OAT, OATP, and MRP Drug Transporters and the Remote Sensing and Signaling Theory.

The coordinated movement of organic anions (e.g., drugs, metabolites, signaling molecules, nutrients, antioxidants, gut microbiome products) between tissues and body fluids depends, in large part, on organic anion transporters (OATs) [solute carrier 22 (SLC22)], organic anion transporting polypeptides (OATPs) [solute carrier organic (SLCO)], and multidrug resistance proteins (MRPs) [ATP-binding cassette, subfamily C (ABCC)]. Depending on the range of substrates, transporters in these families can be considered multispecific, oligospecific, or (relatively) monospecific. Systems biology analyses of these transporters in the context of expression patterns reveal they are hubs in networks involved in interorgan and interorganismal communication. The remote sensing and signaling theory explains how the coordinated functions of drug transporters, drug-metabolizing enzymes, and regulatory proteins play a role in optimizing systemic and local levels of important endogenous small molecules. We focus on the role of OATs, OATPs, and MRPs in endogenous metabolism and how their substrates (e.g., bile acids, short chain fatty acids, urate, uremic toxins) mediate interorgan and interorganismal communication and help maintain and restore homeostasis in healthy and disease states.

Oleanolic acid induces hepatic injury by disrupting hepatocyte tight junction and dysregulation of farnesoid X receptor-mediated bile acid efflux transporters.

Oleanolic acid (OA) is a naturally occurring pentacyclic triterpene compound that has been reported to cause cholestatic liver injury. However, the regulation and pathogenic role of bile acids in OA-induced development of cholestatic liver injury remains largely unclear. Farnesoid X receptor (FXR) is a metabolic nuclear receptor that plays an important role in bile acid homeostasis in the liver by regulating efflux transporters bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2). The aim of this study was to investigate the effect of OA on hepatocyte tight junction function and determine the role of FXR, BSEP, and MRP2 in the mechanism of impairment of transport of bile acids induced by OA. Both in vivo and in vitro models were used to characterize the OA-induced liver injury. The liquid chromatography-tandem mass spectrometry (LC-MS) was employed to characterize the efflux function of the transporters, and the results showed that OA caused a blockage of bile acids efflux. OA treatment resulted in decreased expression levels of the tight junction proteins zonula occludens-1 and occludin. Immunofluorescence results showed that OA treatment significantly reduced the number of bile ducts and the immunofluorescence intensity. Pretreatment with agonists of FXR and MRP2, respectively, in animal experiments attenuated OA-induced liver injury, while pretreatment with inhibitors of BSEP and MRP2 further aggravated OA-induced liver injury. These results suggest that OA inhibits FXR-mediated BSEP and MRP2, leading to impaired bile acid efflux and disruption of tight junctions between liver cells, resulting in liver damage.

Cyclosporine-A induced cytotoxicity within HepG2 cells by inhibiting PXR mediated CYP3A4/CYP3A5/MRP2 pathway.

Cyclosporine-A (CsA) is currently used to treat immune rejection after organ transplantation as a commonly used immunosuppressant. Liver injury is one of the most common adverse effects of CsA, whose precise mechanism has not been fully elucidated. Pregnane X receptor (PXR) plays a critical role in mediating drug-induced liver injury as a key regulator of drug and xenobiotic clearance. As a nuclear receptor, PXR transcriptionally upregulates the expression of drug-metabolizing enzymes and drug transporters, including cytochrome P4503A (CPY3A) and multidrug resistance-associated protein 2 (MRP2). Our study established CsA-induced cytotoxic hepatocytes in an in vitro model, demonstrating that CsA dose-dependently increased the aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) level secreted in the HepG2 cell supernatant, as well as viability and oxidative stress of HepG2 cells. CsA also dose-dependently decreased the PXR, CYP3A4, CPY3A5, and MRP2 levels of HepG2 cells. Mechanistically, altering the expression of PXR, CYP3A4, CYP3A5, and MRP2 affected the impact of CsA on AST and LDH levels. Moreover, altering the expression of PXR also changed the level of CYP3A4, CPY3A5, and MRP2 of HepG2 cells treated by CsA. Our presented findings provide experimental evidence that CsA-induced liver injury is PXR tightly related. We suggest that PXR represents an attractive target for therapy of liver injury due to its central role in the regulation of the metabolizing enzymes CYP3A and MRP2-mediated bile acid transport and detoxification.

