Visual Overview
Abstract
Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important endogenous substances and the regulation of physiologic function. Although CYP2J2 is very important, few animal models are available to study its function in vivo. In particular, a CYP2J gene knockout (KO) rat model for drug metabolism and pharmacokinetics is not available. In this report, the CRISPR/Cas9 technology was used to delete rat CYP2J3/10, the orthologous genes of CYP2J2 in humans. The CYP2J3/10 KO rats were viable and fertile and showed no off-target effect. Compared with wild-type (WT) rats, the mRNA and protein expression of CYP2J3/10 in liver, small intestine, and heart of KO rats were completely absent. At the same time, CYP2J4 mRNA expression and protein expression were significantly decreased in these tissues. Further in vitro and in vivo metabolic studies of astemizole, a typical substrate of CYP2J, indicated that CYP2J was functionally inactive in KO rats. The heart function indexes of WT and KO rats were also measured and compared. The myocardial enzymes, including creatine kinase–muscle brain type (CK-MB), creatine kinase (CK), and CK-MB/CK ratio, of KO rats increased by nearly 140%, 80%, and 60%, respectively. In conclusion, this study successfully developed a new CYP2J3/10 KO rat model, which is a useful tool to study the function of CYP2J in drug metabolism and cardiovascular disease.
SIGNIFICANCE STATEMENT Human CYP2J2 is involved not only in clinical drug metabolism but also in the biotransformation of important endogenous substances. Therefore, it is very important to construct new animal models to study its function in vivo. This study successfully developed a new CYP2J knockout rat model by using CRISPR/Cas9 technology. This rat model provides a useful tool to study the role of CYP2J in drug metabolism and diseases.
Introduction
Human cytochrome P450 2J2 (CYP2J2) enzyme is involved in the metabolism of about 3% clinical drugs, which mainly include some antihistamines, such as astemizole, ebastine, and terfenadine (Xu et al., 2013; Zanger and Schwab, 2013). CYP2J2 is mainly expressed not only in human heart and liver (Askari et al., 2013; Lu et al., 2018) but also in tumors (Chen et al., 2011). In addition to drug metabolism, CYP2J2 is also involved in metabolism of endogenous substances such as arachidonic acid (AA), linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, and vitamin D3 (Xu et al., 2013). AA is a polyunsaturated fatty acid with important physiologic functions (Chen et al., 2011; Xu et al., 2013). Epoxyeicosatrienoic acids (EETs) are derived from CYP2J2-mediated AA metabolism (Chen et al., 2011). CYP2J2 can protect cardiac function in myocardial infarction–induced heart failure by increasing the concentration of circulating EETs and promoting angiogenesis through the Jagged1/Notch1 signaling pathway (Zhao et al., 2018). In addition, CYP2J2 inhibitor can significantly reduce the proliferation and migration and promote apoptosis of tumor cells by inhibiting the biosynthesis of EETs (Chen et al., 2011; Lu et al., 2018). Therefore, CYP2J2 plays a crucial role in drug metabolism and diseases.
In 2013, the Cyp2j locus was deleted through fusion bacterial artificial chromosome–mediated recombination, and the role of Cyp2j in hypoxic pulmonary vascular response was explored in this knockout mouse model (Zhou et al., 2013). In 2015, the role of CYP2J in pulmonary vascular remodeling and hypertension in mice was also discussed (Beloiartsev et al., 2015). The anti-inflammatory effect of CYP2J in macrophage is also revealed by a CYP2J4 knockout (KO) rat model (Behmoaras et al., 2015). However, a CYP2J3/10 KO animal model is not available. A CYP2J KO rat model is important for pharmacological research, especially for drug metabolism and pharmacokinetic (DMPK) studies. Rats possess larger body size and have more body fluids, which are conducive to DMPK studies (Lu et al., 2017). Moreover, it is reported that the rat model shows advantages over the mouse model for the study of cardiovascular diseases (Iannaccone and Jacob, 2009; Zhao et al., 2018). As the third generation of artificial nuclease technology, the CRISPR/CRISPR-associated 9 (Cas9) system has become the most important gene editing tool (Shao et al., 2014; Wang et al., 2016). CRISPR/Cas9 has made an important breakthrough in the construction of transgenic animal models and targeted gene therapy (Shao et al., 2014). CRISPR/Cas9, as a new gene editing technology, has the characteristics and advantages of accurate targeting, simple operation, simultaneous knockout of multiple target genes, and no species restriction (Shao et al., 2014; Li et al., 2019). In recent years, scientists have made important progress in the construction of rat DMPK models by using the CRISPR/Cas9 technology (Li et al., 2019). For example, in terms of rat DMPK models, we have successfully constructed Cyp2e1 (Wang et al., 2016), Cyp3a1/2 (Lu et al., 2017), Mdr1a/b (Liang et al., 2019), and Slco1b2 (Ma et al., 2020) gene KO rats by using the CRISPR/Cas9 system. These rat DMPK models are further used in the study of drug metabolism and transport, pharmacodynamic, toxicity, and disease-related molecular mechanism, strengthening the relationship between DMPK and pharmacology/toxicology, and promoting the new development of drug metabolism in innovative animal models (Qin et al., 2017; Ma et al., 2019).
