An inducer of mouse liver S9 and its induction method

Abstract

The present invention discloses an inducer for mouse liver S9 and an inducing method thereof. The inducer comprises octyl cyanoacrylate and etaconazole; and through optimization experiments, the optimal inducer dosage and induction time are obtained, and an effective method for inducing liver S9 is obtained; the experimental results show that the inducing method for mouse liver S9 according to the present invention has a strong inducing effect on the expression of typical metabolic enzymes of chemical substances, especially on metabolic enzymes 2E, 2C, and 4A which have no inducing effect in the traditional inducing method. Moreover, the inducing agent method of the present invention is economical and easily available, and is applicable to the safety evaluation of industrial chemicals, drugs, and environmental chemicals.

Description

Technical Field

[0001] The present invention belongs to the field of proteases, and in particular relates to an inducer of mouse liver S9 and an inducing method thereof. Background Art

[0002] In safety evaluation tests such as mutagenicity and carcinogenicity of chemical substances, a considerable part of chemical substances need to be metabolically activated to cause mutagenicity and carcinogenicity. Therefore, in the non-clinical safety evaluation of new drugs or new chemical substances, an appropriate activation system should be used in short-term in vitro tests. Mouse liver S9 is the mitochondrial supernatant of mouse liver tissue homogenate, which contains a large number of chemical substance metabolic enzymes and is often used as a biotransformation activation system in in vitro tests. The traditionally used in vitro metabolic activation system is rat-induced liver S9, and its inducers are of two types: one is a polychlorinated biphenyl mixture, which has strong carcinogenicity and seriously pollutes the environment, and has been prohibited from production in many countries, and is expensive as a scientific research reagent; the other is the combination of phenobarbital and β-naphthoflavone, and the purchase of phenobarbital is strictly controlled as an anesthetic. In addition, there is also a major problem with classical inducers, that is, they have no induction effect on cytochrome P450 (CYP) subtypes 2E, 2C, and 4A, which play a major role in the metabolic activation of chemical substances. Summary of the Invention

[0003] The purpose of the first aspect of the present invention is to provide an inducer.

[0004] The purpose of the second aspect of the present invention is to provide the application of the above inducer.

[0005] The purpose of the third aspect of the present invention is to provide an inducing method of mouse liver S9.

[0006] The purpose of the fourth aspect of the present invention is to provide the application of the above inducing method in the preparation of mouse liver S9.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In the first aspect of the present invention, a mouse liver S9 inducer is provided, and the mouse liver S9 inducer contains octocrylene and difenoconazole.

[0009] In some embodiments of the present invention, octocrylene and difenoconazole in the mouse liver S9 inducer are independent of each other. When in use, octocrylene is first used for induction, and then difenoconazole is used for induction.

[0010] In the second aspect of the present invention, the application of the inducer described in the first aspect of the present invention in inducing mouse liver S9 or in the preparation of inducing mouse liver S9 is provided.

[0011] In some embodiments of the present invention, octyl cyanoacrylate and etaconazole in the mouse liver S9 inducer are independent of each other. When in use, octyl cyanoacrylate is used for induction first, and then etaconazole is used for induction.

[0012] The third aspect of the present invention provides a method for inducing mouse liver S9, which includes the following steps: first, use octyl cyanoacrylate in the inducer described in the first aspect of the present invention to induce a mouse, and then use etaconazole for induction. That's it.

[0013] In some embodiments of the present invention, the dosage of octyl cyanoacrylate is 50-100 mg / kg, and continuous administration for induction is carried out for 2-4 days.

[0014] In some embodiments of the present invention, the administration method of octyl cyanoacrylate is oral administration.

[0015] In some embodiments of the present invention, the oral administration includes oral ingestion and gavage.

[0016] In some embodiments of the present invention, the oral administration is gavage.

[0017] Of course, those skilled in the art can also reasonably select other oral administration methods for administration according to the actual usage requirements and the selection of experimental animals.

[0018] In some embodiments of the present invention, the dosage of etaconazole is 3-5 mg / kg, and administration for induction is carried out for 2-4 days.

[0019] In some embodiments of the present invention, the administration method of etaconazole is injection.

[0020] In some embodiments of the present invention, the injection includes intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intra-articular injection, subconjunctival injection, and epidural injection.

[0021] In some embodiments of the present invention, the injection is intraperitoneal injection.

[0022] Of course, those skilled in the art can also reasonably select other injection methods for administration according to the actual usage requirements and the selection of experimental animals.

