Gancao (Glycyrrhizae Radix) provides the main contribution to Shaoyao-Gancao decoction on enhancements of CYP3A4 and MDR1 expression via pregnane X receptor pathway in vitro
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Chinese herbal formula Shaoyao Gancao decoction (SGD) is often used as an adjuvant with chemotherapeutic agents to treat cancer. Due to the herb-drug interactions, the alternations of drug metabolic enzyme and drug transporters induced by SGD deserve to be explored. We aimed to investigate the effect of SGD on the pregnane X receptor (PXR)-mediated transcriptional regulation of cytochrome P450 3A4 (CYP3A4) and drug transporter multidrug resistance protein 1 (MDR1) in vitro. Besides, we assessed the contribution of constituent herbs to SGD on the regulation of CYP3A4 and MDR1.
The dual luciferase reporter gene system containing the hPXR expression plasmid and the reporter gene plasmid of CYP3A4 or MDR1 was co-transfected to HepG2 and Caco2 cells. Luciferase activities were determined using a Dual-luciferase reporter assay kit. The gene expression of CYP3A4 and MDR1 in the hPXR-transfected LS174T cells were assessed by real-time qPCR. Finally, the contribution of constituent herbs from SGD was evaluated.
SGD, Shaoyao and Gancao concentration-dependently increased promoter activities of CYP3A4 and MDR1 in vitro. Moreover, SGD, Shaoyao and Gancao up-regulated CYP3A4 and MDR1 mRNA in hPXR-transfected LS174T cells. As the herbal constituent of SGD, Gancao possesses significantly higher levels of metabolic enzyme and drug transporters compared with Shaoyao.
SGD tends to enhance CYP3A4 and MDR1 expression via PXR pathway, especially Gancao provides the main contribution. This study highlights a potential in vitro mechanism for SGD on the regulation of drug metabolic enzymes and drug transporters.
KeywordsShaoyao Gancao decoction Pregnane X receptor Cytochrome P450 3A4 Multidrug resistance protein 1 Traditional Chinese medicine
Dulbecco’s modified eagle medium
fetal bovine serum
Multidrug resistance protein 1
Pregnane X receptor
Shaoyao Gancao decoction
Ultra-performance liquid chromatography
Chinese herbal medicine becomes the main adjuvant combined with chemotherapy to improve cancer patients’ quality of life [1, 2]. An overall prevalence of herbal formulas use is estimated from 13 to 63% among cancer patients in the United States . Herbal medicine can enhance the response rate or chemosensitivity, reduce side-effects of chemotherapy, and prolong the survival time of cancer patients [4, 5, 6]. To date, a number of herbs and herbal formulas as an adjuvant have been reported to be beneficial to cancer patients, such as PHY906 (Huangqin Tang) , Liu Jun Zi Tang (Rikkunshito in Japanese) , and Shaoyao Gancao decoction (shakuyaku-kanzo-to in Japanese) .
Shaoyao Gancao decoction (SGD), a classical analgesic prescription, is composed of Shaoyao (Paeoniae Radix Alba) and Gancao (Glycyrrhizae Radix et Rhizoma) in the ratio of 1:1. It is originated from the Treatise on Febrile Diseases, and is widely used in Asia to relieve menstrual pain, muscle spasm, and muscle pain [10, 11]. In Japan and China, doctors prefer to prescribe SGD with chemotherapeutic agent paclitaxel to relieve paclitaxel-induced myalgia and arthralgia [9, 12], and painful peripheral neuropathy [13, 14]. However, in view of the narrow therapeutic window of chemotherapeutic agents, this concomitant use increases the risk of clinically relevant herb–antineoplastic agent interactions . While reducing the toxic side effects of paclitaxel, SGD also increases the metabolism of paclitaxel . Our previous study has proved that pretreatment with SGD for 14 consecutive days significantly decreases the area under the curve and increases the total clearance of intravenous paclitaxel in rats . Most known herb-drug interactions are due to changes in metabolic routes, which is related to altered expression or functionality of drug metabolic enzymes and/or drug transporters [3, 16, 17]. Therefore, it is quite necessary to evaluate effects of SGD on drug metabolic enzymes and drug transporters.
