SI23294A - Cell system for quick sensing and determination of genotoxicity - Google Patents

Abstract

A cell system for quick sensing and determination of genotoxicity is a biosensor cell system with metabolically active human cells stably transfected with a reporter gene which is operatively bound to a gene promoter which responds to DNA damage and quickly solves the problem of quick and precise detection and quantification of genotoxic substances and other samples. In this context, the stably transfected metabolically active human cells are preferably HepG2 cells, the reporter gene is preferably a gene that codes for a fluorescent DsRed2 protein, and the promoter that responds to DNA damage is preferably a p21 gene promoter. A construct, which HepG2 cells are transfected with, is composed of a coding sequence of nucleotides coding for DsRed protein, operatively bound in cis configuration to a p21 promoter region. A method for measurement of genotoxicity consists of the following steps: contact of p21-HepG2- DsRed cells with a tested chemical or sample, incubation at 37 degrees Celsius and 5 % CO2, measurement of fluoresence of the expressed DsRed protein with a microtiter plate reader after 24 and 48 hours, determination of cell density with the MTT test and calculation of normalized values of induced fluorescence, which is a qualitative and a quantitative indicator of genotoxic activity of the tested chemical or sample.

Description

The subject of the invention is a method for determining the genotoxicity of chemicals and other samples and a cellular system for this use. The invention describes a biosensor system for detecting DNA damage in metabolically active cells in culture.

The problem solved by the invention is a method that enables the rapid and accurate measurement of genotoxicity by means of a microfiber spectrofluorimeter. The biosensor system consists of metabolically active cells transfected with a construct with a reporter gene for a fluorescent DsRed2 protein operatively linked to the p21 promoter operating region of the gene that specifically responds to DNA damage. Due to the DsRed reporter gene whose product fluoresces outside the autofluorescence range of cellular components and cyclic organic chemicals, the measurement results are not subject to autofluorescence errors and do not require additional steps to determine the autofluorescence fraction or the use of more sophisticated fluorescence measurement methods such as flow cytometry or fluorescence.

In general, it is important for the protection of human health and the environment that the factors causing DNA damage are identified in a timely manner. In most countries, legislation requires the submission of data confirming that the chemical is not genotoxic before it goes on the market. Different methods are available to detect genotoxicity, but most are time-consuming and expensive. The best known standard method is the Ames test, which measures mutagenicity using bacterial strains that are defective for the synthesis of an essential amino acid. The bacteria are grown in a medium free of essential amino acid in the presence of the test chemical. If bacteria can grow and form colonies in the presence of the test chemical, it means that mutations have occurred and that the chemical is mutagenic. Other tests are the in vitro micronucleus test and the mouse lymphoma test, which detect chromosome damage, the latter mutations. These methods take several days to several weeks and require large quantities of test chemical. Moreover, these methods are not suitable for rapid screening.

Genotoxic substances are those that can cause damage to genomic DNA. "Damage" means that the function of DNA is destroyed or impaired so that the DNA does not express the gene or express it only partially or incorrectly. The damage is done in different ways: by chemical alteration of the nucleotides that make up the DNA, or by breaking the phosphodiester bonds that bind the nucleotides, or by misfolding the pairs of nucleic bases. Due to the importance of DNA functions, organisms have developed complex defense mechanisms against DNA damage, namely cell cycle arrest, repair of DNA damage, and apoptosis, all of which contribute to maintaining genomic stability. In bacteria, DNA damage triggers the well-known SOS response, which activates about 20 different genes (Walker, Ann. Rev. Biochem, 54, 425, 1985). An even greater number of genes are involved in the response to DNA damage in yeast (Weinhart and Hartwell, Science, 421, 317, 1988; Rowley et al., Nature 356, 353, 1992) and mammalian cells (Holbrook and Fornace, New Biologist 3, 825,1991).

