Impact of COVID-19 and Nationwide Lockdowns on Vegetable Prices: Evidence from Wholesale Markets in China.

In this paper, we employ a combination of time regression discontinuity design method (T-RD) and the difference-in-difference method (DID) to identify and quantify the causal effects of the strict lockdown policy on vegetable prices using multiple-year daily price data from 151 wholesale markets of Chinese cabbage. We find that the lockdown policy caused a large and immediate surge in price and price dispersion of Chinese cabbage, though they fluctuated smoothly for the same period in normal years. The DID results further show that the price surge peaked in the fourth week of lockdown but gradually came down to the level of a normal year by week 11. However, the price rose again (though to a much smaller extent) in response to the resurgence of COVID-19 in a few provinces in early-mid April but quickly returned to the normal level in week 15 when the lockdown measures were largely removed. We also find that the supply chain disruption is the driving factor for the price hike. Policy implications are drawn.

Pyrrole-Hemoglobin Adducts, a More Feasible Potential Biomarker of Pyrrolizidine Alkaloid Exposure.

Pyrrolizidine alkaloids (PAs) are naturally occurring phytotoxins widely distributed in about 3% of flowering plants. The formation of PA-derived pyrrole-protein adducts is considered as a primary trigger initiating PA-induced hepatotoxicity. The present study aims to (i) further validate our previous established derivatization method using acidified ethanolic AgNO3 for the analysis of pyrrole-protein adducts and (ii) apply this method to characterize the binding tendency, dose-response, and elimination kinetics of pyrrole-protein adducts in blood samples. Two pyrrole-amino acid conjugates, (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP)-cysteine (7-cysteine-DHP) and 9-histidine-DHP, were synthesized and used to demonstrate that acidified ethanolic AgNO3 derivatization can cleave both S-linkage and N-linkage of pyrrole-protein adducts. Subsequently, using precolumn AgNO3 derivatization followed by ultra-high-pressure liquid chromatography/mass spectrometry analysis, we quantified pyrrole-protein adducts in monocrotaline-treated rat blood protein fractions, including hemoglobin (Hb), plasma, albumin, and plasma residual protein fractions, and found that the amount of pyrrole-Hb adducts was significantly higher than that in all plasma fractions. Moreover, elimination half-life of pyrrole-Hb adducts was also significantly longer than pyrrole-protein adducts in plasma fractions (12.08 vs 2.54-2.93 days). In addition, we also tested blood samples obtained from five PA-induced liver injury patients and found that the amount of pyrrole-protein adducts in blood cells was also remarkably higher than that in plasma. In conclusion, our findings for the first time confirmed that the AgNO3 derivatization method could be used to measure both S- and N-linked pyrrole-protein adducts and also suggested that pyrrole-Hb adducts with remarkably higher level and longer life span could be a better biomarker of PA exposure.

Intestinal and hepatic biotransformation of pyrrolizidine alkaloid N-oxides to toxic pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are among the most significant groups of phytotoxins present in more than 6000 plants in the world. Hepatotoxic retronecine-type PAs and their corresponding N-oxides usually co-exist in plants. Although PA-induced hepatotoxicity is known for a long time and has been extensively studied, the toxicity of PA N-oxide is rarely investigated. Recently, we reported PA N-oxide-induced hepatotoxicity in humans and rodents and also suggested the association of such toxicity with metabolic conversion of PA N-oxides to the corresponding toxic PAs. However, the detailed biochemical mechanism of PA N-oxide-induced hepatotoxicity is largely unknown. The present study investigated biotransformation of four representative cyclic retronecine-type PA N-oxides to their corresponding PAs in both gastrointestinal tract and liver. The results demonstrated that biotransformation of PA N-oxides to PAs was mediated by both intestinal microbiota and hepatic cytochrome P450 monooxygenases (CYPs), in particular CYP1A2 and CYP2D6. Subsequently, the formed PAs were metabolically activated predominantly by hepatic CYPs to form reactive metabolites exerting hepatotoxicity. Our findings delineated, for the first time, that the metabolism-mediated mechanism of PA N-oxide intoxication involved metabolic reduction of PA N-oxides to their corresponding PAs in both intestine and liver followed by oxidative bioactivation of the resultant PAs in the liver to generate reactive metabolites which interact with cellular proteins leading to hepatotoxicity. In addition, our results raised a public concern and also encouraged further investigations on potentially remarkable variations in PA N-oxide-induced hepatotoxicity caused by significantly altered intestinal microbiota due to individual differences in diets, life styles, and medications.

