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Correspondence Jose J. G. Marin, Department of Physiology and Pharmacology, Campus Miguel de Unamuno E.D. S-09, 37007-Salamanca, Spain Tel: +34 923 294674 Fax: +34 923 294669 e-mail: firstname.lastname@example.org
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.
One of the most important roles of the liver is to carry out the secretion into bile of a large variety of structurally unrelated compounds. For some of them, commonly known as cholephilic compounds, the hepatobiliary pathway is the major route for their elimination from the body. Among these substances are several endogenous anions, such as bile acids (BAs) and biliary pigments, mainly biliverdin and bilirubin. During intra-uterine life, the elimination of cholephilic organic anions (COAs) occurs through the excretory pathway constituted by the fetal liver–placenta–maternal liver trio (Fig. 1). When the last element of this trio cannot carry out its task in an efficient manner, as happens in gestations complicated by intrahepatic cholestasis of pregnancy (ICP) – a pregnancy-specific disorder that mainly occurs during the third trimester of pregnancy and is characterized primarily by pruritus, altered liver enzymes and, less frequently, jaundice, the overall excretion of these compounds is impaired and they accumulate first in the mother but then in the placenta and finally in the foetus. The elimination of some COAs is carried out without biotransformation. In these cases, only transport mechanisms of uptake (phase 0) and secretion (phase III) are involved in the detoxification process (Fig. 2) (1). In contrast, other COAs do undergo chemical modifications during their intracellular residence. These consist of oxidation/reduction reactions (phase I) and/or conjugation with polyatomic groups (phase II) (Fig. 2). The present review provides an overview of current information regarding the molecular bases and regulation of the excretory pathway formed by the fetal liver–placenta–maternal liver trio.
Phase I enzymes
In addition to biliverdin reductase and other phase I enzymes (Fig. 2), an important role in these reactions is played by cytochrome P450 enzymes (CYP), such as CYP1, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, CYP11, CYP17 and CYP27, whose expression is particularly high in the liver. Among them, CYP1, CYP2 and CYP3 are mainly involved in the detoxification of xenobiotic substrates (2). In cholestatic liver diseases, it is noteworthy that CYP3A4 is able to catalyse BA hydroxylation at the 6α, 1β and C22 positions, converting them into more hydrophilic compounds that can be eliminated more easily from the body (3). BA synthesis and metabolism in the liver of human foetuses and neonates are significantly different from those occurring in adults (4). For instance, the presence of relatively high proportions of hyocholic acid, often greater than those of cholic acid, and several 1β-hydroxy-cholanoic acid isomers indicates that C1, C4 and C6 hydroxylations are important reactions in BA metabolism during that period of life (5). Hyocholic acid and hyodeoxycholic acid are formed from lithocholic acid and chenodeoxycholic acid by CYP3A4.
Recently, a detailed picture of the ontogenic changes, from the fetal period to senescence, in mRNA levels of enzymes involved in BA synthesis (namely: Cyp7a1, Cyp8b1, Cyp27, Cyp3a11, 5α-reductase and 5β-reductase) has been reported (6).
The human fetal liver expresses several CYPs (CYP1A1, CYP1B1, CYP2C8, CYP2E1, CYP3A4, CYP3A5 and CYP3A7) (7). The ontogenic development of CYP in the liver has been reviewed recently (8). Some of these enzymes (CYP1A1, CYP1A2, CYP2C, CYP2D6, CYP2E1, CYP2F1, CYP3A4, CYP3A5, CYP3A7 and CYP4B1) are also expressed in the human placenta more abundantly in early than in late gestation (9). However, the CYP-mediated detoxifying ability of the maternal liver increases during gestation (e.g. this is because of CYP3A4, CYP2D6 and CYP2C9). This does not include CYP1A2 and CYP2C19, whose activities are decreased during pregnancy (10).
Biliverdin reductase activity can be detected both in placenta and in fetal liver in rodents (11). The relative abundance of its mRNA in rats is higher in fetal liver and placenta than in adult liver (12).
Phase II enzymes
The aim of phase II detoxification reactions is to increase the molecular weight and water solubility of compounds, which facilitates their excretion in bile and urine. Sulphation, glucuronidation and glutathione conjugation represent the three most prevalent classes of phase II metabolism, while other phase II reactions (e.g. amino acid conjugation, acetylation and methylation) are less common metabolic pathways for xenobiotics (13) (Fig. 2). In contrast, under physiological conditions, BAs are mainly conjugated with glycine or taurine, whereas sulphation occurs under cholestatic conditions, as reflected by the appearance of sulphated BAs in the serum and urine of patients with cholestatic liver diseases (14, 15), including ICP (16). Glucuronidation of BAs, which is catalysed by the UDP-glucuronosyltransferases (UGT) UGT2B4 and UGT2B7 (17, 18), is almost selective for 6α-hydroxylated BAs such as hyocholic acid and hyodeoxycholic acid (19). This biotransformation is very poor in healthy individuals, while these derivatives represent up to 8% of the BA pool in the plasma and 35% in the urine of cholestatic patients (20, 21). In contrast, the very active enzyme UGT1A1 is responsible for conjugating bilirubin to permit its secretion into bile (22).
