Comparative Pharmacokinetic Study of Two Pyrrolizidine Alkaloids Lasiocarpine and Heliotrine in Rats

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Materials
• Animals and dosing formulation
• Pharmacokinetic studies
• Solution and sample preparation for pharmacokinetic studies
• LC-MS/MS conditions for pharmacokinetic studies
• Method validation
• Contributorsʼ Statement
• References

Abstract

Lasiocarpine (LAS) and heliotrine (HEL) are two different ester types of toxic pyrrolizidine alkaloids (PAs): open-chain diester and monoester. However, the pharmacokinetics of these two types of PAs in rats have not been reported. In the present study, two LC-MS/MS methods for determining LAS and HEL were established and validated. The methods exhibited good linearity, accuracy, and precision and were then applied to a comparative pharmacokinetic study. After intravenous administration to male rats at 1 mg/kg, the AUC_0-t values of LAS and HEL were 336 ± 26 ng/mL × h and 170 ± 5 ng/mL × h. After oral administration at 10 mg/kg, the AUC_0-t of LAS was much lower than that of HEL (18.2 ± 3.8 ng/mL × h vs. 396 ± 18 ng/mL × h), while the C_max of LAS was lower than that of HEL (51.7 ± 22.5 ng/mL × h vs. 320 ± 26 ng/mL × h). The absolute oral bioavailability of LAS was 0.5%, which was significantly lower than that of HEL (23.3%). The results revealed that the pharmacokinetic behaviors of LAS differed from that of HEL.

Introduction

Pyrrolizidine alkaloids (PAs) are a class of plant toxic secondary metabolites with diverse structures. To date, over 660 PAs and PA N-oxides have been identified from more than 6,000 plants belonging to several families, such as Asteraceae, Boraginaceae, Fabaceae, and Orchidaceae [1], [2], [3]. It is estimated that about 50% of the PAs and PA N-oxides have been found to exhibit hepatotoxicity [4], carcinogenicity [5], and occasionally pulmonary toxicity [6]. PA-induced liver damage can result in hepatic sinusoidal obstruction syndrome in humans and animals [7]. PAs are a group of esters consisting of a necine base and one or two necic acids ([Fig. 1 a]) [8], [9]. Based on the ester types, PAs are classified as monoesters, open-chain diesters, and macrocyclic esters ([Fig. 1 a]) [10]. There are two necessary structural features for PAs to exert toxicity [11], [12], [13]: (i) a double bond at the 1,2-position of the necine base; (ii) at least 7- or 9-position of the necine base esterified with a necic acid. The toxicity of PAs with different ester types may vary dramatically [14], [15], [16]. However, due to a large number of naturally occurring PAs, it is impossible to acquire comprehensive in vivo data to explain the toxicity of all PAs.

Previous studies have revealed that the rat is the most sensitive species [17] and is frequently used to investigate the toxicity of PAs. Therefore, the pharmacokinetic behaviors of PAs in rats may provide valuable information for toxicity studies. However, the pharmacokinetic or toxicokinetic studies in rats have mainly focused on macrocyclic PAs, such as senecionine [18], monocrotaline [19], usaramine [20], riddelliine [21], and seneciphylline [22]. Few pharmacokinetic data about monoesters or open-chain diesters were reported. Lasiocarpine (LAS) is an open-chain diester ([Fig. 1 b]) and is classified as “potentially carcinogenic to humans” (Group 2B) by the International Agency for Research on Cancer (IARC) [23]. Heliotrine (HEL) is a monoester ([Fig. 1 b]) and is classified as “not classifiable as to its carcinogenicity to humans” (Group 3) by IARC [23], [24], [25]. These two PAs were mainly found in Heliotropium genus plants, which belong to the Boraginaceae family [26], [27], [28].

In this study, metabolite screening studies for LAS and HEL in rat plasma were performed; two LC-MS/MS methods to quantify LAS and HEL in rat plasma were then developed and validated. The validated methods were applied to the comparative pharmacokinetic studies of LAS and HEL in rats after intravenous or oral administration of LAS and HEL to male Sprague-Dawley rats.

Results and Discussion

First, the metabolite screening studies were performed for LAS and HEL. The detailed identification process of each metabolite is described in Supporting Information, and the proposed metabolic pathways are summarized in [Fig. 2]. Briefly, 9 metabolites were identified in rat plasma after oral administration of LAS, including LAS included hydrolysis of ester at C9 position (LAS-M1), hydrolysis of ester at C9 position + hydroxylation (LAS-M2), hydrolysis of ester at C9 position + N-oxidation (LAS-M3), hydrolysis of ester at C7 position + demethylation (LAS-M4 and LAS-M5), hydrolysis of ester at C7 position (LAS-M6), demethylation (LAS-M7), demethylation + N-oxidation (LAS-M8), and N-oxidation (LAS-M9). Meanwhile, 8 metabolites of HEL were identified in rat plasma, including desaturation + demethylation (HEL-M1), demethylation (HEL-M2), hydroxylation (HEL-M3, HEL-M4, and HEL-M6), demethylation + N-oxidation (HEL-M5), N-oxidation (HEL-M7), and demethylation + dihydroxylation (HEL-M8).

