The effect of acetaminophen on the expression of BCRP in trophoblast cells impairs the placental barrier to bile acids during maternal cholestasis.

Acetaminophen is used as first-choice drug for pain relief during pregnancy. Here we have investigated the effect of acetaminophen at subtoxic doses on the expression of ABC export pumps in trophoblast cells and its functional repercussion on the placental barrier during maternal cholestasis. The incubation of human choriocarcinoma cells (JAr, JEG-3 and BeWo) with acetaminophen for 48h resulted in no significant changes in the expression and/or activity of MDR1 and MRPs. In contrast, in JEG-3 cells, BCRP mRNA, protein, and transport activity were reduced. In rat placenta, collected at term, acetaminophen administration for the last three days of pregnancy resulted in enhanced mRNA, but not protein, levels of Mrp1 and Bcrp. In fact, a decrease in Bcrp protein was found. Using in situ perfused rat placenta, a reduction in the Bcrp-dependent fetal-to-maternal bile acid transport after treating the dams with acetaminophen was found. Complete biliary obstruction in pregnant rats induced a significant bile acid accumulation in fetal serum and tissues, which was further enhanced when the mothers were treated with acetaminophen. This drug induced increased ROS production in JEG-3 cells and decreased the total glutathione content in rat placenta. Moreover, the NRF2 pathway was activated in JEG-3 cells as shown by an increase in nuclear NRF2 levels and an up-regulation of NRF2 target genes, NQO1 and HMOX-1, which was not observed in rat placenta. In conclusion, acetaminophen induces in placenta oxidative stress and a down-regulation of BCRP/Bcrp, which may impair the placental barrier to bile acids during maternal cholestasis.

The role of reduced intracellular concentrations of active drugs in the lack of response to anticancer chemotherapy.

A major difficulty in the treatment of cancers is the poor response of many tumors to pharmacological regimens. This situation can be accounted for by the existence of a variety of complex mechanisms of chemoresistance (MOCs), leading to reduced intracellular concentrations of active agents, changes in the molecular targets of the drugs, enhanced repair of drug-induced modifications in macromolecules, stimulation of anti-apoptotic mechanisms, and inhibition of pro-apoptotic mechanisms. The present review focuses on alterations in the expression and appearance of the genetic variants that affect the genes involved in reducing the amount of active agents inside tumor cells. These alterations can occur through two mechanisms: either by lowering uptake or enhancing efflux (so-called MOC-1a and MOC-1b, respectively), or by decreasing the activation of prodrugs or enhancing inactivation of active agents through their biotransformation (MOC-2). The development of chemosensitizers that are useful in implementing the pharmacological manipulation of these processes constitutes a challenge to modern pharmacology. Nevertheless, the important physiological roles of the most relevant genes involved in MOC-1a, MOC-1b, and MOC-2 make it difficult to prevent the side effects of chemosensitizers. A more attainable goal in this area of pharmacological enquiry is the identification of proteomic profiles that will permit oncologists to accurately predict a lack of response to a given regimen, which would be useful for adapting treatment to the personal situation of each patient.

Novel artemisinin derivatives with potential usefulness against liver/colon cancer and viral hepatitis.

Antitumor and antiviral properties of the antimalaria drug artemisinin from Artemisia annua have been reported. Novel artemisinin derivatives (AD1-AD8) have been synthesized and evaluated using in vitro models of liver/colon cancer and viral hepatitis B and C. Cell viability assays after treating human cell lines from hepatoblastoma (HepG2), hepatocarcinoma (SK-HEP-1), and colon adenocarcinoma (LS174T) with AD1-AD8 for a short (6h) and long (72h) period revealed that AD5 combined low acute toxicity together with high antiproliferative effect (IC50=1-5µM). Since iron-mediated activation of peroxide bond is involved in artemisinin antimalarial activity, the effect of iron(II)-glycine sulfate (ferrosanol) and iron(III)-containing protoporphyrin IX (hemin) was investigated. Ferrosanol, but not hemin, enhanced antiproliferative activity of AD5 if the cells were preloaded with AD5, but not if both compouds were added together. Five derivatives (AD1>AD2>AD7>AD3>AD8) were able to inhibit the cytopathic effect of bovine viral diarrhoea virus (BVDV), a surrogate in vitro model of hepatitis C virus (HCV), used here to evaluate the anti-Flaviviridae activity. Moreover, AD1 and AD2 inhibited the release of BVDV-RNA to the culture medium. Co-treatment with hemin or ferrosanol resulted in enhanced anti-Flaviviridae activity of AD1. In HepG2 cells permanently infected with hepatitis B virus (HBV), AD1 and AD4, at non-toxic concentrations for the host cells were able to reduce the release of HBV-DNA to the medium. In conclusion, high pharmacological interest deserving further evaluation in animal models has been identified for novel artemisinin-related drugs potentially useful for the treatment of liver cancer and viral hepatitis B and C.

