Neuro-inflammatory effects of photodegradative products of bilirubin.

Phototherapy was introduced in the early 1950's, and is the primary treatment of severe neonatal jaundice or Crigler-Najjar syndrome. Nevertheless, the potential biological effects of the products generated from the photodegradation of bilirubin during phototherapy remain unknown. This is very relevant in light of recent clinical observations demonstrating that the use of aggressive phototherapy can increase morbidity or even mortality, in extremely low birthweight (ELBW) infants. The aim of our study was to investigate the effects of bilirubin, lumirubin (LR, its major photo-oxidative product), and BOX A and B (its monopyrrolic oxidative products) on the central nervous system (CNS) using in vitro and ex vivo experimental models. The effects of bilirubin photoproducts on cell viability and expression of selected genes were tested in human fibroblasts, three human CNS cell lines (neuroblastoma SH-SY5Y, microglial HMC3, and glioblastoma U-87 cell lines), and organotypic rat hippocampal slices. Neither bilirubin nor its photo-oxidative products affected cell viability in any of our models. In contrast, LR in biologically-relevant concentrations (25 µM) significantly increased gene expression of several pro-inflammatory genes as well as production of TNF-α in organotypic rat hippocampal slices. These findings might underlie the adverse outcomes observed in ELBW infants undergoing aggressive phototherapy.

Repeated AAV-mediated gene transfer by serotype switching enables long-lasting therapeutic levels of hUgt1a1 enzyme in a mouse model of Crigler-Najjar Syndrome Type I.

Adeno-associated virus (AAV) -mediated gene therapy is a promising strategy to treat liver-based monogenic diseases. However, two major obstacles limit its success: first, vector dilution in actively dividing cells, such as hepatocytes in neonates/children, due to the non-integrating nature of the vector; second, development of an immune response against the transgene and/or viral vector. Crigler-Najjar Syndrome Type I is a rare monogenic disease with neonatal onset, caused by mutations in the liver-specific UGT1 gene, with toxic accumulation of unconjugated bilirubin in plasma, tissues and brain. To establish an effective and long lasting cure, we applied AAV-mediated liver gene therapy to a relevant mouse model of the disease. Repeated gene transfer to adults by AAV-serotype switching, upon neonatal administration, resulted in lifelong correction of total bilirubin (TB) levels in both genders. In contrast, vector loss over time was observed after a single neonatal administration. Adult administration resulted in lifelong TB levels correction in male, but not female Ugt1-/- mice. Our findings demonstrate that neonatal AAV-mediated gene transfer to the liver supports a second transfer of the therapeutic vector, by preventing the induction of an immune response and supporting the possibility to improve AAV-therapeutic efficacy by repeated administration.

Dissimilar expression of multidrug resistance mdr1 and bcrp by the replication of hepatitis C virus: role of the nonstructural 5A protein.

Multidrug resistance associated with the overexpression of ATP-dependent binding cassette (ABC) proteins is widely accepted as an important cause of treatment failure in patients with neoplastic or infectious diseases. Some of them play also a pivotal role in detoxification processes. Herein, we investigated the effect of hepatitis C virus (HCV) replication and nonstructural 5A (NS5A) protein on the expression and functional activity of two ABC transport proteins: MDR1 and BCRP. RT-quantitative real-time polymerase chain reaction (qPCR) was carried out for mdr1 and bcrp mRNAs in both Huh7 cells expressing NS5A and Huh7.5 cells containing either full-length- or subgenomic-HCV replicon systems. The functional activity of these pumps was studied by performing a dye efflux assay with DiOC2 and Rhodamine 123. A dose-dependent down-regulation of mdr1 expression was documented in Huh7 cells expressing the NS5A protein, as well as in both replicon systems. In contrast, a significant increase of bcrp expression in both systems was recorded, which were in full agreement with the dye efflux assay results. These results warrant further in vivo studies in HCV patients with cholestasis and/or patients that are refractive to the pharmacotherapy due to the activity of these pumps.

HCV, HBV and alcohol - the Dionysos study.

