WO2005045426A2

WO2005045426A2 - Gene expression profiling of hazardous compounds and corresponding database clustering algorithm

Info

Publication number: WO2005045426A2
Application number: PCT/EP2004/012721
Authority: WO
Inventors: Ronny Blust; Wim De Coen; Bart Naudts
Original assignee: Aic
Priority date: 2003-11-10
Filing date: 2004-11-10
Publication date: 2005-05-19
Also published as: WO2005045426A3

Abstract

The present invention relates to a method for identifying and characterizing hazardous and/or toxic compounds in a sample, using a battery of prokaryotic or eukaryotic cells comprising a biological reporter system. In a further aspect, the present invention relates to kits for performing any of the methods according to the present invention.

Description

GENE EXPRESSION PROFILING OF HAZARDOUS COMPOUNDS AND CORRESPONDING DATABASE CLUSTERING ALGORITHM

FIELD OF THE INVENTION

The present invention relates to the field of genetic engineering, molecular biology, (environmental) toxicology and pharmacology. In a first aspect the present invention relates to methods for identifying, characterizing and quantifying hazardous compounds and diagnostic kits for performing such methods. In another aspect, the invention relates to methods to test and assess potential effects and/or efficacy of chemical compounds.

BACKGROUND OF THE INVENTION

Assessing the potential adverse effects of environmental pollutants on man and biota is one of the crucial elements of the environmental risk assessment process. Since the 1950s an increasing number of chemicals have been produced and released into the environment by several types of industries. Various examples are known where products have been synthesized and used for decades because of their interesting physico-chemical properties, e.g. heat resistance, chemical stability, whereas afterwards it became clear that the same compounds were problematic for various habitats and ecosystems. Examples of such compounds are organo-chlorine chemicals, e.g. DDT, organo-bromine, e.g. flame retardants or organo-fluor compounds, e.g. CFC. Also the strongly intensified agriculture and cattle breeding are responsible for the supply of contaminated material which have a toxic influence on the cattle or on the end consumer, e.g. pesticides and PCB's.

Next to exposure through water and food, additional exposure of humans occurs through atmospheric distribution of pollutants originating from industrial activities such as incinerators and transport activities. Though many industries have become more aware of possible environmental impacts, generally persistent chemicals keep accumulating in water, soil and air, and threaten the global health of various ecosystems.

In addition to synthetic pollutants, also biological toxins, i.e. mycotoxins, may be found in the food chain. These can be generated by detrimental microorganisms during the different food treatment processes and may have an ominous influence on livestock and on people.

BESTATIGUNGSKOPIE The major problem for new as well as for existing chemicals is the poor knowledge of the impact and effects of these chemicals on health and environment. Very few of these chemicals have been comprehensively tested for acute or chronic toxicity. This is the case, not only for individual chemicals, but also for chemical mixtures, irrespective of their use e.g. in production or in products, or of their distribution in waste or in wasted material. As a result of the intensive use of chemicals in our daily life, humans and biota are exposed during a full life cycle to a mixture of pollutants, usually at low concentrations.

Today bioassays for testing toxicity of compounds or drugs use living representative indicator organisms, i.e. mammalian, microbial and fish, as biological monitors.

The simplest and most convenient of these systems utilize strictly unicellular microorganisms since they are most easily maintained and manipulated. For instance, bacterial assays have been developed which are colorimetric, luminescent or fluorescent assays comprising the expression of reporter genes encoding an assayable product.

US 5,731,163 describes the use of bacterial cells, which contain a stress-inducible promotor operably linked to a reporter system (lux gene complex). The invention provides lyophilized bacterial cells which are then rehydrated and submitted to a sample, after which luminescence is measured.

US 5,683,868 relates to biosensors comprising genetically engineered cells (prokaryotic as well as eukaryotic) wherein the luminescence gene complex is under the control of a stress- inducible promoter.

US2002/0028445 describes a process and a device for detecting toxic and mutagenic effects of chemicals and mixtures of substances. The method is based on the use of either natural luminescent microorganisms (Vibrio, Photobacterium, sp. are cited) or genetically engineered microorganisms carrying an appropriate plasmid vector and enabled for bioluminescence (e.g. E.coli with LUX- gene complex). In particular, the microorganisms are immobilized in matrices. WO 00/47761 relates to a high throughput method for testing toxicological compounds in cultured prokaryotic or eukaryotic organisms or cells. The method consists of using wildtype and mutant organisms or cells and to evaluate the growth of the wildtype and mutant organisms or cells submitted to toxicological samples. The mutations in the organisms or cells are mutations affecting a particular gene function category (e.g. heat shock, oxidative stress, cell adhesion, cell migration, etc.). Growth is determined using turbidimetry, dose- response curves of mutant sensitivity to the compound are generated and calculated relative to its parent.

The prior art thus describes several and different types of short-term bioassays usually detecting toxicity of isolated compounds or detecting the presence of specified or unspecified compounds in simple solutions.

However, there is still a growing need for reliable methods which can rapidly and efficiently detect sources of pollution in complex mixtures and assess their biological effect.

SUMMARY

The aim of the present invention is to provide for fast alternative methods for identifying persistent bio-accumulating and/or toxic compounds (i.e. (substituted) Polycyclic Aromatic Hydrocarbon (PAHs), polychlorinated biphenyls (PCBs) organo-chlorine pesticides, heavy metals, mycotoxins and combinations thereof), for instance in environmental samples. Another aim of the invention is to develop a quality evaluation system which is based on very sensitive biological responses in order to evaluate in a fast and efficient manner the "hidden" quality of raw materials, components and end products of the food chain. A further aim of the invention is to provide tools for the assessment of ecotoxicological quality of surface waters, water quality in water purification plants, quality of exhaust air in incineration plants, etc...

