5 orders of magnitude lower than does either the reference synthetic oestrogen diethylstilboestrol (DES) or oestradiol (E2). In comparison with E2 or DES, RES is only a weak and partial agonist in a yeast hER‐α transcription assay and in cos‐1 cell assays employing transient transfections of ER‐α or ER‐β associated with two different ER‐response elements. Resveratrol was also concluded to be inactive in immature rat uterotrophic assays conducted using three daily administrations of 0.03–120 mg kg−1/day−1 RES (administered by either oral gavage or subcutaneous injection). These data weaken the suggestion that the oestrogenicity of RES may account for the reported cardiovascular protective effects of red wine consumption, and they raise questions regarding the extent to which oestrogenicity data derived for a chemical using MCF‐7 cells (or any other single in vitro assay) can be used to predict the hormonal effects likely to occur in animals or humans. Copyright

Use of cDNA microarrays to probe and understand the toxicological consequences of altered gene expression

Genomic sciences offer the ability to measure quantitative modulation of transcription in cells and tissues under a wide variety of conditions. We have developed a series of custom cDNA microarrays specifically to investigate toxicity processes. Around 600 marker genes for toxicity were selected and representative cDNA clones were obtained, amplified and purified by polymerase chain reaction (PCR), before being immobilised on nylon membranes. A detailed database on biochemical function, role in disease and allelic variation has been assembled for each gene. Applications in our laboratory include mechanistic investigation of a number of toxic endpoints such as hepatotoxicity and endocrine disruption.

Obstacles to the prediction of estrogenicity from chemical structure: assay-mediated metabolic transformation and the apparent promiscuous nature of the estrogen receptor.

Information on structure-activity relationships (SAR) and pathways of metabolic activation would facilitate the preliminary screening of chemicals for estrogenic potential. Published crystallographic studies of the estrogen receptor (ER) imply an essential role of the two hydroxyl groups on estradiol (17beta-E(2)) for its binding to ER. The influence of these hydroxyl groups on ER binding and estrogenicity was evaluated by the study of 17beta-E(2) with one or both of these hydroxyl groups removed (17beta-desoxyestradiol and 3, 17beta-bisdesoxyestradiol, respectively). 6-Hydroxytetralin (17beta-E(2) with its C- and D-rings removed) and other synthetic estrogens were also studied. The estrogenicity assays comprised a yeast ER-mediated transcription assay, mammalian cell transcription assays incorporating either ER alpha or ER beta, and the immature rat uterotrophic assay. With the exception of 6-hydroxytetralin in the uterotrophic assay, all the chemicals were active in all the assays. Hydroxylation of the two desoxy compounds to estradiol was shown to occur in immature female rats, but metabolism was not implicated in the responses observed in the ER-binding and yeast systems. It is concluded that the 3-hydroxyl and 17beta-hydroxyl groups of 17beta-E(2) are not absolute requirements for estrogenicity. It would therefore be of value to the derivation of SAR for estrogenicity were the crystal structure of the bisdesoxy-E(2)/ER complex to be evaluated.

Identification of a possible association between carbon tetrachloride–induced hepatotoxicity and interleukin‐8 expression

Hepatotoxicants can elicit liver damage by various mechanisms that can result in cell necrosis and death. The changes induced by these compounds can vary from gross alterations in DNA repair mechanisms, protein synthesis, and apoptosis, to more discrete changes in oxidative damage and lipid peroxidation. However, little is known of the changes in gene expression that are fundamental to the mechanisms of hepatotoxicity. We have used DNA microarray technology to identify gene transcription associated with the toxicity caused by the hepatotoxicant carbon tetrachloride. Labeled poly A+ RNA from cultured human hepatoma cells (HepG2) exposed to carbon tetrachloride for 8 hours was hybridized to a human microarray filter. We found that 47 different genes were either upregulated or downregulated more than 2‐fold by the hepatotoxicant compared with dimethyl formamide, a chemical that does not cause liver cell damage. The proinflammatory cytokine interleukin‐8 (IL‐8) was upregulated over 7‐fold compared with control on the array, and this was subsequently confirmed at 1 hour and 8 hours by Northern blot analyses. We also found that carbon tetrachloride caused a time‐dependent increase in interleukin‐8 protein release in HepG2 cells, which was paralleled by a decrease in cell viability. These data demonstrate that carbon tetrachloride causes a rapid increase in IL‐8 mRNA expression in HepG2 cells and that this increase correlates with a later and significant increase in the levels of interleukin‐8 protein. These results illustrate the potential of microarray technology in the identification of novel gene changes associated with toxic processes.