目的研究0.01%、0.05%、0.1%二甲亚砜(DMSO)对细胞色素P450(CYP450)酶系中3A4、2C9两个亚型基因及蛋白表达水平的影响。方法 Chang肝脏细胞经处理后,分为空白对照组(仅含培养液)、DMSO组(分别采用0.01%、0.05%、0.1%DMSO处理)、睾酮组(...目的研究0.01%、0.05%、0.1%二甲亚砜(DMSO)对细胞色素P450(CYP450)酶系中3A4、2C9两个亚型基因及蛋白表达水平的影响。方法 Chang肝脏细胞经处理后,分为空白对照组(仅含培养液)、DMSO组(分别采用0.01%、0.05%、0.1%DMSO处理)、睾酮组(分别采用1、10、100μmol/L睾酮处理)、利福平组(分别采用1、10、100μmol/L利福平处理)、DMSO+睾酮组(分别采用0.01%、0.05%、0.1%DMSO+10μmol/L睾酮处理)、DMSO+利福平组(分别采用0.01%、0.05%、0.1%DMSO+10μmol/L利福平处理)。利用逆转录定量PCR的方法,测定CYP3A4、CYP2C9基因水平的表达。利用蛋白免疫印迹法,测定CYP3A4、CYP2C9蛋白水平的表达。结果 0.1%DMSO组CYP3A4 m RNA高于空白对照组(P<0.01),且0.1%DMSO组CYP3A4蛋白的表达也高于空白对照组。0.01%、0.05%DMSO组CYP3A4 m RNA表达与空白对照组比较差异无统计学意义(P>0.05),但0.01%、0.05%DMSO组CYP3A4蛋白的表达高于空白对照组。0.01%、0.05%、0.1%DMSO组CYP2C9 m RNA表达与空白对照组比较差异无统计学意义(P>0.05),0.01%、0.05%、0.1%DMSO组CYP2C9蛋白表达与空白对照组比较差异无统计学意义(P>0.05)。0.01%、0.05%DMSO+10μmol/L睾酮组CYP3A4 m RNA的表达与空白对照组比较差异无统计学意义(P>0.05),但0.01%、0.05%DMSO+10μmol/L睾酮组CYP3A4蛋白的表达高于睾酮组。0.01%、0.05%、0.1%DMSO+10μmol/L利福平组CYP2C9 m RNA的表达与利福平组比较差异无统计学意义(P>0.05),但0.01%、0.05%、0.1%DMSO+10μmol/L利福平组CYP2C9蛋白的表达高于利福平组。结论0.01%、0.05%、0.1%三个浓度的DMSO在体外肝细胞实验中对CYP3A4的表达有一定的影响,而对CYP2C9的表达影响不明显,当DMSO作为药物溶剂的时候,可能会影响药物单用时的实验结果。展开更多
The cytochrome P450 enzyme CYP3A4 is highly expressed in the intestinal mucosa, where it plays a critical role in the first-pass metabolism of orally administered drugs and thereby influences systemic bioavailability ...The cytochrome P450 enzyme CYP3A4 is highly expressed in the intestinal mucosa, where it plays a critical role in the first-pass metabolism of orally administered drugs and thereby influences systemic bioavailability and drug re- sponse. For example, approximately 45% of an oral dose of the sedative/hypnotic agent midazolam can be inactiva- ted by CYP3A4-dependent 1 -and 4-hydroxylation, necessitating a higher oral dose than an intravenous dose to a- chieve adequate pharmacological response. Unfortunately, there is substantial inter- individual variability in the level of CYP3A4 expressed in the small intestine and this contributes to unpredictability in patient response to this drug and the many other high first-pass CYP3A4 substrates. Over the last 20 years, we have sought to identify and characterize genetic and environmental sources of variable intestinal CYP3A4 expression, with the goal of using that information to guide individual drug dosing decisions in clinical practice. Interestingly, unlike some of the other members of the drug-metabolizing human P450 family, there is only limited variation in the CYP3A4 gene and cod- ing or other highly deleterious variants are rare, despite the fact that CYP3A4 activity in vivo is thought to be highly heritable. More common variation, such as the CYP3A4 ＊ 1Gand CYP3A4 ＊ 22alleles, may influence gene expres- sion, but effect sizes are relatively small. Thus, much of our attention today is focused on the regulation of CYP3A4. With colleagues at St Jude Children＇s Research Hospital, we showed that vitamin D is an important reg- ulator of intestinal CYP3 A4 via a vitamin D receptor - mediated process and that its effects may be exerted predomi- nantly in the duodenum and jejunum, where CYP3A4 expression is most prominent. We have postulated that variation in vitamin D levels and its regulation contribute to variation in intestinal CYP3A4 function. The metabo- lism of 25 - hydroxy vitamin D3 （25 OHD3 ） plays a central role in regulating the effects of the hormone in the body and involves both bioaetivation and catabolic processes. The contributions of CYP2R1, CYP27A1, CYP27B1 and CYP24A1 to these processes are well established. Less well defined is the contribution of enzymes like CYP3A4, SUET and UGT that are known primarily for their roles in drug metabolism, but that can also biotransform 25OHD3. We investigated the oxidation and conjugation of 25OHD3 by these enzymes to better understand their contribution to the regulation of 25OHD3 and to certain pathologies such as drug-induced osteomalaeia and altered drug clearance in patients with liver diseaseE4-61. Results from experiments with cultured primary human hepato- eytes indicate that 25OHD3 is rapidly cleared from hepatic tissue through sulfation by SULT2A1, glueuronidation