title: The G671V variant of MRP1/ABCC1 links doxorubicin-induced acute cardiac toxicity to disposition of the glutathione conjugate of 4-hydroxy-2-trans-nonenal.
abstract: OBJECTIVE
  Doxorubicin-induced acute cardiotoxicity is associated with the Gly671Val (G671V; rs45511401) variant of multidrug resistance-associated protein 1 (MRP1). Doxorubicin redox cycling causes lipid peroxidation and generation of the reactive electrophile, 4-hydroxy-2-trans-nonenal (HNE). Glutathione forms conjugates with HNE, yielding an MRP1 substrate, GS-HNE, whose intracellular accumulation can cause toxicity.
  
  
  METHODS
  We established stable HEK293 cell lines overexpressing wild-type MRP1 (HEKMRP1), G671V (HEKG671V), and R433S (HEKR433S), a variant not associated with doxorubicin-induced cardiotoxicity and investigated the sensitivity of HEKG671V cells to doxorubicin and transport capacity of G671V toward GS-HNE.
  
  
  RESULTS
  In ATP-dependent transport studies using plasma membrane-derived vesicles, the Vmax (pmol/min/mg) for GS-HNE transport was the lowest for G671V (69±4) and the highest for R433S (972±213) compared with wild-type MRP1 (416±22), whereas the Km values were 2.8±0.4, 6.0 or more, and 1.7±0.2 µmol/l, respectively. In cells, the doxorubicin IC50 (48 h) was not different in HEKMRP1 (463 nmol/l) versus HEKR433S (645 nmol/l), but this parameter was significantly lower in HEKG671V (181 nmol/l). HEKG671V retained significantly (approximately 20%) more, whereas HEKR433S retained significantly less intracellular doxorubicin than HEKMRP1. Similarly, HEKG671V cells treated with 1.5 µmol/l of doxorubicin for 24 h retained significantly more GS-HNE. In cells treated with 0.5 µmol/l of doxorubicin for 48 , glutathione and glutathione disulfide levels and the glutathione/glutathione disulfide ratio were significantly decreased in HEKG671V versus HEKMRP1; these values were similar in HEKR433S versus HEKMRP1.
  
  
  CONCLUSIONS
  These data suggest that decreased MRP1-dependent GS-HNE efflux contributes to increased doxorubicin toxicity in HEKG671V and potentially in individuals carrying the G671V variant.
authors.affiliation: Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky, USA.
authors.forename: Paiboon
authors.initials: P
authors.lastname: Jungsuwadee
authors.forename: Tianyong
authors.initials: T
authors.lastname: Zhao
authors.forename: Elzbieta I
authors.initials: EI
authors.lastname: Stolarczyk
authors.forename: Christian M
authors.initials: CM
authors.lastname: Paumi
authors.forename: D Allan
authors.initials: DA
authors.lastname: Butterfield
authors.forename: Daret K
authors.initials: DK
authors.lastname: St Clair
authors.forename: Mary
authors.initials: M
authors.lastname: Vore
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004317:Doxorubicin / Q000493:pharmacokinetics* / Q000633:toxicity; D015870:Gene Expression; D014644:Genetic Variation; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D057809:HEK293 Cells; D006321:Heart / Q000187:drug effects; D006801:Humans; D015227:Lipid Peroxidation; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000493:pharmacokinetics*; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D000447:Aldehydes; D004317:Doxorubicin; D005978:Glutathione; D019803:Glutathione Disulfide; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C547259:4-hydroperoxy-2-nonenal; C421109:multidrug resistance-associated protein 1