Transport of the placental estriol precursor 16α-hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) by stably transfected OAT4-, SOAT-, and NTCP-HEK293 cells.

16α-Hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) mainly originates from the fetus and serves as precursor for placental estriol biosynthesis. For conversion of 16α-OH-DHEAS to estriol several intracellular enzymes are required. However, prior to enzymatic conversion, 16α-OH-DHEAS must enter the cells by carrier mediated transport. To identify these carriers, uptake of 16α-OH-DHEAS by the candidate carriers organic anion transporter OAT4, sodium-dependent organic anion transporter SOAT, Na(+)-taurocholate cotransporting polypeptide NTCP, and organic anion transporting polypeptide OATP2B1 was measured in stably transfected HEK293 cells by LC-MS-MS. Furthermore, the study aimed to localize SOAT in the human placenta. Stably transfected OAT4-HEK293 cells revealed a partly sodium-dependent transport for 16α-OH-DHEAS with an apparent Km of 23.1 ± 5.1 µM and Vmax of 485.0 ± 39.1 pmol/mg protein/min, while stably transfected SOAT- and NTCP-HEK293 cells showed uptake only under sodium conditions with Km of 319.0 ± 59.5 µM and Vmax of 1465.8 ± 118.8 pmol/mg protein/min for SOAT and Km of 51.4 ± 9.9 µM and Vmax of 1423.3 ± 109.6 pmol/mg protein/min for NTCP. In contrast, stably transfected OATP2B1-HEK293 cells did not transport 16α-OH-DHEAS at all. Immunohistochemical studies and in situ hybridization of formalin fixed and paraffin embedded sections of human late term placenta showed expression of SOAT in syncytiotrophoblasts, predominantly at the apical membrane as well as in the vessel endothelium. In conclusion, OAT4, SOAT, and NTCP were identified as carriers for the estriol precursor 16α-OH-DHEAS. At least SOAT and OAT4 seem to play a functional role for the placental estriol synthesis as both are expressed in the syncytiotrophoblast of human placenta.

[Pyrrolizidine alkaloids and seneciosis in farm animals. Part 2: clinical signs, species-specific sensitivity, food residues, feed contamination, limit values]. / Pyrrolizidinalkaloide und die Seneciose bei Tieren. Teil 2: Klinik, Speziesunterschiede, Rückstandsverhalten, Futtermittelkontamination und Grenzwerte.

At the forefront of pyrrolizidine alkaloid (PA) poisoning is the chronic ingestion of contaminated hay, which causes liver damage resulting in an ongoing fatal liver cirrhosis or in the veno-occlusive disease in liver or lung, respectively. The symptomatology of PA-poisoning is not identical for all animal species, and also includes central nervous symptoms. In affected horses significantly elevated levels of hepatogenic serum enzymes and an increase of the retention time for bromosulfophthalein indicates the fatal outcome of the intoxication. Chronic seneciosis of horses is incurable. Rabbits, Japanese quails, and guinea pigs are regarded as poison-resistant species. Sheep and in particular goats are insensitive unless extremely high amounts of plants which exceed the animal's body weight by several-fold are ingested. In contrast, pigs, cattle, and horses as well as chicken and likewise man are very sensitive to poisonings by PA-containing plants. In sensitive animal species a very small amount of contaminated dry hay is needed to exceed the daily dose of 1µg/kg body weight PA which is taken as harmless for man by health authorities. Therefore, all feed with visible pieces of Senecio jacobaea plants are not acceptable as animal fodder and should be destroyed.

[Pyrrolizidine alkaloids and seneciosis in farm animals. Part 1: occurrence, chemistry and toxicology]. / Pyrrolizidinalkaloide und die Seneciose bei Tieren. Teil 1: Vorkommen, Chemie, Toxikologie.

