Publications by authors named "Da-Peng Dai"

48 Publications

Functional characterization of the defective CYP2C9 variant CYP2C9*18.

Pharmacol Res Perspect 2021 02;9(1):e00718

The Key laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, P. R. China.

Cytochrome P450 2C9 (CYP2C9) is one of the most important drugs metabolizing enzymes and accounts for the metabolism of about 13%-17% of clinical drugs. Like other members in CYP2 family, CYP2C9 gene exhibits great genetic polymorphism among different races and individuals. CYP2C9*18 is one CYP2C9 allelic variant identified in a Southeast Asian population and is estimated to cause the amino acid substitutions of I359L and D397A in CYP2C9 enzyme simultaneously. Limited by the low expression level in bacteria and COS-7 cells, no valuable enzyme kinetics have been reported on this CYP2C9 variant. In this study, the baculovirus-based system was used for the high expression of recombinant CYP2C9 s in insect cells. As a result, together with I359L substitution, D397A could significantly decrease the protein expression of CYP2C9.18 in insect cells, although substitution of D397A alone had no effect on the expression of CYP2C9 in vitro. As compared with that of wild-type enzyme, both CYP2C9.18 variant and D397A variant could decrease more than 80% of the catalytic activity of CYP2C9 enzyme toward three probe substrates, suggesting that caution should be exercised when patients carrying CYP2C9*18 taking medicines metabolized by CYP2C9 enzyme with a narrow therapeutic window.
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http://dx.doi.org/10.1002/prp2.718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842875PMC
February 2021

The high expression of MTH1 and NUDT5 promotes tumor metastasis and indicates a poor prognosis in patients with non-small-cell lung cancer.

Biochim Biophys Acta Mol Cell Res 2021 01 21;1868(1):118895. Epub 2020 Oct 21.

Peking University Fifth School of Clinical Medicine, Beijing Hospital, Beijing, PR China; The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, NO.1 DaHua Road, Dong Dan, Beijing 100730, PR China. Electronic address:

MutT Homolog 1 (MTH1) is a mammalian 8-oxodGTPase for sanitizing oxidative damage to the nucleotide pool. Nudix type 5 (NUDT5) also sanitizes 8-oxodGDP in the nucleotide pool. The role of MTH1 and NUDT5 in non-small-cell lung cancer (NSCLC) progression and metastasis remains unclear. In the present study, we reported that MTH1 and NUDT5 were upregulated in NSCLC cell lines and tissues, and higher levels of MTH1 or NUDT5 were associated with tumor metastasis and a poor prognosis in patients with NSCLC. Their suppression also restrained tumor growth and lung metastasis in vivo and significantly inhibited NSCLC cell migration, invasion, cell proliferation and cell cycle progression while promoting apoptosis in vitro. The opposite effects were observed in vitro following MTH1 or NUDT5 rescue. In addition, the upregulation of MTH1 or NUDT5 enhanced the MAPK pathway and PI3K/AKT activity. Furthermore, MTH1 and NUDT5 induce epithelial-mesenchymal transition both in vitro and in vivo. These results highlight the essential role of MTH1 and NUDT5 in NSCLC tumor tumorigenesis and metastasis as well as their functions as valuable markers of the NSCLC prognosis and potential therapeutic targets.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118895DOI Listing
January 2021

Effects of rare alleles on stable warfarin doses in Chinese Han patients with atrial fibrillation.

Pharmacogenomics 2020 09 7;21(14):1021-1031. Epub 2020 Sep 7.

Cardiovascular Department, Beijing Hospital, National Centre of Gerontology, Beijing, 100730, China.

Gene polymorphisms are critical in warfarin dosing variation. Here, the role of rare alleles on warfarin doses in Chinese Han patients was investigated. A retrospective study recruited 681 warfarin treated atrial fibrillation patients. The genetic and clinical data were collected. Dose-related variables were selected by univariate analyses and the warfarin-dosing algorithm was derived by multivariate regression analysis. Three rare alleles (*13, *16 and *60) were associated with lower stable doses. Inclusion of the rare alleles in the prediction model added an extra 3.7% warfarin dose predictive power. *13, *16 and *60 was associated with lower stable warfarin doses in Chinese patients. The algorithm including rare alleles tends to more accurately predict stable warfarin doses.
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http://dx.doi.org/10.2217/pgs-2020-0051DOI Listing
September 2020

An identification and functional evaluation of a novel CYP2C9 variant CYP2C9*62.