Many phase II enzymes are already expressed in fetal human liver early during gestation. However, for most of them there is a progressive evolution towards neonatal levels during intra-uterine life. For instance, regarding UGT activity, a first stage is characterized by the appearance of transcripts, which is followed by a marked upregulation until birth (23). Maximal activity is already reached in the neonatal life at 20 months of age (24). A complete description of the ontogeny of phase II enzymes can be found in a recent review (8).
In the maternal human liver, the activity of phase II enzymes, in general, increases during gestation (e.g. UGT1A4 and UGT2B7) (10) and can be found elevated even some time after delivery, such as N-acyl-transferase-2 (NAT2) (25).
The human placenta also expresses several phase II enzymes. These include UGT1A, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15 and UGT2B17 (26, 27) as well as sulphotransferases (SULT) SULT2A1 and SULT2B1 (28, 29) and NAT1 and, at a lower degree of expression (1000-fold), NAT2 (30). Some phase II enzymes are already expressed at high levels since very early during gestation (e.g. epoxide hydrolase) (31). Interestingly, the glutathione-S-transferase (GST) activity is markedly reduced in spontaneous miscarriages at 28–36 weeks of gestation (32).
Phase 0 transporters
Several transport systems are involved in phases 0 and III of the elimination of fetal COAs through the placenta–maternal liver pathway. Regarding phase 0, both sodium-dependent and sodium-independent transporters are involved. Sodium/taurocholate-cotransporting polypeptide (NTCP, gene symbol SLC10A1), which is not expressed in the liver of lower vertebrates, is largely responsible for sodium-dependent BA uptake by mammalian hepatocytes (33). However, sodium-dependent transporters do not seem to play a role in COA transfer across the placenta (34, 35). Recently, four new members of the SLC10 family have been described, and are referred to as P3 (SLC10A3), P4 (SLC10A4), P5 (SLC10A5) and sodium-dependent organic anion transporter (SOAT; SLC10A6). Experimental data supporting the carrier function of P3, P4 and P5 are currently unavailable (36).
In studies carried out using vesicles from plasma membrane from rat liver, Na+-dependent transport of taurocholate was found only during late gestation, and was lower in fetal and neonatal compared with adult livers (37). Rat Ntcp mRNA can be detected late in gestation, at the time when Na+-dependent BA transport is found (38). Rat Ntcp mRNA in fetal liver is very low (<1% of adult) at the midgestational stage (39). At birth, these levels are between 30 (40) and 50% (39) of adult levels. They increase rapidly during the first post-natal day (41). Thus, Ntcp is at levels close to those in adult liver but has a lower apparent molecular mass. This difference persists until 4 weeks of age. Results from N-glycanase digestion suggested that this difference could be fully accounted for by N-linked glycosylation (41). The adult phenotype of polarized expression is achieved for rat Ntcp at day 5 post-natal (42).
Regarding the maternal liver, the expression of Ntcp has been reported to remain unchanged (43) or decreased by approximately half during pregnancy (44).
Several organic anion-transporting polypeptides (OATPs) classified within the SLCO family of transporters (45) participate in the sodium-independent uptake of COA by the liver. Although OATP1A2, OATP1B1 and OATP1B3 (formerly designated OATP-A, OATP-C and OATP8 respectively) are able to transport BAs (46) and, some of them, also unconjugated bilirubin (47, 48), their relevance in the overall function of COA uptake is not similar. Thus, owing to the low expression of OATP1A2 in normal adult liver cells its role in COA uptake is probably less relevant than that of OATP1B1 and OATP1B3 (48). Although both OATP1B1 and OATP1B3 are highly expressed in the human liver and are able to transport BAs, their mechanisms of action are different. OATP1B1 may be involved in uptake processes, whereas OATP1B3 may mediate the extrusion of organic anions (49).
OATP2B1 (formerly designated OATP-B) is also localized at the basolateral membrane of hepatocytes but this carrier is not able to transport BAs (50), although when uptake experiments are conducted at neutral pH, OATP2B1 is able to mediate the transport of the natural steroid conjugates oestrone-3-sulphate and dehydroepiandrosterone sulphate (DHEAS) (51).
In rats, Oatp1a1 (52) and Oatp1b2 (39) can be detected in the fetal liver during the third week of gestation. At birth, their mRNA levels were still very low (≈10%) as compared with those in adult liver (39, 42). Rat (52) and mouse (53) Oatp1a1 expression increases dramatically after weaning, reaching adult levels by 30 (54) and 45 (53) days of age respectively. Whereas Oatp1a4 mRNA first appears in fetal liver 1 day before birth at a very low level, expression of rat Oatp1a4 mRNA at birth is very low (<0.01% of that found in adults) (39) and increases during the first weeks of age (42). In maternal rat liver, the expression of Oatp1a1 remains unchanged during pregnancy (43) but that of Oatp1a4 is decreased (55).
Several transport proteins belonging to the SLC22A family are able to transport organic anions (OATs) or cations (OCTs), and may therefore play a role in the uptake by the liver of a large variety of compounds (56) including some COAs and their derivatives (57).
The expression of rat Oat2 mRNA in fetal liver is very low (<1% of adult levels) at the midgestational stage, increasing up to ≈50% of the adult levels at the end of gestation (39).
