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Published in final edited form as: Chem Res Toxicol. 2021 Nov 17;34(12):2534–2539. doi: 10.1021/acs.chemrestox.1c00300

Role of CYP2A6 in methimazole bioactivation and hepatotoxicity

Jianhua Li †,§, Zahir Hussain †,§, Junjie Zhu †, Saifei Lei †, Jie Lu †, Xiaochao Ma †,*
PMCID: PMC8939439  NIHMSID: NIHMS1786020  PMID: 34788025

Abstract

Methimazole (MMI) is a widely used antithyroid drug, but it can cause hepatotoxicity by unknown mechanisms. Previous studies showed that hepatic metabolism of MMI produces N-methylthiourea leading to liver damage. However, the specific enzyme responsible for the production of the toxic metabolite N-methylthiourea is still unclear. In this study, we screened cytochromes P450 (CYPs) in N-methylthiourea production from MMI. CYP2A6 was identified as the key enzyme in catalyzing MMI metabolism to produce N-methylthiourea. When mice were pretreated with a CYP2A6 inhibitor, formation of N-methylthiourea from MMI was remarkably reduced. Consistently, the CYP2A6 inhibitor prevented MMI-induced hepatotoxicity. These results demonstrated that CYP2A6 is essential in MMI bioactivation and hepatotoxicity.

Keywords: Methimazole, bioactivation, CYP2A6, hepatotoxicity

Graphical Abstract

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INTRODUCTION

Over 75 years, antithyroid drugs remain the most preferred method of treating hyperthyroidism such as Graves’ disease worldwide.1 These drugs are commonly known as thionamides which include propylthiouracil (6-propyl-2-thiouracil, PTU), methimazole (1-methyl-2-mercaptoimidazole, MMI) and carbimazole (1-ethoxycarbonyl-3-methyl-2-mercaptoimidazole, an MMI prodrug). These thionamides exert their antithyroid effects by directly inhibiting the de novo synthesis of thyroid hormone.1 MMI is commonly used in most parts of Asia and Europe1 and also has become the first-line anti-hyperthyroid drug in the United States2 due to overt toxicity of PTU, especially in children.3

Although MMI is generally well-tolerated during anti-hyperthyroid therapy,1 hepatotoxicity of MMI has been reported, which can lead to the discontinuation of therapy.4-6 Hyperthyroid patients withdrawing their antithyroid medications due to liver toxicity must receive alternative treatments such as radioactive iodine intake or surgical removal of thyroid gland (www.thyroid.org). However, these alternative approaches make these patients rather hypothyroid forever, which further make them depend on lifelong thyroid hormone supplementation (www.thyroid.org). Therefore, it is necessary to develop mechanism-based strategies to predict and prevent MMI hepatotoxicity.

MMI hepatotoxicity is associated with its metabolism. 7, 8 A previous work found that cytochrome P450 (CYP) inhibitors attenuate MMI-induced liver toxicity.7 In a follow-up study conducted by the same research group, N-methylthiourea was determined as the toxic metabolite of MMI.8 These data suggest that CYP-mediated production of N-methylthiourea in MMI metabolism leads to hepatotoxicity. CYPs are a superfamily of enzymes that functions as monooxygenases, which are involved in endobiotic metabolism as well as xenobiotic metabolism.9 However, among these large number of CYPs, it is unknown which specific subtype metabolizes MMI and contributes to its liver toxicity.

In this study, we first established a liquid chromatography-mass spectroscopy-based assay of N-methylthiourea. We next screened and investigated the role of hepatic CYPs in the formation of N-methylthiourea from MMI. We found that CYP2A6, orthologous to mouse Cyp2a5,10 can potently catalyze the formation of N-methylthiourea from MMI. Our further study used a CYP2A6 inhibitor to determine the role of CYP2A6 in N-methylthiourea production and MMI hepatotoxicity in mice.

EXPERIMENTAL PROCEDURES

Chemicals and Reagents.

MMI was purchased from Alfa Aesar Inc. (MA, USA). N-methylthiourea and trans-2-phenylcyclopropylamine were purchased from Acros Organics (NJ, USA). Buthionine sulfoximine (BSO) and reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (MO, USA). p-nitrophenacyl bromide was purchased from Fisher Scientific (NJ, USA). Human liver microsome (HLM) and the recombinant human CYPs were purchased from XenoTech (KS, USA). All the solvents for ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) analysis were of the highest grade commercially available.