Chem Biol Interact 2020 Aug 9;327:109168. Epub 2020 Jun 9.

Cardiovascular Department, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, 100730, PR China. Electronic address:

Warfarin is the most commonly used anticoagulant in the clinical treatment of thromboembolic diseases. The dose of warfarin varies significantly within populations, and the dose is closely related to the genetic polymorphisms of the CYP2C9 and VKORC1 genes. In this study, a new CYP2C9 nonsynonymous mutation (8576C > T) was detected after the genetic screening of 162 patients took warfarin. This mutation, named as the new allele CYP2C9*62, can result in an arginine to cysteine amino acid substitution at position 125 of the CYP2C9 protein (R125C). When expressed in insect cells, the protein expression of CYP2C9.62 was significantly lower than that of the wild-type, and its metabolic activity was also significantly decreased after the addition of three typical CYP2C9 probe drugs, suggesting that the new mutant can dramatically affect the metabolism of CYP2C9 drugs in vitro.
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http://dx.doi.org/10.1016/j.cbi.2020.109168DOI Listing
August 2020

The Pol β variant containing exon α is deficient in DNA polymerase but has full dRP lyase activity.

Sci Rep 2019 07 9;9(1):9928. Epub 2019 Jul 9.

Genome Integrity and Structural Biology Laboratory, National Institutes of Health, NIEHS, 111 T.W. Alexander Drive, P.O. Box 12233, Research Triangle Park, North Carolina, NC, 27709, USA.

DNA polymerase (Pol) β is a key enzyme in base excision repair (BER), an important repair system for maintaining genomic integrity. We previously reported the presence of a Pol β transcript containing exon α (105-nucleotide) in normal and colon cancer cell lines. The transcript carried an insertion between exons VI and VII and was predicted to encode a ~42 kDa variant of the wild-type 39 kDa enzyme. However, little is known about the biochemical properties of the exon α-containing Pol β (exon α Pol β) variant. Here, we first obtained evidence indicating expression of the 42 kDa exon α Pol β variant in mouse embryonic fibroblasts. The exon α Pol β variant was then overexpressed in E. coli, purified, and characterized for its biochemical properties. Kinetic studies of exon α Pol β revealed that it is deficient in DNA binding to gapped DNA, has strongly reduced polymerase activity and higher Km for dNTP during gap-filling. On the other hand, the 5'-dRP lyase activity of the exon α Pol β variant is similar to that of wild-type Pol β. These results indicate the exon α Pol β variant is base excision repair deficient, but does conduct 5'-trimming of a dRP group at the gap margin. Understanding the biological implications of this Pol β variant warrants further investigation.
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http://dx.doi.org/10.1038/s41598-019-45846-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616571PMC
July 2019

Repair pathway for PARP-1 DNA-protein crosslinks.

DNA Repair (Amst) 2019 01 12;73:71-77. Epub 2018 Nov 12.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, NC, 27709, USA. Electronic address:

Poly(ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in many different processes of DNA and RNA metabolism, including DNA repair. Previously, PARP-1 was found capable of forming a covalent DNA-protein crosslink (DPC) at the apurinic/apyrimidinic (AP) site in double-stranded DNA. The C1´ atom of the AP site participates in Schiff base formation with a lysine side chain in PARP-1, and a covalent bond is formed upon reduction of the Schiff base. The PARP-1 DPC is formed in vivo where DPC formation correlates with AP site induction by a monofunctional alkylating agent. Here, we examined repair of PARP-1 DPCs in mouse fibroblasts and found that a proteasome inhibitor, MG-132, reduces repair resulting in accumulation of PARP-1 DPCs and increased alkylating agent cytotoxicity. Using a model DNA substrate mimicking the PARP-1 DPC after proteasomal degradation, we found that repair is completed by a sub-pathway of base excision repair (BER). Tyrosyl-DNA phosphodiesterase 1 was proficient in removing the ring-open AP site sugar at the phosphodiester linkage, leaving an intermediate for processing by other BER enzymes. The results reveal proteasomal degradation of the PARP-1 DPC is active in mouse fibroblasts and that a model repair intermediate is processed by the BER machinery.
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http://dx.doi.org/10.1016/j.dnarep.2018.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312470PMC
January 2019

Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells.

Proc Natl Acad Sci U S A 2018 04 2;115(16):4218-4222. Epub 2018 Apr 2.

