EP1153137A2 - Test toxicologique a haut rendement utilisant des organismes et cellules de culture - Google Patents

Test toxicologique a haut rendement utilisant des organismes et cellules de culture

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Publication number
EP1153137A2
EP1153137A2 EP00908601A EP00908601A EP1153137A2 EP 1153137 A2 EP1153137 A2 EP 1153137A2 EP 00908601 A EP00908601 A EP 00908601A EP 00908601 A EP00908601 A EP 00908601A EP 1153137 A2 EP1153137 A2 EP 1153137A2
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EP
European Patent Office
Prior art keywords
cell
sample
mutant
yeast
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00908601A
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German (de)
English (en)
Inventor
Spencer B. Farr
Bryan A. Shiloff
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Phase-1 Molecular Toxicology Inc
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Phase-1 Molecular Toxicology Inc
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Publication of EP1153137A2 publication Critical patent/EP1153137A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity

Definitions

  • the present invention provides methods and diagnostic kits for identifying and characterizing toxic compounds. These methods and diagnostic kits measure reduced growth, as revealed by reduced relative viability of mutant as compared to wildtype, in samples of cultured organisms or cells exposed to the test compounds.
  • testing could be for a newly developed chemical or drug, as mandated by the Environmental Protection Agency ("EPA") or Food and Drug Administration (“FDA”).
  • EPA Environmental Protection Agency
  • FDA Food and Drug Administration
  • an environmental sample could be suspected of containing a toxin, and such testing could establish its presence.
  • testing methods have focused on bioassay procedures.
  • a wide variety of living things have been used in such assays, ranging from single cells to multicellular, complex organisms such as plants and animals. The large majority of toxicity testing, particularly that required by the government, involves toxicity tests in animals.
  • Toxicity testing in animals is expensive, time consuming, is relatively inaccurate, and can result in animal suffering.
  • a two year toxicity test performed in rats was estimated to cost $800,000 in 1991. Louis J. Casarett et al., Casarett and Doull's Toxicology 37 (4th ed. 1991).
  • the time required for animal toxicity tests varies with the species, short term tests run 3 months to 2 years and long term tests last two to seven years. Even after spending that amount of money and investing that amount of time, the large number of variables present in animal testing, such as their diet, has been shown to effect the accuracy of the findings.
  • animal rights activists have made the general public aware of the issue of animal suffering during these tests, causing companies to seek out alternative testing methods. In short, animal testing has proven to be a very inefficient method of determining whether a sample is toxic, and there is an urgent need for a quick, inexpensive and reliable means of determining toxicity.
  • the prior art describes several different types of short term bioassays, usually detecting mutagenicity or carcinogenicity.
  • the Ames assay and U.S. Patent No. 4,256,832, issued to Findl et al. are two examples of assays which detect reversion of a mutant organism to wildtype. As such reversion is generally the result of DNA mutation, positive results indicate an effect at the DNA level.
  • U.S. Patent No. 4,997,757, issued to Schiestl describes a yeast bioassay which measures the ability of the sample to induce genome arrangement. Again, this bioassay looks only at effects at the DNA level and may well miss non-mutagenic or non-carcinogenic toxins.
  • the present invention are methods and kits for measuring mutant hypersensitivity assay using high throughput screening methodology to evaluate the mechanisms of toxicity of chemicals.
  • the assay is performed in multi-well plates, such as those having 96 wells, making the process conducive to testing many compounds in a short period of time.
  • the assay is versatile in that it can test compounds for ability to cause, for example, DNA damage, ability to mutate genetic material (mutagenicity), the ability to cause cancer (carcenogenicity), cause protein or membrane damage, energy depletion, mitochondrial damage, as well as the more general genotoxicity.
  • toxicity as used in this disclosure, is intended to encompass all of these types of effects.
  • the assay can detect oxidative stress, protein damage, cell cycle disruption, energy charge and depletion, microtubule disruption or onset of metabolic competency through overexpression of human gene inserts encoding metabolism genes or incorporation of S9 fraction.
  • wildtype (wt) yeast and respective mutants are dosed with the desired chemical and yeast growth is determined using turbidimetry.
  • Dose response curves are generated and mutant sensitivity to the compound relative to its parent (relative sensitivity) calculated. Relative sensitivities which are statistically significant indicate a hypersensitivity of the mutant to the test compound.
