INDIVIDUAL METHOD PREDICTIVE OF THE DNA-BREAKING GENOTOXIC EFFECTS OF CHEMICAL OR BIOCHEMICAL AGENTS

Abstract

A predictive method of cell toxicity after exposure to chemical elements breaking DNA directly or indirectly (particularly certain metals, pesticides and certain active substances for chemotherapy) and which is based on the determination and cross-checking of a plurality of cellular and enzymatic parameters and criteria.

Claims

1-15. (canceled)

16. A method for evaluating the sensitivity of a tissue sampled from a subject to a DNA-breaking toxic effect of at least one chemical agent or biochemical agent, the method comprising: establishing a working concentration for said at least one chemical or biochemical agent, or of chemical and/or biochemical agents; sampling, after establishing the working concentration, cells from a tissue to be evaluated of a subject; dispersing and/or amplifying, after the sampling, said cells to obtain a cell sample; bringing, for a predetermined period of time, and after the dispersing and/or the amplifying, said cell sample into contact with said at least one chemical agent or biochemical agent in the working concentration; and detecting, after the bringing, a number of DNA double-strand breaks, and/or a biomarker representing said number, and/or a number of micronuclei.

17. The method of claim 16, further comprising determining a diagnostic score which represents said sensitivity of said tissue to the DNA-breaking toxic effect of said at least one chemical agent or biochemical agent, using said number of DNA double-strand breaks, and/or said number of micronuclei, and said working concentration.

18. The method of claim 16, wherein the detection is carried out using a technique selected from the group consisting of immunofluorescence, cytogenetic testing, and pulsed-field electrophoresis.

19. The method of claim 16, wherein detecting said biomarker comprises detecting a biomarker selected from the group consisting of pH2AX, 53BP1, Phospho-DNAPK, and MDC1.

20. The method of claim 16, wherein detecting said biomarker comprises detecting biomarker pH2AX, and a number and size of nuclear foci of said biomarker.

21. The method of claim 16, further comprising performing counterstaining suitable for locating the cell nuclei to quantify the micronuclei (MN).

22. The method of claim 16, wherein: in the detecting, a working concentration is a previously determined reference concentration C.sub.ref, the number of DNA double-strand breaks is determined by pH2AX immunofluorescence, and, after DAPI counterstaining, the number of micronuclei (MN) is detected, then N.sub.pH2AX(24 h, C.sub.ref) and N.sub.MN(24 h, C.sub.ref) are determined on said cell sample.

23. The method of claim 22, wherein: it is inferred that a genotoxic risk is low and/or described as Group I if for the cell sample N.sub.pH2AX(24 h, C.sub.ref)?2 or N.sub.MN(24 h, C.sub.ref)?2%, and it is inferred that the genotoxic risk is very high and/or described as Group III if for the cell sample N.sub.pH2AX(24 h, C.sub.ref)>8, or N.sub.MN(24 h, C.sub.ref)>10%, for all the other cases, it is inferred that the genotoxic risk is intermediate and/or described as Group II.

24. The method of claim 22, wherein said working concentration is a previously determined reference concentration C.sub.ref.

25. The method of claim 24, wherein said previously determined reference concentration C.sub.ref is performed by: preparing a cell sample by dispersion and/or amplification of reference cells (sensitivity Group I), and subdividing the cell sample into a plurality of fractions, applying a plurality of concentrations of the at least one chemical agent or biochemical agent under test, said concentrations being chosen within a concentration range of said at least one chemical agent or biochemical agent, said concentration range between nM to mM, for a predetermined period of time, determining, for each of fraction of the cell sample, a number of pH2AX foci per cell and/or a number of micronuclei per cell.

26. The method of claim 25, wherein the determination of, for each of fraction of the cell sample, the number of pH2AX foci per cell and/or the number of micronuclei per cell is performed by determining: via pH2AX immunofluorescence with DAPI counterstaining, a mean number (N.sub.pH2AX(t, C)) of nuclear foci obtained with the marker pH2AX at observation times t and at concentration C, a mean number (N.sub.MN(t, C)) of micronuclei per 100 cells at the observation times t and at the concentration C, a standard error a on each respective determination, and the reference concentration C.sub.ref as a concentration with N.sub.pH2AX(24 h, C.sub.ref)+2?=2 or N.sub.MN(24 h, C.sub.ref)+2?=2%.

