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. Author manuscript; available in PMC: 2022 Sep 21.
Published in final edited form as: Xenobiotica. 2021 Aug 9;51(9):1047–1059. doi: 10.1080/00498254.2021.1961920

Metabolic map of the antiviral drug podophyllotoxin provides insights into hepatotoxicity

Dongxue Sun a,b, Xiaoxia Gao b,c, Qiao Wang d, Kristopher W Krausz b, Zhongze Fang b,e, Youbo Zhang b,f, Cen Xie b,g, Frank J Gonzalez b
PMCID: PMC9491014  NIHMSID: NIHMS1834953  PMID: 34319859

Abstract

1. Podophyllotoxin (POD) is a natural compound with antiviral and anticancer activities. The purpose of the present study was to determine the metabolic map of POD in vitro and in vivo.

2. Mouse and human liver microsomes were employed to identify POD metabolites in vitro and recombinant drug-metabolizing enzymes were used to identify the mono-oxygenase enzymes involved in POD metabolism. All in vitro incubation mixtures and bile samples from mice treated with POD were analyzed with ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry.

3. A total of 38 novel metabolites, including six phase I metabolites and 32 phase II metabolites, of POD were identified from bile and feces samples after oral administration, and their structures were elucidated through interpreting MS/MS fragmentation patterns.

4. Nine metabolites, including two phase I metabolites, five glucuronide conjugates, and two GSH conjugates were detected in both human and mouse liver microsome incubation systems and the generation of all metabolites were NADPH-dependent. The main phase I enzymes involved in metabolism of POD in vitro include CYP2C9, CYP2C19, CYP3A4, and CYP3A5.

5. POD administration to mice caused hepatic and intestinal toxicity and the cellular damage was exacerbated when aminobenzotriazole, a broad-spectrum inhibitor of CYPs, was administered with POD, indicating that POD but not its metabolites induced hepatic and intestinal toxicities.

6. This study elucidated the metabolic map and provides important reference basis for the safety evaluation and rational for the clinical application of POD.

Keywords: CYP, podophyllotoxin, metabolism, liver microsomes, recombinant drug-metabolizing enzymes, hepatic and intestinal toxicity

Introduction

Since the early 19th century, the extract of Podophyllum peltatum, Podophyllum emodi, also known as podophyllin, have been used to treat a variety of diseases and conditions as scrofula, syphilis, gonorrhea, and coughing (Kelly and Hartwell 1954). Due to its medicinal value, the chemical components of podophyllin aroused the interest of pharmaceutical chemists, and podophyllotoxin (POD) was first isolated from this herb in the 1880s (Podwissotzki 1882; Podwyssotzki 1880). Subsequently, the planar structure and configuration of POD was established (Borsche and Niemann 1932a; Borsche and Niemann 1932b; Hartwell and Schrecker 1951; Schrecker and Hartwell 1956; Späth and others 1932), and its synthesis was achieved in the 1960s (Gensler and Gatsonis 1962; Gensler and Gatsonis 1966). POD can inhibit the growth of epithelial cells infected by the human papilloma virus (HPV) (Longstaff and Von Krogh 2001), and thus, it was initially used to treat genital warts (Canel and others 2000). As the primary compound, POD was then revealed to have antimitotic and antitumor activities, and showed more potency than podophyllin extracts (Hartwell and Shear 1947; Sullivan 1947). However, clinical development of POD was impeded due to its severe side effects. Because of its poor selectivity against tumor cells and narrow therapeutic window, POD has been implicated in many poisoning cases as a result of either overdose or accidental ingestion of herbs containing POD. Major disturbances induced by POD include clinical symptoms in the gastrointestinal tract such as vomiting, diarrhea, abdominal pain, and abnormal hepatic functions, and sometimes even neurological disorders (Dobb and Edis 1984; Filley and others 1982; Kao and others 1992). Etoposide and teniposide, two semi-synthetic derivatives of POD and DNA topoisomerase-II inhibitors, were approved by the FDA and are presently used as anticancer agents (Damayanthi and Lown 1998). POD is mainly used as a first-line treatment for condyloma acuminate as an externally applied agent (Giri and Narasu 2000).

Drug metabolism, an important contributor to the clearance of drugs and determination of the dosage, can detoxify toxic compounds or activate drugs to chemically reactive electrophilic derivatives which may be potentially toxic or produce oxidative stress (Walsh and Miwa 2011). Drug-induced toxicity is one of the main reasons limiting the clinical use of many drugs (Hornberg and Mow 2014). Whether the toxicity of POD is due to the parent compound or its metabolites is still not clear. A previous study showed that POD possesses strong inhibitory effects on CYP2C9 and CYP3A4 in a concentration-dependent manner (Song and others 2011). However, there are no comprehensive studies on POD metabolism and the metabolic enzymes involved in its metabolism, which is a prerequisite for a better understanding of the mechanism of POD-induced toxicity. The aims of this study were to elucidate the metabolic pathway of POD in the mouse and to identify the drug-metabolizing enzymes that involved in POD metabolism, and reveal the role of metabolism in POD toxicity.

Materials and methods

Chemicals and reagents

POD was purchased from the Chinese National Institutes for Food and Drug Control (No.111645-200602, Beijing, China) with a purity of > 99.0% as detected with HPLC. NADPH, UDPGA, alamethicin, and GSH were obtained from Sigma-Aldrich (St. Louis, MO). Human liver microsomes (HLMs), mouse liver microsomes (MLMs), human recombinant cytochromes P450 (CYP) CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP2C9*2, CYP2D6, CYP2A6, CYP2B6, CYP3A5, CYP3A4, CYP3A7, CYP2C19, CYP2E1, CYP4A11, CYP4F12, and flavin-containing monooxygenases (FMO) FMO1, FMO3 and FMO5) were all purchased from Corning Life Sciences (Tewksbury, MA, USA). All of the other reagents were top commercial grade.