It is known that it is possible to measure changes in gene expression and reporter systems have been developed for this purpose. Expression of genes that respond to DNA damage can be used as an indicator of genotoxicity. The most well known are bacterial systems in which SOS response genes have been used to detect genotoxicity (Ben-lsrael and Ben-lsrael, Appl. Env. Microbiol, 64, 4346, 1998; Ouillardet et al. Biochemie, 64, 797, 1982; Ptitsyn et al., Appl. Env. Microbiolo., 63, 4377, 1997; Oda et al., Mutat. Res., 147, 219, 1985). The Saccharomyces cerevisiae yeast system known as GreenScreen® has recently been developed (Cahill et al., Mutagenesis, 19, 105, 2004). In the GreenScreen test, the RAD54 promoter is associated with the gene for green fluorescent protein (GFP). DNA damage induces RAD54, which also increases the synthesis of GFP, which can be quantitatively measured. Another test (Car-Tox (L)) was developed in 1995 by Todd et al (Todd et al., Fundam. Appl. Toxicol, 28, 118, 1995) with a series of 14 recombinant HepG2 cell lines, each containing a specific stress promoter bound to chloramphenicol acetyl transferase (CAT) reporter gene. Four genes that respond to DNA damage are included in this system: the GADD45 growth arrest and DNA damage protein (GADD45) and 153, the early c-fos response protein, and the 53 kDA tumor suppressor response element P53RE.

The most important route of eukaryotic response to DNA damage is the activation of tumor suppressor and p53 transcription factor via phosphorylation by DNA damage-responsive kinases (Zhou and Elledge 2000, Nature 408, 433, 2000). Activated p53 induces the expression of genes involved in DNA repair, cell cycle arrest and apoptosis, which either repair DNA damage or trigger apoptosis in overly damaged DNA (Sionov and Haupt, Oncogene, 18, 6145, 1999). US Patent Nos. 6,344,324 and WO 2005/113802 disclose a method for detecting changes in GADD 153 gene expression or GADD45 (activated by p53) via expression of the EGFP reporter protein as an indicator of the induction of DNA damage. US 6,344,324 describes that injury detection is possible via induced EGFP expression in a cell line. This document describes a reporter system that combines a reporter gene amplified green fluorescent protein (EGFP) with a GADD153 promoter. This construct is stably inserted into the genome of the cancer cell line. They found that EGFP expression was detectable after contact with the toxin, but not in all cells and only after a few days after maximal expression after 6 days. The test describes the detection of expression after four days by flow cytometry. Significant differences in the responsiveness of the transfected cells have been demonstrated and, although the system allows the detection of damage, it is still time consuming and does not produce sufficiently reliable results. Document W02005 / 113802 describes, however, a system where a construct having the GADD45a gene promoter is operatively linked to the EGFP reporter gene and is inserted into the TK6 lymphoblastoid cell line. The response of the system is good, the results are obtained within 48 hours, and its main disadvantage is that it does not allow accurate detection of indirectly acting genotoxic substances that require metabolic activation.

Recent toxicogenomic studies have shown that the GADD45 and 153 genes are relatively nonspecific and their response is also caused by non-genotoxic carcinogens (Ellinger-Ziegelbauer et al., Mutat. Res. 575, 61, 2005), and is therefore not a reliable indicator of genotoxicity. This study showed that only a few genes specifically respond to genotosic carcinogens-induced damage: cyclin-dependent kinase 1A inhibitor (CDKN1A), cyclin Gl, and 06-methylguanine-DNA methyltransferase (MGMT). These genes responded with increased expression only after exposure to genotoxic carcinogens.

The cyclin-dependent kinase 1A (CDKN1A) inhibitor: p21 (Wafl / Cipl) was found to be a p53-regulated suppressor gene. Several groups independently cloned p21 cDNA using different strategies. Using yeast hybrid screening, p21 was found to be a CDK-binding protein and was termed CIPI as a "CDK-interacting protein" (Harper et al., Celi, 75, 805, 1993). Microsequencing of the interaction of the protein with CDK led to its cloning by PCR (Xiong et al., Nature 366, 701, 1993). They found that p21 expression was directly induced by p53 because the entire promoter is under p53 control. Therefore, it was also called wafl (wilde-type p53-activated factor) (ElDeiry et al., Celi, 75, 817, 1993). They also found that p53-dependent regulation of p21 is critical in responding to DNA damage (Macleod et al., Genes Dev., 9, 935, 1995).