Two LC-MS/MS methods for LAS and HEL were then developed and validated to compare the pharmacokinetics of LAS and HEL in rats.

The acceptable selectivity results for the quantitation of LAS and HEL were detected in rat plasma. The representative chromatograms are shown in [Figs. 3] and [4].

The mean matrix factors (MFs) normalized by IS for LAS were 90.1% with a CV of 2.1% at low QC concentration, while 101.9% with a CV of 1.4% at high QC concentration. The mean IS-normalized MFs for HEL were 96.0% with a CV of 4.6% at low QC concentration, while 99.4% with a CV of 1.0% at high QC concentration. The results are listed in [Table 1].

The calibration curves of LAS exhibited good linearity with the correlation coefficient (r) greater than 0.995 over the range of 0.5 – 1000 ng/mL in rat plasma. The mean accuracy bias ranged from − 7.8% to 5.8%, while the CV ≤ 4.3%. The typical regression equation was y = 0.0332x − 0.000759 (r = 0.9986). The acceptable LLOQ of LAS was 0.5 ng/mL. The calibration curves of HEL were fitted by linear regression using a weighting factor of 1/x². The good linearity was over the range of 0.5 – 1000 ng/mL in rat plasma. The mean accuracy bias was − 2.4 – 2.0%, with the CV ≤ 6.2%. The typical equation was y = 0.0228x + 0.00152 (r = 0.9994). The acceptable LLOQ of HEL was 0.5 ng/mL. No carryover effects were observed in all runs for LAS and HEL.

The within-run and between-run accuracy and precision of the methods all met acceptance criteria and are summarized in [Table 2]. For the method of LAS, the within-run and between-run accuracy were − 6.1 – 4.6% and − 8.6 – 5.4%; the within-run and between-run precision were all ≤ 6.9%. For the method of HEL, the within-run and between-run accuracy were − 0.1 – 2.5% and − 3.5 – 1.1%; the within-run and between-run precision were all ≤ 5.2%.

The overall recoveries of LAS ranged from 92.3% to 94.5% at three QC concentration levels. The overall recoveries of HEL ranged from 84.2% to 95.3% at three QC concentration levels. The results are shown in [Table 1], indicating that the protein precipitation solvent MeOH/ACN (1/1, v/v) resulted in high and reliable recoveries for LAS and HEL in rat plasma.

Bench-top stability (room temperature, 8 h), three freezing-thawing cycles (− 70 °C to room temperature), and long-term stability (− 70 °C, 57 days) were investigated for both LAS and HEL. The processed samples were assessed in the autosampler (~ 5 °C) up to 26 h for LAS and 30 h for HEL. LAS and HEL were stable in rat plasma samples stored under the abovementioned conditions. The results of stability are shown in [Table 3].

The pharmacokinetic studies of LAS and HEL were conducted in male Sprague-Dawley (SD) rats. The reported pharmacokinetic studies for different PAs were investigated after PO administration of 5.7 – 40 mg/kg to rats [18], [19], [20], [21], [22]. In this study, a dose of 10 mg/kg was selected for PO administration to rats, while a relatively low dose of 1 mg/kg was chosen for IV administration to rats. The concentrations of LAS or HEL in rat plasma samples were quantified using the validated LC-MS/MS methods.

The concentration-time profiles of LAS are plotted in [Fig. 5], and the pharmacokinetic parameters are presented in [Table 4]. After a single IV administration, the total plasma clearance (CL) of LAS was 2.98 ± 0.26 L/h/kg. The volume of distribution (V_d) was 0.820 ± 0.093 L/kg, which was close to the total body water (0.7 L/kg), indicating that LAS may distribute evenly throughout the blood and tissues. The absolute oral bioavailability (F, normalized-dose AUC_0-t after PO divided by that after IV administration) was 0.5%. The metabolic profiles of LAS were different after IV and PO administration (Table 1S, Supporting Information). After IV administration, the major detected metabolite was N-oxidation (LAS-M7), and the MS peak area ratio of LAS-M7/LAS was 5.0%. Besides, LAS-M3 (hydrolysis of ester at C9 position + N-oxidation) and LAS-M6 (hydrolysis of ester at C7 position) were detected, and their MS peak area ratios were less than 0.2%. After PO administration, the MS peak area ratios of LAS-M7/LAS and LAS-M6/LAS were 3.0 and 0.4, which were significantly greater than that in plasma after IV administration. In addition, more hydrolysis metabolites and N-oxidation metabolites were detected.