by UGT1A4 and 4-hydroxylation by CYP3A4,with minimal 1 α- and 24-hydroxylation activity. Moreover, perturbation of the CYP3A4 pathway by inducers or inhibitors of the enzyme leads to metabolic switching. Metabolites from these processes circulate in blood, providing a means to test for changes in enzyme function in vivo. Results from studies conducted with healthy volunteers and patients being treated with rifampin for tuberculosis show selective induction of 25OHD3 oxidation to 4β,25（ OH）zD3 by the potent enzyme inducer, with no change in the efficiency of the lot- and 24-hydroxylation reactions. Interestingly, the inductive effect was most obvious in tuberculosis patients supple- mented with vitamin D3 , suggesting additional, positive effects of the hormone on CYP3A4 expression. Preliminary studies in patients with liver disease reveal changes in the sulfation pathway, with greater accumulation of 25OHD3- 3-sulfate in plasma in patients with mild and moderate forms of the disease, despite a reduction in 25OHD3 levels, and reductions in the parent molecule and all metabolites in plasma from patients with severe disease. This raises the possibility of a reduction in biliary secretion of 25OHD3 conjugates in patients with liver disease and accumula- tion in plasma until their synthesis is significantly compromised in late stages of the disease. This mayhelp explain a reduction in the expression of intestinal CYP3A4 that is also observed in patients with moderate and severe liver disease. Moreover, variation in the biliary secretion of 25OHD3 conjugates may contribute to differences in consti- tutive CYP3A4 expression in individuals with normal hepatic and intestinal function. These conjugates can be de- tected in human bile and are converted to 25OHD3 and 1,25（ OH）zD3 and induce the transcription of VDR target genes in an intestinal cell culture model. Ongoing studies suggest that genetic variation in vitamin D regulatory genes, the diet and sunlight exposure all contribute to inter-individual differences in systemic 25OHD3 concentra- tions. It remains to be seen whether these same factors affect intestinal CYP3A4 function and drug response and can be the target of new precision medicine tests in clinical practice.展开更多
文摘目的研究0.01%、0.05%、0.1%二甲亚砜(DMSO)对细胞色素P450(CYP450)酶系中3A4、2C9两个亚型基因及蛋白表达水平的影响。方法 Chang肝脏细胞经处理后,分为空白对照组(仅含培养液)、DMSO组(分别采用0.01%、0.05%、0.1%DMSO处理)、睾酮组(分别采用1、10、100μmol/L睾酮处理)、利福平组(分别采用1、10、100μmol/L利福平处理)、DMSO+睾酮组(分别采用0.01%、0.05%、0.1%DMSO+10μmol/L睾酮处理)、DMSO+利福平组(分别采用0.01%、0.05%、0.1%DMSO+10μmol/L利福平处理)。利用逆转录定量PCR的方法,测定CYP3A4、CYP2C9基因水平的表达。利用蛋白免疫印迹法,测定CYP3A4、CYP2C9蛋白水平的表达。结果 0.1%DMSO组CYP3A4 m RNA高于空白对照组(P<0.01),且0.1%DMSO组CYP3A4蛋白的表达也高于空白对照组。0.01%、0.05%DMSO组CYP3A4 m RNA表达与空白对照组比较差异无统计学意义(P>0.05),但0.01%、0.05%DMSO组CYP3A4蛋白的表达高于空白对照组。0.01%、0.05%、0.1%DMSO组CYP2C9 m RNA表达与空白对照组比较差异无统计学意义(P>0.05),0.01%、0.05%、0.1%DMSO组CYP2C9蛋白表达与空白对照组比较差异无统计学意义(P>0.05)。0.01%、0.05%DMSO+10μmol/L睾酮组CYP3A4 m RNA的表达与空白对照组比较差异无统计学意义(P>0.05),但0.01%、0.05%DMSO+10μmol/L睾酮组CYP3A4蛋白的表达高于睾酮组。0.01%、0.05%、0.1%DMSO+10μmol/L利福平组CYP2C9 m RNA的表达与利福平组比较差异无统计学意义(P>0.05),但0.01%、0.05%、0.1%DMSO+10μmol/L利福平组CYP2C9蛋白的表达高于利福平组。结论0.01%、0.05%、0.1%三个浓度的DMSO在体外肝细胞实验中对CYP3A4的表达有一定的影响,而对CYP2C9的表达影响不明显,当DMSO作为药物溶剂的时候,可能会影响药物单用时的实验结果。
文摘The cytochrome P450 enzyme CYP3A4 is highly expressed in the intestinal mucosa, where it plays a critical role in the first-pass metabolism of orally administered drugs and thereby influences systemic bioavailability and drug re- sponse. For example, approximately 45% of an oral dose of the sedative/hypnotic agent midazolam can be inactiva- ted by CYP3A4-dependent 1 -and 4-hydroxylation, necessitating a higher oral dose than an intravenous dose to a- chieve adequate pharmacological response. Unfortunately, there is substantial inter- individual variability in the level of CYP3A4 expressed in the small intestine and this contributes to unpredictability in patient response to this drug and the many other high first-pass CYP3A4 substrates. Over the last 20 years, we have sought to identify and characterize genetic and environmental sources of variable intestinal CYP3A4 expression, with the goal of using