Methods for evaluating hepatotoxicity caused by test substances and cells

Abstract

This disclosure provides a highly sensitive and accurate method for evaluating the hepatotoxicity of a test substance in vitro. This disclosure also provides cells used in such an evaluation method. [Solution] A method for evaluating the hepatotoxicity of a test substance using cells, comprising the steps of: culturing cells in which the activity of glutathione reductase is reduced; culturing cells in the presence and absence of the test substance; and measuring the concentration of oxidized glutathione, reduced glutathione, and / or total glutathione in each cell after culturing.

Description

[Technical Field]

[0001] This disclosure relates to a method for evaluating hepatotoxicity caused by a test substance and to cells. [Background technology]

[0002] Because the liver plays a central role in metabolism, it tends to generate reactive oxygen species (ROS) during the metabolic processes of drugs and chemicals. In the liver, oxidative stress is mainly caused by the generation of ROS via metabolic enzymes and by the covalent bonding of reactive metabolites to proteins. Oxidative stress refers to a state in which the balance between the production of ROS within cells and the antioxidant defense mechanism is disrupted. Normally, this balance is maintained in the body, but oxidative stress can be caused by factors such as ultraviolet radiation, radiation, air pollution, tobacco, drugs, and the ingestion of oxidized substances, and can be a factor in various diseases and aging. Oxidative stress has also been reported as a contributing factor to hepatotoxicity.

[0003] As a method for evaluating the hepatotoxicity of chemical substances in vitro, for example, Patent Document 1 discloses a method for evaluating hepatotoxicity performed in a culture vessel formed from a substrate in which part or all of the culture surface contains a 4-methyl-1-pentene polymer. According to Patent Document 1, using the above culture vessel enhances the drug metabolism function of hepatocytes and increases their toxicity sensitivity, thereby enabling highly sensitive toxicity evaluation of chemical substances. Non-Patent Documents 1 to 4 also report research results on oxidative stress, etc. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2023-358 [Non-patent literature]

[0005] [Non-Patent Document 1] Sonia Sentellas, et al., “GSSG / GSH ratios in cryopreserved rat and human hepatocytes as a biomarker for drug-induced oxidative stress,” “Toxicology in Vitro,” Vol. 28, (Netherlands), Elsevier, 2014, pp. 1006-1015. [Non-Patent Document 2] Laia Tolosa, et al., “Long-term and mechanistic evaluation of drug-induced liver injury in upcyte human hepatocytes,” “Archives of Toxicology,” Vol. 93, (Germany), Springer Nature, 2019, pp. 519-532. [Non-Patent Document 3] Takafumi Tomida and others, “A modified multiparametric assay using HepaRG cells for predicting the degree of drug-induced liver injury risk”, “Journal of Applied Toxicology”, Vol. 37, (USA), Wiley, 2017, p. 382-390 [Non-Patent Document 4] Xuelian Jia, et al., “Mechanism-driven modeling of chemical hepatotoxicity using structural alerts and an in vitro screening assay,” “Journal of Hazardous Materials,” Vol. 436, (Netherlands), Elsevier, August 15, 2022, pp. 1-31. [Overview of the project] [Problems that the invention aims to solve]

[0006] As mentioned above, oxidative stress is a contributing factor to hepatotoxicity. Normally, glutathione exists as reduced glutathione (also known as GSH) in the cells of mammals and other living organisms, but it is converted from GSH to oxidized glutathione (also known as GSSG) in response to stimuli such as oxidative stress. As oxidative stress increases, GSH concentration decreases and GSSG concentration increases, so it is thought that the degree of oxidative stress caused by a test substance, such as a chemical substance (referred to as the degree of oxidative stress), can be determined based on the intracellular glutathione concentrations.

[0007] Incidentally, the inventors of this invention have found that evaluating the hepatotoxicity of a test substance using conventional in vitro evaluation methods requires complicated procedures or insufficient detection sensitivity, leading to misassessment of the toxicity of the test substance. For example, a substance that actually induces or promotes oxidative stress (referred to as an oxidative stress-positive compound) may be mistakenly evaluated as a substance that does not induce oxidative stress (referred to as an oxidative stress-negative compound) due to insufficient detection sensitivity. Thus, a method that can evaluate the hepatotoxicity of a test substance with high sensitivity and accuracy has not yet been developed.

[0008] This disclosure is made in view of the above-mentioned circumstances and aims to provide a highly sensitive and accurate method for evaluating the hepatotoxicity of a test substance in vitro. This disclosure also aims to provide cells used in such an evaluation method. [Means for solving the problem]

[0009] Disclosure 1 is a method for evaluating the hepatotoxicity of a test substance using cells, (a) A step of culturing the above cells in which the activity of glutathione reductase is reduced, (b) A step of culturing the cells in the presence and absence of the test substance, (c) A method for evaluating hepatotoxicity, comprising the step of measuring the concentration of oxidized glutathione, reduced glutathione, and / or total glutathione in each of the above cells after culture.

[0010] The present disclosure 2 is the evaluation method of the present disclosure 1, in which the above step (a) is a step of culturing the above cells in the presence of a glutathione reductase inhibitor, a step of culturing cells in which the gene encoding glutathione reductase has been knocked down, or a step of culturing cells in which the activity of the selected glutathione reductase has been decreased.

[0011] The present disclosure 3 is the evaluation method of the present disclosure 2, in which the above glutathione reductase inhibitor is at least one selected from the group consisting of carmustine, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)-phenylthiocarbamylsulfanyl]propionic acid (2-AAPA), and butein.

[0012] The present disclosure 4 is the evaluation method of the present disclosure 2 or 3, in which the concentration of the above glutathione reductase inhibitor in the cell culture solution is 10 to 3000 μM.

[0013] The present disclosure 5 is the evaluation method of any one of the present disclosures 1 to 4, in which the above step (a) and the above step (b) are performed simultaneously.

[0014] The present disclosure 6 is the evaluation method of the present disclosure 5, in which the above step (a) and the above step (b) are steps of culturing the above cells in the presence of a glutathione reductase inhibitor in the presence and absence of the above test substance, respectively.

[0015] The present disclosure 7 is the evaluation method of any one of the present disclosures 1 to 6, in which the above cells are cultured in a state of adhering to a culture vessel.