These aims are met by the embodiments of the invention.

Therefore, the present invention relates in a first aspect to a method for identifying a hazardous and/or toxic compound in a sample comprising the steps of: a) providing a battery of prokaryotic or eukaryotic cells each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product, b) determining the gene expression profile of a known toxic compound by submitting said compound to said battery of cells, c) introducing the gene expression profile of said battery of cells, obtained in step b) in a database, d) repeating steps b) and c) for at least one additional known hazardous and/or toxic compound, e) determining the gene expression profile of the unknown toxic compound in said sample by submitting said sample to said battery of cells, and f) comparing the gene expression profile obtained in step e) with the response profiles recorded in the database of step c) thereby identifying said unknown compound in said sample.

In a further embodiment, the invention relates to a method as described above wherein said sample comprises a mixture of hazardous and/or toxic compounds, further characterized in that said method comprises steps g) to i) instead of steps e) and f) : g) fractionating said mixture of hazardous and/or toxic compounds thereby obtaining a number of fractions, h) determining the gene expression profile of each of the obtained fractions by submitting each of said fractions to said battery of cells, and i) comparing the gene expression profiles obtained in step h) with the response profiles recorded in the database of step c) thereby identifying said unknown compound.

In a preferred embodiment said mixture of hazardous and/or toxic compounds is fractionated by using a toxicity identification evaluation (TIE) methodology. This methodology will be explained into more detail below.

In a further embodiment, the invention provides a method for identifying a hazardous and/or toxic compound in a sample which further comprises the step of classifying the identified hazardous and/or toxic compound into a toxicity class. The invention also relates to any of the methods described above wherein the unknown compounds are identified quantitatively and qualitatively.

According to a further embodiment, the invention relates to a database comprising gene response profiles of known toxic compounds obtained by performing steps a) to d) of the methods for identifying a hazardous and/or toxic compound in a sample herein described. The invention also relates to an electronic carrier comprising said database.

In yet another aspect, the invention relates to a method for determining the condition of an environment comprising the steps of: a) extracting a sample from said environment, b) generating a gene expression profile for said extracted sample by submitting said sample to a battery of prokaryotic or eukaryotic cells each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product, and c) determining the condition of said environment by analyzing said generated gene expression profile.

Preferably said sample is taken from said environment e.g. by introducing a sampler for a suitable incubation time in said environment and by subsequently extracting said sample from said sampler. Suitable examples of samplers include Empore discs.

In a preferred embodiment, said method further comprises the step of classifying said environment into a pollution category.

Preferably, the method for determining the condition of an environment further comprises the step of identifying a hazardous and/or toxic compound in said environmental sample preferably by applying a method of the present invention, as explained above.

According to another further aspect, the invention relates to a database comprising gene response profiles of known ^"toxic compounds or of environmental samples that have been obtained by a method according to the present invention. The invention also relates to an electronic carrier comprising said databases. The invention further relates to a kit for identifying unknown hazardous and/or toxic compounds in a sample comprising a battery of prokaryotic or eukaryotic cells, each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product, and an electronic carrier according to the invention.

The present methods and kits to identify the presence or absence of hazardous and/or toxic compounds and/or to determine the condition of an environment have several important advantages over methods and kits using classical chemical analyses. The present methods and kits are fast, accurate, require relatively little labor and are time- and cost-effective. In addition, with the present methods and kits automated high throughput analyses can be performed.

The methods and kits envisaged by the present invention are useful both in environmental pollution, in guarding of food security, product-control in chemical, pharmaceutical, biotechnological, cosmetic and other industries.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 represents the fold induction profile in response to pentachlorophenol. Figure 2 represents the fold Induction profile for 3-Methyl cholantrene.

Figure 3a shows the dose response profiles, i.e. fold induction, of the C18 fraction obtained from a sample of surface water in Belgium. To establish the profiles, a battery of prokaryotic cells was used. In Figure 3b, the viability of these cells is plotted against the fold induction. Figure 4 shows the gene profiles of the water C18 fractions from a typically polluted sample. To establish the profiles, a battery of mammalian cells was used.

DETAILED DESCRIPTION OF THE INVENTION

Both environment and mankind suffer from pollution from industrial, household and agricultural activities. As a result, polluted sites contain a wide variety of chemicals mainly dependent on the input received and on geochemical conditions. Proto-oncogenic, growth arrest, xenobiotic response and other stress related defence systems are specifically induced depending on the pollution load at the sites under study. Until date, evaluation of toxic or hazardous compounds in environmental samples relied on the extrapolation of the known effects of the compounds identified through chemical analysis of the samples. However, it became clear that chemical analysis alone is insufficient to assess the quality of for instance surface waters, the water quality in water purification plants, the quality of exhaust air and/or gasses in incineration plants, etc.; or to investigate safe use of raw materials for use in chemical, pharmaceutical biotechnology, food and feed, personal care and cosmetics and environmental industries.

A problem to be solved by the present invention is thus to provide for fast and sensitive tools and methods to test and assess the potential toxic effects and/or efficacy of chemical compounds.