Cytochrome P450 (CYP450) occupies an important role in the metabolism and detoxification process of drug and other endo-or xeno-biotics. The largest fraction of the CYP450 CYP3A4, representing 40% of the total hepatic and 80% of the total intestinal CYPs [18, 19], is involved in the metabolism of approximately 50% of marketed drugs . Drug transporter multidrug resistance protein 1 (MDR1) encodes P-glycoprotein, an efflux pump, and then plays an important role in the absorption and presystemic elimination of many xenobiotics . Furthermore, the regulation of CYP3A4 and MDR1 is mediated primarily through the activation of nuclear xenobiotic pregnane X receptor (PXR) [22, 23]. The ethanol extracts of Shaoyao or Gancao have been shown to activate human PXR and induce CYP3A4 reporter constructs in HepG2 cells . The aqueous extracts of Shaoyao or Gancao have been reported to induce P- glycoprotein-mediated drug transport [23, 25, 26]. Therefore, we speculate that the mechanism underlying the interaction from SGD might be related to regulation of CYP3A4 and/or MDR1 expression via PXR pathway.
Rifampicin (RIF), pregnenolone 16α-carbonitrile(PCN), phenobarbital (PB), dimethyl sulfoxide (DMSO), diethyl pyrocarbonate (DEPC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RIF, PCN, and PB were dissolved in DMSO at concentrations appropriate for the specific studies in which they were used. Trypsin and Taq DNA polymerase were purchased from Gibco BRL (Grand Island, NY, USA). Reverse transcription system, dual-luciferase reporter assay system, pGL4.17-Luc and pGEM-T constructs were purchased from Promega (Madison, WI, USA).
Preparation of SGD, SY and GC
Shaoyao (Paeoniae Radix Alba, the root of Paeonia lactiflora Pall.) and Gancao (Glycyrrhizae Radix et Rhizoma, the root and rhizome of Glycyrrhiza uralensis Fisch.) were purchased from Xiangya Hospital of Central south university (Changsha, China). The crude herbs were authenticated by Prof. SY Hu, Department of Chinese Herbal Medicine of Central South University. Voucher specimens (No. 20140505 and No. 20140506) were deposited at the Laboratory of Ethnopharmacology in Xiangya Hospital. Crude drugs of SGD (SY: GC, 1:1, w/w) 200 g were immersed in 400 ml distilled water for 30 min and boiled for 40 min, then filtered. The residue was boiled and filtered once more. The filtrates from each time were combined and centrifuged at 3000 rpm for 30 min at room temperature. The supernatant was filtered through a 0.22-μm filter. The concentration of the original SGD was adjusted to 500 mg crude drug per milliliter, and then diluted into 0.1, 1, 2 mg/ml. SY (Paeoniae Radix Alba 100 g) or GC (Glycyrrhizae Radix et Rhizoma 100 g) was immersed in 400 ml distilled water, and processed as SGD. The concentration of the original SY and GC was adjusted to 250 mg crude drug per milliliter, and then diluted into 0.05, 0.5, 1 mg/ml. Decoctions were maintained at 4 °C for further use.
Meanwhile, six key compounds (gallic acid, albiflorin, paeoniflorin, glycyrrhizin, benzoic acid and liquiritigenin) from SGD for quality control using a Waters Acquity ultra-performance liquid chromatography (UPLC) system have been reported in our previous studies [27, 28, 29]. Among the six compounds, gallic acid, albiflorin, paeoniflorin and benzoic acid are for SY, while glycyrrhizin and liquiritigenin are for GC.
Three cell lines were used in this study including HepG2 (human Caucasian hepatocellular carcinoma), Caco2 (human colon adenocarcinoma) and LS174T (human colon adenocarcinoma). Three cell lines were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The HepG2 line was maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose medium (HyClone), Caco2 and LS174T lines were maintained in DMEM/F12 medium (HyClone), at 37 °C in a humidified atmosphere containing 5% CO2. All media were supplemented with 10% fetal bovine serum (FBS, Gibco), added with 100 U/ml penicillin and 100 U/ml streptomycin (Beyotime, Shanghai, China). The medium was replaced every 3 days.