[0016] The present disclosure 8 is the evaluation method of any one of the present disclosures 2 to 7, in which the exposure time of the above glutathione reductase inhibitor to the above cells is 1 to 30 hours.

[0017] Disclosure 9 is an evaluation method from any one of Disclosures 1 to 8, wherein step (c) is a step of measuring at least two concentrations selected from the group consisting of oxidized glutathione concentration, reduced glutathione concentration and total glutathione concentration in each of the cells after culture, and the evaluation method further includes step (d) below. (d) A step of evaluating the hepatotoxicity of the test substance using the concentrations of at least two cells measured in step (c) above.

[0018] Disclosure 10 is an evaluation method of Disclosure 9, wherein the at least two concentrations are oxidized glutathione concentration and reduced glutathione concentration, and step (d) is a step of evaluating the hepatotoxicity of the test substance by comparing the ratio of the oxidized glutathione concentration to the reduced glutathione concentration in each cell.

[0019] Disclosure 11 is a cell with low glutathione reductase activity, used in any one of the evaluation methods described in Disclosures 1 to 10.

[0020] Disclosure 12 is the cells of Disclosure 11, obtained by culturing them in the presence of a glutathione reductase inhibitor. [Effects of the Invention]

[0021] This disclosure provides a highly sensitive and accurate method for evaluating the hepatotoxicity of a test substance in vitro. Furthermore, this disclosure also provides cells used in such an evaluation method. [Modes for carrying out the invention]

[0022] In this specification, cell culture includes not only the proliferation, growth, or maintenance of cells in a living state, but also processes such as cell passaging, differentiation induction, aggregate formation, and self-organization induction.

[0023] [Evaluation Method] The evaluation method disclosed herein is a method for evaluating the hepatotoxicity of a test substance using cells. The above evaluation method includes at least the following steps (a) to (c), but may also include other steps. (a) A step of culturing cells in which glutathione reductase activity has decreased. (b) The process of culturing cells in the presence and absence of the test substance. (c) A step of measuring the concentration of oxidized glutathione, reduced glutathione, and / or total glutathione in each of the above cells after culture.

[0024] Step (c) is performed after steps (a) and (b). Steps (a) and (b) may be performed with step (a) preceding step (b) or simultaneously, but it is preferable that they be performed simultaneously, as will be described later.

[0025] The cells used in the above evaluation method are cells derived from living organisms. Examples of living organisms include mammals such as humans, rats, mice, hamsters, guinea pigs, rabbits, dogs, monkeys, cats, pigs, cows, goats, and sheep. These cells may be, for example, tissue cells, serum, plasma, blood cells, urine, feces, saliva, pancreatic juice, or other bodily fluids or secretions collected from living organisms, primary cultured cells derived from living organisms, or cultured cell lines derived from living organisms (e.g., cancerous cells). Preferably, these cells are cells that can be found in the liver. Cells that can be found in the liver include hepatic parenchymal cells, which are essential for liver function, and non-parenchymal hepatic cells, which support the proliferation and survival of hepatic parenchymal cells. These cells may be either of these, or may include both. Furthermore, these cells may be embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).

[0026] The types of cultured cell lines mentioned above are not particularly limited, but examples include HepG2, HuH-7, HePaRG, Fa2N-4, THLE, HeLa, PLC-PRF-5, AML-12, HepaMN, MCF-7, LNCaP, CHO, etc. Liver-derived cells are preferred, and HepG2 is particularly preferred.

[0027] The test substances targeted by the above evaluation method are not particularly limited, but examples include small molecule compounds, large molecule compounds, proteins, nucleic acids, etc. As mentioned above, conventional in vitro evaluation methods may incorrectly evaluate compounds that are actually oxidative stress-positive as oxidative stress-negative due to insufficient detection sensitivity, but the evaluation method disclosed herein can effectively prevent such misevaluations. Whether a substance is inherently an oxidative stress-positive or oxidative stress-negative compound can be inferred, for example, based on academic papers in Non-Patent Documents 1 to 4.

[0028] -Process (a)- Step (a) described above is a step in which cells with reduced glutathione reductase activity are cultured. Note that glutathione reductase is contained within cells. GSSG produced within cells is regenerated into GSH by glutathione reductase, but when glutathione reductase activity is reduced, the regeneration of GSSG produced within cells into GSH is suppressed. This increases the sensitivity of the test substance used in step (b), making it possible to effectively evaluate the hepatotoxicity of the test substance (for example, whether the test substance is an oxidative stress-positive compound, as will be described later).

[0029] The above step (a) is preferably one of the following: a step of culturing cells in the presence of a glutathione reductase inhibitor (a1), a step of culturing cells in which the gene encoding glutathione reductase has been knocked down (a2), or a step of culturing cells in which the activity of a selected glutathione reductase has been reduced (a3). Due to the simplicity of the process, the above step (a) is preferably step (a1). Specifically, for example, it is preferable to seed cells in a culture vessel and cultivate the cells in the presence of a glutathione reductase inhibitor.

[0030] Here, if the evaluation method described above includes steps (a1), (b), and (c), the cells to be cultured in step (a1) are the cells seeded in a culture vessel. In this case, if step (b) is performed simultaneously with step (a1), the cells to be cultured in step (b) are the same cells seeded in a culture vessel as in step (a1). If step (b) is started after step (a1), the cells to be cultured are those that are in the process of being cultured or have been cultured in step (a1). Since culturing cells in the presence of a glutathione reductase inhibitor reduces the activity of glutathione reductase within the cells, step (a1) is included in step (a) (the step of culturing cells with reduced glutathione reductase activity).

[0031] If the above evaluation method includes steps (a2), (b), and (c), the cells to be cultured in step (a2) are cells in which the gene encoding glutathione reductase has been knocked down. In this case, if step (b) is performed simultaneously with step (a2), the cells to be cultured in step (b) are the same as those in step (a2), with the gene encoding glutathione reductase knocked down. If step (b) is started after step (a2), the cells are those that are in the process of being cultured or have been cultured in step (a2). In step (a2), gene knockdown can be performed by known genetic engineering techniques, and the recombinant cells can be used as cells in which the gene encoding glutathione reductase has been knocked down.