Another problem to be solved is to provide for fast and sensitive tools and methods to evaluate the effect of bio-available fractions of pollutants on biota rather than the total. Another problem to be solved is to provide for fast and sensitive tools and methods to identify, within a complex mixture of chemical compounds, at least one compound that causes a biological effect.

Another problem to be solved is to provide for fast and sensitive tools and methods to evaluate and determine the condition of an environment and its level of pollution.

Applicant has solved these problems by providing methods comprising highly sensitive biosensor systems for detecting and characterizing hazardous or toxic compounds in samples of low or high complexity.

The biosensors according to the invention are thus a battery of transgenic pro- or eukaryotic cells, each comprising a nucleic acid comprising the promoter sequence of known toxicological relevant receptors and a reporter gene (i.e. luciferase, GFP...) which produces a measurable or assayable product. These methods and corresponding assays utilize the fundamental component of an individual cell's response to toxicity in an assay plate, for instance a conventional 96-well plate, rather than using live animals. The presence of a toxic compound induces receptor/reporter gene activation which can be rapidly measured, i.e. within minutes/hours. The reporter gene has been genetically designed for rapidly and easily providing an assayable reporter gene product activity. In certain embodiments, display and attachment at the surface of the detector-organism enables on line and easy detection of the reporter gene product. Readout of the results can be carried out rapidly and simply with the intact organism, without the necessity of disruption of the cell or extraction of the polypeptide or enzyme to be measured. The assay can be performed easily in the laboratory or in the field, by personnel with minimal training.

The Gene Profile Assay Technology used in the embodiments of the present invention measures gene activity after exposure to stressors. This is in contrast to conventional genotoxicity assays which measure DNA damage. The endpoints, or inducing agents of the promoters used in the constructs may be, but are not limited to, oxidative stress, osmotic stress, protein misfolding or protein perturbation, DNA damage, growth arrest, metal ion presence, membrane effects or presence of aromatic hydrocarbons. The gene profiling technique is a powerful mechanistic tool. The generated profiles are specific for the mode of action of single compounds.

The prokaryotic constructs according to the invention are expressible in a prokaryotic host cell selected from the group comprising Gram-positive and Gram-negative bacteria, for instance E. coli. Examples of cells for use in a battery of prokaryotic cells are for instance the cells used in the Pro-Tox assays (Xenometrix) which are E. coli K12 hosts comprising bacterial stress gene reporter constructs driving expression of the bacterial beta-galactosidase gene.

The eukaryotic host cell for expression of eukaryotic constructs is preferably selected from the group comprising yeast or mammalian cells or cell lines derived there from. Depending on the presumed cytotoxity of the samples to be tested, the most suitable cell or cell line may be chosen. For instance when analyzing environmental samples in general, a human liver cell line may be chosen, such as HepG2 or specific intestinal cells or cell lines, ie gut or colon cell lines. In cases where the samples to be tested comprise raw materials or end products from cosmetic industries or products used in personal care, cell lines derived from the cornea or the skin may be preferred. Otherwise, samples containing high concentrations of heavy metals may be best evaluated in cell lines derived from mammalian kidney. An example of mammalian cells for use in a battery of mammalian cells is for instance the cell line used in the CAT-Tox assay (Xenometrics) which is a human liver cell line (HepG2) comprising mammalian stress gene promoter constructs driving expression of the chloramphenicol acetyltransferase (CAT) gene.

According to the present invention, the reporter gene is preceded by an inducible promoter. An inducible promoter is used to control the expression of the above-described reporter gene. The term "inducible promoter" refers to the promoter of a gene that is activated in a cell when a specific inducer is present. Such a promoter preferably is a promoter of a toxicological relevant receptor. Inducible promoters used in the present invention must be operatively linked to the chimeric gene. The term "operative linkage" refers to the positioning of the promoter relative to the reporter gene encoding the assayable product such that transcription of the gene is regulated by the promoter. Such positioning is well known in the art and involves positioning the promoter upstream (5') of the gene so that no transcription termination signals are present between the promoter and the gene. A promoter sequence 'operatively linked' to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence.

Examples of suitable inducible promoters, i.e. promoters of known toxicological relevant receptors, for use according to the present invention in prokaryotic cells may comprise, but are not limited to promoters such as those described in EP 651,825, which is incorporated herein by reference.

Examples of suitable inducible promoters, i.e. promoters of known toxicological relevant receptors, or response elements for use in eukaryotic cells according to the present invention may comprise, but are not limited to promoters or response elements such as those described in WO 94/17208, which is incorporated herein by reference.

In a preferred embodiment, a promoter sequence of known toxicological relevant receptors is used to control the expression of above-described reporter gene. In example 1 , a selection of prokaryotic and eukaryotic promoters is given which are incorporated in an exemplary battery of prokaryotic and mammalian cells. In addition to the promoters described above, new promoters of toxicological relevant receptors may be discovered and characterized and may also be employed in the methods and kits of this invention.

It should be clear that the pollutants present in a complex sample, for example taken from water or soil, are not for a hundred per cent in a biologically available form. For instance, metals can be present in complexes and polycarbons strongly tend to bind to particles because they are not very soluble in water. Indeed only a much smaller fraction of the pollutants which are present may effectively contribute to the biological effect of the sample. It is therefore preferred that only the fraction which is biologically available is taken in account to measure or predict biotoxicity of samples.