Pyrrolizidine alkaloids belong to a class of phytotoxins which are present in more than 6000 plant species. The disease seneciosis in farm animals represents the severe poisoning by pyrrolizidine alkaloids from plants of the genus Senecio. This form of poisoning has been known since the end of the 19th century in Germany, the USA, Canada and New Zealand, and is mainly caused by Senecio jacobaea and related Senecio spp. in farm animals, including poultry. Animal poisoning by pyrrolizidine alkaloids is of worldwide importance. In Germany poisoning of horses and cattle by Senecio jacobaea, which was earlier named Schweinsberg disease, is of renewed relevance for veterinary medicine. The disease occurs almost entirely as a consequence of chronic poisoning and in general ends fatally. The ultimate cause is the formation of toxic metabolites of pyrrolizidine alkaloids in the liver, and their covalent binding to nucleic acids and proteins leading to liver cirrhosis. Because many pyrrolizidine alkaloids possess mutagenic, and a few also carcinogenic properties, European and international authorities are concerned about possible residue levels in food of animal origin. The review addresses in its first part several aspects, being the occurrence, the chemistry, and the toxicology of pyrrolizidine alkaloids as well as animal intoxications by poisonous plants. In the second part (46) clinical characteristics of animal seneciosis, the therapeutic interventions, the significant species differences and a critical assessment of so-called nontoxic amounts of Senecio plants in animal fodder with reference to cumulative lethal toxin doses are presented.

Silibinin pretreatment protects against ochratoxin A-mediated apoptosis in primary rat hepatocytes.

The inhibitory effect of silibinin on ochratoxin A (OTA)-mediated apoptosis on primary rat hepatocytes was investigated. Rat hepatocytes were prepared by two different methods: the classical enzymatic digestion method by collagenase perfusion and a new EDTA-perfusion method. The EDTA-perfusion method yielded hepatocytes, which were stably cultivated without DNA fragmentation for up to 96 h, whereas the collagenase-prepared hepatocytes showed apoptosis events as early as from the start of preparation even in the absence of OTA. Treatment with 12.5 µmol/l OTA of cultured hepatocytes prepared under ETDA perfusion developed DNA-laddering after 24-36 h. Lipopolysaccharide (LPS) of 0.1 up to 12.5 µg/ml showed no apoptotic DNA-effects under these conditions. A low concentration of 26 µmol/l silibinin given prior to OTA slightly prevented OTA-mediated DNA-laddering, whereas a five times higher concentration of silibinin (130 µmol/l) completely inhibited OTA-mediated apoptosis. Under the same conditions, caspase-3 activity in hepatocytes increased in a time-dependent manner under OTA exposure within 12-24 h but was blocked by 130 µmol/l silibinin. In contrast, LPS incubation for 12 and 24 h did not alter caspase-3 activity. To measure viability of OTA-/LPS-treated hepatocytes, the MTT-test and Live/Dead kit were applied. The results demonstrated that the used OTA concentration of 12.5 µmol/l only moderately decreased viability for up to 24 h but showed cytotoxic effects depending on longer incubation times (≥36 h). In contrast, LPS up to 12.5 µg/ml exhibited no cytotoxic effects up to 48 h. In summary, our results showed contrasting effects on apoptosis in primary rat hepatocytes by OTA (produces apoptosis) versus LPS (produces no apoptosis), also depending on the method of hepatocyte preparation. Silibinin at 130 µmol/l showed significant hepatoprotective and antiapoptotic effects against OTA-mediated cell damage on cultured rat hepatocytes.

Cloning and heterologous expression of the ovine (Ovis aries) P-glycoprotein (Mdr1) in Madin-Darby canine kidney (MDCK) cells.

P-glycoprotein (P-gp) plays a crucial role in the multidrug resistance of pathogenic helminths in sheep (Ovis aries) as well as in antiparasitic drug pharmacokinetics in the host. We cloned sheep P-gp cDNA and expressed it stably in Madin-Darby canine kidney (MDCK) cells. The open reading frame consists of 3858 nucleotides coding for a 1285 amino acids containing protein. The sequence shows high homology to the orthologs of other mammalian species, especially cattle. Both ruminant DNA sequences show a 9 bp insertion that is lacking in all other investigated sequences. Expressed in MDCK cells, the protein displays a size of 170 kDa on Western analysis. Transfection of MDCK cells with sheep P-gp resulted in 10- to 50-fold resistance to the cytotoxic P-gp substrates colchicin and daunorubicin, and in reduced digoxin accumulation.

Brain penetration of ivermectin and selamectin in mdr1a,b P-glycoprotein- and bcrp- deficient knockout mice.

