[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.

[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.

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.

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.

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.

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.

Membrane transport of conjugated and unconjugated bile acids into hepatocytes is susceptible to SH-blocking reagents.

The present study indicates that SH-groups are essential for the uptake of [3H]taurocholate and [14C]cholate into isolated rat hepatocytes. Several sulfhydryl-modifying reagents viz. p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), dithio-bis(5-nitropyridine) (DTNP), bromosuccinimide and HgCl2 inhibited uptake of bile acids in a concentration-dependent manner. PCMBS was the most effective inhibitor in the uptake of taurocholate, while NEM is preferentially blocking the cholate uptake. PCMBS inhibited both the sodium- dependent and the sodium-independent bile acid uptake. Two different moieties of SH-groups seemed to be important for bile acid transport. One group was susceptible to DTNP and NEM, whereas PCMBS was able to block another type of SH-groups in addition. Cell viability was altered by SH-blockers, except by PCMBS. Efflux studies with 86Rb+ demonstrated that the transmembrane potential of hepatocytes was less effected by 100 microM PCMBS in contrast to 100 microM HgCl2. Efflux of tetra[3H]phenylphosphonium and of [3H]aflatoxin in PCMBS-treated hepatocytes documented membrane integrity during at least 10 min. PCMBS did not reduce cellular ATP levels significantly (minus 7%) nor did it markedly increase the amount of the Trypan-blue stained hepatocytes (plus 8.5%). The blocking effect of PCMBS was immediate and was completely reversed by the addition of 500 microM dithiothreitol (DTT), indicating a specific interaction with sulfhydryl-groups. This antagonizing effect of DTT depends on the concentration and exposure time of PCMBS. Six other thiols viz. 2-mercaptoethanol, 1,2-dimercaptoethane, 1,4-dimercaptobutane, 1,6-dimercaptohexane, L-cysteine and L-glutathione were less effective. The results suggest that free SH-groups on the outer surface of hepatocytes play an important role in the uptake process for conjugated and unconjugated bile acids.

Hepatocellular uptake of aflatoxin B1 by non-ionic diffusion. Inhibition of bile acid transport by interference with membrane lipids.

Aflatoxin B1 permeates isolated rat hepatocytes by non-ionic diffusion. Its uptake is neither saturable nor influenced by metabolic energy and not inhibited by treatment of cells with proteases. The initial rate of aflatoxin B1 uptake measured at 7 degrees C is between 40 and 50% compared to that at 37 degrees C. However, after an incubation period of 7 minutes identical equilibrium uptake is reached at both temperatures. The apparent activation energies, calculated for aflatoxin B1 uptake by Arrhenius diagrams ranged between 1.69 and 4.5 kcal/mol. A Q10 value of 1.34 was calculated for a temperature interval of 7-17 degrees C but decreased to 1.05 for the interval of 27-37 degrees C. Liposomes or lipoproteins added to the cell suspension inhibited the aflatoxin B1 uptake into hepatocytes. Liposomes mainly composed of unsaturated fatty acids bind twice as much aflatoxin B1 as those composed of saturated ones, indicating that the lipophilicity of the mycotoxin is crucial in the determination of its uptake into liver cells. At concentrations above 5 micrograms/ml, aflatoxin B1 inhibited the carrier-mediated uptake of cholic acid and of phalloidin into hepatocytes. This effect was reversible and abolished by washing the cells after preincubation with aflatoxin. In concentrations below 5 micrograms/ml the uptake of phallotoxin and cholic acid was however stimulated by 15-25%. These results indicate, that a carrier-mediated uptake into hepatocytes via the multispecific bile salt transporter is not responsible for the organoselective clearance of aflatoxins by the liver. On the other hand, the cholestatic effect of aflatoxin B1 results at least partially from the inhibition of the multispecific bile acid transport system. This inhibition may arise from affinity of aflatoxins to lipid domains of the cell membrane.

Comparative investigations on the uptake of phallotoxins, bile acids, bovine lactoperoxidase and horseradish peroxidase into rat hepatocytes in suspension and in cell cultures.

Two alternative uptake mechanisms for phallotoxins by liver cells are debated: carrier-mediated uptake and receptor-mediated endocytosis. We have compared the properties of hepatocellular uptake of the phallotoxins, phalloidin and demethylphalloin, with the uptake of cholate as a substrate for carrier-mediated uptake and compared with iodinated bovine lactoperoxidase or iodinated horseradish peroxidase, as the latter are known to be taken up by vesicular endocytosis. Uptake of phallotoxins and [14C]cholate uptake into isolated hepatocytes is independent of extracellular calcium but inhibited by A23187 or by monensin. Uptake of bovine lactoperoxidase strictly depends on external Ca2+, was insensitive to A23197 and was not inhibited by monensin. No mutual uptake inhibition between phalloidin or cholate and peroxidases was seen, indicating independent permeation pathways in hepatocytes. However, high concentrations of cytochalasin B inhibited the uptake of either phalloidin, cholate or bovine lactoperoxidase. Horseradish peroxidase uptake, which was taken as an indicator for fluid pinocytosis, was low in isolated hepatocytes and could not account for the amount of phalloidin or cholate taken up. In cultured rat hepatocytes, uptake of phallotoxins decreased within 1 day to 10% of the uptake seen in freshly isolated hepatocytes. The results indicate different mechanisms for hepatocellular phallotoxin/bile-acid uptake and peroxidase internalization. As monolayer cultures of hepatocytes rapidly lost the carrier-mediated uptake of phallotoxins and bile acids, freshly isolated hepatocytes might be a more suitable experimental model than cultured cells for kinetic studies on this transport system.

