INDICATOR STEM CELL LINE/LIVING ORGANISM (NON-HUMAN) AND A METHOD FOR DETECTION OF THE GENOTOXIC POTENTIAL OF AQUATIC SAMPLES OR AQUEOUS SOLUTIONS OF TEST COMPOUNDS

Abstract

Indicator stem cell line/transgenic (non-human) living organism for non-destructive, self-signalizing visualization of nuclear structures, which allows the identification of nuclear and chromosome anomaliesincluding micronucleias consequence of exposure to genotoxic compounds, wherein the stem cell line is a transgenic stem cell line from a (non-human) animal with nuclei labelled with a fluorescent protein fused to a chromatin-associated protein and/or the living organism is a transgenic (non-human) living organism in which a cell lineage has nuclei labelled with a fluorescent protein fused to a chromatin-associated protein.

Claims

1. Indicator stem cell line/transgenic (non-human) living organism for non-destructive, self-signalizing visualization of nuclear structures, which allows the identification of nuclear and chromosome anomalies including micronucleias consequence of exposure to genotoxic compounds, wherein the stem cell line is a transgenic stem cell line from a (non-human) animal with nuclei labelled with a fluorescent protein fused to a chromatin-associated protein and/or the living organism is a transgenic (non-human) living organism in which a cell lineage has nuclei labelled with a fluorescent protein fused to a chromatin-associated protein.

2. The stem cell line/living organism of claim 1 wherein the fluorescent protein is fused to a histone in the labelled nuclei.

3. The stem cell line/living organism of claim 2 wherein the fluorescent protein is fused to the histone 2B in the labelled nuclei.

4. The stem cell line/living organism according to claim 1 wherein the fluorescent protein is the green fluorescent protein (GFP).

5. The stem cell line according to claim 1, wherein the transgenic stem cells are pluripotent and tissue-regenerating.

6. The stem cell line according to claim 1 wherein said transgenic stem cell line is from a vertebrate.

7. The stem cell line according to claim 6 wherein said transgenic stem cell line is from fish.

8. The stem cell line according to claim 7 wherein said transgenic stem cell line is from Koi carp (Cyprinus carpio haematopterus).

9. The stem cell line according to claim 1 wherein said transgenic stem cell line is from the (non-human) animal brain.

10. The stem cell line according to claim 1, which is deposited, in accordance with the Budapest Treaty, at the Deutsche Sammlung von Mikroorganis-men und Zellkulturen (DSMZ) under the number DSM ACC3285.

11. The indicator living organism according to claim 1, wherein said transgenic living organism has a cell lineage such as germ cells or erythroid cells showing a lineage specific expression of a histone fused fluorescent protein.

12. The indicator living organism according to claim 11, wherein the indicator organism is a fish.

13. The transgenic fish according to claim 12, wherein the MN visualization takes place in translucent embryos or ex vivo by preparation of blood smears.

14. The transgenic fish according to claim 11, wherein the fish is Medaka (Oryzias latipes).

15. A method for the detection of the genotoxic potential of aquatic samples or aqueous solutions of test compounds by using the stem cell line of claim 1, the method comprising: the use of a transgenic stem cell line from a (non-human) animal with nuclei labelled with fluorescent protein fused to a chromatin-associated protein growing as a monolayer on at least one cell culture plate and cell-culture media to be supplemented with different concentrations of the test compounds, pipetting these media to the exposure wells on the at least one special coated cell culture plate and comparing micronucleus frequency with positive and negative controls after cell division by non destructive visual scoring micronuclei in each test group and the controls.

16. The method of claim 15 wherein there is an exposure period after the pipetting of the dilution media to the exposure wells on the at least one special coated cell culture plate, preferably about 6-16 hours.

17. The method according to claim 15 wherein the exposure takes place in a temperature-controlled environment, preferably between 26-28 Celsius.

18. The method according to claim 15 wherein the exposure medium is replaced by a recovery medium before the scoring.

19. The method according to claim 15 wherein the scoring of micronuclei shall take place earliest 2 hours and latest 12 hours after the end of the exposure period.

20. The method according to claim 15 wherein after the non-destructive scoring process a differential fluorescent measurement is used to distinguish between unaffected cells and carcinogenic transformed cells for direct reflection of the carcinogenic potential of the test compounds.