27. The method of claim 25, wherein the reference cells are chosen from cell lines HF19, IMR90, 48BR, 70BR, 142BR, 155BR, 1BR3, 149BR, and MRC9.

28. The method of claim 16, wherein said at least one chemical agent is chosen from the group consisting of a metallic or non-metallic anion, a non-metallic cation, an organic anion, an organic cation, a zwitterionic compound, an optionally neutral inorganic compound, an optionally neutral organic compound, an organometallic compound, and an insoluble compound.

29. The method of claim 16, wherein said at least one chemical agent or biochemical agent is in at least one of: dissolved form in a liquid medium, particle form, nanoparticle form, fixed on a cell membrane, or in gaseous form.

30. The method of claim 16, wherein said at least one biochemical agent is chosen from the group consisting of a peptide, an antibody, an antigen, a virus, a virus fragment, and a cell fragment.

31. The method of claim 16, wherein cells from said sampled tissue are isolated and/or amplified, said amplified cells being the cell sample.

32. The method of claim 31, further comprising: determining, on said cell sample, a mean number (N.sub.pH2AX(t)) of nuclear foci obtained with a marker pH2AX at observation times between a time t0 in a non-exposed state to said at least one chemical agent or biochemical agent, and at least one observation time t4 after contacting said cell sample with said at least one chemical agent or biochemical agent for a predetermined period of time, said contacting serving as genotoxic exposure, determining a sensitivity group of the sample to the genotoxic exposure, using at least the determined mean numbers N.sub.pH2AX(t), determining a mean number (N.sub.MN(t)) of micronuclei observed at the times t per 100 cells [as a %] on said cell sample, at least at the time t0 and at the time t4.

33. The method of claim 32, wherein t4 comprises a fixed value which represents a time for which a level of DNA breaks attains a residual value thereof.

34. The method of claim 33, wherein t4 is approximately 24 hours.

35. A method for evaluating the sensitivity of a tissue sampled from a subject to a DNA-breaking toxic effect of a combination of chemical agents and/or biochemical agents, the method comprising: establishing a working concentration for said chemical agents and/or biochemical agents included in said combination of chemical agents and/or biochemical agents; sampling, after establishing the working concentration, cells from a tissue to be evaluated of a subject; dispersing and/or amplifying, after the sampling, said cells to obtain a cell sample; bringing, for a predetermined period of time, and after the dispersing and/or the amplifying, said cell sample into contact with said combination of chemical agents and/or biochemical agents in the working concentration; and detecting, after the bringing, a number of DNA double-strand breaks, and/or a biomarker representing said number, and/or a number of micronuclei.

Description

DRAWINGS

[0070] FIG. 1 shows the variations (A), (B), and (C) of the number of pH2AX foci 24 hours after contacting the cell samples with glyphosate (CAS No. 1071-83-6) at a given concentration according to this glyphosate concentration for the fibroblast lines 1BR3 (FIG. 1 (A)), 149BR (FIG. 1 (B)) or 04PSL (FIG. 1 (C)).

[0071] FIG. 2 shows the variations (A), (B) and (C) of the number of pH2AX foci 24 hours after contacting the cell sample with 5FU at a given concentration according to this 5FU concentration for the fibroblast lines MRC9 (FIG. 2 (A)), 03HLS (FIG. 2 (B)) and GM02718 (FIG. 2 (C)).

DESCRIPTION

[0072] An embodiment with a plurality of alternative embodiments suitable for a human patient is described herein.

[0073] Test Preparation

[0074] Before sampling any cells and before handling any sampled cells, the respective operators (belonging for example to a cytological analysis laboratory) are informed (typically by the physician) of the patient's potential HIV or hepatitis C infection status so that said operators can take suitable increased biological safety measures when sampling, handling and managing the cell culture.

[0075] Then, the operator takes a tissue sample used for preparing the cell sample from the patient. Preferably, a skin sample is taken by biopsy; this sample may be advantageously carried out according to a method known as skin punch biopsy. The tissue sample is placed in DMEM medium+20% (sterile fetal calf serum). The tissue sample is transferred without delay to a specialized laboratory, in the knowledge that the sample must not remain more than 38 hours at ambient temperature.

