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.

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.

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.

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).

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.

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.

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".

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.

[Antibiotic usage and antibiotic sales in Germany in 2011--the situation of drug usage in veterinary medicine]. / Antibiotikaverbrauch und Antibiotikaverkauf in Deutschland im Jahr 2011--Zur Situation des Arzneimitteleinsatzes in der Veterinärmedizin.

To appreciate the significance of sales data of antibiotics, data on antibiotic consumption (which were collected for selected food-producing animals within the representative VetCAb pilot study in Germany in 2011) were extrapolated for the total German animal population, and subsequently compared to the antibiotic sales data, which were collected throughout Germany in 2011 for the first time and which were published by the Federal Office of Consumer Protection and Food Safety (BVL). The extrapolation was realized based on the average dosage and the number of treatment units determined in the VetCAb pilot project for each active ingredient and animal species. It was extrapolated that 1302 tons of antimicrobial substances were consumed for treating cattle, pigs and broilers--this amount is accompanied by the amount of 1706 tons sold for all animal species. In Germany, tetracyclines and beta-lactams are the most commonly used and sold antimicrobial drugs. In third place are the potentiated sulfonamide, in fourth and fifth place, macrolides and polypeptides. To describe and evaluate the antibiotic usage for the several food-producing animals, detailed information is needed. Therefore, the number of treatments per active ingredient per animal and per day (treatment unit), and the number of treatment days per active ingredient per animal (treatment frequency) were calculated in the VetCAb project. These values represent scientifically collected orientation values for the treatment frequency of food-producing animal with antimicrobial substances in Germany. For example, a fattening pig is treated at 4.2 days (median) with one active ingredient within its 115-day fattening period. A dairy cow is treated at 3.5 days with one active ingredient within one year. A broiler is treated at 10 days with one active ingredient within its 39-day production period.

Validation of the hepatocyte-like HPCT-1E3 cell line as an in vitro model for the prediction of acute in vivo toxicity.

In a pilot study, we tested 20 randomly-selected chemicals for their cytotoxicity toward the HPCT-1E3 cell model, in order to prove the ability of this in vitro model to predict human acute in vivo toxicity. The study revealed that, in contrast to most other in vitro models, results from the HPCT-1E3 cell-based system show better correlation with the more-relevant human acute lethal doses, whereas results from most other systems have a high predictivity for human lethal serum concentrations. For the prevalidation of the HPCT-1E3 model as a surrogate for regulatory acute in vivo toxicity tests, we have now expanded the list of tested chemicals to 57 substances, and have compared the results with data from the HepG2 cell assay. Again, a better correlation of HPCT-1E3 IC50 values with human oral lethal doses, as compared to correlation with human lethal serum concentrations, was observed after the pooling of all the tested substances (r(2) = 0.53 [P < 0.001] and r(2) = 0.41 [P = 0.009], respectively). Therefore, the HPCT-1E3 in vitro model may be a valuable tool for prediction of human oral toxicity, and may help to further reduce the number of animals used for in vivo toxicity tests.

[Compilation of antibiotic use in farm animals via veterinary surgeries]. / Erfassung des Antibiotikaeinsatzes bei Lebensmittel liefernden Tieren in der tierärztlichen Praxis.

A feasibility study with 20 voluntarily participating veterinary surgeries was carried out in order to test, if the consumption of antibiotics in livestock can be determined systematically. Information about the statutory documents on the application of drugs of the participating surgeries were entered in a central database and analysed systematically. Surgeries that treat only livestock used significantly more antibiotics (number of treatment units per veterinarian) per veterinarian than surgeries that treat also small animals. The comparison of small and large surgeries showed that veterinarians in small surgeries treated fewer pigs and more cattle than their colleagues in large surgeries (number of treatment units per veterinarian). All in all, tetracyclines counted for more than 50% of all substances used (regarding the amount), followed by beta-lactams (25%) and sulfonamides incl. trimethoprim (11%). In poultry, polypeptides and beta-lactams were used most frequently. While cephalosporines were used only in cattle in a noteworthy frequency, fluoroquinolones were applied to poultry in almost 12% of all applications (application of one substance to one animal at one day). In total, it was shown, that harmonized documentation of consumption of antibiotics is feasible, but the relation of antibiotics to the treated population is problematic which has to be considered in the future.The number of applications is more suitable to assess the antibiotic use than the amount in kg, because the latter is dependent of the dosage. The impact of highly dosed substances like e.g. tetracyclines is overestimated by regarding the amount, while substances with low dosages are underestimated.