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase.

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.

Microelectrode measurement of cell membrane potential in isolated hepatocytes attached to collagen.

Isolated spherical rat hepatocytes attached to collagen-coated cover slips generate a mean membrane potential (Em) of -78 +/- 9 mV as measured with high-resistance microelectrodes. The recordings were biphasic and were stable for upto 20 minutes. The correlation between external potassium concentration and Em was not linear. Several potassium-channel blockers did not effect the membrane potential. Addition of ouabain added to the incubation solution slowly depolarized the cells. The results indicate a high potassium permeability of the isolated spherical hepatocytes attached to collagen.

Two distinct types of SH-groups are necessary for bumetanide and bile acid uptake into isolated rat hepatocytes.

Substances that block SH-groups were studied in respect to their effects on the uptake of the loop diuretic bumetanide and the bile acids cholate and taurocholate into isolated rat hepatocytes. SH-blockers, e.g., p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), dithiobis-nitropyridine (DTNP) and dithiobis-2-nitrobenzoic acid (DTNB) reduced bumetanide transport in a concentration-dependent manner. Inhibition of the organic mercurial PCMBS was reversed by the addition of 500 microM dithiothreitol (DTT), indicating an interaction of this substance with free SH-groups. NEM irreversibly blocked SH-groups by covalent binding and was the most effective inhibitor of bumetanide and cholate uptake. In contrast, PCMBS was the most effective inhibitor of taurocholate uptake. Photoaffinity studies with [3H]bumetanide and [3H]7,7-azotaurocholate were performed with isolated rat hepatocytes in the presence of PCMBS and DTNP. Binding of the photolabels was not reduced by SH-group blockers. Newly synthesized sulfhydryl-modifying reagents such as dithio-sulfonate-ethyl-nitrobenzoic acid (DTSNB) and dithio-octyl-nitrobenzoic acid (DTONB), are derivatives of the alkylating agent DTNB. DTSNB is regarded as a selective blocker for SH-groups in a hydrophilic environment, while DTONB is more lipophilic abd interacts with SH-groups in the transmembrane domain of transport proteins. The IC50-values of these blockers for bumetanide uptake (DTSNB 250 microM, DTONB 141 microM) and for cholate uptake (DTSNB 250 microM, DTONB 115 microM) were almost identical. These findings support the concept of a common uptake mechanism for cholate and bumetanide and indicate that two distinct moieties of SH-groups are required for the uptake of both organic anions. One of these is probably located on the outer surface and the other within the membrane of hepatocytes.

Driving forces in hepatocellular uptake of phalloidin and cholate.

Active uptake of phalloidin and cholate in isolated rat liver cells depends upon both Na+ gradient and membrane potential. Omission of Na+ or inhibition of the (Na+ + K+)-ATPase diminished both phalloidin and cholate uptake. Dissipation of the sodium, potassium or proton gradient by monensin, nigericin, gramicidin and valinomycin blocked phalloidin uptake and also caused reduction of cholate transport. Chelation of Ca2+ and Mg2+ by EGTA or incubation of liver cells with NH4Cl neither influenced phalloidin nor cholate uptake. Hyperpolarization of liver cells by the lipophilic anions NO3- or SCN- enhanced phalloidin but reduced cholate uptake. Depolarization induced by a reversed K+ gradient reduced both kinds of transport. The results indicate that sodium ions and the membrane potential are driving forces for phalloidin and cholate uptake in hepatocytes.

Bumetanide is not transported by the Ntcp or by the oatp: evidence for a third organic anion transporter in rat liver cells.

The loop diuretic bumetanide which inhibits hepatic bile acid uptake competitively according to its transport kinetics has been proposed to serve as a substrate of a multispecific bile acid transport system in liver parenchymal cells. However, when the in vitro transcripts of two cloned hepatic bile acid uptake carriers, the Ntcp (Na+/taurocholate cotransporting polypeptide) and the oatp (organic anion transporting polypeptide), was expressed for three days in Xenopus laevis oocytes [3H]bumetanide uptake was not increased although bile acid uptake was stimulated. The data presented show that bumetanide is taken up by a third organic anion transport system which is different from the cloned bile acid transporters.

A membrane-bound form of protein disulfide isomerase (PDI) and the hepatic uptake of organic anions.