In this report, the CRISPR/Cas9 technology is applied to the construction of the innovative rat CYP2J3/10 KO rat model, which can efficiently get the KO rats, significantly reduce the modeling cost, and shorten the modeling time. Further pharmacokinetic studies of astemizole, a typical substrate of CYP2J, confirmed that the KO rats lost the CYP2J metabolic function in vivo. This rat model provides a useful tool to study the role of CYP2J in drug metabolism and diseases.
Materials and Methods
Materials and Chemicals.
Oligos (60 bp, containing CYP2J3 or CYP2J10 knockout target sites) and all primers for polymerase chain reaction (PCR) were ordered from Biosune Biotechnology Co. LTD. (Shanghai, China). Ethidium bromide was obtained from TianGen biotechnology Company (Beijing, China). Trizol, TA cloning kit, Prime Script RT Reagent Kit, SYBR Premix Ex Taq, and in vitro Transcription T7 Kit were bought from Takara (Dalian, China). The mMessage mMachine SP6 kit was bought from Thermo Scientific (Waltham, MA). ELISA kits for the detection of CYP2J3, CYP2J4, and CYP2J10 proteins were purchased from Jiangsu Meimian industrial Co., Ltd. (Jiangsu, China). Astemizole was bought from MeilunBio (Dalian, China), and O-desmethyl astemizole was purchased from Toronto Research Chemicals (Toronto, Canada). Glucose 6-phosphate, glucose 6-phosphate dehydrogenase, NADP, and tribromoethanol were bought from Sigma (St. Louis, MO). Tris base was purchased from Amresco (Solon, OH). The internal standard (mebendazole) for liquid chromatography–tandem mass spectrometry (LC-MS/MS) was purchased from Aladdin (Shanghai, China).
Animals.
All wild-type (WT) male and female Sprague-Dawley (SD) rats used for gene targeting were purchased from the National Rodent Laboratory Animal Resources. All WT rats used in this research were littermate control of KO rats. Both WT and KO rats were kept under a specific pathogen-free facility with access to sterile water and rodent chow cubes, with 12-hour light-dark cycles. The research was conducted in accordance with the Declaration of Helsinki and/or with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health. All the animal experiment protocols in this study were approved by the Ethics Committee on Animal Experimentation of East China Normal University (Shanghai, China).
Generation of CYP2J3/10 Knockout Rat Model.
The CYP2J3/10 KO rat model was generated through CRISPR/Cas9 system, and the gene targeting method was reported in our previous studies with some modifications (Wang et al., 2016; Lu et al., 2017). The sequence fragments of CYP2J3 and CYP2J10 were submitted to a CRISPR Design Tool (http://tools.genome-engineering.org) to get target sites [an 18 bp DNA sequence with a protospacer adjacent motif site (5′-NGG-3′) in the 3′ end]. Oligo DNA sequence (60 bp) containing 18 bp target sequence and T7 promoter was synthesized in vitro. By overlapping PCR, oligos (60 bp) were linked to the pGS3-T7-gRNA vector for single guide RNA (sgRNA) transcription. sgRNAs were produced by using T7 Transcription Kit and purified with phenol/chloroform extraction method. Expression vectors for Cas9 were digested with NotI and then extracted by phenol/chloroform extraction methods. Cas9 mRNA was transcribed by using the mMessage mMachine SP6 kit and recovered by lithium chloride precipitation. The final concentrations of CYP2J3 sgRNA, CYP2J10 sgRNA, and Cas9 mRNA for microinjection were 30 ng/μl, 30 ng/μl, and 50 ng/μl, respectively. Cas9 online designer (http://cas9.wicp.net/) was used to evaluate potential off-target sites for the two sgRNAs, and off-target sites with a score ≥0.25 were selected for validation, using T7 endonuclease 1 (T7E1) assay. All primer pairs used in the generation of CYP2J KO rat model are listed in Table 1, including primer pairs for genotyping and off-target evaluation.