ABCC2 is involved in the hepatocyte perinuclear barrier for small organic compounds.

Small organic molecules are believed to freely diffuse across nuclear pores but this may not be so if this route is blocked during protein and nucleic acid transfer. Here we have investigated the existence of transport mechanisms across the nuclear envelope (NE) of hepatocytes. Using nuclei isolated from rat liver cells, evidence for the existence of ATP-dependent transporters of organic compounds was found. In rat hepatocyte NE, with negligible contamination by other membranes, the presence of mature and glycosylated ABCC2, but not other ABC export pumps, was detected. ABCC2 was localized in the same membranes as the conjugating enzyme UGT1A1. Human ABCC2 ORF was tagged with V5 and transfected to human hepatoma cells. ABCC2-V5 protein was detected at perinuclear ER vesicles and at the NE. Both compartments expressing ABCC2-V5 were able to exclude calcein. ABCC2 abundance at the NE of rat hepatocytes was modified by treatments able to increase or reduce the expression of canalicular ABCC2. The sensitivity to mitoxantrone was higher for hepatocytes obtained from TR- rats whose NE lacked ABCC2. Incubation with mitoxantrone after depletion of ATP resulted in a marked accumulation of mitoxantrone in the nucleus of wild-type, but not TR-, hepatocytes. In sum, ABCC2 is present at the NE and perinuclear ER where, in combination with the activity of conjugating enzymes, this pump may be involved in the perinuclear barrier for small organic molecules, playing a role in protecting DNA from genotoxic compounds and in the control of intranuclear concentrations of ligands for nuclear receptors.

Characterization of the role of ABCG2 as a bile acid transporter in liver and placenta.

ABCG2 is involved in epithelial transport/barrier functions. Here, we have investigated its ability to transport bile acids in liver and placenta. Cholylglycylamido fluorescein (CGamF) was exported by WIF-B9/R cells, which do not express the bile salt export pump (BSEP). Sensitivity to typical inhibitors suggested that CGamF export was mainly mediated by ABCG2. In Chinese hamster ovary (CHO cells), coexpression of rat Oatp1a1 and human ABCG2 enhanced the uptake and efflux, respectively, of CGamF, cholic acid (CA), glycoCA (GCA), tauroCA, and taurolithocholic acid-3-sulfate. The ability of ABCG2 to export these bile acids was confirmed by microinjecting them together with inulin in Xenopus laevis oocytes expressing this pump. ABCG2-mediated bile acid transport was inhibited by estradiol 17ß-d-glucuronide and fumitremorgin C. Placental barrier for bile acids accounted for <2-fold increase in fetal cholanemia despite >14-fold increased maternal cholanemia induced by obstructive cholestasis in pregnant rats. In rat placenta, the expression of Abcg2, which was much higher than that of Bsep, was not affected by short-term cholestasis. In pregnant rats, fumitremorgin C did not affect uptake/secretion of GCA by the liver but inhibited its fetal-maternal transfer. Compared with wild-type mice, obstructive cholestasis in pregnant Abcg2(-/-) knockout mice induced similar bile acid accumulation in maternal serum but higher accumulation in placenta, fetal serum, and liver. In conclusion, ABCG2 is able to transport bile acids. The importance of this function depends on the relative expression in the same epithelium of other bile acid exporters. Thus, ABCG2 may play a key role in bile acid transport in placenta, as BSEP does in liver.

Acetaminophen-induced stimulation of MDR1 expression and activity in rat intestine and in LS 174T human intestinal cell line.