[0032] If the above evaluation method includes steps (a3), (b), and (c), the cells to be cultured in step (a3) ​​are cells with reduced glutathione reductase activity, and the cells to be cultured in step (b) are the same cells to be cultured in step (a3). In this case, if step (b) is performed simultaneously with step (a3), the cells to be cultured in step (b) are the same cells with reduced glutathione reductase activity as in step (a3), and if step (b) is started after step (a3), the cells are those that are in the process of being cultured or have been cultured in step (a3). In step (a3), for example, a cell line with low glutathione reductase activity can be specifically selected from cultured cell lines, and this cell line can be used as the selected cells with reduced glutathione reductase activity. To determine whether the activity of glutathione reductase in cells has decreased, the enzyme activity of the cell line before and after selection can be measured to confirm that the glutathione reductase activity of the cells in question has decreased.

[0033] The above process (a1) will be explained further below.

[0034] Examples of glutathione reductase inhibitors include low-molecular-weight compounds such as carmustine (CAS registry number: 154-93-8), 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)-phenylthiocarbamylsulfanyl]propionic acid hydrate (hereinafter referred to as "2-AAPA"), butein (CAS registry number: 487-52-5), LY83583 (CAS registry number: 91300-60-6), and lomustine (CAS registry number: 13010-47-4), as well as antibodies against glutathione reductase. Among these, the glutathione reductase inhibitor is preferably at least one selected from the group consisting of carmustine, 2-AAPA, and butein, with carmustine being more preferred. Using such a glutathione reductase inhibitor allows for more sensitive evaluation of the hepatotoxicity of the test substance. The glutathione reductase inhibitors listed above may be used individually or in combination of two or more.

[0035] In step (a1) described above, the method for culturing cells is not particularly limited as long as the cells are cultured while exposed to a glutathione reductase inhibitor. For example, a glutathione reductase inhibitor prepared to a predetermined concentration may be added to the culture medium before seeding and culturing the cells, or the glutathione reductase inhibitor may be added to the culture medium in which the cells are being cultured to a predetermined concentration before culturing.

[0036] The above cells may be cultured with the cells adhered to the culture vessel (adherent culture) or with the cells suspended in the culture medium (suspension culture). For example, considering the simplification and efficiency of the evaluation method, it is preferable to culture the cells by adherent culture. That is, it is preferable that the above cells be cultured with the cells adhered to the culture vessel.

[0037] The concentration of cells in the culture vessel (cell culture medium) containing the above cells is, for example, 1 × 10⁻⁶ 4 cells / cm 2 ~30×10 4 cells / cm 2 It is preferable that it be 2 × 104 cells / cm 2 ~25×10 4 cells / cm 2 This is preferable.

[0038] The concentration of the glutathione reductase inhibitor in the cell culture medium is preferably 10 to 3000 μM, and more preferably 10 to 1000 μM. This allows for more sensitive evaluation of the hepatotoxicity of the test substance. From the viewpoint of further enhancing this effect, when carmustine is used as the glutathione reductase inhibitor, the concentration of carmustine in the cell culture medium is more preferably 50 to 3000 μM, particularly preferably 100 to 1000 μM, and most preferably 150 to 600 μM. When 2-AAPA is used as the glutathione reductase inhibitor, the concentration of 2-AAPA in the cell culture medium is more preferably 50 to 500 μM, and particularly preferably 75 to 300 μM. When butein is used as the glutathione reductase inhibitor, the concentration of butein in the cell culture medium is more preferably 10 to 500 μM, and particularly preferably 25 to 100 μM.

[0039] The preparation of a cell culture medium containing a glutathione reductase inhibitor may be done by directly adding the glutathione reductase inhibitor to the cell culture medium at a predetermined concentration, or by preparing a stock solution by dissolving it in an organic solvent or the like to a concentration 10 to 5000 times higher than the predetermined concentration, and then adding 1 / 5000 to 1 / 10 of the stock solution to the cell culture medium.

[0040] The above organic solvents are not particularly limited, but examples include dimethyl sulfoxide (DMSO), methanol, ethanol, acetone, and acetonitrile.

[0041] The above cells may be cultured using, for example, commercially available culture media or culture kits. The culture media are not particularly limited and include, for example, basic cell culture media, differentiation media, and media specifically for primary cell culture. Specific media include, for example, Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), α-MEM, Glasgow MEM, M199, Ham's F-12, Williams E Medium (WME), RPMI1640, IMDM, F-10, MCDB medium, IMDM, DMEM / F12, and mixed media of these. In addition, the media may contain, for example, serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, nitrogen-containing components, sugars, salts, minerals, vitamins, and metal components.

[0042] There are no particular restrictions on the frequency of changing the culture medium during the culture period, but it is preferable to change the medium daily. The culture temperature is also not particularly restricted, for example, 25-40°C.

[0043] In step (a1) above, it is preferable that the exposure time of the glutathione reductase inhibitor to the cells is 1 to 30 hours. When the exposure time is within this range, the effect of using the glutathione reductase inhibitor is exhibited even more significantly. From the viewpoint of exhibiting this effect more effectively, it is more preferable that the exposure time of the glutathione reductase inhibitor to the cells is 1 to 24 hours.

[0044] -Process (b)- Step (b) described above is a step of culturing cells in the presence and absence of the test substance. Specifically, step (b) described above is a step of culturing cells in the presence of the test substance (b1) and a step of culturing cells in the absence of the test substance (b2).

[0045] It is preferable that the conditions in steps (b1) and (b2) are the same except for the presence or absence of the test substance (for example, the container used for cell culture, the culture period, the concentration of cells in the cell culture medium, the type and concentration of glutathione reductase inhibitor in the cell culture medium, the exposure time of the glutathione reductase inhibitor to the cells, etc.). For example, step (b2) may be performed first, and step (b1) may be performed when evaluating the test substance. In this case, step (c) may be performed on the cells obtained in step (b2) before the cells obtained in step (b1). Alternatively, step (b1) may be performed first, and step (b2) may be performed at any time. In this case, step (c) may be performed on the cells obtained in step (b1) before the cells obtained in step (b2).