A reporter construct composed of the p21 regulatory transcriptional region covalently bound in the cis configuration to a measurable gene product and a method for detecting potential therapeutic factors capable of inhibiting tumor cell growth through p21 activation was disclosed by Vogelstein and Kinzler in US2002 patent / 142442. The authors proceeded from the assumption that a factor that activates p21 expression is useful for cancer treatment because p21 is involved in cell cycle arrest. Therefore, the method disclosed by Vogelstein and Kinzler refers to an approach where a potential therapeutic chemical is incubated with cancer cells containing a p21 transcriptional regulatory region reporter region bound in a cis configuration to a gene that codes for a measurable product and measuring the formation of this product. A factor that enhances the cellular formation of a measurable product is considered to be potentially therapeutic which will activate p21 expression and thereby inhibit tumor cell growth.

However, to date, there is no reliable method available to detect the genotoxicity of chemicals in the short term and which could be commercially useful for high-throughput screening. There is still a need for simple, rapid and inexpensive methods for the identification of genotoxic substances, in particular substances capable of causing DNA damage, mutations and other genetic damage in eukaryotic cells and especially in human cells. In addition, methods for improved toxicity testing and with safer materials are needed.

Therefore, the purpose of the invention is a method that will improve the detection and determination of the genotoxicity of chemicals and other samples. In particular, the purpose of the invention is to provide a test system that is safe, fast, inexpensive, and reliable at the same time. These purposes are achieved by the method defined in claim 1.

One aspect of the present invention is to exploit changes in the activation and expression of a specific p53-activated gene as an indicator of DNA damage, and to use this as a tool for the early detection and determination of DNA damage caused by genotoxic factors.

The invention provides a method for detecting and / or determining genotoxicity, consisting of the following steps:

a) Contact of the test substance with a biosensor system consisting of:

a. Metabolically active cells transfected with the construct containing

b. A nucleotide sequence encoding for DsRed or a derivative thereof, operably linked in a cis configuration to

c. The p21 transcriptional regulatory region, or functional equivalent thereof, wherein the p21 regulatory region is activated in response to DNA damage

b) Measurement of DsRed expression

c) Determining after a certain incubation time whether there is a change in the expression of DsRed protein in the test cells as a result of contact with said test substance compared to a control not in contact with said test substance or any genotoxic substance, wherein an increase in DsRed expression means that the test substance causes DNA damage.

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In other words, the genotoxicity of a substance can be determined by incubating the substance with cells containing a reorter construct where the reporter protein is expressed by activation via p21 in response to DNA damage. The expression of the reporter is measured, usually by measuring the fluorescence of the reporter, e.g. the fluorescence index (MFI) is determined and compared with the standard value obtained for cells that have not been in contact with the test substance. If the expression value, e.g. An MFI higher than the standard value is an indicator of the genotoxicity of a substance and a quantitative assessment of the potency of genotoxicity can be made based on the difference between the two values.

It has been shown that genotoxicity can be reliably and in a short time detected by a system consisting of host cells transfected with a construct with a specific regulatory region and a specific reporter gene that primarily integrates a stable integrated construct into the host cells. Reliable results are obtained within 24 to 48 hours.

In the description presented herein, "DNA damage" means any damage or alteration of genomic DNA that results in a malfunction of DNA that results in the disabling, reduced or incorrect expression of the gene. The damage can be caused by various changes, such as bond breaks as described above.

"Genotoxic" in this specification means an adverse effect on DNA that causes damage.

"P21 transcriptional regulatory region" refers to a regulatory region that is responsible for the initiation of p21 expression and is activated by p53. The sequence of the p21 transcriptional regulatory region is known (see ElDeiry et al., Celi, 75, 817, 1993). The functionally equivalent transcriptional regulatory region of p21 is any sequence that has a homology with p21 of at least 70%, preferably at least 80%, and preferably at least 85%, and allows the initiation of gene expression in response to activation by p53. The version may have a codon in use that is silent swap and / or conservative base pairs that do not change function.

The present invention relates to the use of the p21 promoter as a p53-activated transcriptional regulatory region. The promoter region of the p21 gene is known and any variant containing the transcriptional regulatory activity of p21 can be used. The p21 promoter variants or derivatives are well known in the art and can be made by known methods and methods.

