WO1999045390A1

WO1999045390A1 - Composition and device for the detection and elimination of endocrine hormones and/or hormone disrupting substances in and from fluids

Info

Publication number: WO1999045390A1
Application number: PCT/BE1998/000029
Authority: WO
Inventors: Frank Comhaire
Original assignee: Frank Comhaire
Priority date: 1998-03-03
Filing date: 1998-03-03
Publication date: 1999-09-10

Abstract

The present invention relates to a composition for the detection of an endocrine hormone and/or endocrine hormone disrupting substance in a fluid, wherein said composition comprises a hormone receptor compound as a non-living material for forming a complex with said hormone and/or hormone disrupting substance. In that way a composition is provided which allows to perform an almost instantaneous detection of hormones and/or hormone disrupters in fluids. The composition of the present invention also comprises a detector, which is preferably a non-living material, for generating an observable signal upon formation of the complex. The present invention also relates to a composition and a device for eliminating an endocrine hormone and/or endocrine hormone disrupting substance from a fluid, either before or after digestion of the fluid.

Description

- 1 -

Composition and device for the detection and elimination of endocrine hormones and/or hormone disrupting substances in and from fluids.

The present invention relates to a composition for the detection of a hormone and/or hormone disrupting substance in a fluid, as described in the preamble of the first claim

An endocrine hormone disrupting substance is an exogenous agent with an inherent hormonal activity, which is capable of interfering with the production, release, transport, metabolism, bonding, action or elimination of natural hormones in the body of humans and animals, responsible for the maintenance of homeostasis and the regulation of development processes The present invention relates in particular to a composition and a device for the detection and elimination of endocrine hormone disrupting substances of the pseudo- or xeno-oestrogen anti- oestrogen, pseudo-androgen and anti-androgen type, which imitate, respectively antagonise the effect of female and male hormones

Over the recent years, a number of articles have been published in which an association is suggested of human and wildlife health problems with exposure to environmental chemicals More particularly, an association is suggested with those chemicals which interfere with the reproduction system, i e the endocrine hormone disrupting compounds, as well as the hormones themselves Examples of evidence of such association include increasing incidence of testicular cancer in young men, decline in sperm count, sperm quality and semen volume, increasing incidence of cryptorchidism (maldescended testes), increasing incidence of breast cancer in man and women Related - 2 -

observations have been cited for animals For the sake of simplicity, in the following text, the endocrine hormone disrupting compounds will be referred to as hormone disrupters

The hormone disrupters and also the hormones themselves, are most often diffuse substances, which may enter the nutritional chain through various mechanisms, for example through the food, the packing material of food, they may be present in the water supply, or may enter the nutritional chain in any other way In that way, the hormones or hormone disrupters may enter the organism of human beings and other living organisms, become complexated by the corresponding receptor compounds of the human being or the living organism, and subsequently interfere with e g the reproduction system

Several tests have been developed for the detection and measurement of the oestrogenic content of fluids The most promising test is the GL/AXO yeast test, which is disclosed in Routledge, E J and Sumpter J P , "Oestrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen", Environmental Toxicology and Chemistry, Vol 5, nr 3, 1996, pp 241-248

This known GLAXO yeast test however has the disadvantage that it is rather time consuming The known method is namely based on the activity exhibited by living yeast cells To perform the test, first a culture of a genome of a recombinant Saccharomyces cerevisiae phylum organism must be grown, whereby the DNA sequence of the human oestrogen receptor is integrated into the genome The genome also comprises Lac-Z gene expression plasmids with oestrogen response sequences The growing of such a culture may take one or a few days After a sufficient amount of the saccharomyces cerevisiae phylum of the above described genome has been grown, it is contacted with a xeno- oestrogen containing medium The xeno-oestrogen is complexated by the built-in oestrogen receptor, following which the metabolism of the cells is activated Complexation of the xeno-oestrogen is followed by plasmid - 3 -

expression of a Lac-Z gene The Lac-Z gene at its turn, codes for the synthesis of the β-galactosidase enzyme It is only the synthesis of the β- galactosidase which involves a colour transition of the medium from yellow to red, thus visualising the presence of xeno-oestrogen in the added medium

