The livestock photosensitizer, phytoporphyrin (phylloerythrin), is a substrate of the ATP-binding cassette transporter ABCG2

Hepatogenous photosensitization occurs in livestock following damage to the liver or biliary apparatus that results in impaired excretion of phytoporphyrin (phylloerythrin), a photosensitizer. Based on earlier observations that porphyrin-based photosensitizers are substrates of the ATP-binding cassette transporter ABCG2, we examined the ability of the hepatic transporters ABCB1 (P-glycoprotein) and ABCG2 to transport phytoporphyrin. Transport of phytoporphyrin was blocked by the ABCG2-specific inhibitor fumitremorgin C (FTC) in human embryonic kidney cells transfected with full length human ABCG2, while no transport by cells transfected with human ABCB1 was noted. FTC-inhibited transport of phytoporphyrin was also demonstrated in ABCG2-expressing LLC-PK1 pig kidney cells, consistent with the idea that the pig orthologue, like human ABCG2, transports the photosensitizer. ABCG2 expression was confirmed by immunohistochemistry in the hepatocytes of cow, pig and sheep livers. We conclude that phytoporphyrin is a substrate for ABCG2 and that the transporter is likely responsible for its biliary excretion.     

Therapeutic implications of the MDR-1 gene

Drug transporters significantly influence drug pharmacokinetics and pharmacodynamics. P-glycoprotein (P-gp), the product of the MDR1 (ABCB1) gene, is among the most well-characterized drug transporters, particularly in veterinary medicine. A number of clinically relevant, structurally and functionally unrelated drugs are substrates for P-gp. P-gp is expressed by a variety of normal tissues including the intestines, renal tubular cells, brain capillary endothelial cells, biliary canalicular cells, and others, where it functions to actively extrude substrate drugs. In this capacity, P-gp limits oral absorption and central nervous system entry of many substrate drugs. A number of MDR1 polymorphisms have been described in human patients, some of which result in altered drug pharmacokinetics and susceptibility to diseases such as Parkinson's disease, inflammatory bowel disease, refractory seizures, and others. An MDR1 polymorphism in herding breed dogs, including collies and Australian shepherds, has been demonstrated to be the cause of ivermectin sensitivity in these breeds. Recent evidence suggests that this polymorphism, a 4-bp deletion mutation, results in increased susceptibility to the toxicity of several drugs in addition to ivermectin. Furthermore, data in rodent models suggest that P-gp may play an important role in regulating the hypothalamic-pituitary-adrenal axis.     

Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.     

Comparison of chemotherapeutic drug resistance in cells transfected with canine ABCG2 or feline ABCG2

ABCG2 (ATP binding cassette subfamily G, member 2) mediates resistance to a variety of cytotoxic agents. Although human ABCG2 is well characterized, the function of canine ABCG2 has not been studied previously. Feline ABCG2 has an amino acid substitution in the adenosine triphosphate‐binding domain that decreases its transport capacity relative to human ABCG2. Our goal was to compare canine ABCG2‐mediated chemotherapeutic drug resistance to feline ABCG2‐mediated chemotherapeutic drug resistance. HEK‐293 cells stably transfected with plasmid containing canine ABCG2, feline ABCG2 or no ABCG2 were exposed to carboplatin, doxorubicin, mitoxantrone, toceranib or vincristine, and cell survival was subsequently determined. Canine ABCG2 conferred a greater degree of chemotherapy resistance than feline ABCG2 for mitoxantrone. Neither canine nor feline ABCG2 conferred resistance to doxorubicin, vincristine or toceranib. Canine, but not feline, ABCG2 conferred resistance to carboplatin, a drug that is not reported to be a substrate for ABCG2 in other species.     

