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Metabolism of androstenone, 17β-estradiol and dihydrotestosterone in primary cultured pig hepatocytes and the role of 3β-hydroxysteroid dehydrogenase in this process.

Chen G, Bai Y, Ren L, Zhu D, Li Y, Fang M, Al-Kateb H, Doran O - PLoS ONE (2015)

Bottom Line: The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor.Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol.The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Agro-product Quality and Safety, Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.

ABSTRACT
Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

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Ion chromatograms and mass spectra (inset) of 17β-estradiol and its metabolites.(A) 17β-estradiol in the medium in absence of isolated hepatocytes; (B) 17β-estradiol in the medium in the presence of isolated hepatocytes. No 17β-estradiol was found after 6 h of cell culture; (C) identified 17β-estradiol metabolite estrone-3-glucuronide (m/z− = 445.2190). The samples were analyzed in ESI– ionization mode; (D) identified 17β-estradiol metabolite β-estradiol-3-glucuronide (m/z− = 447.2298) in ESI– ionization mode; (E) a mixture of authentic standards β-estradiol-17-glucuronide and β-estradiol-3-glucuronide.
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pone-0113194-g002: Ion chromatograms and mass spectra (inset) of 17β-estradiol and its metabolites.(A) 17β-estradiol in the medium in absence of isolated hepatocytes; (B) 17β-estradiol in the medium in the presence of isolated hepatocytes. No 17β-estradiol was found after 6 h of cell culture; (C) identified 17β-estradiol metabolite estrone-3-glucuronide (m/z− = 445.2190). The samples were analyzed in ESI– ionization mode; (D) identified 17β-estradiol metabolite β-estradiol-3-glucuronide (m/z− = 447.2298) in ESI– ionization mode; (E) a mixture of authentic standards β-estradiol-17-glucuronide and β-estradiol-3-glucuronide.

Mentions: Abundance of 17β-estradiol precursor ion (m/z− = 271.2106) in the medium before cell culturing is shown in Figure 2A. 17β-Estradiol was completely metabolized by pig hepatocytes after 6 h of incubation in the medium (Figure 2B). Investigation of 17β-estradiol metabolites by LC-TOF-MS established the presence of two compounds with m/z− 445.2190 and 447.2298, which correspond to molecular ions of glucuronide-conjugated estrone (E1-G) (Figure 2C) and glucuronide-conjugated estradiol (E2-G) (Figure 2D) respectively. The ratio of E1-G to E2-G was 5∶1. When the cell culture medium was hydrolyzed by β-glucuronidase, both estrone and 17β-estradiol were detected. The ratio estrone: 17β-estradiol was similar to the ratio E1-G: E2-G. Using β-estradiol-3′-glucuronide and β-estradiol-17′-glucuronide authentic standards, this study established that glucuronidation of E2-G occurred at 3′-hydroxyl group (Figure 2E).


Metabolism of androstenone, 17β-estradiol and dihydrotestosterone in primary cultured pig hepatocytes and the role of 3β-hydroxysteroid dehydrogenase in this process.

Chen G, Bai Y, Ren L, Zhu D, Li Y, Fang M, Al-Kateb H, Doran O - PLoS ONE (2015)

Ion chromatograms and mass spectra (inset) of 17β-estradiol and its metabolites.(A) 17β-estradiol in the medium in absence of isolated hepatocytes; (B) 17β-estradiol in the medium in the presence of isolated hepatocytes. No 17β-estradiol was found after 6 h of cell culture; (C) identified 17β-estradiol metabolite estrone-3-glucuronide (m/z− = 445.2190). The samples were analyzed in ESI– ionization mode; (D) identified 17β-estradiol metabolite β-estradiol-3-glucuronide (m/z− = 447.2298) in ESI– ionization mode; (E) a mixture of authentic standards β-estradiol-17-glucuronide and β-estradiol-3-glucuronide.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4295843&req=5

pone-0113194-g002: Ion chromatograms and mass spectra (inset) of 17β-estradiol and its metabolites.(A) 17β-estradiol in the medium in absence of isolated hepatocytes; (B) 17β-estradiol in the medium in the presence of isolated hepatocytes. No 17β-estradiol was found after 6 h of cell culture; (C) identified 17β-estradiol metabolite estrone-3-glucuronide (m/z− = 445.2190). The samples were analyzed in ESI– ionization mode; (D) identified 17β-estradiol metabolite β-estradiol-3-glucuronide (m/z− = 447.2298) in ESI– ionization mode; (E) a mixture of authentic standards β-estradiol-17-glucuronide and β-estradiol-3-glucuronide.
Mentions: Abundance of 17β-estradiol precursor ion (m/z− = 271.2106) in the medium before cell culturing is shown in Figure 2A. 17β-Estradiol was completely metabolized by pig hepatocytes after 6 h of incubation in the medium (Figure 2B). Investigation of 17β-estradiol metabolites by LC-TOF-MS established the presence of two compounds with m/z− 445.2190 and 447.2298, which correspond to molecular ions of glucuronide-conjugated estrone (E1-G) (Figure 2C) and glucuronide-conjugated estradiol (E2-G) (Figure 2D) respectively. The ratio of E1-G to E2-G was 5∶1. When the cell culture medium was hydrolyzed by β-glucuronidase, both estrone and 17β-estradiol were detected. The ratio estrone: 17β-estradiol was similar to the ratio E1-G: E2-G. Using β-estradiol-3′-glucuronide and β-estradiol-17′-glucuronide authentic standards, this study established that glucuronidation of E2-G occurred at 3′-hydroxyl group (Figure 2E).

Bottom Line: The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor.Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol.The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Agro-product Quality and Safety, Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.

ABSTRACT
Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

Show MeSH
Related in: MedlinePlus