It is the aim of the present invention to provide a composition which allows to perform an almost instantaneous detection of hormones and hormone disrupters in fluids, in particular chemicals with oestrogenic, pseudo-oestrogenic, anti-oestrogenic, androgenic, pseudo- androgenic and/or anti- androgenic activity or the like

This is obtained according to the present invention with the measures described in the characterising part of the first claim

Upon complexation of the hormone and/or hormone disrupter by the receptor molecule as a non-living material, the chemical and physical properties of the receptor as well as the hormone and/or hormone disrupter change, which is reflected in an altered interaction of the complex with its environment This altered interaction of the complex with its environment can be detected directly, with several techniques known to the man skilled in the art Because the altered interaction as such can be detected directly, the time consuming interference of a living organism for making a detectable compound is not necessary With the present invention, use is thus made of the chemical affinity of the hormone and/or hormone disrupter for the receptor molecule itself There is no need to wait for the subsequent, biological response of an interfering organism to such chemical affinity

This is a quite unexpected effect, since a receptor molecule is in fact a polypeptide chain, only a fraction of which constitutes the hormone bonding site The ability of the hormone bonding site to accommodate a hormone or hormone disrupter has been found to depend not only on the chemical composition of the bonding site, but also - 4 _

on its spatial configuration The spatial configuration at its turn, and the ability of the hormone bonding site to maintain this configuration, is however found to depend strongly on the physiological, and thus chemical, environment of the hormone bonding site The man skilled in the art will as a consequence not consider to isolate a receptor molecule from its normal environment and use it outside of a living organism, in a different chemical environment

Also, the person skilled in the art is used to find out about lacking or altered physiological responses, when trying to find out on malfunctioning of the hormonal organism The formation of receptor- hormone complexes namely often initiates a number of subsequent, often immediately observable, physiological responses in an organism The man skilled in the art will in general look for the physiological responses or the lack thereof, when solving the problem whether or not the homπone- receptor complex formation has taken place in the regular way He is thus rather used to rather look for the biological reaction of the organism to the hormone-receptor complex formation Because the biological reaction is usually observable, the man skilled in the art does not have any incentive to look for and the formation of the complex itself Surprisingly it has been found that with the composition of the present invention, the sensitivity of the test towards hormones and/or hormone disrupters can be increased Namely, with the composition of the present invention, the formation of the hormone/hormone disrupter - receptor complex itself is detected Because of the high affinity of the hormone and/or hormone disrupter for the receptor molecule, the complex formation can hardly be adversely effected by side reactions With the yeast test known from the art on the other hand, the compound synthesised by a living organism, following formation of the hormone/hormone disrupter - receptor complex, is detected During such synthesis, undesired side reactions may take place, especially if several - 5 -

reaction steps are involved, which often results in a loss of sensitivity of the test

Because according to the present invention, the hormone/hormone disrupter - receptor complex itself is detected, the composition can be used for a quantitative determination of the amount of hormone/hormone disrupter in the fluid Thereby, there is no need to pretreat the sample fluid containing the hormone and/or hormone disrupter, so as to adapt it to the living conditions of the yeast culture

Moreover, because the composition of the present invention does not make use of a living organism, it is suitable for use in a large variety of fluids The composition of the present invention can be uses in a large variety of solvents or mixtures of solvents and reagents in which either the living yeast cells would be destroyed, or alternatively their hormone bonding capacities would be destroyed or inhibited, resulting in an inactivation of the test According to the invention, the wording "fluid" is meant to include human as well as animal body fluids, for example blood, plasma, urine, or extracts, or concentrated extracts thereof, as well as other fluids, for example water, milk, etc