MOH Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, 100730 Beijing, People's Republic of China;

Reactive oxygen species formed within the mammalian cell can produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can cause base mispairing during gene expression. Here we found that administration of 8-oxoGTP in MTH1-knockdown cells results in increased 8-oxoG content in mRNA. Under this condition, an amber mutation of the reporter luciferase is suppressed. Using second-generation sequencing techniques, we found that U-to-G changes at preassigned sites of the luciferase transcript increased when 8-oxoGTP was supplied. In addition, an increased level of 8-oxoG content in RNA induced the accumulation of aggregable amyloid β peptides in cells expressing amyloid precursor protein. Our findings indicate that 8-oxoG accumulation in mRNA can alter protein synthesis in mammalian cells. Further work is required to assess the significance of these findings under normal physiological conditions.
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http://dx.doi.org/10.1073/pnas.1718363115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910836PMC
April 2018

DNA polymerase β: A missing link of the base excision repair machinery in mammalian mitochondria.

DNA Repair (Amst) 2017 12 28;60:77-88. Epub 2017 Oct 28.

Genome Integrity and Structural Biology Laboratory, National Institutes of Health, NIEHS, 111 T.W. Alexander Drive, P.O. Box 12233, Research Triangle Park, NC 27709, USA. Electronic address:

Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) β. Pol β was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol β. Mitochondria from pol β-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol β-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol β and thus pol β plays a role in preserving mitochondrial genome stability.
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http://dx.doi.org/10.1016/j.dnarep.2017.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919216PMC
December 2017

Effects of CYP2C19 Variants on Fluoxetine Metabolism in vitro.

Pharmacology 2017 12;100(1-2):91-97. Epub 2017 May 12.

School of Pharmacy, Wenzhou Medical University, Wenzhou, China.

Aims: CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31 CYP2C19 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of fluoxetine in vitro.

Methods: The wild-type and 30 CYP2C19 variants were expressed in insect cells and each variant was characterized using fluoxetine as the substrate. Reactions were performed at 37°C with 20-1,000 µmol/L substrate for 30 min. By using ultra-high performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of norfluoxetine were determined.

Results: Among the CYP2C19 variants tested, T130M showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 9.56 to 77.77%). In addition, CYP2C19*3 and *35FS could not be detected because they have no detectable enzyme activity.

Conclusion: In China, the assessment of CYP2C19 variants in vitro offers valuable information relevant to the personalized medicine for CYP2C19-metabolized drug.
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http://dx.doi.org/10.1159/000475598DOI Listing
January 2018

Systematic screening for CYP3A4 genetic polymorphisms in a Han Chinese population.

Pharmacogenomics 2017 Mar 17;18(4):369-379. Epub 2017 Feb 17.

The MOH Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, Beijing 100730, P.R. China.

Aim: To systematically investigate the genetic polymorphisms of the CYP3A4 gene in a Han Chinese population.

Materials & Methods: The promoter and exons of CYP3A4 gene in 1114 unrelated, healthy Han Chinese subjects were amplified and genotyped by direct sequencing.

Results: In total, five previously reported alleles (*1G, *4, *5, *18B and *23) were detected, of which one allele (*23) was reported for the first time in Han Chinese population. Additionally, seven novel exonic variants were also identified and designated as new alleles CYP3A4*28-*34.

Conclusion: This study provides the most comprehensive data of CYP3A4 polymorphisms in Han Chinese population and detects the largest number of novel CYP3A4 alleles in one ethnic group.
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http://dx.doi.org/10.2217/pgs-2016-0179DOI Listing
March 2017

Adipose-specific deletion of exacerbates obesity and insulin resistance in a mouse model of diet-induced obesity.

FASEB J 2017 06 27;31(6):2533-2547. Epub 2017 Feb 27.

The Ministry of Health (MOH) Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, Beijing, China;