21. The method according to claim 15 wherein the cell-culture media is lyophilised to be rehydrated with aqueous solutions in different concentrations of the test compounds.

22. The method according to claim 15, wherein the transgenic stem cell line from cells of a (non-human) animal with nuclei labelled with fluorescent protein fused to a chromatin-associated protein is the stem cell line, which is deposited, in accordance with the Budapest Treaty, at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under the number DSM ACC3285.

23. A kit suitable for the detection of the genotoxic potential of samples by the method according to claim 15, which comprises: at least one vial with a cryo-conserved transgenic stem cell line from a (non-human) animal with nuclei labelled with fluorescent protein fused to a chromatin-associated protein, at least one special coated cell culture plate to be pre-pared and pre-cultured with the stem cell line for a couple of days in the customary procedure, lyophilised cell-culture media to be rehydrated with aqueous solutions in different concentrations of the test compounds; and positive control agents.

24. A kit suitable for the detection of the genotoxic potential of samples by the method according to claim 15, which comprises: a cryo-conserved transgenic stem cell line from a (non-human) animal with labelled nuclei with fluorescent protein fused to a chromatin-associated protein conserved as a monolayer on at least one special coated cell culture plate ready to use after thawing for micronucleus testing and lyophilised cell culture media to be rehydrated with aqueous solutions in different concentrations of the test compounds; and positive control agents.

Description

MATERIAL AND METHODS

[0013] This new and inventive method for genotoxic potential assessment of aquatic samples or aqueous solutions employs an innovative transgenic stem cell line of animal (non-human) origin and/or a cell lineage (of the erythrocytic series) of a transgenic non-human animal (in this case the medaka, Oryzias latipes) for the visualisation of nuclear structures. In this cell line and/or cell lineage, a fluorescent protein is fused to a histone in order to restrict the signal to nuclei, preferentially to the histone 2B. The initial fluorescent protein reporter of choice is the enhanced green fluorescent protein (eGFP), but other GFP derivatives (e.g. YFP, EBFP) as well as derivatives from other fluorescent proteins such as DsRed (e.g. mRFP, mCherry) may be employed. The transgenic stem cells are pluripotent and tissue-regenerating, and are preferably derived from vertebrates, especially from fish. The donor organism used in the first cell line prototype was the Koi carp (Cyprinus carpio haematopterus), and for this first assembly stem cells were obtained from the brain. The first transgenic fish prototype employed in this method was the Japanese rice fish or medaka (Oryzias latipes) in which a cell lineage e.g. the erythrocytic series has nuclei labelled with a histone-associated fluorescent protein.

[0014] The stem cell line employed in the original method described herein is deposited, in accordance with the Budapest Treaty, at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), under the number DSM ACC3285.

[0015] The presented new method for the detection of the genotoxic potential of aquatic samples and/or aqueous solutions of test compoundsby using a transgenic stem cell line from a (non-human) animal and/or a cell lineage of a (non-human) animal with nuclei labelled with fluorescent protein fused to a chromatin-associated proteincomprises cell lines with fluorescence-labelled nuclei growing as a monolayer on cell culture plates with cell-culture media to be supplemented with different concentrations of the potentially genotoxic test compounds, pipetting these media to the exposure wells on at least one cell culture plate and comparing micronucleus frequencies with positive and negative controls after cell division, by non-destructive visually scoring micronuclei in each test group and the controls. It also comprises the usage of cell lineages of transgenic (non-human) animals with fluorescence-labelled nuclei for the in vivo and/or ex-vivo scoring of MN after exposure to samples/aqueous solutions.

[0016] The invention covers also test kits suitable for the detection of the genotoxic potential of samples by the presented method. One test kit comprises at least one vial with cryo-conserved transgenic stem cells from a (non-human) animal with nuclei labelled with a fluorescent protein fused to a chromatin-associated protein, at least one cell culture plate to be prepared and pre-cultured with the stem cell line for a few days in the customary procedure, lyophilised cell-culture media to be rehydrated with aqueous solutions in different concentrations of the test compounds, and positive control agents.

