ATP-dependent transport of glutathione conjugate of 7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles.
Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 microM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis-Menten kinetics (Km 46 microM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis-Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 microM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 microM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 microM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.
Srivastava
S K
SK
Cancer Research Laboratory, Mercy Cancer Institute, The Mercy Hospital of Pittsburgh, 1400 Locust Street, Pittsburgh, PA 15219, USA.
Hu
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X
Xia
H
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Bleicher
R J
RJ
Zaren
H A
HA
Orchard
J L
JL
Awasthi
S
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Singh
S V
SV
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism* / Q000494:pharmacology; D001648:Bile Canaliculi / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D001692:Biological Transport; D002273:Carcinogens / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D005978:Glutathione / Q000378:metabolism* / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007700:Kinetics; D051379:Mice; D013237:Stereoisomerism
D001580:Benzopyrenes; D002273:Carcinogens; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C113118:glutathione-BPDE conjugate; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D005978:Glutathione