The concentration-time profiles of HEL are plotted in [Fig. 4], and the pharmacokinetic parameters are presented in [Table 4]. After a single IV administration, CL of HEL was 5.86 ± 0.17 L/h/kg. The V_d was 2.90 ± 0.14 L/kg, which was 4.1-fold greater than the total body water, demonstrating extensive distribution into tissues. After PO administration, HEL was absorbed with a C_max of 320 ± 26 ng/mL at 0.75 ± 0.00 h, and the AUC_0-t value was 396 ± 18 ng/mL × h. The absolute oral bioavailability was 23.3%. Similar to the results of LAS, the metabolic profiles were different after IV and PO administration of HEL (Table 2S, Supporting Information). After IV administration, HEL-M2 (demethylation), HEL-M3 and HEL-M4 (demethylation + hydroxylation), HEL-M6 (hydroxylation), and HEL-M7 (N-oxidation) were detected, and their MS peak area ratios to HEL were less than 1.8%. After PO administration, the major detected metabolites were HEL-M2 (demethylation), HEL-M3 to M4 (demethylation + hydroxylation), and their MS peak area ratios to HEL were 50.5%, 22.9%, and 10.8%. In addition, more demethylation metabolites were detected.

Statistical analysis was performed to compare the pharmacokinetic parameters of LAS and HEL. Most pharmacokinetic parameters excluding T_1/2 in PO groups showed significant differences (P values < 0.05) after both IV and PO administration. After IV administration of LAS or HEL, HEL was eliminated from the plasma more quickly than LAS (CL: 5.86 ± 0.17 L/h/kg vs. 2.98 ± 0.26 L/h/kg). The CL value of HEL was also larger than the CL values reported for other PAs, such as monocrotaline (1.93 L/h/kg) [19], usaramine (2.77 L/h/kg) [20], SCN (1.39 L/h/kg) [18], and SCP (0.991 L/h/kg) [22].

These results indicate that both LAS and HEL may be extensively metabolized in the gastrointestinal (GI) flora or microsomes, and the metabolites can be absorbed into the body. LAS was absorbed into the body more quickly than HEL (T_max: 0.074 ± 0.054 h vs. 0.75 ± 0.00 h). The absolute oral bioavailability of LAS was 0.5%, which was significantly lower than that of HEL (23.3%). Compared to the major metabolites of LAS and HEL, the ester bond at the C7 position of LAS can be extensively hydrolyzed in the GI tract, whereas no esterification occurred at the C7 position of HEL, which may cause LAS to be less bioavailable than HEL. One reason for the low oral bioavailability may be due to the extensive metabolism in the GI tract, and other reasons need to be further investigated.

In summary, the LC-MS/MS methods of LAS and HEL in rat plasma were developed and validated. The validated methods were applied to investigate the pharmacokinetic profiles of LAS and HEL in rats after IV or PO administration of LAS and HEL to male SD rats.

Materials and Methods

Materials

LAS (purity 100%) and HEL (purity 99%) were both purchased from Phytolab GmbH & Co. Senecionine (SCN, purity > 98%) and seneciphylline (SCP, purity > 98%) were both purchased from Chengdu Biopurify Phytochemicals Ltd. The structures are shown in [Fig. 1]. LC-MS grade formic acid was obtained from J & K Technology Co., Limited. Deionized H₂O was prepared by a Milli-Q ultra-pure water purification system (Millipore). All other reagents were HPLC-grade, provided by Sigma-Aldrich.

Animals and dosing formulation

The animal studies followed the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences (IACUC number: 2022-01-YY-20, approved on February 22nd, 2022). Male SD rats (220 – 260 g, 6 – 9 weeks) were purchased from HFK Bio-technology Co. Ltd. (Beijing, China). Rats were housed at 20 – 26 °C (relative humidity of 40 – 70%) and under a 12-h light/dark cycle with free access to diet and water.

Both LAS and HEL were completely dissolved in saline to obtain 0.2 mg/mL of dosing solution for IV and 1 mg/mL for PO.

Pharmacokinetic studies

Twenty rats were randomly divided into four groups (n = 5 per group) after a 12-h fast. For IV groups, both compounds were administered at a dose of 1 mg/kg with 5 mL/kg. For PO groups, both compounds were administered at a dose of 10 mg/kg with 10 mL/kg.

For LAS, blood samples (~ 50 µL) were collected via saphenous vein into heparin-containing tubes at 0.05, 0.25, 0.5, 0.75,1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 6 and 8 h after IV administration, while at 0.05, 0.17, 0.33, 0.67, 1, 1.17, 1.67, 2, 2.5, 3, 4, 6 and 8 h after PO administration. For HEL, blood samples (~ 50 µL) were collected via saphenous vein into heparin-containing tubes at 0.05, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 12 h after IV administration, while at 0.083, 0.25, 0.5, 0.75,1, 1.5, 2, 2.5, 3, 4, 6, 8 and 12 h after PO administration. Plasma samples were harvested after centrifugation for 5 min at 11 363 × g and stored − 70 °C before analysis.

Pharmacokinetic parameters were calculated using non-compartmental analysis in Phoenix WinNonlin (version 8.3, Certara). Statistical analysis of pharmacokinetic parameters between LAS and HEL was performed by Studentʼs t-test in Prism (version 8.4.2, GraphPad Software), and P values < 0.05 were considered statistically significant.

