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 micronuclei—as 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.
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This invention presents a reporter gene technology for non-destructive, self-signalizing visualisation of nuclear structures, which allows the identification of nuclear and chromosome anomalies—including micronuclei—as consequence of exposure to genotoxic compounds. This technology for visualising micronuclei can be applied in vitro as well as in vivo toxicological models which are transgenic for histone associated fluorescent protein. Furthermore, a corresponding transgenic stem cell line based method for the detection of the genotoxic potential of aquatic samples or aqueous solutions of test compounds is presented. This cell line has been employed in the development of kits suitable for the detection of the genotoxic potential of samples by the inventive method.
The micronucleus test is a toxicological assay to assess the genotoxic potential of substances based on the formation of micronuclei (MN). It is recognized as one of the most important tools for assessing the genotoxicity of environmental samples and newly developed chemicals, and is therefore recommended by the OECD for drug screening and ecotoxicological assessment of genotoxic potential.
The formation of micronuclei may be induced either by deterring the migration of whole chromosomes to the cell poles during anaphase (aneugens) or by promoting the formation of acentric chromosomes (clastogens).
Micronuclei represent damages transmitted to daughter cells after mitosis, hence assessing somatic inherited genetic damage that may not be assessed in the traditional chromosome aberrations test scored in metaphase cells (OECD TG 473). Thus, assessment of MN has become a mandatory criterion in environmental risk assessment and drug screening. Two major versions of the test have become standards in toxicological testing, the in vivo mammalian erythrocyte micronucleus test (OECD TG 474) and the in vitro mammalian cell micronucleus test (OECD TG 487).
In spite of its importance due to metabolic and pharmacokinetic factors difficult to reproduce in in vitro systems, the in vivo mammalian erythrocyte micronucleus test presents limitations that may only be overcome with in vitro testing. Many compounds (for instance topically applied or inhaled pharmaceuticals) are not systemically absorbed in living organisms, so that bioavailability for the target tissue (in this case, the bone marrow) may be low or negligible. Moreover, inter-individual variability in spontaneous MN frequency poses a severe difficulty for the in vivo system, demanding a thorough identification of basal MN rates for valid results to be obtained. Besides, the increasing ethical considerations regarding animal testing with vertebrates, and public and political pressure for the development of in vitro alternatives in toxicological assays, favour the usage of cell culture-based systems for the identification of the genotoxic potential of samples.
The in vitro micronucleus test (MNvit) basically consists of exposing cultured cells to a dilution series of a substance that potentially induces MN formation and comparing MN frequency with positive (i.e. aneugens and clastogens) and negative (i.e. solvent) controls after cell division. MN frequency is estimated by visually scoring micronuclei in each test group and the controls after nuclei-specific staining (e.g. Giemsa, acridine orange, propidium iodide) and slide preparation. Asides from initial culturing of cells (including extraction from the donor organism in the case of primary cells), this time-consuming process encompasses exposure, harvesting, staining and scoring. An automatic scoring technique of MN frequency by flow cytometry has been validated, but the method is cost- and knowledge-intensive, requiring excellence in both personnel and laboratory facilities in harvesting, staining and scoring of cells.
Different techniques may be used in cell harvesting and slide preparation, as long as high-quality cell preparations for scoring are obtained and cytoplasm is retained for MN assignment. To date, validity of the MNvit assay using various rodent cell lines (CHO, V79, CHL/IU, and L5178Y) and human lymphocytes has been demonstrated, but generally cell types (and/or lines) with a low, stable background MN frequency are recommended for the assay. In cell lines specifically, background MN frequency has been shown to vary from around 0.3-2% (CHL/IU, V79) up to approximately 4.7% (in the human HeLA cell line).
Slide preparation for visual scoring requires fixation of harvested cells, so that post-exposure evaluation reflects established effects only. Although some methods employing (supra)vital staining have been established for flow cytometry scoring, survival of cells after sorting is usually limited and does not allow for posterior monitoring of cell status and proliferation, hence implying that post-exposure data on cell survival rates, promotion of neoplasia in cultured cells and other related information could not be assessed until now.
Thus, a method for scoring MN in living cells that may be observed after scoring would enable the investigation of long-lasting effects of genotoxic compounds, and the provision of easy-to-handle kits containing cells with fluorescence-labelled nuclei suitable for the assessment of genotoxic potential—without need for harvesting or staining of cells—would greatly simplify routine laboratory work in genotoxic survey.
This invention presents an indicator stem cell line as well as an indicator living organism (non-human) for non-destructive, self-signalizing visualisation of nuclear structures, which allows the identification of nuclear and chromosome anomalies as consequence of exposure to genotoxic compounds.
The inventive new stem cell line is from the Koi carp (Cyprinus carpio haematopterus) brain (KCB) with nuclei successfully labelled with green fluorescent protein fused to the histone 2B (H2B-GFP), thus presenting enormous potential for assessing the cytogenetic status of cells. A background MN frequency around 0.6% and the ability to induce micronuclei in this cell line with both aneugens and clastogens has been successfully demonstrated in a series of preliminary experiments (see below), allowing for the development of kits of cells with fluorescence-labelled nuclei ready for sample exposure and scoring of nuclei/micronuclei.
Furthermore, this invention also presents a living organism (non-human), which is a transgenic fish, namely medaka (Oryzias latipes), as a living indicator organism with cell lineages (such as germ cells or erythroid cells) showing a lineage specific histone associated fluorescent protein expression. MN visualisation is possible in translucent embryos or by ex vivo preparation of blood smears.
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.
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.
The presented new method for the detection of the genotoxic potential of aquatic samples and/or aqueous solutions of test compounds—by 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 protein—comprises 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.
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.
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.
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.
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 PBS—phosphate-buffered saline), 1% penicillin (3.125 mg/ml in PBS) and 1% amphotericin (250 μg/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.
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).
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.
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.
These experiments were performed six times for each chemical agent.
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.
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.
Table 1. Concentrations of colchicine and 4NQO employed in the validation of the method for MN frequency assessment.
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.
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.
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.
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
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.
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.
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.
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 μM) than colchicine (1.77% at 0.375 μM), and a higher maximal frequency of fragmented nuclei (12.56% at 0.9 μM) than colchicine (6.85% at 0.5 μM).
FIG. 2. MN frequency after exposure to different concentrations of (a) colchicine and (b) 4NQO.
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 μM, 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 μM group, the MN frequency further decreases for 0.5 μM, and both results must be considered of little significance due to the small numbers of cells/nuclei assessed (see table 2).
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 μM for colchicine and between 0.6 and 0.9 μM for 4NQO), as evidenced by the correlation between concentrations and fragmented nuclei frequencies in both cases (FIG. 3. a, b).
FIG. 3. Frequency of fragmented nuclei in relation to different concentrations of (a) colchicine and (b) 4NQO.
The coefficients of determination (R2) 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 μM 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.
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 μM group (with the exception of the 0.375 μM 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 μM 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 μM, 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 μM and higher) induced both cytotoxicity and genotoxicity, which would explain the relative stabilization of MN frequency for 4NQO in this range (FIG. 2. b).
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 μM. Lofti & Santelli (2006) observed MN frequencies up to 4.7% after 72 hours exposure to colchicine at 20 ng/mL (around 0.05 μM) 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).
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 μM) 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 μM (Brüsehafer 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 μg/L in the water with a flow-through system (Diekmann et al 2004).
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.
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.
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.