  • the assay therefore provides an inexpensive, reliable, short term toxicity test which is an excellent alternative to animal testing and which provides valuable information about the mechanism of action of a compound.
  • the present invention has applications to the pharmaceutical industry, environmental testing and clinical studies.
  • Fig. 1 A reports the undosed/dosed turbidity of wildtype (parental) yeast strains and mutant yeast strains in the presence varying concentrations of acetylsalicyclic acid (aspirin).
  • Fig. 1 B shows the relative sensitivity of the wildtype (parental) yeast strains to the mutant yeast strains, as calculated from the data of Fig. 1A. None of the sensitivity data fields are shaded, indicating all results are statistically insignificant.
  • Fig. 2A reports the undosed/dosed turbidity of wildtype (parental) yeast strains and mutant yeast strains in the presence varying concentrations of cimetidine (Tagamet ® ).
  • Fig. 2B shows the relative sensitivity of the wildtype (parental) yeast strains to the mutant yeast strains, as calculated from the data of Fig. 2A. None of the sensitivity data fields are shaded, indicating all results are statistically insignificant.
  • Fig. 3A reports the undosed/dosed turbidity of wildtype (parental) yeast strains and mutant yeast strains in the presence varying concentrations of oiigomycin (antibiotic).
  • Fig. 3B shows the relative sensitivity of the wildtype (parental) yeast strains to the mutant yeast strains, as calculated from the data of Fig. 3A. None of the sensitivity data fields are shaded, indicating all results are statistically insignificant.
  • Fig. 4A reports the undosed/dosed turbidity of wildtype (parental) yeast strains and mutant yeast strains in the presence varying concentrations of actinomycin D (a DNA binder).
  • Fig. 4B shows the relative sensitivity of the wildtype (parental) yeast strains to the mutant yeast strains, as calculated from the data of Fig. 4A.
  • the shaded data fields in Fig. 4B indicate statistically significant relative sensitivities.
  • Fig. 5A reports the undosed/dosed turbidity of wildtype (parental) yeast strains and mutant yeast strains in the presence varying concentrations of carboplatin (an antineoplastic alkylating agent).
  • Fig. 5B shows the relative sensitivity of the wildtype (parental) yeast strains to the mutant yeast strains, as calculated from the data of Fig. 5A.
  • the shaded data fields in Fig. 5B indicate statistically significant relative sensitivities.
  • Fig. 6A reports the undosed/dosed turbidity of wildtype (parental) yeast strains and mutant yeast strains in the presence varying concentrations of EMS
  • Fig. 6B shows the relative sensitivity of the wildtype (parental) yeast strains to the mutant yeast strains, as calculated from the data of Fig. 6A.
  • the shaded data fields in Fig. 6B indicate statistically significant relative sensitivities.
  • Fig. 7A reports the undosed/dosed turbidity of wildtype (parental) yeast strains and mutant yeast strains in the presence varying concentrations of 5-FU (5- fluorouracil, an antimetabolite).
  • Fig. 7B shows the relative sensitivity of the wildtype (parental) yeast strains to the mutant yeast strains, as calculated from the data of Fig. 7A.
  • the shaded data fields in Fig. 7B indicate statistically significant relative sensitivities.
  • Fig. 8A reports the undosed/dosed turbidity of wildtype (parental) yeast strains and mutant yeast strains in the presence varying concentrations of MMS (methyl methane sulfonate, an alkylating agent).
  • Fig. 8B shows the relative sensitivity of the wildtype (parental) yeast strains to the mutant yeast strains, as calculated from the data of Fig. 8A. The shaded data fields in Fig. 8B indicate statistically significant relative sensitivities.
  • Fig. 9A reports the undosed/dosed turbidity of wildtype (parental) yeast strains and mutant yeast strains in the presence varying concentrations of PMA (phorbol 12-acetate-13-myristate, a carcinogen).
  • Fig. 9B shows the relative sensitivity of the wildtype (parental) yeast strains to the mutant yeast strains, as calculated from the data of Fig. 9A. The shaded data fields in Fig. 9B indicate statistically significant relative sensitivities.
  • Fig. 10 is a flow chart documenting a preferred embodiment of the assay of the present invention.
  • kits and methods of this invention utilize mutant organisms or cells where the mutation results in an alteration in at least one of multiple gene function categories.
  • Organisms which can be used in the present method, include any prokaryotic and eukaryotic unicellular organism such as the bacteria, E. coli, or the yeast, Saccharomyces cerevisiae. Use of cultured cell lines, particularly from mammalian species, is also contemplated for the present assay.