Uricosuric Agents Affect Plasma and Kidney Concentration of Adefovir via Inhibition of Oat1 and Mrp2 in Rats.

Uricosuric agents lower serum uric acid levels by increasing urinary excretion via inhibition of urate transporter 1 (URAT1), urate reabsorption transporter in the renal proximal tubules. Probenecid and benzbromarone have been used as uricosurics, but these drugs inhibit organic anion transporters (OATs) in addition to URAT1. In this study, we investigated whether uricosuric agents interacted with adefovir, known as a substrate for OAT1, using Sprague-Dawley (SD) rats. Furthermore, involvement of other transporters, multi-drug resistance protein 2 (MRP2) in this interaction was examined using Mrp2-deficient rats. Probenecid and lesinurad increased plasma adefovir concentrations and decreased kidney-to-plasma partition coefficient (Kp) in these rats, presumably by inhibiting Oat1. Although benzbromarone had no effect on plasma adefovir concentration, it increased the Kp to 141% in SD rats. Since this effect was abolished in Mrp2-deficient rats, together with the MRP2 inhibition study, it is suggested that benzbromarone inhibits Mrp2-mediated adefovir excretion from the kidney. In contrast, dotinurad, a novel uricosuric agent that selectively inhibits URAT1, had no effect on the plasma and kidney concentrations of adefovir. Therefore, due to the lack of interaction with adefovir, dotinurad is expected to have low drug-drug interaction risk mediated by OAT1, and also by MRP2.

Molecular and Clinical Links between Drug-Induced Cholestasis and Familial Intrahepatic Cholestasis.

Idiosyncratic Drug-Induced Liver Injury (iDILI) represents an actual health challenge, accounting for more than 40% of hepatitis cases in adults over 50 years and more than 50% of acute fulminant hepatic failure cases. In addition, approximately 30% of iDILI are cholestatic (drug-induced cholestasis (DIC)). The liver's metabolism and clearance of lipophilic drugs depend on their emission into the bile. Therefore, many medications cause cholestasis through their interaction with hepatic transporters. The main canalicular efflux transport proteins include: 1. the bile salt export pump (BSEP) protein (ABCB11); 2. the multidrug resistance protein-2 (MRP2, ABCC2) regulating the bile salts' independent flow by excretion of glutathione; 3. the multidrug resistance-1 protein (MDR1, ABCB1) that transports organic cations; 4. the multidrug resistance-3 protein (MDR3, ABCB4). Two of the most known proteins involved in bile acids' (BAs) metabolism and transport are BSEP and MDR3. BSEP inhibition by drugs leads to reduced BAs' secretion and their retention within hepatocytes, exiting in cholestasis, while mutations in the ABCB4 gene expose the biliary epithelium to the injurious detergent actions of BAs, thus increasing susceptibility to DIC. Herein, we review the leading molecular pathways behind the DIC, the links with the other clinical forms of familial intrahepatic cholestasis, and, finally, the main cholestasis-inducing drugs.

Utilizing Estra-1,3,5,16-Tetraene Scaffold: Design and Synthesis of Nitric Oxide Donors as Chemotherapeutic Resistance Combating Agents in Liver Cancer.

A new series of nitric oxide-releasing estra-1,3,5,16-tetraene analogs (NO-∆-16-CIEAs) was designed and synthesized as dual inhibitors for EGFR and MRP2 based on our previous findings on estra-1,3,5-triene analog NO-CIEA 17 against both HepG2 and HepG2-R cell lines. Among the target compounds, 14a (R-isomer) and 14b (S-isomer) displayed potent anti-proliferative activity against both HepG2 and HepG2-R cell lines in comparison to the reference drug erlotinib. Remarkably, compound 14a resulted in a prominent reduction in EGFR phosphorylation at a concentration of 1.20 µM with slight activity on the phosphorylation of MEK1/2 and ERK1/2. It also inhibits MRP2 expression in a dose-dependent manner with 24% inhibition and arrested the cells in the S phase of the cell cycle. Interestingly, compound 14a (estratetraene core) exhibited a twofold increase in anti-proliferative activity against both HepG2 and HepG2-R in comparison with the lead estratriene analog, demonstrating the significance of the designed ∆-16 unsaturation. The results shed a light on compound 14a and support further investigations to combat multidrug resistance in chemotherapy of hepatocellular carcinoma patients.