Preferably, the receptor compound is applied to, more preferably immobilised to, a solid carrier material, so as to facilitate its storage and allow a more precise dosing of the receptor compound

According to the invention "applied to" includes all kinds of physical mixing of the receptor compound and the carrier material, as well as the bonding of the receptor compound on the carrier material through physical or chemical bonding By immobilising the receptor compound to the carrier material, the presence of hormone disrupters can be determined in situ, without the risk of washing out of the receptor compound from the composition Also, the immobilisation of the receptor molecule to the carrier may assist in maintaining one or more spatial configurations of the hormone bonding site so as to maintain the energetically most favourable configuration for accommodating the - 6 -

hormone and/or hormone disrupter Preferably, the carrier material is suitable for oral ingestion This allows the presence of hormones and/or hormone disrupters not only to be detected in the organism containing the hormones, but also to be removed from the organism during intestinal passage Namely, a hormone receptor compound is mostly a protein which not only shows a high affinity and high specificity for the corresponding hormone, but also for the corresponding hormone disrupter Thereby, most often, the affinity of the receptor towards the hormone is higher than the affinity towards the hormone disrupter The receptor compound is preferably chosen from the group of receptor compounds for oestrogeπs, aπdrogens and mixtures thereof The hormone receptor can either be the natural receptor protein, produced by a cloned gene, or a truncated protein with similar hormone bonding capacity, equally produced by cloned genes The composition of the present invention preferably comprises a detector, which is a non-living material, for producing a observable signal upon the formation of the complex between the hormone and/or hormone disrupting substance to the hormone receptor In that way the presence of hormones and/or hormone disrupting substances in the fluid can be detected directly, without requiring the interference of a living biological material which must undergo a number of reactions after formation of the hormone/ hormone disrupter- receptor complex The type of detector used will depend on parameters such as for example the nature of the carrier material, the nature of the hormone/hormone disrupter, the hormone receptor, the nature of the fluid etc

According to a preferred embodiment of the invention, the indicator is releasibly encapsulated in the carrier material The composition of the present invention may contain a hormone receptor compound which is selective to only one type - 7 -

of hormone e g oestrogens or androgens As has been outlined above, such a receptor compound for oestrogens is also capable of complexating the corresponding hormone disrupters, e g anti-oestrogens and xeno- oestrogens The composition may also contain a mixture of hormone receptor compounds, whereby each receptor is selective to a specific hormone, e g a mixture of oestrogen and androgen receptors

As has been outlined above, hormone disrupters may enter the human nutritional chain through the food or the water supply It is therefore also an aim of the present invention to provide a composition for eliminating hormones and/or hormone disrupters from fluids, after they have been ingested through bonding of the hormone disrupter during intestinal passage

Thereto the receptor compound is preferably applied to a carrier material that is suitable for oral administration, for example bacteria, fungi or trace compounds Such carrier materials can for example be supplied as a medicine, or through daily food so as to remove hormone disrupters from the human or animal body or the food

It is a further aim of the present invention to provide a device for eliminating hormone disrupting substances from fluids, before the fluid is ingested In that way for example an amount corresponding to the daily need of water for drinking and preparing food, can be filtered daily, or with a regular frequency, and stored until use

The filter device of the present invention preferably comprises a first solid filter, whereby hormone receptor compound is immobilised on the surface of the filter If so desired, the filter material can be adapted to the nature of the hormone receptor compound The filter material can for example be pretreated so as to activate its surface and obtain a stronger bonding between the hormone receptor compound and the filter material In that way a washing out of the hormone receptor compound from the filter material, upon contact of the filter with the fluid, can be prevented - 8 -

The filter material can be applied to a grid, whereby the liquid to be filtered is directed to flow through the grid It is also possible to store the filter material in a column and direct the fluid to be filtered through the column Preferably, the filter device also comprises a second filter for removing bacterial contamination material from the already filtered fluid

