Developmental toxicity induced by chelerythrine in zebrafish embryos via activating oxidative stress and apoptosis pathways.

Chelerythrine (CHE), a natural benzophenanthridine alkaloid, possesses various biological and pharmacological activities, such as antimicrobial, antitumor and anti-inflammatory effects. However, its adverse side effect has not been fully elucidated. Therefore, this study was designed to investigate the developmental toxicity of CHE in zebrafish. We found that CHE could lead to a notably increase of the mortality and malformation rate, while lead to reduction of the hatching rate and body length. CHE also could affect the normal developing processes of the heart, liver and phagocytes in zebrafish. Furthermore, the reactive oxygen species (ROS) and apoptosis levels were notably increased. In addition, the mRNA expressions of genes (bax, caspase-9, p53, SOD1, KEAP1, TNF-α, STAT3 and NF-κB) were significantly increased, while the bcl2 and nrf2 were notably inhibited by CHE. These results indicated that the elevation of ROS and apoptosis were involved in the developmental toxicity induced by CHE. In conclusion, CHE exhibits a developmental toxicity in zebrafish, which helps to understand the potential toxic effect of CHE.

Sanguinarine exposure induces immunotoxicity and abnormal locomotor behavior in zebrafish.

Sanguinarine (C20H14NO4+), a plant alkaloid and pesticide, works well a fungicidal and insecticidal applications. The prospect that sanguinarine may have potentially toxic effects on aquatic organisms has been brought to light by its use in agriculture. The first evaluation of the immunotoxic and behavioral effects of sanguinarine exposure on larval zebrafish was done in this work. Firstly, zebrafish embryos exposed to sanguinarine had shorter body length, larger yolk sacs, and slower heart rates. Secondly, the number of innate immune cells was significantly reduced. Thirdly, alterations in locomotor behavior were observed as exposure concentrations increased. Total distance travelled, travel time, and mean speed were all reduced. We also found significant changes in oxidative stress-related indicators and a significant increase in apoptosis in the embryos. Further studies revealed aberrant expression of some key genes in the TLR immune signaling pathway including CXCL-c1c, IL8, MYD88, and TLR4. At the same time, the expression of the pro-inflammatory cytokine IFN-γ was upregulated. To sum up, our results suggest that sanguinarine exposure may cause immunotoxicity and aberrant behavior in larval zebrafish.

Cardiotoxicity of sanguinarine via regulating apoptosis and MAPK pathways in zebrafish and HL1 cardiomyocytes.

Sanguinarine, a plant phytoalexin, possesses extensive biological activities including antimicrobial, insecticidal, antitumor, anti-inflammatory and anti-angiogenesis effect. But its cardiotoxicity has rarely been studied. Here, we assess the cardiotoxicity of sanguinarine in vivo using larval zebrafish from 48 hpf to 96 hpf. The results show that sanguinarine caused severe malformation and the dysfunction of the heart including reductions of heart rate, red blood cell number, blood flow dynamics, stroke volume and increase of SV-BA distance, subintestinal venous congestion. Further studies showed that apoptosis in the zebrafish heart region was observed after sanguinarine exposure using TUNEL assay and AO staining method. In addition, the genes, such as sox9b, myl7, nkx2.5 and bmp10, which play crucial parts in the development and the function of the heart, were changed after sanguinarine treatment. caspase3, caspase9, bax and bcl2, apoptosis-related genes, were also altered by sanguinarine. Further studies were performed to study the cardiotoxicity in vitro using cardiomyocytes HL1 cell line. The results showed that remarkable increase of apoptosis and ROS level in HL1 cells were induced by sanguinarine. Moreover, the MAPK pathway (JNK and P38) were notably enhanced and involved in the cardiotoxicity induced by sanguinarine. Our findings will provide better understanding of sanguinarine in the toxic effect on heart.

Developmental toxicity caused by sanguinarine in zebrafish embryos via regulating oxidative stress, apoptosis and wnt pathways.