In vitro metabolism of POD

For metabolism in vitro, the incubation system (200 μL) contained 50 mM Tris–HCl buffer solution (pH = 7.4), 5 mM MgCl2, 0.5 mg protein/mL HLMs or MLMs, 10 μM POD, 2.5 mM freshly prepared GSH, and 1 mM NADPH. After one hour incubation at 37°C, the reaction was terminated with 200 μL cold 50% aqueous acetonitrile containing 5 μM chlorpropamide as an internal standard. The mixture was centrifuged at 15,000×g for 15 min, and a 5 μL aliquot of the supernatant was injected into the UPLC-ESI-Q-TOF-MS for analysis. The incubation system (200 μL) of recombinant phase I enzymes was similar to the liver microsomes incubation system, including 50 mM Tris–HCl buffer solution (pH = 7.4), 5 mM MgCl2, 50 nM CYPs or FMOs, 10 μM POD, 2.5 mM GSH, and 1 mM NADPH. The reaction was incubated at 37°C for 30 min and was terminated with 200 μL cold 50% aqueous acetonitrile containing 5 μM chlorpropamide. The metabolites were then analyzed by UPLC-ESI-Q-TOF-MS.

Co-activation of phase I enzymes with UDPGA and GSH in liver microsomes

A dual-activity incubation system (200 μL) containing 50 mM Tris–HCl buffer solution (pH 7.4), 2 mM MgCl2, 0.5 mg protein/mL HLMs or MLMs, 1 mM freshly prepared NADPH, 5 mM GSH and/or 1 mM UDPGA, 25 μg/mL alamethicin, and 10 μM POD was used (Fang and others 2012b). After 1 h incubation at 37°C, the reaction was terminated with 200 μL cold 50% aqueous acetonitrile containing 5 μM chlorpropamide. The mixture was centrifuged at 15,000×g for 15 min, and a 5 μL aliquot of the supernatant was injected into a UPLC-ESI-Q-TOF-MS for analysis.

In vivo metabolism of POD

Metabolism of POD in vivo was carried out using 6- to 8-week-old C57BL/6N mice. All studies with mice were in accordance with animal study protocols approved by the National Cancer Institute Animal Care and Use Committee and in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny and others 2010; McGrath and others 2010). The mice were maintained in a standard 12 h light/dark cycle environment with water and chow provided ad libitum before the experiment. Fourteen mice including seven control and seven POD-treated mice were used. POD dissolved in corn oil was administered to mice by oral gavage at a dose of 100 mg/kg BW. The control group was given corn oil. Feces samples (from 0 h to 24 h) were collected with metabolic cages (Metabowls, Jencons Scientific USA, Bridgeville, PA), and bile samples were collected and centrifugated for 15 min at 8,000 g. Serum was obtained for biochemistry analysis. Blood samples obtained from orbital venous plexus and centrifuged for 15 min at 8,000 × g to get serum for biochemistry analysis. To investigate the effect of 1-aminobenzotriazole (ABT, a non-specific CYP inhibitor) on POD metabolism and intestinal toxicity, 15 mice were divided into three groups including control group (corn oil), POD-treated group, and ABT-POD-treated group. The control group was administered corn oil alone by oral gavage. The POD-treated group was administered 100 mg/kg BW POD by oral gavage. ABT-POD-treated mice were intraperitoneally injected 100 mg/kg ABT and then administered POD by gavage 1 h later. Mice were placed in metabolic cages (Jencons Scientific USA, Bridgeville, PA) for 24h. Feces samples (from 0 h to 24 h) were collected, and blood samples were obtained from orbital venous plexus and centrifuged for 15 min at 8,000 × g. The mice were then killed to harvest gall bladders. A 2 μL of bile sample was mixed with 200 μL 66% aqueous acetonitrile containing 5 mM chlorpropamide. After vortex and centrifugation at 15,000 × g for 15 min, 5 μL aliquot of the supernatants was injected into a Waters UPLC–ESI-Q-TOF-MS system (Waters Corporation, Milford, MA) for analysis. Liver and ileum sections were removed immediately for mRNA quantitation and histological analysis.

UPLC-ESI-QTOFMS and triple quadrupole MS analysis

The bile samples were separated and analyzed using a Waters Acquity UPLC system coupled to a Waters Synapt HDMS Q-TOF mass spectrometer under the following conditions: capillary volts 3kV, sample cone 40V, source temperature 150°C, desolvation temperature 400°C, cone and desolvation gas flow 50 and 900 L/h, respectively. Data was acquired in centroid mode in both positive and negative electrospray ionization modes, using sulfadimethoxine as the Lock Mass. Mass range acquired was 50 – 900 Amu at 0.3 second scans. Chromatography was carried out using a Waters Acquity BEH C18 column (2.1x50 mm) under acidic conditions using a water (A) and acetonitrile (B) containing 0.1% formic acid. The following gradient was used: initial conditions 98% (A) for 0.5 min, to 80% (A) at 6.5 minutes, to 70% (A) at 8.0 min, to 1% (A) at 8.5 min, held for one min, returning to initial conditions for two min for column equilibration. Total run time was 11.5 min. Column temperature was maintained at 40°C. All samples were injected at 5 μL.

Histopathology

Neutral formalin (10%) was used to fix small blocks of mouse liver or intestine tissues. Then, the samples were embedded in paraffin and stained with hematoxylin and eosin (H&E), and the slides were observed by microscopic examination. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) kits (Catachem In., Oxford, CT) were used to test serum ALT, AST and ASP levels.

