Comparison of cytochrome P450 activity and mRNA expression in canine vs. human hepatocytes after acetaminophen, diclofenac, or valproic acid exposure

Abstract

Importance

Although extensive studies have been conducted on cytochrome P450 (CYP) enzymes in rodents and humans, research on canine CYP enzymes is limited. The lack of species-specific metabolic research on dogs presents a major challenge in predicting toxicity and adverse drug reactions.

Objective

This study aimed to examine the interspecies differences in CYP enzyme activity and mRNA expression between canine and human hepatocytes following treatment with acetaminophen (AAP), diclofenac (Dic), or valproic acid (VPA).

Methods

We determined the 24-h exposure half-maximal inhibitory concentration (IC₅₀) values of AAP, Dic, and VPA in canine and human hepatocytes. Based on these IC₅₀ concentrations, we compared drug-induced alterations in various parameters, including immunocytochemistry, transcriptomic profiles (RNA-seq), and CYP activity, to assess changes at the gene and protein levels.

Results

AAP and VPA increased CYP2J2 mRNA expression by 4.7- and 7.76-fold, respectively, whereas Dic increased CYP1A1 mRNA expression by 14.25-fold in canine hepatocytes. AAP, VPA, and Dic decreased CYP26B1 mRNA expression in canine hepatocytes by 0.03-, 0.12-, and 0.17-fold, respectively. Dic and VPA increased CYP1A1 mRNA expression by 5.53- and 6.66-fold, respectively, whereas AAP, VPA, and Dic decreased CYP4F22 mRNA expression by 0.03-, 0.13-, and 0.13-fold, respectively, in human hepatocytes.

Conclusions and Relevance

The observed differences between species in CYP activity and mRNA levels in response to drug exposure highlight the importance of accurate and precise experimental models for the development of new medications.

INTRODUCTION

The liver has diverse functions in metabolism, protein synthesis, and bile production [1] and plays a crucial role in drug metabolism. Cytochrome P450 (CYP) enzymes, which serve as oxidizers, reducers, and hydrolyzers [2], play a pivotal role in this process. Understanding CYP activity, which aids in the conversion of fat-soluble chemicals into water-soluble compounds for excretion, is of the utmost importance. The potential benefits of understanding CYP activity include a better understanding of drug duration, drug-drug interactions, and unexpected side effects. In vitro studies on CYP450 enzyme activity are essential during the early phases of drug development to assess the potential of new chemical entities to inhibit or induce these enzymes [3]. CYP450 enzymes are classified across species using a family/subfamily/individual enzyme naming system [4]. Although CYP activity has been extensively studied in rodents as a surrogate for humans [5,6] relatively little is known about CYP activity in dogs. Legal permission for veterinarians to prescribe approved human drugs to animals under certain circumstances further highlights the relevance of this study. Dogs contain enzymes from the same subfamilies as those found in other species (CYPs 1, 2, and 3), and specific dog CYP450 isozymes (CYP1A1/2, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26) have been identified. Orthologs among species are evolutionarily related, share significant amino acid similarities, and may have similar substrate profiles [7,8]. However, there are notable differences in substrate specificity among orthologous P450 enzymes across species [5]. Dog CYP2B11, orthologous to human CYP2B6, metabolizes midazolam in dogs but not in humans [9]. Rifampin induces CYP3A12 expression in dogs and affects CYP3A4 expression in humans. Conversely, dexamethasone, which induces CYP3A4 in humans, exhibits a weaker induction of CYP3A activity in dogs [10]. Understanding the species-specific responses of CYPs to pharmaceuticals is crucial for ensuring their safety and efficacy in a particular species.

