Development of relevant assay system to identify steroidogenic enzyme inhibitors.
Steroidogenesis in the adrenals, testes, and ovaries plays an important roles in hormonal homeostasis in vivo and is mediated by various enzymes, such as acid cholesterol esterase (CEase), neutral CEase, CYP11A, 3beta-hydroxysteroid dehydrogenase (HSD), CYP21, CYP11B, CYP17, CYP19 and 17beta-HSD. Compounds that are potent inhibitors of one or more steroidogenic enzymes can thus cause serious endocrine toxicity so that relevant assay systems for detecting such enzyme inhibition are useful for safety assessment. Methods already reported involve incubation of radiolabeled steroid precursors with enzyme sources followed by separation of products by TLC or other methods. However, it is unclear whether the systems in use are kinetically-optimized (i.e., reactions performed in the linear range in terms of both reaction time and substrate concentrations), so that they might not be applicable for appropriate inhibition studies. The purpose of this present study was to establish kinetically-optimized methods for rat adrenal, testis, and ovary tissues with radio-HPLC. After development of HPLC methods for separation of precursor substrates and derived metabolites, we investigated the time course and substrate concentration dependence of individual reactions, using radiolabeled substrates and enzyme sources (mitochondria-lysosomal, microsomal, and cytosolic fractions from rat adrenal, testis and ovary tissues). Linearity of metabolism was confirmed in terms of both reaction time and substrate concentrations, and we conclude that this approach can be utilized to assess the inhibition profiles of compounds impacting on steroidogenic enzyme activity.
Nishizato
Yohei
Y
Pharmacokinetics Research Laboratories, Dainippon Sumitomo Pharma Co. Ltd., 3-1-98, Kasugade Naka, Konohana-ku, Osaka 554-0022, Japan.
Imai
Satoki
S
Yabuki
Masashi
M
Kido
Hidekatsu
H
Komuro
Setsuko
S
D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D008297:Male; D015394:Molecular Structure; D010053:Ovary / Q000201:enzymology; D051381:Rats; D013256:Steroids / Q000096:biosynthesis* / Q000737:chemistry; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology
D002021:Buffers; D065607:Cytochrome P-450 Enzyme Inhibitors; D013256:Steroids; D003577:Cytochrome P-450 Enzyme System