Real-time PCR analysis

Total RNA from frozen liver or intestine mucosa was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed to cDNA using qScript cDNA SuperMix (Gaithersburg, MD). Real-time PCR primer sequences are listed in Supplementary Table 1. Quantitative PCR analysis was performed on an Agilent Mx3005P Real-time PCR using SYBR®Green as probe. The reaction amount of each mRNA was calculated utilizing the ΔΔCT method and the mRNA levels were normalized to their corresponding Actb mRNA.

Statistical analysis

Experiment values were presented as mean ± SEM. Statistical analysis was calculated using Prism version 7.0 (GraphPad Software, San Diego, CA). Statistical significance between two groups was analyzed using two-tailed Student’s t-test and P values of less than 0.05 were considered to be significant.

Results

In vitro metabolism of POD in HLMs and MLMs

POD metabolism by HLMs and MLMs was carried out using UPLC-ESI-QTOFMS. Only POD could be detected in the positive ionization mode while the metabolites were mainly detected in the negative ionization mode. The MS and MS/MS data were processed with Masslynx 4.1 software. Nine metabolites, including two phase I metabolites (M1 and M2), five glucuronide conjugates (M3-1 to M3-3 and M4-1 to M4-2), and two GSH conjugates (M5-1 and M5-2) were detected both in HLM and MLM incubations (Table 1 and Figure 1A). All phase I metabolites appeared in the NADPH-supplemented incubation systems, but were absent in the NADPH-free incubation systems, indicating that the formation of these metabolites was NADPH-dependent. In the presence of NADPH and UDPGA, M3 and M4 were detected but were not found in the system only with NADPH or UDPGA (Supplementary Figure 1 and 2). Formation of GSH conjugates M5-1 and M5-2 were only observed in the incubation system in the presence of both GSH and NADPH (Supplementary Figure 1 and 2). These results suggest that all metabolites were CYP dependent, and the metabolic pathway was deduced (Figure 1A).

Table 1.

UPLC-HRMS data for POD metabolites detected in HLM and MLM

No. Metabolic pathway Formula m/z Error
(ppm)		 Rt
(min)		 Fragments
M1 −C C21H22O8 401.1223 −3.4 6.12 339.1248, 324.0984, 293.0478, 277.0391, 265.0268, 249.0378, 237.0583, 210.0623, 172.0535, 145.0987
M2-1 Demethylation C21H20O8 399.1090 2.5 5.08 384.0882, 369.0609, 341.0717, 269.0538, 226.0280, 175.044
M3-1 Demethylation+glucuronide conjugation C27H28O14 575.1309 6.9 5.89 399.1091, 384.0837, 369.0566, 355.1077, 349.0902, 325.0632, 270.0258, 201.0399, 186.0386
M3-2 Demethylation+glucuronide conjugation C27H28O14 575.1417 2.8 6.62 399.1094, 384.0913, 369.0634, 341.0645, 270.0529, 254.0692, 239.0599, 227.022, 187.0471, 113.0248
M3-3 Demethylation+glucuronide conjugation C27H28O14 575.1431 5.2 6.40 399.1091, 384.0836, 355.0571, 341.0668, 339.0911, 325.0579, 311.0639, 297.0636, 271.7139
M4-1 −C+glucuronide conjugation C27H30O14 577.1569 2 4.62 401.1230, 383.1215, 339.122, 324.1000, 309.0774, 293.049, 281.0708, 266.0593, 215.0320, 172.052, 129.2002, 113.023
M4-2 −C+glucuronide conjugation C27H30O14 577.1570 2.2 5.57 559.1455, 401.123, 383.1007, 339.1230, 294.0520, 281.1129, 266.1015, 215.0153, 172.0510, 113.0530
M5-1 −C+GSH C31H37N3O14S 706.1939 2.9 3.25 433.0876, 418.0645, 371.0806, 272.0831, 254.1130, 210.0193, 190.0050
M5-2 −C+GSH C31H37N3O14S 706.1981 8.9 3.65 433.0949, 415.0855, 387.0832, 371.0866, 337.2072, 306.0751, 283.2010, 247.0037, 242.0829, 221.1189
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Figure 1.

Figure 1.

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Proposed metabolic pathways of POD in vitro and fragmentation patterns of its representative metabolites. (A) Proposed metabolic pathways of POD in HLMs and MLMs, (B) Proposed fragmentation pattern of its representative metabolite M1 in negative mode.

The chromatographic and mass fragmentation of POD was then investigated. POD eluted at 8.35 min with a protonated molecular ion at m/z 415.2281 in the positive mode. The most abundant peak was at m/z 397.1272 due to a dehydration reaction. The MS/MS spectrum of POD gave major fragment ions at m/z 397.1272, 351.1203, 317.1050, 282.0893, 247.0611, and 229.0515, 185.0612, 145.0660, and 117.0722 (Figure 1A). As noted, the POD metabolites were observed in the negative mode.

M1 was produced through cleavage of the methylenedioxy group and eluted at 6.12 min. It yielded a quasi-molecular ion of [M-H]− = 401.1223 m/z, which gave a mass error of −3.4 ppm with the matched molecular formula C21H22O8. Its chemical structure was deduced base on the MS/MS fragmentation ions (Figure 1B), which further supported cleavage of the methylenedioxy group.

M2 eluted at 5.08 min with a deprotonated molecular ion at m/z 399.10, which was 2 Da lower than M1. Its elemental composition was supposed to be C21H20O8, indicating that M2 is a demethylated metabolite of POD. The possible demethylated site should be one of the methoxyl groups of the parent compound. The MS/MS fragments and the proposed metabolic pathway are shown in Table 1 and Figure 1A.

M3-1, M3-2 and M3-3 eluted at 5.89, 6.62 and 6.40 min, respectively, and possessed a deprotonated molecular ion at m/z 575.14, which were 176 Da higher than M2. The MS/MS fragments of 384.09, 369.06, and 341.06 were similar with that of M2, further indicating demethylation combined with glucuronidation. The possible demethylation and glucuronidation site should be one of the methoxyl groups of POD.

