Absorption difference between hepatotoxic pyrrolizidine alkaloids and their N-oxides – Mechanism and its potential toxic impact

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Absorption difference between hepatotoxic pyrrolizidine alkaloids and their N-oxides – Mechanism and its potential toxic impact Mengbi Yanga,b,1, Jiang Maa,b,1, Jianqing Ruana,b, Chunyuan Zhanga,b, Yang Yeb, Peter Pi-Cheng Fuc, Ge Lina,b,∗ a

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Joint Research Laboratory for Promoting Globalization of Traditional Chinese Medicines between the Chinese University of Hong Kong and Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China c National Center for Toxicological Research, U.S. Food and Drug Administration, USA b

ABSTRACT

Ethnopharmacological relevance: Pyrrolizidine alkaloids (PAs) are a group of phytotoxins widely present in about 3% of flowering plants. Many PA-containing herbal plants can cause liver injury. Our previous studies demonstrated that PA N-oxides are also hepatotoxic, with toxic potency much lower than the corresponding PAs, due to significant differences in their toxicokinetic fates. Aim of study: This study aimed to investigate the oral absorption of PAs and PA N-oxides for better understanding of their significant differences in toxicokinetics and toxic potency. Materials and methods: The oral absorption of PAs and PA N-oxides in rats and in rat in situ single pass intestine perfusion model was investigated. The intestinal permeability and absorption mechanisms of five pairs of PAs and PA N-oxides were evaluated by using Caco-2 monolayer model. Results: The plasma concentrations of total PAs and PA N-oxides within 0–60 min were significantly lower in rats orally treated with a PA N-oxide-containing herbal alkaloid extract than with a PA-containing herbal alkaloid extract at the same dose, indicating that the absorption of PA N-oxides was lower than that of PAs. Using the rat in situ single pass intestine perfusion model, less cumulative amounts of retrorsine N-oxide in mesenteric blood were observed compared to that of retrorsine. In Caco-2 monolayer model, all five PAs showed absorption with Papp AtoB values [(1.43–16.26) × 10−6 cm/s] higher than those of corresponding N-oxides with Papp −6 cm/s. A further mechanistic study demonstrated that except for senecionine N-oxide, retrorsine N-oxide, and lycopsamine NAtoB values lower than 1.35 × 10 oxide, all PAs and PA N-oxides investigated were absorbed via passive diffusion. While, for these 3 PA N-oxides, in addition to passive diffusion as their primary transportation, efflux transporter-mediated active transportation was also involved but to a less extent with the efflux ratio of 2.31–3.41. Furthermore, a good correlation between lipophilicity and permeability of retronecine-type PAs and their N-oxides with absorption via passive diffusion was observed, demonstrating that PAs have a better oral absorbability than that of the corresponding PA N-oxides. Conclusion: We discovered that among many contributors, the lower intestinal absorption of PA N-oxides was the initiating contributor that caused differences in toxicokinetics and toxic potency between PAs and PA N-oxides.

1. Introduction Pyrrolizidine alkaloids (PAs) are common secondary metabolites of plants widely distributed in the world (Fu et al., 2004; Mattocks, 1986). More than 660 PAs and their N-oxides have been identified in about 3% of flowering plants (Smith and Culvenor, 1981), and about half of them have been reported hepatotoxic. Apart from herbal preparations and dietary supplements directly derived from PA- or PA N-oxide-containing plants, a large number of foodstuffs such as green crops, honey, milk, and eggs, have also been reported to be contaminated by PAs and PA N-oxides (Edgar et al., 2015; Zhu et al., 2018). To date, over fifteen thousand acute human PA-poisoning cases have been documented