P-glycoprotein, which is encoded by the multi-drug resistance gene (MDR1), highly restricts the entry of ivermectin into the brain by an ATP-driven efflux mechanism at the blood-brain barrier. In dogs with a homozygous MDR1 mutation though, ivermectin accumulates in the brain and provokes severe signs of neurotoxicosis and even death. In contrast to ivermectin, selamectin is safer in the treatment of MDR1 mutant dogs, suggesting that selamectin is transported differently by P-glycoprotein across the blood-brain barrier. To test this, we applied selamectin to mdr1-deficient mdr1a,b(-/-) knockout mice and wild-type mice. Brain penetration, organ distribution, and plasma kinetics were analyzed after intravenous, oral, and dermal spot-on application in comparison with ivermectin. We found that in vivo both macrocyclic lactone compounds are substrates of P-glycoprotein and that these strongly accumulate in the brain of mdr1a,b(-/-) knockout mice compared with wild-type mice at therapeutic doses of 12 mg/kg selamectin and 0.2 mg/kg ivermectin. However, selamectin accumulates to a much lesser degree (5-10 times) than ivermectin (36-60 times) in the absence of P-glycoprotein. This could explain the broader margin of safety of selamectin in MDR1 mutant dogs. In liver, kidney, and testes, ivermectin and selamectin accumulated less than four times as much in mdr1a,b mutant mice as in wild-type mice. Breast cancer resistance protein (Bcrp)-deficient bcrp(-/-) knockout mice were also included in the application studies, but showed no differences in brain concentrations or organ distribution of either ivermectin or selamectin compared with wild-type mice. This indicates that Bcrp is not a relevant efflux carrier for these macrocyclic lactone compounds in vivo at the blood-brain barrier.

ESI+ MS/MS confirmation of canine ivermectin toxicity.

Ivermectin is a semisynthetic macrocyclic lactone anthelmintic of the avermectin family derived from Streptomyces fermentation products. Avermectins are used as antiparasitic agents in domestic animals; although considered relatively safe, one must consider animal species, breed, weight, and age in dosage determinations.In January 2006, two canines were presented to the UK Livestock Disease Diagnostic Center after dying from suspected ivermectin overdoses [30-50 mg/kg body weight]. To confirm this clinical diagnosis we developed a rapid, sensitive semiquantitative ElectroSpray Ionization-Mass Spectrometry (ESI/MS) method for ivermectin in canine tissue samples. Pharmaceutical ivermectin contains two ivermectins differing by a single methyl group, and each compound forms interpretation-confounding adducts with tissue Na(+) and K(+) ions. We now report that ivermectin administration was clearly confirmed by comparison with standard and dosage forms of ivermectin, and simple proportionalities based on mass spectral intensity of respective molecular ions allowed semiquantitative estimates of injection site tissue concentrations of 20 and 40 microg/g tissue (wet weight) in these animals, consistent with the history of ivermectin administration and the clinical signs observed.There is a distinct need for both rapid detection and confirmation of toxic exposures in veterinary diagnostics, whether for interpretation of clinical cases antemortem or for forensic reasons postmortem. It is vital that interpreters of analytical results have appropriate guidance in the scientific literature and elsewhere so as to enable clear-cut answers. The method presented here is suitable for routine diagnostic work in that it allows rapid extraction of ivermectin from tissue samples, avoids the need for high-performance liquid chromatography and allows ready interpretation of the multiple ivermectin species seen by ESI(+) MS/MS in samples originating from veterinary dosage forms.

Cloning and molecular characterization of the orphan carrier protein Slc10a4: expression in cholinergic neurons of the rat central nervous system.

We report on the cloning and molecular characterization of the rat carrier Slc10a4 and its cellular localization in the CNS by immunohistochemistry. Slc10a4 is the rat counterpart of the human orphan carrier SLC10A4, which was recently reported to be highly expressed in brain and placenta. Both carriers belong to the solute carrier family SLC10, formerly named the "sodium/bile acid cotransporter family." So SLC10A4/Slc10a4 has a phylogenetic relationship to the Na+/taurocholate cotransporting polypeptide Ntcp (Slc10a1) and the apical sodium-dependent bile acid transporter Asbt (Slc10a2). The rat Slc10a4 protein consists of 437 amino acids and exhibits a seven transmembrane domain topology with N(exo)/C(cyt)trans-orientation of the N- and C-terminal ends. Expression of the Slc10a4 protein was detected in motor regions of the spinal cord and rhombencephalon, as well as in mesopontine cholinergic neurons, the medial habenula, cholinergic areas of the forebrain, and the gut myenteric plexus. Co-localization studies with the cholinergic marker proteins choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and high-affinity choline transporter (CHT1) demonstrated expression of Slc10a4 in cholinergic neurons. Despite its close phylogenetic relationship to Ntcp, Slc10a4 showed no transport activity for the Ntcp substrates taurocholate, estrone-3-sulfate, dehydroepiandrosterone sulfate, and pregnenolone sulfate when expressed in HEK293 cells or Xenopus laevis oocytes. Slc10a4 also did not transport choline, which is a substrate of CHT1. Although the functional properties of Slc10a4 could not be elucidated in this study, Slc10a4 is regarded as a new marker protein for cholinergic neurons in the rat CNS.