First evidence of pyrrolizidine alkaloid N-oxide-induced hepatic sinusoidal obstruction syndrome in humans.

Pyrrolizidine alkaloids (PAs) are among the most potent phytotoxins widely distributed in plant species around the world. PA is one of the major causes responsible for the development of hepatic sinusoidal obstruction syndrome (HSOS) and exerts hepatotoxicity via metabolic activation to form the reactive metabolites, which bind with cellular proteins to generate pyrrole-protein adducts, leading to hepatotoxicity. PA N-oxides coexist with their corresponding PAs in plants with varied quantities, sometimes even higher than that of PAs, but the toxicity of PA N-oxides remains unclear. The current study unequivocally identified PA N-oxides as the sole or predominant form of PAs in 18 Gynura segetum herbal samples ingested by patients with liver damage. For the first time, PA N-oxides were recorded to induce HSOS in human. PA N-oxide-induced hepatotoxicity was further confirmed on mice orally dosed of herbal extract containing 170 µmol PA N-oxides/kg/day, with its hepatotoxicity similar to but potency much lower than the corresponding PAs. Furthermore, toxicokinetic study after a single oral dose of senecionine N-oxide (55 µmol/kg) on rats revealed the toxic mechanism that PA N-oxides induced hepatotoxicity via their biotransformation to the corresponding PAs followed by the metabolic activation to form pyrrole-protein adducts. The remarkable differences in toxicokinetic profiles of PAs and PA N-oxides were found and attributed to their significantly different hepatotoxic potency. The findings of PA N-oxide-induced hepatotoxicity in humans and rodents suggested that the contents of both PAs and PA N-oxides present in herbs and foods should be regulated and controlled in use.

The Presystemic Interplay between Gut Microbiota and Orally Administered Calycosin-7-O-ß-D-Glucoside.

Presystemic interactions with gut microbiota might play important roles in the holistic action of herbal medicines in their traditional oral applications. However, research interests usually focus on biologic activities of the in vivo available herb-derived components and their exposure in circulation. In this study, we illustrated the importance of studying the presystemic interplay with gut microbiota for understanding the holistic actions of medicinal herbs by using calycosin-7-O-ß-D-glucoside (C7G), the most abundant flavonoid and chemical marker in Astragali Radix, as a model compound. When C7G was orally administrated to rats, calycosin-3'-O-glucuronide (G2) was the major circulating component in the blood together with a minor calycosin but not C7G. Rat gut microbiota hydrolyzed C7G in vitro rapidly and produced its aglycone calycosin. Calycosin exhibited higher permeability than C7G and further underwent extensive glucuronidation to yield 3'-glucuronide as the dominant metabolite. Bioactivity assays revealed that G2 exhibited similar or more potent proangiogenic effects than calycosin in human umbilical vein endothelial cells in vitro and in the vascular endothelial growth factor receptor tyrosine kinase inhibitor II-induced blood vessel loss model in zebrafish. More interestingly, the incubation of C7G with gut microbiota from both normal and colitic rats showed a probiotics-like effect through stimulating the growth of the beneficial bacteria Lactobacillus and Bifidobacterium. In conclusion, C7G interacts reciprocally with gut microbiota after oral dosing, which makes it not only an angiogenic prodrug but also a modulator of gut microbiota.

Proteomic study of pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome in rats.

