Maria A. Serrano

Role of organic anion-transporting polypeptides, OATP-A, OATP-C and OATP-8, in the human placenta-maternal liver tandem excretory pathway for foetal bilirubin.

Recent functional studies have suggested that, in addition to simple diffusion, carrier-mediated transport may play an important role in foetal unconjugated bilirubin (UCB) uptake by the placenta. We have investigated the role of organic anion-transporting polypeptides (OATPs) in UCB transport by the placenta-maternal liver tandem. RNA was obtained from human liver (hL), human placenta (hPl) at term, and purified (> 80%) cytokeratin-7-positive mononucleated human trophoblast cells (hTCs). By analytical reverse transcription (RT)-PCR, agarose gel electrophoresis separation and sequencing, the mRNA of OATP-A ( SLC21A3 ) and OATP-8 ( SLC21A8 ) was identified in hL, hPl and hTCs, whereas that of OATP-C ( SLC21A6 ) was detectable only in hL. Real-time quantitative RT-PCR revealed that in hL the abundance of mRNA was OATP-8 > OATP-C >> OATP-A, whereas in hPl and hTCs this was OATP-8 >> OATP-A >> OATP-C. Expression levels for these OATPs were hL >> hTCs > hPl. Injection of mRNA of OATP-A, OATP-C or OATP-8 or RNA from hL, hPl or hTCs into Xenopus laevis oocytes conferred on them the ability to take up [(3)H]17 beta-D-glucuronosyl oestradiol ([(3)H]E(2)17 beta G) and [(3)H]UCB, although in the case of OATP-A mRNA, the induced uptake of [(3)H]UCB was very low. Cis -inhibition of [(3)H]E(2)17 beta G and [(3)H]UCB uptake by both unlabelled E(2)17 beta G and UCB was found in all cases. The affinity and efficiency of [(3)H]UCB transport was OATP-C > OATP-8. Kinetic parameters for [(3)H]UCB uptake induced by RNA from hTCs resembled most closely those of OATP-8. In conclusion, our results suggest that OATP-8 may play a major role in the carrier-mediated uptake of foetal UCB by the placental trophoblast, whereas both OATP-8 and OATP-C may substantially contribute to UCB uptake by adult hepatocytes.

Beneficial effect of ursodeoxycholic acid on alterations induced by cholestasis of pregnancy in bile acid transport across the human placenta

BACKGROUND/AIMS The existence of impairment in bile acid transport across the placenta during intrahepatic cholestasis of pregnancy and the effect of ursodeoxycholic acid treatment (1 g/day) were investigated. METHODS Kinetic parameters were calculated from experiments carried out on membrane vesicles obtained from basal (TPMb, fetal-facing) and apical (TPMa, maternal-facing) trophoblast plasma membranes. Bile acid uptake was measured using varying concentrations of [14C]-glycocholate and a rapid filtration technique. RESULTS The maximal velocity of transport (Vmax), the apparent affinity constant (Kt) and the efficiency (Ef) of transport (Vmax/Kt) of the anion:bile acid exchanger located at the TPMb were reduced in intrahepatic cholestasis of pregnancy. Ursodeoxycholic acid induced a reversal of Vmax, Kt and Ef to normal values. Owing to the 3-fold increase in Vmax, with no change in Kt, intrahepatic cholestasis of pregnancy induced an enhancement in Ef of ATP-independent bile acid transport across TPMa. Both Vmax and Ef were restored to normal values by ursodeoxycholic acid. Finally, in ATP-dependent bile acid transport across TPMa, a reduction in the Ef due to an increase in Vmax together with a more pronounced increase in Kt was found. This impairment was also reversed by ursodeoxycholic acid. CONCLUSIONS These results suggest that placenta bile acid transport systems are impaired in intrahepatic cholestasis of pregnancy. Moreover, together with the confirmed beneficial effect for intrahepatic cholestasis of pregnancy patients, such as the relief of pruritus and the improvement in biochemical markers of cholestasis, ursodeoxycholic acid treatment restores the ability of the placenta to carry out vectorial bile acid transfer.

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.

No Correlation between the Expression of FXR and Genes Involved in Multidrug Resistance Phenotype of Primary Liver Tumors