Drug-induced liver injury (DILI)-causing drugs are well-known substances that have been clinically reported to cause liver damage. AAP belongs to the antipyretic and analgesic classes and can induce liver injury as a result of the toxic metabolite (NAPQI) formed in cases of overdose. Dic is a non-steroidal anti-inflammatory drug that causes various mechanisms of liver toxicity, including hepatocyte toxicity and cholestatic injury, through COX inhibition and metabolic processes. VPA is an anticonvulsant used to treat epilepsy that can cause hepatocyte necrosis through specific mechanisms, such as mitochondrial dysfunction. All three drugs (AAP, Dic, and VPA) are widely used and frequently subject to market withdrawal or warnings owing to DILI. The toxic effects of these drugs were similar in humans and dogs, indicating comparable manifestations of liver injury across these species. These drugs were selected to verify whether the experimental model could predict and validate diverse real-world liver toxicity patterns and mechanisms of drug metabolism. Therefore, we hypothesized that CYP activity may differ between dogs and humans treated with various drugs. To investigate this, unbiased mRNA sequencing was performed to analyze CYP expressions in canine and human hepatocytes treated with AAP, Dic, or VPA. Our study aimed to determine whether human medications could be prescribed to companion animals without preclinical and clinical studies on these animals.

METHODS

Establishment of immortalized primary canine hepatocytes

Primary Beagle dog hepatocytes (LONZA, Switzerland) were transduced with a lentivirus carrying simian virus 40 (SV40) T antigen (Applied Biological Materials, Canada) and human telomerase reverse transcriptase (hTERT) (Applied Biological Materials) for 72 h at 37°C with 5% CO₂. Immortalized primary canine hepatocytes retain a morphology similar to that of their primary counterparts.

Cell culture

Canine hepatocytes were maintained on a dish coated with Applied Cell Extracellular Matrix (Applied Biological Materials, Canada) using HCM Basal Medium supplemented with HCM SingleQuot Supplements (LONZA) at 37°C in a 5% CO₂ incubator.

HepaRG cells (Thermo Fisher Scientific, USA), an established human hepatocyte-like cell line [7], were maintained on a dish coated with Applied Cell Extracellular Matrix (Applied Biological Materials) using William’s E Medium (Thermo Fisher Scientific) supplemented with HepaRG™ Thaw, Plate & General Purpose Medium Supplement (Thermo Fisher Scientific) at 37°C in a 5% CO₂ incubator.

Acetaminophen (AAP; Sigma Aldrich, USA), diclofenac (Dic, Sigma Aldrich), and valproic acid (VPA, Sigma Aldrich) were dissolved in 10% methanol to prepare a 100 mM stock solution. Canine hepatocytes were treated with 10 mM AAP, 0.5 mM Dic, or 10 mM VPA as the final concentration for 24 h, and RNA-seq, CYP activity, and CYP immunocytochemistry were conducted. HepaRG cells were treated with 5 mM AAP, 1 mM Dic, or 10 mM VPA as the final concentration for 24 h, and RNA-Seq and CYP assay were conducted.

Cell viability

Cell viability was assessed by measuring the reduction of the water-soluble yellow dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble blue formazan product. The half-maximal inhibitory concentration (IC₅₀), the concentration of the drug that inhibits cell proliferation by 50%, was determined by treating cells with AAP, Dic, or VPA for 24 h, followed by adding 100 μL of MTT working solution (1 mg/mL in phosphate-buffered saline [PBS]) to each well for 4 h. The resulting formazan crystals were then dissolved by adding 100 μL DMSO and measured spectrophotometrically at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, USA). The percentage cell proliferation was calculated as follows: (absorbance of treated cells/absorbance of control cells) × 100. Cell viability was plotted, and the IC₅₀ was calculated to determine the optimal drug dosage.

Immunocytochemistry

Cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.01% Triton X-100 in PBS, and blocked with 1% BSA. The cells were then incubated with primary antibodies against phalloidin 488 (Thermo Fisher Scientific) or CYP26B1 (Abcam, UK). Subsequently, the cells were incubated in the dark for 1h with a secondary antibody solution containing Alexa Fluor® 488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific). Nuclear counterstaining was performed using DAPI (10μg/mL; Thermo Fisher Scientific). Finally, stained cells were photographed using a confocal microscope (ZEISS LSM 800; Germany).