[0076] The following step represents the isolation and/or amplification of the sampled tissue.

[0077] In one embodiment, on receipt, the tissue sample (typically the biopsy) is established in the form of an amplifiable cell line without a viral or chemical transformation agent according to an ancillary procedure well known to culture laboratories, as underlined by the publication of Elkind et al. The radiobiology of cultured mammalian cell, Gordon and Breach (1967). Once the number of cells is sufficient (typically after 1 to 3 weeks), the first experiments are carried out using the process according to the invention. A cell sample is prepared: The cells are inoculated on glass coverslips in Petri dishes. A portion of these coverslips are contaminated with metals or pesticides or any other DNA-breaking chemical or biochemical agent at different concentrations. A further portion is not contaminated; it represents the spontaneous state. During contamination, the cells remain in the culture incubator at 37? C.

[0078] For the contaminated cells, characteristics are acquired corresponding to the state after an incubation time with the DNA-breaking chemical or biochemical agent. Said characteristics are represented by foci corresponding to the marker pH2AX. The cells on glass coverslips are then fixed, lysed and hybridized. The following procedure, known per se (Bodgi et al, A single formula to describe radiation-induced protein relocalization: towards a mathematical definition of individual radiosensitivity, J Theor Biol. 21 p 333:135-45.2013):

[0079] the cells are fixed in 3% paraformaldehyde and 2% sucrose for 15 minutes at ambient temperature and permeabilized in 20 mM HEPES buffer solution (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid) at pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose, 0.5% Triton X-100 (a non-ionic surfactant having the formula t-Oct-C6H4-(OCH2CH2)?OH where x=9-10, CAS No. 9002-93-1, supplied for example by Sigma Aldrich) for 3 minutes. The glass coverslips are then washed in phosphate buffer saline (known as the acronym PBS) before immunological staining. The incubation took place for 40 min at 37? C. in PBS supplemented with 2% bovine serum albumin (known as the acronym BSA or fraction V, supplied for example by Sigma Aldrich) and was followed by a wash with PBS. Anti-pH2AX primary antibodies are used at a concentration of 1:800. The incubations with anti-mouse FITC or anti-rabbit TRITC secondary antibodies (1:100, supplied by Sigma Aldrich) were performed at 37? C. in 2% BSA for 20 minutes. Glass coverslips were treated with Vectashield? containing DAPI (4,6-Diamidino-2-phenylindole) to label the nucleus. Staining with DAPI also makes it possible, indirectly, to determine the number of cells in phase G1 (nuclei with homogenous DAPI staining), in phase S (nuclei with numerous pH2AX foci), in phase G2 (nuclei with heterogeneous DAPI staining) and metaphases (visible chromosomes).

[0080] The results are acquired using these coverslips on an immunofluorescence microscope (Olympus model for example). The reading may be direct (typically by counting the foci on at least 50 cells in G0/G1 for each point) or using dedicated image analysis software, or on an automated microscope; preferably the software or automated microscope methods are calibrated with manual determinations.

[0081] In order to obtain results of sufficient statistical reliability to serve as a basis for diagnosis, at least 3 independent series of experiments (radiation) are performed and the mean of each of the numbers of foci for the times defined is calculated.

[0082] Determination of Biological and Clinical Parameters

[0083] General and Markers Used

[0084] The invention is based, inter alia, on the use of data acquired for one of the two markers pH2AX on non-contaminated (spontaneous state) and contaminated cells. The method is based on the study of the labeling with this marker for a given contamination time: the samples are labeled after a predetermined time interval from discontinuing contamination, and the immunofluorescence thereof is studied.

[0085] The means obtained for each point and each dose with each marker are calculated with the standard errors of the mean (referred to as SEM) given that the sampling is n=3 (no Gaussian type standard error SE).

[0086] pH2AX denotes the phosphorylated forms in serine 439 of variant X of histone H2AX marking, according to the applicant's observations, the number of DNA double-strand breaks (DSBs) that are recognized by the main and faithful repair mode, suture. The marker pH2AX is essentially nuclear in the form of nuclear foci only and only the number and size of the foci shall be analyzed.