Population-based studies on the natural history of chronic viral liver disease that consider co-morbidity factors, such as alcohol or metabolic diseases, are lacking. We report here the contribution of ethanol intake and non-organ-specific autoantibodies (NOSA) to the course of chronic viral disease in the Dionysos cohort. As reported elsewhere, the Dionysos study was performed in two towns of Northern Italy, started in 1992 with 10 years of follow-up in 2002, and allowed us to quantify the burden of chronic liver disease in Northern Italy. We followed 139 subjects with chronic hepatitis C virus (HCV) infection and 61 with chronic hepatitis B virus (HBV) infection for a median (IQR) time of 8.4 (1.0) and 8.3 (0.9) years, respectively. The incidence and remission rates of steatosis were 9.0 and 29.7 per 1,000 person-years in the HCV cohort and 4.0 and 30.4 per 1,000 person-years in the HBV cohort. Progression to cirrhosis and hepatocarcinoma was more common in the HCV than in the HBV cohort. In the HCV cohort, ethanol intake was an independent predictor of liver cirrhosis and of death rate in both cohorts. We found no association between baseline NOSA and 8.4-year mortality. We conclude that morbidity and mortality rate of HBV and HCV infection in the general population is lower than that reported in secondary care populations, blood donors, or clinical series, and that ethanol intake >30 g/day is the most important and evitable risk factor for cirrhosis and death in patients with chronic HCV or HBV infection.

A protective effect of breastfeeding on the progression of non-alcoholic fatty liver disease.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disease characterised by accumulation of large-droplet fat in hepatocytes with possible progression to inflammation and fibrosis. Breastfeeding has benefits for child health, both during infancy and later in life, reducing the risk of manifestations of the metabolic syndrome. Here we investigated the association between early type of feeding (breastfed versus formula-fed and duration of breastfeeding) and later NAFLD development. STUDY DESIGN: We investigated 191 young Caucasian children (3-18 years old) with NAFLD consecutively enrolled between January 2003 and September 2007 in our centre. 48% of these children (n = 91) had been breastfed for a median (interquartile range) time of 8 (7) months. RESULTS: After correction for age, waist circumference, gestational age and neonatal weight, the odds of non-alcoholic steatohepatitis (NASH) (OR 0.04, 95% CI 0.01 to 0.10) and fibrosis (OR 0.32, 95% CI 0.16 to 0.65) were lower in breastfed versus not breastfed infants. Moreover, the odds of NASH (OR 0.70, exact 95% CI 0.001 to 0.87) and fibrosis (OR 0.86, exact 95% CI 0.75 to 0.98) decreased for every month of breastfeeding. CONCLUSIONS: This observational study suggests that earlier feeding habits might affect the clinical expression of NASH from 3 to 18 years later, with an apparent drug-like preventive effect of breastfeeding.

The expression levels of the translational factors eEF1A 1/2 correlate with cell growth but not apoptosis in hepatocellular carcinoma cell lines with different differentiation grade.

Despite the involvement of the elongation factors eEF1A (eEF1A1 and eEF1A2) in the development of different cancers no information is available on their possible contribution to the biology of hepatocellular carcinoma (HCC). We investigated the expression of both forms of eEF1A in HepG2 and JHH6 cell lines considered to be a good in vitro model of HCC at different stage of differentiation. Our data indicate that the mRNA amount of eEF1A1 is increased in both cell lines as compared to normal liver tissue, but eEF1A2 mRNA level is markedly increased only in JHH6. Moreover, the less differentiated cell line JHH6 displays higher EEF1A1 and EEF1A2 mRNAs levels and an higher nuclear-enriched/cytoplasm ratio of EEF1A protein compared to the better differentiated HepG2 cell line. Over-expression depends only partially on gene amplification. The more abundant mRNA levels and the higher nuclear-enriched/cytoplasm ratio of eEF1A in JHH6 neither correlate with apoptosis resistance nor with proliferation rate in sub-confluent cells. However, in confluent cells, a clear tendency to maintain JHH6 into the cell cycle was observed. In conclusion, we document the increased mRNA levels of EEF1A genes in HCC cell lines compared to normal liver. Additionally, we show the increased nuclear-enriched/cytoplasmic protein ratio of eEF1A and the marked raise of the expression of both eEF1A forms in JHH6 compared to HepG2, suggesting the possibility that eEF1A forms might become a relevant markers related to HCC tumor phenotype.

Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin.