that information to guide individual drug dosing decisions in clinical practice. Interestingly, unlike some of the other members of the drug-metabolizing human P450 family, there is only limited variation in the CYP3A4 gene and cod- ing or other highly deleterious variants are rare, despite the fact that CYP3A4 activity in vivo is thought to be highly heritable. More common variation, such as the CYP3A4 ＊ 1Gand CYP3A4 ＊ 22alleles, may influence gene expres- sion, but effect sizes are relatively small. Thus, much of our attention today is focused on the regulation of CYP3A4. With colleagues at St Jude Children＇s Research Hospital, we showed that vitamin D is an important reg- ulator of intestinal CYP3 A4 via a vitamin D receptor - mediated process and that its effects may be exerted predomi- nantly in the duodenum and jejunum, where CYP3A4 expression is most prominent. We have postulated that variation in vitamin D levels and its regulation contribute to variation in intestinal CYP3A4 function. The metabo- lism of 25 - hydroxy vitamin D3 （25 OHD3 ） plays a central role in regulating the effects of the hormone in the body and involves both bioaetivation and catabolic processes. The contributions of CYP2R1, CYP27A1, CYP27B1 and CYP24A1 to these processes are well established. Less well defined is the contribution of enzymes like CYP3A4, SUET and UGT that are known primarily for their roles in drug metabolism, but that can also biotransform 25OHD3. We investigated the oxidation and conjugation of 25OHD3 by these enzymes to better understand their contribution to the regulation of 25OHD3 and to certain pathologies such as drug-induced osteomalaeia and altered drug clearance in patients with liver diseaseE4-61. Results from experiments with cultured primary human hepato- eytes indicate that 25OHD3 is rapidly cleared from hepatic tissue through sulfation by SULT2A1, glueuronidation by UGT1A4 and 4-hydroxylation by CYP3A4,with minimal 1 α- and 24-hydroxylation activity. Moreover, perturbation of the CYP3A4 pathway by inducers or inhibitors of the enzyme leads to metabolic switching. Metabolites from these processes circulate in blood, providing a means to test for changes in enzyme function in vivo. Results from studies conducted with healthy volunteers and patients being treated with rifampin for tuberculosis show selective induction of 25OHD3 oxidation to 4β,25（ OH）zD3 by the potent enzyme inducer, with no change in the efficiency of the lot- and 24-hydroxylation reactions. Interestingly, the inductive effect was most obvious in tuberculosis patients supple- mented with vitamin D3 , suggesting additional, positive effects of the hormone on CYP3A4 expression. Preliminary studies in patients with liver disease reveal changes in the sulfation pathway, with greater accumulation of 25OHD3- 3-sulfate in plasma in patients with mild and moderate forms of the disease, despite a reduction in 25OHD3 levels, and reductions in the parent molecule and all metabolites in plasma from patients with severe disease. This raises the possibility of a reduction in biliary secretion of 25OHD3 conjugates in patients with liver disease and accumula- tion in plasma until their synthesis is significantly compromised in late stages of the disease. This mayhelp explain a reduction in the expression of intestinal CYP3A4 that is also observed in patients with moderate and severe liver disease. Moreover, variation in the biliary secretion of 25OHD3 conjugates may contribute to differences in consti- tutive CYP3A4 expression in individuals with normal hepatic and intestinal function. These conjugates can be de- tected in human bile and are converted to 25OHD3 and 1,25（ OH）zD3 and induce the transcription of VDR target genes in an intestinal cell culture model. Ongoing studies suggest that genetic variation in vitamin D regulatory genes, the diet and sunlight exposure all contribute to inter-individual differences in systemic 25OHD3 concentra- tions. It remains to be seen whether these same factors affect intestinal CYP3A4 function and drug response and can be the target of new precision medicine tests in clinical practice.