The reporter protein of the present invention is a DsRed protein, and a DsRed or functional derivative is present in the construct. The DsRed protein is known, is fluorescent, and is used as a reporter protein. Versions are also known. Functional versions of the DsRed sequence in this specification are sequences that encode for DsRed and may have silent and / or conservative substitutions of base pairs that do not alter the function of the DsRed protein.

It has been found that the system of the present invention enables reliable and rapid detection of genotoxic substances. This is due to the combination of host cells used, specific promoter and specific reporter gene, which alone in this combination give reliable results.

The assay contains the p21 transcriptional regulatory region. This specific regulatory region allows for high sensitivity and system specificity. Cell viability and apoptosis are highly regulated in cells. Inactivation of p53 inactivates apoptosis and cell arrest, which increases genetic instability, a common feature in the development of many human neoplasias. Because apoptosis is an important factor in ensuring genetic stability, a cell responds to p53 activation by activating the expression of genes containing p53 binding sites. Thus, p53-induced genes play the role of tumor suppressors, and most studies have focused on this aspect of the role of p53-activated genes. The gene whose expression activates p53 is p21 or WAF1 whose entire promoter region is under the control of p53 in mammalian cells. The p21 promoter has proven to be very useful with the construct of the present invention because it is very sensitive to cell damage. This sensitivity is due to the fact that p53 induces p21 directly. It is assumed that there is at least one strong binding site in the transcriptional regulatory region. Instead of indirect activation, as in the methods known to date, the present invention utilizes direct activation of the p21 promoter.

It is known that p53-dependent regulation of p21 is critical for responding to DNA damage, and this property has been used to identify genotoxic substances by the method of the present invention based on the detection of changes in gene expression under the control of the p21 promoter.

In the construct used in the present invention, the transcriptional regulatory region p21 is covalently bound in the cis configuration to a gene for a determinable product, in this case DsRed.

The constructs that bind the p21 promoter to the reporter protein have already been described, for example, in US2002 / 142442, where they have been used for another purpose and have used another reporter protein; luciferase activity. The construct described in US2002 / 142442 was used to detect potential therapists who • would suppress tumor cell growth by activating p21 expression. However, we found that these constructs were not used to determine genotoxic effects.

Many reporter genes are known, such as for β-galactosidase, luciferase, chloramphenicol acetyl transferase, but all of these require a substrate to monitor their expression in the form of their activity. Therefore, an additional step is required when conducting tests with these reporters. Reporters based on fluorescent proteins such as green fluorescent protein (GFP) and its derivatives have the advantage that their expression can be measured and evaluated directly. However, GFP has been shown to have an emission spectrum in the wavelength range that coincides with the autofluorescence of many organic compounds. Therefore, when using these known constructs, the results may be too high or false positive. In contrast, the present invention used a fluorescent protein produced by Discosoma sp strains; DsRed encoded by pCLEF35. The variant is the DsRed2 protein, which contains a series of silent base pair substitutions and is suitable for achieving high expression in human cells. DsRed2 contains six amino acid substitutions: A105V, I161T, and S197A, which contribute to the faster occurrence of fluorescence in transfected cells and R2A, K5E, and K9T when they prevent protein aggregation.

The excitation maximum of DsRed is at 563 nm and the emission maximum at 582 nm, ie in the range of wavelengths that do not coincide with the autofluorescence of proteins. In contrast, the EGFP excitation peak at 395 and the second excitation peak at 457 and the emission peak at 510 nm.

The p21 promoter and the DsRed reporter protein gene are operatively linked so that the reporter protein is expressed under the control of the p21 promoter. They are linked covalently in the cis configuration.

We have found that the best results are obtained if the construct of the present invention is stably introduced into the genome of the hepatocyte cell line, especially HepG2. The stably transformed HepG2 cells containing the construct of the present invention produce a reliable result in a short time. Therefore, the use of a HepG2 cell line stably transformed with the p21-DsRed2 gene is preferably included.

The system is a cost-effective tool for the detection of genotoxic substances. The system can be used for high-throughput screening of chemicals to find those that cause DNA damage.

The present invention relates to a method that detects at least one genotoxic substance where the sample is incubated with the detection system described. The incubation time is long enough for expression of the reporter protein if

the sample is genotoxic and the upper limit is not critical. One advantage of the present invention is that reliable results are obtained with the system of the present invention within 24 hours and at the latest within 48 hours.