In this respect, additional systems may be used that properly reflect what is bio-available in a sample. For instance in bio-mimetic Solid Phase Extraction (SPE), membranes are used that all have the same advantageous characteristic in that they are all able to withhold the bio- available fraction of samples. A possible system for use in SPE is an Empore 47 mm disk for which the following membranes are available: C18, C8, C2, Chelating membranes, SDB-RPS membranes. Example 3 shows an experiment wherein a water sample is subjected to extraction on an Empore C18 membrane prior to submission to a battery of prokaryotic and/or eukaryotic cells.

In one embodiment, the methods of the invention thus also relate to the bio-available fraction of pollutants, as mimicked for instance by solid phase extraction, to the stress gene induction profile of a set of bacterial and mammalian transgenic cell systems.

According to a further embodiment, the invention provides methods for identifying a toxic compound in a sample, as described above, but characterized in that said sample comprises a mixture of toxic compounds. According to a specific embodiment, the invention thus relates to a method as described earlier wherein said sample comprises a mixture of hazardous and/or toxic compounds characterized in that said method comprising the following steps: a) providing a battery of prokaryotic or eukaryotic cells each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product, b) determining the gene expression profile of a known toxic compound by submitting said battery of cells to said compound, c) introducing the gene expression profile of said battery of cells, obtained in step b) in a database, d) repeating steps b) and c) for at least one additional known toxic compound, e) fractionating the mixture of hazardous and/or compounds (for instance using TIE techniques) thereby obtaining a number of fractions, f) determining the gene expression profile of the obtained fractions by submitting said fractions to said battery of cells, and g) comparing the gene expression profile obtained in step f) with the response profiles recorded in the database of step c) thereby identifying said unknown compound.

In this respect, the battery of biosensors is coupled to a series of miniaturized chemical separation techniques (based on the toxicity identification evaluation (TIE) methodology) which allows to distinguish the toxic effect of distinct classes of chemical compounds within complex mixtures. This also allows determining the toxicological importance of a certain type of pollution in an objective manner and with fast and inexpensive methods. Industry as well as (local) authorities are especially interested and demanding for such detection methods, able to characterize complex matrices in an unique biological manner, and thus to obtain a better founding of so-called hidden security and quality of, among others, foods and raw materials, as well as of the validation and guarding of production processes.

The TIE methodology according to the invention uses a variety of physico-chemical fractionation methods, i.e. ion chromatography, HPLC, to separate the original sample into fractions of distinct chemical composition. Each of these separate fractions is submitted to the battery of cells to allow determination of the toxic effect response.

By using, for instance complexing agents such as EDTA, the physico-chemical characteristics of the sample will change, as well as its toxicity. On the other hand, if an untreated sample turns out to be very toxic, which toxicity disappears after addition of EDTA, this suggests that the toxicity of the sample is mainly due dot the presence of metals.

A brief summary of toxicity identification evaluations is given below. TIE Phase I stands for the identification of the Toxicant Group. The objective of Phase I is to identify the general chemical group of the causative toxicants. This is accomplished by chemically or physically manipulating the sample to neutralize, alter, or remove specific groups of chemicals. Bioassay tests are then conducted on the manipulated sub-samples to assess which treatments have removed or mitigated toxicity. In some cases, identification of the toxicant's chemical group alone provides enough information for the elimination or effective control of toxicity to an acceptable level.

TIE Phase II stands for the identification of Specific Causative Toxicants. Other situations require specific causative toxicants, not just their group, to be identified. The goal of Phase II is to isolate the causative toxicants and tentatively identify them. Phase II methods are toxicant specific. In some cases, a tentatively identified causative toxicant provides the client with sufficient information to eliminate or control a sample's toxicity to an acceptable level.

TIE Phase III stands for Confirmation of the Causative Toxicants. Phase III evaluations provide the "weight of evidence" that causative toxicants have been accurately identified. Phase III confirmations can involve the correlation of data, sample spiking techniques, mass balance determinations and species sensitivity testing. Upon the successful completion of Phase III, sufficient data will be available to assure that causative toxicants have been accurately identified.

Until now, this methodology has not been applied for identification of hazardous or toxic compounds in food chain. It is evident that this methodology is extremely valuable to determine black-and-white, in case of contamination, the nature of, the source or origin of, and the importance of certain types of pollution within the food chain.

The present invention can be applied on samples of varying origin.

The DNA sequence, which encodes the assayable reporter gene product, may be preceded by a segment of a transmembrane sequence for anchoring and exposing said assayable product in the cell membrane of a host cell towards the extracellular medium. The DNA segment encoding a transmembrane amino acid sequence may be preceded by a targeting DNA sequence for targeting said assayable product to said host cell membrane. As a result, the assayable product of the reporter gene will be targeted to, and displayed and attached at the surface of the detector-organism, being prokaryotic or eukaryotic, in order to allow a correct exposure of the reporter gene product to the extracellular space. Advantageously, the inducible reporter system according to the invention allows on-line and immediate detection of the molecular response of the reporter protein without prior lysis or additional manipulation of the cell population.

Another element in the nucleic acid construct comprised in the battery of prokaryotic or mammalian cells of the invention is the presence of a targeting DNA sequence. Such targeting DNA sequence is intended to indicate a sequence encoding a polypeptide capable of targeting the polypeptide, encoded by the reporter gene, to a particular cellular location, for instance the host cell membrane. It will be clear from the present invention that the term "host cell membrane" may refer to different cell structures depending on the type of host cell applied according to the invention. In one example, this term refers to the inner membrane of Gram-positive bacteria or to the cell membrane of Gram-negative bacteria. In other examples, the term transmembrane amino acid sequence is used instead of targeting DNA sequence. The expression "transmembrane amino acid sequence" specifically relates to an amino acid sequence capable of transporting a polypeptide through the membrane of a host cell and to assure an efficient membrane anchoring and correct exposure of the polypeptide to the external surface of the host cell. DNA sequences encoding a transmembrane amino acid sequence are well known and have been identified in several prokaryotic organisms, including Gram-positive and Gram-negative bacteria.