RNA isolation and RNA-seq analysis

A total of 1 μg RNA was isolated from canine hepatocytes and HepaRG cells using the phenol/chloroform extraction method. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent, USA). Each cDNA library was prepared using a QuantSeq 3’ mRNA-seq Library Prep Kit (Lexogen, Austria). The entire process, including sequencing, mapping, and normalization, was conducted according to the manufacturer’s instructions. Differentially expressed gene (DEG) analysis between groups, along with visualization of hierarchical heat maps and volcano plots, was performed using Excel-based Differentially Expressed Gene Analysis (ExDEGA; E-biogen, Inc., Korea).

Functional annotation analysis and gene ontology

To explore the functional annotation of DEGs in AAP-, Dic-, and VPA-treated cells compared with untreated cells, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted using the Database for Annotation, Visualization, and Integrated Discovery Bioinformatics Resources 6.8. Additionally, upstream regulators such as transcription factors, canonical pathways, and causal network analysis were investigated using Ingenuity Pathway Analysis (Qiagen, USA).

CYP protein levels (ELISA)

CYP protein levels were measured using ELISA kits for CYP 1A1, CYP 2J2, and CYP26B1 (canine CYPs) and CYP1A1 and CYP4F22 (human CYPs), according to the manufacturer’s instructions (MyBioSource, USA; Thermo Fisher Scientific). Cell culture media were centrifuged at 1,000 × g for 15 min to remove debris. A volume of 100 μL of standards or samples was added to the coated wells; 100 μL of PBS was added to the blank control well. For samples, 10 μL of Balance Solution was mixed into 100 μL and well mixed. Next, 50 μL of Conjugate was added to each well except the blank control and mixed thoroughly. Plates were incubated for 1 h at 37°C, then washed manually. A volume of 50 μL each of Substrate A and Substrate B was added to all wells and incubated for 15–20 min at 37°C. Then, 50 μL of Stop Solution was added to each well. The absorbance was measured at 450 nm using a microplate reader.

Statistical analysis

The results were presented as the means ± SD unless noted otherwise. Exact p values were reported, and significance was defined as p < 0.05. Statistical analyses were performed using one-way analysis of variance, followed by Duncan’s multiple range test, using GraphPad Prism 8.0 software (GraphPad Software, USA).

RESULTS

Assessment of IC₅₀ of canine hepatocytes under AAP, Dic, and VPA treatment

To assess the toxicity of AAP, Dic, and VPA in the liver, we exposed canine hepatocytes to various concentrations of these drugs. Canine hepatocytes exhibited a dose-dependent decrease in cell viability; however, the IC₅₀ of these drugs varied. AAP (10 mM) and VPA (10 mM) decreased cell viability by 50%, whereas Dic (0.5 mM) caused 50% cell death (Fig. 1A and B). Drug-mediated apoptosis was linked to structural changes, such as a shift from a spindle to a round shape, caused by disruption of the cytoskeleton (Fig. 1C). The IC₅₀ value of each drug was used for immunocytochemistry, RNA-seq, and CYP activity experiments to determine alterations at the gene level.

Fig. 1. Analysis of IC₅₀ of canine hepatocytes under acetaminophen, diclofenac, or valproic acid treatment. (A) Cells were treated for 24 h with 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, or 10 mM [-2, -1.6, -1.3, -1, -0.6, -0.3, 0, 0.4, 0.7, and 1 log(mM)], and viability was measured by MTT assay. IC₅₀ values selected for downstream assays were 10 mM AAP, 0.5 mM Dic, and 10 mM VPA. The Y-axis represents cell viability (%). (B) Cell morphology under a light microscope; scale bar = 100 µm (40×). (C) Phalloidin-FITC (cytoskeleton) and DAPI (nuclei); scale bar = 20 µm.

Ctl, control; AAP, acetaminophen; Dic, diclofenac; VPA, valproic acid.