  • the mutations present in the organisms or cell lines can be of any type, including lack of function or overexpression, as long as the ultimate phenotypic result is an unwanted adverse and hypersensitive effect which is toxicologically relevant.
  • Some gene function categories contemplated for the mutants for use in the kits and methods of the present invention include mutations which sensitize cells to, cause cells to exhibit, or disrupt a cell's function in acute phase stress, cell adhesion, AH-response, anti-apoptosis and apoptosis, antimetabolism, anti- proliferation, arachidonic acid release, ATP depletion, cell cycle disruption, cell matrix disruption, cell migration, cell proliferation, cell regeneration, cell-cell communication, cholestasis, differentiation, DNA damage, DNA replication, early response genes, endoplasmic reticulum stress, estogenicity, fatty liver, fibrosis, general cell stress, glucose deprivation, growth arrest, heat shock, hepatotoxicity, hypercholesterolemia, hypoxia, immunotox, inflammation, invasion, ion transport, liver regeneration, cell migration, mitochondrial function, mitogenesis, multidrug resistance, nephrotoxicity, oxidative stress, peroxisome damage, recombination, ribotoxic
  • Appendix A yeast
  • Appendix B mimmalian
  • Mutations affecting each of these gene function categories have been isolated and the gene or gene-associated sequence has been isolated and sequenced. Many mutants are available commercially, or can be easily constructed using standard genetic methods well known to one of ordinary skill, such as those described in Molecular Cloning — a Laboratory Manual, J. Sambrook et al., eds. (2d ed. 1989) and/or Molecular Biomethods Handbook, Ralph Rapley and John Walker, eds. (1998).
  • mutants for use in the assay of the present invention at any species level - bacterial, yeast or mammalian - is well within the purview of one of ordinary skill in this art.
  • many samples to be tested for cytotoxicity can be added directly to cells, the true form of the agent in the body is sometimes better represented if the sample if pre-treated with S9 fraction obtained from liver, or microsomes obtained from endoplasmic reticulum. See generally EM Gillam, Human cytochrome P450 enzymes expressed in bacteria: reagents to probe molecular interactions in toxicology, 25 Clin. Exp. Pharmacol.
  • the S9 fraction and microsomes are available commercially from Xenotech (Kansas City, MS) and In vitro Technologies (IVT, Baltimore, MD). Incubation of the fraction or microsomes with the sample is done according to the protocol included with the commercial product. This process allows the sample to be in the form it would be in after metabolism in the organism, thus it is in the form that needs to be tested for an effect on the organism. If the metabolic activation step is used, only the resulting metabolites are actually tested in the assay, with non-treated sample included as a control. Pre-treatment with microsomes from various sources, such as human liver or yeast is contemplated.
  • Reduced growth by the mutant strain when dosed by the test sample indicates a potential toxic effect as well as the mechanism of action of the sample on living organisms.
  • the method of measuring the growth of the organism depends on the particular one used. Bacteria and yeast growth can be followed using media turbidity measurements. Growth of cultured cell lines can be measured using radiolabelled compounds such as carbon 14 or 3H-thymidine incorporation or analysis using fluorescent dyes or colometric methods.
  • MTT or XTT assay measuring the reduction of a tetrazolium salt, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltertazolum bromide, to a formazan product during growth
  • calcein AM Trypan blue
  • neutral red uptake or colony formation method calcein AM
  • the first step of the present assay involves the selection of the mutant to be used in the assay. This decision is made based on the type of toxicity which is to be tested. For example, if a substance is to be tested for mutagenicity, then a mutation in a gene or genes encoding DNA damage or repair would be the most likely candidate. Thus, if no hypersensitivity to the substance is seen by the mutant through a reduced growth rate, it is likely that the substance does not have a mutagenic effect. If hypersensitivity of multiple DNA-related mutants is seen, it is likely that the substance does have a mutagenic effect, particularly if the hypersensitivity is not seen in mutations of other gene function catagories.
  • Yeast is an ideal organism for the present assay system because of its ease of cultivation, ease of generating gene knock-out mutants and extensive publicly available information about mutants.
  • a yeast mutation hypersensitivity ("YMH") assay of the present invention could utilize one or more of literally hundreds of yeast mutations.