The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships.

The solute carrier family 10 (SLC10) comprises two sodium-dependent bile acid transporters, i.e. the Na(+)/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT; SLC10A2). These carriers are essentially involved in the maintenance of the enterohepatic circulation of bile acids mediating the first step of active bile acid transport through the membrane barriers in the liver (NTCP) and intestine (ASBT). Recently, four new members of the SLC10 family were described and referred to as P3 (SLC10A3), P4 (SLC10A4), P5 (SLC10A5) and sodium-dependent organic anion transporter (SOAT; SLC10A6). Experimental data supporting carrier function of P3, P4, and P5 is currently not available. However, as demonstrated for SOAT, not all members of the SLC10 family are bile acid transporters. SOAT specifically transports steroid sulfates such as oestrone-3-sulfate and dehydroepiandrosterone sulfate in a sodium-dependent manner, and is considered to play an important role for the cellular delivery of these prohormones in testes, placenta, adrenal gland and probably other peripheral tissues. ASBT and SOAT are the most homologous members of the SLC10 family, with high sequence similarity ( approximately 70%) and almost identical gene structures. Phylogenetic analyses of the SLC10 family revealed that ASBT and SOAT genes emerged from a common ancestor gene. Structure-activity relationships of NTCP, ASBT and SOAT are discussed at the amino acid sequence level. Based on the high structural homology between ASBT and SOAT, pharmacological inhibitors of the ASBT, which are currently being tested in clinical trials for cholesterol-lowering therapy, should be evaluated for their cross-reactivity with SOAT.

Immunotoxic activity of ochratoxin A.

Ochratoxin A (OTA) is an immunosuppressant fungal compound, produced by toxigenic species of Aspergillus and Penicillium fungi in a wide variety of climates and geographical regions. The contamination of food by this mycotoxin takes place primarily during preharvest periods. Almost all types of food can be contaminated. In addition, its chemical stability against heat and during industrial food processing makes OTA one of the most abundant food contaminating mycotoxins. Due in part to its long serum half-life in man, almost 100% of all human blood samples from some geographic regions may be positive for OTA. The immunosuppressant activity of OTA is characterized by size reduction of vital immune organs, such as thymus, spleen, and lymph nodes, depression of antibody responses, alterations in the number and functions of immune cells, and modulation of cytokine production. The immunotoxic activity of OTA probably results from degenerative changes and cell death following necrosis and apoptosis, in combination with slow replacement of affected immune cells, due to inhibition of protein synthesis.

Frequency of the nt230 (del4) MDR1 mutation in Collies and related dog breeds in Germany.

MDR1 (ABCB1) P-glycoprotein exerts a protective function in the blood-brain barrier thereby limiting the entry of many drugs and other xenobiotics to the central nervous system. A nonsense mutation has been described for Collies and related dog breeds which abolishes this function and is associated with increased susceptibility to neurotoxic side effects of several drugs including ivermectin, moxidectin and loperamide. In order to evaluate the occurrence and frequency of this nt230 (del4) MDR1 mutation in Germany, we screened 1500 dogs. Frequency of the homozygous mutated genotype was highest for Collies (33.0%), followed by Australian Shepherd (6.9%) and Shetland Sheepdog (5.7%). Thirty-seven percent of the Wäller dogs and 12.5% of the Old English Sheepdogs were heterozygous for the mutant MDR1 (-) allele. Considering the predominant role of MDR1 P-glycoprotein in drug disposition and in particular for blood-brain barrier protection, MDR1 genotype-based breeding programs are recommended for improving the safety of drug therapy in these canine breeds.

Interference of arachidonic acid and its metabolites with TNF-alpha release by ochratoxin A from rat liver.