Mouse Oct1 is not expressed in the liver during intra-uterine life, but its mRNA is detectable at birth at ≈five-fold lower levels than in the adult liver. These are gradually increased during the first 3 weeks of age, reaching a plateau at around 3 weeks (58). In rat liver slices, Oct1-mediated uptake of the organic cation 1-methyl-4-phenyl-pyridinium was high shortly after birth and similar to adult levels (59).
Although simple diffusion may allow the transfer of COAs in both directions across the placenta, the lipophobicity because of their nature of charged molecules makes them poorly diffusible across cell membranes (60). The first evidence for the existence of carrier proteins involved in COA transport across the human placenta came from functional experiments using membrane vesicles obtained from the basal or fetal-facing pole (34, 61) and apical or maternal-facing pole (62, 63) of plasma membranes obtained from human and rat trophoblasts. The placental phase 0 for fetal COAs involves the uptake of these substances across the basal plasma membrane. Functional evidence suggests that members of the OATP family could be involved in this transport for fetal BAs (64) bilirubin (65) and biliverdin (12). Moreover, the mRNA of some of these proteins has been detected in rat (39, 66) and human (48) placenta. Qualitative and quantitative PCR analyses, plus sequencing in human trophoblastic cells, have revealed the expression of OATP1A2, OATP2B1 and OATP1B3 but not OATP1B1 (35).
OATP4A1 (previously OATP-E), which, in addition to the liver and other tissues (67), is highly expressed in human placenta (68), is believed to be a thyroid hormone carrier. However, OATP4A1 is also able to transport certain BAs (68). The rat orthologue of this transporter, i.e., Oatp4a1 (previously Oatp12), is also abundantly expressed in rat placenta (39). However, the overall role of OATP4A1 in placental phase 0 and phase III for COAs is not clear, because this carrier has been predominantly detected at the apical pole of the human syncytiotrophoblast (68). Although OATP4A1 is not considered to be a major transporter in the hepatobiliary excretory function carried out by the human liver, a role in the fetal liver–placenta–maternal liver excretory pathway cannot be ruled out.
The expression levels of the BA carriers Oatp1a1, Oatp1a4 and Oatp1b2 in rat placenta are very low under physiological conditions (39) but they are upregulated during maternal cholestasis, and even more so when pregnant rats are treated with ursodeoxycholic acid (UDCA) (66). Additional contributors to this function include OATP2B1, expressed at the basal membrane of human trophoblasts (69), and Oatp2b1 (previously Oatp9), whose mRNA is also detected in rat placenta, although at <10% of that found in adult rat liver (39).
Some members of the SLC22 family (70), such as OAT4 (SLC22A11) (71) and OCT3 (SLC22A3) (72), are highly expressed in placenta. Studies using knock-out mice have shown that Oct3 is required for the uptake of cationic drugs by the placenta (72), provides the possibility of using inhibitors of OCT3 to prevent fetal intoxication when pregnant women need to be treated with cationic drugs. OCT3 is expressed on the basal membrane of human trophoblast cells (73), which does not necessarily mean that it mediates only mother-to-placenta transport. In fact, OCT3 has been described to behave as a bidirectional organic cation transporter (74). Thus, considering the maternal–placental relationship, OCT3 can be involved in placental uptake, i.e., phase 0 processes. However, considering the fetal liver–placenta–maternal liver excretory pathway, this transporter (and probably some others) could actually also work in the opposite direction and hence contribute to the overall phase III carried out by this trio.
The expression of organic solute transporter (OST)-α in placental cells suggests that this protein may also participate in a transport system similar to that described in other epithelia, facilitating the diffusion of important steroid-related molecules across the human trophoblast (35).
Phase III transporters
Several transport systems account for phase III processes in adult hepatocytes and probably also, in part, in trophoblastic placental cells. Most, but probably not all these carriers, are ATP-dependent transporters and most of them belong to the superfamily of ATP-binding cassette (ABC) proteins. Based on their export ability across the canalicular membrane into bile or across the basolateral membrane into blood, they can be classified as phase IIIa or phase IIIb transporters respectively (Fig. 2).
The multidrug resistance family
The ABCB subfamily includes several transporters located in the canalicular plasma membrane of hepatocytes and in the plasma membrane of placental epithelium. Among them is the P-glycoprotein or multidrug resistance protein (MDR1; gene symbol ABCB1), which is probably involved in the transport of organic and inorganic cations (75). In frozen sections of fetal human tissues, MDR1 was detected in bile canaliculi. No differences in MDR1 levels were found at different stages of fetal development, which were similar than in adults (76).
Another member of the ABCB family is the previously known as the sister of the P-glycoprotein, the bile salt export pump (BSEP, gene symbol ABCB11). This is the major mechanism responsible for BA pumping into bile by the liver. However, BSEP does not seem to play a major role in the placental transfer of these compounds. Thus, although BSEP is indeed expressed in human placenta (35, 77, 78), the abundance of this protein in freshly isolated trophoblastic cells and placental cell lines is too low (35) to support a major role of BSEP in the previously described ATP-dependent BA transport across the apical membrane of the human trophoblast (63). However, higher mRNA levels have been found in early stages of pregnancy (78).