During incubation, the cell line is exposed to the test sample. If the sample causes DNA damage, it triggers a cascade of reactions regulated by activated p53. P53 activates the p21 promoter and, under the control of this promoter, a reporter protein is produced that produces a fluorescent signal.

Another advantage of using the system of the present invention is that the expression of the reporter protein occurs directly with DNA damage. Therefore, the system is more sensitive and accurate than other known systems. An expression cassette consisting of a DNA sequence encoding a DsRed2 reporter protein, the sequence of which is operatively linked to the human p21 promoter, is activated so that its expression is activated in response to DNA damage (Figure 1).

Any known vector, such as a plasmid, cosmid or phage, may be used as the recombinant vector of the present invention. These vectors allow replication of the expression cassette. In addition, recombinant vectors enable the transfection of cells with an expression cassette and may also allow the expression of a reporter protein.

The recombinant vector used in the present invention may be such that it reproduces autonomously in the cytosol of the cell or may be such that it is incorporated into the genome of the host cell. Vectors and methods for constructing vectors with known. Elements that enable DNA replication are required in recombinant vectors. Suitable elements are known and obtainable from various commercially available vectors such as pCEP4 (Invitrogen, 3 Fountain Drive, Inchinnan Business Park, Paisley, PA4 9RF, UK) pEGFPN1 (BD Biosciences Clontech UK, 21 and Betvveen Towns Road, Cowley, Oxford, OX4LY, United Kingdom) or pCI and pSI (Promega UK Etc, Delta House, chilvworth Science Park, SouthamptonS016 7NS, UK). Preferably, the recombinant pp21-DsRed2 vector shown in Figure 1 is used in the method of the present invention.

Metabolically active host cells are an integral part of the biosensor system of the present invention. Preference is given to eukaryotic cells. Cells may be mammals including cells of human origin as well as other vertebrate and invertebrate cell lines.

Preference is given to human cell lines. Preferably, the host cell lines are human cells with fully functional p53. Preferably, the host cells are metabolically active, expressing phase I and phase II metabolic enzymes in an inducible form.

The inventors have found that human hepatoma HepG2 cells are a particularly suitable cell line for use in the method of the present invention. Although the inventors do not want to hypothesize, they believe that HepG2 cells are most appropriate because they have fully functional p53 and express more phase I and II metabolic enzymes in an inducible form.

The host cells used to express the DNA-encoded reporter protein are preferably stably transfected, although the use of unstable (transiently) transfected cells is also possible.

Stably transfected cells can be obtained by the procedure described in Example 1. Preferably, the cells are human, for example HepG2. Such transfected cells are best for use in the method of the present invention, for determining whether the test substance induces DNA damage.

The best biosensor system for the method of the present invention consists of HepG2 cells transformed with the p21-DsRed2 vector. These cells here are called p21-HepG2-DsRed.

The method of the present invention is best performed by multiplying cells transfected with the recombinant p21-DsRed2 vector, incubating them for a certain time with a substance likely to damage DNA, and measuring the expression of a reporter protein that emits light directly on the cells.

In the next step of the method of the present invention, the expression of a reporter protein is determined, which is usually done by measuring the mean fluorescence index (MFI). Methods for measuring MFIs and their use are known. Because the emission wavelength is outside the range of autofluorescence, the measured value can be attributed to the expression of a reporter protein, thereby largely excluding the false positive results.

The methods for detecting and quantifying fluorescence are generally known, one method being described in Example 2 (hereinafter).

The cells are best grown in a medium with low fluorescence. This avoids the need to rinse the cells before measurement, thus reducing the number of operations compared to known methods. For example, it is best to grow human cells in a low-fluorescence medium such as minimal essential medium without phenol red. The use of such a medium in fluorescence measurement reduces the signal-to-background ratio.

It is best to incubate the cells with the test substance on microtiter plates and measure fluorescence and absorbance directly on the wells of the microtiter plate. Suitable microtiter plates are known and commercially available. For example, the microtiter plate is black with a transparent bottom and 96 wells. Fluorescence and absorbance can be measured with a suitable microtiter plate reader. Microtiter plate readers are well known in the art and are commercially available. An example is the Tecan Infinitie 2000 (Tecan UK) microtiter plate reader.