If so desired, the device may comprises a third filter, which precedes the first filter, for removing other undesired substances from the fluid before it is contacted with the filter material baring the hormone receptor compound Such undesired compounds can for example be other chemical compounds, dust particles, bacterial contamination In that way a too early contamination of the filter with the hormone receptors can be precluded The filter device of the present invention may be a full-flow filter, that is suitable for a continuous use The filter device may also be a slow continuous filter, for filtering fixed amounts of fluid

The invention is further elucidated in the following description According to the present invention, several types of carrier materials generally known to the man skilled in the art may be used The carrier material will usually be selected, taking into account the application, the nature of the hormone receptor, and the nature of the hormone receptor - carrier material bond An example of a preferred carrier material is a strip made of a plastic material, for example nylon, with a plastic backing so as to improve the adhering of the receptor compound to the strip Such strips present the advantage that they allow an in situ detection of the hormone/hormone disrupter, by immersing the strip in the fluid containing the hormone/hormone disrupter Upon formation of the hormone/hormone - 9 -

disrupter - receptor complex, an observable signal, for example a colour transition is produced by the indicator

Other suitable carrier materials include wells of microplates of a matrix material coated with the receptor compounds The solid carrier material may for example also be silica or glass beads, organic polymers for example ion exchange resins, for example latex beads, polystyrene beads, hydrazide beads, agarose, tnsacryl, HW-65F support (Pierce) Very suitable carrier materials are acidic cation exchange resins with a carboxylic functionality, for example Amberlite" exchange resins Such carrier materials may be added as such to the hormone/hormone disrupter containing fluid In case of a composition suitable for oral administration, the carrier material can for example comprise bacteria, fungi, trace compounds, or mixtures thereof, or other materials suitable for oral administration If so desired, the surface of the carrier material can be pretreated, for example coated with an adhesive, so as to increase its affinity for the receptor compound, and to increase the bond strength of the receptor compound If so desired, the receptor compound may be applied to the carrier material through a spacer compound In case of the device for removing hormone/hormone disrupters from a fluid, the solid filter material may for example be surface activated silica or glass beads, organic polymers for example ion exchange resins, for example latex beads, polystyrene beads, hydrazide beads, agarose, tnsacryl, HW-65F support (Pierce) Very suitable carrier materials for the filter include acidic cation exchange resins with a carboxylic functionality, for example Amber te* exchange resins The carboxylic acid functionality facilitates the bonding of the protein receptor molecule The presence of a pore system with large pore diameters, permits an easy entrance of the protein receptor compounds and provides, together with the carboxylic acid functionality, a high bonding efficiency of the protein receptor compound Also, the ion exchange resin itself may be - 10 -

expected to exhibit adsorbent capacities In that way, other undesired substances that were less effectively removed during a preliminary purification of the liquid, may be adsorbed on the hormone receptor carrier material If so desired, the fluid containing the hormone and/or hormone disrupters can be first concentrated, for example by evaporating part of the fluid, by extraction methods, or any other method known to the man skilled in the art, before being contacted with the composition for detecting their presence, or the device for their removal The hormone receptor compound is mostly a protein which comprises a plurality of specific, structurally separate hormone bonding sites, whereby each site constitutes only a fraction of the total receptor protein Mostly, each hormone bonding site usually bonds only one single molecule of a hormone disrupting compound The hormone bonding site preferably comprises a COOH-termmated region of the protein for complexatioπ of the hormone and/or hormone disrupter The hormone bonding site may comprise a hydrophobic pocket for bonding specific parts of the hormone and/or hormone disrupter compound, for example the steroid ring, or the entire hormone and/or hormone disrupter compound The molecular weight of the hormone receptor compound can vary within a wide range, and is often about 80 to 100 kd