Protein disulfide isomerase (PDI) was considered to be involved in the hepatic uptake of certain organic anions because the protein is photoaffinity labeled by photolabile derivatives of the bile acid taurocholate. Several lines of evidences including photoaffinity labeling experiments indicated a close relationship between the uptake of bile acids and the organic anion bumetanide. The possible involvement of PDI in hepatic transport processes of these organic anions was tested with polyclonal antibodies raised against a PDI-beta-galactosidase fusion protein. Western blot analysis and immunofluorescence of intact hepatocytes showed that protein disulfide isomerase is located in sinusoidal rat liver plasma membranes. This protein is immunologically identical with microsomal PDI prepared from bovine liver. The plasma membrane form of PDI is, however, not labeled by photoactivated bumetanide as revealed by two-dimensional gel electrophoresis. These results indicate that, although a membrane-bound form of the PDI is present in the sinusoidal plasma membrane of rat hepatocytes, this protein is not involved in the hepatocellular uptake of the organic anion bumetanide.

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.

Secretion of plasma proteins and its insulin-dependent regulation in rat hepatocyte-hepatoma hybrid cells.

Hepatocyte-hepatoma hybrid cells were obtained by fusion of primary rat hepatocytes with Fao hepatoma cells. Synthesis and secretion of the liver-specific plasma proteins albumin and transferrin were preserved in these proliferating hybrids, called hepatocytoma cells. In the hybrid clone 1E3, secretion of albumin was 75% and that of transferrin was 45% of the secretion in normal hepatocytes. The level of albumin mRNA was fivefold higher in hepatocytoma cells than in normal hepatocytes. Essentially no albumin and transferrin secretion or albumin mRNA was found in the parental Fao hepatoma cells. Insulin decreased the albumin mRNA level in hybrid cells, while it increased the level in normal hepatocytes. This indicates an inverse effect of the hormone due to this regulation in immortalized hepatocytoma cells compared to normal hepatocytes.

Immortalization of rat hepatocytes by fusion with hepatoma cells. II. Studies on the transport and synthesis of bile acids in hepatocytoma (HPCT) cells.

Hepatocytoma (HPCT) cells were screened for the uptake of bile acids, the organic anion bumetanide and phalloidin. In addition, the rate of bile acid synthesis and the ability to conjugate exogenously added free bile acids was investigated. The unconjugated bile acid cholate is transported via carrier-mediated uptake into HPCT cells of the clone 1E3. This transport is expressed less by HPCT clones 1F9 and 2E9. Cholate uptake is 40% sodium-dependent, temperature-sensitive, and reveals a km-value of 47 +/- 9 pmol/l and a Vmax-value of 94 +/- 29 pmol x min-1 x mg-1. However, all of the HPCT clones lack carrier-mediated uptake of glycocholate and taurocholate. The clones which possess cholate transport are sensitive to phalloidin and take up bumetanide. In higher cell passages cholate transport and the sensitivity to phalloidin are reduced. This process is not prevented by a selection procedure with HAT-medium, nor is it stimulated by nicotinamide, dimethyl sulfoxide, or sodium butyrate. The cells of the 1E3, 1F9 and 2E9 clones, but not Fao hepatoma cells synthesize bile acids endogenously even after 60 passages, and conjugate exogenously added cholate with taurine and glycine. These results indicate that liver-specific properties of freshly isolated hepatocytes which are lacking in the parental hepatoma cells are maintained in HPCT cells.

Functional characterization of the hepatic sodium-dependent taurocholate transporter stably transfected into an immortalized liver-derived cell line and V79 fibroblasts.

Bile acids are taken up into liver parenchymal cells by active, carrier-mediated transport. This transport is lost during cell transformation in permanent growing liver tumor cell lines. In order to establish bile acid uptake in a permanent mammalian cell culture system, we transfected the cDNA from the cloned rat liver Na(+)-taurocholate cotransporting polypeptide (Ntcp) in Chinese hamster lung fibroblasts (V79 cells) and in a "hepatocyte-like" cell line HPCT-1F3 with three different gene transfer methods (calcium phosphate precipitation, lipofection, electroporation). A stable integration of the cDNA in both cell genomes was observed. However, in V79 fibroblasts, a permanent functional expression of taurocholate transport was not achieved. The sodium-dependent uptake of taurocholate was expressed permanently only in HPCT-1E3 cells, if the Ntcp was transfected by electroporation. In this cell line (HPCT-1E3-TC-6/2), substrate specificity, sodium- and energy dependence, as well as the kinetic parameters of the transfected single transporter were measured. The sodium-dependent taurocholate uptake was inhibited by addition of non-labeled bile acids, bumetanide, sulfobromophthalein and oligomycin. Pretreatment with 10 mM Na(+)-butyrate of this cell culture for 22 h stimulated taurocholate uptake twofold. Neither butyrate-stimulated cells nor unstimulated cells transport glycocholate or cholate. Besides taurocholate a fluorescence-labeled taurocholate derivative, NBD-taurocholate, was taken up by the HPCT-1E3-TC cells. In conclusion, the specific gene transfer with the electroporation technique in combination with the "right" cell line, HPCT-1E3, has been successful for the permanent and functional expression of the Ntcp. This allowed direct monitoring of the solitary sodium-dependent taurocholate transport system in a "liver cell-like" environment.