Milk secretion of nitrofurantoin,as a specific BCRP/ABCG2 substrate,in assaf sheep: modulation by isoflavones1

Studies on residues in milk used for human consumption have increased due to health concerns and priority interest in the control of potentially risky drugs. The protein BCRP/ABCG2, present in the mammary epithelia, actively extrudes drugs into milk and can be modulated by isoflavones. Nitrofurantoin is a specific BCRP substrate which is actively excreted into milk by this transporter. In this research, we studied nitrofurantoin transport into milk in four experimental groups: G1‐calves fed forage with isoflavones; G2‐calves fed forage with isoflavones and administered exogenous genistein and daidzein; G3‐calves fed forage without isoflavones; G4‐calves fed forage without isoflavones and administered exogenous genistein and daidzein. Results show increased levels of nitrofurantoin in milk from calves without isoflavones (G3) and decreased nitrofurantoin residues in milk when isoflavones were present, either by forage (G1 and G2) or by exogenous administration (G4). The values of C_max in milk were significantly lower in those groups with isoflavones in forage (G1, G2). Plasma levels were low and unmodified among the groups. Inter‐individual variation was high. All these results seem to point to a feasible control of drug secretion into milk through isoflavones in the diet when the drug is a good BCRP/ABCG2 substrate.     

Interaction of mammary bovine ABCG2 with AFB1 and its metabolites and regulation by PCB 126 in a MDCKII in vitro model

The ATP‐binding cassette efflux transporter ABCG2 plays a key role in the mammary excretion of drugs and toxins in humans and animals. Aflatoxins (AF) are worldwide contaminants of food and feed commodities, while PCB 126 is a dioxin‐like PCB which may contaminate milk and dairy products. Both compounds are known human carcinogens. The interactions between AF and bovine ABCG2 (bABCG2) as well as the effects of PCB 126 on its efflux activity have been investigated by means of the Hoechst H33342 transport assay in MDCKII cells stably expressing mammary bABCG2. Both AFB1 and its main milk metabolite AFM1 showed interaction with bABCG2 even at concentrations approaching the legal limits in feed and food commodities. Moreover, PCB 126 significantly enhanced bABCG2 functional activity. Specific inhibitors of either AhR (CH233191) or ABCG2 (Ko143) were able to reverse the PCB 126‐induced increase in bABCG2 transport activity, showing the specific upregulation of the efflux protein by the AhR pathway. The incubation of PCB 126‐pretreated cells with AFM1 was able to substantially reverse such effect, with still unknown mechanism(s). Overall, results from this study point to AFB1 and AFM1 as likely bABCG2 substrates. The PCB 126‐dependent increased activity of the transporter could enhance the ABCG2‐mediated excretion into dairy milk of chemicals (i.e., drugs and toxins) potentially harmful to neonates and consumers.     

Establishment of a cell line for assessing drugs as canine P‐glycoprotein substrates: proof of principle

P‐glycoprotein (P‐gp), encoded by the ABCB1 (MDR1) gene, dramatically impacts drug disposition. P‐gp is expressed in the intestines, biliary canaliculi, renal tubules, and brain capillaries where it functions to efflux substrate drugs. In this capacity, P‐gp restricts oral absorption, enhances biliary and renal excretion, and inhibits central nervous system entry of substrate drugs. Many drugs commonly used in veterinary medicine are known substrates for canine P‐gp (vincristine, loperamide, ivermectin, others). Because these drugs have a narrow therapeutic index, defective P‐gp function can cause serious adverse drug reactions due to enhanced brain penetration and/or decreased clearance. P‐gp dysfunction in dogs can be intrinsic (dogs harboring ABCB1‐1Δ) or acquired (drug interactions between a P‐gp inhibitor and P‐gp substrate). New human drug candidates are required to undergo assessment for P‐gp interactions according to FDA and EMA regulations to avoid adverse drug reactions and drug–drug interactions. Similar information regarding canine P‐gp could prevent adverse drug reactions in dogs. Because differences in P‐gp substrates have been documented between species, one should not presume that human or murine P‐gp substrates are necessarily canine P‐gp substrates. Thus, our goal was to develop a cell line for assessing drugs as canine P‐gp substrates.     