M4-1 and M4-2 eluted at 4.62, and 5.57 min, respectively. The deprotonated molecular ion at m/z 577.15, corresponding to the elemental composition of C27H30O14, was 176 Da higher than that of M1. The MS/MS fragments of 339.12, 324.10, and 293.04, which were similar with that of M1, further indicated that formation of M4-1 and M4-2 were via cleavage of the methylenedioxy group and glucuronidation.

M5-1 and M5-2 eluted at 3.25, and 3.65 min, respectively. It yielded a deprotonated molecular ion of 706.19 m/z, and gave a mass error of 2.9 and 8.9 ppm with the matched molecular formula C31H37N3O14S. The molecular weights of M5-1 and M5-2 were 305 Da higher than that of M1, indicating cleavage of the methylenedioxy group and GSH conjugation of POD. The MS/MS fragmentation behavior confirmed that GSH was attached to the B-ring of POD.

Screening the drug metabolic enzymes (DMEs) involved in the oxidative metabolism of POD

The identity of the human phase I enzymes involved in oxidative metabolism and bioactivation of POD was screened using recombinant human enzymes. M1, M5-1 and M5-2 were detected in the incubation system. CYP2C9, CYP2C19, CYP3A4, and CYP3A5 were found to be involved in the demethylation of methylenedioxyphenyl group to form catechol metabolite M1 (Figure 2A), and also the in the sequential oxidation of catechol to the ortho-benzoquinone intermediate, which were trapped by GSH to yield M5-1 and M5-2 (Figure 2B). Among these, CYP2C9 and 3A5 exhibited higher catalytic activity towards M1 formation, while CYP2C9 as a major enzyme participated in the production of M5-1 and M5-2 (Figure 2). Slightly unfortunately, the demethylated metabolite M2 was not find in the recombinant enzymes incubation systems. The possible reason is that the cleavage of the methylenedioxy group is easier than demethylation of POD, which made M1 easier to be detected by LC-MS/MS technology.

Figure 2.

Figure 2.

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In vitro recombinant enzymes screening for the production of M1 and M5. (A) The CYPs involved in M1, (B) The CYPs involved in M5.

Metabolic behavior of POD in mice

A total of 38 metabolites, including six phase I metabolites and 32 phase II metabolites, were identified from mouse bile samples (Figure 3). Detailed information on these metabolites is shown in Table 2. Among these, nine metabolites were obtained from in vitro metabolism. For the demethylated metabolites, three metabolites were obtained from mouse bile samples. The retention times of M2-2, M2-3, and M2-4 eluted at 6.93, 7.51, and 7.74 min, respectively. The deprotonated molecular ion was at m/z 399.11, which was 14 Da lower than that of POD, and corresponds to the elemental composition of C21H20O8. The MS/MS fragments of 384.09, 369.06, 341.07, 269.05, and 226.03 further indicated M2-2, M2-3, and M2-4 (Figure 4) are the demethylation metabolites of POD.

Figure 3.

Figure 3.

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Metabolic profile of the representative metabolites in bile sample of mice after administration of POD.

Table 2.