[0017] Another test kit comprises cryo-conserved transgenic stem cells from a (non-human) animal with nuclei labelled with fluorescent protein fused to a chromatin-associated protein conserved as a monolayer on at least one special coated cell culture well plate ready to use after thawing for micronucleus testing, lyophilised cell-culture media to be rehydrated with aqueous solutions in different concentrations of the test compounds, and positive control agents.

[0018] Preliminary results with the presented invention show that the inventive cell line is suitable for the MNvit assay, enabling visual scoring of normal cell nuclei, fragmented nuclei and MN by fluorescence microscopy using a Motic AE 21 microscope with special filters for fluorescence microscopy, a Zeiss Axiocam MRc 5 camera and a LQ-HXP 120 (LEJ) compact light source.

[0019] The inventive cells were cryopreserved in a solution consisting of 10% DMSO and 10% FBS (fetal-bovine-serum) in MEM (minimum-essential-medium) at 80 C. The cells were thawed, washed with MEM and transferred to a 96 micro-well plate. In order to avoid evaporation of medium and consequent bias in MN frequency, the outer wells were left empty and the micro-well plate was sealed with duct tape. The inner wells were filled with cells in medium. The medium employed was composed of 86% MEM, 10% FBS, 1% non-essential-amino acids, 1% streptomycin (5 mg/ml in PBSphosphate-buffered saline), 1% penicillin (3.125 mg/ml in PBS) and 1% amphotericin (250 g/ml in deionized water). The cells were cultivated at 26 C. until they formed a moderately dense monolayer. The time until optimal monolayer density was achieved varied from two to five days.

[0020] 8 rows with cells were then exposed to colchicine (aneugen) or to 4NQO (4-Nitroquinoline 1-oxide, clastogen) as positive controls for 12 to 14 hours in a temperaturecontrolled environment (26-28 Celsius).

[0021] Under real conditions in routine applications, some rows with cells will then additionally be exposed to aquatic samples and/or aqueous solutions of potentially genotoxic test compounds in different concentrations. These aqueous solutions can also be used to rehydrate lyophilised cell-culture media (in an instant form as part of a test kit) before being pipetted.

[0022] In the basic test, four different concentrations of each chemical agent (colchicine and 4NQO) were used in each plate, each concentration in two columns, and a total of 7 concentrations (including amounts above the cytotoxic threshold) were tested for validating the assay. Two untreated (i.e. not exposed to genotoxic compounds) columns served as internal (negative) controls of each plate. The pipetting scheme used is shown in FIG. 1.

[0023] These experiments were performed six times for each chemical agent.

[0024] FIG. 1. Pipetting scheme used for the 96 micro-well plate. C (control) stands for wells with untreated (non-exposed) KCB cells and 1 to 4 for cells exposed to different concentrations of colchicine or 4-NQO.

[0025] The concentrations of colchicine and 4NQO employed for these experiments are displayed in table 1, with 4 concentrations per plate, following the scheme presented in FIG. 1.

[0026] Table 1. Concentrations of colchicine and 4NQO employed in the validation of the method for MN frequency assessment.

[0027] It is possible, but not absolutely necessary, to replace the exposure medium by a recovery medium before the scoring. The scoring of micronuclei shall take place earliest 2 hours and latest 12 hours after the end of the exposure period.

[0028] In the basic test, a minimum of two pictures per well, corresponding to at least 24 pictures per concentration or control, were taken under a fluorescence microscope with an objective magnification of 40. Normal cell nuclei, MN and fragmented nuclei in each picture were counted visually with aid of the Cell Counter plugin from the public domain image processing software ImageJ. Finally, the frequencies of MN per concentration were calculated.

[0029] To enable the detection of nucleoplasmic bridges and single chromosomes, a different microscope and camera with a higher resolution were used. For this, cover slips were placed in a 12 micro-well plate where the thawed cells were cultured. The cells were left to grow and proliferate on top of the cover slips at 26 C. for one or two days until they formed a moderately dense monolayer. The medium was removed and the cells were fixed by adding 1 ml of a 4% solution of PFA (Paraformaldehyde) for 20 to 30 minutes at 4 C. Cover slips were then mounted on slides with Roti-Mount FluorCare (Carl Roth). Subsequently, the slides were stored at 4 C. until photographs were taken of normal nuclei, MN, fragmented nuclei and nucleoplasmic bridges by using a Zeiss Axioskop 40 with an objective magnification of 100. A high resolution camera additionally magnified (digitally) the cell/nuclei 10 times.

