Jan A. Schrickx

Ochratoxin A secretion by ATP-dependent membrane transporters in Caco-2 cells

The ATP-dependent membrane transporters, P-gp, MRP2 and BCRP, localized in the luminal membranes of the intestines, liver and kidney, counteract absorption and increase excretion of xenobiotics and drugs. Previously, it has been suggested that the mycotoxin ochratoxin A (OTA) is a substrate for ATP-dependent transporters, and hence the absorption and secretion of OTA in the Caco-2 cell model was investigated. To this end, Caco-2 cells were cultured as confluent monolayers in bicameral inserts and the transepithelial transport of the mycotoxin was assessed. Caco-2 cells secreted OTA to the luminal side in a concentration-dependent manner. This secretory permeability was higher than the absorptive permeability, while the absorptive permeability remained constant for all OTA concentrations tested. The secretion decreased and absorption increased in the presence of the MRP-inhibitor MK571, the P-gp and BCRP inhibitor GF120918, and the BCRP-inhibitor Ko143, suggesting that the secretion of OTA is mediated by MRP2 and BCRP. Cyclosporine A also decreased the secretory permeability, but did not affect absorptive permeability, while PSC833 did neither change absorption nor secretion of OTA. Hence it can be suggested that OTA is a substrate for MRP2 as well as BCRP. These findings are of interest in evaluating mycotoxin absorption after oral ingestion, tissue distribution and particularly excretion pathways, including renal, biliary and mammary gland excretion.

Implications of ABC transporters on the disposition of typical veterinary medicinal products

The ATP-Binding Cassette (ABC) transporters ABCB1, ABCC2 and ABCG2 are efflux transporters that facilitate the excretion of drugs, contribute to the function of biological barriers and maintain low cytoplasmic substrate concentrations in cells. ABC transporters modulate drug absorption, distribution and elimination according to the level of expression in the intestine, liver, kidney, and at biological barriers such as the blood-brain barrier. Moreover individual transporters are known to convey multi-drug resistance to tumour cells. While these diverse functions have been described in laboratory animal studies and in humans, the available information is very limited in animal species that are typical veterinary patients. This brief review summarizes the available data on organ distribution and expression levels in animals, genetic defects in dogs resulting in a non-functional P-gp expression, and describes examples of kinetic investigations directed to assess the clinical relevance of species differences in ABC-transporter expression.

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.

Danofloxacin‐mesylate is a substrate for ATP‐dependent efflux transporters

Next to its broad antimicrobial spectrum, the therapeutic advantages of the fluoroquinolone antimicrobial drug Danofloxacin‐Mesylate (DM) are attributed to its rapid distribution to the major target tissues such as lungs, intestines and the mammary gland in animals. Previous analyses revealed that effective drug concentrations are achieved also in luminal compartments of these organs, suggesting that active transport proteins facilitate excretion into the luminal space. Members of the ATP‐Binding Cassette (ABC) superfamily, including P‐gp, BCRP and MRP2 are known to be expressed in many tissue barriers and in cell‐membranes facing luminal compartments. Hence we hypothesized that DM is a substrate for one of these efflux‐transporters.

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.

Multi-drug resistance in a canine lymphoid cell line due to increased P-glycoprotein expression, a potential model for drug-resistant canine lymphoma

Canine lymphoma is routinely treated with a doxorubicin-based multidrug chemotherapy protocol, and although treatment is initially successful, tumor recurrence is common and associated with therapy resistance. Active efflux of chemotherapeutic agents by transporter proteins of the ATP-Binding Cassette superfamily forms an effective cellular defense mechanism and a high expression of these transporters is frequently observed in chemotherapy-resistant tumors in both humans and dogs. In this study we describe the ABC-transporter expression in a canine lymphoid cell line and a sub-cell line with acquired drug resistance following prolonged exposure to doxorubicin. This sub-cell line was more resistant to doxorubicin and vincristine, but not to prednisolone, and had a highly increased P-glycoprotein (P-gp/abcb1) expression and transport capacity for the P-gp model-substrate rhodamine123. Both resistance to doxorubicin and vincristine, and rhodamine123 transport capacity were fully reversed by the P-gp inhibitor PSC833. No changes were observed in the expression and function of the ABC-transporters MRP-1 and BCRP. It is concluded that GL-40 cells represent a useful model for studying P-gp dependent drug resistance in canine lymphoid neoplasia, and that this model can be used for screening substances as potential P-gp substrates and their capacity to modulate P-gp mediated drug resistance.

Expression of MDR1, MRP2 and BCRP mRNA in tissues of turkeys.

MDR1, MRP2 and BCRP are members of the superfamily of ABC membrane transporters that export a large variety of structurally diverse substances out of the cell, hence being an integral part of various biological barriers. Here we report for the first time the tissue distribution of these ABC efflux transporters in the gastrointestinal tract (crop, proventriculus, duodenum, proximal and distal jejunum, ileum, caecum, colon) as well as in liver, kidney, lung, brain, adrenal gland, ovaries, oviduct and testes in BUT9 turkeys. MDR1 and BCRP mRNA expression was detected in all tissue samples, and the highest levels were measured in the small intestines. The tissue distribution of MRP2 mRNA was less consistent and some tissues seemed to lack any significant expression. Moreover, in consideration of previous findings suggesting that fluoroquinolones are substrates and modulators of ABC transporters, the effect of orally administered danofloxacin mesylate on the levels of MDR1, MRP2 and BCRP mRNA expression was investigated. Danofloxacin treatment resulted in a significant up-regulation of the measured transporters at the transcriptional level in the upper part of gastro-intestinal tract, liver and kidneys as well as in barrier-protected organs, such as the brain. However, despite this significant increase in the transcription levels, the pharmacokinetic parameters after repeated application of danofloxacin mesylate were not significantly altered.

Effects of fluoroquinolone treatment on MDR1 and MRP2 mRNA expression in Escherichia coli-infected chickens.

Current knowledge about the expression of ABC transport proteins suggests that their expression is regulated by a variety of factors, including pathological conditions, and in particular inflammatory reactions to infection. As ABC transporters are major determinants of absorption, distribution and excretion of many antimicrobials, modulation of their activity may result in increased or decreased tissue levels of drugs, affecting the efficacy of treatment. As fluoroquinolones have been identified as modulators and substrates of a number of drug transporters, we evaluated the effect of danofloxacin mesylate and enrofloxacin treatment on the levels of expression of MDR1 and MRP2 mRNAs in the intestines and livers of broilers with experimentally induced colibacillosis. MDR1 mRNA expression was significantly decreased in infected animals and was partly restored over 5 days of treatment with orally administered danofloxacin mesylate or enrofloxacin. Changes in the level of expression of MRP2 mRNA were less prominent. The study suggests that the treatment of colibacillosis with fluoroquinolones, which resulted in a significant clinical improvement of the animals, also restored the expression of drug transporters. This is of clinical importance as these ABC transporters significantly contribute to the functionality of important biological barriers, protecting the bird and specific tissues from pathogens and bacterial toxins.