The well-known analgesic and antipyretic drug N-acetyl-p-aminophenol (acetaminophen; APAP) has been previously reported to affect MDR1 expression in rat liver. In this study, we have investigated the effect of subtoxic doses of APAP on MDR1 expression and activity in rat intestine and human intestinal cells. Administration of APAP at increasing doses of 0.2, 0.3, and 0.6g/kg b.w., i.p. over three consecutive days, induced MDR1 expression in rat duodenum (+240%) and ileum (+160%) as detected by western blotting. This was accompanied by preserved localization of the protein at the surface of the villus, as detected by confocal immunofluorescence microscopy. MDR1 activity was increased by 50% in APAP treated rats, as evaluated by serosal to mucosal secretion of rhodamine 123 in everted intestinal sacs. Treatment with APAP also decreased by 65% the portal vein concentrations of digoxin found in anesthetized rats after intraduodenal administration of this drug, which is consistent with an APAP-induced increased efficacy of intestinal barrier for digoxin net absorption. Exposure of LS 174T human colon adenocarcinoma cells to subtoxic APAP concentration (5mM) induced an increase in MDR1 mRNA expression (+46%), which was accompanied with an enhanced ability (+78%) to reduce intracellular content of rhodamine 123. Taken together these data suggest the existence of APAP-induced stimulation of MDR1 transcription in the intestinal epithelium. These findings are of clinical relevance, as co-administration of APAP with other MDR1 substrates could indirectly inhibit the net intestinal absorption of these drugs, leading to changes in their pharmacokinetics and therapeutic efficacy.

In vitro and in vivo interaction of moxidectin with BCRP/ABCG2.

The study characterizes the interaction between BCRP/ABCG2 and moxidectin by means of cellular transport, and pharmacokinetic studies in Bcrp1 (-/-) and wild-type mice. Milbemycin moxidectin ([(3)H]-moxidectin) was tested for its ability to be transported across MCDK-II epithelial monolayer cultures transfected with BCRP. In a second approach, accumulation assays by BCRP-expressing Xenopus laevis oocytes were carried out. Finally, pharmacokinetic studies were performed in order to establish the role of the transporter in milk secretion and tissue distribution. The efflux was negligible in polarized cells but moxidectin was efficiently transported in BCRP-expressing X. laevis oocytes. The transport was blocked by an acridone derivative, a novel BCRP inhibitor. Moxidectin secretion into breast milk was decreased in Bcrp1-knockout mice and the milk to plasma ratio was 2-fold higher in wild-type mice after i.v. administration. Drug accumulation in intestinal content, bile, and intestine was higher in wild-type mice but the plasma concentration was not different. Moxidectin is identified as a BCRP substrate since its Bcrp1-mediated secretion into breast milk and the involvement of Bcrp1 in intestinal and bile secretion has been demonstrated. This interaction has pharmacokinetic and toxicological consequences. The most important toxicological consequences of the interaction between BCRP and moxidectin may be related with the presence of drug residues in milk.

Importance and limitations of chemotherapy among the available treatments for gastrointestinal tumours.

Gastrointestinal tumours constitute one of the worldwide leading causes of death. One important limitation in the battle against these types of cancer is their lack of sensitivity to currently available chemotherapy and the development of drug resistance during treatment. The mechanisms responsible for this refractivity include a reduction in drug uptake, enhanced drug export, intracellular inactivation of the effective agent, alteration of the molecular target, an increase in the activity of the target route to be inhibited or the appearance or stimulation of alternative routes, enhanced repair of drug-induced modification in the target molecules, and activation/inhibition of intracellular signalling pathways, which leads to a negative balance between apoptosis/survival of tumour cells. A better understanding of these mechanisms is needed in order to develop both accurate tests to predict the lack of response to chemotherapy and novel approaches aimed to overcome the drug resistance of gastrointestinal tumours. The complexity of this issue is further increased owing to the existence of marked differences among the types of primary malignant gastrointestinal tumours and the diversity of tissues from which metastatic cells can access the gut. Moreover, inter-individual variability plus the fact that sensitivity/refractivity may change during the evolution of the tumour further complicate the overall situation. The present article reviews anti-cancer agents used either alone or, more frequently, combined in regimens, as neoadjuvant or postsurgical adjuvant chemotherapy within the context of the available curative and palliative therapeutic options used to treat the most common types of cancer of the gastrointestinal tract and pancreas.

Molecular bases of the fetal liver-placenta-maternal liver excretory pathway for cholephilic compounds.