Farnesoid X receptor (FXR) has been recently reported to enhance chemoresistance through bile acid-independent mechanisms. Thus, FXR transfection plus activation with GW4064 resulted in reduced sensitivity to cisplatin-induced toxicity. This is interesting because primary tumors of the liver, an organ where FXR is expressed, exhibit marked refractoriness to pharmacological treatment. Here we have determined whether FXR is upregulated in hepatocellular carcinoma (HCC), cholangiocarcinoma (CGC) and hepatoblastoma (HPB) and whether this is related with the expression of genes involved in mechanisms of chemoresistance. Using RT-QPCR and Taqman low density arrays we have analyzed biopsies from healthy livers or surgically removed tumors from naive patients and cell lines derived from HCC (SK-HEP-1, Alexander and Huh7), CGC (TFK1) and HPB (HepG2), before and after exposure to cisplatin at IC50 for 72 h. In liver tumors FXR expression was not enhanced but significantly decreased (healthy liver > HCC > HPB ≈ CGC). Except for CGC, this was not accompanied by changes in the proportions of FXR isoforms. Changes in 36 genes involved in drug uptake/efflux and metabolism, expression/function of molecular targets, and survival/apoptosis balance were found. Changes affecting SLC22A1, CYP2A1 and BIRC5 were shared by HCC, CGC and HPB. Similarity in gene expression profiles between cell lines and parent tumors was found. Pharmacological challenge with cisplatin induced changes that increased this resemblance. This was not dependent upon FXR expression. Thus, although FXR may play a role in inducing chemoresistance under certain circumstances, its upregulation does not seem to be involved in the multidrug resistance phenotype characteristic of HCC, CGC and HPB.

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.

Cisplatin-induced chemoresistance in colon cancer cells involves FXR-dependent and FXR-independent up-regulation of ABC proteins.

Export pumps often limit the usefulness of anticancer drugs. Here we investigated the effect of cisplatin on the expression of ABC proteins in human colon cancer cells. Short-term incubation of Caco-2 and LS174T cells with cisplatin resulted in up-regulation of several ABC pumps, in particular MRP2 and BCRP. In partially cisplatin-resistant cells (LS174T/R) obtained by long-term exposure to cisplatin, MRP2 and BCRP up-regulation was more marked. This was further enhanced when these cells were cultured under maintained stimulation with cisplatin. The MRP2 promoter (MRP2pr) was cloned, and partially deleted constructs linked to reporter genes were generated. Transfection of LS174T and LS174T/R cells with these constructs revealed the ability of cisplatin to activate MRP2pr. The intensity of this response was dependent on the conserved MRP2pr region. Basal MRP2pr activity was higher in LS174T/R cells, in which the expression of the transcription factors c/EBPβ, HNF1α, HNF3β, and HNF4α, but not PXR, p53, c-Myc, AP1, YB-1, NRF2, and RARα was enhanced. Up-regulation was particularly high for FXR (200-fold) and SHP (50-fold). In LS174T/R cells, GW4064 induced the expression of FGF19, SHP, OSTα/β, but not MRP2 and BCRP, although the sensitivity of these cells to cisplatin was further reduced. In LS174T cells, GW4064-induced chemoresistance was seen only after being transfected with FXR+RXR, when BCRP, but not MRP2, was up-regulated. Protection of LS174T cells against cisplatin was mimicked by transfection with BCRP. In conclusion, in colon cancer cells, cisplatin treatment enhances chemoresistance through FXR-dependent and FXR-independent mechanisms involving the expression of BCRP and MRP2, respectively.

Relationship between Bile Acid Transplacental Gradients and Transport across the Fetal-Facing Plasma Membrane of the Human Trophoblast

ABSTRACT: Bile acids and bilirubin are synthesized by the fetal liver very early on during intrauterine life. The main fate of these compounds is to be transferred to the mother. This excretory role of the placenta is primarily determined by the ability of the trophoblast to transport them, which is believed to occur mainly by carrier-mediated processes. The aim of this study was to investigate the role of the cholephilic organic anion exchanger located in the fetal-facing plasma membrane of the human trophoblast in placental “biliary-like‘’ function. No relationship between the magnitude of transplacental gradients for total bile acids and bilirubin was found. However, transport studies, which were carried out by using perified plasma membrane vesicles derived from the fetal-facing pole of the human trophoblast, revealed that [14C]taurocholate transport was affected by both another bile acid (taurochenodeoxycholic acid) and a non-bile acid cholephilic organic anion (bromosulfophthalein). On plotting the ability of different major bile acid species to inhibit radiolabeled taurocholate uptake by these vesicles versus their concentrations in fetal serum or the magnitude of their transplacental gradients, inverse relationships were found. Lower fetal serum concentrations and transplacental gradients were found for bile acid species with higher abilities to affect this transport and presumably to interact with the carrier. By contrast, the magnitude of the transplacental gradient for bile acid species was not correlated with their hydrophobic/hydrophilic balance, as would be expected if diffusion across the lipidic structures of the placental barrier would be the major pathway for the flux of bile acid across this organ. In summary, these results indicate that carriers located in the basal plasma membrane may play an important role in the control of the qualitative and quantitative fetal-maternal bile acid exchange. Moreover, they suggest that although both bile acids and bilirubin may share this pathway for access to the trophoblast, other additional mechanisms are probably responsible in part for the control of the magnitude of their transplacental gradients.