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.

Fungicide prochloraz and environmental pollutant dioxin induce the ABCG2 transporter in bovine mammary epithelial cells by the arylhydrocarbon receptor signaling pathway.

The molecular mechanisms by which environmental pollutants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or widely used imidazole fungicide prochloraz display their toxic effects in vertebrates are still not well understood. Using computer analysis, we recently identified nuclear aryl hydrocarbon receptor (AhR) binding sites termed "dioxin response elements" (DREs) in the 5'-untranslated region (5'-UTR) of efflux transporter ABCG2 (Accession No. EU570105) from the bovine mammary gland. As these regulatory motifs mediate regulation of target genes by AhR agonists including TCDD and prochloraz, we have systematically investigated the effect of both contaminants on functional ABCG2 transport activity in primary bovine mammary epithelial cells. TCDD or prochloraz doubled ABCG2-mediated Hoechst H33342 secretion. This effect was almost completely reversed by specific ABCG2 inhibitor Ko143. In further mechanistic studies, we showed that this induction was due to binding of activated AhR to DRE sequences in the ABCG2 5'-UTR. Receptor binding was significantly reduced by specific AhR antagonist salicyl amide. Induction of AhR by TCDD and prochloraz resulted in a time- and dose-dependent increase of ABCG2 gene expression and transporter protein levels. As ABCG2 represents the main mammary transporter for xenobiotics including drugs and toxins, exposure to prevalent AhR agonists may enhance transporter-mediated secretion of potential harmful compounds into milk. Through identification of mammary ABCG2 as a novel target gene of pesticide prochloraz and dioxin, our results may therefore help to improve the protection of breast-feeding infants and the consumer of dairy products.

Antiepileptic drugs reduce the efficacy of methotrexate chemotherapy through accelerated degradation of the reduced folate carrier by the ubiquitin-proteasome pathway.

BACKGROUND/AIMS: Concurrent treatment with methotrexate (MTX) and enzyme-inducing antiepileptic drugs including phenobarbital (PB) reduces the efficacy of MTX chemotherapy in cancer patients. We have shown that Reduced folate carrier (Rfc1)-mediated uptake of MTX, an essential determinant of MTX chemotherapy, is significantly reduced by PB via protein kinase C (PKC). However, whether PB treatment affects Rfc1 activity through regulation of carrier protein stability and the mechanisms involved remain unclear. METHODS/RESULTS: Protein turnover assays using hepatocytoma cells demonstrated that Rfc1 is a long-lived protein that is mainly degraded by the ubiquitin-proteasome proteolytic pathway under basal conditions. Pretreatment with PB significantly reduced Rfc1-mediated MTX uptake and shortened the carrier protein half-life. This effect was abolished by the specific PKC inhibitor Gö6976. Inhibition of proteasomes with MG-132 significantly elevated Rfc1 protein levels and induced colocalization of Rfc1 and ubiquitin particularly in submembranous cellular compartments. Finally, we demonstrated that PB treatment resulted in enhanced levels of Rfc1 polyubiquitin conjugates. CONCLUSIONS: Our results demonstrate that PB treatment causes downregulation of Rfc1 activity through PKC-dependent accelerated degradation of the Rfc1 protein by the ubiqutin-proteasome pathway. This regulatory mechanism may therefore involve clinically relevant drug resistance in patients concurrently receiving MTX and enzyme-inducing antiepileptic drugs.

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.

Dioxin mediates downregulation of the reduced folate carrier transport activity via the arylhydrocarbon receptor signalling pathway.