(Kakar et al., 2010; Lin et al., 2011; Robinson et al., 2014; Schneider et al., 2012; Willmot and Robertson, 1920). There are two types of toxic PAs: retronecine-type and otonecinetype (Fu et al., 2004; Mattocks, 1986). In retronecine-type PAs, there are two forms: PAs and their corresponding PA N-oxides, while otonecine-type PAs do not have the N-oxide form (Fig. 1). PAs exert their toxicity via metabolic activation catalyzed by cytochrome P450 enzymes in the liver. Exposure to PAs may develop liver injury such as hepatic sinusoidal obstruction syndrome (HSOS), cirrhosis, and cancer (Edgar et al., 2015; Field et al., 2015; Fu et al., 2004; Gao et al., 2015; Lin et al., 2011; Ruan et al., 2015; Yang et al., 2017). PAs and PA N-oxides generally coexist in plants (Cao et al., 2008;

Corresponding author. School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, SAR, Hong Kong. E-mail address: [email protected] (G. Lin). 1 Author Contributions: These authors contributed equally to this work. ∗

https://doi.org/10.1016/j.jep.2019.112421 Received 29 May 2019; Received in revised form 22 October 2019; Accepted 19 November 2019 0378-8741/ © 2019 Published by Elsevier B.V.

Please cite this article as: Mengbi Yang, et al., Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2019.112421

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2. Materials and methods 2.1. Chemicals Senecionine, seneciphylline, and senkirkine were purchased from Extrasynthesese. Lycopsamine and lycopsamine N-oxide were from PhytoLab. Riddelliine was a gift from Dr. Po-Chan from the U.S. National Toxicology Program. Seneciphylline N-oxide, riddelliine Noxide, and senecionine N-oxide were synthesized as previously described (Chou et al., 2003; Ruan et al., 2012). Clivorine was isolated in our laboratory as previously reported (Lin et al., 2003). Alkaloid extracts of roots of PA N-oxide-containing Gynura pseudochina and PAcontaining Gynura segetum were previously prepared in our laboratory (Yang et al., 2017). Other chemicals, including retrorsine, isatidine (retrorsine N-oxide), DMSO, collagen, trypan blue, HEPES, verapamil, atenolol and propanolol were purchased from Sigma Chemical Company (St. Louis, MO). 2.2. Animals and treatment Male Sprague−Dawley rats (180–200 g) were purchased from the Laboratory Animal Services Centre at The Chinese University of Hong Kong. All animal experiments were approved by Animal Experimental Ethics Committee of The Chinese University of Hong Kong. Rats were randomly divided into two groups (n = 4/group). Alkaloid extracts of roots of PA N-oxide-containing Gynura pseudochina and PA-containing Gynura segetum were orally administered to two groups of rats at the same dose (0.2 mmol total amount of PAs or PA N-oxides/kg bodyweight). Blood samples (0.25 mL) were collected from the treated rats via lateral tail vein into heparinized tubes at 15, 30, and 60 min post dosing, and centrifuged at 4000 g for 10 min to obtain plasma samples. To each plasma sample, three-volume of methanol was added to precipitate proteins and centrifuged at 15,000 g for 10 min. The supernatant was then collected for the subsequent analysis of PAs and PA Noxides by ultra-high-performance liquid chromatography (UHPLC)tandem mass spectrometry (MS/MS). 2.3. In situ single-pass intestinal perfusion Male Sprague−Dawley rats (280–320 g) were fasted overnight with free access to water prior to the experiment. The rats were anesthetized with an intraperitoneal injection of 75 mg/kg ketamine and 10 mg/kg xylazine and placed on a 37 °C surface. The right jugular vein was cannulated (0.4 mm i.d., 0.8 mm o.d.). The abdomen was opened by midline incision of 3–4 cm. A 7- to 11-cm-long jejunum to ileum segment of the small intestine was cannulated on two ends with PVC tubing (3 mm i.d., 5 mm o.d.), and flushed with perfusion buffer. The mesenteric vein from the specified intestinal segment was cannulated with a polyethylene tubing (0.86 mm i.d., 1.27 mm o.d.) for blood collection. Donor blood was infused through the right jugular cannulation with the rate adjusted based on the outflow from the mesenteric blood (~0.15 mL/min). The intestinal perfusions were initiated by infusing retrorsine or retrorsine N-oxide (30 μM) in perfusion buffer (2.7 mM KCl, 1.3 mM KH2PO4, 8.1 mM Na2HPO4, 0.9 mM CaCl2, and 0.4 mM MgCl2 with pH 7.2 at 37 °C) with 10 μg/mL phenol red as a non-absorbable marker at 0.3 mL/min. The blood from the mesenteric vein was continuously collected at 5 min intervals for 45 min. Samples were collected from the outflow of the perfusate every 5 min up to 45 min. An aliquot (100 μL) of plasma or perfusate sample was extracted with 200 μL methanol followed by centrifugation at 20,000 g for 10 min. The supernatant was collected for UHPLC-MS/MS analysis.