Sanguinarine, derived from the root of Sanguinaria canadensis, have multiple biological activities, such as antimicrobial, insecticidal, antitumor, anti-inflammatory and anti-angiogenesis effect, but little is known about its toxicity on normal embryonic development. Here, we study the developmental toxicity using zebrafish model. Notably, sanguinarine caused a significant increase of the malformation rate and decrease of hatching rates and body length of zebrafish embryos. Sanguinarine also impaired the normal development of heart, liver and nerve system of zebrafish embryos. Further, the ROS level and MDA concentrations were remarkably increased, while the activity of T-SOD was decreased. In addition, obvious increase of apoptosis were observed by AO staining or TUNEL assay. Further studies showed that the oxidative stress-, apoptosis-related genes were changed, while genes of nrf2 and wnt pathways were inhibited by sangunarine. To sum up, our study will be helpful to understand the adverse effect of sanguinarine on embryonic development and the underlying molecular mechanism.

Effects and possible mechanisms of sanguinarine on the competition between Raphidiopsis raciborskii (Cyanophyta) and Scenedesmus obliquus (Chlorophyta): A comparative toxicological study.

The phytogenic algicide sanguinarine shows strong inhibitory effects on some bloom-forming cyanobacteria and exhibits great potential in cyanobacterial bloom mitigation. To evaluate the possible ecological effects of sanguinarine on microalgae, the effects and possible mechanisms of sanguinarine on the competition between bloom-forming cyanobacterium Raphidiopsis raciborskii (formerly named Cylindrospermopsis raciborskii) and green alga Scenedesmus obliquus were investigated through co-culture competition test and comparative toxicological study including growth characteristics, chlorophyll fluorescence transients, activities of antioxidant enzymes, and lipid peroxidation. The results of Raphidiopsis-Scenedesmus co-culture competition test showed that sanguinarine decreased the competition ability of R. raciborskii, which benefitted S. obliquus in winning the competition. Toxicological studies have shown that sanguinarine exhibited high inhibitory effects on the growth and photosynthesis of R. raciborskii but no obvious toxicity on S. obliquus at concentrations of no more than 80 µg L-1. Oxidative damage partially contributed but was not the primary mechanism for the toxicity of sanguinarine on R. raciborskii. The results presented in this study indicate that sanguinarine may be a good algicidal candidate in mitigation of Raphidiopsis-based water bloom.

Sanguinarine caused larval lethality and growth inhibition by suppressing energy metabolism in silkworms, Bombyx mori.

Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05-0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests.

Intracellular Accumulation as an Indicator of Cytotoxicity to Screen Hepatotoxic Components of Chelidonium majus L. by LC-MS/MS.

A novel strategy was developed to identify hepatotoxic compounds in traditional Chinese medicines (TCMs). It is based on the exposure of HL-7702 cells to a TCM extract, followed by the identification and further determination of potential hepatotoxic compounds accumulated in the cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a case study, potential hepatotoxic components in Chelidonium majus L. were screened out. Five alkaloids (sanguinarine, coptisine, chelerythrine, protopine, and chelidonine) were identified by LC-MS/MS within 10 min, and their intracellular concentrations were first simultaneously measured by LC-MS/MS with a run time of 4 min. A cell viability assay was performed to assess the cytotoxicity of each alkaloid. With their higher intracellular concentrations, sanguinarine, coptisine, and chelerythrine were identified as the main hepatotoxic constituents in Ch. majus. The study provides a powerful tool for the fast prediction of cytotoxic components in complex natural mixtures on a high-throughput basis.

Protopine attenuates inflammation stimulated by carrageenan and LPS via the MAPK/NF-κB pathway.