Dioxins such as 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) are common environmental contaminants known to regulate several genes via activation of the transcription factor aryl hydrocarbon receptor (AhR) associated with the development of numerous adverse biological effects. However, comparatively little is known about the molecular mechanisms by which dioxins display their toxic effects in vertebrates. The 5' untranslated region of the hepatocellular Reduced folate carrier (Rfc1; Slc19a1) exhibits AhR binding sites termed dioxin responsive elements (DRE) that have as yet only been found in the promoter region of prototypical TCDD target genes. Rfc1 mediated transport of reduced folates and antifolate drugs such as methotrexate (MTX) plays an essential role in physiological folate homeostasis and MTX cancer chemotherapy. In order to determine whether this carrier represents a target gene of dioxins we have investigated the influence of TCDD on functional Rfc1 activity in rat liver. Pre-treatment of rats with TCDD significantly diminished hepatocellular Rfc1 uptake activity in a time- and dose-dependent manner. In further mechanistic studies we demonstrated that this reduction was due to TCDD-dependent activation of the AhR signalling pathway. We additionally showed that binding of the activated receptor to DRE motifs in the Rfc1 promoter resulted in downregulation of Rfc1 gene expression and reduced carrier protein levels. As downregulation of pivotal Rfc1 activity results in functional folate deficiency associated with an elevated risk of cardiovascular diseases or carcinogenesis, our results indicate that deregulation of this essential transport pathway represents a novel regulatory mechanism how dioxins display their toxic effects through the Ah receptor.

Prediction of acute toxicity in HPCT-1E3 hepatocytoma cells with liver-like transport activities.

A battery of in vitro methods has been developed for the prediction of acute oral toxicity, to reduce the number of animals used for this purpose. However, the results of these tests correlate more closely with lethal serum concentrations than with lethal doses. To address this issue, we have further evaluated the HPCT-1E3 model, which may be better able to emulate toxicokinetic factors that occur in vivo, due to the presence in these hepatocytoma cells of endogenous transmembrane carriers and a basal activity of xenobiotic metabolism. IC50 values produced by using the MTT test after a 48-hour incubation with 20 randomly-selected MEIC substances, correlated better with human oral LD50 values than with LC50 data, supporting this hypothesis. As with other models, the toxicity of receptor-specific rather than cytotoxic substances, for example digoxin, was underpredicted. When digoxin was removed from the correlation analysis, the coefficient of determination (r(2)) improved to 0.81, and none of remaining chemicals were wrongly predicted by more than one order of magnitude. IC50 values obtained with HepG2 cells under similar conditions (MEIC Test No. 3, 24 hours, MTT) correlated with human LD50 data with a r(2) value of 0.55. A direct comparison of HPCT-1E3 and HepG2 cells further suggested that the differences between them may be due to transport processes. In conclusion, the HPCT-1E3 model may be valuable in improving the prediction of lethal doses, rather than lethal serum concentrations.

Downregulation of the reduced folate carrier transport activity by phenobarbital-type cytochrome P450 inducers and protein kinase C activators.

The sodium dependent reduced folate carrier (Rfc1; Slc19a1) provides the major route for cellular uptake of reduced folates and antifolate drugs such as methotrexate (MTX) into various tissues. Despite its essential role in folate homeostasis and cancer treatment, little is known about Rfc1 regulation. A barbiturate recognition box, which as yet has only been found in the promoter region of xenobiotic metabolizing enzymes, particularly those of the CYP450 enzyme family, was predicted in the 5' untranslated region of rat rfc1 cDNA. We have therefore investigated the regulation of Rfc1 by phenobarbital (PB)-type CYP450 inducers on the functional, transcriptional and translational level in a suitable in vitro model for rat liver. A decrease of >75% in substrate uptake was observed following treatment (48 h) with 1-10 times therapeutic plasma concentrations of PB-type CYP450 inducers like PB, carbamazepine, chlorpromazine, clotrimazole and with 0.1-1 ng/ml of the constitutive androstane receptor agonist TCPOBOP. This was not associated with reduced mRNA and protein levels. Further mechanistic investigations revealed that short-term treatment (2 h) of cells with protein phosphatase 1/2A inhibitor okadaic acid (80.5 ng/ml) and proteinkinase C inducer phorbol 12-myristate 13-acetate (PMA; 0.62 microg/ml) almost abolished Rfc1 mediated MTX uptake. Finally, the reduction in Rfc1 activity caused by PB, TCPOBOP and PMA was reversed by simultaneous incubation with the specific PKC inhibitor bisindolylmaleimide (BIM; 21 ng/ml). These results demonstrate that clinically relevant concentrations of PB-type CYP450 inducers cause a significant PKC-dependent reduction in Rfc1 uptake activity at the posttranscriptional level.