Method Development for N-methylthiourea Detection.

Because the metabolite N-methylthiourea cannot be detected directly by UPLC-QTOFMS, p-nitrophenacyl bromide was used as a trapping reagent, which reacts with N-methylthiourea to form 2-(methylamino)-4-(p-nitrophenyl)thiazole (Figure 1A). Briefly, 50 μM of N-methylthiourea and 5 mM of p-nitrophenacyl bromide were incubated under room temperature for 1 h with shaking. 2-(methylamino)-4-(p-nitrophenyl)thiazole, the trapped N-methylthiourea, was verified by the UPLC-QTOFMS.

Figure 1. UPLC-QTOFMS analysis of N-methylthiourea, the toxic metabolite of MMI.

Figure 1.

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(A) Trapping reaction between N-methylthiourea and P-nitrophenacyl bromide. (B, C) Chromatograms of trapped N-methylthiourea from the incubations of MMI with HLM (B) and standard (C), respectively. Trapped N-methylthiourea was detected by UPLC-QTOFMS.

MMI Metabolism with HLM and Recombinant Human CYPs.

The incubations with HLM were performed in 1×PBS (pH 7.4) containing 100 μM MMI, 100 μg of HLM, and 1 mM NADPH in a final volume of 97.5 μL followed by incubation at 37 °C for 1 h. The reactions were terminated with an equal volume of acetonitrile, vortexed for 30 s and centrifuged at 15,000 rpm for 10 min. Next, the supernatant was collected and 5 mM of p-nitrophenacyl bromide was added as a trapping agent for N-methylthiourea bringing the final volume to 200 μL followed by incubation for another 1 h under shaking. The resultant mixture was centrifuged to collect the supernatant. Two microliters of the supernatant were injected into the UPLC-QTOFMS system to measure the levels of trapped N-methylthiourea. Incubations in the absence of MMI, HLM, NADPH or p-nitrophenacyl bromide were performed as controls. The incubations with recombinant CYPs were performed in 195 μL volume under the same assay conditions as described above for HLM. These reactions contained 15 pmol of each recombinant human CYP (Control, 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 4A11 or 4F2). All the reactions were performed in triplicate, and the levels of trapped N-methylthiourea were measured by UPLC-QTOFMS. To validate the functionalities of these CYPs, classic probes including 7-ethoxy-resorufin, coumarin, efavirenz, warfarin, dextromethorphan, chlorzoxazone, midazolam were used for CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1 and 3A4 assays, respectively. These assays were performed in duplicates and the corresponding products were measured by UPLC-QTOFMS.

Animals and Treatments.

Swiss Webster mice (male, 8 weeks old, purchased from Taconic) were used to determine the effects of CYP2A6 inhibitor (trans-2-phenylcyclopropylamine) on MMI metabolism and hepatotoxicity. For MMI metabolism, mice were first treated with trans-2-phenylcyclopropylamine (30 mg/kg, in saline, i.p.). Ten min later, the mice were treated with MMI (40 mg/kg, in saline, p.o.) and were sacrificed at 0.5 and 1 h to collect livers (n = 3 at each time point). For MMI hepatotoxicity, mice were treated with MMI (40 mg/kg, in saline, p.o.) or BSO (667 mg/kg, in saline, i.p.) or trans-2-phenylcyclopropylamine (30 mg/kg, in saline, i.p.) or in combinations. BSO is a specific inhibitor of γ-glutamylcysteine synthetase and decreases endogenous glutathione (GSH) levels.11 BSO is needed in the development of MMI hepatotoxicity in mice.7, 8 Similar as the previous work,7, 8 MMI treatment was performed 1 h after BSO treatment in BSO+MMI (n = 5) and BSO+MMI+ CYP2A6 inhibitor groups (n = 6). BSO+MMI+CYP2A6 inhibitor group received trans-2-phenylcyclopropylamine 10 min earlier of MMI treatment. Mice in control or BSO or MMI group (n = 4) received saline at the aforementioned time intervals. Six h after MMI treatment, blood and liver samples were collected for evaluation of liver damage. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.

Liver Function Tests.

Hepatotoxicity was evaluated by analyzing serum alanine aminotransaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities (Pointe Scientific Canton, MI), and total bile acids (Cell Biolabs, Inc., San Diego, CA) according to the protocols supplied by the manufacturers. Serum bilirubin was measured by UPLC-QTOFMS.