Nerve cell injury by unconjugated bilirubin (UCB) has been implicated in brain damage during neonatal hyperbilirubinemia, particularly in the preterm newborn. Recently, it was shown that UCB is a substrate for the multidrug resistance-associated protein 1 (Mrp1), an ATP-dependent efflux pump, which may decrease UCB intracellular levels. To obtain a further insight into the role of Mrp1 in the increased vulnerability of immature cells to UCB, we evaluated the mRNA and the protein levels of Mrp1 throughout differentiation in primary cultures of rat neurons and astrocytes. Furthermore, in order to provide supportive evidence for the role of Mrp1 in the protection of nerve cells from UCB-induced effects, we evaluated cell susceptibility to UCB when Mrp1 was inhibited with MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl) ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid). The results are the first to demonstrate that Mrp1 is expressed in neurons and that both mRNA and protein levels of Mrp1 increase with cell differentiation. Additionally, inhibition of Mrp1 was associated with an increase in UCB toxic effects, namely cell death, cell dysfunction, and secretion of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, as well as of glutamate. These results point to a novel role of Mrp1 in the susceptibility of premature babies to UCB encephalopathy, and provide a startup point for the development of a new therapeutic strategy.

HBV, HCV, and TTV detection by in situ polymerase chain reaction could reveal occult infection in hepatocellular carcinoma: comparison with blood markers.

OBJECTIVE: To report a retrospective analysis on the presence of hepatitis B virus (HBV), hepatitis C virus (HCV), and transfusion transmitted virus (TTV) sequences in formalin fixed, paraffin embedded liver biopsies from eight patients with hepatocellular carcinoma, in comparison with blood markers. METHODS: A direct in situ polymerase chain reaction (PCR) technique was developed for the detection and localisation of genomic signals in the liver tissue. Conventional serological and molecular methods were used for blood evaluation. RESULTS: In situ PCR showed the presence of one of the three viruses (four HCV, two HBV, and one TTV) in seven of the eight patients. In addition, a co-infection with HBV and HCV was detected in one patient. HCV and HBV sequences were located in the cytoplasm and the nucleus, respectively. When compared with blood markers, these findings were compatible with one occult HBV and two occult HCV infections. CONCLUSIONS: These findings provide further evidence for occult HBV and HCV infections in cancerous tissues from patients with hepatocellular carcinomas. In situ PCR could be an additional tool for evaluating the viral aetiology of hepatocellular carcinoma alongside conventional diagnostic procedures.

Body mass index is a good predictor of an elevated alanine transaminase level in the general population: hints from the Dionysos study.

AIM: To establish the contribution of body mass index (BMI), sex, age, ethanol intake, hepatitis B (HBV) and hepatitis C (HCV) virus infection, coffee and drug consumption, and cigarette smoking to account for an elevated alanine transaminase (ALT) level in the general population. SUBJECTS: A total of 6315 adult subjects from the Dionysos study. METHODS: Logistic regression was used to quantify the contribution of the variables of interest to elevated ALT, defined as a value of ALT>60 U/l. Areas under ROC curves (AUCs) were calculated to assess accuracy. RESULTS: All the variables considered, with the exception of coffee and drug consumption, were significant predictors of elevated ALT at univariable analyses. When significant predictors were employed in a multivariable model, age and cigarette smoking were no longer significant. The AUC was 0.77 (95% CI=0.74-0.80) for the multivariable model and 0.64 (95% CI=0.60-0.68) for the univariable BMI model (p<0.0001 for the comparison). CONCLUSION: BMI is a good predictor of elevated ALT serum activity in the general population. The ability to predict an elevated ALT is however increased substantially by considering sex, ethanol intake, HBV and HCV infection together with BMI.

Evidence for carrier-mediated transport of unconjugated bilirubin across plasma membrane vesicles from human placental trophoblast.