Fig. 1. Chemical structures and names of five representative retronecine-type PAs (A), their corresponding PA N-oxides (B), and two representative otonecine-type PAs (C).

Molyneux et al., 2011; Williams et al., 2011). We previously determined that some of the herbal samples ingested by HSOS patients contained PA N-oxides as the sole or predominant form of PAs, and for the first time demonstrated PA N-oxide-induced HSOS in humans (Yang et al., 2017). We also revealed that the hepatotoxic potency of a PA Noxide was significantly lower than that of its corresponding PA, which could be partially attributed to its remarkably less oral absorbability with lower Cmax and delayed Tmax (Yang et al., 2017). However, previous toxicokinetic studies on PA N-oxides focused mainly on their metabolic conversion to PAs (Powis et al., 1979; Yan et al., 2008), the oral absorption of PA N-oxides was largely unknown. The present study aimed to determine and compare the oral absorbability and absorption mechanism of PAs and PA N-oxides. The results would provide scientific evidence from the toxicokinetic point of view to understand the differences in toxic potency between PAs and PA N-oxides.

2.4. Caco-2 monolayer model Caco-2 cells (ATCC) at passage 45–55 were cultured and seeded on 2

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twelve-well Transwell plates (Corning®) at a density of 1 × 105 cells/ well and cultured for 21–26 days as previously described (Ma et al., 2011). The bi-directional transport study was carried out following the previously reported method (Ma et al., 2011) using PAs or PA N-oxides at 50 μM. Samples were collected at 20, 40, 60, 80 and 100 min after incubation with PAs or PA N-oxides. Inhibition study was conducted after the cells were pre-incubated with verapamil (100 μM) for 15 min following with the same protocol of bi-directional transport study. Only monolayer with Trans-epithelial electrical resistance (TEER) above 350 Ωcm2 before and after the transport study was employed (Hubatsch et al., 2007). The integrity and function of Caco-2 monolayers used in the bi-directional transport assay were validated using two marker compounds, propranolol, and atenolol, as transcellular and paracellular transport marker respectively. In addition, as a marker for the quality of monolayer, TEER value was also monitored before and after the bi-direction transportation assay to confirm the integrity of the Caco-2 monolayer during the entire experiment. The results confirmed the integrity and function of the Caco-2 monolayer model used in the present study and all PAs and PA N-oxides tested did not affect TEER value of the cell monolayer during 100 min exposure (data not shown).

where Concp and Vp represent the plasma concentration of retrorsine or retrorsine N-oxide and volume of the plasma collected, Kb/p (whole blood to plasma partition coefficient) of retrorsine and retrorsine Noxide were determined as 0.80 and 0.82 respectively using the previously reported protocol (Ma et al., 2011) at 2 μM. The appearance of retrorsine or retrorsine N-oxide in the blood (Pblood) was calculated using the following equations:

Pblood =

where dX/dt is the rate of retrorsine or retrorsine N-oxide appeared in mesenteric blood in steady state, r is the radius of the perfused intestine, which was reported to be 0.18 cm (Cummins et al., 2003), l is the length of the perfused intestine, and C0 is the initial concentration of retrorsine or retrorsine N-oxide. Fraction absorbed (Fa) was calculated according to the equation:

Fa = 1

Recovery =

Agilent 6460 Triple Quadrupole LC/MS system was employed for the analysis of PAs and PA N-oxides according to our previously reported method (Ruan et al., 2014, 2012; Yang et al., 2017; Zhu et al., 2018). Chromatographic separation was achieved on an Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm). The mobile phase comprising 0.1% aqueous formic acid (A) and acetonitrile (B) was used with a gradient elution as follows: 0–4.5 min, 15–22.5% B; 4.5–4.6 min, 22.5–85% B; 4.6–6 min, 85% B. The flow rate was 0.4 mL/min. The injection volume was 2 μL. The optimal multiple reaction monitoring (MRM) methods for individual analytes are listed in Supplementary Table S1.

3.1. Absorption of PAs and PA N-oxides in rats and in situ single pass intestine perfusion model Alkaloid extracts of roots of PA N-oxide-containing Gynura pseudochina and PA-containing Gynura segetum were used to compare the absorption difference of PAs and PA N-oxides in rats. The contents of PAs and PA N-oxides in these two alkaloid extracts were determined by UHPLC-MS/MS analysis. The Gynura pseudochina alkaloid extract contained mainly 2 PA N-oxides, seneciphylline N-oxide (0.18 mmol/g extract) and senecionine N-oxide (0.21 mmol/g extract). The Gynura segetum alkaloid extract contained only the two corresponding PAs, seneciphylline (0.53 mmol/g extract) and senecionine (0.50 mmol/g extract). As shown in Fig. 2A, after oral treatment of these two alkaloid extracts, plasma concentrations of total PAs and PA N-oxides were remarkably higher in rats orally dosed with PA-containing alkaloid extract than that dosed with PA N-oxide-containing alkaloid extract within 0–1 h absorption phase. The results indicated a significantly more extensive in vivo absorption in rats after oral administration of PAs compared to their corresponding PA N-oxides at the same molar dose. This finding provided evidence to explain that significantly less absorption after PA N-oxides dosing might be one of the major contributors to the significant difference of toxicokinetics between PAs and PA N-oxides and the less hepatotoxic potency caused by PA N-oxides, which were discovered in our previous findings (Yang et al., 2017). The absorption difference between PAs and PA N-oxides was further confirmed in the rat in situ single pass intestine perfusion model by using the selected one pair of representative PA and PA N-oxide, retrorsine and retrorsine N-oxide. The cumulative amounts of retrorsine in mesenteric blood were higher than those of retrorsine N-oxide within the 0–45 min perfusion period (Fig. 2B) and the calculated permeability (Pblood) of retrorsine was also significantly higher (Fig. 2C). Based on the calculated Pblood values, the fraction absorbed (Fa) in rats of retrorsine and retrorsine N-oxide was about 40% and 18% respectively by a

dC / dT × V A × C0

where dC/dT is the initial slope of the plot of cumulative concentrations versus time; V is the volume of receiver chamber (0.5 mL for the apical side and 1.5 mL for the basolateral side); A is the apparent surface area of the monolayer (1.12 cm2 for the 12-well Transwell plate); C0 is the initial concentration in donor site. The efflux ratio was calculated as follows (Hubatsch et al., 2007): / Papp

AtoB

where Papp BtoA is the Papp from basolateral side to apical side; Papp AtoB is the Papp from apical side to basolateral side. The percentage of recovery was calculated from the following equation (Hubatsch et al., 2007): Recovery % = (Qr + Qd) / C0 × Vd where Qr is the final amount in the receiver side (μmol); Qd is the final amount in the donor side (μmol), Vd is the volume of donor chamber (ml); C0 is the initial concentration in donor site (μmol/ml). 2.6.2. Data analysis for in situ intestine perfusion model The amount of retrorsine or retrorsine N-oxide was calculated by:

Amount =

Css × Tss × Q+ A cum × 100% C0 × Tss×Q

3. Results and discussion

2.6.1. Data analysis for Caco-2 monolayer model The apparent permeability coefficient (Papp) was calculated as follows (Hubatsch et al., 2007):

BtoA

Pblood × 2 rl/Q int

where Acum is the cumulative amount of retrorsine or retrorsine N-oxide in the mesenteric blood, Q is the flow rate of perfusate, Tss is the time of steady state, Css is the concentration of retrorsine or retrorsine N-oxide in the intestine lumen in steady state, and C0 is the concentration of retrorsine or retrorsine N-oxide at the start of perfusion (in the syringe).

2.6. Data analysis

Efflux Ratio = Papp

e

where r is the radius of rat intestine, l is the length of rat small intestine, which was reported as 150 cm (Kwon, 2002), Qint is the flow rate of the rat intestinal fluid, which was reported as 0.0017 mL/s (Kwon, 2002). The recovery was calculated as:

2.5. UHPLC-MS/MS analysis

papp =

dX/dt 2 rlC0

Concp × Vp × Kb/ p (1 Hc) 3

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Table 1 The permeability, recovery and efflux ratio of PAs and PA N-oxides (50 μM) in Caco-2 monolayer model.

Retronecine-type Senecionine Senecionine N-oxide Seneciphylline Seneciphylline Noxide Retrorsine Retrorsine N-oxide Riddelliine Riddelliine N-oxide Lycopsamine Lycopsamine Noxide Otonecine-type Senkirkine Clivorine

Papp (A to B) (10−6 cm/s)

Recovery (%)

16.26 ± 2.21 0.72 ± 0.17*** 15.82 ± 2.46 1.35 ± 0.45***

99.0 ± 16.3 85.6 ± 7.2 102.3 ± 4.8 105.2 ± 4.0

0.77 3.41 0.83 1.23

± ± ± ±

0.03 0.72 0.04 0.45

3.36 0.33 3.00 0.94 1.43 0.64

98.8 ± 4.4 101.7 ± 0.5 99.2 ± 3.7 100.9 ± 8.3 94.7 ± 3.2 99.9 ± 3.8

1.60 2.31 1.24 1.48 0.92 2.68

± ± ± ± ± ±

0.10 0.85 0.08 0.35 0.46 1.62

92.9 ± 1.9 97.4 ± 4.9

1.30 ± 0.48 0.94 ± 0.04

± ± ± ± ± ±

0.29 0.09*** 0.11 0.01*** 0.32 0.29***

5.06 ± 1.07### 7.05 ± 1.85

(A to B)

Efflux ratio

***p < 0.001, significant difference compared with the corresponding PAs. ### p < 0.001, significant difference compared with senecionine. Data are presented as mean ± SD (n = 3).