Recent studies have shown that KIF5B (conventional kinesin heavy chain) mediates glucose transporter type 4 translocation and adiponectin secretion in 3T3-L1 adipocytes, suggesting an involvement of KIF5B in the homeostasis of metabolism. However, the physiologic function of KIF5B in adipose tissue remains to be determined. In this study, adipose-specific knockout (F-K5bKO) mice were generated using the Cre-LoxP strategy. F-K5bKO mice had similar body weights to controls fed on a standard chow diet. However, F-K5bKO mice had hyperlipidemia and significant glucose intolerance and insulin resistance. Deletion of aggravated the deleterious impact of a high-fat diet (HFD) on body weight gain, hepatosteatosis, glucose tolerance, and systematic insulin sensitivity. These changes were accompanied by impaired insulin signaling, decreased secretion of adiponectin, and increased serum levels of leptin and proinflammatory adipokines. F-K5bKO mice fed on an HFD exhibited lower energy expenditure and thermogenic dysfunction as a result of whitening of brown adipose due to decreased mitochondria biogenesis and down-regulation of key thermogenic gene expression. In conclusion, selective deletion of in adipose tissue exacerbates HFD-induced obesity and its associated metabolic disorders, partly through a decrease in energy expenditure, dysregulation of adipokine secretion, and insulin signaling.-Cui, J., Pang, J., Lin, Y.-J., Gong, H., Wang, Z.-H., Li, Y.-X., Li, J., Wang, Z., Jiang, P., Dai, D.-P., Li, J., Cai, J.-P., Huang, J.-D., Zhang, T.-M. Adipose-specific deletion of exacerbates obesity and insulin resistance in a mouse model of diet-induced obesity.
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http://dx.doi.org/10.1096/fj.201601103RDOI Listing
June 2017

Effect of 22 CYP2D6 variants found in the Chinese population on tolterodine metabolism in vitro.

Chem Biol Interact 2017 Feb 10;264:10-15. Epub 2017 Jan 10.

Department of Pharmacology, School of Pharmacy of Wenzhou Medical University, Wenzhou, Zhejiang 325035, China. Electronic address:

Cytochrome P450 2D6 (CYP2D6) is an important member of the cytochrome P450 enzyme superfamily. We recently identified 22 novel variants in the Chinese population using PCR and bidirectional sequencing methods. The aim of this study is to characterize the enzymatic activity of these variants and their effects on the metabolism of the antimuscarinic drug tolterodine in vitro. A baculovirus-mediated expression system was used to express wild-type CYP2D6 and 24 variants (CYP2D6*2, CYP2D6*10, and 22 novel CYP2D6 variants) at high levels. The insect microsomes expressing CYP2D6 proteins were incubated with 0.1-50 μM tolterodine at 37 °C for 30 min and the metabolites were analyzed by high-performance liquid chromatography-tandem mass spectrometry system. Of the 24 CYP2D6 variants tested, 2 variants (CYP2D6*92 and CYP2D6*96) were found to be catalytically inactive, 4 variants (CYP2D6*94, F164L, F219S and D336N) exhibited markedly increased intrinsic clearance values (V/K) compared with the wild-type (from 66.34 to 99.79%), whereas 4 variants (CYP2D6*10, *93, *95 and E215K) exhibited significantly decreased values (from 49.02 to 98.50%). This is the first report of all these rare alleles for tolterodine metabolism and these findings suggest that more attention should be paid to subjects carrying these infrequent CYP2D6 alleles when administering tolterodine in the clinic.
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http://dx.doi.org/10.1016/j.cbi.2017.01.003DOI Listing
February 2017

Oxidized nucleotide insertion by pol β confounds ligation during base excision repair.

Nat Commun 2017 01 9;8:14045. Epub 2017 Jan 9.

Genome Integrity and Structural Biology Laboratory, National Institutes of Health, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.

Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides.
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http://dx.doi.org/10.1038/ncomms14045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228075PMC
January 2017

Role of cytochrome P450 2D6 genetic polymorphism in carvedilol hydroxylation in vitro.

Drug Des Devel Ther 2016 8;10:1909-16. Epub 2016 Jun 8.

Department of Pharmacology, School of Pharmacy, Wenzhou Medical University, Wenzhou, People's Republic of China.

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that catalyzes the metabolism of a great number of therapeutic drugs. Up to now, >100 allelic variants of CYP2D6 have been reported. Recently, we identified 22 novel variants in the Chinese population in these variants. The purpose of this study was to examine the enzymatic activity of the variants toward the CYP2D6 substrate carvedilol in vitro. The CYP2D6 proteins, including CYP2D6.1 (wild type), CYP2D6.2, CYP2D6.10, and 22 other novel CYP2D6 variants, were expressed from insect microsomes and incubated with carvedilol ranging from 1.0 μM to 50 μM at 37°C for 30 minutes. After termination, the carvedilol metabolites were extracted and detected using ultra-performance liquid chromatography tandem mass-spectrometry. Among the 24 CYP2D6 variants, CYP2D6.92 and CYP2D6.96 were catalytically inactive and the remaining 22 variants exhibited significantly decreased intrinsic clearance values (ranging from ~25% to 95%) compared with CYP2D6.1. The present data in vitro suggest that the newly found variants significantly reduced catalytic activities compared with CYP2D6.1. Given that CYP2D6 protein activities could affect carvedilol plasma levels, these findings are greatly relevant to personalized medicine.
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http://dx.doi.org/10.2147/DDDT.S106175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907640PMC
May 2017

Effects of 22 CYP2D6 Genetic Variations Newly Identified in Chinese Population on Olanzapine Metabolism in vitro.