IL-25 ameliorates acute cholestatic liver injury via promoting hepatic bile acid secretion.

Cholestasis caused by bile secretion and excretion disorders is a serious manifestation of hepatopathy. Interleukin (IL)-25 is a member of the IL-17 cytokine family, which involves in mucosal immunity and type 2 immunity via its receptor-IL-17RB. Our previous studies have shown that IL-25 improves non-alcoholic fatty liver via stimulating M2 macrophage polarization and promotes development of hepatocellular carcinoma via alternative activation of macrophages. These hepatopathy are closely associated with cholestasis. However, whether IL-25 play an important role in cholestasis remains unclear. IL-25 treatment and IL-25 knockout (Il25-/-) mice were injected intragastrically with α-naphthyl isothiocyanate (ANIT) to determine the biological association between IL-25 and cholestasis. Here, we found that IL-25 and IL-17RB decreased in ANIT-induced cholestatic mice. Il25-/- mice showed exacerbated ANIT-induced parenchymal injury and IL-25 treatment significantly alleviated cholestatic liver injury induced by ANIT. We found that IL-25 reduced the level of hepatic total bile acids and increased the expression of multidrug resistance-associated protein 2 (MRP2) and multidrug resistance-associated protein 3 (MRP3) in liver. In conclusion, IL-25 exhibited a protective effect against ANIT-induced cholestatic liver injury in mice, which may be related to the regulation on bile acids secretion. These results provide a theoretical basis for the use of IL-25 in the treatment of cholestatic hepatopathy.

Genotype-Phenotype Association in ABCC2 Exon 18 Missense Mutation Leading to Dubin-Johnson Syndrome: A Case Report.

We report a case of a patient with Dubin-Johnson syndrome confirmed by a genetic study. A 50-year-old woman who had symptoms of intermittent right upper quadrant abdominal pain was diagnosed with calculous cholecystitis at another institute and was presented to our hospital for a cholecystectomy. She had no history of liver disease, and her physical examination was normal. Abdominal computed tomography showed a gallbladder stone with chronic cholecystitis. During a laparoscopic cholecystectomy for cholecystitis, a smooth, black-colored liver was noted, and a liver biopsy was performed. The biopsy specimen showed coarse, dark brown granules in centrilobular hepatocytes via hematoxylin and eosin staining. We performed a genetic study using the blood samples of the patient. In the adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2) mutation study, a missense mutation in exon 18 was noted. Based on the black-colored liver without nodularity, conjugated hyperbilirubinemia, the liver biopsy results of the coarse pigment in centrilobular hepatocytes, and the ABCC2 mutation, Dubin-Johnson syndrome was diagnosed. The patient was managed with conservative care using hepatotonics. One month after follow-up, total bilirubin and direct bilirubin remained in a similar range. Another follow-up was planned a month later, and the patient maintained her use of hepatotonics.

Ex vivo gadoxetate relaxivities in rat liver tissue and blood at five magnetic field strengths from 1.41 to 7 T.

Quantitative mapping of gadoxetate uptake and excretion rates in liver cells has shown potential to significantly improve the management of chronic liver disease and liver cancer. Unfortunately, technical and clinical validation of the technique is currently hampered by the lack of data on gadoxetate relaxivity. The aim of this study was to fill this gap by measuring gadoxetate relaxivity in liver tissue, which approximates hepatocytes, in blood, urine and bile at magnetic field strengths of 1.41, 1.5, 3, 4.7 and 7 T. Measurements were performed ex vivo in 44 female Mrp2 knockout rats and 30 female wild-type rats who had received an intravenous bolus of either 10, 25 or 40 µmol/kg gadoxetate. T1 was measured at 37 ± 3°C on NMR instruments (1.41 and 3 T), small-animal MRI (4.7 and 7 T) and clinical MRI (1.5 and 3 T). Gadolinium concentration was measured with optical emission spectrometry or mass spectrometry. The impact on measurements of gadoxetate rate constants was determined by generalizing pharmacokinetic models to tissues with different relaxivities. Relaxivity values (L mmol-1 s-1 ) showed the expected dependency on tissue/biofluid type and field strength, ranging from 15.0 ± 0.9 (1.41) to 6.0 ± 0.3 (7) T in liver tissue, from 7.5 ± 0.2 (1.41) to 6.2 ± 0.3 (7) T in blood, from 5.6 ± 0.1 (1.41) to 4.5 ± 0.1 (7) T in urine and from 5.6 ± 0.4 (1.41) to 4.3 ± 0.6 (7) T in bile. Failing to correct for the relaxivity difference between liver tissue and blood overestimates intracellular uptake rates by a factor of 2.0 at 1.41 T, 1.8 at 1.5 T, 1.5 at 3 T and 1.2 at 4.7 T. The relaxivity values derived in this study can be used retrospectively and prospectively to remove a well-known bias in gadoxetate rate constants. This will promote the clinical translation of MR-based liver function assessment by enabling direct validation against reference methods and a more effective translation between in vitro findings, animal models and patient studies.

