Chapman D E, Holbrook D J, Chaney S G, Hall I H, Lee K H
Curriculum in Toxicology, University of North Carolina, Chapel Hill 27599.
Biochem Pharmacol. 1989 Nov 15;38(22):3913-23. doi: 10.1016/0006-2952(89)90668-0.
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.
倍半萜内酯（STL）海伦内酯和土木香内酯是小鼠肝微粒体混合功能氧化酶（MFO）、氨基比林脱甲基酶（APD）、苯胺羟化酶（ANH）和7-乙氧基异吩恶唑酮脱乙基酶（ERD）有效的体外抑制剂。0.5 mM的海伦内酯和土木香内酯浓度可使APD和ERD受到50 - 60%的抑制，ANH受到20 - 30%的抑制。底物（氨基比林）浓度从0.5 mM增加到25 mM时，STL对APD的抑制作用降低了12 - 32%。在低氨基比林浓度（0.5 mM）下，海伦内酯衍生物2,3,11,13 - 四氢海伦内酯（四氢海伦内酯）也能抑制APD。STL与氧化型微粒体产生I型结合光谱；海伦内酯和土木香内酯的Ks值分别为161和9 microM。这些结果表明，STL对MFO系统的抑制作用部分源于STL与细胞色素P - 450底物结合位点的结合。据报道，蛋白质半胱氨酰残基的不可逆烷基化是STL抑制几种不同酶的原因，海伦内酯和土木香内酯与谷胱甘肽反应的二级速率常数分别为25.1和1.80 mM⁻¹·hr⁻¹。四氢海伦内酯不与谷胱甘肽反应。然而，在经STL处理（0.5 mM）的微粒体中随后加入3.0 mM硫醇，即L - 半胱氨酸、N - 乙酰半胱氨酸或谷胱甘肽，可使海伦内酯和土木香内酯对APD和ERD的抑制作用逆转50 - 80%。通过将STL与微粒体和NADPH生成系统共同孵育，硫醇逆转STL对APD抑制作用的能力降低了20 - 43%。此外，在向小鼠肝微粒体中加入海伦内酯（1.0 mM）或土木香内酯（0.5 mM）后，未观察到巯基反应性化合物对MFO系统的既定影响，即对NADPH - 细胞色素c还原酶的抑制作用以及细胞色素P - 450向细胞色素P - 420的转化。这些结果表明，STL对MFO酶的抑制作用可能不依赖于STL对巯基的反应性。相反，我们认为STL与细胞色素P - 450底物结合位点的结合以及随后STL的代谢可能有助于对MFO系统的抑制。