Pharmacology 2016 2;98(3-4):124-33. Epub 2016 Jun 2.

Department of Pharmacology, School of Pharmacy, Wenzhou Medical University, Wenzhou, China.

The objective of this study was to assess the catalytic activity of 22 novel CYP2D6 allelic variants (2D6*87-*98, R25Q, F164L, E215K, F219S, V327M, D336N, V342M, R344Q, R440C and R497C) to olanzapine in vitro. Their protein products expressed in Spodoptera frugiperda 21 (Sf21) insect cells were incubated with olanzapine 100-2,000 μmol/l for 30 min. The kinetic parameters of Km, Vmax and intrinsic clearance were determined by 2-hydroxymethylolanzapine, the metabolite of olanzapine mediated by CYP2D6, using ultra-performance liquid chromatography tandem mass spectrometry. Results showed that the kinetic parameters of 2 alleles, CYP2D6*92 and 2D6*96, could not be detected; 17 allelic variants, CYP2D6*87-*88, 2D6*90-*91, 2D6*93-*95, 2D6*97, R25Q, F164L, E215K, F219S, V327M, V342M, R344Q, R440C and R497C, significantly reduced the intrinsic clearance of olanzapine; 2 variants, CYP2D6*89 and 2D6*98, increased the intrinsic clearance of olanzapine; no difference was found in intrinsic clearance of D336N. Furthermore, 6 alleles, CYP2D6*87, 2D6*88, 2D6*91, 2D6*93, 2D6*97 and R497C, exhibited higher Km values in a range of 120.80-217.56% relative to wild-type CYP2D6*1. The research demonstrated the metabolic phenotype of the 22 novel CYP2D6 variants for olanzapine that were different from probe drugs we used previously and might provide beneficial information to the personalized medicine of olanzapine.
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http://dx.doi.org/10.1159/000446807DOI Listing
March 2017

Assessment of 25 CYP2D6 alleles found in the Chinese population on propafenone metabolism in vitro.

Can J Physiol Pharmacol 2016 Aug 17;94(8):895-9. Epub 2016 Mar 17.

c The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing 100730, China.

Cytochrome P450 enzyme 2D6 (CYP2D6) is an important member of the cytochrome P450 enzyme superfamily, with more than 100 CYP2D6 allelic variants being previously reported. The aim of this study was to assess the catalytic characteristics of 25 alleles (CYP2D6.1 and 24 CYP2D6 variants) and their effects on the metabolism of propafenone in vitro. Twenty-five CYP2D6 alleles were expressing in 21 Spodoptera frugiperda (Sf) insect cells, and each variant was evaluated using propafenone as the substrate. Reactions were performed at 37 °C with 1-100 μmol/L propafenone for 30 min. After termination, the product 5-OH-propafenone was extracted and used for signal collection by ultra-performance liquid chromatography (UPLC). Compared with wild type CYP2D6.1, the intrinsic clearance (Vmax and Km) values of all variants were significantly altered. Three variants (CYP2D6.87, CYP2D6.90, CYP2D6.F219S) exhibited markedly increased intrinsic clearance values (129% to 165%), whereas 21 variants exhibited significantly decreased values (16% to 85%) due to increased Km and (or) decreased Vmax values. These results indicated that the majority of tested alleles had significantly altered catalytic activity towards propafenone hydroxylation in this expression system. Attention should be paid to subjects carrying these rare alleles when treated with propafenone.
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http://dx.doi.org/10.1139/cjpp-2015-0509DOI Listing
August 2016

Effects of CYP2C19 variants on methadone metabolism in vitro.

Drug Test Anal 2017 Apr 19;9(4):634-639. Epub 2016 May 19.

School of Pharmacy, Wenzhou Medical University, Wenzhou, China.