[0087] Counterstaining with DAPI (a DNA marker known to those skilled in the art) makes it possible to locate the nucleus to situate the cytoplasmic or nuclear location to quantify the micronuclei, which are complementary cell markers to the data on the foci.

[0088] Biological and Clinical Parameters

[0089] The definition and determination are performed as indicated of: [0090] NpH2AX(t), the mean numbers of nuclear foci obtained with the markers pH2AX at the observation times t0 (non-contaminated) and t4, in the knowledge that the determination of the parameter NpH2AX(t) is mandatory within the scope of the method according to the invention, [0091] NMN(t) the number of micronuclei observed spontaneously (at t=t0, i.e. without contamination) or at t=t4 after contamination per 100 cells (as a %).

[0092] The process according to the invention demonstrates that the tissue sensitivity to a given metal varies according to the tissue of interest. For example, astrocytes contaminated with 100 ?M of aluminum exhibit less breaks (HA cells, 2 H2AX foci) compared to endothelial cells for the same concentration (HMEC cells, 3.7 H2AX foci) (see table 1). Furthermore, for some metals, the inventors demonstrated that there was a correspondence between the single toxicity scale proposed and certain clinical signs described for example in the case of lead (saturnism) or in the case of cadmium (Itai-Itai disease) (see table 3).

[0093] The process according to the invention makes it possible to also demonstrate that cells contaminated with copper exhibited for the highest concentration tested (1 mM) a number of DNA breaks visualized by H2AX foci ranging from 2 to 21 foci according to the cell type tested (see tables 1 and 2).

[0094] It is noted that the process according to the invention is so sensitive that it makes it possible to characterize the impact on a tissue of DNA-breaking chemical agents in very low concentrations, which are of the order of magnitude of the regulatory limit values for certain chemical agents in drinking water; these limit values are for example of the order of 2 mM (2 mg/L) for copper, 200 ?m for aluminum, 5 ?m for cadmium, 10 ?M for Pb.

[0095] Predictive Evaluation

[0096] This targets the prediction of clinical parameters using the biological data measured.

[0097] A quantitative diagnosis directly derived from the mathematical value of the scores or mathematical formulas correlating the scores; this relates to the following criterion:

[0098] (i) Patient classification in a Group I, II or III (criterion referred to as GROUP):

[0099] The definition of the sensitivity groups (GROUP) helps the physician determine based on the scores according to the invention and the clinical profile of the patient analogies with known genetic syndromes. These groups were initially defined in the publication by Joubert et al. (Int. J. Radiat. Biol. 84(2), p. 107-125 (2008), cited above.

[0100] According to the present invention, it is considered that:

[0101] If for the cell sample NpH2AX(24 h, Cref)<=2 or NMN(24 h, Cref)<=0.5%, preferably NMN(24 h, Cref)<=1% or even more preferentially NMN(24 h, Cref)<=2%, then the genotoxic risk is considered to be low or described as Group I

[0102] If for the cell sample NpH2AX(24 h, Cref)>8 or NMN(24 h, Cref)>10%, then the genotoxic risk is considered to be very high and described as Group III

[0103] For all other cases, the genotoxic risk is considered to be intermediate and described as Group II.

EXAMPLES

Example 1

[0104] Determination, on Control Fibroblast Lines, of the Reference Concentration of Glyphosate (Chemical Agent)

[0105] Commercial 1BR3 and 149 BR control fibroblasts were amplified according to the recommendations of the supplier (SIGMA-ALDRICH) until the number of cells sought was obtained. After obtaining a sufficient number of cells (generally after one to 3 weeks), the first experiments were conducted using the process according to the invention. The cells were inoculated on glass coverslips in Petri dishes. A portion of these coverslips was then contacted with the medium under test comprising glyphosate (CAS No. 1071-83-6) at a given concentration presented in table 1 hereinafter.

[0106] Table 1 presents detection of the number of pH2AX foci 24 hours after contacting 1BR3, 149 BR control fibroblast cells and 04PSL cells with glyphosate according to the glyphosate concentration used.