UPLC-HRMS data for POD metabolites detected in mouse bile

No. Metabolic pathway Formula m/z Error
(ppm)		 Rt
(min)		 Fragments bile feces
M1 Cleavage of
methylenedioxy		 C21H22O8 401.12
23		 −3.4 6.12 339.1248, 324.0984, 293.0478, 277.0391, 265.0268, 249.0378, 237.0583, 210.0623, 172.0535, 145.0987 √
M2-2 Demethylation C21H20O8 399.1090 2.5 6.93 384.0882, 369.0609, 341.0717, 269.0538, 226.0280, 175.0440 √ √
M2-3 Demethylation C21H20O8 399.1099 4.7 7.51 384.1006, 369.0568, 355.0383, 341.0587, 325.0635, 253.0500, 214.0280, 187.0390, 158.0360 √ √
M2-4 Demethylation C21H20O8 399.1078 0.5 7.74 384.0793, 369.0598, 355.0764, 341.0656, 325.0624, 297.0826, 282.0378, 269.0437, 254.0561,226.0895, 187.0443 √ √
M3-1 Demethylation+glucuronide conjugation C27H28O14 575.1441 6.9 5.88 399.1091, 384.0837, 369.0566, 355.1077, 349.0902, 325.0632, 270.0258, 201.0399, 186.0386 √
M3-2 Demethylation+glucuronide conjugation C27H28O14 575.1417 2.8 6.63 399.1094, 384.0913, 369.0634, 341.0645, 270.0529, 254.0692, 239.0599, 227.0220, 187.0471, 113.0248 √
M3-3 Demethylation+glucuronide conjugation C27H28O14 575.1431 5.2 6.41 399.1091, 384.0836, 355.0571, 341.0668, 339.0911, 325.0579, 311.0639, 297.0636, 271.7139 √
M3-4 Demethylation+glucuronide conjugation C27H28O14 575.1426 4.3 4.96 531.1584, 399.1072, 369.0678, 355.1063, 340.0962, 325.0691, 299.0407, 269.0428, 221.855, 113.0601 √
M3-5 Demethylation+glucuronide conjugation C27H28O14 575.1423 3.8 5.55 399.1129, 384.0775, 369.0723, 355.0593, 340.1182, 325.0668, 298.3300, 269.0538, 201.0249, 185.1393, 175.0196 √
M3-6 Demethylation+glucuronide conjugation C27H28O14 575.1446 7.8 5.71 399.1090, 384.0869, 369.0633, 355.0855, 341.0823, 325.0712, 269.0506, 255.0427, 242.0083, 174.9917 √
M3-7 Demethylation+glucuronide conjugation C27H28O14 575.1435 5.9 6.76 399.1115, 384.0853, 369.0585, 355.0851, 341.0630, 325.0765, 319.1001, 187.0376 √
M4-1 Cleavage of methylenedioxy+glucuronide conjugation C27H30O14 577.1569 2 4.63 401.1230, 383.1215, 339.1220, 324.1000, 309.0774, 293.0490, 281.0708, 266.0593, 215.0320, 172.0520, 129.2002, 113.0230 √
M4-2 Cleavage of methylenedioxy+glucuronide conjugation C27H30O14 577.157 2.2 5.58 559.1455, 401.1230, 383.1007, 339.1230, 324.1000, 294.0520, 281.1129, 266.1015, 215.0153, 172.0510, 113.0530 √
M5-1 Cleavage of methylenedioxy+GSH C31H37N3O14S 706.1939 2.9 3.25 433.0876, 418.0645, 371.0806, 272.0831, 254.1130, 210.0193, 190.0050 √
M5-2 Cleavage of methylenedioxy+GSH C31H37N3O14S 706.1981 8.9 3.65 433.0949, 415.0855, 387.0832, 371.0866, 337.2072, 306.0751, 283.2010, 247.0037, 242.0829, 221.1189 √
M6-1 2 × Demethylation C20H18O8 385.0952 7.3 6.2 370.0685, 355.0446, 327.0551, 255.0263, 173.0360 √
M6-2 2 × Demethylation C20H18O8 385.0897 −6.8 6.55 370.0659, 311.0406, 277.0762, 254.0294, 187.0810 √
M7 Demethylation+sulfate conjugation C20H18O8 479.0662 2.8 7.41 399.1030, 384.0828, 369.0601, 340.0986 , 325.0631, 252.2453, 187.7986 √
M8-1 2 × Demethylation+glucuronide conjugation C26H26O14 561.127 4.5 4.6 √
M8-2 2 × Demethylation +glucuronide conjugation C26H26O14 561.1243 −0.3 5.31 385.0927, 370.0681, 355.0548, 337.0338, 309.0448, 269.0330 √
M8-3 2 × Demethylation+glucuronide conjugation C26H26O14 561.1266 3.8 5.48 495.1594, 385.0953, 370.0692, 311.0600 √
M8-4 2 × Demethylation+glucuronide conjugation C26H26O14 561.1276 5.6 5.59 448.1119, 385.0969, 370.0578, 355.0551, 326.0879, 311.0695, 270..2240 √
M9-1 Glucuronide conjugation C28H30O14 589.1582 4.1 6.14 545.1631, 413.1216, 398.1004, 369.1363, 339.1353, 309.1948, 299.1623, 269.3391, 202.0482, 188.1066, 175.0220, 113.0258 √
M9-2 Glucuronide conjugation C28H30O14 589.1586 4.8 6.32 413.1219, 383.0779, 368.0705, 355.1013, 340.0582, 325.0591, 284.1261, 266.8437, 250.1859, 227.3869, 175.027, 113.0285 √
M9-3 Glucuronide conjugation C28H30O14 589.1575 3 6.7 413.1189, 398.0909, 383.0685, 369.1264, 354.1086, 339.0630, 324.0875, 299.0606, 255.0750, 203.0353, 175.0061, 145.1546, 113.0138 √
M9-4 Glucuronide conjugation C28H30O14 589.1573 2.6 7.01 √
M9-5 Glucuronide conjugation C28H30O14 589.16 7.2 7.09 413.1212, 398.0994, 383.0744, 369.0588, 355.0831, 340.0855, 325.0072, 286.3051, 269.0588, 203.0353, 175.0380 √
M9-6 Glucuronide conjugation C28H30O14 589.1583 4.3 7.21 413.1237, 398.1023, 383.0129, 339.0854, 309.0428, 186.0380, 175.0291 √
M10-1 Cleavage of methylenedioxy+glucuronide conjugation+methylatio n C28H32O14 591.1715 0.2 4.93 514.2815, 415.1417, 353.1461, 338.1165, 323.0939, 186.0668, 175.0242 √
M10-2 Cleavage of methylenedioxy+glucuronide conjugation+methylatio n C28H32O14 591.1737 3.9 6.29 514.2841, 415.1274, 414.1250, 413.1176, 399.1145, 383.0753, 296.6187, 157.0182, 99.0161 √
M11-1 Cleavage of methylenedioxy+GSH+ glucuronide conjugation C37H45N3O20S 882.2272 3.7 4.14 706.1192, 575.1458, 399.1082, 355.1165, 306.0752, 272.0855, 254.0826, 210.0868, 128.3057 √
M11-2 Cleavage of methylenedioxy+GSH+ glucuronide conjugation C37H45N3O20S 882.228 4.6 3.73 706.2347, 575.1404, 399.1087, 355.1194, 306.0722, 272.0764, 210.0845, 177.0395 √
M11-3 Cleavage of methylenedioxy+GSH+ glucuronide conjugation C37H45N3O20S 882.2292 6 3.86 706.1868, 272.0910, 254.0812 √
M11-4 Cleavage of methylenedioxy+GSH+ glucuronide conjugation C37H45N3O20S 882.2292 6 3.56 575.1255, 306.0772, 272.0910, 254.0812 √
M12-1 Methylation + Cleavage of methylenedioxy +GSH+ glucuronide conjugation C38H47N3O20S 896.2437 4.6 3.74 589.1584, 413.1256, 369.1298, 306.0741, 272.078 √
M12-2 Methylation + Cleavage of methylenedioxy +GSH+ glucuronide conjugation C38H47N3O20S 896.2438 4.7 4.84 750.9995, 720.2079, 589.153, 369.1283, 306.0712, 272.09, 254.0798, 210.0905, 143.0298 √
M12-3 Methylation + Cleavage of methylenedioxy +GSH+ glucuronide conjugation C38H47N3O20S 896.2453 6.4 4.36 720.1957, 589.1576, 369.1283, 306.0788, 272.1041, 254.0677, 210.0823, 179.0457, 159.9807, 143.0401, 128.0094 √
M12-4 Methylation + Cleavage of methylenedioxy +GSH+ glucuronide conjugation C38H47N3O20S 896.2457 6.8 4.07 599.1248, 590.1661, 514.1617, 417.2951, 373.2826, 369.1425, 306.0735, 272.0733, 254.0791, 160.0085, 143.0451 √
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Figure 4.

