A higher throughput and physiologically relevant two-compartmental human ex vivo intestinal tissue system for studying gastrointestinal processes.

A majority of the preclinical intestinal screening models do not properly reflect the complex physiology of the human intestinal tract, resulting in low translational value to the clinical situation. The often used cell lines such as Caco-2 or HT-29 are not well suited to investigate the different processes that predict oral bioavailability in real life, or processes involved in general gut health aspects. Therefore, highly realistic models resembling the human in vivo situation are needed; application of ex vivo intestinal tissue is an interesting and feasible alternative. After previously using porcine intestinal tissue as a predictive model for human intestinal absorption, we now have successfully applied human intestinal tissue into a newly developed InTESTine™ two-compartmental disposable device suitable for standard 6- or 24-well plate format. With this set-up we demonstrated (regional differences in) drug absorption, by using a subset of compounds with known varying Fa (fraction absorbed) values. A rank-order relationship of R2 = 0.85 could be established between the Fa and Papp of these commercially available drugs. Additionally, comparison between the InTESTine system and the established Ussing chamber technology showed a correlation of R2 = 0.94 (10 drugs) with respect to Papp values, indicating good comparison of both models. Besides absorption, intestinal wall metabolism of testosterone (CYP3A4) was determined by showing a linear formation (R2 = 0.99; up to 165 min) of the main metabolites androstenedione and 6Beta-hydroxytestosterone, indicating no loss of metabolic capacity of the intestinal tissue within the system. Enteroendocrine responses were assessed of the satiety hormones GLP-1 and PYY after stimulation with rebaudioside A and casein, resulting in significantly increased secretion to the luminal side as well as to the basolateral side. Incubation with the probiotic strain LGG showed to enhance the viability of the tissue by showing to decrease the LDH secretion compared to blank intestinal tissue. In conclusion, we show that human ex vivo intestinal tissue mounted in the higher throughput InTESTine 6- 24-transwell plate system is easy to handle and a suitable system to study diverse functional GI processes.

Proteomics of human liver membrane transporters: a focus on fetuses and newborn infants.

BACKGROUND: Hepatic membrane transporters are involved in the transport of many endogenous and exogenous compounds, including drugs. We aimed to study the relation of age with absolute transporter protein expression in a cohort of 62 mainly fetus and newborn samples. METHODS: Protein expressions of BCRP, BSEP, GLUT1, MCT1, MDR1, MRP1, MRP2, MRP3, NTCP, OCT1, OATP1B1, OATP1B3, OATP2B1 and ATP1A1 were quantified with LC-MS/MS in isolated crude membrane fractions of snap-frozen post-mortem fetal and pediatric, and surgical adult liver samples. mRNA expression was quantified using RNA sequencing, and genetic variants with TaqMan assays. We explored relationships between protein expression and age (gestational age [GA], postnatal age [PNA], and postmenstrual age); between protein and mRNA expression; and between protein expression and genotype. RESULTS: We analyzed 36 fetal (median GA 23.4 weeks [range 15.3-41.3]), 12 premature newborn (GA 30.2 weeks [24.9-36.7], PNA 1.0 weeks [0.14-11.4]), 10 term newborn (GA 40.0 weeks [39.7-41.3], PNA 3.9 weeks [0.3-18.1]), 4 pediatric (PNA 4.1 years [1.1-7.4]) and 8 adult liver samples. A relationship with age was found for BCRP, BSEP, GLUT1, MDR1, MRP1, MRP2, MRP3, NTCP, OATP1B1 and OCT1, with the strongest relationship for postmenstrual age. For most transporters mRNA and protein expression were not correlated. No genotype-protein expression relationship was detected. DISCUSSION AND CONCLUSION: Various developmental patterns of protein expression of hepatic transporters emerged in fetuses and newborns up to four months of age. Postmenstrual age was the most robust factor predicting transporter expression in this cohort. Our data fill an important gap in current pediatric transporter ontogeny knowledge.

Development of a mechanistic biokinetic model for hepatic bile acid handling to predict possible cholestatic effects of drugs.