  • Gene function categories of particular interest include, for example, cell rescue, defense, cell death and aging, cell growth, cell division, and DNA synthesis, heat shock proteins, mitochondrial, peroxisomal, DNA- associated, immunosuppressent, cyclins and cell cycle control proteins, ATP- binding cassette proteins, cytoskeletal, metabolism and apoptosis.
  • Appendix A of the present specification lists particular genes of interest in these and a "other relevant mutants" category.
  • Rad 6 mutants are defective in repair of damaged DNA, mutagenesis and sporulation.
  • the Rad 6 gene encodes a ubiquitin conjugating enzyme (E2) that is necessary for post-replication repair of damaged DNA and sporulation.
  • Rad 6 exhibits an increase in transcription in response to DNA damage and during meiosis.
  • Rad 6 encodes a protein of 172 amino acids which is a homologue of the mammalian ubiquitin carrier protein. Rad 6 mRNA levels have been shown to increase during sporulation and after exposure to UV light. However, no effect on transcription was observed after heat shock or starvation. These results suggest that Rad 6 is involved in genetic recombination repair rather than in the heat shock or stress response.
  • a mammalian homologue of the Rad 6 gene is known - HHR6B - and cells lines mutant in this gene are available from commercial sources, such as the American Tissue Culture Collection (ATCC, Bethesda, Md.)
  • the Rad 18 gene encodes a 66 kDa zinc finger protein that contains a nucleotide binding motif and the protein is required for repair of DNA damage and mutagenesis. Like Rad 6, Rad 18 is also involved in post-replication repair and similarly, an increase in transcription is observed as a response to DNA damages. Rad 6 has been shown to form a heterodimeric complex with Rad 18 that has ubiquitin conjugating activity. The Rad 6-Rad 18 complex has been demonstrated to contain ATP hydrolytic activity in addition to binding single stranded DNA (ssDNA). Furthermore, Rad 6 alone has no binding affinity towards ssDNA but it is not essential that Rad 18 form a complex with Rad 6 in order for Rad 18 to bind ss DNA. Rad 18 might possibly target damaged sites on the DNA followed by Rad 6- directed ubiquitin mediated proteolysis.
  • Rad 51 is a structural and functional homologue of the bacterial strand exchange protein RecA.
  • the Rad 51 gene product forms a complex with replication protein A (RPA) and ssDNA, and the protein is required for recombination and for x- ray damage repair.
  • double stranded break (DSB) repair is dependent upon the Rad 51 gene.
  • a mammalian equivalent of this gene is also known, HHR51.
  • the Rad 52 gene is necessary for mitotic recombination and repair of x-ray damage and DNA DSB.
  • Rad 52 appears to bind Rad 51 to form a heterodimeric complex, which has an increased affinity toward ssDNA.
  • Rad 52 protein enhances DNA strand exchange by causing Rad 51 protein to form a complex with RPA and ss DNA.
  • formation of the Rad 51-RPA-ssDNA complex is an important intermediate in the recombination mechanism.
  • a mammalian equivalent of this gene has been identified - HHR52.
  • Positive test compounds were selected by category of DNA damage. Some compounds tested thus far include antimetabolites such as hydroxyurea, methotrexate, and 5-fluorouracil. Alkylating agents such as cyclophosphamide, mitomycin C and ethyl methane sulfonate have also been tested. Also, examples of topoisomerase inhibitors, which include etopodide and camptothecin have been tested. Other compounds such as the DNA binding compound Actinomycin D have been use in the assay. In addition innocuous compounds which should show no
  • DNA damage effects are screened as negative controls.
  • Well characterized compounds provide a test set of chemicals that can be compared against unknowns.
  • Control sets are introduced in the assay for verification and validation.
  • the data can be analyzed using several methods.
  • One method looks at the IC 50 values for the parent and respective mutant strains.
  • the IC 50 value may not be the best measure of mutant hypersensitivity especially when greater sensitivity is observed at concentrations other than IC 50.
  • Mutant hypersensitivity can be observed throughout the dose response curve or conversely, mutant hypersensitivity might be seen in a narrow range of dosing concentrations. Therefore, plots of differential sensitivity (proliferation of mutant/proliferation of wt) or relative sensitivity (1 -differential sensitivity) are constructed to evaluate the genotoxic effects of compounds. With the assay of the present invention it may be possible to determine the mechanism of DNA damage that occurs which can be largely dependent upon the mutant(s) that display hypersensitivity.