Histological Analysis.

Liver tissues were first fixed in 4% formaldehyde phosphate solution overnight followed by dehydrated and embedded in paraffin. Paraffin blocks were cut into 4 μm sections and stained with hematoxylin-eosin (H&E).

Sample Preparation for UPLC-QTOFMS Analysis.

To measure N-methylthiourea from the liver samples, liver tissues were homogenized in water (50 mg tissue in 200 μL water), and then 200 μL of acetonitrile was added to 100 μL aliquot of the liver homogenate. The resulting mixtures were vortexed for 2 min and centrifuged at 15,000 rpm for 10 min. 200 μL of supernatant was then mixed with 5 mM trapping agent and incubated for 2 h under shaking. To measure bilirubin, 20 μL of serum sample was vortexed with 80 μL methanol:acetonitrile (1:1) for 2 min followed by centrifugation at 15,000 rpm for 10 min. Each supernatant from all samples mentioned above was transferred to an auto sampler vial and injected into the UPLC-QTOFMS system to analyze the metabolite of interest.

UPLC-QTOFMS Analysis.

Metabolites were separated by an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) followed by QTOFMS analysis using a SYNAPT G2 mass spectrometer (Waters Corporation, Milford, MA). Column temperature was maintained at 50 °C. The mobile phases for metabolite analysis were A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) and were delivered at a flow rate of 0.5 mL/min with a gradient elution (0.0-1.0 min, 2% B; 1.0-3.5 min, 2-95% B; 3.5-5 min, 95% B; 5-5.1 min, 95-2% B; 5.1-6 min, 2% B). The QTOFMS system was operated in positive mode with electrospray ionization. The detailed mass parameters were the same as previously reported.12

Statistical Analysis.

All data were expressed as means ± SD. Statistical analysis was performed with one-way ANOVA or two-tailed Student’s t-test, where applicable. P value < 0.05 was considered statistically significant.

RESULTS

N-methylthiourea Detection by UPLC-QTOFMS.

We developed an UPLC-QTOFMS method to detect N-methylthiourea by using P-nitrophenacyl bromide as a trapping reagent (Figure 1A). The trapped N-methylthiourea, from both the incubations of MMI with HLM (Figure 1B) and standard N-methylthiourea (Figure 1C), is readily detected by UPLC-QTOFMS, showing the same retention time at 3.1 min. The MS/MS spectra of MMI-derived N-methylthiourea also matched with standard N-methylthiourea because they share the same fragmental ions at m/z 190, 162 and 134 (Supplementary Figure 1).

Determination of MMI Catalyzing CYP Subtype.

Once the authentic standard of N-methylthiourea could be detected as described above, we tried to optimize the assay condition of MMI catalysis in terms of N-methylthiourea formation using HLM as an enzyme source. HLM contains most of drug metabolizing enzymes including various subtypes of CYP and is widely used to screen drug metabolic reactions in vitro.9, 13 As shown in Figure 2A, HLM-mediated production of N-methylthiourea from MMI is NADPH-dependent and can only be detected after adding the trapping agent in the assay mixtures. Next, to identify specific contribution of CYP subtypes, MMI was incubated with various recombinant CYP isozymes respectively using the same assay condition as HLM. The functionalities of major CYPs were verified by using the corresponding probes (Supplementary Figure 2). As shown in Figure 2B, CYP2A6 could potently produce N-methylthiourea while the other subtypes tested produced very minor amounts of it. These data suggest that CYP2A6 is the main enzyme that catalyzes MMI towards N-methylthiourea.

Figure 2. N-methylthiourea production in the incubation of MMI with HLM or recombinant CYPs.

Figure 2.

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N-methylthiourea was trapped by P-nitrophenacyl bromide and detected by UPLC-QTOFMS. (A) Formation of N-methylthiourea from MMI in the incubation with HLM. NADPH was used as a cofactor of CYP-mediated N-methylthiourea production. (B) Determination of CYP subtype for N-methylthiourea production from MMI. All the data are expressed as means ± SD (n = 3).

Inhibition of CYP2A6 Decreases N-methylthiourea Production from MMI.

