Summary

Since 1992 several scientists have been suggesting that a number of reproductive disorders in men and female are, as in wildlife, caused by anthropogenic industrial compounds that have the capacity to interfere with the body's endocrine (hormone) system. Testicular cancer, urethral abnormalities and the decrease in sperm count and quality in the male and breast cancer and endometriosis in the female are disorders of the reproductive system which have increased in many countries over a short period of time. Therefore they rather seem to reflect changes in environmental factors or "life style" than genetic factors. During 2000, a candidate list of 562 substances has been identified by the European Commission and a priority list of actions has been developed in order to further evaluate the role of these substances in endocrine disruption. Indeed, most of the listed compounds have been described to bind to the estrogen receptor, but due to the complexity of the endocrine system, numerous possible levels of interaction are possible. In addition, most of these compounds are hydrophobic and persistent, making their half life in body tissues and environmental matrices high.

In this project we proposed to tackle the problem of endocrine disruption in a multidisciplinary way. We anticipated to the recommendations from the European Commission and US-EPA and focused on two important aspects: detection and prevention. The first research objective consisted of

1. the development of new test systems to study the influence of xenohormones on the neuro-endocrine regulation of male fertility, and
2. the implementation and validation of a reference assay for estrogenic compounds used worldwide.

Samples from Flemish waters were to be screened with these assays. In order to reduce human exposure we proposed to

1. investigate the feasibility of using estrogen receptor proteins in a bio-extraction cartridge as part of a filtration system to eliminate estrogens from tap water, and to
2. study the bioadsorptive and/or metabolisation potential of selected strains of microorganism for the development of a health food type of product preventing the uptake of ingested compounds from the gastro-intestinal tract.

We successfully developed an assay based on immature mouse Sertoli cells. Using these so-called nurse cells of spermatogenesis, we were able to study the effects of estrogens on the production of inhibinB, one of the most important proteins involved in the gonadal-pituitary-hypothalamo feedback system regulating male fertility. Secondly, for reasons of animal welfare and experimental reproducibility and sensitivity we effectively explored the use of immortile Sertoli cells in the development of an estrogen bioassay with a high relevance for male fertility. Thirdly, because of the unforeseen problems encountered with the use of a previously validated in vitro experimental setup based on hypothalamic brain explants, we decided to focus on the role of estrogens at the level of the pituitary which is under direct and close control of the hypothalamus. The influence of the model estrogenic compound 17-estradiol on the LH secretion by an immortalized pituitary cell line was investigated in vitro. Similarly, some important aspects of the particular guinea pig pituitary control of fertility were elucidated. This rodent is a potentially attractive in vivo model, because several aspects of its reproductive function are more similar to that of primates e.g. the more similar anatomical distribution of hypothalamic GnRH neurons as compared to other rodents. Fourthly, we extensively optimised and validated a reference yeast assay for estrogenic substances and checked the interlaboratory variability for a number of substances including pesticides, monomers and detergent components. At this moment, this yeast assay is being used for the screening of waters deriving from the upper Schelde basin.

In search for tools to reduce involuntary human exposure to estrogenic compounds we investigated the use of estrogen receptors to extract compounds from water using the same selective mechanisms by which they exert their biological function in the body. We successfully cloned several chimaeric human estrogen receptors, determined their binding capacity, scaled-up the production of the most optimal ones and try to purify them. Although it was seen that the receptors could be used in laboratory circumstances, we had to conclude that the proteins were too unstable to be applied in a household purification system. We subsequently modified our experimental paradigm in order to develop a bio-extraction module for the elimination of estrogenic compounds using catalytic manganese dioxide particles. The results are promising but further experiments are needed to investigate and optimise the estrogen removal efficiency of this material in combination with selected microorganism strains. Finally we examined the hypothesis that the uptake of certain endocrine disrupters via the gastro-intestinal tract in the body could be prevented by the presence of certain strains of Lactobacilli. These strains were tested for their capacity of adsorbing model compounds in a gastro-intestinal simulator (SHIME), developed several years ago in our laboratories. At the same time the fate and transformation of hormone disrupters in the intestinal tract was investigated.

The European Commission has emphasized the need for further research data on the endocrine disruptive capacity of more than 550 compounds. The results obtained in this life98 project will make it possible to address a number of the priority actions listed in EC document COM(2001)262.

It is difficult to estimate the economic cost of the decline in fertility, the rise in certain cancers or other endocrine disruptions in animals or man. In 1991 scientists hypothesised that hormone disruption could be the cause of declines in the populations of some wildlife species which have occurred over the past 50 years.Extinction of species is a major environmental issue, next to the obvious social and relational impact which involves impaired fertility or cancer.