Bioactivation of 3-n-butylphthalide via sulfation of its major metabolite 3-hydroxy-NBP: mediated mainly by sulfotransferase 1A1.
3-n-Butylphthalide (NBP) [(±)-3-butyl-1(3H)-isobenzofuranone] is an anti-cerebral-ischemia drug. Moderate hepatotoxicity has been observed in clinical applications. One of the major metabolites, 3-N-acetylcysteine-NBP, has been detected in human urine, indicating the formation of a reactive metabolite. We elucidated the formation mechanism of the reactive metabolite and its association with the hepatotoxicity of NBP. The in vitro incubations revealed that 3-glutathione-NBP (3-GSH-NBP) was observed only in fresh rat liver homogenate rather than in liver microsomes, liver cytosol, or liver 9,000g supernatant supplemented with NADPH and GSH. We also detected 3-GSH-NBP when 3'-phosphoadenosine-5'-phosphosulfate was added in GSH-fortified human liver cytosol (HLC). The formation of 3-GSH-NBP was 39.3-fold higher using 3-hydroxy-NBP (3-OH-NBP) as the substrate than NBP. The sulfotransferase (SULT) inhibitors DCNP (2,6-dichloro-4-nitrophenol) and quercetin suppressed 3-GSH-NBP formation in HLC by 75 and 82%, respectively, suggesting that 3-OH-NBP sulfation was involved in 3-GSH-NBP formation. Further SULT phenotyping revealed that SULT1A1 is the major isoform responsible for the sulfation. Dose-dependent toxicity was observed in primary rat hepatocytes exposed to 3-OH-NBP, with an IC50 of approximately 168 μM. Addition of DCNP and quercetin significantly increased cell viability, whereas l-buthionine-sulfoximine (a GSH depleter) decreased cell viability. Overall, our study revealed the underlying mechanism for the bioactivation of NBP is as follows. NBP is first oxidized to 3-OH-NBP and further undergoes sulfation to form 3-OH-NBP sulfate. The sulfate spontaneously cleaves off, generating highly reactive electrophilic cations, which can bind either to GSH to detoxify or to hepatocellular proteins to cause undesirable side effects.
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People's Republic of China.
Xingxing
X
Diao
Xiaoyan
X
Pang
Cen
C
Xie
Zitao
Z
Guo
Dafang
D
Zhong
Xiaoyan
X
Chen
D000111:Acetylcysteine / Q000378:metabolism; D000818:Animals; D015239:Arylsulfotransferase / Q000378:metabolism*; D001572:Benzofurans / Q000378:metabolism* / Q000493:pharmacokinetics / Q000633:toxicity; D001711:Biotransformation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects; D006801:Humans; D006900:Hydroxylation; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D018696:Neuroprotective Agents / Q000378:metabolism* / Q000493:pharmacokinetics / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D013457:Sulfur Compounds / Q000378:metabolism*
C000592574:3-hydroxy-3-n-butylphthalide; D001572:Benzofurans; D018696:Neuroprotective Agents; D013457:Sulfur Compounds; C027125:3-n-butylphthalide; D015239:Arylsulfotransferase; C117544:SULT1A1 protein, human; D005978:Glutathione; D000111:Acetylcysteine