In order to confirm the role of CYP2A6 for N-methylthiourea production, trans-2-phenylcyclopropylamine, a CYP2A6 inhibitor,14 was used both in in vitro and in vivo studies. As shown in Figure 3A, the inhibitor suppressed the production of N-methylthiourea by 83% in the in vitro incubation system with CYP2A6. In mice pretreated with trans-2-phenylcyclopropylamine, the production of N-methylthiourea in the liver also decreased significantly (Figure 3B). These data indicate that CYP2A6 is essential in catalyzing MMI to produce N-methylthiourea.

Figure 3. Inhibition of CYP2A6 decreases N-methylthiourea production from MMI.

Figure 3.

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Trans-2-phenylcyclopropylamine was used as a CYP2A6 inhibitor. (A) Effect of CYP2A6 inhibitor on N-methylthiourea production in the incubation of MMI with recombinant CYP2A6. (B) Formation of N-methylthiourea from MMI in the liver of mice pretreated with (+) or without (−) CYP2A6 inhibitor. Liver samples were collected at 0.5 and 1 h after MMI treatment. N-methylthiourea was extracted from the liver and trapped by P-nitrophenacyl bromide. Trapped N-methylthiourea was detected by UPLC-QTOFMS. All the data are expressed as means ± SD (n = 3). ****p < 0.0001.

Inhibition of CYP2A6 Attenuates MMI-induced Hepatocellular Injury.

Like previous work,8 mice were pretreated with BSO to deplete GSH and used as a model of MMI-induced hepatocellular injury. When serum levels of ALT and AST activities were measured, BSO+MMI group showed 113.4- and 55-fold higher values, respectively compared to control, whereas BSO or MMI alone showed no changes, indicating pronounced hepatocellular injury by MMI in GSH-depleted mice pretreated with BSO (Figure 4A, B). Expectedly, BSO+MMI group pretreated with the CYP2A6 inhibitor showed 91.7% and 88.1% lower values, respectively, of these transaminase activities (Figure 4A, B). Consistently, the H&E staining images of the liver sections showed large areas of hepatocyte necrosis and hemorrhage around the central vein of hepatic lobules in the BSO+MMI group, while the CYP2A6 inhibitor reversed all these phenotypes (Figure 4C). Taken together, these data suggest that CYP2A6 plays an important role in MMI-induced hepatocellular injury.

Figure 4. Suppression of CYP2A6 attenuates MMI-induced hepatocellular injury.

Figure 4.

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Mice were treated with vehicle, BSO, MMI, BSO+MMI, or BSO+MMI+CYP2A6 inhibitor. BSO was used to deplete GSH in the liver. Trans-2-phenylcyclopropylamine was used as a CYP2A6 inhibitor. Six h after MMI treatment, blood and liver samples were collected for evaluation of liver damage. (A, B) Serum activities of ALT and AST. All the data are expressed as means ± SD (n = 4 to 6). ****p < 0.0001. (C) Representative liver sections (200 X) with H&E staining. Blue lines indicate necrosis areas with hemorrhage. CV, central vein.

CYP2A6 is in part Responsible for MMI-induced Cholestatic Injury.

In addition to hepatocellular injury, clinical reports also highlighted the implication of MMI in cholestatic injury.15-17 We therefore measured the biomarkers associated with cholestatic injury, including serum bilirubin, bile acids, and ALP activities. As shown in Figure 5A-C, BSO+MMI group showed 87.2-, 18.5- and 1.6-fold increase in these values, respectively compared to control, indicating cholestatic damage by MMI in GSH-depleted mice. Interestingly, BSO+MMI group pretreated with the CYP2A6 inhibitor showed 93.4% and 99.7% decreased levels of bilirubin and bile acids compared to BSO+MMI group (Figure 5A, B) but no significant changes in ALP activities (Figure 5C). These results suggest that CYP2A6 is in part responsible for MMI-induced cholestatic injury.

Figure 5. Role of CYP2A6 in MMI-induced cholestatic injury.

Figure 5.

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Mice were treated with vehicle, BSO, MMI, BSO+MMI, or BSO+MMI+CYP2A6 inhibitor. Six h after MMI treatment, blood samples were collected for evaluation of cholestatic biomarkers in sera including bilirubin (A), total bile acids (B), and ALP activities (C). All the data are expressed as means ± SD (n = 4 to 6). ****p < 0.0001; ns, not significant.

DISCUSSION

In the present study, we screened 16 subtypes of CYP enzymes for their role in the production of toxic metabolite N-methylthiourea from MMI. CYP2A6 was identified as the key enzyme for this reaction. In addition, we found that suppression of CYP2A6 prevents MMI-induced hepatocellular injury (Figure 6).