We investigated the anti-inflammatory activity of protopine (PTP) and sought to determine its mechanism of action in LPS-stimulated BV2 cells and a carrageenan (CA)-induced mouse model. Treatment with PTP (5, 10, and 20 µM) significantly suppresses the secretion of NO and PGE2 in a concentration-dependent manner without affecting cell viability by downregulating iNOS and COX-2 expression in LPS-induced BV2 cells. PTP also attenuates the production of pro-inflammatory chemokines, such as MCP-1, and cytokines, including TNF-α, IL-1ß and IL-6, and augments the expression of the anti-inflammatory cytokine IL-10. In addition, PTP suppresses the nuclear translocation of NF-κB by hindering the degradation of IκB and downregulating the expression of mitogen-activated protein kinases (MAPKs), including p38, ERK1/2 and JNK protein. Furthermore, PTP treatment significantly suppresses CA-induced paw oedema in mice compared to that seen in untreated mice. Expression of iNOS and COX-2 proteins is also abrogated by PTP (50 mg/kg) treatment in CA-induced mice. PTP treatment also abolishes IκB phosphorylation, which hinders the activation of NF-κB. Collectively, these results suggest PTP has potential for attenuating CA- and LPS-induced inflammatory symptoms through modulation of MAPKs/NF-κB signaling cascades.

Characterization of the cytotoxicity of selected Chelidonium alkaloids in rat hepatocytes.

Phytomedicinal preparations containing extracts of the plant Chelidonium majus (Greater Celandine) have been used in the therapy of upper abdominal disorders. C. majus alkaloids (CAL) were suspected to be responsible for reported cases of liver symptoms including cases of acute liver failure in patients upon treatment with certain C. majus preparations. Based on these reports, a safe oral daily dose limit of not more than 2.5 mg CAL was established in the EU. However, C. majus extracts and individual CAL were not able to elicit similar adverse effects when given orally to pigs or rats. We found that CAL differ considerably in their cytotoxicity in rat hepatocytes in culture. The cationic congeners chelerythrine, coptisine and sanguinarine were the most toxic ones (EC20 values ≤2 µM) while the neutral congeners chelidonine, dihydrosanguinarine and protopine were less toxic, with a rank order of toxicity of coptisine > chelerythrine > sanguinarine > chelidonine > protopine > dihydrosanguinarine. Calculation of octanol-water partition coefficients revealed that the most cytotoxic CAL in hepatocytes were the cationic polar ones. At cytotoxic concentrations sanguinarine led to a marked decrease in reduced and oxidized intracellular glutathione while the much less cytotoxic dihydrosanguinarine did not. After glutathione depletion with menadione, CAL toxicity was only slightly enhanced. Comparison of the cytotoxic concentrations to reported liver levels in experimental animals suggests that the latter were too low to cause hepatotoxicity, probably due to an extremely low oral availability of certain CAL.

Sanguinarine in Chelidonium majus induced antifeeding and larval lethality by suppressing food intake and digestive enzymes in Lymantria dispar.

Our previous studies had identified that both crude extracts and total alkaloid from Chelidonium majus exerted a significant antifeeding and larval lethality on Lymantria dispar. Moreover, sanguinarine, chelidonine, berberine hydrochloride and coptisine were the main alkaloid in C. majus exerting toxicity to L. dispar. In this paper, we evaluated the insecticidal and antifeeding activities of each alkaloid on the 3rd instar L. dispar larvae by bioassay. Meanwhile, the effects of alkaloids from C. majus on the activities and mRNA levels of three main digestive enzymes in L. dispar larvae were investigated. The results indicated that sanguinarine possessed the strongest insecticidal activity with a LD50 value of 4.963 µg/larva, and the coptisine showed little lethality to 3 rd instar L. dispar larvae among four alkaloids from C. majus. The insecticidal capacity of four alkaloids on 3rd instar L. dispar larvae was in the following decreasing order of sanguinarine > chelidonine > berberine hydrochloride > coptisine. Similarly, except coptisine, the other three alkaloids significantly reduced food intakes of third instar L. dispar larvae and suppressed activities of three digestive enzymes (α-amylase, lipase and total protease) simultaneously. Finally, qRT-PCR analysis revealed that the transcriptions of α-amylase, lipase and serine protease were affected by sanguinarine. Especially, at 48 h after treatment, the mRNA expressions of those digestive enzymes were significantly suppressed by sanguinarine. In conclusion, we suggested that alkaloids from C. majus induced antifeeding and larval lethality on L. dispar larvae by suppressing food intake and digestive enzymes in L. dispar. Our findings provide a novel insight into evaluating the antifeeding and insecticidal properties of C. majus, which afford a new strategy for integrated pest management programs as well.