Acute regulation of apical ABC transporters in the gut. Potential influence on drug bioavailability.

The extensive intestinal surface offers an advantage regarding nutrient, ion and water absorptive capacity but also brings along a high exposition to xenobiotics, including drugs of therapeutic use and food contaminants. After absorption of these compounds by the enterocytes, apical ABC transporters play a key role in secreting them back to the intestinal lumen, hence acting as a transcellular barrier. Rapid and reversible modulation of their activity is a subject of increasing interest for pharmacologists. On the one hand, a decrease in transporter activity may result in increased absorption of therapeutic agents given orally. On the other hand, an increase in transporter activity would decrease their absorption and therapeutic efficacy. Although of less relevance, apical ABC transporters also contribute to disposition of drugs systemically administered. This review article summarizes the present knowledge on the mechanisms aimed to rapidly regulate the activity of the main apical ABC transporters of the gut: multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). Regulation of these mechanisms by drugs, drug delivery systems, drug excipients and nutritional components are particularly considered. This information could provide the basis for controlled regulation of bioavailability of therapeutic agents and at the same time would help to prevent potential drug-drug interactions.

A Case of Dubin-Johnson Syndrome Presenting as Neonatal Cholestasis With Paucity of Interlobular Bile Ducts.

Dubin-Johnson syndrome (DJS) is a rare autosomal recessive disorder that typically manifests in young adulthood as jaundice with conjugated hyperbilirubinemia. We report a case presenting as neonatal cholestasis with the unexpected histologic finding of paucity of interlobular bile ducts, a feature that is not typically seen in DJS. The diagnosis was confirmed by absent canalicular multidrug-resistance-associated protein 2 (MRP2) immunohistochemical staining on liver biopsy tissue and molecular genetic testing that demonstrated heterozygous mutations in the ATP-Binding Cassette Subfamily C Member 2 (ABCC2) gene, including a novel missense mutation. This report describes a case of DJS with atypical clinicopathologic findings and suggests that DJS should be considered in patients with neonatal cholestasis and bile duct paucity.

The permeability characteristics and interaction of main components from Si-Ni-San in a MDCK epithelial cell monolayer model.

1. Si-Ni-San (SNS) possesses extensive therapeutic effects, however, the extent to which main components are absorbed and the mechanisms involved are controversial. 2. In this study, MDCK cell model was used to determine the permeability characteristics and interaction between the major components of Si-Ni-San, including saikosaponin a, paeoniflorin, naringin and glycyrrhizic acid. 3. The transport of the major components was concentration-dependent in both directions. Moreover, the transport of paeoniflorin, naringin and glycyrrhizic acid was significantly reduced at 4 °C or in the presence of NaN3. Additionally, the efflux of paeoniflorin and naringin were apparently reduced in the presence of P-gp inhibitor verapamil. The transport of glycyrrhizic acid was clearly inhibited by the inhibitors of MRP2, indicating that MRP2 may be involved in the transport of glycyrrhizic acid. However, the results indicated that saikosaponin a was absorbed mainly by passive diffusion. Furthermore, the combined incubation of four major components had a powerful sorbefacient effect than a single drug used alone which may be regulated by tight junctions. 4. Taken together, our study provides useful information for pharmacological applications of Si-Ni-San and offers new insights into this ancient decoction for further researches, especially in drug synergism.

ABCC2 c.-24 C>T single-nucleotide polymorphism was associated with the pharmacokinetic variability of deferasirox in Chinese subjects.