Figure 6. Proposed mechanisms of MMI-induced hepatotoxicity.

Figure 6.

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MMI undergoes bioactivation by CYP2A6 to produce N-methylthiourea, which causes hepatocellular injury when GSH level is insufficient. Cholestatic injury is facilitated by hepatocellular injury and is also caused by MMI through an unknown mechanism.

Hepatotoxicity of MMI is associated with its reactive metabolites formed in the host liver.6, 8 It has been proposed that hepatic CYPs oxidize MMI to form N-methylthiourea (N-methylthiourea), which causes hepatocellular injury.8 However, it remains unknown which specific CYP contributes to N-methylthiourea production from MMI. The current work filled this knowledge gap by illustrating the essential role of CYP2A6 in MMI bioactivation and hepatotoxicity. Our data are consistent with previous reports showing the role of Cyp2a5 in MMI-induced nasal toxicity in mice.18, 19

The expression and activity of CYP2A6 in humans are highly variable due to its extensive polymorphisms as well as interactions with non-genetic factors.20, 21 More than 40 genetic variants of CYP2A6 gene have been identified.20 While most of these variants show loss-of-function, others such as CYP2A6*1X2A, CYP2A6*1X2B and CYP2A6*1B alleles show gain-of-function in compared to wild type CYP2A6*1A allele.20, 22-24 Among the non-genetic factors, certain drugs such as phenobarbital, dexamethasone, rifampin, as well as female gender and older age are associated with the increased expression and activity of CYP2A6.10, 20, 25, 26 Thus, the risk of MMI hepatotoxicity is likely to be associated with the variation of CYP2A6 but future clinic work is needed to confirm such correlation.

It is important to note that without GSH depletion by BSO, MMI alone showed only marginal toxicity in mouse model, suggesting oxidative stress by its toxic metabolites is critical in MMI-induced hepatocellular injury.7 Therefore, the preconditions which reduce GSH level in the liver such as fasting, malnutrition, and chronic liver injury,7, 27 will put patients in danger when using MMI, especially in patients having gain-of-function mutation of CYP2A6 or taking CYP2A6 inducers as mentioned above.20

According to clinical reports, MMI may also cause cholestatic jaundice, and its mechanism is unclear.4, 28 When markers for cholestasis such as bilirubin, bile acids, and ALP were measured from the same mouse sera used in this study, BSO+MMI group showed significant elevation of bilirubin and bile acids, suggesting cholestatic injury occurred. Under physiological condition, bilirubin and bile acids are excreted from hepatocytes into bile via various efflux transporters.29, 30 Under pathological conditions, such as bile duct blockage and/or hepatocellular injury, bilirubin and bile acids accumulate in the liver and release into blood.29-31 Our data showed the occurrences of hepatocellular injury in MMI+BSO group, suggesting that MMI-induced hepatocellular injury may contribute to its cholestatic injury (Figure 6). However, failure to reduce ALP by CYP2A6 inhibitor suggests the existence of another mechanism of MMI-induced cholestatic injury, and this needs to be clarified in the future work.

In summary, the current work determined CYP2A6 as the key enzyme for MMI bioactivation and hepatotoxicity. Our data also suggest that CYP2A6 is a potential target for prediction and prevention of MMI hepatotoxicity in the clinic.

Supplementary Material

1

FUNDING INFORMATION

This work was supported by the startup funding for Xiaochao Ma at the University of Pittsburgh School of Pharmacy, and in part by the National Institute of Allergy and Infectious Diseases (R01AI131983) and the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK126875).

ABBREVIATIONS

ALT

alanine aminotransferase

AST

aspartate aminotransferase

ALP

alkaline phosphatase

BSO

buthionine sulfoximine

CV

central vein

CYP

cytochrome P450

GSH

glutathione

H&E

hematoxylin-eosin

FMO

flavin-containing monooxygenases

HLM

human liver microsome

MMI

methimazole (1-methyl-2-mercaptoimidazole)

NADPH

reduced β-nicotinamide adenine dinucleotide phosphate

PTU

propylthiouracil (6-propyl-2-thiouracil)

UPLC-QTOFMS

ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry

Footnotes

SUPPORTING INFORMATION

MS/MS analysis of trapped N-methylthiourea; and verifications of CYP activities

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Supplementary Materials

1
NIHMS1786020-supplement-1.pdf (345.1KB, pdf)

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