Primer pairs used in this study
Real-Time PCR.
Male SD rats (about 8 weeks) were sacrificed with carbon dioxide asphyxiation. Tissues were isolated as soon as possible and stored at −80°C for further processing. All tissues were adequately ground under the atmosphere of liquid nitrogen and then transferred into tubes. Trizol was added into tubes for the lysis of tissues, and total mRNA was extracted according to the manufacturer’s instructions. Total RNA was then reverse-transcribed into cDNA using the Takara RR036A RT kit. Real-time PCR was performed by using a Stratagene Mx3005P with SYBR Premix Ex Taq. All primer pairs used for real-time PCR are listed in Table 1. To detect the mRNA expression of CYP2J3, the primer pair (CYP2J3-Q-PCR forward and reverse) was designed on the downstream sequence of the sgRNA target site, and exon 1 to exon 3 of CYP2J3 gene were amplified. To detect the mRNA expression of CYP2J10, the primer pair (CYP2J10-Q-PCR forward and reverse) was designed on the downstream sequence of the sgRNA target site, and exon 1 to exon 2 of CYP2J10 gene were amplified. The PCR products for CYP2J3 and CYP2J10 mRNA detection were 268 bp and 235 bp, respectively.
Enzyme-Linked Immunosorbent Assay.
ELISA was conducted to detect the protein expression of CYP2J isoforms in rats. Male SD rats (about 8 weeks) were sacrificed with carbon dioxide asphyxiation. Tissues (liver, small intestine, and heart) were isolated as soon as possible and stored at −80°C for further processing. PBS (1 ml) was added into tissues (100 mg), and then tissues were homogenized under the temperature of 4°C. The homogenate was then centrifuged at 4°C, 3000g for 20 minutes. The supernatant was collected for further use. The method of ELISA detection is according to the product instructions with some modification. Standard samples and test samples (tissues supernatant) were added into 96-well plate, respectively, and the enzyme-labeled reagent (100 μl) was added into each well. Then, the plate was incubated at 37°C for 60 minutes and then washed with washing buffer for 5 times. Developer A (50 μl) and developer B (50 μl) were added into each well, and then the plate was shaken gently to mix, followed by incubation at 37°C for 15 minutes in the dark. After incubation, the reaction was stopped by adding the stop solution (50 μl). Finally, the absorbance (optical density value) of each well was measured at 450 nm wavelength after setting the zero point by using the blank control well.
Metabolic Capacity of CYP2J in WT and KO Rats In Vitro.
Methods for the preparation of rat liver microsomes (RLMs; from 8 weeks, male rats) were modified from our previous studies (Wang et al., 2010; Wang and Yeung, 2011). Astemizole was used as the probe substrate, and the reaction of astemizole O-demethylation was monitored for the evaluation of CYP2J metabolic activity in vitro (Lu et al., 2018). The incubation mixture contained an NADPH regenerating system (5 mM glucose 6-phosphate, 0.4 U/ml glucose 6-phosphate dehydrogenase, and 1 mM NADP), astemizole (0.2–10 μM), and 0.1 mg/ml of RLM in 0.05 M Tris-HCl buffer (pH 7.4). The incubation was stopped by precooled acetonitrile after 20-minute incubation. The internal standard (mebendazole) was added, and the supernatant of the incubation mixture was then transferred to the autosampler vial after a protein precipitation process for LC-MS/MS analysis. According to the standard Michaelis-Menten equation, Km and Vmax were analyzed by GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA), and the Vmax/Km ratio was defined as intrinsic clearance (CLint).
Pharmacokinetic Analysis of Astemizole in WT and KO Rats.