Bsep mRNA is detectable in mice at day 15 of gestation and during human embryonic life (79). Although rat Bsep mRNA can be first detected in fetal liver at the 15th day of gestation, the expression levels are still very low (<0.01% with respect to adult). These progressively increase to 30% of adult levels at birth. Bsep protein can be detected in fetal rat liver at day 20 of gestation (39). Bsep expression increases rapidly after birth to reach adult levels by 4 weeks (80). The adult phenotype of polarized expression is achieved for rat Bsep at day 12 post-natal (42). No changes in Bsep mRNA levels were seen after weaning in this species (6). BSEP has also been detected by immunohistochemical staining in human fetal liver, where the localization was intracellular and canalicular (81).
MDR3 (ABCB4), known as Mdr2 in rodents, is able to transport some toxins that are especially threatening to the liver, although its major role is the translocation of phosphatidylcholine from the inner to the outer leaflet of the lipid bilayer plasma membrane, which is critical for neutralizing the detergent effect of BAs present in bile at high concentrations (82).
Human MDR3 is expressed at the midgestational stage in fetal liver, although at significantly lower levels compared with adults (81). The immunohistochemical staining of human MDR3 was faint and only occasional canalicular patterns could be seen (81).
The expression of MDR3, as well as of another member of this family, MDR1, has been detected in the placenta throughout pregnancy (83). The abundance of mRNA of both MDR1 and MDR3 decreases during trophoblast differentiation and syncytion formation (83). Placental MDR1 expression is enhanced in early pregnancy, which presumably helps to protect the foetus from xenobiotic toxicity at a time when it is most vulnerable to such a challenge (84). At term, the expression of MDR1 is found at similar levels in the liver and human trophoblastic cells, whereas the expression of MDR3 in the human placenta is barely detectable (35).
Like MDR3, FIC1, the product of the ATP8B1 gene, seems to be involved in maintaining appropriate membrane structure and integrity. However, FIC1 is not an ABC protein but a member of the type 8 subfamily of P-type ATPases, whose function is to flip the excess of aminophospholipids from the outer to the inner leaflet of the plasma membrane (85). Surprisingly, the abundance of FIC1 mRNA in the human placenta is higher than that in the liver (35, 78). The physiological role of FIC1 in the placental transport/barrier processes is not known but it may well be a regulatory one for maintaining membrane asymmetry.
The multidrug resistance-associated protein family
Regarding multidrug resistance-associated proteins (the MRP family), the level of expression of MRP1 (ABCC1), MRP3 (ABCC3), MRP4 (ABCC4), MRP5 (ABCC5), MRP6 (ABCC6), MRP7 (ABCC10) and MRP8 (ABCC11) is very low in adult hepatocytes, but for some of them a marked upregulation in response to cholestasis (86, 87) and endotoxaemia (88) has been found. This, together with their presence at the basolateral plasma membrane, has led to the suggestion that they may serve as a compensatory overflow system under cholestatic conditions when the function of the canalicular export pumps is impaired.
MRP2 (ABCC2) plays an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, sulphate and glucuronate, such as conjugated bilirubin. MRP2 can also transport uncharged compounds in cotransport with glutathione. Unlike other members of the MRP family, MRP2 is specifically expressed in the apical membrane domain of polarized cells, including hepatocytes and placental syncytiotrophoblasts (89).
In rat fetal liver, the expression of Mrp2 is lower (<20%) than that in the adult liver. These levels increase during the first month of extra-uterine life (80).
The preference of MRP3 for glucuronidated compounds, together with its presence in tissues that have a high glucuronidating capacity, suggests that this transporter has a role in the disposal of xenobiotics and metabolic waste products after their conjugation with glucuronic acid (90). The expression of Mrp3 in fetal rat liver is very high. Thus, at birth, the levels of Mrp3 mRNA are two-fold higher than those found in adult liver (40).
MRP4 can mediate the efflux of glutathione from hepatocytes into blood by cotransport with monoanionic BAs (91). Under physiological conditions, sinusoidal MRP4 may compete with canalicular BSEP for BAs and thereby play a key role in determining the hepatocyte concentration of BAs. However, in cholestasis MRP4 may become a key pathway for the efflux of BAs from hepatocytes into the blood.
MRP5 is a low-affinity cyclic nucleotide transporter expressed ubiquitously, albeit at a low level in the liver (90), whereas MRP6 is also a primary active transporter for organic anions (92), which has a widespread tissue distribution, with high levels of mRNA in the liver (93). Mrp6 mRNA and protein in fetal rat liver are detectable at the midgestational stage (42). At day 15 of gestation, the abundance of rat Mrp6 mRNA in the fetal liver is ≈5% of adult levels (39).
MRP7 and MRP8 are lipophilic anion pumps able to confer resistance to chemotherapeutic agents. Moreover, MRP7 is competent in the transport of oestradiol 17β-d-glucuronide (E217βG) (94). In the case of murine Mrp7, the highest levels of transcript have been detected in the heart, liver, skeletal muscle and kidney (95). MRP8 is able to transport a diverse range of lipophilic anions, including cyclic nucleotides, oestradiol 17β-d-glucuronide, steroid sulphates such as DHEAS and oestrone-3-sulphate, glutathione conjugates such as leukotriene C4 and dinitrophenyl-S-glutathione and monoanionic BAs (94).
