Characterization of multidrug resistance-associated protein 2 in the hepatocellular disposition of 4-hydroxynonenal.
4-hydroxynonenal (4HNE) is a major product of peroxidative membrane lipid destruction and exerts a variety of deleterious actions through formation of covalent adducts with cellular nucleophiles. Consequently, a number of cellular enzyme systems exist that are capable of detoxifying this reactive aldehyde by oxidation, reduction, or conjugation with glutathione. In this investigation we characterize the multidrug resistance-associated protein 2 (MRP2) as the primary transmembrane transport protein in hepatocytes responsible for extracellular export of 4HNE-glutathione conjugate (HNE-SG) from the intracellular site of its formation. Suspensions of freshly isolated hepatocytes (10(6) cells/ml) prepared from either wild-type (WT) Wistar rats or TR(-) rats possessing a mutated Mrp2 gene were incubated with 4HNE (50 nmol/10(6) cells). The formation of 4HNE metabolites, 4-hydroxynonenoic acid (HNA) and HNE-SG, was quantified in the intracellular and extracellular fractions. These studies demonstrated that freshly isolated hepatocytes from both WT and TR(-) rats formed and exported the oxidized metabolite (HNA) to similar extents. Likewise, both populations of hepatocytes displayed nearly identical rates of glutathione conjugation with 4HNE. However, the rate of HNE-SG export from TR(-) hepatocytes was approximately fourfold less than that of WT hepatocytes. In TR(-) hepatocytes, HNE-SG accumulated and remained predominantly intracellular throughout the time course, suggesting an absence of compensatory export by other hepatocellular transporters. In conclusion, these data demonstrate that although WT and TR(-) hepatocytes are similar in their conjugative and oxidative metabolism of 4HNE, export of 4HNE-SG is mediated by the MRP2 transporter, a transport system distinct from that involved in HNA efflux.
Reichard
John F
JF
Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Doorn
Jonathan A
JA
Simon
Franz
F
Taylor
Melinda S
MS
Petersen
Dennis R
DR
D018528:ATP-Binding Cassette Transporters; D000447:Aldehydes / Q000378:metabolism* / Q000633:toxicity; D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005231:Fatty Acids, Unsaturated / Q000032:analysis / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006880:Hydroxy Acids / Q000032:analysis / Q000378:metabolism; D009154:Mutation; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000737:chemistry
C406867:4-hydroxynonenoic acid; D018528:ATP-Binding Cassette Transporters; C423013:Abcc2 protein, rat; D000447:Aldehydes; D002352:Carrier Proteins; D005231:Fatty Acids, Unsaturated; D006880:Hydroxy Acids; D005978:Glutathione; C027576:4-hydroxy-2-nonenal