Astemizole (20 mg/kg) was administered through gavage for all WT and KO rats (8 weeks, male), and blood samples (300 μl) were collected into heparinized centrifuge tubes at 5, 15, 30, 45, 60, 120, 180, 240, 300, and 420 minutes through caudal vein with capillary tubes. Blood samples were centrifuged at 5500g for 15 minutes at 4°C, and the plasma was transferred into new tubes and frozen at −80°C for further detection. Protein precipitation was applied for the pretreatment of plasma samples. Acetonitrile (300 μl) and internal standard (mebendazole, 10 μl) were added to 100 μl plasma in order, followed by the centrifugation at 4°C, 14,000g for 15 minutes. The supernatant (70 μl) was transferred into the autosampler vial for LC-MS/MS detection.
Methods for LC-MS/MS Analysis.
For the LC-MS/MS detection, a 1290 HPLC-6460 triple quadrapole mass spectrometer coupled with electrospray ionization ion source (Agilent Technologies) was employed. For the detection of astemizole and its metabolite O-demethylastemizole in both RLM incubation mixture and plasma, the mobile phase, consisting of water (A) with 0.1% formic acid and acetonitrile (B) with 0.1% formic acid, was applied. Astemizole and O-demethylastemizole were eluted by gradient with a flow rate of 0.3 ml/min as follows: 0–1 minute, 20% acetonitrile; 1–3.5 minutes, 20%–80% acetonitrile; 3.5–4.5 minutes, 80% acetonitrile; 4.5–5 minutes, 80%–20% acetonitrile; 5–8 minutes, 20% acetonitrile. The detection of the ions was performed in a positive multiple reaction monitoring mode, monitoring the transition of m/z 459.2–218.2 for astemizole, m/z 445.2–121.2 for O-demethylastemizole, and m/z 296.1–264.1 for internal standard mebendazole.
Clinical Chemistry Analysis of WT and KO Rats.
Blood from 8-week-old male rats was collected through caudal vein, and the serum was prepared by the following steps: standing at 4°C for 30 minutes, centrifugation at 4°C, 1500g for 15 minutes. Then the serum supernatant was transferred into a new tube and stored at −80°C for further use. Serum samples were sent to ADICON Clinical Laboratories (Shanghai, China) for the detection of biochemical criterions, including albumin, globulin, albumin/globulin ratio, total proteins, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, aspartate aminotransferase/alanine aminotransferase ratio, direct bilirubin, indirect bilirubin, total bilirubin, triglycerides, low-density-lipoprotein cholesterol, high-density-lipoprotein cholesterol, total cholesterol, high-density-lipoprotein cholesterol/low-density-lipoprotein cholesterol ratio, creatine kinase (CK), creatine kinase–muscle brain type (CK-MB), and CK-MB/CK ratio.
Statistical Analysis.
All data are presented as means ± S.D. Statistical analysis between different groups was performed using two-tailed t test, and P values less than 0.05 were considered to indicate statistical significance. The pharmacokinetic parameters of astemizole were calculated by WinNonlin professional version 5.2.1 (Pharsight Mountain View, CA) based on the noncompartmental method.
Results
Generation of CYP2J3/10 Knockout Rat Model.
According to the results from “CRISPR DESIGN”, 5′-CCCCAAGAACTACCCGCC-3′ (followed with AGG) and 5′-GGTGTAACAGGGCGATTC-3′ (followed with AGG) were selected as target sites for rat CYP2J3 and CYP2J10, respectively. The quality of CYP2J3 and CYP2J10 sgRNA was good enough for injection, which was determined through agarose gel electrophoresis (Fig. 1, A and B). After coinjection of CYP2J3 sgRNA, CYP2J10 sgRNA, and Cas9 mRNA into one-cell fertilized eggs of SD rats, five progenies were born. For identification of gene modifications in the F0 generation progeny, sequence flanking target sites were amplified through PCR, and the products were digested with T7E1. For CYP2J3, PCR products of three progenies (F0-2#, 3#, 4#) were detected with T7E1 cleavage, which indicated the potential of gene modification around the target sites (Fig. 1C). The PCR product was inserted into the T vector, and the sequencing of the T vector further revealed that all three progenies contained the same 10 bp deletion at the target site (Fig. 1D). For CYP2J10, amplification of sequence flanking the target site was not successful for F0-3#, 5# until the product size was enlarged to more than 3 kb. This demonstrated a large-scale deletion possibility, and direct sequencing of these products showed an 1199 bp deletion around the target site for both F0-3# and F0-5# (Fig. 1, C and D). Thus, F0-3# was selected for the breeding of homozygous KO rats, and finally F2 progenies with 10 bp deletion for CYP2J3 and 1199 bp deletion for CYP2J10 were gained.