physiologically based method (Kwon, 2002). The recovery was 94.8 ± 7.1% for retrorsine and 93.9 ± 7.0% for retrorsine N-oxide, and no metabolites were detected in both the blood and perfusate, indicating no significant metabolism during the perfusion. The results further confirmed that the absorbability of retrorsine N-oxide was significantly lower than that of retrorsine. 3.2. Different permeability and transport mechanism of PAs and PA Noxides in Caco-2 monolayer model In order to systematically investigate the absorption difference and the underlying mechanisms between PAs and PA N-oxides, Caco-2 monolayer model was applied to test permeability and transport mechanism of five pairs of retronecine-type PAs (retrorsine, seneciphylline, senecionine, riddelliine and lycopsamine), their corresponding PA N-oxides, and two otonecine-type PAs (clivorine and senkirkine). Papp values of all PAs and PA N-oxides were analyzed by the linear appearance rate of the analytes on the receiver side, with all fulfilling the sink condition. The results (Table 1) demonstrated a moderate to good absorption range of all PAs with relatively higher Papp AtoB values (1.43–16.26) × 10−6 cm/s, while a poor to moderate absorption of all PA N-oxides with Papp AtoB values lower than 1.35 × 10−6 (Artursson et al., 2001; Artursson and Karlsson, 1991). The recovery of all PAs and PA N-oxides in permeability assay was above 85%, giving an acceptable accuracy for the Papp values (Hubatsch et al., 2007). Comparing each pair of PA and PA N-oxide, significant absorption difference between PAs and their corresponding PA N-oxides was identified and further conformed that all PAs had remarkably higher permeability than their corresponding N-oxide form. To further evaluate the potential involvement of efflux transportation in the absorption, bi-directional transport assay on Caco-2 monolayer model was conducted to obtain efflux ratios. The results found that among all PAs and PA N-oxides tested, 3 PA N-oxides, senecionine N-oxide, retrorsine N-oxide, and lycopsamine N-oxide, had efflux ratios higher than 2.0 (Table 1), suggesting that efflux transportation might be involved in the absorption of these 3 PA N-oxides and most likely in the absorption of senecionine N-oxide, because its efflux ratios were all high than 2.0 (ranging from 2.58 in three independent experiments), while for retrorsine N-oxide and lycopsamine N-oxide, their efflux ratios obtained from triplicated experiments were around 2.0 ranging from 1.37 to 1.16, respectively, indicating a marginal effect of active transportation if involved (International Transporter Consortium et al.,

Fig. 2. Plasma concentrations of total PAs and PA N-oxides in rats orally treated with PA-containing Gynura segetum and PA N-oxide-containing Gynura pseudochina alkaloid extracts at 0.2 mmol PA or PA N-oxides/kg (A). Permeability of retrorsine and retrorsine N-oxide (30 μM) in rat in situ single-pass intestine perfusion model: cumulative amount of retrorsine and retrorsine N-oxide in mesenteric blood (B), and permeability of retrorsine and retrorsine N-oxide (C). *p < 0.05, **p < 0.01 compared with PA N-oxide group.

4

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transportation could also significantly reduce the absorbability of some PA N-oxides is unknown and warranted for further study. In addition, the absorbability between retronecine-type and otonecine-type PAs was also compared. For instance, senecionine (retronecine-type) and senkirkine (otonecine-type) had the same necic acid but different structures of necine base (Fig. 1), while, the absorbability (Table 1) of senecionine was significantly higher but its log P value (Fig. 3A) was remarkably lower than that of senkirkine, indicating that comparing different types of PAs, necine base might play more important role than lipophilicity in determining absorbability of PAs. Therefore, the results demonstrated that the developed correlation between lipophilicity (log P value) and permeability of PAs and PA Noxides could be used with cautions mainly for the prediction of absorbability of retronecine-type PAs and their N-oxides. Further investigations on the establishment of permeability-structure relationship and the prediction of absorbability between retronecine-type and otonecine-type PAs are warranted.

Table 2 Effect of verapamil on the efflux transportation of senecionine N-oxide. Papp (×10−6 cm/s)

Senecionine N-oxide Senecionine N-oxide + verapamil (0.1 mM)

Efflux Ratio

A to B

B to A

0.72 ± 0.17 1.11 ± 0.06***

2.39 ± 0.08 1.16 ± 0.43***

3.41 ± 0.72 1.05 ± 0.42***

***p < 0.001, significant difference compared with the senecionine N-oxide group in the absence of verapamil. Data are presented as mean ± SD (n = 3).