In prokaryotic cells, targeting sequences are well known and have been identified in several membrane proteins and periplasmic proteins, including in the £. coli lipoprotein (Lpp). Generally, as is the case for Lpp, the protein domains serving as localization signals are relatively short. The E. coli Lpp targeting sequence includes the signal sequence and a part of the outer membrane protein amino acid sequence, in particular the first 9 amino acids of the mature protein. These amino acids are found at the amino terminus of Lpp. Other secreted proteins from which targeting sequences may be derived include TraT, OsmB, KlpB and BlaZ. Lipoprotein 1 from Pseudomonas aeruginosa or the PA1 and PCN proteins from Haemophilus influenza as well as the 17 kDa lipoprotein from Rickettsia rickettsij and the H.8 protein from Neisseria gonorrhea and the like may be used.

According to another embodiment, the transgenic pro- or eukaryotic cells of the present invention may comprise a nucleic acid as described above, comprising an artificial DNA sequence, being created between the promoter and the DNA sequence encoding the reporter gene. More preferably, the artificial DNA sequence precedes the targeting DNA sequence of the reporter gene. As used herein, the term "artificial DNA sequence" or "artificial gene" refers to a DNA sequence or gene that provides a defective transcription or translation of the reporter gene. Such reporter gene is preceded by a functional inducible promoter which provides a defective transcription or translation of said reporter gene. In particular the artificial sequence comprises a mutational hotspot sequence. As used herein, a "mutational hotspot sequence" refers to a gene sequence, which has certain DNA base sequences that are highly susceptible for mutation, induced by e.g. carcinogenic chemicals or compounds. By cloning specific mutational hotspot sequences downstream a promoter of a known toxicological relevant receptor, inducible by the corresponding toxic compound, and in front of a reporter gene, the induction of the reporter gene for surface display becomes dependent on the presence of the said toxic compound in the sample to be analyzed. The action of the toxic compound is needed to restore proper translation of the reporter gene cloned behind the hotspot sequence. Thus, the artificial sequence can reveal the presence or the absence of a specific toxic compound in a sample.

The mutational hotspot sequence may be susceptible to a stress-inducing compound inducing a point mutation, a frame shift mutation, a nucleotide transition, a nucleotide transversion.

The transgenic pro- or eukaryotic cells within the battery thus each comprise a nucleic acid construct each independently responding to a specific toxic compound, herewith generating a gene expression profile within the battery of cells for said toxic compound. The expressions "gene expression profile", "response profile", "toxic effect response" and "fingerprint" are used interchangeable herein, and are representative for the specific toxic compound and the battery used. Preferably, at least 10 different prokaryotic or eukaryotic constructs are comprised in the battery, more preferably, at least 20, 25, 30, 35, 40, 45, 50 or 100 different pro- or eukaryotic constructs are herein comprised.

According to the invention, at least two known hazardous or toxic compounds are exposed to the battery of pro- or eukaryotic cells and the gene expression profiles are stored in a database. The invention thus specifically relates to the database, comprising the gene expression profiles of known hazardous or toxic compounds. The invention further relates to the use of the database in the methods of the invention.

Examples of known hazardous and/or toxic compounds for which such a response file is generated and stored in the database, comprise but are not limited to PCB congeners; polybromo- and perfluorchemicals; mycotoxines such as aflotoxine, deoxynivalenol, zearalenon, fumosin and T-2 toxin; compounds which are hormonally active such as estradiol, nonylfenol ad atrazine.

According to one embodiment, the Gene Profiling set up of a mammalian battery of cells, for instance a CAT-Tox battery of cells, comprises different cell lines per row of an assay plate, each cell line comprising a different stress-inducible promoter. The sample to be analysed is added in increasing doses in the columns of the assay plate. For each dosage, three replica plates may be made. Two plates may be pooled after lysis of the cells for measuring CAT activity. One plate may be used for measuring cell viability/density. In another embodiment where a battery of prokaryotic cells are used and the reportergene is Lacz, the three plates may be analysed separately for beta-gal activity and cell viability/density. For each dose of the sample, a Fold Induction (FI) is calculated. For instance:

Fold induction = CAT/GAL activity at Dose x / CAT/GAL activity at Dose y.

As an example illustrating Gene Profiling results of a CAT-Tox battery of cells after exposure to 3-methyl cholantrene are represented in Figure 2 the Gene Profiling results of a Pro-Tox battery of cells after exposure to pentachlorophenol are represented in Figure 1. Other methods may be used to calculate the fold induction. For instance, kinetic measurement of the production of reporter gene product may be used. Here, the product may be calculated using Michaelis Menten and/or Eadie-Hofstee equations. Fold induction is again measured as the concentration of the product at dose x relative to the concentration of product at dosis 0. Example 3 provides the results of gene profiling of a mammalian battery of cells using these equations.

Further, bioinformatics will be used to identify the most optimal gene expression profile (fingerprint) for each of the given hazardous and/or toxic compounds. At this moment, more than 150 gene profiles of known toxic or hazardous compounds are available in an in-house database.