Generation of CYP2J3/10 knockout rat model. (A) Characterization of precursors for CYP2J3 (lane 1) and CYP2J10 (lane 2) sgRNA. (B) Characterization of sgRNA for CYP2J3 (lane 1) and CYP2J10 (lane 2). (C) Detection of mutations in the F0 generation using T7E1 assay. T7E1−, before T7E1 digestion; T7E1+, after T7E1 digestion;, mutant band. (D) DNA sequencing flanking CYP2J3 and CYP2J10 target loci in F0 rats. “.”, nucleotide deletion; “∆”, the number of changed nucleotide; “X”, the number of each genotype in four clones.
Off-Target Analysis.
Off-target effects of CRISPR/Cas9 system, targeting CYP2J3 and CYP2J10, were searched on a genomewide sgRNA off-target searching tool (http://cas9.wicp.net/) and then evaluated by T7E1 cleavage assay. Off-target sites with a relatively high off-target score (≥0.25) were selected for T7E1 treatment (Table 2). Agarose gel electrophoresis results showed no off-target effect in all high potential off-target sites (Fig. 2).
Potential off-target sites evaluated through T7E1
Off-target analysis of CYP2J3 (A) and CYP2J10 (B) sgRNA. T7E1−, before T7E1 digestion; T7E1+, after T7E1 digestion; OT, off-target site.
Expression of CYP2J in Tissues of WT and KO Rats.
The mRNA expression of CYP2J3, CYP2J4, and CYP2J10 in rat liver, small intestine, and heart was detected through specific primer pairs (Table 1). Agarose gel electrophoresis results verified that no mRNA expression of CYP2J3, CYP2J4, and CYP2J10 was detected in above tissues of KO rats, whereas it was highly expressed in WT control of these corresponding tissues (Fig. 3A).
The mRNA (A) and protein (B) expression of CYP2J3, CYP2J4, and CYP2J10 in liver, small intestine, and heart of WT and KO rats. N.D., not detected. The data are expressed as means ± S.D. (n = 6). ***P< 0.001 compared with WT group.
The protein levels of CYP2J3, CYP2J4, and CYP2J10 in rat liver, small intestine, and heart were detected by ELISA. CYP2J3 was not detected in the liver of KO rats, whereas CYP2J4 and CYP2J4 decreased by about 80% and 90% (Fig. 3B), respectively. CYP2J3 and CYP2J10 in the small intestine of KO rats were undetectable, whereas CYP2J4 decreased by around 75% (Fig. 3B). In the heart of KO rats, CYP2J3, CYP2J4, and CYP2J10 decreased about 90%, 60%, and 90% (Fig. 3B), respectively.
Metabolic Capacity of CYP2J in WT and KO Rats In Vitro.
To assess the metabolic function of CYP2J in the KO rat in vitro, astemizole was chosen as a probe substrate to characterize the metabolic activity of CYP2J in RLMs. In the RLMs of WT rats, the O-desmethylastemizole was formed effectively, compared with that in RLMs of KO rats (Fig. 4, A–C). The apparent Km values for the metabolism of astemizole in the RLMs of WT and KO rats were 8.2 and 9.7 μM, respectively. The Vmax of astemizole metabolism in KO rats was decreased to 40% of that in WT rats, and the CLint was also decreased to 35% of that in WT rats (Fig. 4, B and C). All these results indicated that the CYP2J activity of KO rats was significantly decreased in vitro.
Effects of CYP2J3/10 deletion on the metabolic capacity of CYP2J. (A) Saturation curve of astemizole metabolism in RLMs from WT and KO rats. (B) The Vmax of astemizole metabolism in RLMs from WT and KO rats. (C) The CLint of astemizole metabolism in RLMs from WT and KO rats. (D) Pharmacokinetic profiles of astemizole in WT and KO rats. (E) The concentration-time curve of astemizole metabolite (O-desmethyl astemizole) in WT and KO rats. The data are expressed as means ± S.D. (n = 5). *P< 0.05; ***P< 0.001 compared with WT group.