Drug-induced liver injury (DILI) is a common reason for drug withdrawal from the market. An important cause of DILI is drug-induced cholestasis. One of the major players involved in drug-induced cholestasis is the bile salt efflux pump (BSEP; ABCB11). Inhibition of BSEP by drugs potentially leads to cholestasis due to increased (toxic) intrahepatic concentrations of bile acids with subsequent cell injury. In order to investigate the possibilities for in silico prediction of cholestatic effects of drugs, we developed a mechanistic biokinetic model for human liver bile acid handling populated with human in vitro data. For this purpose we considered nine groups of bile acids in the human bile acid pool, i.e. chenodeoxycholic acid, deoxycholic acid, the remaining unconjugated bile acids and the glycine and taurine conjugates of each of the three groups. Michaelis-Menten kinetics of the human uptake transporter Na+-taurocholate cotransporting polypeptide (NTCP; SLC10A1) and BSEP were measured using NTCP-transduced HEK293 cells and membrane vesicles from BSEP-overexpressing HEK293 cells. For in vitro-in vivo scaling, transporter abundance was determined by LC-MS/MS in these HEK293 cells and vesicles as well as in human liver tissue. Other relevant human kinetic parameters were collected from literature, such as portal bile acid levels and composition, bile acid synthesis and amidation rate. Additional empirical scaling was applied by increasing the excretion rate with a factor 2.4 to reach near physiological steady-state intracellular bile acid concentrations (80µM) after exposure to portal vein bile acid levels. Simulations showed that intracellular bile acid concentrations increase 1.7 fold in the presence of the BSEP inhibitors and cholestatic drugs cyclosporin A or glibenclamide, at intrahepatic concentrations of 6.6 and 20µM, respectively. This simplified model provides a tool for a first indication whether drugs at therapeutic concentrations might cause cholestasis by inhibiting BSEP.

Regional Expression Levels of Drug Transporters and Metabolizing Enzymes along the Pig and Human Intestinal Tract and Comparison with Caco-2 Cells.

Intestinal transporter proteins and metabolizing enzymes play a crucial role in the oral absorption of a wide variety of drugs. The aim of the current study was to characterize better available intestinal in vitro models by comparing expression levels of these proteins and enzymes between porcine intestine, human intestine, and Caco-2 cells. We therefore determined the absolute protein expression of 19 drug transporters and the mRNA expression of 12 metabolic enzymes along the pig intestinal tract (duodenum, jejunum, ileum; N = 4), in human intestine (jejunum; N = 9), and Caco-2 cells. Expression of the included transporters and enzymes was in general well comparable between porcine and human intestinal tissue, although breast cancer resistance protein, monocarboxylate transporter 5, multidrug resistance protein (MRP) 1, MRP1, MRP3 (∼2-fold), and organic anion-transporting polypeptide (OATP) 4A1 (∼6-fold) was higher expressed in pig compared with human jejunum. Alternatively, expression level of relevant transporter proteins (glucose transporter 1, OATP4A1, MRP2, MRP1, and OATP2B1) was significantly higher (3- to 130-fold) in Caco-2 cells compared with human jejunum. Moreover, all examined CYPs showed at least a fivefold lower gene expression in Caco-2 cells compared with human jejunum, with the smallest differences for CYP1A1 and CYP3A5 and the largest difference for CYP3A4 (871-fold higher expression in human jejunum compared with Caco-2 cells). In conclusion, a comprehensive overview is provided of the expression levels of clinically relevant transporter proteins and metabolic enzymes in porcine and human intestinal tissue and Caco-2 cells, which may assist in deciding upon the most suitable model to further improve our understanding of processes that determine intestinal absorption of compounds.

Bioactivation of dibrominated biphenyls by cytochrome P450 activity to metabolites with estrogenic activity and estrogen sulfotransferase inhibition capacity.