Analysis of differential mRNA expression in canine hepatocytes under various drug treatments

mRNA sequencing was performed to analyze gene expression in canine hepatocytes exposed to hepatotoxic drugs (AAP, Dic, and VPA). When canine hepatocytes were treated with the IC₅₀ of each drug, various changes in gene expression patterns, either upregulation or downregulation, were observed. In total, 17,915 genes, excluding long non-coding RNA, were detected among the 35,531 genes in the canine reference genome. A comparison between untreated hepatocytes and those treated with AAP, Dic, or VPA revealed 455 (upregulated: 241, downregulated: 214), 329 (upregulated: 68, downregulated: 261), or 480 (upregulated: 330, downregulated: 150) DEGs with statistical significance (p < 0.05; |log₂ fold| ≥ 4), respectively (Fig. 2A and B). KEGG pathway analysis indicated that AAP regulates transcription, Dic regulates O-glycan biosynthesis, and VPA regulates arachidonic acid metabolism (Fig. 2C). These findings suggest that each drug regulated distinct genes and their related functional pathways.

Fig. 2. Transcriptome profile of DEGs in canine hepatocytes under acetaminophen, diclofenac, or valproic acid treatment. Canine hepatocytes were treated with 10 mM AAP, 0.5 mM Dic, or 10 mM VPA, and mRNA sequencing was conducted. (A) Hierarchical clustering heatmap of canine hepatocytes. Each row and column represents an individual library sample and gene product (gene symbols not shown). Colors represent the relative degree of gene expression (log₂-transformed); red indicates higher expression levels and blue indicates lower expression levels. (B) Volcano plot illustrating DEGs (red dots: upregulated genes; green dots: downregulated genes) defined by |log₂ fold| ≥ 2 on the X-axis and –log₁₀(p value) ≥ 1.3 on the Y-axis. (C) KEGG pathway analysis. The X-axis indicates the fold enrichment of DEGs and canonical KEGG terms. Color and size represent statistical significance.

AAP, acetaminophen; Dic, diclofenac; VPA, valproic acid; MAPK, mitogen-activated protein kinase; ECM, extracellular matrix; NF, nuclear factor; DEG, differentially expressed gene; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Involvement of CYPs under various drug treatments in canine hepatocytes

The expression of the drug-metabolizing CYP enzymes was examined in canine hepatocytes to determine which specific enzymes were influenced by drug interactions. AAP and VPA increase CYP2J2 expression, whereas Dic increases CYP1A1 expression. Interestingly, CYP26B1 expression was downregulated upon treatment with all three drugs (Fig. 3A). Notably, the actual CYP protein levels measured using ELISA revealed a different pattern compared with CYP gene expression. A significant increase in CYP1A1 levels was observed after VPA treatment, whereas elevated CYP2J2 levels were attributed to AAP and VPA treatments (Fig. 3B). In contrast, CYP26B1 protein levels did not decrease under the influence of the drugs, unlike the gene expression levels (Fig. 3C). These findings suggest that CYP response depend on specific drugs and may differ between mRNA and protein levels.

Fig. 3. CYP modulation in canine hepatocytes under acetaminophen, diclofenac, or valproic acid treatment. Canine hepatocytes were treated with 10 mM AAP, 0.5 mM Dic, or 10 mM VPA. (A) DEGs of CYPs defined by |log₂ fold| ≥ 2 are shown. The numerical values indicate fold changes at the gene level. (B) Measurement of CYP protein levels using ELISA. Protein levels of CYP1A1, CYP2J2, and CYP26B1 isoforms were assessed using specific ELISA kits. Data are presented as the mean ± standard deviation (n = 3). p < 0.05 vs. the control group. (C) Drug-induced CYP26B1 expression in canine hepatocytes determined by fluorescence microscopy; scale bar = 20 µm.

AAP, acetaminophen; CYP2J2, cytochrome P450 family 2 subfamily J member 2; CYP1A1, cytochrome P450 family 1 subfamily A member 1; CYP26B1, cytochrome P450 family 26 subfamily B member 1; Dic, diclofenac; VPA, valproic acid; Ctl, control; DEG, differentially expressed gene; CYP, cytochrome P450; ELISA, enzyme-linked immunosorbent assay.