Figure 4.

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Proposed fragmentation patterns of representative metabolites in mice after administration of POD.

For the demethylated and glucuronidated metabolites, seven were obtained in mice. M3-1, M3-2, and M3-3, M3-4 to M3-7 also showed a deprotonated molecular ion of 575.14 m/z (shown in Table 2) with the matched molecular formula C27H28O14. The fragment pathway of M3-4 to M3-7 (Figure 4), which was similar with that of M3-1 to M3-3, indicating demethylation and glucuronidation. Because POD has only three methoxy groups, an isomerization between methoxy and methylenedioxy groups of POD was proposed to happen when undergoing metabolism.

M6-1 and M6-2 were phase I metabolites with a deprotonated molecular ion of 385.09 m/z, which gave a mass error of 7.3 and −6.8 ppm, respectively. The molecular formula of these metabolites were proposed as C20H18O8 according to their fragmentation patterns (Figure 4). Finally, M6-1 and M6-2 were deduced as di-demethylated metabolites of POD.

M7 eluted at 7.41 min. The deprotonated molecular ion was at m/z 479.0662, which was 80 Da higher than that of M2, corresponding to the elemental composition of C21H20O11S. The MS/MS fragments of 399.103, 384.0828, 369.0601, and 325.0631 further indicated formation of the M7 via demethylation combined with sulfate conjugation.

M8-1 to M8-4 were eluted at 4.60, 5.31, 5.48, and 5.59 min, respectively. They possessed the deprotonated molecular ions at m/z 561.12, which were 14 Da lower than M3 and 176 Da higher than M6. The elemental composition of the metabolites was proposed as C26H26O14, and the MS/MS fragments of 385.09, 370.06, and 355.05 were similar with that of M6, further indicating di-demethylation combined with glucuronidation. Formation of M8-1 to M8-4 were proposed to undergo isomerization between the methoxy and methylenedioxy groups of POD.

M9-1 to M9-6 at m/z 589.15 were detected at 6.14, 6.32, 6.70, 7.01, 7.09, and 7.21 min, respectively. Their elemental composition was C28H30O14, indicating glucuronidation of POD, which was supported by the observation of a typical fragment ion at m/z 413.12 formed by neutral loss of the glucuronic moiety in the MS/MS spectrum (Figure 4).

M10-1 and M10-2 eluted at 4.93 and 6.29 min, respectively. The deprotonated molecular ion at m/z 591.17, corresponding to the elemental composition of C28H32O14, was 14 Da higher than that of M4. The MS/MS fragments of 415.12, 399.11, and 383.07 further indicated that the formation of M10-1 and M10-2 were via cleavage of the methylenedioxy group and glucuronidation and methylation; the tentative fragmentation patterns are proposed in Figure 4.

M11-1 to M11-4 were detected at 4.14, 3.73, 3.86, and 3.56 min, respectively. The deprotonated molecular ions at m/z 882.22 were 176 Da higher than M5, indicating cleavage of the methylenedioxy group and GSH conjugation and glucuronidation of POD. The presence of m/z 706.11 and 575.14 fragment ions suggested cleavage of the glucuronide and GSH moiety (Figure 4).

M12-1 to M12-4 at m/z 896.24 eluted at 3.74, 4.84, 4.36, and 4.07 min, respectively. The molecular weight of M12 was 14 Da higher than that of M11, and the elemental composition was proposed to be C38H47N3O20S, indicating methylation of M11. The proposed structure was supported by the observation of a typical fragment ion at m/z 720.20 and 589.15 formed by cleavage of glucuronide and GSH moiety (Figure 4).

The CYP inhibitor ABT reduced metabolite generation

Concentrations of the main metabolites of POD in bile samples with or without ABT, a broad-spectrum CYP inhibitor, were detected using UPLC-MS/MS. There was a significant decrease of most metabolites in the ABT-POD group except for M9-4 (Figure 5), suggesting that generation of these metabolites were all CYP-dependent. M9-4 exhibited an equal concentration in the POD and ABT-POD groups and a exhibited a CYP-independent conversion in vivo, indicating that the glucuronidation conjugation site of was the hydroxyl group of POD.

Figure 5.

Figure 5.

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Relative concentration of representative metabolites in mice after treatment with POD or POD and ABT. (A) M1, (B) M2, (C) M3, (D) M4, (E) M5, and (F) M6. horizontal bar represents the peak area of metabolites; **p <0.01, ***p <0.001 versus POD group.