We measure the MFI and compare it with the standard value to determine if the test substance has genotoxic activity, if the necessary genotoxic activity is also quantitatively evaluated. Genotoxicity is assessed by the value of x / y, where x is the MFI in the presence of the test substance and y is the standard MFI value measured in cells not in contact with the test substance. If the MFI of cells of the test substance exposed is statistically significantly different from the MFI of control, non-exposed cells and the x / y ratio is greater than 1.5, the test substance is considered to be genotoxic if the value of the x / y ratio is less than 1.5 or the difference between the MFIs of the exposed and non-exposed cells are not statistically significant, the substance is considered to be non-toxic.

However, the MFI value depends not only on the excitatory fluorescence of the reporter protein in response to genotoxic damage but also on cell density. The higher the cell density, the higher the fluorescence and the lower the density, the lower the fluorescence. To correct this dependence, we divide the fluorescence data by the density of the cells to obtain "fluorescence units", that is, the average fluorescence per cell. This value is independent of cell density and is therefore more reliable.

Cell density can be determined in ways known to those skilled in the art, for example, by the MTS assay. The MTS test is a colorimetric method for identifying the number of viable cells and for determining the cytotoxicity of a test substance. The test measures the formation of a soluble formazan product that is directly proportional to the number of live cells in culture. The absorbance value (determined at 492 nm) reflecting cell density is then used to normalize the fluorescence signals. When setting up the experiment, the number of cells not exposed and exposed is equal to the value of the absorbance of formazan exposed • · • · cells against non-exposed at the same time giving information about the effect of the test substance on cell viability. The invention will now be explained by way of example. Methods for measuring fluorescence and cell density are described in the examples.

Example 1:

Vector construction and cell transfection:

The example lists the components used to construct the p21-DsRed2 vector according to the first and second aspects of the invention, describes the construction of the biosensor (transfected cells according to the third aspect of the invention), and the selection of response clones.

1. System components:

a. Promoter - human p21 (CDKN1A; Cap20; CDKN1; CIPI; MDA-6; P21; SDI1; WAF1; P21CIP1) gene

The source of the p21 promoter is the WWP-LUC plasmid provided to us by prof. Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore, Maryland, USA). The plasmid has the pBluescript (KS +) vector as described by El-Deiry et al. (Celi, 75, 817,1993).

b. Fluorescent Protein - DsRed2:

The DsRed2 source is the pCLEF35 DsRed2 plasmid (Invivogen). pCLEF35 DsRed2 encodes a fast version of Discosoma sp. a rapidly evolving red fluorescent protein (DsRed). DsRed2 contains a series of silent base-pair substitutions corresponding to high expression in human cells (excitation maximum = 563 nm, emission maximum = 582 nm). In addition to these changes, DsRed2 has six amino acid substitutions: A105V, O161T, and S197A, which contribute to the faster occurrence of red fluorescence in transfected cells and R2A, K5E, and K9T, which prevent protein aggregation.

2. Design of the biosensor reporting cassette (Figure 1)

The construction of the recombinant vector containing the p21 promoter in the reporter cassette was performed in several steps using the plasmid pEGFP-N1 (Clontech) as a framework. pEDFP is a mammalian expression vector that has an immediate early cytomegalovirus promoter / promoter (CMV) for high transcription of the genes behind it. EGFP sequences were changed to Kozak's general starting sites to increase translational efficiency in the eukaryotic cell. There is a multiple cloning site (MSC) between the CMV promoter and EFGP coding sequences. SV40 polyadenylation signals along the EGFP direct the proper production of EFGP mRNA from the 3 'end. The vector framework also contains an SV40 replication initiator for replication in mammalian cells expressing the SV40 T antigen. Neomycin Resistance Cassette (Neor), which consists of it

The SV40 early promoter, the neomycin / kanamycin Tn5 resistance gene, and the polyadenylase signal from the Herpes simplex virus thymidine kinase gene (HSV TK) allow selection of stably transfected cells using G418.

Stage I: (Removal of CMV promoter from pEGFP-Nl plasmid) - CMV promoter was excised from pEGFP-Nl plasmid by Nhel and Sall restriction enzymes and top end ligation. This non-promoter plasmid was named pEGFP-N1 without CMV.