The expression plasmid for the human PXR receptor pcDNA3.1-PXR was kindly provided by Prof. Guo Wang (Institute of Clinical Pharmacology, Central South University, Changsha, China), containing the full-length human PXR .
The CYP3A4-Luc reporter constructs containing the basal promoter (− 362 ~ + 53 bp) with proximal PXR response element and the distal xenobiotic responsive enhancer module (− 7836 ~ − 7208 bp) of the CYP3A4 gene 5′-flanking region inserted to pGL4.17-Basic reporter vector has been reported previously . The construction of MDR1-Luc reporter gene vector has been described previously [21, 30]. Human MDR1 promoter fragment (− 7975 ~ − 7013 bp) containing the cluster of xenobiotic-responsive enhancer modules was amplified by PCR with primers 5′- TCTGCTAGCAGTGTTTCTTGT-3′, containing a natural NheI site, and 5′-AATCTCGAGCATATAAGGCAACTGTTTTGT-3′, introducing an XhoI site. The NheI/XhoI-digested PCR fragment was ligated between the NheI/XhoI sites of pGL4.17-Basic luciferase reporter vector.
Cell transient transfections
Transient transfections of HepG2 and Caco2 cells were performed as described previously . In brief, prior to cell transfection for 24 h, HepG2 or Caco2 cells were seeded in 24-well and 6-well plates at a density of 1 × 105 cells per well. Transfections were performed in triplicate using 600 ng CYP3A4/MDR1-Luc, 100 ng pcDNA3.1-PXR (or 100 ng pcDNA3.1), 10 ng internal control PRL-SV40, and 5 μl lipofectamine 2000 (Invitrogen) in a well. Four to six hours after transfection, HepG2 cells were washed in DMEM, replaced with DMEM + 10% FBS medium and treated with drugs 24 h later, while Caco2 cells were washed in DMEM/F12, replaced with DMEM/F12 + 10% FBS medium and treated with drugs 24 h later.
LS174T cells (1.2 × 105 per well) were seeded into 24-well and 6-well plates and cultivated for 24 h. Then LS174T cells were transfected with 100 ng/well PXR expression plasmids or pcDNA3.1 vector or weren’t transfected. Four to six hours after transfection, LS174T cells were washed in DMEM/F12, replaced with DMEM/F12 + 10% FBS medium and treated with drugs 24 h later.
Western blot analysis
After transfection, we firstly detected expression of PXR protein in non-transfected, vector-transfected, and PXR-transfected cells by western blot. As for HepG2 or Caco2 cells in 6-well plates, we set non-transfected cells as the blank group, cells transfected with 600 ng pGL4.17-CYP3A4 (or MDR1) + 100 ng pcDNA3.1 + 10 ng PRL-SV40 as the vector group, cells transfected with 600 ng pGL4.17-CYP3A4 (or MDR1) + 100 ng pcDNA3.1-PXR + 10 ng PRL-SV40 as the trans-PXR group. As for LS174T cells in 6-well plates, we set non-transfected cells as the blank group, cells transfected with 100 ng pcDNA3.1 + 10 ng PRL-SV40 as the vector group, cells transfected with 100 ng pcDNA3.1-PXR + 10 ng PRL-SV40 as the trans-PXR group.
Western blot analysis was performed according to the standard protocol. Following 48 h transfection, transfected cells were washed with cold phosphate buffer saline once and lysed using 50 μl radioimmunoprecipitation assay lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. After 30 min on ice, lysates were clarified by centrifugation at 12000 × rpm for 15 min at 4 °C. Amounts of protein 50–100 μg were loaded on a 10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes. Following a blocking incubation with TBST containing 5% non-fat dried milk at room temperature for 2 h, membranes were incubated at 4 °C overnight with primary antibody (1:1000 dilution) to PXR (rabbit, no. 15607–1-AP, Proteintech) or β-actin (mouse, no. 60008–1-Ig, Proteintech), followed by incubation at room temperature for 1 h with the goat anti-mouse or anti-rabbit horseradish peroxidase conjugated secondary antibody (1,3000 dilution; cat no. ab97051, Proteintech). The membranes were analyzed using ECL detection reagent (Thermo). β-actin was used as an internal control. All experiments were conducted in triplicate.