Metabolic inhibition exacerbated POD-induced hepatotoxicity and diarrhea

Hepatic and gastrointestinal toxicity by POD is the main limitation for its clinical application. To study the role of metabolism in POD poisoning in vivo, ABT, a non-specific and non-toxic mechanism-based inhibitor of CYP enzymes, was used combined with POD to investigate the metabolism-dependent toxicity of POD. ALT, AST, ALP and hepatic and intestinal histology were measured to evaluate hepatocellular and gastrointestinal toxicity after 24 hours. Serum ALT, AST and ALP sharply increased in the POD and ABT/POD-treated groups, and they all increased more than 30% in ABT/POD-treated groups than that in POD-treated group (Figure 6A-C). Microscopic examination of liver sections revealed no visible lesion in the liver of control mice (Figure 6D), while some pathological changes as vascular congestion and dilatation, mononuclear cellular infiltration, swelling, and necrotic hepatocytes were obviously observed in POD and ABT/POD-treated mice, and ABT/POD-treated mice showed more severely acute hepatic damage than POD-treated mice (Figure 6E-F). In H&E-stained colonic tissue sections (Figure 6G-I), POD and ABT/POD-treated mice show more histological damage, as cellular infiltration, goblet cell depletion, distortion to crypt architecture, compared to the control group. The goblet cell depletion and distortion of crypt architecture in ABT/POD-treated mice were more severe than in the POD-treated mice. The mRNA expression of some related pro-inflammatory cytokines in ileum were measured. The expression of Il6, Ccl2, Ccl3, Lcn2, and Icam1 mRNAs increased significantly in ileum tissue of both POD- and ABT/POD-treated mice, and expression of Il6, Ccl2, and Lcn2 mRNAs were higher in ABT/POD-treated mice than in POD-treated mice (Figure 6J).

Figure 6.

Figure 6.

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The hepatic and intestinal toxicities in mice after treatment with control vehicle (corn oil), POD, and POD+ABT, respectively. (A-C) serum ALT, AST, and ALP levels in mice. (D-F) light microscopic examination of H&E-stained liver sections of mice treated with vehicle (D), POD (E), and POD & ABT (F); scale bar, 50 μm, 20 x. (G-I), light microscopic examination of H&E-stained colonic sections of mice treated with vehicle (G), POD (H), and POD & ABT (I); scale bar, 50 μm, 20×. (J) qPCR analysis of mRNA in mice ileum after treatment with vehicle (D), POD (E), and POD & ABT. Data are presented as mean ± SEM; n = 5/group. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle group, by One-way ANOVA test.

Discussion

Drug metabolism has a major influence on the safety and efficiency of drugs (Lin and Lu 1997). POD, a naturally occurring lignan with good antineoplastic and antiviral activites (Tian and others 2007; Yang and others 1994a), was first reported to have a curative effect on condylomata acuminata (Kaplan 1942) and is still used as an effective treatment. The initial aim for the possible clinical utility of POD was as an antitumor agent, but this was abandoned because of its poor selectivity and unacceptable gastrointestinal side effects (Tian and others 2007). Oxidative damage and inhibition of mitosis are likely to be the underlying poisoning mechanisms observed in the animals treated with POD (Yang and others 1994b). Until now, detailed metabolic information of POD and the toxicity caused by the parent compound or its metabolites was hitherto unknown. Therefore, a UPLC-HRMS-based study was used in the present study to elucidate the metabolic pathways of POD combined with in vitro and in vivo models.

POD processes a methylenedioxyphenyl group, which has been considered as a structure alert. Many methylenedioxyphenyl-containing drugs or chemicals could be demethylated to yield a catechol metabolite and then undergo further bioactivation to a reactive ortho-quinone intermediate by CYP-mediated metabolism (Fang and others 2012a; Gan and others 2009; Zhao and others 2007). These compounds are of considerable toxicological significance due to their capacity for the mechanism-based inactivation of CYPs and induction of neurotoxic and hepatotoxic effects (Murray 2000). The first task of the current study was to analyze the metabolites of POD formed by HLM and MLM, and the inter-species differences in the metabolism of POD. A total of nine metabolites were identified from the incubation systems, and the formation of these metabolites was NADPH-dependent. Consistent with previous findings, POD was mainly demethylated on the methylenedioxyphenyl group to form the catechol metabolite M1, and then further underwent oxidation of the catechol to the reactive ortho-benzoquinone intermediate, which was trapped by GSH to yield M5-1 and M5-2. No inter-species differences were found with the nine metabolites identified in both HLMs and MLMs. Thus, the mouse model is a reasonable surrogate for predicting metabolism and toxicity of POD in humans. One important task in the study of drug metabolism is determining the enzymes involved in POD metabolism. The phase I enzymes catalying POD metabolism include CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Among these, CYP2C9 and CYP3A5 exhibited the highest catalytic activity towards cleavage of the methylenedioxyphenyl group. However, based on their average hepatic expression, CYP3A4 was likely the principal CYP tmediating POD demethylation and bioactivation to reactive ortho-quinone intermediate.

POD is extensively metabolized in mice as indicated by a total of 36 metabolites including six phase I metabolites and 30 phase II metabolites identified in mouse bile and feces. The primary routes of POD biotransformation involved methylenedioxy cleavage, demethylation, glutathione conjugation and glucuronidation. Among the metabolites found, the demethylated metabolite M2-2, methylenedioxy cleavage and glucuronide metabolites M4-1 and M4-2, demethylated and glucuronidated metabolites M3-3, M3-5, and M3-6, and the glucuronide metabolite M9-5 are the major biliary metabolites. After administration of ABT, a broad-spectrum CYP inhibitor, the concentrations of most metabolites decreased significantly in mouse bile samples, except for M9-4. The concentration of M9-4 is equal in POD and ABT+POD-treated mice, which showed that it is not the result of CYP metabolism and thus directly conjugated at the POD hydroxyl group.

ABT is a well-tolerated non-specific suicide substrate inhibitor of CYPs and no obvious hepatotoxicity has observed in previous studies, as suggested by no significant increased livers enzyme levels in serum and no histopathological changes (Huang and others 2020; Meschter and others 1994; Pandey and others 2020; Zong and others 2016). However, slight increases in liver weights were often encountered after long-term or repeated ABT treatment which might be related to the enhanced synthesis of microsomal enzymes, a common effect for suicide substrate inhibitors of CYPs (Meschter and others 1994). Therefore, ABT is widely used as a co-treatment to investigate the metabolism-dependent toxicity of drugs or chemicals under controlled laboratory conditions.