Metabolic Capacity of CYP2J in WT and KO Rats In Vivo.
To further assess the metabolic function of CYP2J in the KO rat in vivo, the pharmacokinetics behavior of astemizole was monitored and compared between WT and KO rats. After oral administration of astemizole, the metabolic rate of KO rats was much slower, and the area under the curve (AUC) was 4.6 times higher than that of WT rats (Fig. 4, D and E, and Table 3). At the same time, the Cmax of astemizole in KO rats was significantly increased, with 2.4 times of that in WT rats (Table 3). CYP2J3/10 KO also prolonged the half-life (3.4 times of that in WT rats) and mean retention time (1.4 times of that in WT rats) and decreased the clearance (CL/F) and apparent volume of distribution (Vd/F) values (Table 3). Therefore, the pharmacokinetic results were consistent with the data of the in vitro metabolic activity test, indicating that CYP2J lost its metabolic function in KO rats.
Pharmacokinetic parameters of astemizole in WT and KO rats
All data are expressed as means ± S.D. (n=5).
Clinical Biochemical Criterion Analysis of WT and KO Rats.
In addition to drug metabolism, CYP2J2 is also a great contributor to the biotransformation of physiologic substances and plays an important role in human health. The loss of CYP2J genes in rats may cause some physiologic changes, and thus the clinical chemistry analysis was carried out. Compared with WT rats, CYP2J3/10 KO in rats showed no significant difference of serum protein, alkaline phosphatase, transaminase, bilirubin, triglyceride, and cholesterol (Fig. 5A). Since CYP2J2 is necessary for cardiac function, the biomarkers of myocardial function were also measured and compared. The results showed that the values of CK-MB, CK, and CK-MB/CK ratio of KO rats significantly increased by almost 140%, 80%, and 60%, respectively, indicating that KO rat cardiomyocytes had the potential of injury (Fig. 5B).
Effects of CYP2J3/10 deletion on clinical biochemical criterions in KO rats. (A) Effects on serum protein, alkaline phosphatase, transaminase, bilirubin, triglyceride, and cholesterol. (B) Effects on creatine kinases. ALB, albumin; ALT, alanine amino transferase; AP, alkaline phosphatase; AST, aspartate amino transferase; D-BIL,direct bilirubin; GLB, globulin; HDL-CHOL, high-density-lipoprotein cholesterol; ID-BIL, indirect bilirubin; LDL-CHOL, low-density-lipoprotein cholesterol; T-BIL, total bilirubin; T-CHOL, total cholesterol; TP, total proteins; TRIG, triglycerides. All data are expressed as means ± S.D. (n = 5). *P< 0.05; **P< 0.01 compared with WT group.
Compensatory Expression of Other Cytochrome P450 Isoforms and Nuclear Receptors in CYP2J KO Rats.
The deficiency of CYP2J3/10 may affect the expression of other cytochrome P450 (P450) isoforms and nuclear receptors in rats. Thus, mRNA expression of other main P450 isoforms and nuclear receptors in the liver, small intestine, and heart were measured by real-time PCR. Cyp1a2 mRNA expression in the WT liver decreased by about 50% of that in the KO rat liver, whereas the expression of Cyp3a2 significantly increased by about 80% (Fig. 6A). No significant change was observed in the small intestinal expression of all tested P450 isoforms and nuclear receptors (Fig. 6B). In the heart tissue of KO rats, Cyp1a2, Cyp2e1, Cyp3a1, and Cyp3a2 were upregulated by about 60%, 140%, 180%, and 260%, respectively (Fig. 6C).
Compensatory expression of other P450 isoforms and nuclear receptors in CYP2J3/10 KO rats. (A) Compensatory expression in the liver. (B) Compensatory expression in the small intestine. (C) Compensatory expression in the heart. All data are expressed as means ± S.D. (n = 5). *P< 0.05; **P< 0.01 compared with WT group.