Herbal treatment in HepG2 or Caco2 cells and detecting luciferase activity
Co-transfected HepG2 or Caco2 cells were treated with rifampicin (10 μM), PCN (10 μM), SGD (0.1, 1, and 2 mg/ml), SY (0.05, 0.5, and 1 mg/ml), GC (0.05, 0.5, and 1 mg/ml), or the solvent control (0.1% DMSO) for 24 h, respectively. Vector control (VC) group was transfected with pcDNA3.1-PXR + PRL-SV40 + pGL4.17 (or pcDNA3.1 + PRL-SV40 + pGL4.17-CYP3A4) and treated with 0.1% DMSO. The blank group was co-transfected with pcDNA3.1-PXR + PRL-SV40 + pGL4.17-CYP3A4 and treated with nothing. The firefly luciferase activity was detected using the dual-luciferase reporter assay (Promega) according to the manufacturer’s protocol. The Renilla luciferase activity was used as an internal control. Each experiment was repeated three times in triplicate. The efficiency of transfection in each treatment groups was adjusted by the ratio of firefly luciferase activity to Renilla luciferase activity.
Real-time qPCR analysis of CYP3A4 and MDR1 mRNA in LS174T
To determine CYP3A4 and MDR1 mRNA induction, LS174T cells were transfected with 100 ng/well PXR expression plasmids or pcDNA3.1 vector or weren’t transfected. Following 24 h transfection, appropriate cell samples were exposed to phenobarbital (1 mM), SGD (1 mg/ml), SY (1 mg/ml), GC (1 mg/ml) or the solvent control (0.1% DMSO) for 48 h, respectively. Then cells were harvested and the mRNA levels of CYP3A4, MDR1 were measured by real-time qPCR.
Primer sequences used for real-time qPCR
All data are expressed as means ± S.E.M of three independent experiments performed in triplicates. The data were first tested for the normality of distribution with Shapiro-Wilk test. The distribution of samples is approximately normal. Differences between groups were analyzed using a one-way ANOVA with Welch correction, followed by the Games Howell test. All statistical analyses were performed using SPSS 23.0 (International Business Machines Corp., Armonk, NY, USA). Values of p < 0.05 are considered to be statistically significant.
SGD and its constituent herbs significantly enhance the CYP3A4 promoter activities via hPXR in HepG2 and Caco2 cells
However, in both transfected HepG2 and Caco2 cells, PCN treatment doesn’t cause a significant increase in CYP3A4 luciferase activities when compared with the DMSO group. Previous studies have found rifampicin activates human but not rodent PXR, while PCN activates rodent but not human PXR [33, 34]. Consistent with the previous report , similar activation of hPXR and inductions of CYP3A4 and MDR1 by rifampicin are observed in HepG2, Caco-2 cells, except PCN.
SGD and its constituent herb GC markedly elevate the MDR1 promoter activities via hPXR in HepG2 and Caco2 cells
Then, we examined whether the three decoctions induce the activation of MDR1-Luc reporter constructs through hPXR in HepG2 and Caco2 cells. We successfully transfected PXR-MDR1 plasmid constructs in HepG2 and Caco2 cells (Fig. 2). Twenty-four hours following transfection, HepG2 and Caco2 cells were separately treated with rifampicin (10 μM, hPXR agonist), PCN (10 μM, rPXR agonist), SGD (0.1, 1, and 2 mg/ml), SY (0.05, 0.5, and 1 mg/ml) and GC (0.05, 0.5, and 1 mg/ml) for 24 h, detected dual luciferase activity.
In a word, in HepG2 and Caco2 cells, SGD and its constituent prescription GC significantly increase promoter activities of CYP3A4 and MDR1 via PXR in a concentration-dependent manner. While SY doesn’t significantly increase promoter activities of MDR1 in transfected Caco2 cells. Like rifampicin, 1 mg/ml GC is a strong agonist of PXR, followed by SGD, and SY ranges the weakest.