CYP2C19 is an important member of the cytochrome P450 (CYP450) enzyme super family and is responsible for clearing approximately 10% of commonly used clinical drugs that undergo phase I metabolism. Genetic polymorphisms of CYP2C19 significantly influence the efficacy and safety of some drugs, which might cause undesirable adverse effects or cure failure at standard dosages. The aim of this study was to clarify the catalytic activities of 31 CYP2C19 alleles on the oxidative in vitro metabolism of methadone. Insect microsomes expressing the CYP2C19 alleles were incubated with 50-2000 μM methadone for 30 min at 37 °C and terminated by cooling to -80 °C immediately. Methadone and its metabolite EDDP were analyzed by an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system. Of the 31 tested CYP2C19 allelies variants, CYP2C19*1 is the wild-type. Compared with CYP2C19*1, two CYP2C19 variants (CYP2C19*3 and *35FS) had no detectable enzyme activity, one variant L16F exhibited slightly increased intrinsic clearance values, and one variant N277K showed no significant difference. In addition, 26 variants exhibited significantly decreased values (from 1.48% to 80.40%). These findings suggest that more attention should be paid in clinical administration of methadone to individuals carrying these CYP2C19 alleles. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/dta.1997DOI Listing
April 2017

In vitro metabolism of phenytoin in 36 CYP2C9 variants found in the Chinese population.

Chem Biol Interact 2016 Jun 6;253:93-9. Epub 2016 May 6.

Department of Pharmacology, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China. Electronic address:

Cytochrome P450 2C9 (CYP2C9) is an important member of the cytochrome P450 enzyme superfamily, with 57 CYP2C9 allelic variants being previously reported. Recently, we identified 22 novel alleles (*36 -*56 and N418T) in the Han Chinese population. This study aims to assess the catalytic activities of wild-type (CYP2C9*1) and 36 CYP2C9 allelic variants found in the Chinese population toward phenytoin (PHT) in vitro. Insect microsomes expressing CYP2C9*1 and 36 CYP2C9 variants were incubated with 1-200 μM phenytoin for 30 min at 37 °C. Then, these products were extracted and the signal detection was performed by HPLC-MS/MS. The intrinsic clearance (Vmax/Km) values of all variants, with the exception of CYP2C9*2, CYP2C9*11, CYP2C9*23, CYP2C9*29, CYP2C9*34, CYP2C9*38, CYP2C9*44, CYP2C9*46 and CYP2C9*48, were significantly different from CYP2C9*1. CYP2C9*27, *40, *41, *47, *49, *51, *53, *54, *56 and N418T variant exhibited markedly larger values than CYP2C9*1 (>152.8%), whereas 17 variants exhibited smaller values (from 48.6% to 99.9%) due to larger Km and/or smaller Vmax values than CYP2C9*1. The findings suggest that more attention should be paid on subjects carrying these infrequent CYP2C9 alleles when administering phenytoin in clinic.
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http://dx.doi.org/10.1016/j.cbi.2016.04.040DOI Listing
June 2016

Functional characterization of 22 novel CYP2D6 variants for the metabolism of Tamoxifen.

J Pharm Pharmacol 2016 Jun 25;68(6):819-25. Epub 2016 Apr 25.

Department of Pharmacology, School of Pharmacy of Wenzhou Medical University, Wenzhou, Zhejiang, China.

Objectives: This study aimed to assess the catalytic characteristics of 24 CYP2D6 allelic isoforms found in Chinese Han population on the metabolism of tamoxifen in vitro.

Methods: Recombinant CYP2D6 microsomes of distinguished genotypes were used to characterize the corresponding enzyme activity towards tamoxifen. About 5-2500 μm tamoxifen was incubated for 30 min at 37 °C. Using high-performance liquid chromatography to detect the products, the kinetic parameters Km , Vmax and intrinsic clearance (Vmax /Km ) of N-desmethyltamoxifen were determined.

Key Findings: Of the 24 tested allelic variants, the differences of intrinsic clearance value were shown as follows: CYP2D6.89 was much higher than wild-type CYP2D6.1, 2 allelic isoforms (CYP2D6.88 and D336N) exhibited similar intrinsic clearance values as the wild-type enzyme, two variants displayed weak or no activity, while the rest 19 variants showed significantly reduced intrinsic clearance values ranging from 7.46 to 81.11%.

Conclusion: The comprehensive assessment of CYP2D6 variants provides significant insights into allele-specific activity towards tamoxifen in vitro, suggesting that most of the carriers of these alleles might be paid more attention when using CYP2D6-mediated drugs clinically.
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http://dx.doi.org/10.1111/jphp.12556DOI Listing
June 2016

Effect of CYP2D6 genetic polymorphism on the metabolism of citalopram in vitro.