Exposure of humans and wildlife to xenobiotics, such as halogenated biphenyls, that interfere with the endogenous estrogen balance may lead to endocrine disruption. Such compounds may either mimic or block estradiol's action by agonistic or antagonistic action, respectively. They may also affect endogenous estradiol concentrations by induction or inhibition of enzymes that metabolize estradiol. In the present study, we demonstrate that estrogenic metabolites of two brominated biphenyls, 2,2'-dibromobiphenyl (2,2'-DBB) and 4,4'-dibromobiphenyl (4,4'-DBB), are formed by rat liver microsomal cytochrome P450 (CYP) activity. Bioactivation of 2,2'-DBB and 4,4'-DBB yielded various mono- and dihydroxylated bromobiphenyl metabolites, which were collected by preparative HPLC and analyzed by LC/MS. Several of the metabolites bound to the estrogen receptor (ER) activated the ER and inhibited human estrogen sulfotransferase (hEST). Seven monohydroxylated metabolites were positively identified using synthetic monohydroxylated reference compounds. These synthetic monohydroxylated bromobiphenyls also bound to and activated the ER and inhibited hEST. The highest ER affinity was observed for 4-OH-2,2'-DBB, with an EC50 of 6.6 nM. The highest ER activation was observed for 4-OH-3,4'-DBB (EC50 of 74 nM) while 4-OH-4'-MBB and 4-OH-2,2'-DBB induced a supramaximal (as compared to estradiol) ER activation. The strongest hEST inhibition was found with 4-OH-3,4'-DBB (EC50 = 40 nM). In conclusion, we show that two dibrominated biphenyls are bioactivated by CYP activity into very potent estrogenic metabolites and inhibitors of hEST. These findings are of vital importance for accurate risk assessment of exposure to environmental contaminants, such as halogenated biphenyls. Neglecting bioactivation through biotransformation will lead to underestimation of health risks of this class of xenobiotics.

The chemical interaction between the estrogen receptor and monohydroxybenzo[a]pyrene derivatives studied by fluorescence line-narrowing spectroscopy.

A novel approach is presented for studying the chemical interaction between receptor binding sites and ligands. Monohydroxylated polyaromatic compounds were found to be environmentally sensitive ligands when applying a special mode of fluorescence: fluorescence line-narrowing spectroscopy (FLNS). With this technique, solvent dependencies and ligand-receptor interactions can be studied in great detail, due to the high spectral resolution and the fact that at cryogenic temperatures (4 K), no solvent reorientation effects complicate the interpretation. The FLN spectrum of a ligand bound to the receptor is compared to the spectra of the free ligand in solvent mixtures that mimic the functionalities present within the receptor's binding site. It is shown that for the well-known estrogen receptor (ER), the orientations of two xenoestrogenic ligands 3- and 9-hydroxybenzo[a]pyrene (3- and 9-OH-BaP) can be determined. The FLN results clearly indicate that an H-bond accepted by HIS524 plays a major role in the binding of these ligands to the ER. Furthermore, the spectra indicated a pi-pi stacking aromatic interaction for 9-OH-BaP with PHE404. These results are in line with molecular modeling studies published earlier.

Formation of estrogenic metabolites of benzo[a]pyrene and chrysene by cytochrome P450 activity and their combined and supra-maximal estrogenic activity.

Metabolism of polycyclic aromatic hydrocarbons (PAHs) has been studied intensively, and potential metabolites with estrogenic activity have been identified previously. However, little attention has been paid to the metabolic pathways in mammalians and to the combined effect of individual metabolites. Several hydroxylated metabolites of benzo[a]pyrene (BaP) and chrysene (CHN) were formed by rat liver microsomal cytochrome P450 (CYP) activity, some of which possess estrogenic activity. All mono- and several dihydroxylated metabolites of BaP and CHN were tested for ER affinity and estrogenic activity in a proliferation assay (E-screen) and in a reporter-gene assay (ER-CALUX). Twelve estrogenic metabolites were identified with EC50 values ranging from 40nM to 0.15mM. The combined effect of a mixture of seven PAH-metabolites was also studied in the ER binding assay. At concentrations that show little activity themselves, their joint action clearly exhibited significant estrogenic activity. BaP itself exhibited estrogenicity in the ER-CALUX assay due to bio-activation into estrogenic metabolites, probably via aryl hydrocarbon receptor (AhR) induced CYP activity. Furthermore, 2-hydroxy-CHN (2-OHCHN) induced supra-maximal (400%) estrogenic effects in the ER-CALUX assay. This effect was entirely ER-mediated, since the response was completely blocked with the ER-antagonist ICI182,780. We showed that 2-OHCHN increased ER-concentration, using ELISA techniques, which may explain the observed supra-maximal effects. Co-treatment with the AhR-antagonist 3',4'-dimethoxyflavone (DMF) enhanced ER-signaling, possibly via blockage of AhR-ER inhibitory cross-talk.

Prediction of ligand binding affinity and orientation of xenoestrogens to the estrogen receptor by molecular dynamics simulations and the linear interaction energy method.