[0030] Given the significantly better representation of the nuclear morphology when using a transient microscope, it seems to be promising to test the visualization of micronuclei of isolated cells after trypsinization and transfer to standard microscope slights

Results

[0031] The detection of MN and a significant, dose-dependent increase in MN frequency could be verified in KCB cells labelled with the H2B-GFP fusion protein by using fluorescence microscopy.

[0032] Genotoxic potential assessment of the tested substances (colchicine and 4NQO) was performed by visually scoring MN and calculating their frequency in relation to the total number of nuclei scored. The counting results for the experiments are summed up in table 2.

[0033] Table 2. Total number of micronuclei and fragmented nuclei, as well as their frequencies (relative to the total number of assessed cells/nuclei) in the negative controls and in 12-14 hours exposure of cells to different concentrations of colchicine and 4NQO.

[0034] Cells exposed to colchicine and 4NQO clearly presented higher MN frequencies than the control (table 2, FIG. 2. a, b), and also higher frequencies of fragmented nuclei (table 2). Exposure to 4NQO induced higher maximal MN frequency (3.66% at 1.2 M) than colchicine (1.77% at 0.375 M), and a higher maximal frequency of fragmented nuclei (12.56% at 0.9 M) than colchicine (6.85% at 0.5 M).

[0035] FIG. 2. MN frequency after exposure to different concentrations of (a) colchicine and (b) 4NQO.

[0036] In the experiments with 4NQO, both the MN frequency and the frequency of fragmented nuclei rise with increasing concentrations. After the exposure to colchicine, overall MN frequency has risen in a dose-dependent manner up to 0.125 M, where a shift in MN frequency shows decreasing values with increased concentrations. Although this pattern is interrupted by the high MN frequency of the 0.375 M group, the MN frequency further decreases for 0.5 M, and both results must be considered of little significance due to the small numbers of cells/nuclei assessed (see table 2).

[0037] The frequency of fragmented nuclei seems to present a geometric progression with increased concentrations, at least up to a certain point (which seems to be close to 0.5 M for colchicine and between 0.6 and 0.9 M for 4NQO), as evidenced by the correlation between concentrations and fragmented nuclei frequencies in both cases (FIG. 3. a, b).

[0038] FIG. 3. Frequency of fragmented nuclei in relation to different concentrations of (a) colchicine and (b) 4NQO.

[0039] The coefficients of determination (R.sup.2) close to 95% indicate a very good fit of the data to the exponential trend line displayed in FIGS. 3 a and b, although in the case of 4NQO (FIG. 3. b) the frequency of fragmented nuclei for the 1.2 M group had to be left out for the analysis to be meaningful. In any case, FN frequencies in both cases show a dose-response relationship to the concentration of these substances.

DISCUSSION

[0040] Cells not treated with a chemical agent (controls) clearly show a lower MN frequency than cells treated with either colchicine or 4NQO. The MN frequency initially rises as the concentration of the genotoxic substance is increased (table 2; FIG. 2), and for colchicine it decreases in concentrations higher than 0.188 M group (with the exception of the 0.375 M group). For 4NQO, on the other hand, no decrease in higher concentration ranges has been observed, although a dose-dependent increase for concentrations higher than 0.6 M could not be observed. These results indicate that a cytotoxic threshold may have been achieved at these concentrations, which in the case of colchicine leads to a higher mortality rate in affected cells (hence the diminishing MN frequency in concentrations higher than 0.188 M, FIG. 2. a). The stabilization of MN frequency and increase in FN rates in 4NQO indicate that the higher concentrations (especially in the range of 0.6 M and higher) induced both cytotoxicity and genotoxicity, which would explain the relative stabilization of MN frequency for 4NQO in this range (FIG. 2. b).