  • Rad 6 and Rad 18 may be hypersensitive to a compound whereas Rad 51 and Rad 52 may show no effect. That would indicate toxicity related to recombination repair as opposed to post- replication repair. Therefore, insight into the molecular mechanism of toxicity can be gained for target compounds.
  • the discussion above of the YMH assay can be summarized by the flow chart of Figure 10.
  • the yeast strain of interest is selected where both the desired mutant and the parental strain is obtained. Cultures of both the wildtype and mutant are grown, and then dosed with the test substance. The dosed yeast, both mutant and wildtype are separately incubated in a 96 well plate. The plate is read for optical density and this data is used to detect any differences in growth in response to exposure to the test substance. The data is analyzed using various standard toxicity values, plotting methods, and comparisons. Finally, through particular selection of mutants and differential results in various gene function categories, the assay could provide information as to the mechanism of toxicity of the test substance.
  • a final embodiment of the present invention is a diagnostic kit which includes a battery of mutant organisms, and the parental strain for those mutants, where the mutants are selected to determine at what subcellular function the sample compound has its effect.
  • An example of such a kit would include the Rad 6, Rad 18, Rad 51 , and Rad 52 mutants and the parental strains. This kit would be useful in determining whether sample, suspected of having a DNA associated toxic effect, works through a double strand breaks or disruption of DNA replication.
  • YM-1 stocks were prepared by innoculating a 15 ml sterile culture tubes containing 2.5 ml of YM-1 media. The tubes were incubated for about 18hrs in a shaking water bath set at 30° C and at least 137rpm. 2.5 ml of C media was placed in a 15 ml culture tube and this tube was innoculated with 100 ⁇ l of the YM-1 stocks prepared above. These tubes were incubated for about 4 hr. in a shaking water bath set at 30° C and at least 137 rpm. While these tubes were incubating, a dosing plate which contains the compound solution and dilutions was prepared.
  • a dosing plate containing a serial dilution profile for various compounds that will be used to dose each yeast strain.
  • This can be accomplished by constructing a masterplate that contains enough solution in each well such that multiple secondary plates can be produced by transferring 30 ⁇ l of each well in the master plate to corresponding wells in each of the secondary plates. When finished, all secondary plates will be identical, containing 30 ⁇ l per well of the desired compounds and concentrations.
  • Stock dilutions should be made by solubilizing the compounds in water, DMSO, ethanol, or other appropriate liquid.
  • DMSO or ethanol concentrations should not exceed about 0.1% including cells.
  • C media as diluent.
  • Compounds solubilized in DMSO or ethanol should be diluted using C media containing 0.1% DMSO or ethanol.
  • DMSO and ethanol control wells should contain a final concentration of 0.1% of the appropriate solvent.
  • Up to 14 compounds can be tested at one time if the assay is done in a 96 well plate. A plate of this size can accommodate six different dosing concentrations with no replicates. In general, a maximum of seven compounds are screened against six yeast strains at six different compound concentrations performed in duplicate.
  • the 96 well microplates that are utilized for the assay are usually prepared with a single yeast strain per plate. Conversely, one compound can be tested against multiple strains on each plate.
  • the data is now ready for analysis. First, make any necessary background corrections to the data. Corrections can be made by subtracting the media blank absorbance from all absorbance values prior to generating dose response curves or calculating differential mutant sensitivity. From the spectophotometric data, calculate the ratio of absorbance (dosed/undosed) for mutant and wild type strains at each compound dilution.
  • Dosed/Undosed Abs @ 600nm for dosed yeast/Abs @ 600 nm for undosed yeast.
  • the relative sensitivity is a measure of the effect of a compound on the mutant relative to the parent strain. Dose response curves can be compared between wild type and mutant strains.
  • yeast nitrogen base 1.5 g NaOH 1.25 g ammonium sulfate 5 g dextrose
  • a cell line having a desired mutation is selected for use in the assay.
  • the genes listed in Appendix B have been identified as appropriate for the present invention.
  • cell lines deficient in genes homologous to those preferred for the yeast assay, as listed in Appendix A are also preferred for the mammalian embodiment of the present invention.
  • the cell line is grown in appropriate media, such as DME + 10% calf serum (Dulbecco's Modification of Eagle's Basal Medium, Irvine Scientific, Santa Ana, CA), with appropriate characteristics for maintenance of the mutant's growth, if necessary.
