ABC-transporters in the pig

The ATP-Binding Cassette (ABC) of transmembrane proteins comprises a large superfamily of efflux transporters with diverse physiological functions. Three members, ABCB1, ABCC2 and ABCG2, are predominantly expressed at the luminal membranes of excretory organs like the liver and the kidneys, as well as at biological barriers, as for example the intestinal barrier and the blood-brain barrier. It has been postulated that not only many drugs but also various toxins are substrates for these transporters, which in turn represent an innate defence mechanism of the organism against many undesirable food/feed components as well as toxins. In the first part of the thesis, three different substances were studied in a defined cell culture model comprising Caco-2 cells that resemble the intestinal barrier and allow the prediction of drug/toxin absorption and passage into barrier-protected tissues. Three compounds, that are relevant to pigs were selected: the antibiotic oxytetracycline was chosen due to its long-standing and common use in pig health care, despite its low oral bioavailability. In contrast, danofloxacin, a modern fluoroquinolone was selected as it gains importance in the therapy of infectious diseases in pigs. The third compound, the mycotoxin ochratoxin A, was included in these tests as it is known that pigs are the most sensitive animal species, and hence insight into the organ distribution of the toxin might contribute to the understanding of this species-specific sensitivity. All three compounds were found to be substrates for one or more ABC transporters and hence these findings confirmed that more detailed information on the tissue distribution of ABC transporters, particularly ABCB1, ABCC2 and ABCG2 in pigs, is needed. Subsequently the expression of P-gp (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2) was investigated by immunohistochemistry and quantitative RT- PCR analysis in a wide range of porcine tissues. A comparison of these findings with investigations in other species indicated a lower level of expression of P-gp in porcine kidneys and BCRP in porcine liver, as compared to human and rodent data. These findings suggest that the differences in the kinetics of the mentioned therapeutics and the mycotoxin are partly attributable to species-differences at the level of membrane transporters. Sequence analysis of porcine transporters demonstrated a high degree of homology with the corresponding human ABC proteins, which was higher than that for mice and rats. These findings confirm the good correlation between the kinetic data obtained from humans and pigs, and suggest that bioassays in pigs or with pig tissues may serve as an elegant model for kinetic studies with drugs and toxins affecting humans. Finally, an easy and rapid model, based on isolated porcine lymphocytes is presented, that allows to predict directly the function of P-gp. These functional studies quantify P-gp dependent Rhodamin 123 transport and allow to identify not only substrates but also inhibitors of P-gp. The latter might be involved in drug-drug interactions and interactions between drugs, toxins and dietary components. In conclusion, the presented results provide for the first time detailed insight into the levels of expression and function of ABC transporters in pigs

Expression of drug efflux transporters in poultry tissues.

Multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 2 (MRP2) are two prominent members of the super-family of ATP-binding cassette (ABC) transporters that carry a wide range of substrates across biological membranes, using ATP as energy source. The level of expression of these efflux transporters in different tissues has hitherto been studied mainly in mammals, and only P-glycoprotein (P-gp), the product of the MDR1 gene, has been described in chickens as of yet. The aim of this study was to describe the levels of expression of MDR1 and MRP2 mRNAs in different tissues of chickens, as these transporters play an important role in the absorption, distribution and excretion of drugs and toxins. In the gastro-intestinal tract, the highest levels of MDR1 mRNA expression were found in the small intestines, followed by the colon, whereas lower levels were found in the crop, proventriculus and the caeca. High MDR1 expression was also measured in the excretory organs such as liver, kidney and lungs. In contrast to rodents and humans, relatively low levels were found in the adrenals and in the immature sex organs such as testicles and ovaries. MRP2 mRNA expression was high in the liver, kidneys, duodenum and the jejunum, but expression was low in the ileum as well as in the lungs. No MRP2 expression could be detected in the other organs tested. Comparing the findings in chickens with previously published data, in particular those from humans and rodents, an unexpected high degree of similarity in the expression pattern of MDR1 and MRP2 mRNAs was apparent

The feline bile salt export pump: a structural and functional comparison with canine and human Bsep/BSEP.

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Deoxynivalenol-induced cytotoxicity, cytokines and related genes in unstimulated or lipopolysaccharide stimulated primary porcine macrophages.

The cytotoxicity of deoxynivalenol (DON) as well as the induction of cytokines and related genes was investigated in porcine pulmonary alveolar macrophages (PAM) in absence or presence of lipopolysaccharides (LPS). IC(20) values were 1.1, 0.4 and 1.0microM DON in the MTT, neutral red and alamar blue assay, respectively, and did not differ significantly in the presence of LPS. The mRNA expression of tumour necrosis factor (TNF)-alpha peaked at 3h, whereas LPS and DON showed synergistic effects resulting in an approximately 20-fold increase at 500nM DON as compared to untreated controls. The supernatant concentrations of TNF-alpha showed similar synergistic effects. The expression of interleukin (IL)-1beta was significantly induced by DON (except for 12h) and LPS. An induction of the mRNA expression of IL-6 by DON was evident only at 3h, whereas the supernatant concentrations of LPS stimulated PAM incubated with 500nM DON were significantly decreased at most time points. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression did not seem to contribute to the effects of DON in porcine macrophages. The results of the present investigation suggest a contribution of cytokines, especially TNF-alpha and IL-1beta, induced by DON in porcine macrophages to the effects observed in vivo

Interactions of deoxynivalenol and lipopolysaccharides on cytokine excretion and mRNA expression in porcine hepatocytes and Kupffer cell enriched hepatocyte cultures.