Exposure to environmental estrogens has been proposed as a risk factor for disruption of reproductive development and tumorigenesis of humans and wildlife (McLachlan, J. A.; Korach, K. S.; Newbold, R. R.; Degen, G. H. Diethylstilbestrol and other estrogens in the environment. Fundam. Appl. Toxicol. 1984, 4, 686-691). In recent years, many structurally diverse environmental compounds have been identified as estrogens. A reliable computational method for determining estrogen receptor (ER) binding affinity is of great value for the prediction of estrogenic activity of such compounds and their metabolites. In the presented study, a computational model was developed for prediction of binding affinities of ligands to the ERalpha isoform, using MD simulations in combination with the linear interaction energy (LIE) approach. The linear interaction energy approximation was first described by Aqvist et al. (Aqvist, J.; Medina, C.; Samuelsson, J. E. A new method for predicting binding affinity in computer-aided drug design. Protein Eng. 1994, 7, 385-391) and relies on the assumption that the binding free energy (DeltaG) depends linearly on changes in the van der Waals and electrostatic energy of the system. In the present study, MD simulations of ligands in the ERalpha ligand binding domain (LBD) (Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P. J.; Agard, D. A.; Greene, G. L. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998, 95, 927-937), as well as ligands free in water, were carried out using the Amber 6.0 force field (http://amber.scripps.edu/). Contrary to previous LIE methods, we took into account every possible orientation of the ligands in the LBD and weighted the contribution of each orientation to the total binding affinity according to a Boltzman distribution. The training set (n = 19) contained estradiol (E2), the synthetic estrogens diethylstilbestrol (DES) and 11beta-chloroethylestradiol (E2-Cl), 16alpha-hydroxy-E2 (estriol, EST), the phytoestrogens genistein (GEN), 8-prenylnaringenin (8PN), and zearalenon (ZEA), four derivatives of benz[a]antracene-3,9-diol, and eight estrogenic monohydroxylated PAH metabolites. We obtained an excellent linear correlation (r(2) = 0.94) between experimental (competitive ER binding assay) and calculated binding energies, with K(d) values ranging from 0.15 mM to 30 pM, a 5 000 000-fold difference in binding affinity. Subsequently, a test set (n = 12) was used to examine the predictive value of our model. This set consisted of the synthetic estrogen 5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (THC), daidzein (DAI), equol (EQU) and apigenin (API), chlordecone (KEP), progesterone (PRG), several mono- and dihydroxylated PAH metabolites, and two brominated biphenyls. The predicted binding affinities of these estrogenic compounds were in very good agreement with the experimental values (average deviation of 0.61 +/- 0.4 kcal/mol). In conclusion, our LIE model provides a very good method for prediction of absolute ligand binding affinities, as well as binding orientation of ligands.

Detection of estrogenic potency in wastewater and surface water with three in vitro bioassays.

A study was performed to optimize sample preparation and application of three in vitro assays for measuring estrogenic potency in environmental extracts. The three assays applied were an estrogen receptor (ER)-binding assay and two reporter gene effect assays: a yeast estrogen screen (YES) and the ER-mediated chemically activated luciferase gene expression (ER-CALUX) assay. All assays were able to detect estrogenicity, but the amounts of material needed for the assays differed greatly between the three assays (ER-binding assay >> YES > ER-CALUX). In addition, in the ER-binding assay, both agonists and antagonists give an estrogenic response, resulting in higher estradiol equivalency (EEQ) levels than both the ER-CALUX and the YES assay for the same samples. The EEQs found in wastewater treatment plants (WTPs) with the ER-CALUX assay were in the range of 4 to 440 and 0.11 to 59 pmol/L for influent and effluent, respectively. Water extracts from four large rivers had levels ranging from 0.25 to 1.72 pmol/L. Extracts from suspended matter and sludge contained estrogenic potency of 0.26 to 2.49 and 1.6 to 41 pmol EEQ/g dry weight, respectively. In WTPs, the average reduction of estrogenic potency in effluent compared to influent was 90 to 95% in municipal WTPs and about 50% in industrial WTPs. In influent, 30% of the ER-CALUX activity could not be explained by the calculated potencies based on chemical analysis of a number of known (xeno)estrogens; in effluent the unexplained fraction was 80%. These first results of analyzing estrogenic potency in WTP water and surface water in The Netherlands indicate that further studies are warranted to investigate the actual risks for aquatic systems.