In vitro inhibition of mouse hepatic mixed-function oxidase enzymes by helenalin and alantolactone.
The sesquiterpene lactones (STL) helenalin and alantolactone were effective in vitro inhibitors of the mouse hepatic microsomal mixed-function oxidase (MFO) enzymes, aminopyrine demethylase (APD), aniline hydroxylase (ANH) and 7-ethoxyresorufin deethylase (ERD). Helenalin and alantolactone concentrations of 0.5 mM produced a 50-60% inhibition of APD and ERD, and a 20-30% inhibition of ANH. An increase in substrate (aminopyrine) concentration from 0.5 to 25 mM decreased STL inhibition of APD by 12-32%. APD was also inhibited at low aminopyrine concentrations (0.5 mM) by the helenalin derivative 2,3,11,13-tetrahydrohelenalin (tetrahydrohelenalin). The STL produced type I binding spectra with oxidized microsomes; Ks values for helenalin and alantolactone were 161 and 9 microM respectively. These results suggest that STL inhibition of the MFO system results, in part, from STL binding to the substrate-binding site of cytochrome P-450. It has been reported that the irreversible alkylation of protein cysteinyl residues is responsible for STL inhibition of several different enzymes, and second-order rate constants for the reaction of helenalin and alantolactone with glutathione were 25.1 and 1.80 mM-1.hr-1 respectively. Tetrahydrohelenalin did not react with glutathione. However, the subsequent addition of 3.0 mM thiols, i.e. L-cysteine, N-acetylcysteine or glutathione, to STL-treated (0.5 mM) microsomes reversed helenalin and alantolactone inhibition of APD and ERD by 50-80%. The ability of thiols to reverse STL inhibition of APD was decreased 20-43% by the coincubation of STL and microsomes with an NADPH-generating system. In addition, established effects of sulfhydryl-reactive compounds on the MFO system, i.e. inhibition of NADPH-cytochrome c reductase and conversion of cytochrome P-450 to cytochrome P-420, were not observed after addition of helenalin (1.0 mM) or alantolactone (0.5 mM) to mouse hepatic microsomes. These results suggest that STL inhibition of MFO enzymes may not be dependent upon the reactivity of the STL towards sulfhydryl groups. Instead, we suggest that STL binding to the substrate-binding site of cytochrome P-450 and subsequent metabolism of the STL may contribute to inhibition of the MFO system.
Chapman
D E
DE
Curriculum in Toxicology, University of North Carolina, Chapel Hill 27599.
Holbrook
D J
DJ
Chaney
S G
SG
Hall
I H
IH
Lee
K H
KH
D000633:Aminopyrine N-Demethylase / Q000037:antagonists & inhibitors*; D000815:Aniline Hydroxylase / Q000037:antagonists & inhibitors*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors*; D019363:Cytochrome P-450 CYP1A1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D007783:Lactones; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D012717:Sesquiterpenes / Q000378:metabolism / Q000494:pharmacology*; D045787:Sesquiterpenes, Eudesmane; D045789:Sesquiterpenes, Guaiane; D014118:Toxins, Biological
D065607:Cytochrome P-450 Enzyme Inhibitors; D007783:Lactones; D012717:Sesquiterpenes; D045787:Sesquiterpenes, Eudesmane; D045789:Sesquiterpenes, Guaiane; D014118:Toxins, Biological; C001329:helenalin; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D000815:Aniline Hydroxylase; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D000633:Aminopyrine N-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase; D005978:Glutathione; C004363:alantolactone; D005033:Ethylmaleimide