RESUMEN
OBJECTIVES: Distribution of fluoroquinolones to the retina is normally restricted by ABCG2 at the blood-retinal barrier. As the cat develops a species-specific adverse reaction to photoreactive fluoroquinolones, our goal was to investigate ABCG2 as a candidate gene for fluoroquinolone-induced retinal degeneration and blindness in cats. METHODS: Feline ABCG2 was sequenced and the consensus amino acid sequence was compared with that of 10 other mammalian species. Expression of ABCG2 in feline retina was assessed by immunoblot. cDNA constructs for feline and human ABCG2 were constructed in a pcDNA3 expression vector and expressed in HEK-293 cells, and ABCG2 expression was analyzed by western blot and immunofluorescence. Mitoxantrone and BODIPY-prazosin efflux measured by flow cytometry and a phototoxicity assay were used to assess feline and human ABCG2 function. RESULTS: Four feline-specific (compared with 10 other mammalian species) amino acid changes in conserved regions of ABCG2 were identified. Expression of ABCG2 on plasma membranes was confirmed in feline retina and in cells transfected with human and feline ABCG2, although some intracellular expression of feline ABCG2 was detected by immunofluorescence. Function of feline ABCG2, compared with human ABCG2, was found to be deficient as determined by flow cytometric measurement of mitoxantrone and BODIPY-prazosin efflux and enrofloxacin-induced phototoxicity assays. CONCLUSION: Feline-specific amino acid changes in ABCG2 cause a functional defect of the transport protein in cats. This functional defect may be owing, in part, to defective cellular localization of feline ABCG2. Regardless, dysfunction of ABCG2 at the blood-retinal barrier likely results in accumulation of photoreactive fluoroquinolones in feline retina. Exposure of the retina to light would then generate reactive oxygen species that would cause the characteristic retinal degeneration and blindness documented in some cats receiving high doses of some fluoroquinolones. Pharmacological inhibition of ABCG2 in other species might result in retinal damage if fluoroquinolones are concurrently administered.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Enfermedades de los Gatos/inducido químicamente , Enfermedades de los Gatos/genética , Fluoroquinolonas/efectos adversos , Degeneración Retiniana/veterinaria , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Compuestos de Boro/metabolismo , Gatos , Secuencia Conservada/genética , ADN Complementario/genética , Dermatitis Fototóxica/complicaciones , Dermatitis Fototóxica/genética , Dermatitis Fototóxica/veterinaria , Técnica del Anticuerpo Fluorescente , Fluoroquinolonas/química , Células HEK293 , Humanos , Mitoxantrona/farmacología , Biología Molecular , Datos de Secuencia Molecular , Prazosina/análogos & derivados , Prazosina/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/complicaciones , Degeneración Retiniana/genética , TransfecciónRESUMEN
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.