Unconjugated bilirubin (UCB) is currently believed to cross the placenta only by passive diffusion. To assess whether carrier-mediated transport might be involved, the uptake of [(3)H]-UCB by basal (bTPM) and apical (aTPM) plasma membrane vesicles from human placental trophoblast at term was investigated. In both types of vesicles, the uptake of [(3)H]-UCB into an osmotically sensitive space was temperature-dependent, independent of the presence of Na(+), and not affected by changes in membrane potential. The uptake of [(3)H]-UCB by aTPM, but not bTPM, was activated by ATP hydrolysis and inhibited by vanadate. Thus, the exact contribution of both inside out and right-side out bTPM to UCB uptake could not be distinguished, while only inverted aTPM were expected to carry out ATP-dependent UCB uptake. In bTPM and aTPM, uptake of free (unbound) [(3)H]-UCB (B(f)) consisted of a dominant, saturable, presumably carrier-mediated process and a diffusional component that became predominant only at B(f) near or above aqueous solubility limit for UCB (70 nM ). For bTPM, K(m)=7.2 nM; V(max)=9.8 pmol/20s/mg protein; and diffusion coefficient (K(D))=0.14 ml/20s/mg protein. For aTPM in the presence of 9.5m M ATP, K(m)=18 n M; V(max)=131 pmol/20s/mg protein; and K(D)=0.47 ml/20s/mg protein. The uptake of [(3)H]-UCB by bTPM was cis-inhibited by estrone-3-sulfate and estradiol-17 beta-glucuronide and trans-stimulated by unlabelled UCB and bromosulphopthalein. ATP-dependent UCB uptake by aTPM was cis-inhibited by doxorubicin, cholic acid, methotrexate and pronenecid. These findings suggest the presence of distinct transporters in the two domains of human placental trophoblast that could cooperate to transfer UCB from the foetus to the maternal circulation.

In situ polymerase chain reaction detection of transfusion-transmitted virus in liver biopsy.

The potential role of transfusion-transmitted virus (TTV) infection in determining liver damage is poorly understood and no information exists about TTV replication within hepatocytes. In this study, we assess TTV in situ PCR in liver tissue. Twenty-one patients with different degrees of liver damage were studied by both serum TTV-DNA detection and in situ TTV PCR analysis and extractive PCR in liver biopsy paraffin sections (FFPE). Extractive PCR and in situ PCR detected TTV-DNA both in serum and liver tissue of five patients. The presence of TTV in serum matched with that found in the liver and TTV sequences were never found independently in liver or serum. Four out of five TTV-DNA-positive patients have not other known cause of liver damage while in one a coinfection from HCV was observed. Our data indicate that in situ PCR appears to be a reliable tool for the detection of TTV-DNA in FFPE, and may help detecting unknown origin of liver damage.

Preparation of an antibody recognizing both human and rodent MRP1.

Based on the high level of identity among human, mouse, and rat MRP1 protein sequence, we produced a specific polyclonal antibody (MRP1-A23) against a synthetic polypeptide covering the C-terminus of the human protein. Western blot analysis showed a reactivity against human MRP1 similar to that obtained with the monoclonal QCRL1 antibody. Differently from other available antibodies against human MPR1, MRP1-A23 also detected both rat and mouse MRP1. No cross-reactivity was observed with either human or mouse MRP2 while MRP1-A23 weakly cross-reacted with rat MRP2 in the protein region ranging from 1512 to 1533 amino acids. These data indicate that MRP1-A23 allows specific MRP1 detection in both human and rodent tissues and may provide an important tool in the study of MRP1 expression and function in both experimental and clinical materials.

Tauroursodeoxycholic acid protects hepatocytes from ethanol-fed rats against tumor necrosis factor-induced cell death by replenishing mitochondrial glutathione.

Mitochondrial glutathione (GSH) plays a key role against tumor necrosis factor alpha (TNF)-induced apoptosis because its depletion is known to sensitize hepatocytes to TNF. The present study examined the role of tauroursodeoxycholic acid (TUDCA) administration to chronic ethanol-fed rats on mitochondrial GSH levels and kinetics, mitochondrial membrane physical properties, TNF-induced peroxide formation, and subsequent hepatocyte survival. TUDCA selectively increased the levels of GSH in mitochondria without an effect on cytosolic GSH. This outcome was accompanied by improved initial rate of GSH transport examined at low (1 mmol/L) and high (10 mmol/L) GSH concentrations both in intact mitochondria and mitoplasts prepared from ethanol-fed livers. Assessment of membrane fluidity revealed an increased order parameter in mitochondria and mitoplasts from ethanol-fed rats compared with pair-fed controls, which was prevented by TUDCA administration. Compared with hepatocytes from pair-fed rats, TNF stimulated peroxide generation in hepatocytes from ethanol-fed rats, preceding TNF-induced cell death. Administration of TUDCA to ethanol-fed rats prevented TNF-induced peroxide formation and cell death, an effect that was reversed on depletion of the recovered mitochondrial GSH levels by (R,S)-3-hydroxy-4-pentenoate before TNF treatment. The protective effect of TUDCA against TNF was not because of activation of phosphatidylinositol 3-kinase, discarding a role for a survival-dependent pathway. Thus, these findings reveal a novel role of TUDCA in protecting hepatocytes in long-term ethanol-fed rats through modulation of mitochondrial membrane fluidity and subsequent normalization of mitochondrial GSH levels.