Pyrrolizidine alkaloids (PAs) are a group of phytotoxins that can induce human liver injury, particularly hepatic sinusoidal obstruction syndrome (HSOS). To date, the molecular targets of PA-induced HSOS are largely unknown. In this study, retrorsine (RTS), a known hepatotoxic PA, was used as a representative PA for proteomic studies. Toxicological assessment demonstrated that 35 mg/kg RTS (designated as RTS-L) caused early lesions of HSOS at 24 h after dosing. A proteomic approach revealed 17 up-regulated and 31 down-regulated proteins in RTS-L-treated rats. Subsequently, bioinformatic analysis suggested that two proteins, carbamoyl-phosphate synthase (CPS1) (p < 0.05) and ATP synthase subunit beta (ATP5B) (p < 0.01) were associated with RTS-L intoxication. Using immunohistochemical staining, we further verified the down-regulation of CPS1 and ATP5B in RTS-L-treated rats. These findings indicated that CPS1 and ATP5B were altered in the RTS-induced early lesions of HSOS in rats, and therefore, these two proteins and their involved pathways might play important roles in the initiation of HSOS. To the best of our knowledge, our study using a proteomic approach combined with conventional toxicological assessment is the first systems toxicology study on PA-induced HSOS. The results of this study provide novel findings on protein profiles in response to PA exposure, which can serve as a starting point to further investigate potential protein targets and their interactions with PAs to induce HSOS.

Blood Pyrrole-Protein Adducts--A Biomarker of Pyrrolizidine Alkaloid-Induced Liver Injury in Humans.

Pyrrolizidine alkaloids (PAs) induce liver injury (PA-ILI) and is very likely to contribute significantly to drug-induced liver injury (DILI). In this study we used a newly developed ultra-high performance liquid chromatography-triple quadrupole-mass spectrometry (UHPLC-MS)-based method to detect and quantitate blood pyrrole-protein adducts in DILI patients. Among the 46 suspected DILI patients, 15 were identified as PA-ILI by the identification of PA-containing herbs exposed. Blood pyrrole-protein adducts were detected in all PA-ILI patients (100%). These results confirm that PA-ILI is one of the major causes of DILI and that blood pyrrole-protein adducts quantitated by the newly developed UHPLC-MS method can serve as a specific biomarker of PA-ILI.

Lack of metabolic activation and predominant formation of an excreted metabolite of nontoxic platynecine-type pyrrolizidine alkaloids.

Pyrrolizidine alkaloid (PA) poisoning is well-known because of the intake of PA-containing plant-derived natural products and PA-contaminated foodstuffs. Based on different structures of the necine bases, PAs are classified into three types: retronecine, otonecine, and platynecine type. The former two type PAs possessing an unsaturated necine base with a 1,2-double bond are hepatotoxic due to the P450-mediated metabolic activation to generate reactive pyrrolic ester, which interacts with cellular macromolecules leading to toxicity. With a saturated necine base, platynecine-type PAs are reported to be nontoxic and their nontoxicity was hypothesized to be due to the absence of metabolic activation; however, the metabolic pathway responsible for their nontoxic nature is largely unknown. In the present study, to prove the absence of metabolic activation in nontoxic platynecine-type PAs, hepatic metabolism of platyphylline (PLA), a representative platynecine-type PA, was investigated and directly compared with the representatives of two toxic types of PAs in parallel. By determining the pyrrolic ester-derived glutathione conjugate, our results confirmed that the major metabolic pathway of PLA did not lead to formation of the reactive pyrrolic ester. More interestingly, having a metabolic rate similar to that of toxic PAs, PLA also underwent oxidative metabolisms mediated by P450s, especially P450 3A4, the same enzyme that catalyzes metabolic activation of two toxic types of PAs. However, the predominant oxidative dehydrogenation pathway of PLA formed a novel metabolite, dehydroplatyphylline carboxylic acid, which was water-soluble, readily excreted, and could not interact with cellular macromolecules. In conclusion, our study confirmed that the saturated necine bases determine the absence of metabolic activation and thus govern the metabolic pathway responsible for the nontoxic nature of platynecine-type PAs.