Discussion
CYP2J2 is the only isoenzyme of the human CYP2J subfamily, whereas there are three CYP2J subtypes (CYP2J3, CYP2J4, and CYP2J10) in the rat genome. Compared with human CYP2J2 cDNA, the homology of CYP2J3, CYP2J4, and CYP2J10 cDNA is 79%, 80%, and 78%, respectively (data from National Center for Biotechnology Information). Human CYP2J2 is mainly expressed in the heart and liver, and the expression pattern of CYP2J3 in rats is similar (Xu et al., 2013). CYP2J10 is also expressed in rat aorta (Di Pascoli et al., 2015). However, CYP2J4 shows a different expression pattern compared with human CYP2J2 (Zhang et al., 1998; Xu et al., 2013). Additionally, CYP2J3 shows similar metabolic preference to human CYP2J2 on some endogenous substance, such as AA (Xu et al., 2013). Therefore, CYP2J3 and CYP2J10 were selected in this study to investigate the effect of CYP2J deficiency on the metabolic functions in rats.
To achieve effective target gene editing, we selected the target site at the first exon of CYP2J3 and finally obtained homozygotes with 10 bp deletion around the target site, which causes frame shift mutation of CYP2J3. For CYP2J10 targeting, a preferential target was found and chosen at the second exon. However, the sgRNA targeting CYP2J10 tends to induce the large-scale deletion on both sides of the target site, resulting in homozygous CYP2J10 with 1199 bp deletion. The same phenomenon has been reported in previous studies; that is, even if only one sgRNA is introduced into the zygote, there may be a deletion extending to more than thousands of bases (Kosicki et al., 2018). It is reported that CRISPR/Cas9 system can tolerate a 1–3 base pair mismatch in its target sequence (Shao et al., 2014). Therefore, the off-target effects in CYP2J3/10 KO rat model were evaluated. Under the threshold value of 0.25, only six and two potential off-target sites were selected for CYP2J3 and CYP2J10 sgRNA, respectively, which indicated a low off-target possibility of the target sites used in this study. No off-target effect was observed among these eight potential off-target sites by T7E1 assay. Furthermore, the PCR products were sequenced directly to verify the results from T7E1 assay (data not shown).
Our results showed the mRNA expression of CYP2J3/10 was completely absent in the liver, small intestine, and heart of KO rats. The protein level of CYP2J3 in the liver and small intestine of KO rats was undetectable, whereas it was detected in the heart of KO rats with a very low level (<10%, compared with that in the WT group). Since the protein level of CYP2J3 was measured using ELISA, we speculate that the detected value in the heart of KO rats was the background value after the endogenous CYP2J3 was missing. Similarly, the protein level of CYP2J10 in the small intestine of KO rats was undetectable, and the levels in the liver and heart of KO rats were also very low (<10%, compared with that in the WT group). To evaluate the compensation of CYP2J4 after CYP2J3/10 knockout, the mRNA and protein expression of CYP2J4 in KO rats was measured. The mRNA expression of CYP2J4 was also completely absent in the liver, small intestine, and heart of KO rats. However, the protein expression of CYP2J4 in the liver, small intestine, and heart of KO rats decreased by 80%, 75%, and 60%, respectively. We speculate that CYP2J4, CYP2J3, and CYP2J10 share some regulatory regions and sequence because they are located in order on 5q33 of chromosome 5 and are close to each other. Therefore, the deletion of CYP2J3 and CYP2J10 sequences may interfere with the regulation region and sequence of CYP2J4 and eventually lead to the downregulation of CYP2J4. Our initial aim was to knock out CYP2J3 and CYP2J10, but we unexpectedly decreased the expression of CYP2J4. Such a model may be more conducive to study the function of CYP2J.
To further confirm the deficiency of metabolic function of CYP2J, its metabolic ability was evaluated both in vitro and in vivo. Although other P450 isoforms, such as CYP2D6 and CYP4F12, may be involved in the catalytic action of the conversion of astemizole to O-demethylated astemizole, CYP2J is still the primary drug metabolizing enzyme that mediates this reaction (Matsumoto and Yamazoe, 2001; Matsumoto et al., 2003; Xu et al., 2013). Therefore, astemizole is selected as the probe substrate of CYP2J metabolic capacity phenotype in rats (Matsumoto et al., 2003; Lu et al., 2018). The RLM incubation and pharmacokinetics of astemizole indicated that the metabolic function of CYP2J was decreased in KO rats. The residual metabolic activity of KO group may be related to other P450 isoforms in rats. Therefore, the CYP2J3/10 KO rat model was generated successfully by using CRISPR/Cas9 technology. To our knowledge, it is the first time to obtain CYP2J3/10 KO rats, which are a good tool to study CYP2J-mediated drug metabolism in vivo.
