Endocrine toxicology--contributions of in vitro methods to the 3R concept

Concerns have been raised about the potential of natural and man-made chemicals to interfere with the endocrine system of humans and wildlife adversely. This issue has been addressed by intensive research over the past years. Most studies focused on interactions with sex hormone receptors. An estrogenic or (anti)androgenic potential was demonstrated for a number of natural and anthropogenic compounds. This work focused on the detection of compounds with affinity to the androgen receptor (AR). An AR binding assay based on a recombinant rat AR (fusion protein to thioredoxin) properly ranked strong and weak (anti)androgens and other compounds with affinity to the AR. The data correlated excellently with that obtained in binding studies employing a cytosolic AR preparation from rat prostate. Studies with detergents indicated that these are confounders of the assay. They most likely disturb ligand receptor interactions and/or modification of the receptor protein. A transactivation assay based on the androgen-sensitive PALM cell line detected strong and weak androgens, and was able to detect antiandrogenicity when cells had been stimulated with an androgen. These findings indicate that in vitro methods can contribute successfully to the characterisation of compounds with affinity to sex hormone receptors. Whereas binding assays provide the basic information on affinity to the receptors, transactivation assays can discriminate agonists and antagonists and also provide information on cytotoxicity. These in vitro assays could also reduce animal use in endocrine toxicity testing (a) by using recombinant sex hormone receptors instead of cytosolic receptor preparations, (b) by applying intelligent screening strategies that include (potential) metabolites, as these assays probably have only little or no metabolic capacity and by combining several in vitro systems should equivocal results be obtained in one system, and (c) by using in vitro methods to identify mechanisms of action of effects observed in animal studies. In order to support the acceptance of these methods, a thorough validation and the characterisation of confounders is desirable. Whether receptor binding and transactivation assays can predict the outcome of in vivo screening assays reliably remains to be established.