Metabolic activation of pyrrolizidine alkaloids: insights into the structural and enzymatic basis.

Pyrrolizidine alkaloids (PAs) are natural toxins widely distributed in plants. The toxic potencies of different PAs vary significantly. PAs are mono- or diesters of necine acids with a necine base. On the basis of the necine bases, PAs are classified into three types: retronecine-type, otonecine-type, and platynecine-type. Hepatotoxic PAs contain an unsaturated necine base. PAs exert hepatotoxicity through metabolic activation by hepatic cytochromes P450s (CYPs) to generate reactive intermediates which form pyrrole-protein adducts. These adducts provide a mechanism-based biomarker to assess PA toxicity. In the present study, metabolic activation of 12 PAs from three structural types was investigated first in mice to demonstrate significant variations in hepatic metabolic activation of different PAs. Subsequently, the structural and enzymatic factors affecting metabolic activation of these PAs were further investigated by using human liver microsomes and recombinant human CYPs. Pyrrole-protein adducts were detected in the liver and blood of mice and the in vitro systems treated with toxic retronecine-type and otonecine-type PAs having unsaturated necine bases but not with a platynecine-type PA containing a saturated necine base. Retronecine-type PAs produced more pyrrole-protein adducts than otonecine-type PAs with similar necine acids, demonstrating that the structure of necine base affected PA toxic potency. Among retronecine-type PAs, open-ring diesters generated the highest amount of pyrrole-protein adducts, followed by macrocyclic diesters, while monoesters produced the least. Only CYP3A4 and CYP3A5 activated otonecine-type PAs, while all 10 CYPs studied showed the ability to activate retronecine-type PAs. Moreover, the contribution of major CYPs involved also varied significantly among retronecine-type PAs. In conclusion, our findings provide a scientific basis for predicting the toxicities of individual PAs in biological systems based on PA structural features and on the pattern of expression and the selectivity of the CYP isoforms present.

Do Long-Term Natural Disasters Influence Social Trust? Empirical Evidence from China.

The natural environment is one of the most critical factors that profoundly influences human races. Natural disasters may have enormous effects on individual psychological characteristics. Using China's long-term historical natural disaster dataset from 1470 to 2000 and data from a household survey in 2012, we explore whether long-term natural disasters affect social trust. We find that there is a statistically significant positive relationship between long-term natural disaster frequency and social trust. We further examine the impact of long-term natural disaster frequency on social trust in specific groups of people. Social trust in neighbors and doctors is stronger where long-term natural disasters are more frequent. Our results are robust after we considering the geographical difference. The effect of long-term natural disasters remains positively significant after we divide the samples based on geographical location. Interestingly, the impact of long-term flood frequency is only significant in the South and the impact of long-term drought frequency is only significant in the North.

Space Power in Inclusive Development: Industrial Clusters and Rural Anti-Poverty.

Poverty seriously hinders the inclusive development of mankind and is closely related to economic growth, ecological protection, ecological restoration and sustainable use of resources. Based on the data of economic census and rural fixed observation point, a spatial econometric model is established to test the direct impact and spatial spillover effect of industrial clusters on rural poverty alleviation. The result of household-level is that the number of industrial clusters has a negative effect on poverty, namely the farmers who live in the county with more industrial clusters, may be less likely to become the poor. The number of industrial clusters in other regions also has a negative effect on poverty. By dividing farmers into the poverty and non-poverty group, the study finds that, for the poverty group, the number of industrial clusters has a positive direct and spillover effect on farmers' income. For the non-poverty group, the number of local industrial clusters has a positive direct effect on farmers' income, but the number of industrial clusters in other regions does not have any effects or has a negative direct effect on farmers' income. By classifying the industries, the study discovers that the labor-intensive industrial clusters, such as textiles, manufacture and processing of machinery parts and paper industries, have a positive effect on farmers' income.