TABLE-US-00001 TABLE 1 glyphosate 1BR3 (control cells) 149BR (control cells) 04PSL concentration pH2AX(24) + pH2AX(24) + pH2AX(24) + (?M) pH2AX(24) SEM 2xSEM pH2AX(24) SEM 2xSEM pH2AX(24 h) SEM 2xSEM 3 1.6 0.128 1.856 0.8 0.064 0.928 1.7 0.136 1.972 10 1.6 0.128 1.856 1 0.08 1.16 1.8 0.144 2.088 30 1.9 0.152 2.204 1.5 0.12 1.74 4.1 0.328 4.756 100 2 0.16 2.32 1.8 0.144 2.088 6 0.48 6.96 300 2.3 0.184 2.668 3 0.24 3.48 8.4 0.672 9.744

[0107] After contacting with glyphosate at a given concentration, the cells were stored in the culture incubator at 37? C. 24 hours after contacting with glyphosate at a given concentration as presented in table 1, the mean number of nuclear foci obtained with the marker pH2AX was acquired. The acquisition of the results was carried out using these coverslips on an immunofluorescence microscope (Olympus model). The reading was performed directly by counting the foci obtained with the marker pH2AX on at least 50 cells in G0/G1 for each point or using dedicated image analysis software (imageJ).

[0108] In order to obtain results of sufficient statistical reliability to serve as a basis for diagnosis, 3 independent series of experiments were performed. The mean and standard errors of the mean (SEM or ?) of each of the numbers of foci acquired after 24 hours of contacting the control cells with glyphosate at a given concentration was calculated and presented in table 1.

[0109] As such, for the skin control cell samples 1BR3 and 149BR (see table 1), the reference concentration was determined. This reference concentration was defined as being the concentration giving: NpH2AX(24 h, Cref)+2?=2

[0110] where ? corresponds to the standard error of the measurements of the number of pH2AX foci acquired 24 hours after contacting the control cells with glyphosate at a given concentration, these measurements being carried out on 3 independent experiments of 50 cells (standard error of the mean).

[0111] FIG. 1 represents the variation of the number of pH2AX foci acquired per cell, 24 hours after contacting the control cells 1BR3 (see FIG. 1 (A)) and 149 BR (see FIG. 1 (B)) with glyphosate according to the glyphosate concentration used. The concentration Cref defined by NpH2AX(24 h, Cref)+2?=2 for the 2 control cells lines 149BR and 1BR3 is 100 ?M.

[0112] Test Preparation (Cell Lines 04PSL)

[0113] A skin cell sample from a patient was sampled by biopsy via the skin punch method known to those skilled in the art. The cell sample was then placed in DMEM medium+20% sterile fetal calf serum. The cell sample was then transferred without delay to a specialized laboratory, so that the sample remained not more than 38 hours at ambient temperature.

[0114] On receipt, the cell sample from the biopsy was established in the form of an amplifiable 04PSL cell line according to a procedure well known to culture laboratories and those skilled in the art: using particularly the trypsin dispersion, the cells are once again diluted in replenished medium and so on until the number of cells sought is obtained. After obtaining a sufficient number of cells (generally after one to 3 weeks), the first experiments were conducted using the process according to the invention. The 04PSL line cells were inoculated on glass coverslips in Petri dishes. A portion of these coverslips was then contacted with glyphosate at a concentration of 100 ?M. By way of verification, a further portion of these coverslips was contacted with glyphosate at a given concentration (see table 1, FIG. 1 (C)).

[0115] After contacting with glyphosate at a given concentration, the cells were stored in the culture incubator at 37? C. 24 hours after contacting with glyphosate at a given concentration as presented in table 1, the mean number of nuclear foci obtained with the marker pH2AX was acquired. The acquisition of the results was carried out using these coverslips on an immunofluorescence microscope (Olympus model). The reading was performed directly by counting the foci obtained with the marker pH2AX on at least 50 cells in G0/G1 for each point or using dedicated image analysis software (imageJ).

[0116] In order to obtain results of sufficient statistical reliability to serve as a basis for diagnosis, 3 independent series of experiments were performed. The mean and standard errors of the mean (SEM or ?) of each of the numbers of foci acquired after 24 hours of contacting the control cells with glyphosate at a given concentration was calculated and presented in table 1 and in FIG. 1 (C).

[0117] Determination of Genotoxic Risk of Cell Line 04PSL

[0118] At a glyphosate concentration of 100 ?M, the number of pH2AX foci obtained for the cell line 04PSL is approximately 7; this figure validates the equation 2<NpH2AX(24 h)<8. Consequently, for the cell line 04PSL, the genotoxic risk associated with glyphosate is group II or described as intermediate. The line 04PSL is chemosensitive.