The effects of deoxynivalenol (DON) on the mRNA expression of cytokines and inflammation-related genes, as well as the cytokine secretion of porcine hepatocytes and Kupffer cell enriched hepatocyte cultures (co-cultures), were investigated in the absence or presence of LPS. DON and LPS acted in a synergistic manner with regard to a significantly increased mRNA expression of TNF-alpha in hepatocytes exposed to 500 nM or 2000 nM DON, or non-significant increase in co-cultures after 3h of exposure. TNF-alpha supernatant concentrations were increased due to LPS but did not reflect the synergistic effects with DON as observed at mRNA level. IL-6 mRNA in hepatocyte cultures at 6h paralleled the TNF-alpha supernatant pattern at this time point. In co-cultures and hepatocytes, a DON dose dependent induction of IL-6 mRNA was detected in cells not exposed to LPS. Supernatant concentrations of LPS-induced IL-6 were significantly decreased by 2000 nM DON in both types of cell cultures. Also the mRNA expression of the anti-inflammatory IL-10 was increased by DON to various degrees depending on DON-dose, stimulation with LPS and time point of measurement. After 6h, expression of iNOS was only induced by 2000 nM DON, but not in LPS treated cells. Even if mRNA induction was not paralleled by related supernatant concentrations of TNF-alpha, IL-6 and IL-10 under the conditions of the present investigations, it was clearly demonstrated that DON has the potential to provoke and modulate immunological reactions of porcine liver cells

Interactions of deoxynivalenol and lipopolysaccharides on cytotoxicity protein synthesis and metabolism of DON in porcine hepatocytes and Kupffer cell enriched hepatocyte cultures.

The cytotoxicity of deoxynivalenol (DON), effects on protein synthesis and albumin secretion was investigated in porcine hepatocytes and Kupffer cell-enriched hepatocyte cultures (co-cultures) in the presence and absence of lipopolysaccharides (LPS). Up to 16microM DON did not reduce the metabolic activity of hepatocytes. Lysosomal activity reacted more sensitively as neutral red uptake was decreased starting at 2 or 4microM DON irrespective of LPS exposure. The synthesis of secreted proteins was reduced to 31% and 42%, and of cellular proteins to 47% and 39%, in the absence and presence of LPS, respectively, when hepatocytes were exposed to 2microM DON. Reduced albumin secretion in response to DON was already observed after 3h in hepatocytes as well as co-cultures while LPS-mediated decrease was not evident until 24h, when interactions between DON and LPS resulted from a diminishing difference between LPS stimulated and non-stimulated cultures with increasing concentrations of DON. All observed effects may be biased by the cells' ability to conjugate DON to glucuronic acid as 54% and 64% of DON administered at 5nM were recovered as conjugates after 48h. Glucuronidation rate, as well as total DON recovery, decreased with increasing concentrations of DON, giving rise to assumptions on the formation of undetected metabolites

Masitinib reverses doxorubicin resistance in canine lymphoid cells by inhibiting the function of P-glycoprotein

Overexpression of ABC-transporters including Pgp, MRP1, and BCRP has been associated with multidrug resistance (MDR) in both human and canine oncology. Therapeutic interventions to reverse MDR are limited, but include multidrug protocols and the temporary concomitant use of inhibitors of ABC-transporters. Recently, the use of tyrosine kinase inhibitors has been proposed to overcome MDR in human oncology. One of the tyrosine kinase inhibitors, masitinib, is licensed for veterinary use in the treatment of canine mast cell tumors. Therefore, this study aimed to assess the potential of masitinib to revert MDR in canine malignant lymphoma using an in vitro model with canine lymphoid cell lines. Masitinib had a mild antiproliferative effect on lymphoid cells, inhibited Pgp function at concentrations equal to or exceeding 1 μm and was able to reverse doxorubicin resistance. The current findings provide the rationale for a combined use of masitinib with doxorubicin in the treatment of dogs with doxorubicin-resistant malignant lymphoma but await confirmation in clinical trials

Feline hepatic biotransformation of diazepam: Differences between cats and dogs

Item does not contain fulltextIn contrast to humans and dogs, diazepam has been reported to induce severe hepatic side effects in cats, particularly after repeated dosing. With the aim to elucidate the mechanisms underlying this apparent sensitivity of cats to drug-induced liver injury, in a series of in vitro experiments, the feline-specific biotransformation of diazepam was studied with liver microsomes obtained from cats and dogs and the possible inhibition of the bile salt export pump (Bsep) was measured in isolated membrane vesicles overexpressing feline and canine Bsep. In line with previous in vivo studies, the phase I metabolites nordiazepam, temazepam and oxazepam were measurable in microsomal incubations, although enzyme velocity of demethylases and hydroxylases differed significantly between cats and dogs. In cats, the main metabolite was temazepam, which also could be glucuronidated. In contrast to dogs, no other glucuronidated metabolites could be observed. In addition, in the membrane vesicles an inhibition of the transport of the Bsep substrate taurocholic acid could be observed in the presence of diazepam and its metabolites. It was concluded that both mechanisms, the slow biotransformation of diazepam as well the inhibition of the bile acid efflux that results in an accumulation of bile acids in the hepatocytes, seem to contribute to the liver injury observed in cats following repetitive treatment with diazepam