Potentially toxic endogenous compounds, such as bile acids (BAs) and biliary pigments, as well as many xenobiotics, such as drugs and food components, are biotransformed and eliminated by the hepatobiliary system with the collaboration of the kidney. However, the situation is very different during pregnancy because the fetal liver produces biliary compounds despite the fact that this organ, owing to its immaturity, is not able to eliminate them into bile. Moreover, the excretory ability of the fetal kidneys is also very limited. Thus, during the intra-uterine life, the major route to eliminate fetal BAs and biliary pigments is their transfer to the mother across the placenta. The maternal liver and, to a lesser extent, the maternal kidney, are then in charge of their biotransformation and elimination into faeces and urine respectively. This review describes current knowledge of the machinery responsible for the detoxification and excretion of cholephilic compounds through the pathway formed by the fetal liver-placenta-maternal liver trio.

Insulin internalizes GLUT2 in the enterocytes of healthy but not insulin-resistant mice.

OBJECTIVES: A physiological adaptation to a sugar-rich meal is achieved by increased sugar uptake to match dietary load, resulting from a rapid transient translocation of the fructose/glucose GLUT2 transporter to the brush border membrane (BBM) of enterocytes. The aim of this study was to define the contributors and physiological mechanisms controlling intestinal sugar absorption, focusing on the action of insulin and the contribution of GLUT2-mediated transport. RESEARCH DESIGN AND METHODS: The studies were performed in the human enterocytic colon carcinoma TC7 subclone (Caco-2/TC7) cells and in vivo during hyperinsulinemic-euglycemic clamp experiments in conscious mice. Chronic high-fructose or high-fat diets were used to induce glucose intolerance and insulin resistance in mice. RESULTS AND CONCLUSIONS: In Caco-2/TC7 cells, insulin action diminished the transepithelial transfer of sugar and reduced BBM and basolateral membrane (BLM) GLUT2 levels, demonstrating that insulin can target sugar absorption by controlling the membrane localization of GLUT2 in enterocytes. Similarly, in hyperinsulinemic-euglycemic clamp experiments in sensitive mice, insulin abolished GLUT2 (i.e., the cytochalasin B-sensitive component of fructose absorption), decreased BBM GLUT2, and concomitantly increased intracellular GLUT2. Acute insulin treatment before sugar intake prevented the insertion of GLUT2 into the BBM. Insulin resistance in mice provoked a loss of GLUT2 trafficking, and GLUT2 levels remained permanently high in the BBM and low in the BLM. We propose that, in addition to its peripheral effects, insulin inhibits intestinal sugar absorption to prevent excessive blood glucose excursion after a sugar meal. This protective mechanism is lost in the insulin-resistant state induced by high-fat or high-fructose feeding.

Characterization of WIF-B9/R cells as an in vitro model with hepatocyte-like polarity and enhanced expression of canalicular ABC transporters involved in phase III of hepatic detoxification.

The rat hepatoma/human fibroblast hybrid cell line WIF-B9 was developed to be used in studies requiring maintained hepatocyte-like polarity. To enhance their usefulness in order to investigate hepatic phase III detoxification process, we have characterized a subline of WIF-B9 cells (WIF-B9/R) obtained by exposure to progressively increasing cisplatin concentrations (up to 10 microM) and double sub-clonal selection. As compared to WIF-B9 cells, the cytostatic effect of cisplatin and doxorubicin on WIF-B9/R cells was lower (>10-fold), whereas the ability to reduce cell loading of cisplatin, doxorubicin, rhodamine 123 and calcein was higher. As their parent cells, WIF-B9/R cells express hepatocyte-like polarity. However, they have enhanced stable expression of Mdr1, Mrp1, Mrp2, Mrp3 and BCRP, but not Bsep/BSEP, as determined by real-time quantitative RT-PCR and western blot. Differentiation to hepatocyte-like phenotype was characterized by the formation of canalicular-like structures, containing in their membranes immunocytochemically detectable Mdr1, Mrp2 and BCRP. Functionality of these ABC transporters was evaluated by using specific substrates and inhibitors. Thus, canalicular-like structures were able to concentrate calcein, rhodamine 123 and doxorubicin. Moreover, verapamil, probenecid and Hoechst-33342 inhibited doxorubicin efflux and enhanced its content in WIF-B9/R cells. Probenecid inhibited calcein efflux and increased calcein cell load, but had no effect on cell loading of rhodamine 123, which was increased by verapamil and Hoechst-33342. In conclusion, WIF-B9/R cells are a useful model of polarized cells to study, in the absence of Bsep/BSEP, hepatic phase III of the detoxification process of several compounds whose canalicular transport is mediated by ABC proteins.