[0046] In step (b1) described above, the method for culturing cells in the presence of the test substance is not particularly limited as long as the cells are exposed to the test substance and cultured. For example, the test substance prepared to a predetermined concentration may be added to the culture medium before seeding and culturing the cells, or the test substance may be added to the culture medium in which the cells are being cultured to a predetermined concentration before culturing.

[0047] The concentration at which the above-mentioned test substance is exposed to cells can be appropriately selected based on the solubility and cytotoxicity of the test substance, and, if the test substance is a pharmaceutical or drug candidate compound, the dosage, etc.

[0048] For example, the concentration of the test substance in the cell culture medium varies depending on the type of test substance, but is preferably 0.01 to 10000 μM, and more preferably 0.1 to 1000 μM. This allows for a more sensitive evaluation of the hepatotoxicity of the test substance. As will be described later, the preferred range of the test substance concentration is the same even when steps (a) and (b) are performed simultaneously.

[0049] Here, when adding the test substance to the culture medium, first dissolve the test substance in an organic solvent to prepare a test substance solution so that the concentration becomes about 10 to 10,000 times, and then add it to the culture medium so as to dilute this test substance solution 10 to 10,000 times. As the organic solvent, for example, dimethyl sulfoxide (DMSO), methanol, ethanol, acetone, acetonitrile, etc. are used. Also, there is a method of directly dissolving the test substance in the culture medium without using an organic solvent.

[0050] In the above step (b2), it is preferable to add the same amount of a solvent generally used to dissolve the test substance (for example, dimethyl sulfoxide) as the test substance solution used in the above step (b1).

[0051] In the above step (b1) and the above step (b2), the cell culture may be performed by adherent culture or suspension culture, but as in the above step (a1), it is preferable to perform the cell culture by adherent culture. That is, it is preferable that the above cells are cultured in a state of adhering to the culture vessel.

[0052] The concentration of the cells in the culture vessel containing the above cells (for example, the cell culture vessel at the time of cell seeding) is, for example, 1×10 4 cells / cm 2 ~30×10 4 cells / cm 2 preferably, and 2×10 4 cells / cm 2 ~25×10 4 cells / cm 2 is more preferable. When the above step (a) and the above step (b) are performed simultaneously as described later, the preferable range of the cell concentration is the same.

[0053] The cell culture period can be set appropriately depending on the type of cells and culture medium, culture conditions, etc., but it is preferably 1 to 21 days, and more preferably 1 to 3 days. As will be described later, the preferred range for the cell culture period is the same even when steps (a) and (b) are performed simultaneously, but in particular when steps (a1) and (b) are performed simultaneously, it is preferable to set the cell culture period so that the exposure time of the cells to the glutathione reductase inhibitor falls within the range described above.

[0054] The above cells may be cultured using, for example, commercially available culture media or culture kits. The culture media is not particularly limited, and examples include the media mentioned above in step (a1). There is no particular limit to the frequency of changing the culture medium during the culture period, but it is preferable to change the culture medium daily. The culture temperature is also not particularly limited, for example, 25 to 40°C.

[0055] It is preferable that steps (a) and (b) above are performed simultaneously. In particular, it is more preferable that steps (a1) and (b) above are performed simultaneously. In this case, steps (a) and (b) above, which are included in the evaluation method of this disclosure, can be rephrased as "the step of culturing cells in the presence of a glutathione reductase inhibitor, in the presence and absence of the test substance, respectively."

[0056] When steps (a1) and (b) are performed simultaneously, step (b1) of step (b) corresponds to the step of culturing cells in the presence of the test substance and a glutathione reductase inhibitor. The method of culturing cells in the presence of the test substance and a glutathione reductase inhibitor is not particularly limited as long as the cells are exposed to the test substance and the glutathione reductase inhibitor and cultured. For example, the test substance and the glutathione reductase inhibitor may be added to the culture medium in which cells seeded in wells or plates are cultured to a predetermined concentration, or the glutathione reductase inhibitor may be added to the culture medium in which seeded cells are cultured to a predetermined concentration, and then the test substance may be further added to a predetermined concentration and the culture may be continued, or the test substance and the glutathione reductase inhibitor, each prepared to a predetermined concentration, may be added to the culture medium, and then the cells may be seeded and cultured.

[0057] When steps (a1) and (b) are performed simultaneously, step (b2) of step (b) corresponds to the step of culturing cells in the absence of the test substance and in the presence of a glutathione reductase inhibitor. The method of culturing cells in this case is not particularly limited as long as the cells are cultured in the absence of the test substance and exposed to a glutathione reductase inhibitor. For example, a glutathione reductase inhibitor prepared to a predetermined concentration may be added to the culture medium before seeding and culturing the cells, or the glutathione reductase inhibitor may be added to the culture medium in which the cells are being cultured to a predetermined concentration before culturing.

[0058] When steps (a1) and (b) are performed simultaneously, it is preferable that the concentration of the glutathione reductase inhibitor in the cell culture medium is within the range described above in step (a1). It is also preferable to set the exposure time of the glutathione reductase inhibitor to the cells to be within the preferred range described above.

[0059] -Process (c)- Step (c) above is a step of measuring at least one concentration selected from the group consisting of oxidized glutathione (GSSG) concentration, reduced glutathione (GSH) concentration, and total glutathione concentration in each of the cells after culture. Since the evaluation method of this disclosure is performed by measuring these glutathione concentrations, it does not require complicated procedures compared to, for example, the method in Patent Document 1, and is therefore useful.

[0060] Specifically, step (c) above is a step of measuring at least one concentration (referred to as concentration C1) selected from the group consisting of GSSG concentration, GSH concentration, and / or total glutathione concentration in cells cultured in the presence of the test substance, and at least one concentration (referred to as concentration C2) selected from the group consisting of GSSG concentration, GSH concentration, and total glutathione concentration in cells cultured in the absence of the test substance. As the degree of oxidative stress increases, the GSSG concentration increases and the GSH concentration decreases, so by comparing the above concentration C1 and concentration C2, the hepatotoxicity of the test substance (for example, whether or not the test substance is an oxidative stress-positive compound) can be suitably evaluated. The molar concentration of GSSG in the solution containing the cultured cells is referred to as the GSSG concentration, and the molar concentration of GSH in the said solution is referred to as the GSH concentration. The total glutathione concentration is the sum of the GSSG concentration and the GSH concentration.