We investigated the role of arachidonic acid and its metabolites on the ochratoxin A (OTA) provoked release of proinflammatory and apoptotic cytokine TNF-alpha from blood-free perfused rat liver. OTA induced TNF-alpha release dose- and time-dependently yielding 2600 pg TNF-alpha/ml at 2.5 micromol/l after 90 min without significant release of LDH and lactate. Aristolochic acid, 50 micromol/l, a phospholipase A2 inhibitor, and 10 micromol/l of exogenous arachidonic acid decreased TNF-alpha below normal level. Indomethacin, 10 micromol/l, a potent inhibitor of the cyclooxygenase (COX) pathway, almost doubled TNF-alpha concentrations in the perfusion solution to reach 5500 pg/ml at 90 min. On the other hand, inhibition of lipoxgenase (LPX) by 30 micromol/l nordihydroguaiaretic acid (NDGA) and the cytochrome P-450 (CYP) pathway by 100 micromol/l of metyrapone decreased TNF-alpha below normal levels as well. Concurrent administration of two blockers (COX inhibitor with LPX inhibitor, or COX inhibitor with CYP-450 inhibitor, or LPX inhibitor with CYP-450 inhibitor) blocked TNF-alpha release below normal levels. In addition, 10 micromol/l caffeic acid phenylethyl ester, a NF-(kappa)B inhibitor, blocked OTA mediated TNF-alpha release. In conclusion, arachidonic acid and its cyclooxygenase metabolites are suppressors of OTA mediated TNF-alpha release from liver, whereas LPX and CYP-450-metabolites have the opposite effect. OTA-induced TNF-alpha release is likely to occur via the NF-(kappa)B transcription factor pathway in perfused rat liver.

Development of a PCR-based diagnostic test detecting a nt230(del4) MDR1 mutation in dogs: verification in a moxidectin-sensitive Australian Shepherd.

A subpopulation of dogs of the Collie and Australian Shepherd breeds show increased sensitivity to central nervous actions of ivermectin, doramectin, loperamide, and probably several other drugs. The molecular background for this greater sensitivity is a nonsense mutation in the MDR1 efflux pump, which is part of the functional blood-brain barrier and normally limits drug penetration into the brain. This report describes a rapid PCR-based method for detection of this nt230(del4) MDR1 mutation using a small amount of genomic DNA from blood cells. Thereby, homozygous intact, homozygous mutated, and heterozygous mutated MDR1 genotypes can be clearly differentiated by high resolution polyacrylamide gel electrophoresis. Using this diagnostic test two Collies and one Australian Shepherd were screened for the nt230(del4) MDR1 mutation. The Collies had no history of altered drug sensitivity and showed homozygous intact and heterozygous mutated MDR1 alleles, respectively. However, the Australian Shepherd developed clear signs of neurotoxicity including ataxia, crawling, acoustic and tactile hyperexcitability, and miosis after a single dose of moxidectin (400 microg/kg). For this dog two mutated MDR1 alleles were detected. This report describes for the first time moxidectin neurotoxicosis in a dog with a homozygous MDR1 mutation.

In vitro induction of tumor necrosis factor-α by ochratoxin A (OTA) from rat liver: role of Kupffer cells.

Tumor necrosis factor-α (TNF-α) is released from blood-free perfused rat liver by the fungal metabolite ochratoxin A. Here we have identified Kupffer cells as the sole source of OTA-mediated cytokine release. If single cell preparation of Kupffer cells, hepatocytes, or sinusoidal endothelial cells were prepared from rat livers, only Kupffer cells released TNF-α upon incubation with 2.5 µmol/l OTA. OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blockedin vitro by 15 µmol/l gadolinium chloride. When rats were pretreatedin vivo with the Kupffer cell depleting clodronate liposomes, OTA-mediated TNF-α release was abrogated in the isolated perfused liver model.

Bile acid-oligodeoxynucleotide conjugates: synthesis and liver excretion in rats.

The synthesis of bile acid oligodeoxynucleotide conjugates via the 3-OH group of the bile acids is described. When used in vivo in rats, covalent conjugation of an oligodeoxynucleotide via a linker to cholic acid resulted in an increased biliary excretion of bile acid-oligodeoxynucleotide conjugates compared to unconjugated oligodeoxynucleotides.

Ochratoxin A from a toxicological perspective.