植物ABCG转运蛋白研究进展

ABCG转运蛋白是ABC蛋白家族最庞大的亚族,广泛存在于植物体内。ABCG亚族主要由半分子转运蛋白WBC(white-brown complex)和全分子转运蛋白PDR(pleiotropic drug resistance)组成,其底物类型广泛,包括抗生素、植物激素、木质素单体、脂质及次生代谢产物等,涉及植物生命周期中的多种代谢活动。本文综述了植物ABCG转运蛋白的分子特性、结构及功能方面的研究进展,并对今后有关该蛋白的主要研究方向做了展望。     

Interaction of enrofloxacin with breast cancer resistance protein (BCRP/ABCG2): influence of flavonoids and role in milk secretion in sheep

The ATP-binding cassette (ABC) transporter breast cancer resistance protein (BCRP)/ABCG2 is a high-capacity efflux transporter with wide substrate specificity located in apical membranes of epithelia, which is involved in drug availability. BCRP is responsible for the active secretion of clinically and toxicologically important substrates to milk. The present study shows BCRP expression in sheep and cow by immunoblotting with MAb (BXP-53). Vanadate-sensitive ATPase activity with specific BCRP substrates and inhibitors was measured in bovine mammary gland homogenates. To assess the role of BCRP in ruminant mammary gland we tested the fluoroquinolone enrofloxacin (ENRO). In polarized cell lines, ENRO was transported by Bcrp1/BCRP with secretory/absorptive ratios of 6.5 and 2 respectively. The efflux was blocked by the BCRP inhibitor Ko143. ENRO pharmacokinetics in plasma and milk was studied in sheep after co-administration of drug (2.5 mg/kg, i.v.) and genistein (0.8 mg/kg, i.m.) or albendazole sulfoxide (2 mg/kg, i.v) as BCRP inhibitors. Concomitant administration of BCRP inhibitors with ENRO had no significant effect on the plasma disposition kinetics of ENRO but decreased ENRO concentrations in 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.     

Canine ABCB1 and macrocyclic lactones: heartworm prevention and pharmacogenetics

The impact of drug transporters on drug pharmacokinetics and pharmacodynamics has been increasingly recognized in recent years. P-glycoprotein (P-gp), the product of the ABCB1 (formerly MDR1) gene, is among the most well-characterized drug transporters, particularly in veterinary medicine. P-gp is expressed by a variety of normal tissues, including the intestines, brain capillary endothelial cells, renal tubular cells, and biliary canalicular cells, where it functions to actively extrude substrate drugs. In this capacity, P-gp limits oral absorption and central nervous system entry of many substrate drugs and enhances their excretion from the body. Many drugs used in veterinary medicine are substrates for P-gp, including many chemotherapeutic agents and macrocyclic lactones (avermectins and milbemycin). A 4-base pair deletion mutation in the ABCB1 gene occurs in many herding breed dogs, including collies, Australian shepherds, and Shetland sheepdogs. The mutation (ABCB1-1Delta) renders affected animals extremely susceptible to toxicosis induced by substrate drugs, such as the macrocyclic lactones at doses well below those tolerated by dogs with the wild-type ABCB1 gene. However, at the manufacturer's recommended dose, all FDA-approved heartworm preventive products marketed in the United States are safe, even for dogs with the ABCB1 mutant/mutant genotype.     

Effect of colibacillosis or coccidiosis on expression of breast cancer resistance protein in small intestine and liver of chickens

Breast cancer resistance protein (BCRP, ABCG2) is a member of ABC (ATP‐binding cassette) transporter superfamily that occurs in a variety of tissues including liver and small intestine of animals. As BCRP is involved in drug absorption, distribution, and elimination, modulation of its expression may affect the clinical efficacy of drugs. However, little is known about the effects of coccidiosis or colibacillosis infection on the levels of BCRP expression in chickens. Here, we studied the effect of infection with Escherichia coli (E. coli) or Eimeriida mixture (E. necatrix and E. tenella) on the expression levels of ABCG2 mRNA and BCRP in the different segments of small intestine and liver in chickens. Expression of ABCG2 mRNA or BCRP was detected in the entire small intestine and liver of healthy chickens, and the expression levels in liver and ileum were significantly higher than duodenum and jejunum. Infection with E. coli or Eimeriida mixture resulted in significant decrease in ABCG2 mRNA and BCRP expression in liver, ileum, and jejunum, but not in duodenum, in comparison with noninfection control. The results indicate that coccidiosis or colibacillosis infection inhibits BCRP expression in chickens, which may consequently influence drug distribution and therapeutic efficacy.     