[0061] The above step (c) is preferably a step of measuring at least two concentrations selected from the group consisting of GSSG concentration, GSH concentration, and total glutathione concentration in each of the above-mentioned cells after culture. In this case, the evaluation method of the present disclosure preferably further includes (d) below. (d) A step of evaluating the hepatotoxicity of the test substance using the concentrations of at least two cells measured in step (c) above.

[0062] In step (d) above, it is preferable to calculate the concentration ratio using the two concentrations measured in step (c) above. For example, it is preferable to calculate the ratio of the two concentrations measured for cells cultured in the presence of the test substance (referred to as concentration ratio 1) and the ratio of the two concentrations measured for cells cultured in the absence of the test substance (referred to as concentration ratio 2), and to evaluate hepatotoxicity based on the ratio of these concentration ratios (concentration ratio 1 / concentration ratio 2).

[0063] The two concentrations measured in step (c) above are preferably, for example, GSSG concentration and GSH concentration, and the concentration ratio 1 and concentration ratio 2 calculated in step (d) above are preferably the ratio of GSSG concentration to GSH concentration (for example, GSSG / GSH or GSH / GSSG). Thus, an embodiment in which the at least two concentrations are GSSG concentration and GSH concentration, and step (d) is a step in which the hepatotoxicity of the test substance is evaluated by comparing the ratio of GSSG concentration to GSH concentration in each cell, is a preferred embodiment of the present disclosure. Such an evaluation method can evaluate the hepatotoxicity of the test substance more efficiently and simply.

[0064] One embodiment of a method for evaluating the hepatotoxicity of a test substance is described. In step (d) above, for example, if the ratio of concentration ratio 1, which is the ratio of GSSG concentration to GSH concentration in cells cultured in the presence of the test substance (GSSG / GSH), to the ratio of GSSG concentration to GSH concentration in cells cultured in the absence of the test substance (GSSG / GSH), is greater than or equal to a certain standard value (e.g., 1.5), the test substance can be evaluated as an oxidative stress-positive compound. If it is less than the standard value, the test substance can be evaluated as an oxidative stress-negative compound. As mentioned above, oxidative stress is a contributing factor to hepatotoxicity, so evaluating whether a test substance is an oxidative stress-positive or oxidative stress-negative compound leads to an evaluation of the hepatotoxicity of the test substance.

[0065] The glutathione concentrations in each of the above cells are, for example, "GSH / GSSG-Glo TM Measurements can be taken using the "Assay kit" (manufactured by Promega).

[0066] For example, the supernatant is first collected from each of the cell culture media obtained through steps (a) and (b) above (i.e., the culture medium containing cells cultured in the presence of the test substance and the culture medium containing cells cultured in the absence of the test substance). After removing the supernatant, Lysis Buffer is added to the cells remaining in the culture vessel to lyse the cells. Each of the obtained cell lysates is dispensed into two containers, and the GSH concentration is measured in one container and the GSSG concentration in the other, for example, using the "GSH / GSSG-Glo" method described above. TM Each measurement is performed using the "Assay kit". The activity of lactate dehydrogenase (LDH) that has leaked extracellularly is measured using the collected supernatant. When cells are damaged by oxidative stress, the cell membrane is destroyed and LDH leaks out of the cell. In other words, a high enzymatic activity of LDH in the cell culture medium obtained through steps (a) and (b) above indicates that the cells have been damaged. It is preferable to exclude groups with an LDH enzymatic activity exceeding 20%, with the enzymatic activity of LDH in the cell culture medium when the cells are completely destroyed being defined as 100% (activity%).

[0067] 〔cell〕 The cells of this disclosure are cells with low glutathione reductase activity, which are used in the evaluation method of this disclosure described above. Specifically, it is preferable that the cells are obtained by culturing them in the presence of a glutathione reductase inhibitor. This results in cells with low glutathione reductase activity. More specifically, it is preferable that the cells are obtained by culturing them in the absence of the test substance and in the presence of a glutathione reductase inhibitor. This cell culture step substantially corresponds to step (b2) of step (b) when steps (a1) and (b) are performed simultaneously. For further details, refer to the explanation above.

[0068] The activity of glutathione reductase can be measured by known methods, such as OxiSelect. TMGlutathione reductase activity can be measured using commercially available kits such as the Glutathione Reductase Measurement Assay Kit (CELL BIOLABS, INC.) or the DetectX Glutathione Reductase (GR) Fluorescent Detection Kit (Arbor Assays LLC).

[0069] Using the above-described cells, the hepatotoxicity of a test substance (for example, whether or not the test substance is an oxidative stress-positive compound) can be easily evaluated. A cell culture medium containing the above-described cells or a kit containing the above-described cells is one of the preferred embodiments of this disclosure. [Examples]

[0070] The following are specific examples that further illustrate this disclosure, but this disclosure is not limited to these examples.

[0071] [material] The materials used in this embodiment are shown below. • HepG2 cells: Manufactured by ATCC, catalog number HB-8065 • Dulbecco's Modified Eagle (DMEM) Medium: Manufactured by Thermo Fisher Scientific, catalog number 310534 • Fetal bovine serum (FBS): Thermo Fisher Scientific, catalog number 26140079 • William's E Medium (WME): Manufactured by Thermo Fisher Scientific, catalog number A1217601 • Dimethyl sulfoxide (DMSO): Manufactured by Sigma-Aldrich, catalog number D2650-100ML • Carmustine: Manufactured by Tokyo Chemical Industry Co., Ltd., catalog number C2634 • 2-AAPA: Manufactured by Sigma-Aldrich, catalog number A4111 Buteine: Manufactured by Tokyo Chemical Industry Co., Ltd., catalog number B3803 • Dexamethasone: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number 047-18863 • Troglitazone: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number 205-19483 • Menadione: Manufactured by Fluorochem, catalog number F049845 • Cyclosporine: Cayman Corporation, catalog number 12088 • Nephazodon: Manufactured by Tokyo Chemical Industry Co., Ltd., catalog number N1030 • Ketoconazole: Manufactured by Tokyo Chemical Industry Co., Ltd., catalog number K0045 • Folic acid: Manufactured by Tokyo Chemical Industry Co., Ltd., catalog number F0043 • Carbamazepine: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number 034-23701 • Ciprofloxacin: Manufactured by Tokyo Chemical Industry Co., Ltd., catalog number C2510 • Triton-X100: Manufactured by Nakalai Tesque, catalog number 35501-02 · GSH / GSSG-Glo TM Assay kit: Manufactured by Promega, catalog number V6611 • LDH-Cytotoxicity Test Wako: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number 299-50601 • 96-well transparent bottom plate: Corning Corporation, catalog number 356407