Drug Metab Pharmacokinet 2016 Apr 22;31(2):133-8. Epub 2016 Jan 22.

Department of Pharmacology, School of Pharmacy of Wenzhou Medical University, Wenzhou, Zhejiang, China. Electronic address:

Genetic polymorphisms of CYP2D6 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. We aimed at investigating the role of CYP2D6 in the metabolism of citalopram and identifying the effect of 24 CYP2D6 allelic variants we found in Chinese Han population on the metabolism of citalopram in vitro. These CYP2D6 variants expressed by insect cells system were incubated with 10-1000 μM citalopram for 30 min at 37 °C and the reaction was terminated by cooling to -80 °C immediately. Citalopram and its metabolites were analyzed by high-performance liquid chromatography (HPLC). The intrinsic clearance (Vmax/Km) values of the variants toward citalopram metabolites were significantly altered, 38-129% for demethylcitalopram and 13-138% for citalopram N-oxide when compared with CYP2D6*1. Most of the tested rare alleles exhibited significantly decreased values due to increased Km and/or decreased Vmax values. We conclude that recombinant system could be used to investigate the enzymes involved in drug metabolism and these findings suggest that more attention should be paid to subjects carrying these CYP2D6 alleles when administering citalopram in the clinic.
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http://dx.doi.org/10.1016/j.dmpk.2016.01.001DOI Listing
April 2016

In vitro assessment of 24 CYP2D6 allelic isoforms on the metabolism of methadone.

Drug Test Anal 2017 Feb 9;9(2):216-220. Epub 2016 Mar 9.

The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing, China.

CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme super family, with at least 100 CYP2D6 alleles being previously identified. Genetic polymorphisms of CYP2D6 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. The aim of this study was to clarify the catalytic activities of 24 CYP2D6 alleles on the oxidative in vitro metabolism of methadone. Reactions were incubated with 50-2000  µM methadone for 30 min at 37 °C and terminated by cooling to -80 °C immediately. Methadone and the major metabolite EDDP were analyzed by an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) system. Compared with wild-type CYP2D6*1, most variants showed significantly altered values in V and intrinsic clearance (V /K ). Only three variants (CYP2D6*88, *91 and E215K) exhibited markedly increased intrinsic clearance values, and one variant CYP2D6*94 showed no significant difference. On the other hand, the kinetic parameters of two CYP2D6 variants (CYP2D6*92 and *96) could not be determined because they had no detectable enzyme activity, whereas 18 variants exhibited significantly decreased values. To sum up, this study demonstrated that more attention should be paid in clinical administration of methadone to individuals carrying these CYP2D6 alleles. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/dta.1959DOI Listing
February 2017

Effects of 24 CYP2D6 Variants Found in the Chinese Population on the Metabolism of Risperidone.

Pharmacology 2015 7;96(5-6):290-5. Epub 2015 Nov 7.

School of Pharmacy, Wenzhou Medical University, Wenling, PR China.

Aims: Cytochrome P450 (CYP450) 2D6 is an important member of the P450 enzyme superfamily and responsible for clearing 25% of clinically important drugs. The aim of this study was to assess the catalytic characteristics of 24 CYP2D6 allelic isoforms found in the Chinese population and their effects on the metabolism of risperidone in vitro.

Methods: Insect microsomes expressing wild-type CYP2D6 and 24 CYP2D6 allelic variants were incubated with 20-1,000 μmol/l risperidone for 40 min at 37°C. After termination, risperidone and 9-OH risperidone, the metabolite of risperidone, were precipitated and used for signal collection by ultra-performance liquid-chromatography tandem mass spectrometry.

Results: Among 24 CYP2D6 variants tested, 2 variants (CYP2D6*92 and CYP2D6*96) were found to be with no detectable activity. Two variants (E215K and R440C) exhibited higher intrinsic clearance values than the wild-type protein, while the remaining 20 CYP2D6 allelic variants exhibited significantly decreased clearance values (2.01-87.56%) compared to CYP2D6*1.

Conclusion: These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2D6 alleles when administering risperidone in the clinic. This is the first report of all these novel alleles for risperidone metabolism, providing fundamental data for further clinical studies on CYP2D6 alleles.
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http://dx.doi.org/10.1159/000441007DOI Listing
September 2016

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program.

PLoS One 2015 30;10(9):e0139485. Epub 2015 Sep 30.

HLA Laboratory, Yueyang Red Cross Blood Center, Yueyang, Hunan, China.