High prevalence of celiac disease in Italian general population.

The worldwide increase of celiac disease prompted us to assess its prevalence in the Italian general population. The 3483 inhabitants of Campogalliano were tested for immunoglobulin A anti-endomysial antibodies. Twenty subjects showed antibody positivity and duodenal biopsy detected typical mucosal lesions of celiac disease in 17 of them; the remaining three cases had a normal villous architecture, but the finding of increased gamma/delta intraepithelial lymphocytes in all and the heterodimer DQA1*0501, DQB1*0201 in two of them was consistent with potential celiac disease. Only one patient had an overt malabsorption syndrome, characterized by diarrhea, weight loss, and severe weakness. In eight subjects atypical symptoms of celiac disease, such as dyspepsia and depression, were present, whereas the remaining subjects were silent. Celiac disease was more frequent in younger age groups. Our cross-sectional design study demonstrates that celiac disease prevalence in the Italian general population is 4.9 per 1000 (95% CI 2.8-7.8), increasing up to 5.7 per 1000 (95% CI 3.5-8.8) with the inclusion of potential cases.

Genetic determinants of ethanol-induced liver damage.

BACKGROUND: Although a clear correlation exists between cumulative alcohol intake and liver disease, only some of the alcohol abusers develop signs of ethanol-induced liver damage. To identify some of the genetic variations predisposing persons to alcoholic liver disease (ALD), a genetic study was performed in heavy drinkers from the cohort of the Dionysis study, a survey aimed at evaluating liver disease in the open population of two towns in Northern Italy (6917 individuals). MATERIALS AND METHODS: 158 heavy drinkers (approximately 85% of all heavy drinkers in the population; daily alcohol intake > 120 g in males and >60 g in females) were investigated by the analysis of nine polymorphic regions, mapping in exons III and IX of the alcohol-dehydrogenase (ADH)-2 gene, in exon VIII of the ADH3 gene, in intron VI, in the promoter region of the cytochrome P4502E1 (CYP2E1) gene, and in the promoter region of the tumor necrosis factor-alpha gene. RESULTS: Heavy drinkers with or without ALD significantly differed for the distribution of alleles of the cytochrome P4502E1 (CYP2E1) and alcohol-dehydrogenase-3 (ADH-3) genes. In one town, allele C2 in the promoter region of the CYP2E1 gene had a frequency of 0.06 in healthy heavy drinkers, of 0.19 in heavy drinkers with ALD (p = 0.012), and of 0.33 in heavy drinkers with cirrhosis (p = 0.033). In the other town, whose inhabitants have different genetic derivation, a prominent association between ALD and homozygosity for allele ADH3*2 of ADH3 was found, with a prevalence of 0.31 in heavy drinkers with ALD and of 0.07 in healthy heavy drinkers controls (p = 0.004). CONCLUSIONS. Both heterozygosity for allele C2 of CYP2E1 and homozygosity for allele ADH3*2 of ADH3 are independent risk factors for ALD in alcohol abusers. The relative contribution of these genotypes to ALD is dependent on their frequency in the population. Overall, heavy drinkers lacking either of these two genotypes are 3.2 and 4.3 times more protected from developing ALD and cirrhosis respectively.

DNA oxidative damage in leukocytes correlates with the severity of HCV-related liver disease: validation in an open population study.