Solution and sample preparation for pharmacokinetic studies

Stock solutions of LAS, HEL, SCN, and SCP were prepared in methanol, respectively. Calibration standards of LAS were diluted from the stock solution by the blank rat plasma to get the final concentration of 0.5, 1, 3, 10, 30, 100, 300, and 1000 ng/mL. The separated stock solution was used to prepare QC samples in rat plasma at four concentration levels: 0.5, 1.5, 100, and 750 ng/mL. IS working solution of SCN was diluted to 200 ng/mL in MeOH/H₂O (1/1, v/v). The calibration standards and QC samples of HEL were prepared following the same procedures as LAS. IS working solution of SCP was prepared at 100 ng/mL in MeOH/H₂O (1/1, v/v).

The same sample preparation protocol was adopted to determine that LAS and HEL: plasma proteins were precipitated by the addition of 100-µL MeOH/ACN (1/1, v/v) into the mixture containing 10-µL IS working solution and 10-µL plasma sample. The mixtures were vortexed for 5 min and centrifuged for 5 min at 3220 × g. An aliquot of 60-µL supernatant was sampled and mixed with 120-µL water, and 1 µL aliquot was injected into LC-MS/MS.

LC-MS/MS conditions for pharmacokinetic studies

All experiments were performed on an LC-MS/MS system, respectively. The LC-MS/MS system consisted of a Waters Xevo TQ-S Triple Quadrupole Mass Spectrometer with electrospray ionization (ESI) coupled with a Waters Acquity^™ I-class system. Data acquisition and quantification were conducted using MassLynx 4.1 software.

For analysis of LAS, chromatography was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 µm) at 45 °C. Gradient elution was using 0.1% formic acid with 5 mM ammonium acetate in H₂O (mobile phase A) and 0.1% formic acid in ACN (mobile phase B). The optimal gradient elution program was as follows: 0 – 0.8 min, 20% B to 30% B; 0.8 – 1.1 min, 30% to 80% B; 1.1 – 1.4 min, 80% to 95% B; 1.4 – 2.0 min, 20% B. The elution flow rate was 0.5 mL/min, and the injection volume was 1 µL.

For analysis of HEL, the LC column, mobile phase composition, and elution flow rate were the same as LAS. The gradient elution program was as follows: 0 – 0.6 min, 10% B to 20% B; 0.6 – 1.1 min, 20% to 80% B; 1.1 – 1.4 min, 80% to 95% B; 1.4 – 2.0 min, 10% B. The injection volume was 1 µL.

The MS was operated in the positive ion mode. The optimal MS parameters of LAS were as follows: capillary voltage, 3.0 kV; source temperature and desolvation temperature, 150 °C and 500 °C; desolvation gas flow and cone gas flow, 1000 and 150 L/h. For the determination of LAS, the multiple reaction monitoring (MRM) transitions for quantitation were m/z 412.1 → m/z 120.0 for LAS, and m/z 336.1 → m/z 120.1 for SCN (IS); the collision energies and cone voltages were 28 eV and 40 V for LAS, and 26 eV and 40 V for SCN. For analysis of HEL, the MRM transitions were m/z 314.2 → m/z 138.0 for HEL, and m/z 334.0 → m/z 138.1 for SCP (IS); the collision energies were 17 eV and 40 V for HEL, and 26 eV and 40 V for SCP.

Method validation

The two methods for the determination of LAS and HEL were validated according to the Bioanalytical Method Validation Guidance of the USA FDA [29], respectively.

Selectivity of the method was assessed by comparing the analytes and IS peak areas of blank samples in six individual rats and LLOQ samples. The ratios should be ≤ 20% for analytes and ≤ 5% for ISs.

The matrix effect was evaluated using plasma from six different rats at low and high QC concentration levels. The matrix factor (MF) was calculated by comparing the peak area ratio of analyte to IS in samples with and without the addition of plasma. The CV of IS-normalized MFs at each concentration level for analytes should be less than 15%.

Linearity was assessed by performing a linear regression (weighting 1/x²) using the peak area ratio of each analyte to IS on the non-zero calibration standards. At least 75% of the calibration standards should be within ± 15.0% (± 20.0% at LLOQ) of the nominal concentration.

The carryover was determined using a blank sample injected immediately after the upper limit of quantitation (ULOQ) sample. An acceptable carryover was the peak area of analyte in the carryover sample less than 20% of the peak area in the LLOQ sample, while the peak area of IS in the carryover sample less than 20% of the mean peak area in the calibration standards.

The accuracy and precision were investigated at LLOQ, low, medium, and high QC concentration levels. Six replicates per QC level were analyzed in three independent runs on different days. Within-run accuracy and precision were assessed by analyzing the QCs in an individual run (n = 6), and between-run accuracy and precision were evaluated by analyzing the QCs in all three runs (n = 18). The acceptable precision should be ≤ 15% (20.0% at LLOQ), and the acceptable accuracy should be within ± 15.0% (± 20.0% at LLOQ) of the nominal concentration.

The recovery was evaluated at low, medium, and high QC concentration levels. The recovery values were calculated by dividing the peak area of analyte or IS in the QC samples by the reference samples post-extraction.

The stability experiments of LAS and HEL were assessed using low and high QC samples under different storage conditions: bench-top stability (room temperature, 12 h); at − 70 °C for 57 days; after three freeze and thaw cycles (< − 60 °C to room temperature); and processed samples stored in the autosampler (the temperature was set at 5 °C) for at least 24 h. Samples with the mean accuracy bias within ± 15.0% of nominal concentration at each concentration level and the CV ≤ 15% were considered stable.