SGD and its constituent herbs upregulate expression of CYP3A4 and MDR1 mRNA in LS174T via PXR
Chinese herbal medicine has been shown to have synergistic actions with chemotherapy and reduce their side effects . An overall prevalence of combined utilization is estimated up to 63% among cancer patients in the United States . However, with the increasing co-administration of herbal medicine with chemotherapy, herb-drug interactions have become an important issue in clinical practice. It has been found that traditional herbal formulas cause clinically-relevant interactions and affect the metabolism and pharmacokinetics of chemotherapeutic agents [3, 36, 37]. In the previous study, we observed that pretreatment with SGD for 14 days significantly altered pharmacokinetics of paclitaxel involved in decreasing the area under the curve and increasing the total clearance of intravenous paclitaxel in rats . In general, the reduction in area under the curve and increase of the clearance after co-administration is due to the induction of the metabolic enzyme of drugs and/or the drug transporters . However, studies of SGD on drug metabolic enzymes and drug transporters are rare so far. Therefore, it is urgent to evaluate effects of SGD on drug metabolic enzymes and drug transporters and to uncover the underlying molecular mechanism.
The CYP450 enzyme system is responsible for the biotransformation of more than 90% of the drugs on the market . The inducers of CYP450 can speed up the metabolism of substrates and drugs, including most chemotherapeutic drugs . And the largest fraction of the P450 reactions is catalyzed by CYP3A4 , which is mainly distributed in the liver and intestine and metabolizes at least 50% of marketed pharmaceutical agents . MDR1 encodes P-glycoprotein, an important transmembrane efflux pump protein, to limit the absorption of drugs and contribute to the excretion of drugs in the intestine . The induction of many CYPs and drug transporters occurs by a similar mechanism, where ligand activation of PXR . PXR could be activated by ligands and translocate from the cytoplasm to the nucleus . Then activated PXR binds to specific promoter responsive elements of many xenobiotic enzymes and transporter genes as a heterodimer or heterotetramer with the retinoid X receptor (RXR), thus, induces the transcription [40, 41, 42]. Here, we established SGD and its constituent herbs (SY and GC) as natural potent agonists of PXR and proved that they induce the transcription of intestinal and hepatic CYP3A4 and MDR1 via PXR activation in vitro. Those in vitro results might partially account for the pharmacokinetics alteration of drugs via SGD in vivo.
SGD, an antispasmodic herbal formula, is consisted of Shaoyao (Paeoniae Radix Alba) and Gancao (Glycyrrhizae Radix et Rhizoma). It is widely used in Asia to treat muscle cramps, many kinds of pain, abdominal spasmodic pain, and dysmenorrhea . The previous study has shown that the combination of Shaoyao and Gancao might work “synergistically” to improve the efficacy or reduce the toxicity of individual components . The contribution of constituent herbs should also be assessed. In the present study, the induction of CYP3A4 and MDR1 by SY and GC is in accordance with previous reports [25, 26, 44, 45]. The present study also showed the induction of CYP3A4 and MDR1 via Gancao was much higher than Shaoyao. That indicates Gancao is the main contributor to SGD on the induction of CYP3A4 and MDR1. Excess of SGD or Gancao can also lead to a failure of most drugs since they enhance the drug metabolizing enzymes which could lead to a rapid removal of the drugs before its therapeutic action. SGD or Gancao should be administrated with caution.
In most cases, it is not good for PXR to be activated. Conversely, the activation of PXR by herbs may have favorable effects. Recent studies have revealed much more complex regulatory pathways governed by PXR, such as lipid and glucose metabolism, bile acid metabolism and inflammation . PXR activation prevents lithocholic acid-induced hepatotoxicity, suggesting that PXR agonists may be useful in the treatment of human cholestatic liver disease . A strong inducer of PXR St. John’s wort significantly affects serum concentrations of irinotecan, a chemotherapeutic agent, consequently reducing the antineoplastic effect and toxicity of this CYP3A4 substrate . Overall, these studies highlight the dual features of PXR activation: promoting drug metabolism leads to serious potential drug interactions and treatment failures, and activation of the detoxification system helps protect our body from invading of toxic substances. Therefore, when prescribing herbs with agents, it is necessary to monitor the plasma concentration of agents to guarantees effectiveness and reduce toxicity.