苜蓿皂苷对大鼠肝脏及肝脏细胞低密度脂蛋白受体、三磷酸腺苷结合盒转运体mRNA表达的影响

本试验旨在研究苜蓿皂苷对大鼠肝脏及大鼠肝脏细胞(BRL细胞)胆固醇清除和转运途径中关键基因低密度脂蛋白受体(LDLR)、三磷酸腺苷结合盒转运体G5(ABCG5)、三磷酸腺苷结合盒转运体G8(ABCG8)mRNA表达量的影响,从个体和细胞水平初步探讨苜蓿皂苷调控胆固醇清除和转运的分子机制。采用高脂饲粮建立大鼠高脂模型,测定苜蓿皂苷对正常、高脂大鼠血清指标[总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)含量]和肝脏LDLR、ABCG5、ABCG8 mRNA表达量的影响;采用高糖DMEM培养液建立BRL细胞脂变模型,测定苜蓿皂苷浓度对BRL细胞活性的影响,测定苜蓿皂苷对正常、脂变细胞LDLR、ABCG5、ABCG8 mRNA表达量的影响。结果表明:1)苜蓿皂苷显著降低高脂大鼠血清中TG、TC和LDL-C的含量(P0.05);2)苜蓿皂苷显著上调正常大鼠肝脏LDLR、ABCG5、ABCG8及高脂大鼠ABCG5、ABCG8 mRNA表达量(P0.05);3)添加200、250μg/m L苜蓿皂苷显著提高了BRL细胞的活性(P0.05);4)苜蓿皂苷显著上调正常BRL细胞LDLR、ABCG5、ABCG8 mRNA表达量(P0.05),而对脂变BRL细胞各基因mRNA表达量无显著影响(P0.05)。结果提示,苜蓿皂苷可通过调控LDLR、ABCG5、ABCG8 mRNA的表达来增加肝细胞内胆固醇的清除和转运,从而降低机体胆固醇的含量。     

Brain penetration of emodepside is increased in P‐glycoprotein‐deficient mice and leads to neurotoxicosis

The antiparasitic drug emodepside (EMO) is a substrate of the P‐glycoprotein multidrug efflux carrier (P‐gp; syn. MDR1, ABCB1), which has an important function in protecting the brain from potentially toxic compounds by functional drug efflux at the blood–brain barrier (BBB). Many dogs of the Collie breed and even dogs of many other breeds have a loss‐of‐function 4‐bp deletion mutation in the MDR1 gene. In these dogs, brain penetration of many P‐gp‐transported drugs is increased and so their therapeutic usage is restricted. To elucidate the role of P‐gp at the BBB for the brain penetration of EMO, we applied EMO at 1 mg/kg to mdr1‐deficient (PGP^mut) and mdr1‐intact (PGP^WT) CF1 mice. Whereas in the brain of the PGP^WT mice, EMO was below the detection level of 10 ng/g, its concentration was at 43.7 ng/g in the PGP^mut mice. Furthermore, appearance of neurological toxicity was analyzed in these mice after application of 1 mg/kg EMO using a rotarod setup. In all PGP^mut mice, but not in the PGP^WT mice, the walking performance on the rotarod was impaired by EMO with clear differences in the degree and duration of neurological toxicity. Some of the mice were completely unable to walk on the rotarod already at 2 h after drug application and showed long‐lasting ataxia over >24 h. Others even showed significantly reduced walking performance, but completely recovered within 1 day. In conclusion, P‐gp restricts brain penetration of EMO and prevents neurological toxicity of this drug in mice.     