Regarding the role of MRPs in phase III processes in the placenta, several isoforms with a known ability to transport BAs have been detected. These include MRP1, MRP2 and MRP3 in human placenta (96), and Mrp1, Mrp2 and Mrp3 in rat placenta (66, 39). At least in rats, these three transporters are markedly upregulated during maternal cholestasis (66). Moreover, Mrp4 mRNA levels are 20-fold higher in placenta than in normal liver (97). This could form part of a defensive response by enhancing the barrier against the inverted gradient of BAs, which, under physiological circumstances, favours the flow from the foetus to the mother but that, under cholestatic conditions, favours the entry of these compounds from maternal blood into the trophoblast. These transporters may play a role in returning BAs towards the maternal compartment. Indeed, rat common bile duct ligation results in an increase in serum BA concentrations to above 200 μM in the mother, whereas in fetal blood they are maintained at approximately only seven-fold lower (66).
Determination of mRNA levels by real-time quantitative PCR has revealed that the expression of MRP1 mRNA is higher in human trophoblastic cells than that in the liver. In contrast, the abundance of MRP2 mRNA is higher in the human liver than in trophoblastic cells, whereas MRP3 is similarly expressed in the liver and in human trophoblastic cells. The expression of MRP4 and MRP8 is very low both in liver and in human trophoblastic cells (35). MRP5 is preferentially located in the basal membrane of syncytiotrophoblasts and in the plasma membrane of endothelial cells of chorionic vessels (98).
Breast cancer resistance protein
Also located at the canalicular membrane is the breast cancer resistance protein (BCRP, gene symbol ABCG2). Natural substrates of BCRP include endogenous compounds such as porphyrins and porphyrin-like substances (99, 100) and xenobiotics, such as pheophorbide A, a chlorophyll metabolite used as a photosensitizer in the experimental treatment of tumours (101). In rat liver, at birth Bcrp mRNA levels are ≈200% of those in adults (40).
The expression of BCRP in placenta is very high (102), which explains why this protein was also called ABC placental protein. Indeed, BCRP mRNA is more abundant in human trophoblastic cells than in liver (35). BCRP (protein and mRNA) expression in placental tissue does not decrease significantly with gestational age (84); instead, an increased expression accompanies trophoblast differentiation (83). In the human placenta, BCRP is localized mainly in the syncytiotrophoblast layer and in fetal vessels of the chorionic villi (103). The abundance of BCRP transcripts in the human term placenta has been found to be more than 10-fold higher than those of MDR1 (103). This, together with the ability of BCRP to transport BAs (104) in addition to a broad range of substrates, suggests that this transporter may play an important role in COA transfer across the placenta as well as forming part of the protection and detoxification mechanisms of the fetus (105).
Adenosine triphosphate-independent phase III transporters
Another group of transporters not directly activated by ATP and located at the basolateral membrane of hepatocytes might also favour the elimination from liver cells of potentially toxic COAs, which would subsequently be excreted by the kidney when they cannot be secreted into bile. This is the case of OATP1B3 (49) and the heterodimeric protein OSTα–OSTβ, which is able to transport oestrone 3-sulphate, DHEAS, taurocholate, digoxin and prostaglandin E2 (106). Moreover, OSTα–OSTβ is highly upregulated in several tissues in response to cholestasis (107, 108).
Ontogeny of polarity and phase IIIb transporters
In rat hepatocytes, maturation to a polarized transporter phenotype starts with the expression of Mrp2 and Mrp6 around the end of the second week of gestation and these are followed by the basolateral and canalicular bile salt transporters Ntcp and Bsep, respectively, shortly before birth (42). This is consistent with the absence during intra-uterine life of a functional secretory machinery able to excrete COAs from blood to bile. Therefore, basolateral transporters may constitute the major route for exporting the COAs produced by fetal hepatocytes. This function might be carried out by ATP-dependent and -independent mechanisms. Moreover, expression levels of several OATPs, or at least some of them that are believed to behave as bidirectional transporters (109), have been detected in fetal rat liver (39). Their intracellular localization preceded expression at the plasma membrane; full maturation of polarized Oatp expression in rat liver requires several weeks (42). Except for Oatp4a1, mRNA levels are lower in fetal than in adult liver. However, the expression of basolateral Oatps is immunohistologically detectable at the plasma membrane only after birth. This starts with Oatp1b2 during the first post-natal week, followed by Oatp1a4 after approximately 2 weeks and by Oatp1a1 after 3–4 weeks (42).
Regulation of the elements involved in the excretory function carried out by the fetal liver–placenta–maternal liver trio
The transcriptional and post-transcriptional regulation of hepatobiliary carriers and enzymes in states of health and disease is dependent on multiple factors, such as the levels of BAs, pro-inflammatory cytokines, hormones and many drugs. Hepatocyte nuclear receptors are sensitive to activation by ligands and transcription factors, which play a critical role in the transcriptional regulation of many proteins. In chronic cholestatic liver disease, many alterations could be owing to the activation of these sensors subsequent to the accumulation in liver cells of cholephilic molecules (110). Thus, BAs are able to activate the mitogen-activated protein kinases (MAPK) pathway (111, 112) and they are ligands of the membrane-bound receptor associated with G protein TGR5 (113, 114) and of nuclear receptors, such as farnesoid X receptor α (FXRα; NR1H4) (115–117). In general, under cholestatic conditions, when BA levels are elevated, an activation of the complex adaptive machinery aimed at preventing or reducing BA-induced damage occurs (118). BA-induced damage can be particularly serious in placenta (119) and fetal liver (120).
