The Snail Assay as an Alternative to the Rodent Hershberger Assay for Detecting Androgens and Anti-androgens

Reproductive cancers and disorders and cancers are increasing in men and boys. Scientists suggest these problems may be caused by exposure to environmental pollutants found in residues on food, PVC plastics and in the linings of food cans. By fooling the body into thinking they are sex hormones, these chemical imposters break into the chemical hormone messenger system (endocrine system) and interfere with the signalling, causing reproduction and development to go awry. The government is developing policies towards endocrine disrupting chemicals using animal tests with rats and mice to test chemical safety. Medicines used to treat reproductive diseases and disorders are also put through the same tests in order to discover effective and safe new medicines with which to treat reproductive cancers and other diseases. Around 200,000 rodents and a smaller number of fish are used in chemical and drug testing of this kind annually. Although animal testing is essential for medical practice and the protection of consumers, it generates widespread public concern and reduction, refinement and replacement of vertebrate animals in chemical and drug testing is an acknowledged need. Replacement of rodents used in animal testing with less sentient invertebrate animals (like worms and flies) is one solution. Our common evolutionary heritage means that some of the mechanisms underlying medical conditions are the same in humans, flies and worms and that these animals could be used as alternatives to rats and mice to test chemicals. Currently, however, invertebrate models for reproduction are lacking.
In order to fill this important gap, we will develop a model using the male snail reproductive organs. Snails share many structural and functional features with mammals, from the presence of similar reproductive genes to reproductive organs with similar functions such as the penis and the prostate. Preliminary studies have shown that human male hormones (androgens) stimulate the growth and development of these organs, and of undesireable tumour like growths on the reproductive organs just like in humans. We intend to expand on our existing knowledge of these phenomena and develop a properly validated system for testing pharmaceutical anti-androgens used to treat androgen-dependent cancers and for environmental safety testing of chemicals suspected of having harmful androgenic or anti-androgenic properties. We are hopeful that this assay will lead to a replacement of rodents in some animal tests.

Prostate cancer is the third most common cancer in older men and reproductive disorders and cancers are increasing in younger men and boys. Scientific research has revealed that exposure to endocrine disrupting environmental pollutants may be partly responsible for these trends and this has led to changes in legislation concerning the safety testing of chemicals involving increased numbers of animal tests. The rat Hershberger assay, originally developed to identify the hormones involved in male sexual development, is the gold standard pre-clinical test used both in the development of drugs used to treat hormone-dependent male cancers and for screening suspected endocrine disrupting chemicals for androgenic and anti-androgenic activity. This proposal aims to explore the possibility that snails might be good model organisms for screening chemicals for androgenic and anti-androgenic effects. Our hypothesis is that the reproductive organs and accessory sex glands of snails are functional analogues of parts of the mammalian male reproductive organs and so respond to androgenic and anti-androgenic chemicals in a similar fashion to their mammalian counterparts. The notion that molluscs could be used as potential surrogates for mammals in reproductive tests is supported by the many features their reproductive systems share with those of mammals, from the molecular level to gross anatomy. We will compare the response of reproductive tract of the mollusc (B. glabrata) exposed to androgenic chemicals (in the presence and absence of clinical anti-androgenic drugs or endocrine disrupting chemicals with different modes of action) to known effects of the same chemicals reported in vertebrate tests at different levels of complexity. We will characterise changes in the morphology of reproductive organs, changes in androgen-dependent antigenic markers (such as prostate specific antigens; PSA) with labelled PSA-antibodies on histological sections, and at the genomic level we will characterise differentially expressed genes in tissues of exposed snails using Suppression Subtractive Hybridisation (SSH) approaches combined with the use of a B. glabrata microarray. A properly validated, mechanistically relevant mollusc test that can objectively quantify the response of the male reproductive system to androgens and anti-androgens will have the potential to at least partially replace the use of the rat Hershberger Assay and would be expected to lead to a reduction in the numbers of vertebrate animals needed in reproductive toxicity tests and possibly carcinogenicity testing and, consequently, to a refinement of test protocols in general, minimizing vertebrate animal usage.