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139485PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589403PMC
April 2016

Effect of CYP2D6 variants on venlafaxine metabolism in vitro.

Xenobiotica 2016 25;46(5):424-9. Epub 2015 Sep 25.

a School of Pharmacy, Wenzhou Medical University , Wenzhou , China and.

1. CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme superfamily, we recently identified 22 CYP2D6 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of venlafaxine in vitro. 2. The wild-type and 24 CYP2D6 variants were expressed in insect cells, and each variant was characterized using venlafaxine as the substrate. Reactions were performed at 37 °C with 5-500 μM substrate (three variants was adjusted to 1000 μM) for 50 min. By using high-performance liquid chromatography to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of O-desmethylvenlafaxine were determined. 3. Among the 22 CYP2D6 variants, the intrinsic clearance (Vmax/Km) values of all variants were significantly decreased (from 0.2% to 84.5%) compared with wild-type CYP2D6*1. In addition, the kinetic parameters of two CYP2D6 variants could not be detected because they have no detectable enzyme activity. 4. The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards venlafaxine in vivo.
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http://dx.doi.org/10.3109/00498254.2015.1089364DOI Listing
November 2016

The effect of resveratrol on pharmacokinetics of aripiprazole in vivo and in vitro.

Xenobiotica 2016 22;46(5):439-44. Epub 2015 Sep 22.

a School of Pharmacy, Wenzhou Medical University , Wenzhou , China and.

1. The objective of this study were to investigate the effect of orally administered resveratrol on the pharmacokinetics of aripiprazole (APZ) in rat, and the inhibitory effects of resveratrol on APZ dehydrogenation activity in liver microsomes and human cytochrome P450 3A4 and 2D6. 2. Twenty-five healthy male Sprague-Dawley rats were randomly divided into five groups: A (control group), B (multiple dose of 200 mg/kg resveratrol), C (multiple dose of 100 mg/kg resveratrol), D (a single dose of 200 mg/kg resveratrol) and E (a single dose of 100 mg/kg resveratrol). A single dose of 3 mg/kg APZ administered orally 30 min after administration of resveratrol. In addition, CYP2D6*1, CYP3A4*1, human and rat liver microsomes were performed to determine the effect of resveratrol on the metabolism of APZ in vitro. 3. The multiple dose of 200 or 100 mg/kg resveratrol significantly increased the AUC and Cmax of APZ. The resveratrol also obviously decreased the CL, but without any significant difference on t1/2 in vivo. On the other hand, resveratrol showed inhibitory effect on CYP3A4*1, CYP2D6*1, human and rat microsomes, the IC50 of resveratrol was 6.771, 87.87, 45.11 and 35.59 μmol l(-1), respectively. 4. Those results indicated more attention should be paid when APZ was administrated combined with resveratrol.
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http://dx.doi.org/10.3109/00498254.2015.1088175DOI Listing
November 2016

Effects of 22 Novel CYP2D6 Variants Found in the Chinese Population on the Bufuralol and Dextromethorphan Metabolisms In Vitro.

Basic Clin Pharmacol Toxicol 2016 Mar 26;118(3):190-9. Epub 2015 Sep 26.

The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing, China.

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that metabolizes a large number of therapeutic drugs. To date, more than 100 CYP2D6 allelic variants have been reported. Among these variants, we recently identified 22 novel variants in the Chinese population. The aim of this study was to functionally characterize the enzymatic activity of these variants in vitro. A baculovirus-mediated expression system was used to express wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) at high levels. Then, the insect microsomes containing expressed CYP2D6 proteins were incubated with bufuralol or dextromethorphan at 37°C for 20 or 25 min., respectively. After termination, the metabolites were extracted and used for the detection with high-performance liquid chromatography. Among the 24 CYP2D6 variants tested, two variants (CYP2D6.92 and CYP2D6.96) were found to be catalytically inactive. The remaining 22 variants exhibited significantly decreased intrinsic clearance values for bufuralol 1'-hydroxylation and 20 variants showed significantly lower intrinsic clearance values for dextromethorphan O-demethylation than those of the wild-type CYP2D6.1. Our in vitro results suggest that most of the variants exhibit significantly reduced catalytic activities compared with the wild-type, and these data provide valuable information for personalized medicine in Chinese and other Asian populations.
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http://dx.doi.org/10.1111/bcpt.12478DOI Listing
March 2016

Identification and characterization of a novel CYP2C9 allelic variant in a warfarin-sensitive patient.

Pharmacogenomics 2015 10;16(13):1475-86. