Relationship between covalent binding to microsomal protein and the destruction of adrenal cytochrome P-450 by spironolactone.
Previous investigations have demonstrated that guinea pig adrenal microsomes catalyze an NADPH-dependent activation of spironolactone (SL) resulting in the degradation of cytochrome(s) P-450 and decreases in steroidogenic enzyme activities. Studies were done to evaluate the relationship between the destruction of cytochrome P-450 and the covalent binding to microsomal protein by SL and by 7 alpha-thiospironolactone (7 alpha-thio-SL), an obligatory intermediate in the activation pathway. NADPH-dependent irreversible binding to guinea pig adrenal microsomal protein was demonstrable with 22-14C- and with 35S-labelled SL or 7 alpha-thio-SL as substrates. In the absence of NADPH, there was relatively little binding. NADPH-dependent covalent binding was not demonstrable with hepatic microsomal preparations. The amount of covalent binding to adrenal microsomes was far greater with 7 alpha-thio-SL than with SL and also greater with 35S-labelled than with 14C-labelled substrates. The latter results suggest the possibility of more than one reactive metabolite. Time-course experiments revealed a good correlation between covalent binding and P-450 destruction by SL and by 7 alpha-thio-SL. In addition, the 17 alpha-hydroxylase inhibitor, SU-10'603, and the 17 alpha-hydroxylase substrate, progesterone, prevented both the degradation of cytochrome P-450 and the NADPH-dependent covalent binding by 7 alpha-thio-SL. Reduced glutathione also decreased covalent binding but did not diminish P-450 destruction. The latter results indicate that some of the covalent binding is unrelated to the degradation of cytochrome P-450. However, all of the data are consistent with the hypothesis that 7 alpha-thio-SL is a suicide inhibitor of adrenal cytochrome P-450 and that covalent binding to protein is involved in the degradation of cytochrome P-450.
Colby
H D
HD
Department of Pharmacology and Toxicology, Philadelphia College of Pharmacy and Science, PA 19104.
O'Donnell
J P
JP
Flowers
N L
NL
Kossor
D C
DC
Johnson
P B
PB
Levitt
M
M
D000311:Adrenal Glands / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006168:Guinea Pigs; D008297:Male; D008861:Microsomes / Q000187:drug effects* / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D013148:Spironolactone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity*
D011506:Proteins; D013148:Spironolactone; C033554:7 alpha-thiospironolactone; D009249:NADP; D003577:Cytochrome P-450 Enzyme System