^*p < 0.05.

Comparison of CYP regulation between canine and human hepatocytes (HepaRG) under AAP, Dic, and VPA treatment

We used human hepatocytes (HepaRG) to compare the involvement of CYPs in canine and human hepatocytes under the influence of AAP, Dic, and VPA. When HepaRG cells were treated with various drug concentrations, 5 mM AAP, 1 mM Dic, or 10 mM VPA resulted in a 50% reduction in cell survival (Fig. 4A and B). Drug-mediated apoptosis was associated with morphological shrinkage of cells, leading to a contracted or shrunken appearance as a result of cytoskeletal disruption (Fig. 4C). Analysis of CYP gene expression revealed that Dic and VPA increased CYP1A1 mRNA expression, whereas AAP, Dic, and VPA decreased CYP4F22 expression (Fig. 4D). In the present study, VPA decreased CYP1A1 protein levels, whereas Dic increased CYP4F22 protein levels (Fig. 4E). These results indicate that these drugs induce species-specific changes in CYP protein levels in canine and human hepatocytes and that drugs regulate gene expression and enzymatic activity of CYPs in distinct ways.

Fig. 4. CYP modulation in human hepatocytes under acetaminophen, diclofenac, or valproic acid treatment. (A) HepaRG cells, a human hepatocyte-like cell line, were treated for 24 h with 0–10 mM (10-point dilution: -2, -1.6, -1.3, -1, -0.6, -0.3, 0, 0.4, 0.7, and 1 log(mM)) and viability was measured by MTT assay. IC₅₀ values selected for downstream assays were 5 mM AAP, 1 mM Dic, and 10 mM VPA. The Y-axis represents cell viability (%). (B) Cell morphology under a light microscope; scale bar = 100 µm (40×). (C) Phalloidin-FITC (cytoskeleton) and DAPI (nuclei); scale bar = 20 µm. (D) Differentially expressed genes of CYPs defined by |log₂ fold| ≥ 2 are shown. The numerical values indicate fold changes at the gene level. (E) Measurement of CYP protein levels using ELISA. Protein levels of CYP1A1 and CYP4F22 isoforms were assessed using specific ELISA kits. Data are presented as mean ± standard deviation (n = 3). p < 0.05 vs. the control group.

AAP, acetaminophen; Dic, diclofenac; VPA, valproic acid; IC₅₀, half-maximal inhibitory concentration; Ctl, control; CYP1A1, cytochrome P450 family 1 subfamily A member 1; CYP4F22, cytochrome P450 family 4 subfamily F member 22; CYP, cytochrome P450; ELISA, enzyme-linked immunosorbent assay.

^*p < 0.05.

Analysis of differential mRNA expression in HepaRG under various drug treatments

The mRNA sequencing was performed to analyze gene expression in HepaRG cells exposed to hepatotoxic drugs (AAP, Dic, and VPA). When human hepatocytes were treated with the IC₅₀ of each drug, various changes in gene expression patterns, either upregulation or downregulation, were observed. In total, 29,346 genes, excluding long non-coding RNA, were detected among the 43,424 genes in the human reference genome. A comparison between untreated hepatocytes and those treated with AAP, Dic, or VPA revealed 1,420 (upregulated: 729, downregulated: 691), 1,683 (upregulated: 789, downregulated: 894), or 2,059 (upregulated: 1,055, downregulated: 1,004) DEGs with statistical significance (p < 0.05; |log₂ fold| ≥ 2), respectively (Fig. 5A and B). Moreover, KEGG pathway analysis indicated that AAP regulated fluid shear stress and atherosclerosis, whereas Dic and VPA regulated the mitogen-activated protein kinase signaling pathway (Fig. 5C). These findings suggest that each drug regulated distinct genes and their related functional pathways.