Human Organic Anion Transporting Polypeptides 1B1, 1B3, and 2B1 Are Involved in the Hepatic Uptake of Phenolsulfonphthalein.

Phenolsulfonphthalein (PSP or phenol red), a sulfonphthalein dye, has been used as a diagnostic agent and a pH indicator in cell culture medium. After administered into the body, PSP is excreted into urine and bile. The urinary excretion of PSP is mediated by organic anion transporter 1/3 (OAT1/3) and multidrug resistance protein 2 (MRP2). In biliary excretion, PSP is effluxed from hepatocytes into the bile via MRP2. However, so far, the molecular mechanism for PSP transport from the blood into hepatocytes is unclear. In the present study, six human major hepatic uptake transporters expressed on the basolateral membrane of hepatocytes, namely, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, Na+/taurocholate cotransporting polypeptide (NTCP), organic cation transporter 1 (OCT1), and OAT2, have been investigated to see whether they are involved in the hepatic uptake of PSP. An in vitro cell-based study demonstrated that PSP is a substrate for OATP1B1, OATP1B3, and OATP2B1, with OATP1B3 showing the highest transport efficiency. The K m values for OATP1B1-, OATP1B3-, and OATP2B1-mediated PSP uptake were 11.3 ± 1.5, 7.0 ± 1.5, and 5.1 ± 1.0 µM, respectively. PSP interacts with known OATP substrates/inhibitors. However, the presence of PSP in cell culture medium has no significant effect on OATP's function. In vivo pharmacokinetic study in wild-type and Oatp1b2-knockout mice showed that Oatp1b2-knockout led to elevated plasma concentration and decreased liver accumulation of PSP. Taken together, the present study showed that in the liver, OATP1B1, OATP1B3, and OATP2B1 are involved in the uptake of PSP from the blood into hepatocytes, which, along with MRP2-mediated efflux of PSP from hepatocytes into the bile, constitute the vectorial transport of PSP from the blood to the bile and may play a critical role in the biliary excretion of PSP.

Do Long-Run Disasters Promote Human Capital in China? -The Impact of 500 Years of Natural Disasters on County-Level Human-Capital Accumulation.

Is there a relationship between the frequency of regional natural disasters and long-term human-capital accumulation? This article investigates the long-run causality between natural calamities and human-capital accumulation with macro and micro data. Empirical cross-county analysis demonstrates that higher frequencies of natural calamities are correlated with higher rates of human-capital accumulation. Specifically, on the basis of empirical data of the fifth census in 2000 and China's Labor-Force Dynamics Survey in 2012, this paper exploits the two databases to infer that the high disaster frequency in the years of 1500-2000 was likely to increase regional human-capital accumulation on district level. High natural-calamity frequency reduces the expected rate of returning to physical capital, which also serves to increase human-capital. Thus, experiencing with natural disasters would influence human's preference to human-capital investment instead of physical capital.

Polyplexes by Polymerized Dequalinium and Bifunctional Aptamer for Mitochondrial Targeting Drug Release to Overcome Drug Resistance.

Drug resistance is one of the major obstacles to the success of cancer chemotherapy. Mitochondrial targeting drugs are increasingly thought to be able to eradicate resistant cancer cells. However, immature drug release outside mitochondria and the absence of multifunctional targeting carriers against tumor mitochondria greatly limit the corresponding therapeutic benefits. Here, we synthesized polymerized dequalinium by integrating dequalinium, lysine, and poly(ethylene glycol) for mitochondrial targeting. The polymerized dequalinium exhibited lower cytotoxicity and stronger gene condensing ability than free dequalinium. We designed AS1411-ATP fusion aptamer to load doxorubicin (DOX) for both tumor targeting and ATP-responsive DOX release. The polyplexes by polymerized dequalinium and bifunctional aptamer can target tumor cells via AS1411 and show improved stability, mitochondrial targeting, DOX release in response to mitochondrial ATP, and enhanced apoptosis-inducing effect on DOX-resistant MCF-7/DOX cells. The present study highlights a promising application of the polyplexes in reversing drug resistance in tumor cells via tumor mitochondrial targeting drug release.

