Elucidation of metabolic pathways of 25-hydroxyvitamin D3 mediated by CYP24A1 and CYP3A using Cyp24a1 knockout rats generated by CRISPR/Cas9 system

CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-typ...

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Published inThe Journal of biological chemistry Vol. 296; p. 100668
Main Authors Yasuda, Kaori, Nishikawa, Miyu, Okamoto, Kairi, Horibe, Kyohei, Mano, Hiroki, Yamaguchi, Mana, Okon, Risa, Nakagawa, Kimie, Tsugawa, Naoko, Okano, Toshio, Kawagoe, Fumihiro, Kittaka, Atsushi, Ikushiro, Shinichi, Sakaki, Toshiyuki
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LanguageEnglish
Published United States Elsevier Inc 01.01.2021
American Society for Biochemistry and Molecular Biology
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Abstract CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher Tmax value than others. When 23S,25(OH)2D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)3D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)3D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)3D3 via 23,25(OH)2D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)3D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)3D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 μg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.
AbstractList CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher Tmax value than others. When 23S,25(OH)2D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)3D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)3D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)3D3 via 23,25(OH)2D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)3D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)3D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 μg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.
CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately two-fold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher T value than others. When 23S,25(OH) D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH) D3 nor 25(OH)D3-26,23-lactone were observed. However, when 23S,25R,26(OH) D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH) D3 via 23,25(OH) D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH) D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH) D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 μg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.
CYP24A1-deficient ( Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher T max value than others. When 23 S ,25(OH) 2 D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH) 3 D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23 S ,25 R ,26(OH) 3 D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH) 3 D3 via 23,25(OH) 2 D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH) 3 D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH) 3 D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 μg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.
ArticleNumber 100668
Author Tsugawa, Naoko
Kawagoe, Fumihiro
Mano, Hiroki
Okano, Toshio
Yasuda, Kaori
Okon, Risa
Okamoto, Kairi
Horibe, Kyohei
Sakaki, Toshiyuki
Nishikawa, Miyu
Yamaguchi, Mana
Ikushiro, Shinichi
Nakagawa, Kimie
Kittaka, Atsushi
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  givenname: Miyu
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  givenname: Kairi
  surname: Okamoto
  fullname: Okamoto, Kairi
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  organization: Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, Imizu, Toyama, Japan
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  givenname: Risa
  surname: Okon
  fullname: Okon, Risa
  organization: Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, Imizu, Toyama, Japan
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  givenname: Kimie
  surname: Nakagawa
  fullname: Nakagawa, Kimie
  organization: Laboratory of Hygienic Sciences, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Chuo-ku, Kobe, Japan
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  givenname: Naoko
  surname: Tsugawa
  fullname: Tsugawa, Naoko
  organization: Department of Health and Nutrition, Faculty of Health and Nutrition, Osaka Shoin Women's University, Higashi-Osaka, Japan
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  surname: Okano
  fullname: Okano, Toshio
  organization: Department of Hygienic Sciences, Kobe Pharmaceutical University, Higashinada-ku, Kobe, Japan
– sequence: 11
  givenname: Fumihiro
  surname: Kawagoe
  fullname: Kawagoe, Fumihiro
  organization: Faculty of Pharmaceutical Sciences, Teikyo University, Tokyo, Japan
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  surname: Kittaka
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  surname: Sakaki
  fullname: Sakaki, Toshiyuki
  email: tsakaki@pu-toyama.ac.jp
  organization: Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, Imizu, Toyama, Japan
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Keywords CYP24A1
CRISPR/Cas9
ADX
PTH
VDR
vitamin D
1,25(OH)2D3
metabolism
cytochrome P450
25(OH)D3
ADR
Language English
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Snippet CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the...
CYP24A1-deficient ( Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the...
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SourceType Open Access Repository
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StartPage 100668
SubjectTerms CRISPR/Cas9
CYP24A1
cytochrome P450
metabolism
vitamin D
Title Elucidation of metabolic pathways of 25-hydroxyvitamin D3 mediated by CYP24A1 and CYP3A using Cyp24a1 knockout rats generated by CRISPR/Cas9 system
URI https://dx.doi.org/10.1016/j.jbc.2021.100668
https://www.ncbi.nlm.nih.gov/pubmed/33865853
https://search.proquest.com/docview/2515064690
https://pubmed.ncbi.nlm.nih.gov/PMC8134072
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