Once a gene expression profile is generated for an unknown hazardous and/or toxic compound, that profile is compared to profiles of known substances in the database. A substance having a similar promoter induction/suppression profile as the unknown compound is identified. From these results, the identity of the unknown compound may be inferred. Alternatively, the unknown compound may be classified to belong to a certain group of related chemicals.

In a preferred embodiment, the present invention relates to any of the methods herein described characterized in that the unknown compounds are identified qualitatively and/or quantitatively, either by direct or indirect detection of the assayable product displayed at the surface of the host cell.

According to the present invention, the DNA sequence encoding an assayable product is a reporter gene. This DNA sequence is positioned downstream from the DNA segment encoding the transmembrane sequence. The term "reporter gene" as used herein, refers to nucleic acid sequences encoding assayable proteins. The choice of reporter genes to be used is essentially limitless, as long as a DNA sequence encoding the assayable product has been characterized; and the product of the gene can be detected. Sufficient characterization includes knowledge of the entire coding sequence and availability of a cDNA molecule. For example, the assayable product is /?-galactosidase (encoded by the lacZ gene), chloramphenicol acetyl transferase (encoded by the cat gene), galactose kinase (encoded by the galK gene), /?-glucosidase (encoded by the gus gene), glutathione transferase or luciferase (encoded by the lux gene), or green fluorescent protein (encoded by the GFP gene). Most preferably, the GFP gene is employed, and even more preferably a mutated version of the GFP gene, GFPmut2 is used. In addition, it will be clear that any other gene encoding an assayable protein, including newly identified genes, may be used in accordance with the present invention.

In another embodiment, the invention relates to a method for the classification of the identified hazardous and/or toxic compound or mixtures thereof into toxicity classes. Different toxicity classes may be distinguished according to the present invention, including but not limited to classes comprising compounds or mixtures that a) cause no or negligible, b) low; c) moderate, d) high or e) very high hazards and/or pollution.

In another embodiment, the invention refers to a method for determining the condition of an environment comprising the steps of: a) taking a sample from said environment e.g. by introducing a sampler in said environment for a suitable incubation time, e.g. hours, days or weeks, in said environment, b) extracting said sample from said sampler, c) generating a gene expression profile for said extracted sample by submitting said sample to a battery of prokaryotic or eukaryotic cells each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product, and d) determining the condition of said environment by analyzing said generated gene expression profile.

The generated gene expression profile clearly indicates whether the environment is contaminated with hazardous or polluting compounds. The major advantage of the present method which uses a biological reporter system, compared to a chemical analysis of the environment, is the ease, accuracy and rapidity of the method. By simply analyzing the gene expression profile generated for a certain sample, conclusions can be drawn regarding the pollution level of the environment. Because a biological reporter system is used, it is not required to make suppositions a priori of the possible compounds which could be present in the environment. The battery of cells sensitively reacts whenever a compound is present in the environmental sample which is hazardous and for which the cells are sensitive.

In a further embodiment, the invention provides for the classification of the environment into a pollution category. Different pollution categories may be distinguished according to the present invention, including but not limited to categories of a) no or negligible, b) low; c) moderate, d) high or e) very high pollution level.

The present invention further relates to any of the methods herein described wherein said battery of prokaryotic or eukaryotic cells are provided with (a) specific combination(s) of (fluorogenic) reporter genes.

According to a further embodiment, the present invention also relates to a database comprising gene response profiles of known toxic compounds obtained by performing steps a) to d) of any of the methods described earlier.

The present invention also relates to the electronic carrier comprising a database of gene expression profiles generated by submitting the said hazardous and/or toxic compound to said battery of protox/cattox cells and recording the results.

The present invention also provides diagnostic kits for performing the methods according to the present invention.

A kit according to the invention comprises at least a battery of pro- or eukaryotic cells each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product and an electronic carrier comprising a database of gene expression profiles of hazardous and/or toxic compounds, each of which is generated by submitting the said hazardous and/or toxic compound to said battery of cells.

The methods and kits according to the invention are particularly suitable for the identification and evaluation of hazardous and/or toxic compounds in general, and of mutagens in particular, which may be present in the environment or during food processing. Several categories of hazardous and/or toxic compounds can be identified using the methods of the invention. Potential uses include monitoring of air, soil, water and food quality, manufacturing and fermentation process control, process monitoring and toxicity screening. These applications may benefit many industries including chemical, beverage, food and flavor, cosmetics, agricultural, environmental, regulatory and health care industries. In a preferred embodiment, the present invention relates to a method assay wherein the sample to be analyzed is selected from the group comprising an aqueous solution, water, soil, sediment, sludge, food, beverage or pesticides.

Thus, the present invention further relates to the use of a kit according to the invention in environmental protection, in food industry, in cosmetic industry, in pharmaceutical industry, in biotechnology and/or for detection of hazardous and/or toxic compounds in food processes.

Other advantages and applications of the present invention will become clear form the examples.

Although in some cases individual compounds may not be toxic, sometimes combinations of non-toxic compounds may be toxic. Therefore, it should be understood that the kits and methods of this invention can also be utilized to determine the potential toxicity of more or less complex combinations of known and unknown compounds in an identical manner to that described above.