The dilution integrity of LAS was evaluated using the 5-fold dilution of QC samples (4000 ng/mL). The back-calculated concentrations should be within ± 15.0% of the nominal concentration, while the CV should be ≤ 15%.

Contributorsʼ Statement

Data collection: Feifei Lin; design of the study: Jia Liu, Yang Ye; analysis and interpretation of the data: Feifei Lin, Yingying Wang, Lijuan Zhao; drafting the manuscript: Feifei Lin; critical revision of the manuscript: Jia Liu.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors are grateful to the National Natural Science Foundation of China (81 803 610 and 21 920 102 003), Science and Technology Commission of Shanghai Municipality (20 430 780 300), Key-Area Research and Development Program of Guangdong Province (2020B0 303 070 002), and Shanghai Municipal Science and Technology Major Project.

• References

• 1 Hartmann T, Witte L. Chapter Four – Chemistry, Biology and Chemoecology of the Pyrrolizidine Alkaloids. In: Pelletier SW. ed. Alkaloids: Chemical and biological Perspectives. Oxford: Pergamon Press; 1995: 155-233
  Search in Google Scholar
• 2 Fu PP, Yang Y-C, Xia Q, Chou MW, Cui Y, Lin G. Pyrrolizidine alkaloids-tumorigenic components in Chinese herbal medicines and dietary supplements. J Food Drug Anal 2002; 10: 198-211
  PubMedSearch in Google Scholar
• 3 Schrenk D, Gao L, Lin G, Mahony C, Mulder PPJ, Peijnenburg A, Pfuhler S, Rietjens IMCM, Rutz L, Steinhoff B, These A. Pyrrolizidine alkaloids in food and phytomedicine: Occurrence, exposure, toxicity, mechanisms, and risk assessment – A review. Food Chem Toxicol 2020; 136: 111107
  CrossrefPubMedSearch in Google Scholar
• 4 Molyneux RJ, Gardner DL, Colegate SM, Edgar JA. Pyrrolizidine alkaloid toxicity in livestock: a paradigm for human poisoning?. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2011; 28: 293-307
  CrossrefPubMedSearch in Google Scholar
• 5 He Y, Shi M, Wu X, Ma J, Ng KT, Xia Q, Zhu L, Fu PP, Man K, Tsui SK, Lin G. Mutational signature analysis reveals widespread contribution of pyrrolizidine alkaloid exposure to human liver cancer. Hepatology 2021; 74: 264-280
  CrossrefPubMedSearch in Google Scholar
• 6 Song Z, He Y, Ma J, Fu PP, Lin G. Pulmonary toxicity is a common phenomenon of toxic pyrrolizidine alkaloids. J Environ Sci Health C Toxicol Carcinog 2020; 38: 124-140
  PubMedSearch in Google Scholar
• 7 Zhu L, Zhang CY, Li DP, Chen HB, Ma J, Gao H, Ye Y, Wang JY, Fu PP, Lin G. Tu-San-Qi (Gynura japonica): the culprit behind pyrrolizidine alkaloid-induced liver injury in China. Acta Pharmacol Sin 2021; 42: 1212-1222
  CrossrefPubMedSearch in Google Scholar
• 8 Madge I, Gehling M, Schone C, Winterhalter P, These A. Pyrrolizidine alkaloid profiling of four Boraginaceae species from Northern Germany and implications for the analytical scope proposed for monitoring of maximum levels. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2020; 37: 1339-1358
  CrossrefPubMedSearch in Google Scholar
• 9 Louisse J, Rijkers D, Stoopen G, Holleboom WJ, Delagrange M, Molthof E, Mulder PPJ, Hoogenboom RLAP, Audebert M, Peijnenburg AACM. Determination of genotoxic potencies of pyrrolizidine alkaloids in HepaRG cells using the γH2AX assay. Food Chem Toxicol 2019; 131: 110532
  CrossrefPubMedSearch in Google Scholar
• 10 Ebmeyer J, Rasinger JD, Hengstler JG, Schaudien D, Creutzenberg O, Lampen A, Braeuning A, Hessel-Pras S. Hepatotoxic pyrrolizidine alkaloids induce DNA damage response in rat liver in a 28-day feeding study. Arch Toxicol 2020; 94: 1739-1751
  CrossrefPubMedSearch in Google Scholar
• 11 Ruan J, Yang M, Fu P, Ye Y, Lin G. Metabolic activation of pyrrolizidine alkaloids: insights into the structural and enzymatic basis. Chem Res Toxicol 2014; 27: 1030-1039
  CrossrefPubMedSearch in Google Scholar
• 12 Allemang A, Mahony C, Lester C, Pfuhler S. Relative potency of fifteen pyrrolizidine alkaloids to induce DNA damage as measured by micronucleus induction in HepaRG human liver cells. Food Chem Toxicol 2018; 121: 72-81
  CrossrefPubMedSearch in Google Scholar
• 13 Fu PP. Pyrrolizidine alkaloids: metabolic activation pathways leading to liver tumor initiation. Chem Res Toxicol 2017; 30: 81-93
  CrossrefPubMedSearch in Google Scholar