2010; Hubatsch et al., 2007; Sun et al., 2008). Therefore, further investigation to confirm the efflux transport mechanism was conducted for senecionine N-oxide by co-incubated with verapamil, a typical inhibitor of efflux transporters, especially P-gp (Wandel et al., 1999). The results showed that verapamil completely inhibited the efflux of senecionine N-oxide, by reversing its efflux ratio from 3.41 to 1.05 (Table 2), demonstrating that senecionine N-oxide was a substrate of certain efflux transporters, most likely P-gp, and indeed underwent efflux transporter-mediated active transportation during its oral absorption. However, because verapamil has also been reported to modulate other transporters such as multidrug resistance-associated protein 1 (Loe et al., 2000), further studies are required to identify the involved transport(s) and delineate the detailed mechanism underlying the active transportation of senecionine N-oxide. For these 3 PA N-oxides, passive diffusion was also believed to be their main absorption mechanism, while efflux transporter might be involved in their absorption process but to a much less extent, as evidenced by very low efflux ratios (mean value: 2.31–3.41) (Hubatsch et al., 2007). On the other hand, all other PAs and PA N-oxides had the efflux ratio less than 1.60 and Papp −6 cm/s (Table 1), demonstrating passive AtoB larger than 0.94 × 10 diffusion as their primary absorption mechanism without an involvement of efflux transportation (Artursson et al., 2001; Hodgson, 2010). All the in vivo, in situ and in vitro data suggested that PAs had a higher absorbability than that of the corresponding PA N-oxides. Further study was to identify reasons responsible for the significant different absorbability between PAs and PA N-oxides. As shown in Fig. 3A, with a consideration of the structure-related lipophilicity, a good correlation between lipophilicity (which is represented by log P value calculated by XLOGP3 calculator from PubChem) and permeability of all PAs and PA N-oxides was observed, and a much better correlation was obtained between retronecine-type PAs and PA Noxides with only passive diffusion in their absorption (Fig. 3B). These correlations suggested that the lower absorbability of PA N-oxides was possibly due to the lower lipophilicity of PA N-oxides. However, whether the involvement of efflux transporter-mediated active

4. Conclusion PA N-oxides are less toxic than their corresponding PAs in both animals and humans (Mattocks, 1971; Yang et al., 2017). Our study suggested that the significantly lower intestinal absorption of PA Noxides should be one of the causes of lower hepatotoxicity of PA Noxides in vivo. The absorption difference between PAs and PA N-oxides was mainly due to their differences in the lipophilicity and the involvement of efflux transportation during the absorption of some PA Noxides. The present study explained why the significantly less absorption of PA N-oxide was one, yet the key and significant one, of the contributors to cause the significantly less toxic potency of PA N-oxides than that of the corresponding PAs, and also provided a good reference and scientific base for a better prediction of the toxicity of different PAs and PA N-oxides. Author contributions Ge Lin conceived and planned the study. Mengbi Yang and Jiang Ma conducted experiments and performed data analysis. Jianqing Ruan and Chunyuan Zhang contributed to the in situ single pass intestine perfusion study. Ge Lin, Mengbi Yang, and Jiang Ma wrote the manuscript. Peter Pi-Cheng Fu and Yang Ye synthesized PA N-oxides and provided critical review of this manuscript. Declaration of competing interest All authors declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. This article is not an official U.S. Food and Drug Administration (FDA)

Fig. 3. Correlation between lipophilicity (log P) and Papp AtoB of all PAs and PA N-oxides tested (A) and of retronecine-type PAs and PA N-oxides with absorption via passive diffusion (B). Log P Data were calculated by XLOGP3 calculator from PubChem. Abbreviations: RET, retrorsine; SEP, seneciphylline; SEN, senecionine; RID, riddelliine; CLV, clivorine; SEK, senkirkine; LYC, lycopsamine; RET-N, retrorsine N-oxide; SEP-N, seneciphylline N-oxide; SEN-N, senecionine N-oxide; RID-N, riddelliine N-oxide; LYC-N, lycopsamine N-oxide. 5

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guidance or policy statement. No official support or endorsement by the U.S. FDA is intended or should be inferred.

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