A review on the molecular mechanisms involved in the placental barrier for drugs.

The placenta has traditionally been considered as a highly permeable organ for a large variety of substances with diverse molecular structures that are readily able to cross it from the maternal blood to reach the foetus. This has recommended limiting the use of drugs during pregnancy as far as possible. However, our present knowledge points to the existence of different systems, including plasma membrane carriers, biotransforming enzymes, and export pumps, that determine the selectivity and efficacy of the so-called placental barrier. A good understanding of the molecular bases of these processes and their regulation is crucial: i) to predict interactions between drug-drug, drug-endogenous substances and drug-food components, ii) to analyse the relevance of polymorphisms in the inter-individual variability of conceptus sensitivity to drugs, and iii) to develop novel pharmacological strategies aimed at delivering medicinal drugs to pregnant women, simultaneously minimising the risk of foetal exposure to active agents, and to specifically target drugs to the placenta and/or foetus. The present review does not attempt to offer a complete list of the available medicinal compounds and their ability to cross the placenta but instead to provide the reader with an up-to-date overview of the mechanisms involved in carrying out or preventing the transfer of active drugs across the placenta.

DNA interaction and cytostatic activity of the new liver organotropic complex of cisplatin with glycocholic acid: Bamet-R2

The aim of this study was to investigate the ability of the new liver organotropic complex of cisplatin with glycocholate (GC), Bamet‐R2, to interact with DNA, inhibit its replication and hence reduce tumor‐cell proliferation. Changes in the electrophoretic mobility of the open and covalently closed circular forms of the pUC18 plasmid DNA from Escherichia coli, a shift in the denaturation temperature of double‐stranded DNA, and ethidium‐bromide displacement from DNA binding, were induced by Bamet‐R2 and cisplatin, but not by GC. Neutral‐red retention was used to measure the number of living cells in culture after long‐term (72‐hr) exposure to these compounds and to evaluate the effect on cell viability after short‐term (6‐hr) exposure. Bamet‐R2 and cisplatin, but not GC, induced significant inhibition of cell growth. This effect ranged from mild to strong, depending upon the sensitivity of the different cell types as follows: cisplatin, rat hepatocytes in primary culture < rat hepatoma McA‐RH7777 cells (rH) < human colon carcinoma LS 174T cells (hCC) < mouse hepatoma Hepa 1–6 cells (mH); Bamet‐R2, rat hepatocytes < mH ≈ hCC < rH. DNA synthesis was measured by radiolabeled‐thymidine incorporation into DNA. Bamet‐R2 and cisplatin, but not GC, significantly inhibited the rate of DNA synthesis by these cells. After short‐term exposure to Bamet‐R2 or GC, no acute cell toxicity was observed, except on hCC cells. By contrast, acute toxicity was induced by cisplatin for all cell types studied. The in vivo anti‐tumoral effect was investigated in 3 different strains of mice following s.c. implantation of tumor cells (mouse sarcoma S‐180II cells in Swiss and B6 mice and hCC cells in nude mice). In all 3 models, tumor growth was inhibited by Bamet‐R2 and cisplatin to a similar degree. However, signs of toxicity (increases in blood urea concentrations and decreases in packed blood cell volume and in liver, kidney and body weight) and a reduction in survival rate were observed only during cisplatin administration. In sum, these results indicate that this bile‐acid derivative can be considered as a cytostatic drug whose potential usefulness deserves further investigation. Int. J. Cancer 78:346–352, 1998.© 1998 Wiley‐Liss, Inc.

Sensitivity of bile acid transport by organic anion-transporting polypeptides to intracellular pH.

We investigated the influence of intracellular pH (pHi) on [14C]-glycocholate (GC) uptake by human hepatoblastoma HepG2 cells that express sodium-independent (mainly OATP-A and OATP-8), but not sodium-dependent, GC transporters. Replacement of extracellular sodium by choline (Chol) stimulated GC uptake but did not affect GC efflux from loaded cells. Amiloride or NaCl replacement by tetraethylammonium chloride (TeACl) or sucrose also increased GC uptake. All stimulating circumstances decreased pHi. By contrast, adding to the medium ammonium or imidazole, which increased pHi, had no effect on GC uptake. In Chinese hamster ovary (CHO) cells expressing rat Oatp1, acidification of pHi had the opposite effect on GC uptake, that is, this was reduced. Changes in extracellular pH (pHo) between 7.40 and 7.00 had no effect on GC uptake at pHi 7.30 or 7.45 when pHopHi. Inhibition was not proportional to the pHo-pHi difference. Intracellular acidification decreased V(max), but had no effect on K(m). In sum, sodium-independent GC transport can be affected by intracellular acidification, possibly due both to modifications in the driving forces and to the particular response to protonation of carrier proteins involved in this process.