PURPOSE: Our aim was to evaluate the influence of genetic polymorphisms involved in the metabolism and transportation of deferasirox on deferasirox pharmacokinetics in the Chinese population. METHODS: Thirty-eight healthy Chinese subjects were administered with a single dose of 20 mg kg-1 deferasirox. Allelic discriminations for eight single-nucleotide polymorphisms (SNPs) in UDP-glucuronosyltransferase 1A1, 1A3 (UGT1A1, UGT1A3), multidrug resistance protein 2 (MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2) were performed. The concentrations of deferasirox in the plasma were determined by UPLC-MS/MS. RESULTS: Subjects carrying ABCC2 c.-24 T allele had a 65% higher clearance (CL/F) and 42% lower area under the concentration-time curve from 0 to 72 h (AUC0-72h) as compared with non-carriers (P = 0.008, P = 0.011, respectively). ABCC2 c.-24 T was also associated with 59% shorter half-life (T1/2) and 17% shorter mean residence time (MRT) (P = 0.030, P = 0.014, respectively). ABCC2 1249A was associated with a marginal increase in deferasirox Cmax (P = 0.07). Genetic polymorphisms of UGT1A1, UGT1A3, and ABCG2 did not significantly influence the pharmacokinetics of deferasirox. Subjects with UGT1A1 211GG-(-1352)CC-(-3156)GG haplotype had higher AUC0-72h than others. Since only two subjects were recruited in the GG-CC-GG group, further confirmative studies were warranted. CONCLUSIONS: ABCC2 c.-24 C>T was associated with the pharmacokinetic variability of deferasirox in Chinese subjects. This study revealed an important role of MRP2 in the pharmacokinetics of deferasirox and drew attention to drug combination with MRP2 inhibitors like cyclosporine and methotrexate in deferasirox therapy.

Rifampicin induces clathrin-dependent endocytosis and ubiquitin-proteasome degradation of MRP2 via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K signaling pathways in HepG2 cells.

It was reported that antituberculosis medicines could induce liver damage via oxidative stress. In this study, we investigated the effects of rifampicin (RFP) on the membrane expression of multidrug resistance-associated protein 2 (MRP2) and the relationship between oxidative stress and RFP-induced endocytosis of MRP2 in HepG2 cells. We found that RFP (12.5-50 µM) dose-dependently decreased the expression and membrane localization of MRP2 in HepG2 cells without changing the messenger RNA level. RFP (50 µM) induced oxidative stress responses that further activated the PKC-ERK/JNK/p38 (protein kinase C-extracellular signal-regulated kinase/c-JUN N-terminal kinase/p38) and PI3K (phosphoinositide 3-kinase) signaling pathways in HepG2 cells. Pretreatment with glutathione reduced ethyl ester (2 mM) not only reversed the changes in oxidative stress indicators and signaling molecules but also diminished RFP-induced reduction in green fluorescence intensity of MRP2. We conducted co-immunoprecipitation assays and revealed that a direct interaction existed among MRP2, clathrin, and adaptor protein 2 (AP2) in HepG2 cells, and their expression was clearly affected by the changes in intracellular redox levels. Knockdown of clathrin or AP2 with small interfering RNA attenuated RFP-induced decreases of membrane and total MRP2. We further demonstrated that RFP markedly increased the ubiquitin-proteasome degradation of MRP2 in HepG2 cells, which was mediated by the E3 ubiquitin ligase GP78, but not HRD1 or TEB4. In conclusion, this study demonstrates that RFP-induced oxidative stress activates the PKC-ERK/JNK/p38 and PI3K signaling pathways that leads to clathrin-dependent endocytosis and ubiquitination of MRP2 in HepG2 cells, which provides new insight into the mechanism of RFP-induced cholestasis.

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF's active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5-100 µmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 µmol/L. TER (5 µmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells' uptake of acyclovir and increased the plasma concentration.

Aryl hydrocarbon receptor ligands increase ABC transporter activity and protein expression in killifish (Fundulus heteroclitus) renal proximal tubules.