Fig. 5. Transcriptome profile of DEGs in human hepatocytes under acetaminophen, diclofenac, or valproic acid treatment. HepaRG cells were treated with 5 mM AAP, 1 mM Dic, or 10 mM VPA, and mRNA sequencing was performed. (A) Hierarchical clustering heatmap of human hepatocytes. Each row and column represents an individual library sample and gene product (gene symbols not shown). Colors represent the relative degree of gene expression (log₂-transformed); red indicates higher expression levels and blue indicates lower expression levels. (B) Volcano plot illustrating DEGs (red dots: upregulated genes; green dots: downregulated genes) defined by |log₂ fold| ≥ 2 on the X-axis and –log₁₀(p value) ≥ 1.3 on the Y-axis. (C) KEGG pathway analysis. The X-axis indicates the fold enrichment of DEGs and canonical KEGG terms. Color and size represent statistical significance.

Dic, diclofenac; AAP, acetaminophen; VPA, valproic acid; MAPK, mitogen-activated protein kinase; TNF, tumor necrosis factor; IL, interleukin; DEG, differentially expressed gene; KEGG, Kyoto Encyclopedia of Genes and Genomes.

DISCUSSION

In this study, we compared CYP activity and gene expression in canine and human hepatocytes after exposure to AAP, Dic, and VPA. As the number of companion animals has increased, industries have increasingly focused on medical care, general care, food, and treatment. Currently, companion animals are primarily treated with human medications and often depend on a limited range of veterinary drugs. However, variations in anatomy, physiology, and biochemistry (such as body weight, liver weight, tissue size, blood flow, and metabolic process rates) as well as drug absorption, distribution, metabolism, and excretion occur between animals and humans [11]. Moreover, variations in CYP isoforms among species significantly contribute to differences in drug metabolism [8]. For instance, omeprazole has been shown to induce hepatic CYP1A1 expression in humans but not in mice or rats [12,13,14,15]. In contrast, furafylline inhibits CYP1A2 activity in mice, dogs, and humans, but not in monkeys [16]. In our study, AAP upregulated CYP2J2 mRNA and protein expression in canine hepatocytes and increased CYP1A1 protein expression in human hepatocytes. Dic was associated with the upregulation of CYP1A1 mRNA in canine and human hepatocytes. VPA enhanced CYP1A1 and CYP2J2 protein levels, upregulated CYP2J2 mRNA expression in canine hepatocytes, and increased CYP1A1 mRNA expression in human hepatocytes. Collectively, these findings highlight the necessity of employing appropriate in vitro and in vivo models for nonclinical safety and efficacy evaluations in the development of veterinary pharmaceuticals.

Our results revealed a distinct pattern for CYP proteins and their corresponding mRNAs. Several factors influence gene expression, including mRNA stability, translation efficiency, and post-translational modifications (PTMs), which can affect protein stability [17]. In addition, variations in CYP metabolic activity among individuals have been linked to genetic polymorphisms. These polymorphisms can reduce the ability to induce or synthesize certain forms of CYP, which may alter their catalytic activities [18,19]. Thus, the differences observed between the mRNA and protein levels might be attributable to PTMs and/or genetic polymorphisms. CYP2E1, CYP1A2, CYP2A6, and CYP2D6 catalyze the conversion of AAP to NAPQI [20,21,22]; CYP2C9 hydrolyzes DIC [23]; and CYP2C9 and CYP2A6 mediate the oxidation of VPA to 4-Ene-VPA in the human liver [24,25]. However, different CYPs were regulated by AAP, Dic, and VPA in our study. These discrepancies highlight the limitations of in vitro studies compared with in vivo studies. In this study, we found variations in the major CYP enzymes between canine and human hepatocytes exposed to the same drugs, as well as differences in CYP protein and mRNA expression levels. Our findings suggest a need to develop medications specifically for companion animals. Because our experiments involved exposure to the drugs for 24 h, future studies incorporating multiple time points, proteome-level analyses, and evaluation of PTMs will be essential to capture the dynamic and multifactorial regulatory mechanisms of CYP enzymes.