Report LIFE 98

Introduction

Since industrial activities in the western countries began to expand after World War II, an ever growing number and diversity of man-made chemicals have been released into the environment. Humans and wildlife are exposed to these chemicals through their nutrition, the air and water and through the placenta during the sensitive foetal developmental stage. Since 1992, several scientists have been stating that a number of reproductive disorders in men and female are, as in wildlife, caused by chemicals in the environment interfering with the body's hormone (endocrine) system. The general paradigm was that exposure of individuals during fetal life to compounds mimicking the female hormone 17-estradiol, could mechanistically explain the proven rise in testicular cancer in many countries worldwide and the possible augmentation in the incidence in hypospadias and cryptorchidism in the male. Continuous exposure to hormone like chemicals is also implicated in the occurrence of breast cancer and endometriosis in the female. Although there is no consensus regarding the role of endocrine disruptive chemicals in these effects and no conclusive studies demonstrating that endocrine disrupters initiate or contribute to the development of these effects, results from in vitro and in vivo bioassays have been repeatedly used to identify chemicals that can elicit endocrine responses, thus further implicating all endocrine disrupters in adversely affecting human and wildlife health. Nevertheless, there is little doubt that small disturbances in endocrine function, particularly during certain highly sensitive stages of the life cycle can lead to profound and lasting effects.

Without any doubt, reproduction is one of the most fundamental processes in humans and other vertebrates. The increasing prevalence of infertility in man is illustrated by the steady rise of couples in Europe undergoing medical assisted conception during the last decade. Apart from the high cost, fertility treatment involves serious physical and mental stress. Furthermore, certain techniques of assisted reproduction, which are needed to solve infertility, are potentially dangerous to the mother and the offspring (e.g. genetic defects).

Most research to date in the field of endocrine disruption has focused on estrogenic chemicals. Although the role of xenoestrogens in the alleged decrease of sperm quality is one of the most controversial scientific subjects of the past years, the assays that are available to date to measure estrogenicity all suffer from a lack of relevance for male fertility. The last couple of years, important scientific contributions in the field of endocrinology have revealed a clear cell type and promotor context specificity of estrogen response. This is the experimental paradigm behind the booming research into selective estrogen modulators (SERMs) used for the treatment of osteoporosis, breast cancer and the post-menopausal syndrome. Most in vitro tests nowadays are artificially designed. In many cases the cell system does not endogenously express the estrogen receptor, but is transcribed from a plasmid that is brought into the cell by specific molecular techniques. Nor the cell system, nor the promotor context of most of the developed assays are relevant for male fertility and the latter should thus only be considered as yes/no screens. The National (American) co-ordinators of the OECD Test Guidelines Programme proposed at their 7th Meeting at the end of 1996, that new test guidelines should be developed and/or existing test guidelines should be revised for the testing and hazard characterisation of hormone disrupters. It was recognised that the existing OECD Test Guidelines are probably insufficient as to endpoints addressing endocrine disruption and that reliable (in vitro) screening methods are not yet available for this purpose.

Male fertility is regulated through the hypothalamic-pituitary-gonadal axis via a series of complex feedback mechanisms. Gonadotropin releasing hormone (GnRH) is produced and secreted by the hypothalamus in a pulsatile manner. These pulses of GnRH entrail the production of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) by gonadotropic cells present in the anterior lobe of the pituitary. The latter two peptide hormones affect two different cell types in the testes: the Leydig cells that react on LH with the production of the steroid testosterone and the Sertoli cells that respond to FSH with the production of a number of secretory proteins, the most important being androgen binding protein and inhibin B. Testosterone, after peripheral transformation to 17-estradiol, and inhibin B exert on their turn a negative feedback on the pituitary. Estradiol has also been described as affecting the GnRH pulse generator in the hypothalamus (Figure 1).

This part of the project aimed at the development of a complementary set of test methods to study the impact of hormone disrupting substances on the different levels of the hypothalamic-pituitary-gonadal axis.

In addition we validated an easy in vitro test for estrogenic compounds based on genetically modified yeasts. The latter assay was intended as a control for the effects seen with the other assays described above. In addition we optimised a previously developed estrogen receptor binding assay (ERBA) and a liquid chromatography-mass spectrometry chemical analysis method. Through the use of the bioassays we will be able to study the effects of endocrine disruptive compounds, which may lead to the identification of previously undetected types of hormone disrupters. At this moment, we are screening Flemish surface waters, together with effluents from sewage treatment works and industrial discharges for the presence of estrogenic compounds using the yeast assay together with the LC/MS method and the ERBA.

Although thanks to a number of regulations, the use of several endocrine disrupters will be reduced in the coming years, humans and wildlife are still exposed to them, because of their previous release in the environment (e.g. PCB's, some organochlorine insecticides) and their low biodegradability and important bioaccumulation in the body, and because of their widespread and poorly regulated use in some (developing) countries (e.g. DDT). This makes the development of systems preventing ingestion of those compounds more than necessary. Food and drinking water are generally considered as the major sources by which humans (and wildlife) are exposed to hormone disrupting substances. In this Life98 project we proposed to approach this problem from two angles of incidence.

Firstly, we proposed to develop a water filter system for household use. The elimination of the compounds of concern from surface or drinking water requires a highly specific and very expensive technology because of the low concentrations and the low biodegradability of the chemicals. The core of the filter system would consist of a "biological cartridge" composed of the immobilized estrogen receptor. Estrogenic compounds would thus be eliminated from the water through the same selective mechanisms by which the receptors exert their function in the human body. However, when it became obvious that the cloned receptors were not stable in normal conditions, thus losing their binding characteristics, we were forced to change our experimental setup. We subsequently evaluated the use of manganese oxide particles for their catalytic properties towards the degradation of estrogenic substances.

The second project aimed at preventing the uptake of endocrine disrupters through the principle of adsorbing endocrine disrupting chemicals during their passage through the intestine, thus reducing the amount taken up from the intestinal tract into the body. As an adjunct to the diet, foods or supplements that contain microorganisms that salutarily colonize the human mucosal surfaces have been referred to as probiotics. This good microflora can provide a protective effect only when a proper balance is maintained among all the different bacteria that normally reside in the intestine. Therefore, the most frequently used probiotic genera are Lactobacilli and Bifidobacteria. Health benefits of probiotics are being vigorously investigated today at various medical and research centers around the world. Results from clinical studies are showing that specific probiotic bacteria can alleviate or prevent diverse intestinal disorders and/or diseases. The aim of this research part was to direct the scientific research towards the development of a "health food" that can be applied to reduce the bioavailability of these compounds in the gastrointestinal tract. Our results will lead to a better understanding of internal exposure of humans to endocrine disrupters. Although it is known that micro-organisms adsorb all kinds of compounds, and although Lacobacilli are frequently used in health food products, there has never been a selection of strains with the highest adsorbing capacities for endocrine disrupting substances in view of an application into a health product.

From an environmental viewpoint, our project aims at decreasing the exposure of men to endocrine disrupting compounds. Identification of new hormone disrupters, can result in certain regulatory measures (on a national, European Union or world-wide level) bridling the use and production of particular chemicals in industrial processes or for agricultural or household use. In the long run the project will contribute substantially to the knowledge on the working mechanisms and dispersion of endocrine disrupters in the environment, leading to recommendations for regulatory measures at national, European Union or world-wide level. These recommendations should result in a safer and cleaner environment and should consequently lead to an improvement of the life quality (physical and mental) for all living creatures.