Example 2

[0119] Determination, on Control Fibroblast Lines, of the Reference Concentration of the Chemotherapeutic Drug 5FU (Chemical Agent)

[0120] Commercial MRC9 control fibroblasts were amplified according to the recommendations of the supplier (SIGMA-ALDRICH) until the number of cells sought was obtained. After obtaining a sufficient number of cells (generally after one to 3 weeks), the first experiments were conducted using the process according to the invention. The cells were inoculated on glass coverslips in Petri dishes. A portion of these coverslips was then contacted with the medium under test comprising 5FU at a given concentration presented in table 2 hereinafter.

[0121] Table 2 presents a detection of the number of pH2AX foci 24 hours after contacting MRC9 control fibroblast cells and GM02718 and 03HLS cells with 5FU according to the 5FU concentration used

TABLE-US-00002 TABLE 2 5FU MRC9 (control cells) GM002718 03HLS concentration pH2AX(24 h) + pH2AX(24 h) + pH2AX(24 h) + (?M) pH2AX(24 h) SEM 2xSEM pH2AX(24 h) SEM 2xSEM pH2AX(24 h) SEM 2xSEM 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.16 0.41 1.97 2.70 0.31 3.32 1.40 0.04 1.49 3.00 1.38 0.28 1.93 2.41 0.01 2.43 1.69 0.29 2.26 10.00 0.30 0.11 0.51 2.96 0.46 3.87 1.63 0.03 1.69 30.00 1.18 0.41 2.01 2.38 0.00 2.38 2.00 0.29 2.59

[0122] After contacting with 5FU at a given concentration, the cells were stored in the culture incubator at 37? C. 24 hours after contacting with 5FU at a given concentration as presented in table 2, the mean number of nuclear foci obtained with the marker pH2AX was acquired. The acquisition of the results was carried out using these coverslips on an immunofluorescence microscope (Olympus model). The reading was performed directly by counting the foci obtained with the marker pH2AX on at least 50 cells in G0/G1 for each point or using dedicated image analysis software (imageJ).

[0123] In order to obtain results of sufficient statistical reliability to serve as a basis for diagnosis, 3 independent series of experiments were performed. The mean and standard errors of the mean (SEM or ?) of each of the numbers of foci acquired after 24 hours of contacting the control cells with 5FU at a given concentration was calculated and presented in table 2.

[0124] As such, for the skin control cell samples MRC9 (see table 2, FIG. 2 (A)), the reference concentration was determined. This reference concentration was defined as being the concentration giving: NpH2AX(24 h, Cref)+2?=2

[0125] where ? corresponds to the standard error of the measurements of the number of pH2AX foci acquired 24 hours after contacting the control cells with glyphosate at a given concentration, these measurements being carried out on 3 independent experiments of 50 cells (standard error of the mean).

[0126] FIG. 2 represents the variation of the number of pH2AX foci acquired per cell, 24 hours after contacting the control cells MRC9 (see FIG. 2 (A)) with 5FU according to the 5FU concentration used. The concentration Cref defined by NpH2AX(24 h, Cref)+2?=2 for the control cells line MRC9 is 30 ?M.

[0127] Test Preparation (Cell Lines GM02718 and 03HLS)

[0128] The cell line GM02718 was amplified according to the recommendations of the supplier (Coriell Institute) until the number of cells sought was obtained.

[0129] For the line 03 HLS, a skin cell sample from a patient was sampled by biopsy via the skin punch method known to those skilled in the art. The cell sample was then placed in DMEM medium+20% sterile fetal calf serum. The cell sample was then transferred without delay to a specialized laboratory, so that the sample remained not more than 38 hours at ambient temperature.

[0130] On receipt, the cell sample from the biopsy was established in the form of an amplifiable 03HLS cell line according to a procedure well known to culture laboratories and those skilled in the art: using particularly the trypsin dispersion, the cells are once again diluted in replenished medium and so on until the number of cells sought is obtained.