• 14 Gao L, Rutz L, Schrenk D. Structure-dependent hepato-cytotoxic potencies of selected pyrrolizidine alkaloids in primary rat hepatocyte culture. Food Chem Toxicol 2020; 135: 110923
  CrossrefPubMedSearch in Google Scholar
• 15 Merz KH, Schrenk D. Interim relative potency factors for the toxicological risk assessment of pyrrolizidine alkaloids in food and herbal medicines. Toxicol Lett 2016; 263: 44-57
  CrossrefPubMedSearch in Google Scholar
• 16 EFSA Panel on Contaminants in the Food Chain (CONTAM), Knutsen HK, Alexander J, Barregård L, Bignami M, Brüschweiler B, Ceccatelli S, Cottrill B, Dinovi M, Edler L, Grasl-Kraupp B, Hogstrand C, Hoogenboom LR, Nebbia CS, Oswald IP, Petersen A, Rose M, Roudot AC, Schwerdtle T, Vleminckx C, Vollmer G, Wallace H, Gomez Ruiz JA, Binaglia M. Risks for human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal infusions and food supplements. EFSA J 2017; 15: e04908
  CrossrefPubMedSearch in Google Scholar
• 17 World Health Organization & Food and Agriculture Organization of the United Nations (WHO-FAO). Safety evaluation of certain food additives and contaminants: supplement 2: pyrrolizidine alkaloids, prepared by the eightieth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). WHO Food Additives Series, No. 71-S2. 2020. Accessed May 21, 2022 at: https://www.who.int/publications/i/item/9789240012677
  PubMed
• 18 Wang C, Li Y, Gao J, He Y, Xiong A, Yang L, Cheng X, Ma Y, Wang Z. The comparative pharmacokinetics of two pyrrolizidine alkaloids, senecionine and adonifoline, and their main metabolites in rats after intravenous and oral administration by UPLC/ESIMS. Anal Bioanal Chem 2011; 401: 275-287
  CrossrefPubMedSearch in Google Scholar
• 19 Lin F, Pan A, Ye Y, Liu J. Simultaneous determination of monocrotaline and its N-oxide metabolite in rat plasma using LC-MS/MS: Application to a pharmacokinetic study. Biomed Chromatogr 2021; 35: e5207
  CrossrefPubMedSearch in Google Scholar
• 20 Lin F, Ma Y, Pan A, Ye Y, Liu J. Quantification of usaramine and its N-oxide metabolite in rat plasma using liquid chromatography-tandem mass spectrometry. J Anal Toxicol 2022; 46: 512-518
  CrossrefPubMedSearch in Google Scholar
• 21 Williams L. Toxicokinetics of riddelliine, a carcinogenic pyrrolizidine alkaloid, and metabolites in rats and mice. Toxicol Appl Pharmacol 2002; 182: 98-104
  CrossrefPubMedSearch in Google Scholar
• 22 Long F, Ji J, Wang X, Wang L, Chen T. LC-MS/MS method for determination of seneciphylline and its metabolite, seneciphylline N-oxide in rat plasma, and its application to a rat pharmacokinetic study. Biomed Chromatogr 2021; 35: e5145
  CrossrefPubMedSearch in Google Scholar
• 23 International Agency for Research on Cancer (IARC). IARC monographs on the identification of carcinogenic hazards to humans. Report of the Advisory Group to Recommend Priorities for the IARC Monographs during 2020–2024. Accessed June 01, 2022 at: https://monographs.iarc.who.int/wp-content/uploads/2019/10/IARCMonographs-AGReport-Priorities_2020-2024.pdf
  PubMed
• 24 Luckert C, Braeuning A, Lampen A, Hessel-Pras S. PXR: Structure-specific activation by hepatotoxic pyrrolizidine alkaloids. Chem Biol Interact 2018; 288: 38-48
  CrossrefPubMedSearch in Google Scholar
• 25 Buchmueller J, Sprenger H, Ebmeyer J, Rasinger JD, Creutzenberg O, Schaudien D, Hengstler JG, Guenther G, Braeuning A, Hessel-Pras S. Pyrrolizidine alkaloid-induced transcriptomic changes in rat lungs in a 28-day subacute feeding study. Arch Toxicol 2021; 95: 2785-2796
  CrossrefPubMedSearch in Google Scholar
• 26 Shimshoni JA, Mulder PP, Bouznach A, Edery N, Pasval I, Barel S, Abd-El Khaliq M, Perl S. Heliotropium europaeum poisoning in cattle and analysis of its pyrrolizidine alkaloid profile. J Agric Food Chem 2015; 63: 1664-1672
  CrossrefPubMedSearch in Google Scholar
• 27 Shimshoni JA, Barel S, Mulder PPJ. Comparative risk assessment of three native heliotropium species in israel. Molecules 2021; 26: 689
  CrossrefPubMedSearch in Google Scholar
• 28 Eroksuz Y, Eroksuz H, Ozer H, Ilhan N, Cevik A, Yaman I, Ceribasi AO. Toxicity of dietary Heliotropium dolosum seed to Japanese quail. Vet Hum Toxicol 2002; 44: 264-268
  PubMedSearch in Google Scholar
• 29 U.S. Food and Drug Administration (FDA). Bioanalytical Method Validation: Guidance for Industry. Accessed June 1, 2022 at: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry
  PubMed

