Detoxification of 1-chloro-2,4-dinitrobenzene in MCF7 breast cancer cells expressing glutathione S-transferase P1-1 and/or multidrug resistance protein 1.
We examined the roles of glutathione S-transferase (GST) P1-1 and the glutathione S-conjugate (GS-X) transporter, multidrug resistance protein 1 (MRP1), singly or in combination, in the detoxification of 1-chloro-2,4-dinitrobenzene (CDNB). Derivatives of MCF7 breast carcinoma cells expressing GST P1-1 and MRP1 alone or in combination were developed. Detoxification was measured in cells as formation of the glutathione conjugate of CDNB, S-(2,4-dinitrophenyl)-glutathione (DNP-SG), efflux of DNP-SG, and ultimately protection from CDNB cytotoxicity. MRP1 expression in the absence of GST P1-1 confers a three- to fourfold resistance to CDNB, which is associated with a >10-fold increase in the maximum rate of DNP-SG efflux. DNP-SG efflux in MRP1-expressing MCF7 cells was ATP-dependent and exhibited an apparent Km for DNP-SG of 95 microM. MRP1 expression alone, however, had no effect on DNP-SG formation. Combined expression of GST P1-1 and MRP1 increased the rates of DNP-SG formation when cells were exposed to 10 microM CDNB. Moreover, combined expression of GSTP1-1 with MRP1 moderately augmented MRP1-mediated resistance to CDNB but only during short term (10 min) exposures to CDNB where IC50 values were in the 8-10 microM range. In contrast, expression of GST P1-1 in the absence of MRP1 slightly sensitized cells to the toxicity of CDNB (10 min exposures), despite increasing rates of DNP-SG formation. The sensitization to CDNB in cells expressing GST P1-1 alone was associated with increased intracellular accumulation of DNP-SG, indicating that DNP-SG may contribute to CDNB toxicity. The potential toxicity of DNP-SG is also suggested by the finding that inhibition of DNP-SG formation by prior glutathione depletion confers resistance to CDNB cytotoxicity in MRP1-poor MCF7 cells. Altogether, our results demonstrate that glutathione conjugation and MRP1-mediated conjugate efflux can operate together to confer resistance to CDNB. The data indicate that MRP1-mediated conjugate efflux is required for cytoprotection from CDNB because its conjugate (DNP-SG), when present at high intracellular levels, may also be toxic to cells.
Diah
S K
SK
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina, 27157, USA.
Smitherman
P K
PK
Townsend
A J
AJ
Morrow
C S
CS
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001943:Breast Neoplasms; D002352:Carrier Proteins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003847:Deoxyglucose / Q000494:pharmacology; D004137:Dinitrochlorobenzene / Q000378:metabolism* / Q000633:toxicity*; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008658:Inactivation, Metabolic; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010084:Oxidation-Reduction; D019810:Sodium Azide / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D002352:Carrier Proteins; D003847:Deoxyglucose; D004137:Dinitrochlorobenzene; D005978:Glutathione; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D007527:Isoenzymes; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019810:Sodium Azide; C086860:glutathione transporter; C030190:S-(2,4-dinitrophenyl)glutathione; C496556:GSTP1 protein, human