[0023] In some embodiments of the present invention, the mouse is a 6-8 week old male mouse.

[0024] The fourth aspect of the present invention provides the application of the induction method described in the third aspect of the present invention in the preparation of mouse liver S9.

[0025] In some embodiments of the present invention, the method for preparing mouse liver S9 includes:

[0026] Taking the mouse liver after induction treatment according to the induction method described in the third aspect of the present invention, cutting it into pieces and adding a buffer to make a liver homogenate. After centrifugation, the supernatant is the mouse liver S9.

[0027] In some embodiments of the present invention, the mice are sacrificed specifically 20 - 28 h after the last administration.

[0028] In some embodiments of the present invention, the residual blood in the liver needs to be removed before taking out the liver.

[0029] In some embodiments of the present invention, the method for removing the residual blood in the liver includes washing the liver with a buffer.

[0030] In some embodiments of the present invention, the buffer is 0.1 - 0.2 mol / L KCl buffer (pH 7 - 8).

[0031] In some embodiments of the present invention, the addition amount of the buffer in step (b) is 2 - 4 times the wet weight ratio of the liver.

[0032] In some embodiments of the present invention, the buffer needs to be pre-cooled.

[0033] In some embodiments of the present invention, the centrifugation conditions include: 2 - 6 °C, 8000 - 10000 g, 15 - 25 min.

[0034] The beneficial effects of the present invention are:

[0035] The present invention provides an inducer that can be used to induce liver S9, including octyl cyanoacrylate and etaconazole; and through optimizing the experiments, the optimal inducer dose and induction time are obtained, and an effective method for inducing liver S9 is obtained; the experimental results show that the method for inducing mouse liver S9 described in the present invention has a strong induction effect on the expression of typical metabolic enzymes of chemical substances, especially on metabolic enzymes 2E, 2C, and 4A that have no induction effect in the traditional induction method. Moreover, the inducer method of the present invention is economical and easily available, and is applicable to the safety evaluation of industrial chemicals, drugs, and environmental chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 For the application results of mouse liver S9 obtained by the induction method in the embodiments of the present invention and the traditional induction method in the bacterial reverse mutation test, wherein TA97, TA98, TA100, TA102, TA1535 represent the test strains, 2AF represents 2 - aminofluorene,... represents NMDA.

[0037] Figure 2Induction fold of metabolic enzyme CYP1A, CYP2B, and CYP2C genes in mouse liver S9 obtained by using the induction method of the present invention and the traditional induction method.

[0038] Figure 3 Induction fold of metabolic enzyme CYP1A, CYP2B, CYP2C, CYP2E, CYP3A, and CYP4A genes in mouse liver S9 obtained by using the induction method of the present invention and the traditional induction method. Detailed implementation mode

[0039] The concept of the present invention and the technical effects produced will be clearly and completely described below in conjunction with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0040] Experimental materials

[0041] In the embodiments of the present invention, octocrylene, triadimefon, β-naphthoflavone, 2-aminofluorene, and N-nitrosodimethylamine used were all purchased from SIGMA.

[0042] An induction method for mouse liver S9 established in the following embodiments of the present invention has a strong induction effect on the expression of typical metabolic enzymes of chemical substances.

[0043] Example 1: Selection of inducer dose

[0044] Randomly select 21 male mice aged 6 - 8 weeks, which have been acclimatized and quarantined in the animal room for 1 week before use; stratify and randomly assign the 21 mice into 7 groups, with 3 mice in each group.

[0045] The grouping is as follows:

[0046] Group 1: Oral gavage with octocrylene at 25 mg / kg for 3 consecutive days;

[0047] Group 2: Oral gavage with octocrylene at 50 mg / kg for 3 consecutive days;

[0048] Group 3: Oral gavage with octocrylene at 100 mg / kg for 3 consecutive days;

[0049] Group 4: Intraperitoneal injection of triadimefon at 2.5 mg / kg for 3 consecutive days;

[0050] Group 5: Intraperitoneal injection of triadimefon at 5 mg / kg for 3 consecutive days;

[0051] Group 6: Intraperitoneal injection of triadimefon at 10 mg / kg for 3 consecutive days.

[0052] Group 7 was the negative control group, and the mice in the group were orally gavaged with 10 mL / kg olive oil for 3 consecutive days.

[0053] All the mice in each group were sacrificed 24 h after the last administration to obtain liver tissues.

[0054] Approximately 0.3 g of the obtained liver tissues was cut with surgical scissors for gene expression detection.