[0072] [Test Example 1: Evaluation 1]

[0073] 1. Cell culture HepG2 cells were cultured in cell culture medium (DMEM, 10% FBS) in 4 × 10⁶ units. 4 A cell solution was prepared by adjusting the cell volume to the specified number of cells / well. This cell solution was seeded into a 96-well clear-bottom plate. After seeding, the cells were cultured in a CO2 incubator for 2 days.

[0074] 2. Preparation of exposure solution First, solution X1 was prepared by dissolving the test substance in DMSO so that its concentration was 1000 times the final concentration shown in Table 1 (however, in each control example shown in Table 1, solution X1 consisting only of DMSO was prepared). Separately, the glutathione reductase inhibitor was dissolved in William's E Medium so that its concentration was the final concentration shown in Table 1. Then, the exposure solution was prepared by adding 1 / 1000th the volume of solution X1 to the William's E Medium containing the dissolved glutathione reductase inhibitor.

[0075] [Table 1]

[0076] 3. Exposure The culture medium of the cells cultured in step 1 above was removed, 100 μL of the exposure solution prepared in step 2 above was added, and the cells were incubated at 37°C for 4 hours.

[0077] 4. Calculation of LDH leakage rate After performing step 3 above, the cell supernatant from each well was collected and subjected to measurement of LDH enzyme activity. Specifically, the collected cell supernatant was diluted 10-fold with PBS, and the cytotoxicity of the diluted cell supernatant was measured according to the LDH-cytotoxicity test manual of Wako. The LDH enzyme activity when cells were completely destroyed with Triton-X100 was set as 100% (activity%), and the LDH enzyme activity in the cell supernatant was expressed as a percentage.

[0078] 5. Calculation of reduced glutathione concentration and oxidized glutathione concentration GSH / GSSG-Glo TM Using the Assay Kit, the total glutathione concentration and oxidized glutathione (GSSG) concentration in each well were measured after performing step 3 above, in accordance with the instructions provided with the kit. The reduced glutathione (GSH) concentration was calculated using the following formula. GSH concentration (μM) = Total glutathione concentration (μM) - GSSG concentration (μM)

[0079] 6. Methods for evaluating hepatotoxicity The ratio of GSH to GSH concentrations (GSSG / GSH ratio) was calculated from the GSH and GSSG concentrations in each well, as calculated in step 4 above. The GSSG / GSH ratio for each control case, which used an exposure solution containing DMSO instead of the test substance, was defined as the "DMSO ratio." The GSSG / GSH ratio for each test case relative to the DMSO ratio (GSSG / GSH vs. DMSO) was calculated to evaluate the hepatotoxicity of the test substance (whether or not the test substance is an oxidative stress-positive compound).

[0080] 7. Evaluation Results Table 2 shows the reduced glutathione concentration, oxidized glutathione concentration, GSSG / GSH ratio, GSSG / GSH vs. DMSO ratio, and LDH leakage rate. If the "GSSG / GSH vs. DMSO" ratio was 1.5 or higher, the test substance was evaluated as an oxidative stress-positive compound (+), and if it was less than 1.5, the test substance was evaluated as an oxidative stress-negative compound (-).

[0081] [Table 2]

[0082] Control example 1-A, and test examples 1-A1 and 1-A2 are examples in which step (a) described above was not performed. In this case, the GSSG / GSH ratios of test examples 1-A1 and 1-A2 are all close to 1 relative to the DMSO ratio (i.e., the GSSG / GSH ratio of control example 1-A).

[0083] In contrast, for example, control example 1-B and test examples 1-B1 and 1-B2 are examples in which the above step (a) was performed using carmustine as a glutathione reductase inhibitor. In this case, the GSSG / GSH ratio of test example 1-B1 relative to the DMSO ratio (i.e., the GSSG / GSH ratio of control example 1-B) is larger than the "GSSG / GSH vs. DMSO" ratio of test example 1-A1, which used the same test substance (dexamethasone). Also, the GSSG / GSH ratio of test example 1-B2 relative to the DMSO ratio (i.e., the GSSG / GSH ratio of control example 1-B) is significantly larger than the "GSSG / GSH vs. DMSO" ratio of test example 1-A2, which used the same test substance (troglitazone). A similar trend is observed in examples using other compounds as glutathione reductase inhibitors, and it can be seen that the "GSSG / GSH vs. DMSO" ratio is significantly larger than that of test example 1-A2, especially when the test substance is troglitazone.

[0084] Based on the aforementioned academic papers (Non-Patent Documents 1-4), it is inferred that dexamethasone is inherently an oxidative stress-negative compound, while troglitazone is an oxidative stress-positive compound. Furthermore, troglitazone is highly cytotoxic, causing cell death in cell culture, making evaluation difficult. Nevertheless, it has been found that the evaluation method disclosed here allows for the assessment of the hepatotoxicity of the test substance (particularly whether or not the test substance is an oxidative stress-positive compound) with high accuracy and sensitivity. This suggests that the hepatotoxicity of other test substances can also be assessed with greater accuracy and sensitivity.

[0085] [Test Example 2: Evaluation 2 (Examination of Exposure Time)] The following tests were conducted to investigate the effect of exposure time of the test substance to cells on hepatotoxicity assessment.

[0086] 1. Cell culture Cells were cultured using the method described in 1. of Test Example 1.

