Walther Honscha

Cross-Sectional Study on Antibiotic Usage in Pigs in Germany

To be able to analyze the relationship between the level of resistance and the use of antimicrobials, it is necessary to collect detailed data on antimicrobial usage. For this reason, data on antimicrobial use on 495 pig farms from entire Germany were collected and analyzed. In Germany, each application and dispensing of medicines to food-producing animals is documented in detail obligatorily by the veterinarian. This information was collected retrospectively for the year 2011. The analyses undertook separate examinations on the age groups sow, piglet, weaner and fattening pig; both the route of administration and indication per active ingredient, and active ingredient class, were evaluated. In total, 20,374 kg of antimicrobial substances were used in the study population. Tetracyclines were used in highest amounts, followed by beta-lactams, trimethoprim-sulfonamides and macrolides. Concerning the frequency of using an active substance per animal, polypeptides were most commonly administered. In all age groups, respiratory infections were the main indication for using antimicrobials, followed by intestinal diseases in piglets, weaners and fattening pigs and diseases of reproductive organs in sows. Over a period of 100 days, the median number of treatment days with one antimicrobial substance for piglets was 15 days, for weaners about 6 days, for fattening pigs about 4 days and for sows about 1 day. A multifactorial ANOVA was conducted to investigate which factors are associated with the treatment frequency. The factors “veterinarian” and “age group” were related to the treatment frequency, just as the interaction between “veterinarian” and “farm size” as well as the interaction between “veterinarian” and “age group”.

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.

Feasibility study of veterinary antibiotic consumption in Germany - comparison of ADDs and UDDs by animal production type, antimicrobial class and indication

BackgroundWithin a feasibility study the use of antibiotics in pigs and cattle was determined in 24 veterinary practices in Lower Saxony and on 66 farms in North Rhine-Westphalia in Germany. Focus was laid on the comparison of the Used Daily Doses (UDD) (dose per animal and day prescribed by the veterinarians) with the Defined Animal Daily Doses (ADD) (dose per animal and day calculated by means of recommended dosages and estimated live weights).ResultsFor piglets and calves most of the UDD (50% and 46% of nUDD, respectively) were above the ADD (i.e. UDD/ADD-ratio above 1.25). Regarding sows, fattening pigs, dairy and beef cattle, most of the UDDs (49% to 65% of nUDD) were lower than the respective ADD (i.e. UDD/ADD-ratio below 0.8). In pigs, the UDDs of beta-lactams, fluoroquinolones and cephalosporins, and in cattle, those of macrolides and beta-lactams were often below the ADDs. Tetracyclines were frequently used above the recommended dose.Enteric diseases were more often treated below the recommended dose than respiratory diseases, possibly due to overestimation of the live weight (diarrhea in young animals, respiratory diseases in elder animals) and consequently overestimation of the recommended dose.ConclusionComparisons between UDD and ADD can be used to observe differences between antimicrobials and trends in the usage of antibiotics. But individual treatment comparisons of UDD and ADD must be interpreted carefully, because they may be due to lower live weights than estimated. Correlating such data with data on the occurrence of resistant bacteria in future may help to improve resistance prevention and control.

Subcellular localization and distribution of the reduced folate carrier in normal rat tissues

The reduced folate carrier (Rfc1; Slc19a1) mediated transport of reduced folates and antifolate drugs such as methotrexate (MTX) play an essential role in physiological folate homeostasis and MTX cancer chemotherapy. As no systematic reports are as yet available correlating Rfc1 gene expression and protein levels in all tissues crucial for folate and antifolate uptake, storage or elimination, we investigated gene and protein expression of rat Rfc1 (rRfc1) in selected tissues. This included the generation of a specific anti-rRfc1 antibody. Rabbits were immunised with isolated rRfc1 peptides producing specific anti-rRfc1 antiserum targeted to the intracellular C-terminus of the carrier. Using RT-PCR analysis, high rRfc1 transcript levels were detected in colon, kidney, brain, thymus, and spleen. Moderate rRfc1 gene expression was observed in small intestine, liver, bone marrow, lung, and testes whereas transcript levels were negligible in heart, skeletal muscle or leukocytes. Immunohistochemical analyses revealed strong carrier expression in the apical membrane of tunica mucosa epithelial cells of small intestine and colon, in the brush-border membrane of choroid plexus epithelial cells or in endothelial cells of small vessels in brain and heart. Additionally, high rRfc1 protein levels were localized in the basolateral membrane of renal tubular epithelial cells, in the plasma membrane of periportal hepatocytes, and sertoli cells of the testes. Taken together, our results demonstrated that rRfc1 is expressed almost ubiquitously but to very different levels. The predominant tissue distribution supports the essential role of Rfc1 in physiological folate homeostasis. Moreover, our results may contribute to understand antifolate pharmacokinetics and selected organ toxicity associated with MTX chemotherapy.

Determination of Functional ABCG2 Activity and Assessment of Drug–ABCG2 Interactions in Dairy Animals Using a Novel MDCKII In Vitro Model

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is a member of the ATP-binding cassette (ABC) family of efflux carriers that mediates cellular extrusion of various drugs and toxins. In the mammary gland, ABCG2 is expressed at the apical membrane of alveolar epithelial cells and is induced during lactation. It is well established that ABCG2 plays the main role in active secretion of xenobiotics into milk of humans and mice. In contrast, no detailed information is as yet available about functional activity and substrate spectrum of ABCG2 in dairy animals. Therefore, we cloned full-length ABCG2 from bovine, ovine and caprine lactating mammary gland tissues using rapid amplification of complementary DNA (cDNA) ends polymerase chain reaction. The generated full-length ABCG2 cDNA constructs were stably transduced in MDCKII cells. Functional ABCG2 efflux activity was demonstrated with the Hoechst H33342 accumulation assay using the specific ABCG2 inhibitor Ko143. The established ruminant MDCKII-ABCG2 cell culture models in conjunction with the H33342 transport assay showed interaction of various drugs such as cefalexin and albendazole with bABCG2, oABCG2 or cABCG2. Moreover, the flavonoids equol and quercetin exhibited interaction with all ruminant ABCG2 clones. Altogether, our generated cell culture models allowed rapid and high-throughput screening of potential ruminant ABCG2 substrates and thus increase the understanding of carrier-associated secretion of xenobiotics into milk.

Expression and subcellular localization of efflux transporter ABCG2/BCRP in important tissue barriers of lactating dairy cows, sheep and goats.

Expression of efflux transporter ABCG2/BCRP in tissues barriers has shown to be associated with altered pharmaco- and toxicokinetics of xenobiotics. Until now, little is known about the functional expression of this transporter in dairy animals. We therefore systematically examined the expression and subcellular localization of ABCG2/BCRP in small intestine, colon, lung, liver, kidney and mammary gland in lactating cows, sheep and goats. Carrier expression was investigated by RT-PCR and Western blot analysis showing highest expression of ABCG2/BCRP in small intestine and mammary gland, high levels in liver and moderate amounts of protein in lung, colon and kidney. Regarding subcellular localization, BCRP was predominantly found at the apical plasma membrane of small intestine, colon, bronchial epithelium, bile ducts and overall in endothelial structures in all tested species. In the mammary gland, there was strong apical staining of the alveolar epithelial cells and most of the ducts in all dairy ruminants. We also detected significantly elevated protein expression in lactating mammary gland compared with nonlactating cows, sheep and goats. Our results contribute to the role of BCRP in cytoprotection and disposition in important tissue barriers and may have important implications for veterinary pharmacotherapy of dairy animals using drugs identified as BCRP substrates.

The H+-dependent reduced folate carrier 1 of humans and the sodium-dependent methotrexate carrier-1 of the rat are orthologs

Previously, two different carrier systems for uptake of reduced folates and the antifolate methotrexate (Mtx) were described: the pH‐dependent folate sensitive reduced folate carrier 1 (RFC1) from human, hamster and mouse and a sodium‐dependent and folate insensitive Mtx carrier‐1 (MTX‐1) from rat. It was found that all critical residues of the homologous amino acid sequence were identical. RFC1‐ as well as MTX‐1‐mediated uptake of a marker substrate into suitable human and rat cell lines increased with proton concentration, was sodium‐dependent at neutral pH, and inhibited by folate at acidic pH. It is concluded that RFC1 and MTX‐1 are orthologs.

A novel MDCKII in vitro model for assessing ABCG2-drug interactions and regulation of ABCG2 transport activity in the caprine mammary gland by environmental pollutants and pesticides

The ABC efflux transporter ABCG2 represents the main route for active secretion of xenobiotics into milk. Thus, ABCG2 regulation by aryl hydrocarbon receptor (AhR) ligands including ubiquitously environmental pollutants is of great toxicological relevance. However, no adequate in vitro model is as yet available to study AhR-dependent ABCG2 regulation in dairy animals. In this study, we therefore systematically investigated the effect of various environmental contaminants and pesticides on ABCG2 efflux activity in MDCKII cells stably expressing mammary ABCG2 from dairy goats. The AhR-agonists TCDD, Aroclor 1254, prochloraz, and iprodione caused a dose- and time-dependent increase in EROD activity. Moreover, TCDD and prochloraz significantly stimulated ABCG2 transport activity through a dose- and time-dependent induction of transporter gene expression. AhR inhibitors like CH223191 significantly reversed TCDD- and prochloraz-induced stimulation of ABCG2 efflux activity. In contrast, non-AhR activators such as PCB 101 had no significant effect on EROD activity, ABCG2 gene expression or transporter activity. As we identified various anthelmintics including monepantel as potential ABCG2 substrates this regulatory mechanism may result in increased milk residues of potentially harmful xenobiotics. Thus, MDCKII-cABCG2 cells may represent a suitable in vitro model to study mammary ABCG2 secretory activity and its potential regulation by AhR-activating contaminants.

The Antiepileptic Drugs Phenobarbital and Carbamazepine Reduce Transport of Methotrexate in Rat Choroid Plexus by Down-Regulation of the Reduced Folate Carrier

Intrathecal methotrexate (MTX) has been associated with severe neurotoxicity. Because carrier-associated removal of MTX from the cerebrospinal fluid (CSF) into blood remains undefined, we determined the expression and function of MTX transporters in rat choroid plexus (CP). MTX neurotoxicity usually manifests as seizures requiring therapy with antiepileptic drugs (AEDs) such as phenobarbital (PB). Because we have demonstrated that PB reduces activity of MTX influx carrier reduced folate carrier (Rfc1) in liver, we investigated the influence of the AEDs PB, carbamazepine (CBZ), or gabapentin on Rfc1-mediated MTX transport in CP. Reverse transcriptase-polymerase chain reaction and Western blot analysis showed similar expression of the MTX influx carrier Rfc1 and organic anion transporter 3 or efflux transporter multidrug resistance-associated protein 1 (Mrp1) and breast cancer resistance protein (Bcrp) in rat CP tissue and choroidal epithelial Z310 cells. Confocal microscopy revealed subcellular localization of Rfc1 and Bcrp at the apical and of Mrp1 at the basolateral CP membrane. Uptake, efflux, and inhibition studies indicated MTX transport activity of Rfc1, Mrp1, and Bcrp. PB and CBZ but not gabapentin significantly inhibited Rfc1-mediated uptake of MTX in CP cells. Studies on the regulatory mechanism showed that PB significantly inhibited Rfc1 translation but did not alter carrier gene expression. Altogether, removal of intrathecal MTX across the blood-CSF barrier may be achieved through Rfc1-mediated uptake from the CSF followed by MTX extrusion into blood, particularly via Mrp1. Antiepileptic treatment with PB or CBZ causes post-transcriptional down-regulation of Rfc1 activity in CP. This mechanism may result in enhanced MTX toxicity in patients with cancer who are receiving intrathecal MTX chemotherapy by reduced CSF clearance of the drug.

The ABCG2 Efflux Transporter in the Mammary Gland Mediates Veterinary Drug Secretion across the Blood-Milk Barrier into Milk of Dairy Cows.

In human and mice ATP-binding cassette efflux transporter ABCG2 represents the main route for active drug transport into milk. However, there is no detailed information on the role of ABCG2 in drug secretion and accumulation in milk of dairy animals. We therefore examined ABCG2-mediated drug transport in the bovine mammary gland by parallel pharmacokinetic studies in lactating Jersey cows and in vitro flux studies using the anthelmintic drug monepantel (MNP) as representative bovine ABCG2 (bABCG2) drug substrate. Animals received MNP (Zolvix, Novartis Animal Health Inc.) once (2.5 mg/kg per os) and the concentrations of MNP and the active MNP metabolite MNPSO2 were assessed by high-performance liquid chromatography. Compared with the parent drug MNP, we detected higher MNPSO2 plasma concentrations (expressed as area under the concentration-versus-time curve). Moreover, we observed MNPSO2 excretion into milk of dairy cows with a high milk-to-plasma ratio of 6.75. In mechanistic flux assays, we determined a preferential time-dependent basolateral-to-apical (B > A) MNPSO2 transport across polarized Madin-Darby canine kidney II cells–bABCG2 monolayers using liquid chromatography coupled with tandem mass spectrometry analysis. The B > A MNPSO2 transport was significantly inhibited by the ABCG2 inhibitor fumitremorgin C in bABCG2- but not in mock-transduced MDCKII cells. Additionally, the antibiotic drug enrofloxacin, the benzimidazole anthelmintic oxfendazole and the macrocyclic lactone anthelmintic moxidectin caused a reduction in the MNPSO2 (B > A) net efflux. Altogether, this study indicated that therapeutically relevant drugs like the anthelmintic MNP represent substrates of the bovine mammary ABCG2 transporter and may thereby be actively concentrated in dairy milk.