Correspondence

Publication History

Received: 05 January 2022

Accepted after revision: 27 July 2022

Article published online:
28 September 2022

© 2022. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Hartmann T, Witte L. Chapter Four – Chemistry, Biology and Chemoecology of the Pyrrolizidine Alkaloids. In: Pelletier SW. ed. Alkaloids: Chemical and biological Perspectives. Oxford: Pergamon Press; 1995: 155-233
  Search in Google Scholar
• 2 Fu PP, Yang Y-C, Xia Q, Chou MW, Cui Y, Lin G. Pyrrolizidine alkaloids-tumorigenic components in Chinese herbal medicines and dietary supplements. J Food Drug Anal 2002; 10: 198-211
  PubMedSearch in Google Scholar
• 3 Schrenk D, Gao L, Lin G, Mahony C, Mulder PPJ, Peijnenburg A, Pfuhler S, Rietjens IMCM, Rutz L, Steinhoff B, These A. Pyrrolizidine alkaloids in food and phytomedicine: Occurrence, exposure, toxicity, mechanisms, and risk assessment – A review. Food Chem Toxicol 2020; 136: 111107
  CrossrefPubMedSearch in Google Scholar
• 4 Molyneux RJ, Gardner DL, Colegate SM, Edgar JA. Pyrrolizidine alkaloid toxicity in livestock: a paradigm for human poisoning?. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2011; 28: 293-307
  CrossrefPubMedSearch in Google Scholar
• 5 He Y, Shi M, Wu X, Ma J, Ng KT, Xia Q, Zhu L, Fu PP, Man K, Tsui SK, Lin G. Mutational signature analysis reveals widespread contribution of pyrrolizidine alkaloid exposure to human liver cancer. Hepatology 2021; 74: 264-280
  CrossrefPubMedSearch in Google Scholar
• 6 Song Z, He Y, Ma J, Fu PP, Lin G. Pulmonary toxicity is a common phenomenon of toxic pyrrolizidine alkaloids. J Environ Sci Health C Toxicol Carcinog 2020; 38: 124-140
  PubMedSearch in Google Scholar
• 7 Zhu L, Zhang CY, Li DP, Chen HB, Ma J, Gao H, Ye Y, Wang JY, Fu PP, Lin G. Tu-San-Qi (Gynura japonica): the culprit behind pyrrolizidine alkaloid-induced liver injury in China. Acta Pharmacol Sin 2021; 42: 1212-1222
  CrossrefPubMedSearch in Google Scholar
• 8 Madge I, Gehling M, Schone C, Winterhalter P, These A. Pyrrolizidine alkaloid profiling of four Boraginaceae species from Northern Germany and implications for the analytical scope proposed for monitoring of maximum levels. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2020; 37: 1339-1358
  CrossrefPubMedSearch in Google Scholar
• 9 Louisse J, Rijkers D, Stoopen G, Holleboom WJ, Delagrange M, Molthof E, Mulder PPJ, Hoogenboom RLAP, Audebert M, Peijnenburg AACM. Determination of genotoxic potencies of pyrrolizidine alkaloids in HepaRG cells using the γH2AX assay. Food Chem Toxicol 2019; 131: 110532
  CrossrefPubMedSearch in Google Scholar
• 10 Ebmeyer J, Rasinger JD, Hengstler JG, Schaudien D, Creutzenberg O, Lampen A, Braeuning A, Hessel-Pras S. Hepatotoxic pyrrolizidine alkaloids induce DNA damage response in rat liver in a 28-day feeding study. Arch Toxicol 2020; 94: 1739-1751
  CrossrefPubMedSearch in Google Scholar
• 11 Ruan J, Yang M, Fu P, Ye Y, Lin G. Metabolic activation of pyrrolizidine alkaloids: insights into the structural and enzymatic basis. Chem Res Toxicol 2014; 27: 1030-1039
  CrossrefPubMedSearch in Google Scholar
• 12 Allemang A, Mahony C, Lester C, Pfuhler S. Relative potency of fifteen pyrrolizidine alkaloids to induce DNA damage as measured by micronucleus induction in HepaRG human liver cells. Food Chem Toxicol 2018; 121: 72-81
  CrossrefPubMedSearch in Google Scholar
• 13 Fu PP. Pyrrolizidine alkaloids: metabolic activation pathways leading to liver tumor initiation. Chem Res Toxicol 2017; 30: 81-93
  CrossrefPubMedSearch in Google Scholar
• 14 Gao L, Rutz L, Schrenk D. Structure-dependent hepato-cytotoxic potencies of selected pyrrolizidine alkaloids in primary rat hepatocyte culture. Food Chem Toxicol 2020; 135: 110923
  CrossrefPubMedSearch in Google Scholar
• 15 Merz KH, Schrenk D. Interim relative potency factors for the toxicological risk assessment of pyrrolizidine alkaloids in food and herbal medicines. Toxicol Lett 2016; 263: 44-57
  CrossrefPubMedSearch in Google Scholar