Many widespread and persistent organic pollutants, for example, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and some polychlorinated biphenyls, activate the aryl hydrocarbon receptor (AhR) causing it to translocate to the cell nucleus where it transactivates target genes, increasing expression of a number of xenobiotic metabolizing enzymes as well as some transporters. AhR's ability to target transporters within the kidney is essentially unexplored. We show here that exposing isolated killifish (Fundulus heteroclitus) renal proximal tubules to micromolar ß-naphthoflavone (BNF) or nanomolar TCDD roughly doubled the transport activity of Multidrug resistance-associated proteins Mrp2 and Mrp4, P-glycoprotein (P-gp) and Breast cancer resistance protein (Bcrp), all ATP-driven xenobiotic efflux pumps and critical determinants of renal xenobiotic excretion. These effects were abolished by actinomycin D and cycloheximide and by the AhR antagonist, α-naphthoflavone, indicating that increased transport activity was dependent on transcription and translation as well as ligand binding to AhR. Quantitative immunostaining of renal tubules exposed to BNF and TCDD showed increased luminal membrane expression of Mrp2, Mrp4, P-gp and Bcrp. Thus, in these renal tubules, the four ABC transporters are targets of AhR action.

Effects of Hepatic Ischemia-Reperfusion Injuries and NRF2 on Transcriptional Activities of Bile Transporters in Rats.

BACKGROUND: The effect of hepatic ischemia-reperfusion injury (IRI) on bile transporter (BT) gene expression is unknown. We hypothesized that abnormal expression of BTs during hepatic IRI is dependent on nuclear factor erythroid 2-related factor 2 (NRF2), which contributes to the cholestasis after reperfusion. METHODS: Sham surgery and short (60 min) or long (90 min) periods of warm ischemia time (WIT) with or without reperfusion for 24 h were applied to wild-type Sprague-Dawley rats and Nrf2 knockout rats (n = 5 per group). At each stage of IRI, the serum levels of aminotransferase, total bilirubin, and bile acids were measured. In addition, hepatic tissue was sampled to determine the histologic score of IRI (Suzuki score), measure adenosine triphosphate (ATP), and identify the quantitative real-time polymerase chain reactions of BTs (Oatp1, Ntcp, Mrp2, Bsep, and Mrp3). RESULTS: In short periods of WIT, BT expression increased during the ischemia stage and returned to the baseline after reperfusion. However, in long periods of WIT, BT expression did not increase after ischemia and decreased further after reperfusion. Short WIT did not increase BT expression in Nrf2 knockout animals. The level of BT expression was correlated with the Suzuki score, the serum levels of aminotransferase, bilirubin, and bile acids, and tissue ATP level. Stepwise multiple regression analysis derived equations to predict the Suzuki score (R2 = 76.8, P < 0.001), serum total bilirubin (R2 = 61.2, P < 0.001), and tissue ATP (R2 = 61.1, P < 0.001). CONCLUSIONS: Short WIT induces the transcriptional activities of BT, whereas long WIT depresses them, and the effect was blunted by Nrf2 knockout status. BT expression can be considered a surrogate marker for hepatic IRI.

Design, synthesis and biological study of hybrid drug candidates of nitric oxide releasing cucurbitacin-inspired estrone analogs for treatment of hepatocellular carcinoma.

Development of hybrid drug candidates is well known strategy for designing antitumor agents. Herein, a novel class of nitric oxide donating cucurbitacin inspired estrone analogs (NO-CIEAs) were designed and synthesized as multitarget agents. Synthesized analogs were initially evaluated for their anti-hepatocellular carcinoma activities. Among the tested analogs, NO-CIEAs 17 and 20a exhibited more potent activity against HepG2 cells (IC50 = 4.69 and 12.5 µM, respectively) than the reference drug Erlotinib (IC50 = 25 µM). Interestingly, NO-CIEA 17 exerted also a high potent activity against Erlotinib-resistant HepG2 cell line (HepG2-R) (IC50 = 8.21 µM) giving insight about its importance in drug resistance therapy. Intracellular measurements of NO revealed that NO-CIEAs 17 and 20a showed a significant increase in NO production in tumor cells after 1 h of incubation comparable to the reference prodrug JS-K. Flow cytometric analysis showed that both NO-CIEAs 17 and 20a mainly arrested the HepG2 cells in the G0/G1 phase. Also, In-Cell Based ELISA screening showed that NO-CIEA 17 resulted in a potential inhibitory activity towards the EGFR and MAPK (25% and 29% inhibition compared to untreated control cells, respectively). This data suggests the binding ability of NO-CIEA 17 to the EGFR and ERK to be well correlated along with the docking and cellular studies. Also, treatment of HepG2-R cells with NO-CIEA 17 showed a potential reduction of MRP2 expression in a dose dependent manner providing a significant impact on the chemotherapeutic resistance. Overall, the current study provides a potential new approach for the discovery of a novel antitumor agent against HCC.