  • DME + 10% calf serum Dulbecco's Modification of Eagle's Basal Medium, Irvine Scientific, Santa Ana, CA
  • the cells to be used must be made quiescent, that is, synchronized in growth stage at a low growth rate.
  • the 3H-thymidine remaining unincorporated is extracted by aspirating the media, a careful wash with 1 ml ice cold PBS (phosphate buffered physiological saline solution), which is aspirated from the cells. ! ml of ice cold 5% trichloroacetic acid (TCA) is added to each well, and left at 4°C for 30 minutes. The TCA is aspirated off and the remaining cells are washed one time with PBS. At room temperature, each well receives 0.5 ml of 0.5N NAOH/0.5% SDS. This is mixed by pipetting up and down, then placed in the scintillation vials for counting of the incorporated radioactivity.
  • TCA trichloroacetic acid
  • a bacterial strain is selected that has a mutation in a gene function of interest.
  • genes having homologous functions as those listed as preferred for the yeast assay in Appendix A and the human cell assay listed in Appendix B are also preferred for the bacterial assay. Streak out the selected E. coli mutant and control parental strain on a solid agar plate, containing the appropriate selection agents to maintain the mutation. Incubate overnight at 37° C (or lower if mutant is temperature sensitive). Grow small cultures (5ml) from 5-10 isolated colonies in LB media (Luria-Bertani Medium). Incubate overnight at 37° C. Inoculate 5 ml of LB with 50 ⁇ l of each overnight culture. Incubate for 2 hours with
  • aeration 2.5 ml of bacteria/LB culture prepared above should be combined with an additional 22.5 ml of LB media, for a total volume of 25 ml.
  • 270 ⁇ l of this diluted bacteria solution prepared above is added to each well of a 96 well secondary dosing plate.
  • Each diluted bacterial strain (25 ⁇ l) will be used for a single secondary dosing plate.
  • the secondary dosing plate is produced as discussed above for the yeast assay of Example 1. Final volume in the well will be 300 ⁇ l. It is important that parental and mutant strains have undosed control wells.
  • a control well containing media and compound without bacteria should also be prepared for background correction. The plates are incubated for 18-24 hr. at 37° C.
  • NAM8 REC107, REC102, REC114, MER1, RIM101, NDJ1, CDC54, CP R7,
  • ORC5 ORC4, ORC3, RRR1, SIC1, BUD3, PWP2, STE3, STE2, OPY2, S
  • GSD2 GSD2, GUT2, HEM1, HEM15, HSP10, HSP60, HSP78, HTS1, IDH1, ID
  • MEF2 MET13, MGE1, MGM101, MIP1, MIR1, MIS1, MMM1, MMT1, M
  • MRPL2 MRPL2, MRPL20, MRPL23, MRPL24, MRPL25, MRPL27, MRPL28, MRP
  • PET111 PET112
  • PET117 PET122, PET123, PET127, PET130, PET191
  • TIM44 TIM54, TOM20, TOM22, TOM37, TOM40, TOM6, TOM7, TOM
  • TOM72 TRM1, TUF1, UNG1, VAR1, YAH1, YAL011W, YAT1, YBL013 w,
  • OS2 DOT6, DPB11 , DPB2, DPB3, DST1 , ECM22, ENS2, EST1 , EZL1 , FCP1 ,
  • NHP6B NOT3, NUC2, OAF1, OPI1, ORC1, ORC2, ORC3, ORC4, ORC5, ORC6, PAF1, PCH1, PCH2, PDR1, PDR3, PGD1, PHD1, PHO2, PHO4,
  • RFC4 RFC5, RGM1, RGT1, RIF1, RIF2, RIM1, RIM101, RLF2, RLM1, R
  • RPB8 RPB9, RPC10, RPC19, RPC25, RPC31, RPC34, RPC40, RPC53, RPC82, RPD3, RPO21, RPO31,
  • RPO41 RRN10, RRN11, RRN3, RRN5, RRN6, RRN7, RRN9, RSC4, RSC
  • RSC8 RTG1, RTG3, SAS5, SEF1, SET1, SFH1, SFL1, SGS1, SIG1, SIN
  • TFC5 TFG1, TFG2, THI2, TOA1, TOA2, TOP1, TOP2, TOP3, TRF4, TS
  • CTK2 PCL1, PCL10, PCL2, PCL5, PCL6, PCL7, PCL8, PCL9, PHO80, S
  • A2 SMY1, SMY2, SPA2, SPH1, SPR28, SPR3, SRV2, TCP1, TPM1, TPM2, TUB1, TUB2, TUB3, VPS16, VRP1