Involvement of breast cancer resistance protein (BCRP/ABCG2) in the secretion of danofloxacin into milk: interaction with ivermectin

Danofloxacin, a veterinary fluoroquinolone antimicrobial drug, is actively secreted into milk by an as yet unknown mechanism. One of the main determinants of active drug secretion into milk is the transporter (BCRP/ABCG2). The main purpose was to determine whether danofloxacin is an in vitro substrate for Bcrp1/BCRP and to assess its involvement in danofloxacin secretion into milk. In addition, the role of potential drug-drug interactions in this process was assessed using ivermectin. Danofloxacin was transported in vitro by Bcrp1/BCRP, and ivermectin efficiently blocked this transport. Experiments with Bcrp1(-/-) mice showed no evidence of the involvement of Bcrp1 in plasma pharmacokinetics of danofloxacin. However, the milk concentration and milk-to-plasma ratio of danofloxacin were almost twofold higher in wild-type compared with Bcrp1(-/-) mice. The in vivo interaction with ivermectin was studied in sheep after co-administration of danofloxacin (1.25 mg/kg, i.m.) and ivermectin (0.2 mg/kg, s.c.). Ivermectin had no significant effect on the plasma levels of danofloxacin but significantly decreased danofloxacin concentrations in milk by almost 40%. Concomitant administration of multiple drugs, often used in veterinary therapy, may not only affect their pharmacological activity but also their secretion into milk, because of potential drug-drug interactions mediated by BCRP.     

小肽转运载体2及其在乳腺泌乳中的作用

作为一种潜在的重要的乳腺小肽转运系统,PepT2在乳腺氨基酸氮转运、降低乳汁药物分布以及乳腺疾病治疗方面都起到重要作用.对PepT2在乳腺中作用的深入研究在泌乳生理和临床治疗上都有着非常重要的意义.本文主要从PepT2的蛋白质结构与功能、底物转运特性、表达调控以及其在泌乳生理中的作用4个方面进行简要综述.     

P-glycoprotein in intestines, liver, kidney and lymphocytes in horse

P-glycoprotein (P-gp) is an important drug transporter, which is expressed in a variety of cells, such as the intestinal enterocytes, the hepatocytes, the renal tubular cells and the intestinal and peripheral blood lymphocytes. We have studied the localization and the gene and protein expression of P-gp in these cells in horse. In addition we have compared the protein sequence of P-gp in horse with the protein sequences of P-gp in several other species. Real time RT-PCR and Western blot showed gene and protein expression of horse P-gp in all parts of the intestines, but there was no strict correlation between these parameters. Immunohistochemistry showed localization of P-gp in the apical cell membranes of the enterocytes and, in addition, staining was observed in the intestinal intraepithelial and lamina propria lymphocytes. Peripheral blood lymphocytes also stained for P-gp, and gene and protein expression of P-gp were observed in these cells. There was a high gene and protein expression of P-gp in the liver, with P-gp-immunoreactivity in the bile canalicular membranes of the hepatocytes. Gene and protein expression of P-gp were found in the kidney with localization of the protein in different parts of the nephrons. Protein sequence alignment showed that horse P-gp has two amino acid insertions at the N-terminal region of the protein, which are not present in several other species examined. One of these is a 99 amino acid long sequence inserted at amino acid positions 23–121 from the N-terminal. The other is a six amino acid long sequence present at the amino acid positions 140–145 from the N-terminal. The results of the present study indicate that P-gp has an important function for oral bioavailability, distribution and excretion of substrate compounds in horse.     

Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors

We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half‐maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152‐induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi‐agent protocols is warranted.     

Canine ABCB4: Tissue expression and cDNA structure

The ABCB gene subfamily of ABC (ATP-binding cassette) transporters is responsible for transporting a wide spectrum of molecules including peptides, iron, bile salts, drugs, and phospholipids. In humans, ABCB4 appears to be exclusively expressed on the apical membrane of hepatocytes where it translocates phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane. Functional alterations in the ABCB4 transporter are associated with a number of cholestatic syndromes in humans. Because of its role in biliary lipid homeostasis in humans, investigation of the ABCB4 gene in dogs is warranted. Thus, the full cDNA sequence of canine ABCB4 was elucidated and its mRNA and protein expression levels in tissues were determined. Canine ABCB4 consists of 3804 nucleotides spanning 26 exons and is 89% identical to human ABCB4. Expression of ABCB4 in canine liver supports a potential role for the protein in normal biliary function similar to that in humans. The function of ABCB4 expressed in brain tissue has yet to be determined.