Regarding the co-ordinated regulation of these enzymes and transporters, it should be mentioned that the downregulation of OATPs results in a decreased sensitivity of the liver to chemical toxicity. In this respect, the effect of several inducers of the expression of phase I enzymes is in general opposite to that of OATP expression, with the exception of rodent Cyp3a11 and Oatp1a4, which are upregulated in parallel as a result of the activation of the nuclear receptor PXR (pregnane X receptor) (121) (Table 1).
Table 1. Nuclear receptors involved in the control of major enzymes and transporters accounting for hepatobiliary function
FTF/LRH-1 × 1
HNF4α × 2
ERα × 2
Active forms are heterodimeric form with retinoid X receptor (RXR) (+RXR); homodimeric (× 2) and monomeric (× 1).
Nuclear receptors: CAR, constitutive androstane receptor; ER, oestrogen receptor; FTF/LRH, α-foetoprotein transcription factor/liver receptor homologous; FXR, farnesoid X receptor; HNF, hepatic nuclear factor; LXR, liver X receptor; PPAR, peroxisome proliferator-activated receptor; PXR/SXR, pregnane X receptor/steroid and xenobiotic receptor; VDR, vitamin D receptor. Enzymes and transporters: ABC, ATP-binding cassette; BACS, bile acid-CoA synthetase; BAT, bile acid-CoA: amino acid N-acetyltransferase; BSEP, bile salt export pump; CYP, cytochrome P450 enzymes; FGF, fibroblast growth factor; MDR, multidrug resistance; MRP, multidrug resistance-associated protein; NTCP, sodium/taurocholate-cotransporting polypeptide; OATP, organic anion-transporting polypeptide; OST, organic solute transporter; SULT, sulphotransferases; UGT, UDP-glucuronosyltransferases.
Indirect through HNF4α.
†In combination with other nuclear receptors, such as PXR and CAR.
Human nuclear receptors constitute a group of 49 members whose activation requires interaction with more or less specific ligands (168). Once activated by their ligands, which include steroid hormones, free fatty acids, oxysterols, etc., nuclear receptors modify the transcription rate of a large number of genes that usually contribute to adapting cell metabolism to specific changes (169). Thus, several nuclear receptors are involved in the control of mechanisms involved in the metabolism and transport of substrates detoxified by the hepatobiliary system. These include, in addition to FXR, several isoforms of peroxisome proliferator-activated receptor (PPAR, NR1C), liver X receptors (LXRa and LXRb, NR1H3 and NR1H2 respectively), human xenobiotic receptor SXR (steroid and xenobiotic receptor, NR1I2) and its rodent homologue PXR. These nuclear receptors belong to class II, whose members need to form heterodimers with the retinoid X receptor (RXR; NR2B1) (169). The constitutive androstane receptor (CAR; NR1I3) can be activated by bilirubin (170), although it is also sensitive to certain species of BAs (171). Some of these nuclear receptors have also been detected in the placenta (35).
In contrast to PXR/SXR, which are normally located in the nucleus, CAR resides in the cytoplasm and is translocated to the nucleus only upon activation by binding with the ligand (152). The small heterodimer partner (SHP, NR0B2) is an atypical member of the superfamily of nuclear receptors, because it is able to act directly as a transcriptional repressor (172) and to interact with several other nuclear receptors, in most cases inducing their inactivation (173). However, SHP is also able to stimulate the transcription of nuclear receptors, such as PPARγ (174) and PPARα (175).
The role of nuclear receptors in the transcriptional control of the genes involved in the hepatobiliary function is summarized in Table 1.
The expression of oestrogen receptor-α (ERα) is enhanced in female rat liver during pregnancy and early post-partum (176). In contrast, in mice there is a decrease in the expression of several nuclear receptors involved in the control of lipid metabolism. These include RXRs, PPARs, LXRs and FXR. This could contribute to the alterations in lipid metabolism during late pregnancy (177).
Most receptors involved in COA metabolism and transport have already been found to be expressed in human placenta (35). Moreover, evidence for the expression in rat and human placenta of related nuclear receptors, such as liver receptor homologue-1 (LRH-1) (178), vitamin D receptor (VDR) (179), ERs (180) and PPARs (181, 182), has been reported.
With some exceptions, including RXRα and hepatocyte nuclear factor 4α (HNF4α), which are highly expressed in fetal human liver (183), most nuclear receptors mentioned above have already been found to be expressed during intra-uterine life in human (183, 184) and rat (185–187) livers, but at levels lower than in adult livers. As mentioned, this is not the case for HNF4α, which seems to be the key transcription factor regulating responses to xenobiotics through activation of the PXR gene during fetal liver development (188).