BACKGROUND/AIMS: Oxidative DNA damage, identifiable in the formation of 8-hydroxydeoxyguanosine (8-OHdG), is relevant in the mutagenesis/carcinogenesis process. The aim of this study was to assess 8-OHdG levels in patients with hepatitis C virus (HCV) infection in relation to extent of liver damage and HCV genotype. METHODS: 8-OHdG levels were measured in DNA from circulating leukocytes of 110 anti-HCV positive subjects belonging to the population of the Dionysos study, subgrouped in: 50 anti-HCV+ with persistently normal ALT, 48 with chronic hepatitis and 12 with cirrhosis. Twenty normal subjects served as Controls. 8-OHdG levels were assayed by HPLC/electrochemical detector. RESULTS: 8-OHdG levels rose (P < 0.00001) from Controls to HCV+; chronic hepatitis and cirrhosis were associated with a further increase (P < 0.02 versus HCV+). Genotype 1 was associated with higher levels of 8-OHdG (P < 0.04). Multiple logistic regression analysis showed that, after correction for potential confoundings, 8-OHdG levels correlated (P < 0.02) with presence and extent of liver damage. CONCLUSIONS: An accumulation of 8-OHdG in circulating leukocytes is a reliable marker of the extent of liver damage in HCV+ patients and is present in particular in genotype 1 infection. This genomic damage may contribute to liver carcinogenesis by causing persistent DNA changes.

Abc protein transport of MRI contrast agents in canalicular rat liver plasma vesicles and yeast vacuoles.

The mechanism of excretion into bile of hepatospecific magnetic resonance imaging (MRI) contrast media employed labeled Gd-reagents EOB.DTPA, BOPTA, B 20790 (iopanoate-linked), and B 21690 (glycocholate-linked) for measurement in rat liver canalicular plasma membrane vesicles and yeast vacuoles. The presence of ATP gave threefold greater transport of B 20790 and B 21690 than of EOB.DTPA and BOPTA. In yeast vacuoles the ATP stimulatory effect was eightfold with B 20790 and fivefold greater for B 21690, whereas in YCF1- or YLLO115w-deleted yeast cells the transport was significantly reduced and absent from double mutants, YCF1 and YLLO15w. The transport was similar in wild-type and deletant cells for B 21690; taurocholate gave 85% inhibition. These data suggest that bilary secretion of structurally related MRI agents depend on molecular structure. The findings are suggestive as of possible value for clinical diagnosis of inherited hyperbilirubinemias and other liver disorders.

Detection of MRP1 mRNA in human tumors and tumor cell lines by in situ RT-PCR.

The detection of the multridrug resistance-associated proteins is becoming increasingly important in assessing tumor sensitivity to treatment. In this work we describe a new, rapid, sensitive, and robust method for the detection of MRP1 expression based on direct RT-in situ-PCR technology and fluorochrome-modified (dCTP(Cy3)) nucleotides. MRP1 expression was found in both placenta (BeWo) and liver (Hep G2)-derived tumor cell line as well as in small cell lung carcinoma. In liver-derived cells, MRP1 expression was detected by RT-in situ-PCR but not by in situ hybridization, suggesting a higher sensitivity of in situ amplification for the low level of expression in Hep G2 cells. RT solution PCR confirmed the presence of MRP1 in BeWo and Hep G2 cells, although the level of the gene expression was lower in liver cells. This method represents a viable alternative to conventional immunohistochemistry, and may be useful in the evaluation of MRP1 expression in different tissue or cell lines.

The products of YCF1 and YLL015w (BPT1) cooperate for the ATP-dependent vacuolar transport of unconjugated bilirubin in Saccharomyces cerevisiae.

Since bilirubin-like pigments are present in the environment as degradation products of heme-containing proteins, yeast could have developed a detoxifying system to transport these compounds into their vacuoles. Vacuoles from Saccharomyces cerevisiae showed an ATP-dependent, saturative transport of unconjugated bilirubin (UCB) that was reduced by 60% and 40% in YCF1 and YLL015w-deleted cells, respectively; the double deletant showed no UCB uptake. Conversely, the transport of bile acids (taurocholate) was comparable in wild and deleted stains. These data identify YCF1 and YLL015w, named BPT1 (Bile Pigment Transporter), as the genes responsible for ATP-dependent UCB transport in yeast. Since YCF1 and YLL015w are rather homologous with multidrug resistant proteins (MRPs), they also suggest the involvement of this class of transporters in the ATP-dependent transport of unconjugated bilirubin.