  • DNA repair protein (XRCC1), DNA repair protein XP-D, DNA replication factor C (36kDa), DNA topoisomerase I, DNA topoisomerase II, Dopamine beta-hydroxylase, DRA, Dynein light chain 1 , E2F, Early growth regulated protein 1 , E-Cadherin, ECE-1 (endothelin converting enzyme), Endothelin- 1 , Enolase alpha, Enoyl CoA hydratase, Eotaxin, Epidermal growth factor, Epoxide hydrolase, ERA-B, ERCC 1 (excision repair protein), ERCC 3 (DNA repair helicase II), ERCC 5 (excision repair protein), ERCC 6 (excision repair protein), ERK1 , Erythropoietin, Erythropoietin receptor, E- Selectin, Estrogen receptor, Farnesol receptor, Fas antigen, Fas associated death domain (FADD), Fas ligand, Fas/
  • Peroxisomal acyl-CoA oxidase Peroxisomal fatty acyl-CoA oxidase, Peroxisome assembly factor 1 , Peroxisome assembly factor 2, Peroxisome biogenesis disorder protein-1 , Peroxisome biogenesis disorder protein-11 , Peroxisome biogenesis disorder protein-4, Peroxisome hydratase, Peroxisome proliferator activated receptor alpha, Peroxisome proliferator activated receptor gamma, Phenol sulfotransferase, Phosphoenolpyruvate carboxykinase, Phosphoglyceride kinase, Phospholipase A2, Plasminogen activator inhibitor 2, Platelet derived growth factor B, Platelet endothelial cell adhesion molecule-1 , Poly(ADP- ribose) polymerase, Proliferating cell nuclear antigen gene, Prostaglandin H synthase, Protein kinase C betal , Protein-tyrosine

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Abstract

La présente invention concerne des procédés et des kits de mesure des essais d'hypersensibilité de mutants par utilisation de techniques de recherche systématique à haut rendement destinés à l'évaluation des mécanismes de toxicité de produits chimiques. L'essai se fait dans des plateaux multi-cupules, tels que les plateaux à 96 cupules, ce qui fait que le procédé permet de tester de nombreux composés en peu de temps. L'essai est polyvalent. En effet, il permet de rechercher l'aptitude de composés à provoquer des dégâts dans l'ADN, à provoquer des mutations génétiques (mutagénicité), à provoquer le cancer (carcinogénicité), à provoquer des dégâts aux protéines ou aux membranes, à entraîner des affaiblissements, à provoquer des dégâts au mitochondre, ainsi que plus généralement une propension à la génotoxicité. En effet, aux termes de l'invention, la toxicité recouvre la totalité de ces comportements. De plus, l'essai permet de détecter la contrainte oxydative, les dégâts aux protéines, l'interruption du cycle cellulaire, la charge et les pertes énergétiques, la rupture des microtubules ou le déclenchement de compétence métabolique par sur-expression d'inserts de gènes humain codant pour des gènes du métabolisme ou incorporation de fraction S9. Dans une réalisation préférée de l'invention, on fait le dosage de levure sauvage et de ses différents mutants en employant les produits chimiques désirés, et pour évaluer la croissance des levures, on procède par opacimétrie. On génère des courbes de réponse en fonction des doses, et on calcule la sensibilité du mitant au composé par rapport à ses parents (sensibilité relative). Les sensibilités relatives qui sont statistiquement significative indique une hypersensibilité du mutant au composé de test. Il en découle que cet essai constitue un moyen bon marché et fiable de recherche de la toxicité à court terme, et ce, sans expérimentation animale, les renseignements apportés sur le mécanisme d'action du composé étant tout aussi pertinents. Cette invention s'applique principalement à l'industrie pharmaceutique, aux essais sur l'environnement et aux recherches cliniques.
EP00908601A 1999-02-12 2000-02-11 Test toxicologique a haut rendement utilisant des organismes et cellules de culture Withdrawn EP1153137A2 (fr)

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US24971999A 1999-02-12 1999-02-12
US249719 1999-02-12
PCT/US2000/003557 WO2000047761A2 (fr) 1999-02-12 2000-02-11 Test toxicologique a haut rendement utilisant des organismes et cellules de culture

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