Several of the elements involved in hepatobiliary function are also regulated post-transcriptionally. For instance, cAMP controls the amount of NTCP in the plasma membrane by activating its dephosphorylation. On the contrary, phosphorylated (Ser/Thr) NTCP is retrieved from the plasma membrane (189), and hence sodium-dependent BA transport is reduced (190). The pathway of this control system involves the activation of phosphatidylinositol 3-kinase/protein kinaseCζ (PIK3/PKCζ) followed by the activation of the PIK3/PKB route (191). Moreover, insertion into the plasma membrane of some of the transporters can be modified by exchange with intracellular pools of these transporters (192). This concept is consistent with the existence of endosomal pools containing reversible reservoirs of ABC proteins (193–195).
Several stimuli have been suggested to activate the mechanism controlling the insertion and retrieval of hepatobiliary transporters. Among these stimuli are the osmolarity of the medium, the levels of cAMP and Ca2+ (196), the activation of PKC, PIK3 and MAPKs (197). This has interesting pharmacological implications. Thus, the anticholestatic effect of amidated derivatives of UDCA is due in part to stimulation of the insertion into the canalicular plasma membrane of export pumps, such as BSEP and MRP2 (198).
Effect of maternal cholestasis on phase I/II enzymes
In adult rat liver, extrahepatic obstructive cholestasis exerts different effects on the expression of different CYP isoforms. This varies from absence of change (CYP1A1 and CYP2C6) to a moderate (CYP2B and CYP2E1) or marked (CYP2C11 and CYP3A2) decrease (199). In fetal liver, the expression of neither CYP7A1, CYP27 or CYP8B1, involved in BA synthesis, is affected by maternal cholestasis. In contrast, under these circumstances upregulation of biliverdin reductase has been detected in fetal liver, placenta and maternal liver (120).
In human placentas collected after delivery from patients who had suffered ICP, a reduction in the drug detoxifying ability has been reported (200).
Cholestasis during pregnancy also reduces GST expression in rat placenta (119) as well as in fetal and maternal livers (120).
Hepatobiliary transporters in the aetiopathogenesis of cholestasis of pregnancy
Hepatocellular cholestasis appears in some pregnant women, usually in the third trimester of pregnancy, and resolves soon after delivery. In some cases, probably affecting to 10% of pregnancies, the condition called asymptomatic hypercholanaemia of pregnancy (AHP) remains at the subclinical level and only hypercholanaemia is detected as a sign in biochemical analyses (201, 202). The impairment in bile formation observed in AHP probably reflects a continuum between normal pregnancy and clinically overt ICP. Although the aetiology of ICP is probably multifactorial, involving predisposing dietary, hormonal and genetic factors (203, 204), the impairment of the expression or the function of hepatobiliary transporters seems to be involved in most cases. It is very probable that the co-existence of combined heterozygous alterations affecting several canalicular transporters would determine the predisposition to develop ICP and also the severity of the impairment in biliary function (205). In addition, patients harbouring mutations of hepatobiliary transporters may develop cholestasis during pregnancy as the first manifestation of a potentially progressive liver disease (206). Thus, mutations in ATP8B1, ABCB11 and ABCB4 genes may be involved in the aetiology of ICP (207) whereas, pregnancy may unmask a hitherto undiagnosed Dubin–Johnson syndrome, caused by mutations in the ABCC2 gene (208, 209).
Alterations in the ATP8B1 gene have been associated with progressive type 1 familial intrahepatic cholestasis (PFIC) or PFIC1, also known as Byler's syndrome (210). A variant with a less serious prognosis is type 1 benign recurrent intrahepatic cholestasis (BRIC1). The incidence of ICP in the mothers of children suffering from PFIC1 or BRIC1 is higher than in the rest of the population (211, 212), probably because these women bore heterozygote mutations in the ATP8B1 gene, which predisposed them to ICP (203). Moreover, pregnancy may be a triggering factor of cholestatic episodes in patients with BRIC1 (213). Alterations in the ABCB11 gene accounting for defective BSEP, have been identified in patients with PFIC2 (214). Mutations in this gene have also been associated with BRIC2 (215). Although some variants in the ABCB11 gene are probably not relevant for the development of ICP (206), other mutations in this gene seem to be more frequently and more clearly linked to ICP, because they result in a reduction in the amount of functional BSEP in the canalicular membrane because of protein instability or mistargeting (216).
Homozygous inactivating mutations of the ABCB4 gene result in a lack of normal MDR3, leading to PFIC3 (217). The fact that the heterozygous mother of a child affected with PFIC3 was reported to experience recurrent episodes of ICP first suggested a role for MDR3 in the aetiology of this disease (217). This link has been confirmed in further studies (205, 218, 219). A heterozygous mis-sense mutation in the ABCB4 gene in a patient with ICP with no known family history of PFIC has been reported (219). A total of 51 variant sites were detected in the ABCB4 gene when 21 pregnant women with ICP were investigated (206). Moreover, the findings of this latter study support the hypothesis that MDR3 genetic variation may be involved in the pathogenesis of ICP (206). In a pregnant woman with ICP, a homozygous MDR3 gene mutation (S320F) was also associated with early onset and the severity of the clinical condition (205). Recently, splicing mutations in the ABCB4 gene in up to 20% of ICP women have been described (220).
