The aims of this project are to develop and evaluate tests for toxicological insults to Leydig cells, in particular those leading to changes in steroid hormone formation, cytotoxicity and hyperplasia. This will be achieved by the development and use of (1) Leydig and Sertoli cell lines from transgenic mice carrying a temperature-sensitive T-antigen and (2) established tumour Leydig cell lines. Co-cultures and comparison of these cells with primary Leydig and Sertoli cells and macrophages will be studied in order to develop in-vitro Leydig cell models suitable for toxicity testing.
Leydig cells are the main source of the male androgen, testosterone, which plays an essential role in controlling spermatogenesis. In view of the wide range of pharmaceutical compounds reported as Leydig cell toxicants (1) and the recent discovery of the deterioration in human male reproductive health (viz falling sperm count and associated reproductive disorders)(2), there is an urgent need for developing Leydig cell toxicity tests. Also, because in-vivo toxicity studies are over a 1-2. year period, it is desirable to have acute in-vitro methods which would detect early changes in Leydig cell function. Identification of targets and mechanisms of Leydig cell toxicity will allow the development of specific end points such as cytotoxicity, inhibition or overstimulation of steroidogenesis, specific proteins involved in the control of steroidogenesis, transcription factors, adhesion molecule expression as a marker of abnormal cellular interactions and tissue development, and hyperplasia.
Transgenic mice will be developed and Leydig cell lines (with constitutively active luteinising hormone (LH) receptors) and Sertoli cell lines (lacking anti-mullerian hormone (AMH)) will be produced from such mice (by EUR). These cell lines have been chosen because they may have increased sensitivity to Leydig cell toxicants. Together with mouse MA-10 and rat H540 Leydig cell lines they will be compared to each other and to rat and mouse primary cultures of Leydig cells (by RFHSM and TCD) for maximal retention of normal phenotype. The effects of known Leydig cell toxicants and non-toxicants will be studied in these cells in order to establish both the suitability of the cell lines for toxicity testing and to develop them as suitable in-vitro models. GLAXO will develop a co-culture system of primary Leydig cells together with macrophages and Sertoli cells in order to provide a model system more closely related to the animal models.
The suitability and differences of primary Leydig cells and the developed cell lines will also be studied in this co-culture system. This approach will lead to the development of in-vitro models of Leydig cell toxicity which will have application to the human because many of the proposed end points of toxicological damage are common to the rat, mouse and human.
This approach will also provide a strong basis for any future prevalidation and validation studies on new in-vitro tests which would establish the scientific support of the methods to be employed by the future EU regulations - European Medicines Agency (EMA), European Centre for Validation of Alternative Methods (ECVAM) - and for the proposals of internationally accepted procedures of drug and chemical risk assessment. The nature of these studies is, therefore, clearly placed within the pre-normative research area.
1. Prentice DE & Meikle AN (1995) Human & Experimental Toxicology 14, 562-572. 2. Sharpe, RM & Skakkebaek, NE (1993) Lancet 341, 1392-5.
Funding SchemeCSC - Cost-sharing contracts
3015 GE Rotterdam
SG12 0DP Stevenage