[0055] Using a commercially available kit, total RNA in the liver tissues was extracted according to the instructions of the manual, reverse transcribed to obtain cDNA, and then quantitative PCR detection was performed using the following primers:

[0056] CYP2B10 PCR detection primer pair:

[0057] CYP2B10 forward primer: 5’-ACCCCACGTTCCTCTTCCA-3’ (SEQ ID NO:1);

[0058] CYP2B10 reverse primer: 5’-CAGCAGGCGCAAGAACTGA-3’ (SEQ ID NO:2).

[0059] CYP2E1 PCR detection primer pair:

[0060] CYP2E1 forward primer: 5’-CGTTGCCTTGCTTGTCTGGA-3’ (SEQ ID NO:3);

[0061] CYP2E1 reverse primer: 5’-AAGAAAGGAATTGGGAAAGGTCC-3’ (SEQ ID NO:4).

[0062] It was found that the expression level of CYP2B1 induced by oral gavage of octyl cyanoacrylate was significantly lower at the dose of 25 mg / kg than at 50 - 100 mg / kg, and the expression level of CYP2E1 induced by intraperitoneal injection of etaconazole was significantly lower at the dose of 2.5 mg / kg than at 5 mg / kg. Moreover, an injection volume of 10 mg / kg had a toxic effect on the mice. Therefore, the inducer concentrations were selected as follows: octyl cyanoacrylate 50 - 100 mg / kg, etaconazole 5 mg / kg.

[0063] Example 2: Selection of induction time

[0064] Twelve male mice aged 6 - 8 weeks were randomly selected and had been acclimatized and quarantined in the animal room for 1 week before use; the twelve mice were randomly assigned to 4 groups according to body weight stratification, with 3 mice in each group.

[0065] The grouping is as follows:

[0066] Group 1: Orally gavaged with octocrylene 100 mg / kg for 1 day;

[0067] Group 2: Orally gavaged with octocrylene 100 mg / kg for 3 consecutive days;

[0068] Group 3: Orally gavaged with octocrylene 100 mg / kg for 5 consecutive days;

[0069] Group 4 was the negative control group, and the mice in the group were orally gavaged with 10 mL / kg olive oil for 3 consecutive days.

[0070] Mice in each group were sacrificed 24 h after the last administration to obtain liver tissues.

[0071] Approximately 0.3 g of the obtained liver tissue was taken and cut with surgical scissors for gene expression detection.

[0072] Using a commercially available kit, total RNA in liver tissues was extracted according to the instructions of the manual, reverse transcribed to obtain cDNA, and then quantitative PCR detection was performed using the primers shown in SEQ ID NO:1-2 and 3-4 in the above-mentioned examples.

[0073] It was found that the expression level of CYP2B1 induced by octocrylene was significantly lower on the 1st day of administration than on the 3rd - 5th day. Therefore, for the selection of the induction time: octocrylene was continuously induced for 3 - 5 days.

[0074] Example 3: Preparation of mouse liver S9

[0075] Nine male mice aged 6 - 8 weeks were randomly selected and had been acclimatized and quarantined in the animal room for 1 week before use; the nine mice were randomly assigned to 3 different parallel groups according to body weight stratification, with 3 mice in each group.

[0076] Mice in each experimental group were first orally gavaged with octocrylene 50 mg / kg for 3 consecutive days, and then intraperitoneally injected with etaconazole 5 mg / kg for 3 consecutive days. The mice were sacrificed 24 h after the last administration to obtain the liver.

[0077] After sacrificing the mice, the liver was perfused with pre - cooled 0.15 mol / L KCl buffer (pH 7.4) under sterile conditions and weighed, and then approximately 0.3 g of the liver was cut with surgical scissors for gene expression detection. The remaining part was cut into pieces with surgical scissors and mixed with pre - cooled KCl buffer at a ratio of 3 times the wet weight of the liver, and homogenized into liver homogenate in an ice bath using a homogenizer. The homogenate was placed in a low - temperature high - speed centrifuge, with the temperature controlled at 2 - 6 °C, the centrifugation speed at 9000 g, and the time at 20 min. The supernatant was taken after centrifugation to obtain liver S9.

[0078] In addition, a mouse negative control group and a rat negative control group, as well as a traditional induction control group (since traditional induction uses rats, this group also uses rats) were set up.