Although the genetic background seems to play a key role in predisposing to the development of ICP, other factors probably act as triggers for the functional impairment. Several lines of evidence support a major role of hormonal factors in the development of ICP (203, 221, 222). Thus: (i) ICP frequently appears when placental oestrogen synthesis is maximal, i.e., in the third trimester of pregnancy; (ii) moreover, the symptoms of ICP disappear soon after delivery, when levels of placental hormones reach normal values; (iii) in patients with ICP, this condition re-occurs in 45–70% of the cases; and (iv) the incidence of ICP and the levels of placental hormones are more elevated in twin pregnancies.
Among the placental hormones, steroids (oestrogens and progesterone derivatives) are the main candidates to be involved in the aetiology of ICP. Progesterone metabolites seem to play an even more important role than oestrogens in the aetiopathogenesis of ICP. Because oral contraceptive therapies based on progestin have been found to induce cholestasis in sensitive women (223), a selective defect in the secretion of sulphated progesterone metabolites into bile has been suggested to trigger ICP in genetically predisposed individuals (224). Indeed, the profile of progesterone metabolites in the plasma of patients with ICP is markedly different from that seen in normal pregnancy (225). Thus, sulphated derivatives of two pregnanediol isomers, 5α-pregnane-3α,20α-diol, and 5P-pregnane-3α,20α-diol are elevated in ICP. An interesting question arises as to whether an abnormal accumulation of steroid metabolites in ICP is primary or secondary to cholestasis. Because biliary elimination of sulphated and glucuronated progesterone metabolites is decreased in patients with ICP, it has been suggested that the increase in the serum concentrations of progesterone metabolites might be the consequence rather than the cause of ICP (226). However, this is unlikely because pregnant women with cholestasis because of other causes, such as viral hepatitis, have a normal profile of steroid sulphates in blood. This observation suggests that the presence of a typical pattern of sulphated progesterone metabolites can have a causative role in the aetiopathogenesis of ICP (227). Moreover, in AHP, the absence of clinical manifestations during a mild accumulation in serum of BAs, but not other biliary compounds, is accompanied by enhanced levels of progesterone metabolites in the serum of these pregnant women (202), more specifically, sulphated derivatives of progesterone metabolites, such as 5α-pregnan-3α-ol-20-one (202), which have strong cholestatic ability (228).
The metabolites of steroid hormones may interfere with the normal function of hepatobiliary transporters by both direct and indirect effects: (i) direct inhibition of BA secretion is probably due in part to the trans-inhibition of BSEP (228), upon secretion of hormone metabolites into the bile canaliculi by MRP2 (229), because, at least in rodents, Mrp2 is required for oestrogens and progesterone metabolites to induce the harmful effect (230, 231); (ii) oestrogens are also able to interfere with the microtubular system affecting the normal turnover of insertion/retrieval of rat Bsep in the canalicular membrane (232); (iii) oestrogens may also affect hepatobiliary transporters by modifying membrane fluidity and hence the efficacy of plasma membrane transporters (233); (iv) moreover, oestrogen-induced cholestasis in rats is accompanied at midterm by modifications in the expression of transporters such as rat Ntcp varying from −21 (234) to −80% (234), as well as Oatp1a1, Oatp1a4 and Oatp1b2 varying from −21 (233) to 40% (234). Oestrogen derivatives also markedly inhibit the expression of Mrp2 (235).
Concluding remarks and future perspectives
During pregnancy, the lack of hepatobiliary function of the fetal liver requires the existence of alternative mechanisms to carry out biotransformation and elimination of COAs produced by the fetus. This task is performed mainly by the trio formed by the fetal liver, the placenta and the maternal liver. The former organ expresses, at a rather low degree, most mechanisms of biotransformation, transport and regulation. Only some export pumps involved in phase IIIb detoxification process are highly expressed in fetal liver, probably favouring the transfer of COAs to fetal blood from where they are taken up biotransformed and transported towards the maternal blood by the placenta. This organ and the maternal liver are well equipped to account for an appropriate detoxification procedure. Further improvement of our knowledge on the mechanisms involved in this function and their control is necessary in order to better understand the aetiopathogenesis of several pregnancy-associated diseases, such as ICP, and some alterations of intra-uterine growth and liver maturation, as well as to develop novel pharmacological strategies to treat these patients.
Financial support: This study was supported in part by the Junta de Castilla y Leon (Grants SA013/04; SAN/191/2006 and SA059A05), Spain; Ministerio de Ciencia y Tecnologia, Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (Grant BFI2003-03208), Spain; Fundacion Investigacion Medica Mutua Madrileña (Conv-III, 2006); and Instituto de Salud Carlos III, FIS (Grants CP05/00135, PI051547 and PI070517). The group is a member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educacion y Ciencia, Spain, and the European Regional Development Fund (ERDF) (Grant BFU2005-24983-E/BFI) and belongs to the ‘Centro de Investigacion Biomedica en Red’ for Hepatology and Gastroenterology Research (CIBERehd), Instituto de Salud Carlos III, Spain. Marco Arrese was partially supported by a grant from Fondo Nacional de Desarrollo Científico y Tecnológico de Chile (FONDECYT#1050780).