[0041] Surrales and collaborators (1994) have observed a MN frequency increase from 1% (in controls) to around 15.2% in human lymphocytes exposed for 24 hours to colchicine at 0.06 M. Lofti & Santelli (2006) observed MN frequencies up to 4.7% after 72 hours exposure to colchicine at 20 ng/mL (around 0.05 M) in human skin fibroblasts, which background frequencies range between 0.2 and 0.9%. In terms of in vivo studies, MN frequency in carp (Cyprinus carpio) erythrocytes increased from a background of 2% to around 10% in fish injected with 0.4 mg colchicine/kg body weight (Gustavino et al 2001). In erythrocytes of the European minnow (Phoxinus phoxinus) and of the sailfin molly (Poecilia latipinna), MN frequency increased from a background of 6.25 to 20.76% in fish injected with 10 mg colchicine/kg body weight (Aylon & Garcia-Vasquez 2000).

[0042] As for 4NQO, Valentin-Severin et al (2003) describe how the MN frequency increases from a background of 1% up to 3.5% (at 2 M) in the human liver carcinoma cell line HepG2. In the mouse lymphoma cell line L5178Y, MN frequency increases from around 0.2% (controls) to around 1.8% after 4 hours exposure to 4NQO at approximately 0.25 M (Brsehafer et al 2015). In vivo results with zebrafish (Danio rerio) erythrocytes show an increase in MN frequency from a background around 0.3% to values between 1% and 1.7% when exposed to 4NQO at 2.9 g/L in the water with a flow-through system (Diekmann et al 2004).

[0043] These results show a maximum 20-fold increase in MN frequency in cell lines exposed to colchicine and around 5-fold increase in individuals injected with the aneugen. Exposure of cell lines to 4NQO leads to a maximal 10-fold increase in MN frequency in the cell lines described, and around 6-fold increase in the erythrocytes of fish exposed to the clastogen. In general terms, these results are significantly higher than the 3- (for colchicine) and 4-fold (for 4NQO) increase observed in the cell line presented herein, but are not directly comparable considering the higher concentrations and/or longer exposure periods employed in the literature data.

[0044] The present inventive method simplifies the MNvit assay by avoiding both the harvesting and staining steps involved in alternative methods, allowing for visual and/or automated scoring (through image analysis software) of MN frequency in an easy to handle and low-maintenance cell line. The fluorescence labelled nuclei permit visualization of micronuclei through simple fluorescence microscopy, without need of high-end devices (e.g. flow cytometer) and or expert personnel, and allow for the monitoring of living cells after MN scoring for further information regarding genotoxic effects.

[0045] The presented inventive method is also suitable to distinguish between unaffected cells and carcinogenic transformed cells for direct reflection of the carcinogenic potential of the test compounds by using a differential fluorescent measurement after the non-destructive scoring process.

[0046] The coefficients of determination (R.sup.2) close to 95% indicate a very good fit of the data to the exponential trend line displayed in FIGS. 3 a and b, although in the case of 4NQO (FIG. 3. b) the frequency of fragmented nuclei for the 1.2 M group had to be left out for the analysis to be meaningful. In any case, FN frequencies in both cases show a dose-response relationship to the concentration of these substances.

DISCUSSION

[0047] Cells not treated with a chemical agent (controls) clearly show a lower MN frequency than cells treated with either colchicine or 4NQO. The MN frequency initially rises as the concentration of the genotoxic substance is increased (table 2; FIG. 2), and for colchicine it decreases in concentrations higher than 0.188 M group (with the exception of the 0.375 M group). For 4NQO, on the other hand, no decrease in higher concentration ranges has been observed, although a dose-dependent increase for concentrations higher than 0.6 M could not be observed. These results indicate that a cytotoxic threshold may have been achieved at these concentrations, which in the case of colchicine leads to a higher mortality rate in affected cells (hence the diminishing MN frequency in concentrations higher than 0.188 M, FIG. 2. a). The stabilization of MN frequency and increase in FN rates in 4NQO indicate that the higher concentrations (especially in the range of 0.6 M and higher) induced both cytotoxicity and genotoxicity, which would explain the relative stabilization of MN frequency for 4NQO in this range (FIG. 2. b).