[0079] Among them, the mice in the mouse negative control group were orally gavaged with 10 mL / kg of olive oil for 3 consecutive days, and the rats were sacrificed 24 h after the last administration to obtain the liver, and the other steps were the same as those in the above-mentioned examples. The rats in the rat negative control group were orally gavaged with 0.5 mL of a solution containing 0.5% Tween 20 for 3 consecutive days, and the rats were sacrificed 24 h after the last administration to obtain the liver, and the other steps were the same as those in the above-mentioned examples. In the rat traditional induction control group, phenobarbital tablets were made into a solution with the required concentration using 0.9% sodium chloride injection, and then a solution of β-naphthoflavone with a concentration of 32 mg / mL was prepared with corn oil. On the first day, each rat was intraperitoneally injected with a phenobarbital solution with a concentration of 12 mg / mL at a dose of 2.5 mL / kg, and on the 2nd - 4th days, phenobarbital with a concentration of 32 mg / mL and β-naphthoflavone with a concentration of 32 mg / mL were intraperitoneally injected. The rats were sacrificed 24 h after the last administration to obtain the liver, and the other steps were the same as those in the above-mentioned examples.

[0080] Example 4: Bacterial reverse mutation test

[0081] The mouse liver S9 obtained by the induction method in the above-mentioned examples and the rat liver S9 obtained by the traditional induction method were applied to the classical in vitro bacterial reverse mutation test (TA97, TA98, TA100, TA102, TA1535), and the specific experimental method referred to "GB / T 21786 - 2008 Test method for bacterial reverse mutation of chemicals".

[0082] The results are as Figure 1 shown, and it can be seen from Figure 1 that the mouse liver S9 obtained by the induction method in the above-mentioned examples has a slightly better activation effect on the typical indirect mutagen 2 - amino - fluorene (2AF) than the rat liver S9 obtained by the traditional induction method. For N - nitrosodimethylamine (NMDA), which does not show an activation effect with the TA100 strain for the rat liver S9 obtained by the traditional induction method, an activation effect is shown when using the mouse liver S9 obtained by the induction method in the above-mentioned examples.

[0083] Example 5: Detection of relative fold change in the expression of typical liver metabolic enzyme genes

[0084] Total RNA was extracted from the mouse liver S9 obtained by the induction method in the above-mentioned examples and the mouse liver S9 obtained by the traditional induction method, and cDNA was obtained by reverse transcription; quantitative PCR detection was carried out using the following primers:

[0085] Among them, for mouse genes:

[0086] ACTB PCR detection primer pair:

[0087] ACTB forward primer: 5'-GGCTGTATTCCCCTCCATCG-3' (SEQ ID NO:5);

[0088] ACTB reverse primer: 5'-CCAGTTGGTAACAATGCCATGT-3' (SEQ ID NO:6);

[0089] CYP1A1 PCR detection primer pair:

[0090] CYP1A1 forward primer: 5'-GACCCTTACAAGTATTTGGTCGT-3' (SEQ ID NO:7);

[0091] CYP1A1 reverse primer: 5'-GGTATCCAGAGCCAGTAACCT-3' (SEQ ID NO:8);

[0092] CYP2B10 PCR detection primer pair:

[0093] CYP2B10 forward primer: 5'-ACCCCACGTTCCTCTTCCA-3' (SEQ ID NO:1);

[0094] CYP2B10 reverse primer: 5'-CAGCAGGCGCAAGAACTGA-3' (SEQ ID NO:2);

[0095] CYP2C29 PCR detection primer pair:

[0096] CYP2C29 forward primer: 5'-ATCTGGTCGTGTTCCTAGCG-3' (SEQ ID NO:9);

[0097] CYP2C29 reverse primer: 5'-AGTAGGCTTTGAGCCCAAATAC-3' (SEQ ID NO:10);

[0098] CYP2E1 PCR detection primer pair:

[0099] CYP2E1 forward primer: 5'-CGTTGCCTTGCTTGTCTGGA-3' (SEQ ID NO:3);

[0100] CYP2E1 reverse primer: 5'-AAGAAAGGAATTGGGAAAGGTCC-3' (SEQ ID NO:4);

[0101] CYP3A11 PCR detection primer pair:

[0102] CYP3A11 upstream primer: 5'-GTCAAACGCCTCTCCTTGCTG-3' (SEQ ID NO:11);

[0103] CYP3A11 downstream primer: 5'-GGCTTGCCTTTCTTTGCCTTC-3' (SEQ ID NO:12);

[0104] CYP4A10 PCR detection primer pair:

[0105] CYP4A10 upstream primer: 5'-TTCCCTGATGGACGCTCTTTA-3' (SEQ ID NO:13);

[0106] CYP4A10 downstream primer: 5'-GCAAACCTGGAAGGGTCAAAC-3' (SEQ ID NO:14).