Importance of OATP1B1 and 1B3 in the Liver Uptake of Luteolin and Its Consequent Glucuronidation Metabolites.

Luteolin is a typical flavonoid and broadly distributed in the plants. Oral bioavailability of luteolin is low owing to extensive metabolism. Regioselective glucuronidation by UDP-glucuronosyltransferases (UGTs) and liver uptake by organic anion transporting polypeptides (OATPs) of luteolin and consequent glucuronidation metabolites were studied. Luteolin-3'-O-glucuronide (L-3'-G) and luteolin-7-O-glucuronide (L-7-G) were the major metabolites in human liver microsomes. Further study demonstrated that UGT1A9 played a predominant role in the glucuronidation of luteolin. Transporter study showed that OATP1B1- and 1B3-transfected cells selectively uptake L-3'-G into cells but not luteolin or L-7-G. After intravenous administration of luteolin to mice, the area under the curve of L-3'-G in the plasma was the highest among luteolin, L-3'-G, and L-7-G. In the liver, the concentration of L-3'-G was significantly greater than L-7-G. In conclusion, OATP1B1 and OATP1B3 play an important role in the liver disposition of luteolin and its glucuronidation metabolites.

Suitability evaluation on material specifications and edible methods of Dendrobii Officinalis Caulis based on holistic polysaccharide marker.

BACKGROUND: Dendrobii Officinalis Caulis (DC) is a well-known tonic herbal medicine worldwide and has favorable immunomodulatory activity. Various material specifications of DC are available in herbal markets, and DC is ingested by different edible methods. However, whether these specifications and edible methods are suitable or not remains unknown. METHODS: In this study, we evaluated the suitability of four material specifications (fresh stem, dried stem, fengdou and powder) and three edible methods (making tea, soup and medicinal liquor) based on holistic polysaccharide marker (HPM), the major polysaccharide components in DC. First, the HPMs were extracted from the four specifications of DC by the three edible methods in different conditions. Second, qualitative and quantitative characterization of the extracted HPMs was performed using high performance gel permeation chromatography (HPGPC). Third, immunomodulatory activities of the extracted HPMs were evaluated in vivo. RESULTS: The results showed that the HPMs were found to be quantitatively different from various specification of DC and edible methods. In vivo analysis indicated that the HPMs exerted positive effects on innate immune responses by increment in proliferation of splenocytes, secretion of IL-2 and cytotoxicity activity of NK cells. Moreover, the dosage amount of HPM should be defined as a certain range, but not the larger the better, for exerting strong immunological activities. CONCLUSION: According to the both chemical and biological results, fengdou by boiling with water for 4 h is the most recommended specification and edible method for DC.

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

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 AtoB values lower than 1.35 × 10-6 cm/s. A further mechanistic study demonstrated that except for senecionine N-oxide, retrorsine N-oxide, and lycopsamine N-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.

UGT-dependent regioselective glucuronidation of ursodeoxycholic acid and obeticholic acid and selective transport of the consequent acyl glucuronides by OATP1B1 and 1B3.

Ursodeoxycholic acid (UDCA) is a major effective constituent of bear bile powder, which is widely used as function food in China and is documented in the Chinese pharmacopoeia as a traditional Chinese medicine. UDCA has been developed as the only accepted therapy by the US FDA for primary biliary cholangitis. Recently, the US FDA granted accelerated approval to obeticholic acid (OCA), a semisynthetic bile acid derivative from chenodeoxycholic acid, for primary biliary cholangitis. However, some perplexing toxicities of UDCA have been reported in the clinic. The present work aimed to investigate the difference between UDCA and OCA in regard to potential metabolic activation through acyl glucuronidation and hepatic accumulation of consequent acyl glucuronides. Our results demonstrated that the metabolic fates of UDCA and OCA were similar. Both UDCA and OCA were predominantly metabolically activated by conjugation to the acyl glucuronide in human liver microsomes. UGT1A3 played a predominant role in the carboxyl glucuronidation of both UDCA and OCA, while UGT2B7 played a major role in their hydroxyl glucuronidation. Further uptake studies revealed that OATP1B1- and 1B3-transfected cells could selectively uptake UDCA acyl glucuronide, but not UDCA, OCA, and OCA acyl glucuronide. In summary, the liver disposition of OCA is different from that of UDCA due to hepatic uptake, and liver accumulation of UDCA acyl glucuronide might be related to the perplexing toxicities of UDCA.