CYP2J is also involved in the metabolism of various endogenous substances and may play a role in physiologic functions. Thus, the basic physiologic status of KO rats was evaluated through clinical chemistry analysis. Compared with WT rats, CYP2J3/10 deficiency had no effect on serum protein, alkaline phosphatase, transaminase, bilirubin, triglyceride, and cholesterol. However, myocardial enzyme CK, CK-MB, and their ratio (CK-MB/CK) significantly increased by 140%, 80%, and 60%, respectively. Actually, the myocardial enzyme CK-MB is often used as a biomarker for the diagnosis of myocardial injury (Hueb et al., 2012) because it mainly exists in the heart muscle with relatively high concentration. In addition, the CK-MB index (CK-MB/CK) is a specific parameter to evaluate the relative contribution of cardiomyocyte damage, compared with other nonspecific myocardial cell injury (Saenger and Jaffe, 2007). In this study, CYP2J3/10 deficiency increased serum CK-MB concentration and the ratio of CK-MB/CK. These results suggested that there may be myocardial injury in KO rats, which needs further study.
AA and EETs, as essential polyunsaturated fatty acids, have many biologic functions, especially in human heart (Murray, 2016). CYP2J2 is a principal enzyme that promotes the biotransformation of AA to EETs in myocardial cells (Imig, 2012; He et al., 2017). In recent years, a series of studies have focused on the cardioprotection of the AA-CYP2J2-EET pathway (Ai et al., 2009; Imig, 2012; Ma et al., 2013; Fleming, 2014). In this study, the deletion of CYP2J3/10 was used to simulate the loss of function mutation of CYP2J2 in human body. The results revealed that CYP2J3/10 deficiency had some effects on cardiac function. Therefore, CYP2J3/10 is an important factor, not the only one, to maintain EET concentrations in vivo. Other P450s, such as CYP2C and CYP3A, have also been reported to mediate the biotransformation of AA to EETs (Panigrahy et al., 2010; Mitra et al., 2011). Therefore, the compensatory effect of other P450s in KO rats should be considered.
Last but not least, aging is a major risk factor for myocardial disease, which promotes changes in the structure and function of the heart, leading to deterioration in some patients with acute myocardial infarction (Boon et al., 2013; Roh et al., 2016). Therefore, we speculate that the effect of CYP2J3/10 deficiency on cardiac function of rats may become more and more serious in the process of aging. Further research should be carried out to make sure of this issue.
In conclusion, this study successfully developed a new CYP2J3/10 KO rat model, which provides a useful tool for the study of CYP2J in drug metabolism and diseases. CYP2J3/10 deficiency not only significantly reduced the metabolism of astemizole but also increased myocardial enzymes (CK and CK-MB) and their ratio (CK-MB/CK), which is a typical sign of myocardial injury. The present study provides a preliminary assessment of the role of CYP2J in drug metabolism and its potential impact on cardiac function.
Authorship Contributions
Participated in research design: Liu, Wang.
Conducted experiments: Lu, Chen, Ma, Shang, Zhang.
Performed data analysis: Lu, Wang.
Wrote or contributed to the drafting of the manuscript: Lu, Guo, Wang.
Footnotes
- Received May 10, 2020.
- Accepted August 4, 2020.
This work was supported in whole or part by grants from the National Natural Science Foundation of China [81301908, 81773808, and 81830083] and the Science and Technology Commission of Shanghai Municipality [17140901000, 17140901001, and 18430760400]. This work was also supported from East China Normal University Multifunctional Platform for Innovation [011].
Abbreviations
- AA
- arachidonic acid
- Cas9
- CRISPR-associated 9
- CK
- creatine kinase
- CK-MB
- creatine kinase–muscle brain type
- CLint
- intrinsic clearance
- CYP2J2
- cytochrome P450 2J2
- DMPK
- drug metabolism and pharmacokinetic
- EET
- epoxyeicosatrienoic acid
- KO
- knockout
- LC-MS/MS
- liquid chromatography–tandem mass spectrometry
- P450
- cytochrome P450
- PCR
- polymerase chain reaction
- RLM
- rat liver microsome
- SD
- Sprague-Dawley
- sgRNA
- single guide RNA
- T7E1
- T7 endonuclease 1
- WT
- wild-type
- Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics
