Enhanced reversal of ABCG2-mediated drug resistance by replacing a phenyl ring in baicalein with a meta-carborane.

Success of chemotherapy is often hampered by multidrug resistance. One mechanism for drug resistance is the elimination of anticancer drugs through drug transporters, such as breast cancer resistance protein (BCRP; also known as ABCG2), and causes a poor 5-year survival rate of human patients. Co-treatment of chemotherapeutics and natural compounds, such as baicalein, is used to prevent chemotherapeutic resistance but is limited by rapid metabolism. Boron-based clusters as meta-carborane are very promising phenyl mimetics to increase target affinity; we therefore investigated the replacement of a phenyl ring in baicalein by a meta-carborane to improve its affinity towards the human ABCG2 efflux transporter. Baicalein strongly inhibited the ABCG2-mediated efflux and caused a fivefold increase in mitoxantrone cytotoxicity. Whereas the baicalein derivative 5,6,7-trimethoxyflavone inhibited ABCG2 efflux activity in a concentration of 5 µm without reversing mitoxantrone resistance, its carborane analogue 5,6,7-trimethoxyborcalein significantly enhanced the inhibitory effects in nanomolar ranges (0.1 µm) and caused a stronger increase in mitoxantrone toxicity reaching similar values as Ko143, a potent ABCG2 inhibitor. Overall, in silico docking and in vitro studies demonstrated that the modification of baicalein with meta-carborane and three methoxy substituents leads to an enhanced reversal of ABCG2-mediated drug resistance. Thus, this seems to be a promising basis for the development of efficient ABCG2 inhibitors.

Carboranes as Potent Phenyl Mimetics: A Comparative Study on the Reversal of ABCG2-Mediated Drug Resistance by Carboranylquinazolines and Their Organic Isosteres.

Multidrug resistance is a major challenge in clinical cancer therapy. In particular, overexpression of certain ATP-binding cassette (ABC) transporter proteins, like the efflux transporter ABCG2, also known as breast cancer resistance protein (BCRP), has been associated with the development of resistance to applied chemotherapeutic agents in cancer therapies, and therefore targeted inhibition of BCRP-mediated transport might lead to reversal of this (multidrug) resistance (MDR). In a previous study, we have described the introduction of a boron-carbon cluster, namely closo-dicarbadodecaborane or carborane, as an inorganic pharmacophore into a polymethoxylated 2-phenylquinazolin-4-amine backbone. In this work, the scope was extended to the corresponding amide derivatives. As most of the amide derivatives suffered from poor solubility, only the amide derivative QCe and the two amine derivatives DMQCc and DMQCd were further investigated. Carboranes are often considered as sterically demanding phenyl mimetics or isosteres. Therefore, the organic phenyl and sterically demanding adamantyl analogues of the most promising carborane derivatives were also investigated. The studies showed that the previously described DMQCd, a penta-methoxylated N-carboranyl-2-phenylquinazolin-4-amine, was by far superior to its organic analogues in terms of cytotoxicity, inhibition of the human ABCG2 transporter, as well as the ability to reverse BCRP-mediated mitoxantrone resistance in MDCKII-hABCG2 and HT29 colon cancer cells. Our results indicate that DMQCd is a promising candidate for further in vitro as well as in vivo studies in combination therapy for ABCG2-overexpressing cancers.

Carborane-Based ABCG2-Inhibitors Sensitize ABC-(Over)Expressing Cancer Cell Lines for Doxorubicin and Cisplatin.

The ABCG2 transporter protein, as part of several known mechanisms involved in multidrug resistance, has the ability to transport a broad spectrum of substrates out of the cell and is, therefore, considered as a potential target to improve cancer therapies or as an approach to combat drug resistance in cancer. We have previously reported carborane-functionalized quinazoline derivatives as potent inhibitors of human ABCG2 which effectively reversed breast cancer resistance protein (BCRP)-mediated mitoxantrone resistance. In this work, we present the evaluation of our most promising carboranyl BCRP inhibitors regarding their toxicity towards ABCG2-expressing cancer cell lines (MCF-7, doxorubicin-resistant MCF-7 or MCF-7 Doxo, HT29, and SW480) and, consequently, with the co-administration of an inhibitor and therapeutic agent, their ability to increase the efficacy of therapeutics with the successful inhibition of ABCG2. The results obtained revealed synergistic effects of several inhibitors in combination with doxorubicin or cisplatin. Compounds DMQCa, DMQCc, and DMQCd showed a decrease in IC50 value in ABCB1- and ABCG2-expressing SW480 cells, suggesting a possible targeting of both transporters. In an HT29 cell line, with the highest expression of ABCG2 among the tested cell lines, using co-treatment of doxorubicin and DMQCd, the effective inhibitory concentration of the antineoplastic agent could be reduced by half. Interestingly, co-treatment of compound QCe with cisplatin, which is not an ABCG2 substrate, showed synergistic effects in MCF-7 Doxo and HT29 cells (IC50 values halved or reduced by 20%, respectively). However, a literature-known upregulation of cisplatin-effluxing ABC transporters and their effective inhibition by the carborane derivatives emerges as a possible reason.

2-Carboranylquinazoline: The Path to an ABCG2 Inhibitor.

The role of ATP-binding cassette (ABC) transporter-mediated multidrug resistance (MDR) in anti-cancer therapy is often challenging, frequently leading to inefficiency of treatments. Cancer cells exploit efflux transporters, like the breast cancer resistance protein (BCRP, ABCG2), to secrete chemotherapeutic substances. In this study, an N-phenyl-2-carboranylquinazolin-4-amine (8) was designed as inorganic-organic hybrid BCRP inhibitor. In particular, the ABCG2-transporter inhibitor-prominent scaffold N-phenylquinazolin-4-amine was combined with a boron-carbon cluster (carborane) moiety. Introducing a carborane at 2-position of the quinazoline scaffold resulted in an increased inhibitory activity towards human ABCG2 (hABCG2) compared to its recently published regioisomer N-carboranyl-2-phenyl-quinazolin-4-amine. The carboranylquinazoline 8 further showed the ability to reverse hABCG2-mediated drug resistance in MDCKII-hABCG2 cells by lowering the IC50 value of the BCRP-substrate mitoxantrone, similar to the standard reference and strong inhibitor Ko143, without exhibiting intrinsic toxicity in the lower micromolar ranges. These results make compound 8 a promising scaffold for the design of further BCRP inhibitors.

The More the Better-Investigation of Polymethoxylated N-Carboranyl Quinazolines as Novel Hybrid Breast Cancer Resistance Protein Inhibitors.

The ineffectiveness and failing of chemotherapeutic treatments are often associated with multidrug resistance (MDR). MDR is primarily linked to the overexpression of ATP-binding cassette (ABC) transporter proteins in cancer cells. ABCG2 (ATP-binding cassette subfamily G member 2, also known as the breast cancer resistance protein (BCRP)) mediates MDR by an increased drug efflux from the cancer cells. Therefore, the inhibition of ABCG2 activity during chemotherapy ought to improve the efficacy of the administered anti-cancer agents by reversing MDR or by enhancing the agents' pharmacokinetic properties. Significant efforts have been made to develop novel, powerful, selective, and non-toxic inhibitors of BCRP. However, thus far the clinical relevance of BCRP-selective MDR-reversal has been unsuccessful, due to either adverse drug reactions or significant toxicities in vivo. We here report a facile access towards carboranyl quinazoline-based inhibitors of ABCG2. We determined the influence of different methoxy-substitution patterns on the 2-phenylquinazoline scaffold in combination with the beneficial properties of an incorporated inorganic carborane moiety. A series of eight compounds was synthesized and their inhibitory effect on the ABCG2-mediated Hoechst transport was evaluated. Molecular docking studies were performed to better understand the structure-protein interactions of the novel inhibitors, exhibiting putative binding modes within the inner binding site. Further, the most potent, non-toxic compounds were investigated for their potential to reverse ABCG2-mediated mitoxantrone (MXN) resistance. Of these five evaluated compounds, N-(closo-1,7-dicarbadodecaboran(12)-9-yl)-6,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-quinazolin-4-amine (DMQCd) exhibited the strongest inhibitory effect towards ABCG2 in the lower nanomolar ranges. Additionally, DMQCd was able to reverse BCRP-mediated MDR, making it a promising candidate for further research on hybrid inorganic-organic compounds.

Development of an electronic interface for transfer of antimicrobial administration data in dairy farms.

Surveillance of antimicrobial administration in livestock production is an important factor in global policies to reduce spreading of antimicrobial resistance. In recent years, many studies have been carried out concerning the usage of antimicrobials in animal production and in some countries recording of antimicrobial quantities dispensed to famers is mandatory. On cattle farms, antimicrobial treatments are recorded for fattening calves under 8 months of age and for fattening cattle older than 8 months in Germany and treatment frequencies are then calculated. However, with the entry into force of Regulation (EU) 2019/6 on 01/28/2022, antimicrobial monitoring will gradually be extended to all animal species and age groups. Therefore, an effective, fast and accurate transfer of data on the use of antimicrobials, especially in the field of livestock farming, into corresponding databases is required to determine the treatment frequencies for the individual animal species or types of use. For this purpose, an electronic interface was programmed to transfer the data on antimicrobial use in dairy cattle farms from a herd management software program directly into a database. To test the practicability and effectiveness of this interface, 10 dairy cattle farms from Saxony were initially selected. Based on an in-depth analysis of the treatment frequencies of antimicrobial administration of 7 different age groups of animals after a two-year observation period, the functionality of the electronic interface could be established. The greatest potential for reduction of antimicrobials is in newborn calves, as they represent the age group with the highest treatment frequency.

Borcalein: a Carborane-Based Analogue of Baicalein with 12-Lipoxygenase-Independent Toxicity.

12-Lipoxygenase is crucial for tumour angiogenesis. 5,6,7-Trihydroxy-2-phenyl-4H-1-benzopyran-4-one (baicalein) is a suitable inhibitor for this enzyme but is rapidly metabolised in vivo. Thus, an improvement of the metabolic stability is necessary to enhance the therapeutic efficiency. An emerging approach to enhance metabolic stability of carbon-based pharmaceuticals is the use of metabolically stable, non-toxic boron clusters, such as dicarba-closo-dodecaborane(12)s (carboranes) as phenyl mimetics. Therefore, the unsubstituted phenyl ring of baicalein was replaced by meta-carborane, resulting in borcalein, the carborane analogue of baicalein. This substitution resulted in a decreased inhibitory activity toward 12-lipoxygenase, but led to increased toxicity in melanoma (A375, B16, B16F10) and colon cancer cell lines (SW480, HCT116, CT26CL25) with decreased tumour selectivity in comparison to baicalein. Surprisingly, borcalein displays a different mechanism of cytotoxicity with increased intracellular production of reactive oxygen species (ROS), reactive nitrogen species (RNS) and nitric oxide (NO).

Multidrug-resistant enterobacteria in newborn dairy calves in Germany.

We studied the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in dairy calves as part of a routine health check protocol. In addition, data regarding antimicrobial use (AMU), farm hygiene, and farm management were collected in order to identify possible risks for ESBL occurrence. Ten farms participated in the study with a median of 781 milking cows (319-1701). All calves investigated were younger than two weeks with an average age of 6.8 (±3.9) days. The farms were visited and samples were collected twice at an interval of 7-11 months. Faecal samples diluted 1:10, were plated onto BrillianceTM ESBL agar in duplicates. After 24 hours at 37°C, colonies were counted and total colony forming units (cfu)/ml calculated. Bacteria species were identified biochemically. ESBL-production was phenotypically confirmed using the MICRONAUT-S ß-Lactamases system. Additionally, antimicrobial susceptibility was tested using VITEK® 2 technology. Phylotyping of E. coli isolates and screening for bla genes was performed by PCR. ESBL-producing enterobacteria were detected on all farms and 96.5% of calves investigated shed ESBL-positive bacteria. Of all ESBL-producing isolates, the majority were E. coli (92.9%), followed by Enterobacter cloacae (5.1%) and Klebsiella pneumoniae subsp. pneumoniae (2.0%). The majority of E. coli isolates was clearly assigned to phylogroup C (25.0%), followed by phylogroups A (15.2%) and E (14.1%). CTX-M group 1 was most frequently detected (80.4%). E. cloacae contained blaCTX-M and blaTEM or blaSHV. K. pneumoniae harboured blaSHV only. Besides resistance to penicillins and cephalosporins, the majority of isolates was also resistant to one or more antibiotic classes, with a high proportion being resistant against fluoroqinolones. 52.5% of isolates were further characterised as threefold multidrug resistant gram-negative bacteria (3MDR-GNB) according to the German Commission for Hospital Hygiene and Infection Prevention. None of the isolates were 4MDR-GNB, i.e. none revealed carbapenem-resistance. Penicillins were the most frequently administered antibiotics to calves on most farms and were the predominant substance class at herd level on all farms. Overall, the number of calves treated prior to sampling was rather low (11.7%). Analyses of data regarding the farm management identified weaknesses in biosecurity and cleaning and disinfection. Besides beta-lactam antibiotics being the most commonly used antibiotics no other risk factors could be identified. In summary, the prevalence of ESBL-carriers in dairy calves was exceptionally high and should be motivation to develop strategies for the reduction of multidrug-resistant bacteria in farm animals.

AhR-activating pesticides increase the bovine ABCG2 efflux activity in MDCKII-bABCG2 cells.

In bovine mammary glands, the ABCG2 transporter actively secretes xenobiotics into dairy milk. This can have significant implications when cattle are exposed to pesticide residues in feed. Recent studies indicate that the fungicide prochloraz activates the aryl hydrocarbon receptor (AhR) pathway, increasing bovine ABCG2 (bABCG2) gene expression and efflux activity. This could enhance the accumulation of bABCG2 substrates in dairy milk, impacting pesticide risk assessment. We therefore investigated whether 13 commonly used pesticides in Europe are inducers of AhR and bABCG2 activity. MDCKII cells expressing mammary bABCG2 were incubated with pesticides for up to 72 h. To reflect an in vivo situation, applied pesticide concentrations corresponded to the maximum residue levels (MRLs) permitted in bovine fat or muscle. AhR activation was ascertained through CYP1A mRNA expression and enzyme activity, measured by qPCR and 7-ethoxyresorufin-Ο-deethylase (EROD) assay, respectively. Pesticide-mediated increase of bABCG2 efflux activity was assessed using the Hoechst 33342 accumulation assay. For all assays, the known AhR-activating pesticide prochloraz served as a positive control, while the non-activating tolclofos-methyl provided the negative control. At 10-fold MRL concentrations, chlorpyrifos-methyl, diflufenican, ioxynil, rimsulfuron, and tebuconazole significantly increased CYP1A1 mRNA levels, CYP1A activity, and bABCG2 efflux activity compared to the vehicle control. In contrast, dimethoate, dimethomorph, glyphosate, iprodione, methiocarb and thiacloprid had no impact on AhR-mediated CYP1A1 mRNA levels, CYP1A activity or bABCG2 efflux. In conclusion, the MDCKII-bABCG2 cell model proved an appropriate tool for identifying AhR- and bABCG2-inducing pesticides. This provides an in vitro approach that could reduce the number of animals required in pesticide approval studies.

Evaluation of the hepatocyte-derived cell line BFH12 as an in vitro model for bovine biotransformation.

The knowledge of drug metabolising enzymes (DMEs) in cattle is rather limited. The capability of the bovine foetal hepatocyte-derived cell line BFH12 to serve as model for biotransformation was evaluated. Gene expression analysis of DMEs was performed by reverse transcription PCR (RT-PCR). The presence of efflux transporters was visualised by immunocytochemistry, and functional induction of cytochrome P450 (CYP) 1A was assessed by the ethoxyresorufin-O-deethylase (EROD) assay. The production of bile acids was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RT-PCR revealed the expression of cytochromes 1A1, 1A2, 3A4 and phase II enzymes UGT1A1, UGT1A6 and GSTM1. Immunofluorescence demonstrated efflux transporters ABCG2 and ABCC1. The EROD assay revealed a dose-dependent CYP1A induction after treatment with benzo[a]pyrene (BP). LC-MS/MS analysis of cell culture supernatants showed the production of bile acids including taurocholic acid, tauro-chenodeoxycholic acid, taurodeoxycholic acid and taurolithocholic acid. The results strongly suggest the applicability of the cell line BFH12 for subsequent experiments in the emerging field of bovine biotransformation.

Association of farm-related factors with characteristics profiles of extended-spectrum ß-lactamase- / plasmid-mediated AmpC ß-lactamase-producing Escherichia coli isolates from German livestock farms.

Resistance to ß-lactam antibiotics, including third-generation cephalosporins, is of major concern for animal and human health. In this study, extended-spectrum ß-lactamase (ESBL) / plasmid-mediated AmpC (pAmpC) ß-lactamase -producing Escherichia coli isolates from German livestock farms were characterised and associations of these isolate characteristics with farm-related factors were investigated across different types of livestock. A total of 469 isolates originating from 150 farms (34 broiler farms, 38 fattening pig farms, 43 dairy cattle farms, 35 beef cattle farms) was included in the analyses. ESBL-gene family, phylogroup and phenotypic antimicrobial susceptibility for several antimicrobial agents were determined. This data was used to define different profiles characterising the isolates. Multivariate analyses using a distance-based non-parametric approach were performed to investigate associations between the profiles of the isolates and farm-related factors (e.g. management, husbandry, and environment of the farms). Co-occurrence of ESBL-gene families were not found in any of the isolates analysed. Sixty-eight percent of the isolates carried blaCTX-M variant genes. The frequency of phylogroups was as follows: A (55%), B1 (35%), D (17%) and B2 (3%). The most frequent phenotypic non-wildtype profile was non-wildtype status of solely cefepime (27%). Profiles of isolates from broilers differed substantially from those of other isolates. Associations between farm-related factors and characteristics profiles differed, depending on the isolate characteristics included in the analyses. Some factors describing the farm environment, like waterfowl in the surrounding of the farm, were associated with all tested profiles. The epidemiological method applied defines distances between isolates on basis of isolate characteristics data and is capable of analysing associations between isolate characteristics and epidemiological factors. As additional data, such as plasmid characteristics, gene type, or sequence information could be included in future studies, the method is suitable to identify points of action to reduce the occurrence of antimicrobial resistant bacteria.

Resistance towards monensin is proposed to be acquired in a Toxoplasma gondii model by reduced invasion and egress activities, in addition to increased intracellular replication.

Monensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.

Quantitative proteomic studies in resistance mechanisms of Eimeria tenella against polyether ionophores.

Polyether ionophores are widely used to treat and control coccidiosis in chickens. Widespread use of anticoccidials resulted in worldwide resistance. Mechanisms of resistance development and expansion are complex and poorly understood. Relative proteomic quantification using LC-MS/MS was used to compare sensitive reference strains (Ref-1, Ref-2) with putatively resistant and moderately sensitive field strains (FS-R, FS-mS) of Eimeria tenella after isotopic labelling with tandem mass tags (TMT). Ninety-seven proteins were identified, and 25 of them were regulated. Actin was significantly upregulated in resistant strains in comparison with their sensitive counterparts. On the other hand, microneme protein (MIC4) was downregulated in resistant strains. Optimization of labelling E. tenella sporozoites by TMT might identify further proteins that play a role in the obvious complex mechanism leading to resistance against Monensin.

Assessment of ABCG2-mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model.

In humans, the ATP-binding cassette efflux transporter ABCG2 contributes to the fetoprotective barrier function of the placenta, potentially limiting the toxicity of transporter substrates to the fetus. During testing of chemicals including pesticides, developmental toxicity studies are performed in rabbit. Despite its toxicological relevance, ABCG2-mediated transport of pesticides in rabbit placenta has not been yet elucidated. We therefore generated polarized MDCK II cells expressing the ABCG2 transporter from rabbit placenta (rbABCG2) and evaluated interaction of the efflux transporter with selected insecticides, fungicides, and herbicides. The Hoechst H33342 accumulation assay indicated that 13 widely used pesticidal active substances including azoxystrobin, carbendazim, chlorpyrifos, chlormequat, diflufenican, dimethoate, dimethomorph, dithianon, ioxynil, methiocarb, propamocarb, rimsulfuron and toclofos-methyl may be rbABCG2 inhibitors and/or substrates. No such evidence was obtained for chlorpyrifos-methyl, epoxiconazole, glyphosate, imazalil and thiacloprid. Moreover, chlorpyrifos (CPF), dimethomorph, tolclofos-methyl and rimsulfuron showed concentration-dependent inhibition of H33342 excretion in rbABCG2-transduced MDCKII cells. To further evaluate the role of rbABCG2 in pesticide transport across the placenta barrier, we generated polarized MDCKII-rbABCG2 monolayers. Confocal microscopy confirmed correct localization of rbABCG2 protein in the apical plasma membrane. In transepithelial flux studies, we showed the time-dependent preferential basolateral to apical (B>A) directed transport of [(14)C] CPF across polarized MDCKII-rbABCG2 monolayers which was significantly inhibited by the ABCG2 inhibitor fumitremorgin C (FTC). Using this novel in vitro cell culture model, we altogether showed functional secretory activity of the ABCG2 transporter from rabbit placenta and identified several pesticides like the insecticide CPF as potential rbABCG2 substrates.

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.

The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization.

In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84-86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter.

Effects of ceftiofur treatment on the susceptibility of commensal porcine E.coli--comparison between treated and untreated animals housed in the same stable.

BACKGROUND: Healthy farm animals have been found to act as a reservoir of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli). Therefore, the objective of the study was to determine the input of antimicrobial active ceftiofur metabolites in the stable via faeces and urine after intramuscular administration of the drug to pigs and the elucidation of the Escherichia coli ESBL resistance pattern of treated and untreated pigs housed in the same barn during therapy. METHODS: For determination of the minimal inhibitory concentration (MIC) the method of microdilutionaccording to the recommended procedure of the Clinical and Laboratory Standards Institute was used. Inaddition to that, a qualitative determination was performed by agar dilution. Unsusceptible E. coli speciesselected via agar dilution with cefotaxime were confirmed by MALDI-TOF and ESBL encoding genes wereidentified by PCR. The amounts of ceftiofur measured as desfuroylceftiofur (DFC) in the different probes (plasma, urine, faeces and dust) were analysed by UPLC-MS/MS. RESULTS: In a first experiment two groups of pigs (6 animals per group) were housed in the same barn in two separated boxes. One group (group B) were treated with ceftiofur according to the licence (3 mg/kg administered intramuscularly (i.m.) on three consecutive days, day 1-3). During a second treatment period (day 29-31) an increased rate of ESBL resistant E. coli was detectable in these treated pigs and in the air of the stable. Moreover, the second group of animals (group A) formerly untreated but housed for the whole period in the same stable as the treated animals revealed increased resistance rates during their first treatment (day 45-47) with ceftiofur. In order to investigate the environmental input of ceftiofur during therapy and to simulate oral uptake of ceftiofur residues from the air of the stable a second set of experiments were performed. Pigs (6 animals) were treated with an interval of 2 weeks for 3 days with different doses of ceftiofur (3 mg/kg, 1 mg/kg and 0.3 mg/kg i.m.) as well as with 3 mg/kg per os) and the renal and biliary excretion of ceftiofur as its active metabolite were measured in comparison to the plasma levels. In addition to that, probes of the sedimentation dust and the air of the stable were analysed for drug residues. CONCLUSION: The present study shows that treatment of several animals in a stable with ceftiofur influences the resistance pattern of intestinal Escherichia coli of the treated as well as untreated animals housed in the same stable. During therapy with the drug which was administered by injection according to the licence we detected nameable amounts of ceftiofur and its active metabolites in the dust and air of the stable.

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".

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

BACKGROUND: Within 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). RESULTS: For 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. CONCLUSION: Comparisons 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.

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.