[0131] After obtaining a sufficient number of cells (generally after one to 3 weeks), the first experiments were conducted using the process according to the invention. The GM02718, or 03HLS, line cells were inoculated on glass coverslips in Petri dishes. A portion of these coverslips was then contacted with 5FU at a concentration of 30 ?M. By way of verification, a further portion of these coverslips was contacted with 5FU at a given concentration (see table 2, see FIG. 2 (B) for the cell line GM02718, respectively FIG. 2 (C) for the cell line 03HLS).

[0132] After contacting with 5FU at a given concentration, the cells were stored in the culture incubator at 37? C. 24 hours after contacting with 5FU at a given concentration as presented in table 2, the mean number of nuclear foci obtained with the marker pH2AX was acquired. The acquisition of the results was carried out using these coverslips on an immunofluorescence microscope (Olympus model). The reading was performed directly by counting the foci obtained with the marker pH2AX on at least 50 cells in G0/G1 for each point or using dedicated image analysis software (imageJ).

[0133] In order to obtain results of sufficient statistical reliability to serve as a basis for diagnosis, 3 independent series of experiments were performed. The mean and standard errors of the mean (SEM or ?) of each of the numbers of foci acquired after 24 hours of contacting the control cells with 5FU at a given concentration was calculated and presented in table 2 and in FIG. 2 (B) for the cell line GM02718, respectively FIG. 2 (C) for the cell line 03HLS.

[0134] Determination of the Genotoxic Risk of the Cell Line GM02718, or 03HLS Respectively

[0135] At a 5FU concentration of 30 ?M, the number of pH2AX foci obtained for the cell line GM02718, or 03HLS respectively, is approximately 2.38 or 2.59 foci respectively; this figure validates the equation 2<NpH2AX(24 h)<8. Consequently, for the cell line GM02718, or 03HLS respectively, the genotoxic risk associated with 5FU is group II or described as intermediate. The lines GM02718 and 03HLS are chemosensitive.

Further Examples

[0136] The tables hereinafter summarize the results of numerous experiments which were carried out as described in the Detailed description section above.

[0137] In tables 3 and 4, pH2AX corresponds to the mean number of nuclear foci obtained with the marker pH2AX, 24 hours after contacting the cell sample with the chemical agent at the concentration C (NpH2AX(24 h, C)), and where Micronuclei corresponds to the mean number of micronuclei observed per 100 cells 24 hours after contacting the cell sample with the chemical agent at the concentration C(N.sub.MN(24 h, C)).

[0138] Table 3 presents a detection of the number of ph2AX foci and the number of micronuclei 24 hours after contacting 04PSL, 01PAU, 08HNG, 1BR3 cells with a pesticide breaking agent (Glyphosate, Permethrin, Thiobendazole, PCP, Atrazine) according to the pesticide concentration used.

TABLE-US-00003 TABLE 3 Breaking Concentration [?M] Line agent Markers 0 3 10 30 100 150 300 04PSL Glyphosate micronuclei 1 6 4 10 12 14 pH2AX 0 1.7 1.9 4.8 6.5 8.7 Breaking Concentration [?M] Line agent Markers 0 0.3 1 5 10 01PAU Permethrin micronuclei 2 2 2 4 6 pH2AX 0 0.1 0.2 1.3 1.8 Breaking Concentration [?M] Line agent Markers 0 0.3 1 3 10 08HNG Thiobendazole micronuclei 0 2 4 10 10 pH2AX 0 1.8 2 4.3 6 Breaking Concentration [?M] Line agent Markers 0 0.3 3 10 30 50 100 1BR3 PCP micronuclei 1 2 4 10 10 20 pH2AX 0 1.2 1.5 1.9 4.3 Breaking Concentration [?M] Line agent Markers 0 0.01 0.1 0.3 1 10 20 30 1BR3 Atrazine micronuclei 1 5 6 13.5 15 40 pH2AX 1 1.3 1.8 3.1 3.5

[0139] Table 4 presents a detection of number of pH2AX foci and number of micronuclei 24 hours after contacting control nervous system cell lines Ha (astrocyte cells), Hah (hippocampus astrocyte cells) and Hasp (spinal cord astrocyte cells) with a metallic compound (AlCl.sub.3, Cu, CuCl.sub.2, CuSO.sub.4, Pb(NO.sub.3).sub.2, CdCl.sub.2, Cd-acetate or Cd-acetate-citrate) according to the concentration of said metallic compound used.