Assessing the risk of epidemic dropsy from black salve use.

Epidemic dropsy is a potentially life-threatening condition resulting from the ingestion of argemone oil derived from the seeds of Argemone mexicana Linn. Exposure to argemone oil is usually inadvertent, arising from mustard cooking oil adulteration. Sanguinarine, an alkaloid present in argemone oil, has been postulated as a causative agent with the severity of epidemic dropsy correlating with plasma sanguinarine levels. Cases of epidemic dropsy have also been reported following the topical application of argemone containing massage oil. Black salve, a topical skin cancer therapy also contains sanguinarine, but at significantly higher concentrations than that reported for contaminated massage oil. Although not reported to date, a theoretical risk therefore exists of black salve inducing epidemic dropsy. This literature review explores the presentation and pathophysiology of epidemic dropsy and assesses the risk of it being induced by black salve.

Supramolecular formulation of nitidine chloride can alleviate its hepatotoxicity and improve its anticancer activity.

Nitidine chloride (NC) has demonstrated promising anticancer activity. However, NC has also shown non-specific toxicity in various healthy organs such as the liver. In this study, we aimed to develop a supramolecular formulation of NC and investigate the associated benefits of such a supramolecular formulation on modulating its inherent hepatotoxicity and anticancer activity. The formation of NC-cucurbit[7]uil (NC@CB[7]) complexes was characterized by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy, differential scanning calorimetry and powder X-ray diffraction analysis. As a consequence of the supramolecular complexation, NC@CB[7] showed significantly lower toxicity (IC50: 6.87 ± 0.80 µM) on a liver cell line (LO2), and higher cytotoxicity (IC50: 2.94 ± 0.15 µM) on a breast cancer cell line (MCF-7), when compared with the free drug (IC50 of 3.48 ± 0.49 µM and 7.28 ± 0.36 µM, on these two cell lines, respectively). Investigation of cellular uptakes revealed that CB[7]'s capability in modulating the toxicity/activity of NC was mainly attributed to the drug's different cellular uptake behaviors that were influenced by CB[7]'s complexation. Taken together, we have demonstrated that supramolecular formulation of NC by CB[7] significantly alleviated its hepatotoxicity and improved its anticancer activity in vitro.

Role of OCT2 and MATE1 in renal disposition and toxicity of nitidine chloride.

BACKGROUND AND PURPOSE: Nitidine chloride (NC), a benzophenanthridine alkaloid, has various biological properties including anticancer and analgesic activities. The aim of the present study was to evaluate the role of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1) in the renal disposition and nephrotoxicity of NC. EXPERIMENTAL APPROACH: MDCK cells stably expressing human OCT2 and/or hMATE1 were used to investigate the OCT2- and MATE1-mediated transport of NC. In addition, the accumulation of NC and its potential toxicity were studied in rat primary-cultured proximal tubular (rPCPT) cells and in rats in vivo. KEY RESULTS: NC was found to be a high-affinity substrate of both OCT2 and MATE1 with high cytotoxicity in MDCK-hOCT2/hMATE1 and MDCK-hOCT2 compared to mock cells. The OCT2 inhibitors, cimetidine and (+)-tetrahydropalmatine ((+)-THP), significantly reduced NC accumulation and cytotoxicity in MDCK-hOCT2, MDCK-hOCT2/hMATE1 and rPCPT cells. Severe kidney damage with high levels of blood urea nitrogen and lactate dehydrogenase (LDH), reduced levels of alkaline phosphatase (ALP) and pathological changes were found in rats after 20 days of successive i.v. doses of NC (5 mg·kg(-1) ·day(-1) ). Concomitantly, the concentration of NC in the kidney reached similar high levels at 2 h after the last dose of the 20 day treatment as those observed at 0.5 h after a single i.v. dose of 5 mg·kg(-1) . CONCLUSIONS AND IMPLICATIONS: Our data indicate that NC-induced nephrotoxicity might be mainly attributed to OCT2-mediated extensive renal uptake and weak tubular secretion by MATE1.