The hormone receptor compound can be applied to the carrier material in various ways It can for example be physically mixed with the carrier material, or be immobilised on the carrier material through various types of bonding, for example, adsorption bonding, Van der Waals bonding, ionic bonding or covalent bonding Care should be taken that the bonding is strong enough, so as to avoid a release of the hormone receptor from the carrier upon contact of the composition with the fluid If so desired, the surface of the carrier material can be pretreated so as to - 11 -

activate it and allow a stronger bonding of the hormone receptor compounds

In case a latex type carrier material is used, the bonding between the hormone receptor compound and the carrier will usually be a non-covalent, adsorption bonding In case the carrier material comprises terminal carboxylic acid (-COOH), amine (-NH₂) or sulphine (SH) groups, usually a covalent bonding will take place It is furthermore possible to immobilise the hormone receptor compound on the carrier material through cross-linking An example of a detector suitable for use in the composition for detecting the presence of hormone disrupting substances in a fluid, is a colour indicator which produces a colour transition upon complexation of the hormone/hormone disrupter to the receptor compound, a fluorescent material, a material for producing a luminescent signal, materials inducing a change of the electric charge of the fluid upon complexation etc It is also possible to conjugate the receptor compound with an enzyme, which produces a colour transition after complexation of a hormone disrupting substance The concentration of the hormone disrupting substance can be determined for example by measuring the extinction or absorption at the appropriate wave length, depending on the colour of the solution The concentration of the hormone disrupter will mostly be proportional to the intensity of the produced signal

Other suitable compositions for the detection of hormone and/or hormone disrupters include compositions comprising a second hormone and/or hormone disrupter which bind competitively to the receptor compound Such a composition is mostly used in combination with a solid carrier strip which is coated with a receptor compound

Thereby the second hormone and/or hormone disrupter is for example immobilised onto a carrier material, for example to a liposome If so desired, the second hormone and/or hormone disrupter may be bound to the outer surface of the liposome through spacer - 12 -

molecules The second hormone and/or hormone disrupter is chosen such that the affinity of the receptor to the hormone disrupter is higher than its affinity to the liposome The detector material is preferably encapsulated into the liposome, but may also be applied to the surface of the liposome If such a composition is used, an amount of the above described liposomes is added to the hormone/hormone disrupter containing fluid Upon immersing of the liposomes in the solution, the hormone disrupter and the liposome bind competitively to the receptor compound The unbound liposomes will be able to migrate on the strip, to a lysis zone provided on the strip In the lysis zone, the indicator is released from the liposome The indicator can for example be an acidic or alkaline compound, which will result in a change of the electric charge of the fluid The indicator can also be a dye or a colour indicator The higher the amount of hormone and/or hormone disrupting compound in the fluid, the larger the amount of liposomes that will migrate on the strip, and the more intense the observable signal will be Such a system may be used for the quantitative detection of hormone/hormone disrupters in a fluid

It is also possible to use a liposome immuno aggregate for detecting the presence of hormone/hormone disrupters in a fluid In that case, the multihaptened liposomes compete with the hormone/hormone disrupter of the fluid for forming a complex with the receptor compound If no hormone/hormone disrupter is present in the fluid, aggregates will be formed between the multihaptened liposomes and the receptor compound If the fluid contains hormone/hormone disrupters, these will be complexated by the receptor compound, and less or no aggregates will be formed, depending on the amount of hormone/hormone disrupter in the solution The liposomes can then be removed from the fluid, for example by filtration Thereafter the liposomes can be submitted to a lysis procedure, so as to release the indicator compound, and determine the amount of free liposomes, which will be proportional to the amount of hormone/hormone disrupter in the original fluid - 13 -

The composition of the present invention for the detection of endocrine hormone and/or hormone disrupting substances presents the advantage that there is no need for a specialised staff for growing the yeast culture and carrying out the test The test is available, ready for use Thereby, no special knowledge is required to the person carrying out the test Also, there is no need to first grow a sufficient amount of a living culture so as to allow the test to be performed with sufficient sensitivity, nor is there a need to store and maintain a living cultures of yeast so as to have the test available Such a storage requires rather severe control of the conditions of temperature and humidity