Ochratoxin A (OTA) is a widespread mycotoxin which is produced mainly by the mould fungi Aspergillus ochraceus and Penicillum verrucosum during the storage of cereals, cereal products and other plant-derived products such as herbs, spices, grapes, etc. By carry over from mouldy fodder, ochratoxin A is also found in pork meat, offal and sausages containing pork blood. When ingested as a food contaminant, OTA is very persistent in human beings with a blood half-life of 35 days after a single oral dosage due to unfavourable elimination toxicokinetics. This renders the toxin among the most frequent mycotoxin contaminants in human blood in the EU, the US, Canada, and elsewhere, where it has been investigated. OTA is neither stored nor deposited in the body, but heterogeneous body distribution may impose serious damage to the kidneys. The toxin was classified a 2B cancer compound, being possibly carcinogenic for humans. It was among the strongest carcinogenic compounds in rats and mice. As the toxicological profile also includes teratogenesis, nephrotoxicity, and immunotoxicity, legislation authorities are currently discussing maximal residue levels (MRL) for OTA in various foodstuffs. In the present article arguments are presented which suggest an acceptable daily intake (ADI) of 1.5 ng OTA/kg body weight and a much lower MRL than 5 microgram OTA/kg cereals and cereal products as has been postulated by the EU commission.

Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells.

We have cloned two complementary DNAs (cDNAs), RL-Mtx-1 and RL-Mtx-2, corresponding to the bile acid- sensitive methotrexate carrier from rat liver by direct full-length rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using degenerated primers that were deduced from published sequences of tumor cell methotrexate transporters. When expressed in Xenopus laevis oocytes and cosM6 cells, both clones mediate methotrexate and bumetanide transport. RL-Mtx-1 consists of 2,445 bp with an open reading frame of 1,536 bp. The corresponding protein with 512 amino acids has a molecular weight of 58 kd. RL-Mtx-2 (2,654 bp) differs by an additional insert of 203 bp. This insert is located in frame at position 1,196 of the RL-Mtx-1 and contains the typical splice junction sites at the 5' and 3' end, indicating that the RL-Mtx-2 messenger RNA (mRNA) is generated by alternative splicing. The insert contains a stop codon that shortens the RL-Mtx-2 protein to 330 amino acids (38 kd). Both cDNAs contain the binding site sequence for the dioxin/nuclear translocator responsive element (Ah/Arnt-receptor) in conjunction with a barbiturate recognition sequence (Barbie box). Preliminary results show that the Barbie box acts as a negative regulatory element. The two liver cDNA clones show homologies to the published sequences of folate and the reduced folate carriers, but no homology is found to the transport systems for organic anions like the Ntcp1, oatp1, OAT-K1, and OAT1. Expression of the mRNA for the methotrexate carrier is found in liver, kidney, heart, brain, spleen, lung, and skeletal muscle, but not in the testis as revealed by Northern blot analysis. The highest abundance of the mRNA is found in the kidney.

Ochratoxin A induced TNFα release from the isolated and blood- free perfused rat liver.

Ochratoxin A (OTA) a naturally occuring mycotoxin is nephrotoxic, hepatotoxic, teratogenic, embryotoxic, genotoxic, cancerogenic and immunosuppressive. Focussing on the immunemodulating effects, we found that OTA induces cytokine release from the isolated and bloodfree perfused rat liver. In the present study, we have characterized the liberation of tumor necrosis factor alpha (TNFα) by OTA and we described interactions with various mycotoxins andE.coli lipopolysaccharide (LPS).

Hepatobiliary transport of bile acid amino acid, bile acid peptide, and bile acid oligonucleotide conjugates in rats.

Uptake of drugs by bile acid carriers could account for the selectivity of drug actions in the gut and liver. We have previously shown that conjugation of xenobiotics with bile acids facilitates their transfer to hepatocytes and ileal enterocytes. In this study L-alanine and 2 biooligomers, the tetrapeptide L-(ala)(4) and a 15 mer oligodeoxynucleotide (ODN) were coupled covalently via linker molecules to the 3-position of bile acids. The L-alanine-coupled bile acid conjugates were rapidly taken up by the liver and efficiently eliminated into bile. These compounds mimicked hepatic transport of bile acids. Also in case of the tetrapeptide (ala)(4), bile acid conjugation significantly improved hepatic and intestinal cell uptake and rendered the peptide conjugate resistant to peptidases. Because uptake by isolated hepatocytes was not dependent on sodium ions and was blocked by ochratoxin A, we assume basolateral transport by an oatp-type bile acid carrier. In the case of the 15 mer ODN, normal and bile acid-conjugated oligodeoxynucleotide appeared intact in bile but without marked improvement of hepatocellular uptake and biliary elimination. We conclude that bile acids can deliver small peptides to gut and parenchymal liver cells via bile acid transport pathways, whereas in the case of oligonucleotides an attached bile acid was not sufficient to shuttle them successfully into hepatocytes.