Nitidine chloride inhibits ovarian cancer cell migration and invasion by suppressing MMP-2/9 production via the ERK signaling pathway.

Nitidine chloride (NC) has been demonstrated to exert anti-tumor effects on various types of tumor. However, no studies have investigated the anti­metastatic effect of NC on ovarian cancer cells, and the underlying mechanisms have not yet been clearly established. The present study aimed to determine the effect of NC on the migration and invasion of ovarian cancer cells. Cell viability and proliferation of ovarian cancer cells were assessed by MTT assay. A scratch wound healing assay and Transwell assays were performed to detect migration and invasion of cells, respectively. The expression levels of matrix metalloproteinase (MMP)­2 and 9 were detected at the mRNA and protein level following stimulation with NC. Subsequently, the expression of mitogen­activated protein kinases was detected by western blot analysis. Finally, an inhibitor of extracellular signal­regulated kinase (ERK) was applied to investigate the effect of NC on the expression of MMP­2/9 as well as the migration and invasion of cells. It was found that NC suppressed the proliferation, migration and invasion of A2780 ovarian cancer cells. NC downregulated MMP­2 and MMP­9 in a dose­ and time­dependent manner. In addition, NC was also able to downregulate phosphorylation of ERK. Furthermore, by applying an ERK inhibitor, U0126, the effect of NC on the expression of MMP-2/9 and inhibition of cell migration and invasion was verified. Taken together, these results demonstrated that NC inhibited the migration and invasion of ovarian cancer cells via the ERK signaling pathway.

Isolation and characterization of the alkaloid Nitidine responsible for the traditional use of Phyllanthus muellerianus (Kuntze) Excell stem bark against bacterial infections.

Phyllanthus muellerianus (Kuntze) Excell (family Euphorbiaceae) stem bark methanol extract inhibited the growth of Clostridium sporogenes and Streptococcus pyogenes, responsible for gas gangrene and suppurative and non suppurative diseases, respectively. After the HPLC fingerprint acquisition a bioguided fractionation of the defatted methanol extract allowed the isolation of six fractions whose activity was evaluated against the two pathogen bacteria. A further purification of the most active fraction afforded a pure compound responsible for the very interesting inhibitory activity against C. sporogenes and S. pyogenes (MIC 0.91 µM, MIC 3.64 µM). (1)H NMR and MS analytical techniques allowed the identification of the bioactive as Nitidine; this quaternary ammonium alkaloid was observed in the genus Phyllanthus for the first time. A study on Nitidine counter ion, performed using energy dispersive spectroscopy (EDS) coupled with scanning electron microscopy (SEM) was also carried out.

Hazardous effects of sanguinarine on maturation of mouse oocytes, fertilization, and fetal development through apoptotic processes.

Previously, we reported that sanguinarine, a phytoalexin with antimicrobial, anti-oxidant, anti-inflammatory and pro-apoptotic effects, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, causing decreased embryonic development and cell viability. In the current study, we investigated the deleterious effects of sanguinarine on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre- and postimplantation development both in vitro and in vivo. Notably, sanguinarine significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with sanguinarine during in vitro maturation induced an increase in postimplantation embryo resorption and a decrease in mouse fetal weight. In an in vivo animal model, 1 to 5 µM sanguinarine, provided in drinking water, caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase-3-specific inhibitor effectively blocked sanguinarine-triggered deleterious effects, clearly implying that embryonic injury induced by sanguinarine is mediated by a caspase-dependent apoptotic mechanism.