[0087] 2. Preparation of exposure solution First, solution X2 was prepared by dissolving the test substance in DMSO so that its concentration was 1000 times the final concentration shown in Tables 3 to 10 (however, in each control example shown in Tables 3 to 10, solution X2 consisting only of DMSO was prepared). Separately, the glutathione reductase inhibitor was dissolved in William's E Medium so that its concentration was as shown in Tables 3 to 10. Then, the exposure solution was prepared by adding 1 / 1000th the volume of solution X2 to the William's E Medium containing the dissolved glutathione reductase inhibitor.

[0088] [Table 3]

[0089] [Table 4]

[0090] [Table 5]

[0091] [Table 6]

[0092] [Table 7]

[0093] [Table 8]

[0094] [Table 9]

[0095] [Table 10]

[0096] 3. Exposure The culture medium of the cells cultured in step 1 above was removed, and 100 μL of the exposure solution prepared in step 2 above was added. The cells were incubated at 37°C for a predetermined time. The incubation time (i.e., the exposure time of the test substance and glutathione reductase inhibitor to the cells: in hours) is shown in Tables 3 to 10.

[0097] 4. Calculation of LDH leakage rate The LDH in the cell supernatant was measured using the method described in 5. of Test Example 1, and the percentage that leaked extracellularly was calculated.

[0098] 5. Calculation of reduced glutathione concentration and oxidized glutathione concentration Reduced glutathione and oxidized glutathione concentrations were calculated using the method described in section 4 of Test Example 1. In cases where the percentage of LDH leaked into the cell supernatant was 20% or more, as described in section 4 above, it was considered that there was cell damage, and the concentrations of reduced glutathione and oxidized glutathione were not calculated.

[0099] 6. Methods for evaluating hepatotoxicity Hepatotoxicity was evaluated using the same method as in section 5 of Test Example 1.

[0100] 7. Evaluation Results Tables 11 to 18 show the reduced glutathione concentration, oxidized glutathione concentration, GSSG / GSH ratio, GSSG / GSH vs. DMSO ratio, and LDH leakage rate. If the "GSSG / GSH vs. DMSO" ratio was 1.5 or higher, the test substance was evaluated as an oxidative stress-positive compound (+). If it was less than 1.5, the test substance was evaluated as an oxidative stress-negative compound (-). In cases where the percentage of LDH leaked into the cell supernatant was 20% or higher, the glutathione concentration was indicated as "NC" in the table. In these cases, hepatotoxicity was not evaluated (indicated as " / " in the table).

[0101] [Table 11]

[0102] [Table 12]

[0103] [Table 13]

[0104] [Table 14]

[0105] [Table 15]

[0106] [Table 16]

[0107] [Table 17]

[0108] [Table 18]

[0109] All examples shown in Tables 11 to 14 are examples where step (a) described above was not performed. In contrast, all examples shown in Tables 15 to 18 are examples where step (a) described above was performed, which involved culturing cells in the presence of carmustine (a glutathione reductase inhibitor). Furthermore, based on the aforementioned academic papers, it is presumed that dexamethasone is inherently an oxidative stress-negative compound, while menadione, troglitazone, and cyclosporine are oxidative stress-positive compounds.

[0110] For example, when the test substance is menadione, comparing test examples under identical conditions except for the presence or absence of step (a) above, it is clear that the "GSSG / GSH vs. DMSO" ratio is significantly larger in the examples in Tables 15 to 18 where step (a) is performed compared to the examples in Tables 11 to 14 where step (a) is not performed, indicating that the hepatotoxicity of menadione can be evaluated with high sensitivity and accuracy. Furthermore, from Tables 11 to 14, it can be seen that when step (a) is not performed, the detection sensitivity of glutathione decreases as the cell exposure time increases, and it may not be possible to accurately evaluate the hepatotoxicity of menadione. In contrast, in the examples where step (a) is performed, the detection sensitivity remains high even with long cell exposure times, and the hepatotoxicity of menadione can be evaluated with high sensitivity and accuracy (see Tables 15 to 18).

[0111] Furthermore, when comparing test examples with identical conditions except for the presence or absence of step (a) when the test substance is troglitazone, in the example without step (a), the detection sensitivity was insufficient when the cell exposure time was short, and the hepatotoxicity of troglitazone could not be accurately evaluated (see Tables 11-12). In contrast, in the example with step (a), the hepatotoxicity of troglitazone could be accurately evaluated even with a short cell exposure time (see Tables 15-16). Also, from Tables 13-14 and 17-18, it can be seen that when step (a) is not performed, if the cell exposure time is long, the cells die and the glutathione concentration cannot be measured, or although the glutathione concentration can be measured by lowering the concentration of troglitazone in the cell culture medium, the detection sensitivity of glutathione is significantly lower compared to the example with step (a). By performing step (a) above, it became possible to perform evaluations over a wider concentration range or exposure time compared to the case where step (a) is not performed (see Tables 15 to 18).

[0112] Furthermore, when the test substance is cyclosporine, comparing test examples under identical conditions except for the presence or absence of step (a) above, it can be seen that when step (a) is performed, the detection sensitivity of glutathione is significantly improved when the cell exposure time is 1 hour or more, compared to when step (a) is not performed (see Tables 11 to 18).

[0113] Based on the above, the evaluation method disclosed herein can be suitably applied even when the test substance is exposed to cells for a long period of time, and is therefore an effective method for a wide range of applications. Furthermore, for test substances with strong cytotoxicity, the hepatotoxicity of the test substance can be accurately evaluated even when the test substance is exposed to cells for a long period of time by lowering the concentration of the test substance in the cell culture medium.

[0114] [Test Example 3: Evaluation 3 (Evaluation of the Test Substance)]

[0115] 1. Cell culture The cultures were performed using the method described in 1. of Test Example 1.

[0116] 2. Preparation of exposure solution First, solution X3 was prepared by dissolving the test substance in DMSO so that its concentration was 100 times the final concentration shown in Tables 19 to 21 (however, in each control example shown in Tables 19 to 21, solution X3 consisting only of DMSO was prepared). Separately, the glutathione reductase inhibitor was dissolved in William's E Medium so that its concentration was as shown in Tables 19 to 21. Then, the exposure solution was prepared by adding 1 / 100th the volume of solution X3 to the William's E Medium containing the dissolved glutathione reductase inhibitor.