In order that the invention described herein may be fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

EXAMPLES

Example 1. Selection of a battery of prokaryotic and eukaryotic constructs for

1.1 Origin of promoters used in prokarvotic constructs, e.g. Pro-Tox svstems:

GENE PRODUCTS INDUCING AGENTS katG Hydrogen peroxidase Oxidative stress zwf Glucose-6-P dehydrogenase Oxidative stress soi28 Superoxide inducible Oxidative stress osmY Periplasmic protein Osmotic stress micF Regulation membrane porins Osmotic changes clpB Heat shock chaperonin Protein damage merR Mercury resistance Metals, efc. ada DNA methyl transferase Methylated DNA dinD SOS response enzyme DNA damage nfo Endonuclease IV Abasic DNA sites uspA Universal stress protein DNA damage recA SOS response enzyme DNA damage umuDC Targeted mutagenesis DNA damage sos promoter SOS response DNA damage

1.2 Origin of promoters used in eukaryotic constructs. e.g. CAT-Tox svstems:

GENE PRODUCTS INDUCING AGENTS p53RE p53 response element DNA damage GADD153growth arrest & DNA damage DNA damage, Not to X-ray or phorbol esters GADD 45 growth arrest & DNA damage DNA damage and growth arrest NFKBRE NFKB response element Oxidative damage, inflammation, mitogens, thiols HSP70 heat shock protein 70 kD Denaturants, temperature XRE xenobiotic response element Aromatic Hydrocarbons CYPIAI cytochrome P450 IAI Aromatic Hydrocarbons, dioxins Aromatic Hydrocarbons, phenolic antioxidants,

GSTYa glutathione S transferase electrophiles HMTIIA Metallothioneine IIA Heavy metals, glucocorticoids GRP78 Glucose regulated protein Protein misfolding, RARE Retinoic acid response element Retinoids

CRE C-AMP response element Increased c-AMP level

XHF Collagenase Mitogens, inflamation

FOS c-fos proto-oncogene Tumor induction, oxidative stress, DNA damage

Example 2. Expression profile of the pro- or eukaryotic batteries of cells in response of chemical compounds.

2.1. Expression profile of the prokarvotic battery of cells described in Example 1 in response to toxic compounds

A known toxic compound or a compound to be tested were added to the wells of a plate, wherein each row comprises cells with the same procaryotic construct. In each assay at least 10, 15 or 20 procaryotic constructs were present. In the columns, different concentrations of the compound were added, ranging for instance from 0.2 to 25000 μg/ml. For each of the tested combinations of cells/compound concentration, a "fold induction" was obtained. The fold induction was measured as the activity of the reporter gene product at dose x μg/ml, relative to the reporter gene product at dose 0 μg/ml. The fold inductions are represented in a three dimensional expression profile. Figure 1 represents the Fold induction profile in response to pentachlorophenol.

2.2. Expression profile of the eukaryotic battery of cells described in Example 1 in response to toxic compounds.

A similar set-up was used as described under 2.1 , except in that eukaryotic cells and constructs were used. For instance, in a mammalian construct comprising the CAT reporter gene, cat activity can be measured through a ¹⁴C based assay ¹⁴C-butyryl-coA + cam → (mono or di) ¹⁴C-butyryl-com + coA A Fold Induction profile for 3-Methyl cholantrene is represented in Figure 2.

Example 3: Extraction of bio-available fractions from a sample

In this experiment, an Empore 47 mm disk comprising a C18 membrane was used, preconditioned with MeCI₂ and methanol. 5 ml methanol was added to 500 ml sample and elution was performed with MeCI₂. The residuent on the membrane dried, taken up in 3 ml DMSO and further diluted. By this method, a concentration of 8.3 x can be reached for use as highest concentration towards the cells in the battery.

Figure 3a shows the dose response profiles, ie fold induction, of the C18 fraction obtained from a sample of a surface water in Belgium. Here, a battery of prokaryotic cells were used. In Figure 3b, the viability of the cells is plotted against the fold induction.

Figure 4 shows the gene profiles of the water C18 fractions from a typically polluted sample, but against a battery of mammalian cells. The results are represented in terms of kinetic measurements. The CYP1A1 response does not give a clear indication of toxicity. The gene response profile of Fos shows clear dose-response curves indicating the presence of a toxic compound that induces the Fos promoter. The gene profile of the GADD45 promoter shows that only a 1/16 dilution results in product concentration above the blancs. This clearly suggests high cytotoxicity of the sample: higher doses of the sample cause cell death.

In Table 1 an overview is given of the fold-inductions (FI) of CAT-Tox gene profiles of C18 fractions of surface waters in Belgium. A preliminary classification was made, based on Total FI. FI between 1 and 16 received a quotation 1 ; FI between 17 and 32 received a quotation 2; FI between 33 and 64 received a quotation 3 and a Total FI of more than 64 received a quotation 4.

The classification according to the above-described system (Global Triad) is compared with the calculation of conventional Triad experiments, which also classify polluted surface waters according to a 1 to 4 quotation. Surprisingly, the results obtained by the methods of the invention are quite similar to the results obtained when using conventional methods. However, it should be noted that in these conventional Triads, the methods used for evaluating the toxicity of surface waters take at least 28 days. During that period, a filter is placed in the surface water and after removal of the filter the compounds bound thereon are evaluated by conventional chemical methods. Since in the methods of the present invention, the taking of a sample only takes a short time and the subsequent retrieval of results from the battery of cells takes no more than a few days, it was very surprising that in such a short period, a measure for the presence of toxicity could be obtained. Moreover, since the samples were only 10 times (8.3 x to be more exact) concentrated, these results are indicative for a very sensitive method. Furthermore using the methods and tools of the present invention, similar results were obtained as with conventional methods, indicating that the methods of the present invention are reliable.