II. Level: (p21 promoter insertion) The 5 'region of p21 was excised from the WWP-Luc plasmid by EcoRI and Sall enzymes, yielding about a 2.4 bp long fragment of the p21 promoter region with a p53 recognition site. This fragment was inserted into the multiple clone site of the plasmid obtained in Stage 1.

III. Level: (Replacing the EGFP sequence with the DsRed2 sequence) The DsRed2 coding sequence was cut from pCLR35DsRed2 with Ncol and Nhel and cloned into a pORF-mlL-12 (invivogen) plasmid to obtain the new restriction site required for further cloning. We cut the DsRed2 coding sequence again, this time with Sall and Cbal restriction enzymes, and replaced the coding sequence for EGFP in p21-EGFP with the sequence for DsRed2. The resulting plasmid was named pp21-DsRed2.

3. Cell line:

HepG2 cells (85011430 ECACC, Wiltshire, UK) are derived from hepatocellular carcinoma of a 15 year old boy. These cells secrete plasma proteins and express many liver enzymes (Science, 1980, 209, 497). HepG2 cells are one of the few cell lines with conserved activity of enzymes for the metabolism of foreign matter, which is usually lost during in vitro culture. Cells have conserved wild-type p53, which is indispensable for cells to respond to genotoxic stress in a manner necessary for the reporter's response. Cells were grown in minimal essential medium without phenol red (to avoid any interference with DsRed fluorescence) with the addition of 10% heat-inactivated calf serum.

4. Cell line transfection and isolation of transfected clones

HepG2 cells were trypsinized and centrifuged at 70% confluency. The pellet was resuspended in 125mM sucrose in PBS buffer and the density was adjusted to 3.3 * 10 ^{4 5} cells / pL. 20 pg of plasmid p21-DsRed2 was added to l * 10 ⁶ cells. A 50 pL droplet with plasmid and cells was inserted into a 2 mm gap between the electroporator electrodes (developed at the Faculty of Electrical Engineering, Ljubljana, Slovenia). For each electroporation, a 5 ms long pulse of 8 600 V / cm ^{2 was used} . After electroporation, cells were transferred to 6-well plates. 5 minutes after electroporation, 3 mL of antibiotic-free medium was added to each well. 48 hours after transfection, the medium was replaced with a complete medium containing lmg / mL G418 (Sigma, St Louis, USA). After 14 days of selection, the grown colonies were transferred to 96-well plates. The isolated clones were propagated in culture medium with 0.5 mg / mL G418, frozen at -80 ° C, and then stored in liquid nitrogen for long-term storage.

5. Selection of clones that respond to treatment with the genotoxic substance MMS

Selected clones of p21-DsRed2 transfection cells were planted on 96-well plates (50,000 cells / well) and allowed to attach overnight. The following day, they were treated with 50 pg / mL methyl methane sulfonate (MMS), which is a known genotoxic substance. Clone fluorescence was measured after 24 and 48 hours with a spectrofluorimeter (Tecan, Austria). Cell density / cytotoxicity was determined by MTS assay. DsRed induction was normalized to cell density. Clone 1 of p21-DsRed2 transfected cells was selected for further use because it was the most responsive. The new cell line was named p21-HepG2-DsRed.

Example 2:

Genotoxic substances induce measurable expression of DsRed protein in p21-HepG2-DsRed cells. Cellular biosensor.

The p21-HepG2-DsRed cells were exposed to the following genotoxic substances: methyl methane sulfonate (MMS), cisplatin (CP), and benzo (a) pyrene (BaP). The selected genotoxic substances represent an S _N2 alkylating substance, a bifunctional alkylating substance, and an indirectly acting genotoxic carcinogen that needs metabolic activation to produce a nucleophilic intermediate that binds to a DNA molecule.