The contribution of human OCT1, OCT3, and CYP3A4 to nitidine chloride-induced hepatocellular toxicity.

Nitidine chloride (NC), a quaternary ammonium alkaloid, has numerous pharmacological effects, such as anticancer activity. However, it was found that NC also has hepatocellular toxicity. Because organic cation transporters 1 and 3 (OCT1 and OCT3) might mediate the influx of NC into hepatocytes, multidrug and toxin extrusion 1 (MATE1) probably mediates the efflux of NC from hepatocytes, while cytochrome P450 (P450) enzymes might contribute to NC metabolism, the present study was to evaluate the contribution of OCT1, OCT3, MATE1, and P450 enzymes to NC-induced hepatocellular toxicity. Our results showed that the uptake of NC in Madin-Darby canine kidney (MDCK) cells expressing human (h) OCT1 and OCT3 (MDCK-hOCT1 and MDCK-hOCT3) was significantly higher than that in mock cells; the hOCT1- and hOCT3-mediated uptake followed typical Michaelis-Menten kinetics. Meanwhile, NC was also a substrate of hMATE1, although its transport capacity was much lower than that of OCT1 NC-induced cytotoxicity in MDCK-hOCT1 or MDCK-hOCT3 cells was obviously higher than that in mock cells. Quinidine and (+)-tetrahydropalmatine [(+)-THP], OCT1 and OCT3 inhibitors, significantly reduced the uptake of NC in MDCK-hOCT1 cells, MDCK-hOCT3 cells, and rat primary hepatocytes, but only (+)-THP markedly attenuated the NC-induced toxicity. In addition, P450 enzymes, such as CYP3A4, mediated the metabolism of NC, and NC-induced toxicity in MDCK-hOCT1/hCYP3A4 cells was lower than that in MDCK-hOCT1 cells. Our results indicated that NC is a substrate of hOCT1, hOCT3, and CYP3A4; that OCT1 and OCT3 mediate the uptake of NC in hepatocytes and subsequently cause hepatotoxicity; and that NC-induced toxicity could be attenuated by CYP3A4-mediated metabolism.

NQO1 involves in the imine bond reduction of sanguinarine and recombinant adeno-associated virus mediated NQO1 overexpression decreases sanguinarine-induced cytotoxicity in rat BRL cells.

Although sanguinarine (SANG) can be transformed to dihydrosanguinarine (DHSA) in human and animals, the enzyme involved in the imine bond reduction of SANG is still unknown. In this study, we found that rat NAD(P)H: quinone oxidoreductase 1 expressed by prokaryotic system can transform SANG to DHSA in an NADPH dependent manner. We also found out that there was more DHSA in rAAV-NQO1 infected than rAAV-CYP1A1 and rAAV-control infected BRL cells. SANG decreased rat BRL cell proliferation and augmented cell apoptosis in a time and dose dependent manner. However, the influence of DHSA to BRL cells is not significant difference than SANG. SANG-induced apoptosis was correlated with the up-regulation of Bax/Bcl2 ratio and the down-regulation of Bcl2. SANG can also dose dependently down regulate NQO1 expression, but CYP1A1 expression was a little up regulated. Since CYP1A1 involving in SANG oxidative reactions and NQO1 involving in the transform of SANG to DHSA, we hypothesized that up regulation of NQO1 could reduce SANG cytotoxicity and up regulation of CYP1A1 could increase SANG cytotoxitity. Our further study showed that recombinant adeno-associated virus (rAAV) mediated overexpression of NQO1 significantly increased cell proliferation and decreased Bax/Bcl2 ratio, apoptosis, and cytotoxicity, whereas rAAV mediated CYP1A1 overexpression had opposite effects. These data illustrated that NQO1 involved in the imine bond reduction of sanguinarine and this was a less toxic metabolizing pathway than CYP1A1-metabolizing pathway.