Polymorphisms in potential mercury transporter ABCC2 and neurotoxic symptoms in populations exposed to mercury vapor from goldmining.

BACKGROUND: Artisanal small-scale gold miners have high levels of mercury in human specimens often above recommended threshold values. There are differences reported in the manifestation of neurological symptoms of individuals with a comparable level of exposure to mercury, suggesting a genetic component influencing the susceptibility to mercury neurotoxicity. OBJECTIVE: To investigate associations between polymorphisms (rs1885301, rs717620, rs2273697) in the potential Hg-transporter ABCC2 gene and neurological effects. METHODS: 968 participants from the Philippines, Indonesia, Tanzania and Zimbabwe were included in this study (age 12-59 years). For the statistical analysis the countries were categorized into Africa (Philippines, Indonesia) and Asia (Tanzania, Zimbabwe). Study participants were from three exposure groups: without mercury exposure from goldmining (n = 129); living in mercury-contaminated areas (n = 281) and mercury working exposure (n = 558). To identify chronic inorganic mercury intoxication we applied a modified neurological score sum including eight binary coded parameters (from anamnestic, clinical and neurophysiological examinations). Associations between genotype and neurological score sum, as well as between genotype and separate neurological tests (ataxia of gait, dysdiadochokinesia, ataxia heel to shin, pencil tapping test and matchbox test) were evaluated. RESULTS: We found that ABCC2 genotype were associated with performance on certain neurological tests: for rs1885301, A-allele carriers in the African populations showed significantly worse performance than GG carriers on the pencil tapping test; whereas for rs2273697, A-allele carriers in the African and Asian populations showed a significantly better performance than GG carriers on this test. When including an interaction term between genotype and exposure subgroup, interaction effects were also seen for the pencil tapping test and matchbox tests for rs2273697 in Asia. CONCLUSIONS: The results suggest that certain ABCC2 polymorphisms may influence the neurotoxic effects in mercury-burdened individuals. ABCC2 alleles associated with worse neurological performance in the present study. These alleles have previously been correlated with higher levels of urinary mercury concentrations in the same cohort. Taken together, these associations between ABCC2 genotype, mercury levels, and neurological effects support the hypothesis that some ABCC2 genotypes may have a higher neurotoxic potential, although further functional studies are needed to prove causation.

Sphingosine 1-phosphate receptor 2/adenylyl cyclase/protein kinase A pathway is involved in taurolithocholate-induced internalization of Abcc2 in rats.

Taurolithocholate (TLC) is a cholestatic bile salt that induces disinsertion of the canalicular transporter Abcc2 (Mrp2, multidrug resistance-associated protein 2). This internalization is mediated by different intracellular signaling proteins such as PI3K, PKCε and MARCK but the initial receptor of TLC remains unknown. A few G protein-coupled receptors interact with bile salts in hepatocytes. Among them, sphingosine-1 phosphate receptor 2 (S1PR2) represents a potential initial receptor for TLC. The aim of this study was to evaluate the role of this receptor and its downstream effectors in the impairment of Abcc2 function induced by TLC. In vitro, S1PR2 inhibition by JTE-013 or its knockdown by small interfering RNA partially prevented the decrease in Abcc2 activity induced by TLC. Moreover, adenylyl cyclase (AC)/PKA and PI3K/Akt inhibition partially prevented TLC effect on canalicular transporter function. TLC produced PKA and Akt activation, which were blocked by JTE-013 and AC inhibitors, connecting S1PR2/AC/PKA and PI3K/Akt in a same pathway. In isolated perfused rat liver, injection of TLC triggered endocytosis of Abcc2 that was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Abcc2 substrate dinitrophenyl-glutathione until the end of the perfusion period. S1PR2 or AC inhibition did not prevent the initial decay, but they accelerated the recovery of these parameters and the reinsertion of Abcc2 into the canalicular membrane. In conclusion, S1PR2 and the subsequent activation of AC, PKA, PI3K and Akt is partially responsible for the cholestatic effects of TLC through sustained internalization of Abcc2.