The composition of the present invention has the further advantage that it is not only suitable for detecting the presence of the proper hormone as well as hormone disrupters in a fluid Moreover, because a rather strong complex is formed between the hormone and/or hormone disrupter and the receptor compound, the composition of the present invention can also be used for removing hormones and/or hormone disrupters from a fluid

Representative examples of hormones include oestrogenic and androgenic hormones The following chemicals have been identified as potential hormone disrupters man made and natural chemicals such as pesticides used in agriculture (atrazine, methoxychlor), industrial and domestic detergents (alkyl phenol compounds and their ethoxyethers), plasticizers (bisphenol-A, phtalates), polychlonnated biphenyl compounds (PCB), polycyclic aromatic hydrocarbon compounds (PAH) chemicals having phenolic moieties, for example, 5- octylphenol, 4- nonylphenol, 4-sec-butylphenol, 4-tert-butylphenol, 4-tert-pentylphenol, 4- Isopentylphenol, alkylphenol diethoxylates, biphenyls, for example 4- hydroxybiphenyl, 4, 4'-dιhydroxybιphenyl, phtalates, bisphenol A, bisphenol A diglycidyl ether, tert-butyihydroxyanisole, alkyl polyetheroxylates, or may be metabolised to phenol or contain a functional equivalent of phenol, such as a polar function that can act as a hydrogen bond acceptor, p, p'-DDT, o, - 14 -

p'-DDT, o, p'-DDE, p, p'- DDE; dieldrin, lindane, chlordecone, toxaphene, endosulphan, naturally occurring chemicals such as the phyto-oestrogens present in food, mycotoxins, cournestrol, genistein, daidzein, zearalenol, zearalenone, naturally occurring oestrogens: phyto-oestrogens, oestrogenic myocotoxins, dioxins, PCB's and indole-3-cabinol, vinclozolin and p, p'- DDE.

Claims

- 15 -

CLAI S.

1 Composition for the detection of an endocrine hormone and/or endocrine hormone disrupting substance in a fluid, characterised in that said composition comprises a hormone receptor compound as a non-living material

2 Composition as claimed in claim 1 , characterised in that said composition also comprises an indicator, which is a non-living material for generating an observable signal following complexation of a hormone and/or hormone disrupting substance to said hormone receptor compound

3 Composition as claimed in claim 1 or 2, characterised in that said receptor compound is applied to a solid carrier, which is a non-living material

4 Composition as claimed in any one of claims 1 to 3, characterised in that said carrier material is a material suitable for oral administration

5 Composition as claimed in any one of claims 1 -4, characterised in that the receptor compound is chosen from the group of receptor compounds for oestrogens and androgens, or mixtures thereof 6 Composition as claimed in any one of claims

2-5, characterised in that said indicator is releasibly encapsulated in said carrier material

7 Composition as claimed in claim 6, characterised in that said carrier material comprises a second hormone or hormone disrupter, whereby said second hormone or hormone disrupter and said hormone and/or hormone disrupter to be removed from said fluid bind competitively to the receptor compound

8 Composition for the elimination of a hormone and/or a hormone disrupting substance from fluids, characterised in that said composition comprises a hormone receptor as a non-living material, which is applied to a solid carrier material - 16 -

9. Device for the elimination of a hormone and/or hormone disrupting substance from fluids, characterised in that said device comprises hormone receptor compound as a non-living material, which composition is applied to a liquid permeable filter. 10. Device as claimed in claim 9, characterised in that said device comprises a first filter for removing solid particles from said fluid before contacting said fluid with said fluid permeable filter.

11. Use of a hormone receptor compound as a non-living material for the detection of an endocrine hormone and/or hormone disrupting substance in a fluid.