Interaction between rhein acyl glucuronide and methotrexate based on human organic anion transporters.

Rhein, a major bioactive compound of many medicinal herbs and the prodrug of diacerein, is often used with low dose of methotrexate as drug combination to treat rheumatoid arthritis. In this study, potential drug-drug interaction between methotrexate and rhein was investigated based on organic anion transporters (OAT). Our study demonstrated that rhein acyl glucuronide (RAG), the major metabolite of rhein in the human blood circulation, significantly inhibited the uptake of p-aminohippurate in hOAT1 transfected cells with IC50 value of 691 nM and estrone sulfate uptake in hOAT3 transfected cells with IC50 value of 78.5 nM. As the substrate of both hOAT1 and hOAT3, the methotrexate transport was significantly inhibited by RAG in hOAT1 transfected cells at 50 µM and hOAT3 transfected cells at 1 µM by 69% and 87%, respectively. Further in vivo study showed that after co-administrated with RAG in rats the AUC0-24 values of methotrexate increased from 3109 to 5370 ng/mL*hr and the t1/2 was prolonged by 40.5% (from 7.4 to 10.4 h), demonstrating the inhibitory effect of RAG on methotrexate excretion. In conclusion, rhein acyl glucuronide could significantly decrease the transport of methotrexate by both hOAT1 and hOAT3. The combination use of rhein, diacerein or other rhein-containing herbs with methotrexate may cause obvious drug-drug interaction and require close monitoring for potential drug interaction in clinical practice.

Cytotoxicity of pyrrolizidine alkaloid in human hepatic parenchymal and sinusoidal endothelial cells: Firm evidence for the reactive metabolites mediated pyrrolizidine alkaloid-induced hepatotoxicity.

Pyrrolizidine alkaloids (PAs) widely distribute in plants and can cause hepatic sinusoidal obstruction syndrome (HSOS), which typically presents as a primary sinusoidal endothelial cell damage. It is well-recognized that after ingestion, PAs undergo hepatic cytochromes P450 (CYPs)-mediated metabolic activation to generate dehydropyrrolizidine alkaloids (DHPAs), which are hydrolyzed to dehydroretronecine (DHR). DHPAs and DHR are reactive metabolites having same core pyrrole moiety, and can bind proteins to form pyrrole-protein adducts, which are believed as the primary cause for PA-induced HSOS. However, to date, the direct evidences supporting the toxicity of DHPAs and DHR in the liver, in particular in the sinusoidal endothelial cells, are lacking. Using human hepatic sinusoidal endothelial cells (HSEC) and HepG2 (representing hepatic parenchymal cells), cells that lack CYPs activity, this study determined the direct cytotoxicity of dehydromonocrotaline, a representative DHPA, and DHR, but no cytotoxicity of the intact PA (monocrotaline) in both cell lines, confirming that reactive metabolites mediate PA intoxication. Comparing with HepG2, HSEC had significantly lower basal glutathione (GSH) level, and was significantly more susceptible to the reactive metabolites with severer GSH depletion and pyrrole-protein adducts formation. The toxic potency of two reactive metabolites was also compared. DHPA was more reactive than DHR, leading to severer toxicity. In conclusion, our results unambiguously provided the first direct evidence for the critical role of DHPA and DHR in the reactive metabolites-mediated PA-induced hepatotoxicity, which occurs predominantly in HSEC due to severe GSH depletion and the significant formation of pyrrole-protein adducts in HSEC.