Moreover, using the methods of the present invention, also a more differentiated response could be obtained. For instance the results from Table 1 indicate: - High-High cytotoxicity as indicated by onset of GADD genes - No oxidative stress - No PAH (in conjunction with oxidative stress) - No cell proliferation - Main effect: protein perturbation.

Table 1

Example 4: TIE-pretreatment of mixtures of samples Possible treatments in TIE phase I are (i) Solid Phase Extraction. A C18 column was used to evaluate samples for constituents resembling nonpolar organics (Mount and Anderson-Camahan 1989). After filtration, a 1 L sample was pumped through a prepared C18 column and three solutions (50% MeOH, 100% MeOH, 50% MeOH-50% MeCI2) were added sequentially to elute nonpolar constituents. Contaminants were removed based on their hydrophobicity (least to most). The solid phase elutriate solutions were added to the column in 2 mL aliquots. Post-column elutriates were diluted to 0.1 % and 0.2% v/v with seawater to adjust nonpolar organic concentrations to 50% and 100% runoff. Elutriate blanks (solvent passed through a clean C18 column) were tested to control for solvent toxicity. Filtered pre-C18 samples, post-C18 samples, and column blanks were included in the testing at concentrations of 12, 25, and 56%.

(ii) EDTA Additions

To evaluate samples for constituents resembling divalent metals, EDTA was added to the samples at 3 and 8 mg/L (Norberg-King et al. 1992). Samples were tested at concentrations of 12%, 25%, and 56%; EDTA controls were included.

(iii) Thiosulfate Additions

To evaluate samples for constituents resembling oxidants, 10 and 25 mg/L of sodium thiosulfate was added to the samples (Norberg-King et al. 1992). Samples were tested at concentrations of 12%, 25%, and 56%; thiosulfate controls were included.

Claims

1. Method for identifying a hazardous and/or toxic compound in a sample comprising the steps of: a) providing a battery of prokaryotic or eukaryotic cells each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product, b) determining the gene expression profile of a known toxic compound by submitting said compound to said battery of cells, c) introducing the gene expression profile of said battery of cells, obtained in step b) in a database, d) repeating steps b) and c) for at least one additional known hazardous and/or toxic compound, e) determining the gene expression profile of the unknown toxic compound in said sample by submitting said sample to said battery of cells, and f) comparing the gene expression profile obtained in step e) with the response profiles recorded in the database of step c) thereby identifying said unknown compound in said sample.

2. A method according to claim 1 , wherein said sample comprises a mixture of hazardous and/or toxic compounds characterized in that said method comprises steps g) to i) instead of steps e) and f): g) fractionating said mixture of hazardous and/or toxic compounds thereby obtaining a number of fractions, h) determining the gene expression profile of each of the obtained fractions by submitting each of said fractions to said battery of cells, and i) comparing the gene expression profiles obtained in step h) with the response profiles recorded in the database of step c) thereby identifying said unknown compound.

3. Method according to claim 2, wherein said mixture of hazardous and/or toxic compounds is fractionated by using a toxicity identification evaluation (TIE) methodology.

4. Method according to any of claims 1 to 3, wherein the unknown compounds are identified quantitatively and qualitatively.

5. Method according to any of claims 1 to 4, further comprising the step of classifying the identified hazardous and/or toxic compound into a toxicity class.

6. Method for determining the condition of an environment comprising the steps of: a) extracting a sample from said environment, b) generating a gene expression profile for said extracted sample by submitting said sample to a battery of prokaryotic or eukaryotic cells each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product, and c) determining the condition of said environment by analyzing said generated gene expression profile.

7. Method according to claim 6, further comprising the step of classifying said environment into a pollution category.

8. A method according to claim 6 or 7, further comprising the step of identifying a hazardous and/or toxic compound in said environmental sample.

9. Method according to claim 8, wherein said hazardous and/or toxic compound in said environmental sample is identified by applying a method according to any of claims 1 to 5.

10. Method according to any of claims 1 to 9, wherein said battery of cells comprises a specific combination of (fluorogenic) reporter genes.

11. Database comprising gene response profiles of known toxic compounds obtained by performing steps a) to d) of the method of claim 1 or 2.

12. Database comprising gene response profiles of environmental samples obtained by performing steps a) to b) of the method of claim 6.

13. Electronic carrier comprising a database according to claim 11 or 12.

14. Kit for identifying unknown hazardous and/or toxic compounds in a sample comprising a battery of prokaryotic or eukaryotic cells, each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product, and an electronic carrier of claim 13.

15. Kit for determining the condition of an environment comprising a battery of prokaryotic or eukaryotic cells, each comprising a nucleic acid comprising the promoter sequence of a known toxicological relevant receptor and a reporter gene which produces a measurable or assayable product, and an electronic carrier of claim 13.

16. Use of a kit according to claim 14 or 15 in environmental protection, for instance for detecting the quality of surface water, the quality of water in an purification plant or the quality of exhaust air in an incineration plant.

17. Use of a kit according to claim 14 or 15 in food industry.

18. Use of a kit according to claim 14 or 15 for detection of hazardous and/or toxic compounds in food processes.

19. Use of a kit according to claim 14 or 15 in cosmetic industry.

20. Use of a kit according to claim 14 or 15 in pharmaceutical industry.