Test: We used the following protocol:

In minimal essential phenol-free medium with 10% calf serum, a suspension of p21-HepG2-DsRed cells was prepared at an exponential growth phase of 3 * 10 ⁵ cells / mL. The suspension was divided into 3 mL plastic tubes. To each tube was added 30 µL of test substance of a given concentration (100 times higher concentration than the final concentration) to a ratio of 30 µL of control solvent. The following final MMS concentrations were used: 5, 10, 20, 40, 50 pg / ml; CP: 0.4125, 0.825, 1.65, 3.3, 6.6 pg / ml; BaP: 30, 20, 10, 5, 2, 1, 0.5 and 0.2 pM. From each tube, 100 pL were divided into 6 wells of 96 wells with a transparent bottom (Greiner) and incubated at 37 ° C in a humidified atmosphere incubator and 5% CO ₂ . After 24 hours of incubation, DsRed fluorescence was measured with a Tecan Infinite 200 microtiter plate reader (Tecan UK) at 560 nm excitation and 590 nm emission. The plates were then incubated for a further 24 h and fluorescence was measured again. Cell viability was determined after 24 and 48 hours by adding MTS reagent (Promega) to each well with treated and control cells and incubated for a further 2 hours. The absorbance at 492 nm was then measured to determine the formazan product, which is an indicator of the cell density and toxicity of the chemical.

Transfer the measured fluorescence and MTS absorbance data to Excel worksheets and present graphically. Fluorescence measurements are divided by MTS absorbance data to obtain the average induction of DsRed per cell ("fluorescence units"). We normalize the data to the untreated control (= 1) to obtain a "relative DsRed induction ratio". The MTS absorbance data were normalized to the untreated control, yielding relative cell viability.

Figure 2 shows the relative liveliness (A) and relative induction of DsRed (B) expression in p21-HepG2-DsRed cells after MMS exposure for 24 and 48 hours, respectively.

Figure 3 shows the relative liveliness (A) and relative induction of DsRed (B) expression in p21-HepG2-DsRed cells after CP exposure for 24 and 48 hours, respectively.

Figure 4 shows the relative liveliness (A) and relative induction of DsRed (B) expression in p21-HepG2-DsRed cells after BaP exposure for 24 and 48 hours, respectively.

Claims (10)

1. Cellular rapid genotoxicity detection and detection system, characterized in that it detects and / or quantifies the genotoxicity of chemicals and other samples, consisting of the following stages: a. contact of a chemical or sample with a biosensor system consisting of: i. metabolically active cells with introduced construct ii. a nucleotide sequence encoding for DsRed or a functional derivative operably linked in the cis configuration to iii. transcriptional regulatory region of p21 gene or its functional version activated in response to DNA damage b. measuring DsRed2 expression c. determining, after a period of time, whether there was a change in MFIs in the test cells, where increased expression of the DsRed2 protein as a result of contact with said chemical or sample compared to a control that did not contact the test chemical or sample or any other genotoxic chemical means that said chemical or sample causes DNA damage. A cellular rapid genotoxicity detection and determination system according to claim 1, characterized in that the metabolically competent cells are a human cell line. A cellular rapid genotoxicity detection and determination system according to claim 2, characterized in that the metabolically competent HepG2 host cells are a cell line. Cell system for rapid detection and determination of genotoxicity according to the preceding claims, characterized in that the metabolically active cells are stably transfected with the construct. Cell system for rapid detection and determination of genotoxicity according to claim 1, characterized in that the biosensor system consists of a cell line stably transfected with an expression vector consisting of coding sequences for the DsRed2 protein operatively linked to the p21 promoter. . A cellular system for rapid detection and genotoxicity determination according to the preceding claim, characterized in that the biosensor system consists of p21-HepG2-DsRed cells stably transfected with an expression vector consisting of a DsRed2 coding sequence operatively linked to the p21 promoter, whereby the expression of said DsRed induces protein in response to DNA damage. 7. Cellular system for rapid detection and determination of genotoxicity according to the preceding claims, characterized in that the expression of DsRed is detected spectrofluorimetrically. Cellular system for rapid detection and determination of genotoxicity according to the preceding claims, characterized in that the expression of DsRed is detected by flow cytometry. Cell system for rapid detection and determination of genotoxicity according to the preceding claims, characterized in that the expression of DsRed is detected by fluorescence microscopy Cell system for rapid detection and determination of genotoxicity according to the preceding claims, characterized in that the system consists of metabolically competent cells transfected with a construct consisting of a DsRed coding nucleotide sequence or functional derivative operably linked to the transcriptional regulatory region p21 or a functional version thereof, wherein the p21 regulatory region is activated in response to DNA damage (Biosensor system for the use of the methods of the preceding claims).