• 16 EFSA Panel on Contaminants in the Food Chain (CONTAM), Knutsen HK, Alexander J, Barregård L, Bignami M, Brüschweiler B, Ceccatelli S, Cottrill B, Dinovi M, Edler L, Grasl-Kraupp B, Hogstrand C, Hoogenboom LR, Nebbia CS, Oswald IP, Petersen A, Rose M, Roudot AC, Schwerdtle T, Vleminckx C, Vollmer G, Wallace H, Gomez Ruiz JA, Binaglia M. Risks for human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal infusions and food supplements. EFSA J 2017; 15: e04908
  CrossrefPubMedSearch in Google Scholar
• 17 World Health Organization & Food and Agriculture Organization of the United Nations (WHO-FAO). Safety evaluation of certain food additives and contaminants: supplement 2: pyrrolizidine alkaloids, prepared by the eightieth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). WHO Food Additives Series, No. 71-S2. 2020. Accessed May 21, 2022 at: https://www.who.int/publications/i/item/9789240012677
  PubMed
• 18 Wang C, Li Y, Gao J, He Y, Xiong A, Yang L, Cheng X, Ma Y, Wang Z. The comparative pharmacokinetics of two pyrrolizidine alkaloids, senecionine and adonifoline, and their main metabolites in rats after intravenous and oral administration by UPLC/ESIMS. Anal Bioanal Chem 2011; 401: 275-287
  CrossrefPubMedSearch in Google Scholar
• 19 Lin F, Pan A, Ye Y, Liu J. Simultaneous determination of monocrotaline and its N-oxide metabolite in rat plasma using LC-MS/MS: Application to a pharmacokinetic study. Biomed Chromatogr 2021; 35: e5207
  CrossrefPubMedSearch in Google Scholar
• 20 Lin F, Ma Y, Pan A, Ye Y, Liu J. Quantification of usaramine and its N-oxide metabolite in rat plasma using liquid chromatography-tandem mass spectrometry. J Anal Toxicol 2022; 46: 512-518
  CrossrefPubMedSearch in Google Scholar
• 21 Williams L. Toxicokinetics of riddelliine, a carcinogenic pyrrolizidine alkaloid, and metabolites in rats and mice. Toxicol Appl Pharmacol 2002; 182: 98-104
  CrossrefPubMedSearch in Google Scholar
• 22 Long F, Ji J, Wang X, Wang L, Chen T. LC-MS/MS method for determination of seneciphylline and its metabolite, seneciphylline N-oxide in rat plasma, and its application to a rat pharmacokinetic study. Biomed Chromatogr 2021; 35: e5145
  CrossrefPubMedSearch in Google Scholar
• 23 International Agency for Research on Cancer (IARC). IARC monographs on the identification of carcinogenic hazards to humans. Report of the Advisory Group to Recommend Priorities for the IARC Monographs during 2020–2024. Accessed June 01, 2022 at: https://monographs.iarc.who.int/wp-content/uploads/2019/10/IARCMonographs-AGReport-Priorities_2020-2024.pdf
  PubMed
• 24 Luckert C, Braeuning A, Lampen A, Hessel-Pras S. PXR: Structure-specific activation by hepatotoxic pyrrolizidine alkaloids. Chem Biol Interact 2018; 288: 38-48
  CrossrefPubMedSearch in Google Scholar
• 25 Buchmueller J, Sprenger H, Ebmeyer J, Rasinger JD, Creutzenberg O, Schaudien D, Hengstler JG, Guenther G, Braeuning A, Hessel-Pras S. Pyrrolizidine alkaloid-induced transcriptomic changes in rat lungs in a 28-day subacute feeding study. Arch Toxicol 2021; 95: 2785-2796
  CrossrefPubMedSearch in Google Scholar
• 26 Shimshoni JA, Mulder PP, Bouznach A, Edery N, Pasval I, Barel S, Abd-El Khaliq M, Perl S. Heliotropium europaeum poisoning in cattle and analysis of its pyrrolizidine alkaloid profile. J Agric Food Chem 2015; 63: 1664-1672
  CrossrefPubMedSearch in Google Scholar
• 27 Shimshoni JA, Barel S, Mulder PPJ. Comparative risk assessment of three native heliotropium species in israel. Molecules 2021; 26: 689
  CrossrefPubMedSearch in Google Scholar
• 28 Eroksuz Y, Eroksuz H, Ozer H, Ilhan N, Cevik A, Yaman I, Ceribasi AO. Toxicity of dietary Heliotropium dolosum seed to Japanese quail. Vet Hum Toxicol 2002; 44: 264-268
  PubMedSearch in Google Scholar
• 29 U.S. Food and Drug Administration (FDA). Bioanalytical Method Validation: Guidance for Industry. Accessed June 1, 2022 at: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry
  PubMed

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