The limitations of the present study are clear, and further explorations in the future are demanded. Firstly, our study only focuses on the activation of PXR. Beyond PXR, more nuclear receptors, such as CAR, are needed to be studied in the future. Besides, we illustrate the up-regulation of SGD on CYP3A4 and MDR1 via PXR activation, we didn’t evaluate effects of active compounds from SGD.
This study firstly observed SGD induce intestinal and hepatic CYP3A4 and MDR1 promoter and enhance mRNA expression via activating PXR pathway in vitro. And Gancao plays a predominant role in the induction effects. This study highlights a potential of SGD on the PXR-mediated regulation of drug metabolic enzymes and drug transporters.
We would like to thank Professor Guo Wang (Institute of Clinical Pharmacology, Central South University, Changsha, China) and his colleagues for their kindly gift of the human PXR expression plasmid constructs.
This work was supported by the Natural Science Foundation of China (Grant Nos. 81202781, 81403259, 81774322, 81673719 and 81473573), China Postdoctoral Science Foundation (No. 2017 T100614) and the Fundamental Research Funds for the Central Universities (2012QNZT110).
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DF, HC, YW, PG designed the study, conducted experiments, analyzed the data and prepared the manuscript. TT, RF, JL reviewed and corrected the manuscript. All authors read and approved the final manuscript.
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- 10.Wang Y, Xu C, Wang P, Lin X, Yang Y, Li D, Li H, Wu X, Liu H. Pharmacokinetic comparisons of different combinations of Shaoyao-Gancao-decoction in rats: simultaneous determination of ten active constituents by HPLC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2013;932:76–87.CrossRefGoogle Scholar
- 19.Lim YP, Ma CY, Liu CL, Lin YH, Hu ML, Chen JJ, Hung DZ, Hsieh WT, Huang JD. Sesamin: a naturally occurring Lignan inhibits CYP3A4 by antagonizing the Pregnane X receptor activation. Evid Based Complement Alternat Med 2012; 2012:242810.Google Scholar
- 22.Burk O, Arnold KA, Nussler AK, Schaeffeler E, Efimova E, Avery BA, Avery MA, Fromm MF, Eichelbaum M. Antimalarial artemisinin drugs induce cytochrome P450 and MDR1 expression by activation of xenosensors pregnane X receptor and constitutive androstane receptor. Mol Pharmacol. 2005;67(6):1954–65.CrossRefGoogle Scholar
- 23.Mu Y, Zhang J, Zhang S, Zhou HH, Toma D, Ren S, Huang L, Yaramus M, Baum A, Venkataramanan R, et al. Traditional Chinese medicines Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch) activate pregnane X receptor and increase warfarin clearance in rats. J Pharmacol Exp Ther. 2006;316(3):1369–77.CrossRefGoogle Scholar
- 27.Gan PP, Zhong MZ, Huang X, Sun M, Wang Y. Determination of six major compounds in Shaoyao-Gancao-Tang and its single herb decoctions. Asian J Chem. 2011;23(4):1515–9.Google Scholar
- 28.Gan PP, Huang X, Zhong MZ, Sun M, Qin F, Zhang CH. Simultaneous determination of eight major constituents in the traditional Chinese medicine Shaoyao-Gancao - Tang by UPLC-PDA. J Med Plants Res. 2010;4(24):2615–21.Google Scholar
- 42.Cerveny L, Svecova L, Anzenbacherova E, Vrzal R, Staud F, Dvorak Z, Ulrichova J, Anzenbacher P, Pavek P. Valproic acid induces CYP3A4 and MDR1 gene expression by activation of constitutive androstane receptor and pregnane X receptor pathways. Drug Metab Dispos. 2007;35(7):1032–41.CrossRefGoogle Scholar
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