In order to facilitate a mechanistic bridge between the metabolism and toxicity of POD, integrated analysis of serum transaminases, liver histology, and mRNA expression of pro-inflammatory factors in liver and ileum of POD and ABT-POD treated mice were analyzed and compared. As key indicators of hepatotoxicity, serum ALT, AST and ALP all sharply increased in the POD and ABT/POD-treated mice compared to the control group. The values observed in ABT+POD-treated mice increased more than 30% compared with that in the POD-treated mice, indicating that POD is highly toxic in liver and its metabolism contributes to reduce hepatotoxicity of the compound. Microscopic examination of H&E-stained liver sections showed obvious pathological changes in POD and ABT+POD-treated mice, and ABT/POD-treated mice appeared more severely hepatic damage than POD-treated mice. Similarly, for H&E-stained colon tissue sections, POD- and ABT+POD-treated mice showed severe histological damage, as cellular infiltration, goblet cell depletion, and distortion of crypt architecture, compared to control group. With combined administration of ABT+POD to mice, more severe ileum and colon damage was found compared to POD single treated mice. An increased production of pro-inflammatory cytokines contributes to the development of intestinal inflammation (Jobin and Sartor 2000; Neurath and others 1998). The expression of pro-inflammatory cytokines Il6, Ccl2, Ccl3, Lcn2, and Icam1 mRNAs increased significantly in ileum tissue of both POD- and ABT+POD-treated mice, and the mRNA expression of Il6, Ccl2, and Lcn2 are higher in ABT+POD-treated mice than in POD-treated mice. The above results clearly showed that POD possesses obvious hepatic and intestinal toxicity, and metabolic inhibition significantly increased its toxicity as revealed when ABT is simultaneous administered, indicating that the toxicities of POD might be associated with its native cytotoxicity. POD is a mitotic inhibitor that irreversibly binds to β-tubulin and thus interrupts the dynamic equilibrium between the assembly and disassembly of microtubules, leading to cell cycle arrest at the G2/M phase. Oxidative stress also plays an important role in the toxicity of POD, as POD induced decline in the superoxide dismutase activity and GSH level but increase in the lipid peroxidation (Li and others 2012; Naik and others 2011). However, the exact poisoning mechanism of POD is still unclear and needs further clarification.

POD is toxic as its oral LD50 is 100 mg/kg in mice and 500 mg/kg in rats. Thus, the clinical practicality of POD was largely tempered due to its undesirable adverse reactions such as gastrointestinal toxicity and neurotoxicity which led to the discovery of less-toxic derivatives (Shah and others 2021). The clinically important anticancer drugs, teniposide and etoposide are β-d-thenylidene glucoside and β-d-ethylidene glucoside of 4’-demethylpodophyllotoxin (4’-demethylation at ring E), respectively. The oral LD50 of etoposide is 3800 mg/kg in mice and 1780 mg/kg in rats, and the oral TDLo is 16 mg/kg in humans, while the oral TDLo of teniposide is 9580 mg/kg in humans. Based on these values, POD is more toxic than its derivatives etoposide and teniposide. POD and its derivatives are potent cytotoxic agents that inhibit cell mitosis and DNA synthesis. POD is a tubulin polymerization inhibitor, while etoposide and teniposide appear to function as DNA topoisomerase II inhibitors that cause cell cycle arrest in the S-phase (Ardalani and others 2017). POD, etoposide and teniposide all have the methylenedioxyphenyl group in their structures. However, ring A demethylated metabolites have not been reported for etoposide and teniposide, perhaps due to the steric hindrance effect of glucosides. Instead, etoposide and teniposide are mainly bioactivated by CYPs and peroxidase at ring E to active p-quinones, or to ortho-quinones subsequent to 3’-demethylation, which are covalent poisons of topoisomerase II (Fan and others 2006; Gantchev and Hunting 1998; Haim and others 1987; Haim and others 1986; Smith and others 2014). These structural and metabolic findings could partially explain the differences in efficacy and toxicity of POD and its derivatives. Further investigation and comparison are needed to better understand the related action.

This study investigated POD metabolism and the relationship between its toxicity and metabolism in mice. The metabolites from POD are mainly derived from CYP2C9 and CYP3A5-mediated oxidation and demethylation. POD, but not its metabolites, caused hepatic and intestinal toxicity, which was suggested by use of ABT, a broad-spectrum inhibitor of CYPs. Taken together, these results will be beneficial for the further development of POD or its derivatives.

Supplementary Material

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Acknowledgments

We thank Linda G. Byrd for the assistance with the mouse studies and animal protocols.

Funding

This research work was supported by the National Cancer Institute Intramural Research Program

Abbreviations:

ALP

alkaline phosphatase

ALT

alanine aminotransferase

AST

aspartate aminotransferase

BW

body weight

CYPs

cytochromes

DMSO

dimethylsulfoxide

DNA

deoxyribonucleic acid

FDA

Food and Drug Administration

FMOs

flavin-containing monooxygenases

GSH

r-glutamyl cysteingl glycine

HDMS Q-TOF

high-definition mass spectrum quadrupole-time of flight

HLMs

human liver microsomes

MLMs

mice liver microsomes

NADPH

nicotinamide adenine dinucleotide phosphate

PCR

polymerase chain reaction

POD

podophyllotoxin

SEM

standard error of mean

UDPGA

uridine-5-diphosphoglucuronic acid

UPLC-ESI-Q-TOF–MS

high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrum

UPLC

high-performance liquid chromatography

Footnotes

Disclosure statement

No potential conflict of interest was reported by the author(s).

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