TABLE-US-00004 TABLE 4 Breaking Concentration [?M] Line agent Marker 3 10 30 100 300 1000 Ha AlCl.sub.3 pH2AX 0.34 0.88 1.37 2.18 2.96 6.04 Micronuclei 10 15 16.7 46.7 56.7 70 Cu pH2AX 0.3 0.62 2.55 0 0 Micronuclei 4 5 13 20 50 Hah AlCl.sub.3 pH2AX 0.07 0.68 0.83 1.22 1.77 2.28 Micronuclei 2 2 4 6 20 30 Cu pH2AX 1.01 1.87 2.62 5.45 6.61 Micronuclei 6 10.7 20.7 46.7 73.3 Hasp AlC.sub.3 pH2AX 0.84 0.46 0.74 2.15 1.7 1.62 Micronuclei 2 4 4 7.3 8.7 13 Cu pH2AX 0.29 0.64 1.13 1.78 2.36 Micronuclei 4 7 9 10 16 AlC.sub.3 pH2AX 3.2 3.2 5.7 3.7 4.5 9.9 Micronuclei 1.75 3.75 5 4.33 7.33 11.5 CuCl.sub.2 pH2AX 1.6 1.9 1.6 1.5 5.3 21.4 Micronuclei 4 6.7 5.3 6.7 7.3 4 CuSO.sub.4 pH2AX 1.9 2.3 3.4 6.4 20.1 30.2 Micronuclei 3 5 4.7 5.3 9.3 4 Pb(NO.sub.3).sub.2 pH2AX 4 7.5 15 21 Micronuclei 0 7 12 20 10 CdCl.sub.2 pH2AX 1.9 3.7 8.3 Micronuclei 3.5 9 12.25 Cd- pH2AX 5 5.5 7 acetate Micronuclei 5 10 10 Cd- pH2AX 1 4.75 7 acetate- Micronuclei citrate

[0140] The experimental data presented in table 4 above were used to determine the reference concentration C.sub.ref particularly for Pb(NO.sub.3).sub.2 (C.sub.ref<1 ?M) and CdCl.sub.2 (C.sub.ref=10 ?M). These data were correlated with the clinical signs observed and presented in table 5 hereinafter, particularly for Pb(NO.sub.3).sub.2 and CdCl.sub.2.

[0141] Table 5 presents numerical examples of correspondence between the single toxicity scale according to the invention and the corresponding clinical signs.

TABLE-US-00005 TABLE 5 Chemical Reference Prediction based species concentration C.sub.ref on algorithm Clinical effects observed Lead <1 ?M Group II risk Onset of signs of saturnism above 100 ?g/l of [Salt used: N.sub.pH2AX(24 h) < 2 predicted between blood corresponding to approximately 2 ?M Pb(NO.sub.3).sub.2] 1 and 30 ?M 2 < N.sub.pH2AX(24 h) < 8 Group III risk Immediate lethal effect never actually observed predicted above 30 ?M N.sub.pH2AX(24 h) > 8 Cadmium 10 ?M Group II risk Relating to exposures sustained by some inhabitants [salt used: N.sub.pH2AX(24 h) < 2 predicted between of the district of Toyama (Japan) following CdCl.sub.2] 10 and 100 ?M systemic cadmium poisoning (Itai-Itai disease) 2 < N.sub.pH2AX(24 h) < 8 Group III risk Fatal fume concentrations: 40-50 mg/m.sup.3 i.e. 245 ?M predicted above 100 ?M per m.sup.3 (death in 100 min) N.sub.pH2AX(24 h) > 8 Chromium 3 nM Group II risk Cases of poisoning in Hinkley USA, Erin Brockovich case) [salt used: N.sub.pH2AX(24 h) < 2 predicted between with 1.19 g/l in well water equivalent to 4.6 mM. The Na.sub.2CrO.sub.4] 3 and 30 nM concentration in tap water was estimated at 23 Nm 2 < N.sub.pH2AX(24 h) < 8 Group III risk predicted above 30 nM N.sub.pH2AX(24 h) > 8

[0142] Where the GROUP criterion is defined as follows: GROUP I=absence of clinical signs, GROUP II=presence of clinical signs, and GROUP III=lethal effect.