[0107] For rat genes:

[0108] GAPDH PCR detection primer pair:

[0109] GAPDH upstream primer: 5'-CCCATCACCATCTTCCAGGAG-3' (SEQ ID NO:15);

[0110] GAPDH downstream primer: 5'-GTTGTCATGGATGACCTTGGC-3' (SEQ ID NO:16);

[0111] CYP1A1 PCR detection primer pair:

[0112] CYP1A1 upstream primer: 5'-CATTGTGCCTGCCTCCTACTT-3' (SEQ ID NO:17);

[0113] CYP1A1 downstream primer: 5'-GTTCCTGTGGGTCTCTGCTGT-3' (SEQ ID NO:18);

[0114] CYP2B1 PCR detection primer pair:

[0115] CYP2B1 upstream primer: 5'-GGGAAAGAGGAGTGTGGAAGAA-3' (SEQ ID NO:19);

[0116] CYP2B1 downstream primer: 5'-GAGCAGATGATGTTGGCTGTG-3' (SEQ ID NO:20);

[0117] CYP2C11 PCR detection primer pair:

[0118] CYP2C11 upstream primer: 5’-AGCTTGGTGGCTACTGTAACTGAC-3’ (SEQ ID NO:21);

[0119] CYP2C11 downstream primer: 5’-CAGCAGCAGCAGGAGTCCATAC-3’ (SEQ ID NO:22);

[0120] CYP2E1 PCR detection primer pair:

[0121] CYP2E1 upstream primer: 5’-GGAAGGATGTGCGGAGGTT-3’ (SEQ ID NO:23);

[0122] CYP2E1 downstream primer: 5’-CAGAAATGTGGGGTCAAAAGG-3’ (SEQ ID NO:24);

[0123] CYP3A2 PCR detection primer pair:

[0124] CYP3A2 upstream primer: 5’-GATCCTTTTGTGGAGAAAACCAAG-3’ (SEQ ID NO:25);

[0125] CYP3A2 downstream primer: 5’-TTGGGGTGAGGAATGGAAAG-3’ (SEQ ID NO:26);

[0126] CYP4A1 PCR detection primer pair:

[0127] CYP4A1 upstream primer: 5’-CTCTTACTTCGGAGAATGGAGAA-3’ (SEQ ID NO:27).

[0128] CYP4A1 downstream primer: 5’-GACTTGGATACCCTTGGGTAAAG-3’ (SEQ ID NO:28).

[0129] Calculate the relative expression induction fold of each detected gene, and the results are shown in Figure 2 and Figure 3 , and the results show that the induction method of mouse liver S9 in the embodiments of the present invention has a strong induction effect on the expression of typical metabolic enzymes of chemical substances, especially on the metabolic enzymes 2E, 2C, and 4A that have no induction effect in the traditional induction method.

[0130] The above specific embodiments have described the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the relevant art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of an inducer in inducing mouse liver S9 or in preparing a product for inducing mouse liver S9; The inducer is octyl cyanoacrylate and etaconazole.

2. An induction method for mouse liver S9, characterized in that, It includes the following steps: First, use octyl cyanoacrylate in the inducer to induce mice, and then use etaconazole for induction. Take the liver of the induced mice, cut it into pieces, add buffer to make a liver homogenate. After centrifugation, the supernatant is mouse liver S9; The inducer is octyl cyanoacrylate and etaconazole.

3. The induction method according to claim 2, characterized in that, The dosage of octyl cyanoacrylate is 50 - 100 mg / kg, and continuous administration for induction is carried out for 2 - 4 days.

4. The induction method according to claim 3, characterized in that, The administration method of octyl cyanoacrylate is oral administration.

5. The induction method according to claim 2, wherein The dosage of etaconazole is 3 - 5 mg / kg, and continuous administration for induction is carried out for 2 - 4 days.

6. The induction method according to claim 5, wherein The administration method of etaconazole is injection.

7. The induction method according to claim 2, wherein The buffer includes 0.1 - 0.2 mol / L KCl buffer, and the addition amount of the buffer is 2 - 4 times the wet weight of the liver.

8. Use of the induction method according to any one of claims 2 - 7 in preparing mouse liver S9.

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