[0117] [Table 19]

[0118] [Table 20]

[0119] [Table 21]

[0120] 3. Exposure The culture medium of the cells cultured in step 1 above was removed, 100 μL of the exposure solution prepared in step 2 above was added, and the cells were incubated at 37°C for 4 hours.

[0121] 4. Calculation of LDH leakage rate The LDH in the cell supernatant was measured using the method described in 5. of Test Example 1, and the percentage that leaked extracellularly was calculated.

[0122] 5. Calculation of reduced glutathione concentration and oxidized glutathione concentration Reduced glutathione and oxidized glutathione concentrations were calculated using the method described in section 4 of Test Example 1. In cases where the percentage of LDH leaked into the cell supernatant was 20% or more, as described in section 4 above, it was considered that there was cell damage, and the concentrations of reduced glutathione and oxidized glutathione were not calculated.

[0123] 6. Methods for evaluating hepatotoxicity Hepatotoxicity was evaluated using the same method as in section 5 of Test Example 1.

[0124] 7. Evaluation Results Tables 22 to 24 show the reduced glutathione concentration, oxidized glutathione concentration, GSSG / GSH ratio, and the leakage rates of GSSG / GSH vs. DMSO and LDH. If the "GSSG / GSH vs. DMSO" ratio was 1.5 or higher, the test substance was evaluated as an oxidative stress-positive compound (+). If it was less than 1.5, the test substance was evaluated as an oxidative stress-negative compound (-). In cases where the percentage of LDH leaked into the cell supernatant was 20% or higher, the glutathione concentration was indicated as "NC" in the table. In these cases, hepatotoxicity was not evaluated (indicated as " / " in the table).

[0125] [Table 22]

[0126] [Table 23]

[0127] [Table 24]

[0128] All the examples shown in Table 22 are examples where step (a), which reduces the activity of glutathione reductase contained in the cells, was not performed. In contrast, all the examples shown in Tables 23 and 24 are examples where step (a) was performed, which involved culturing cells in the presence of carmustine (a glutathione reductase inhibitor). Furthermore, from the aforementioned academic papers, it is presumed that folic acid, carbamazepine, and ciprofloxacin are inherently oxidative stress-negative compounds, while nefazodone and ketoconazole are oxidative stress-positive compounds.

[0129] For folic acid, carbamazepine, and ciprofloxacin, which are inherently oxidative stress-negative compounds, all test substances were evaluated as oxidative stress-negative compounds (-) regardless of whether or not step (a) above was performed (see Tables 22-23).

[0130] When the test substance is nefazodone, which is presumed to be an oxidative stress-positive compound, comparing test examples under identical conditions except for the presence or absence of step (a) above, the "GSSG / GSH vs. DMSO" ratio is clearly larger in the example in Table 23 where step (a) is performed compared to the result in Table 22 where step (a) is not performed, indicating that the hepatotoxicity of nefazodone can be evaluated with high sensitivity and accuracy.

[0131] When the test substance is ketoconazole, which is presumed to be an oxidative stress-positive compound, comparing test examples under identical conditions except for the presence or absence of step (a) above, in the example in Table 22 where step (a) is not performed, cytotoxicity was observed at high concentrations of ketoconazole, making evaluation impossible. In the results in Table 23 where step (a) is performed, evaluation is possible even at high concentrations, and the "GSSG / GSH vs. DMSO" ratio is larger when step (a) is performed, indicating that the hepatotoxicity of ketoconazole can be evaluated broadly, with high sensitivity and accuracy.

[0132] From the above, it has been shown that the evaluation method disclosed herein can evaluate multiple oxidative stress-negative or positive compounds as previously reported (see Non-Patent Documents 1-4). Furthermore, compared to the methods reported in Non-Patent Documents 1-4, the evaluation method disclosed herein is highly versatile and can evaluate many test substances with high accuracy using the same method, making it an extremely useful method for evaluating the hepatotoxicity of compounds in pharmaceutical development and other applications.

Claims

1. A method for evaluating the hepatotoxicity of a test substance using cells, The aforementioned cells possess glutathione reductase, (a) A step of culturing the cells in the presence of a glutathione reductase inhibitor (a1), a step of culturing cells in which the gene encoding glutathione reductase has been knocked down (a2), or a step of selecting and culturing cells in which the activity of glutathione reductase has been reduced compared to the original cells of the same origin (a3), (b) A step of culturing the cells in the presence and absence of the test substance, (c) A step of measuring the concentration of oxidized glutathione, reduced glutathione, and / or total glutathione in each of the cells after culture, Includes, Step (a) and step (b) are performed simultaneously. Step (c) is a step of measuring at least two concentrations selected from the group consisting of oxidized glutathione concentration, reduced glutathione concentration, and total glutathione concentration in each cell cultured in the presence and absence of the test substance, The evaluation method further includes a step (d) in which the hepatotoxicity of the test substance is evaluated using the concentration measured in step (c), Step (d) is a method for evaluating hepatotoxicity, comprising: calculating a ratio of two of at least two concentrations measured for cells cultured in the presence of the test substance (concentration ratio 1); and a ratio of two of at least two concentrations measured for cells cultured in the absence of the test substance (concentration ratio 2); and comparing concentration ratio 1 and concentration ratio 2 (wherein in both concentration ratio 1 and concentration ratio 2, the concentrations used as numerators are of the same type, and the concentrations used as denominators are of the same type).

2. The evaluation method according to claim 1, wherein the glutathione reductase inhibitor is at least one selected from the group consisting of carmustine, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)-phenylthiocarbamylsulfanyl]propionic acid (2-AAPA), and butein.

3. The evaluation method according to claim 1, wherein the concentration of the glutathione reductase inhibitor in the cell culture medium is 10 to 3000 μM.

4. The evaluation method according to claim 1, wherein step (a) and step (b) are steps of culturing the cells in the presence of a glutathione reductase inhibitor in the presence and absence of the test substance, respectively.

5. The evaluation method according to claim 1, wherein the cells are cultured while attached to a culture vessel.

6. The evaluation method according to claim 1, wherein the exposure time of the glutathione reductase inhibitor to the cells is 1 to 30 hours.

7. The evaluation method according to any one of claims 1 to 6, wherein the at least two concentrations in step (c) are the oxidized glutathione concentration and the reduced glutathione concentration.

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