[0048] Surrales and collaborators (1994) have observed a MN frequency increase from 1% (in controls) to around 15.2% in human lymphocytes exposed for 24 hours to colchicine at 0.06 M. Lofti & Santelli (2006) observed MN frequencies up to 4.7% after 72 hours exposure to colchicine at 20 ng/mL (around 0.05 M) in human skin fibroblasts, which background frequencies range between 0.2 and 0.9%. In terms of in vivo studies, MN frequency in carp (Cyprinus carpio) erythrocytes increased from a background of 2% to around 10% in fish injected with 0.4 mg colchicine/kg body weight (Gustavino et al 2001). In erythrocytes of the European minnow (Phoxinus phoxinus) and of the sailfin molly (Poecilia latipinna), MN frequency increased from a background of 6.25 to 20.76% in fish injected with 10 mg colchicine/kg body weight (Aylon & Garcia-Vasquez 2000).

[0049] As for 4NQO, Valentin-Severin et al (2003) describe how the MN frequency increases from a background of 1% up to 3.5% (at 2 M) in the human liver carcinoma cell line HepG2. In the mouse lymphoma cell line L5178Y, MN frequency increases from around 0.2% (controls) to around 1.8% after 4 hours exposure to 4NQO at approximately 0.25 M (Brsehafer et al 2015). In vivo results with zebrafish (Danio rerio) erythrocytes show an increase in MN frequency from a background around 0.3% to values between 1% and 1.7% when exposed to 4NQO at 2.9 g/L in the water with a flow-through system (Diekmann et al 2004).

[0050] These results show a maximum 20-fold increase in MN frequency in cell lines exposed to colchicine and around 5-fold increase in individuals injected with the aneugen. Exposure of cell lines to 4NQO leads to a maximal 10-fold increase in MN frequency in the cell lines described, and around 6-fold increase in the erythrocytes of fish exposed to the clastogen. In general terms, these results are significantly higher than the 3- (for colchicine) and 4-fold (for 4NQO) increase observed in the cell line presented herein, but are not directly comparable considering the higher concentrations and/or longer exposure periods employed in the literature data.

[0051] The present inventive method simplifies the MNvit assay by avoiding both the harvesting and staining steps involved in alternative methods, allowing for visual and/or automated scoring (through image analysis software) of MN frequency in an easy to handle and low-maintenance cell line. The fluorescence labelled nuclei permit visualization of micronuclei through simple fluorescence microscopy, without need of high-end devices (e.g. flow cytometer) and or expert personnel, and allow for the monitoring of living cells after MN scoring for further information regarding genotoxic effects.

[0052] The presented inventive method is also suitable to distinguish between unaffected cells and carcinogenic transformed cells for direct reflection of the carcinogenic potential of the test compounds by using a differential fluorescent measurement after the non-destructive scoring process.

[0053] The cited state of the art is presented in the following list of documents: [0054] Aylon F., Garcia-Vasquez, E. 2000. Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test. Mutat Res., 467(2):177-86. [0055] Brsehafer K., Manshian B. B., Doherty A. T., Zai'r Z. M., Johnson G. E., Doak S. H., Jenkins G. J., 2015. The clastogenicity of 4NQO is cell-type dependent and linked to cytotoxicity, length of exposure and p53 proficiency. Mutagenesis pii: gev069. [Epub ahead of print] [0056] Gustavino B., Scornajenghi K. A., Minissi S., Ciccotti E., 2001. Micronuclei induced in erythrocytes of Cyprinus carpio (teleostei, pisces) by X-rays and colchicine. Mutat Res., 494 (1-2):151-9. [0057] Lotfi C. F., Machado-Santelli G. M., 1996. Comparative analysis of colchicine induced micronuclei in different cell types in vitro. Mutat Res., 349(1):77-83. [0058] Surralles J., Antoccia A., Creus A., Degrassi F., Peris F., Tanzarella C, Xamena N., Marcos R., 1994. The effect of cytochalasin-B concentration on the frequency of micronuclei induced by four Standard mutagens. Results from two laboratories. Mutagenesis, 9(4):347-53. [0059] Valentin-Severin I., Le Hegarat L, Lhuguenot J. C, Le Bon A. M., Chagnon M. C, 2003. Use of HepG2 cell line for direct or indirect mutagens Screening: comparative investigation between comet and micronucleus assays. Mutat Res, 536(1-2):79-90.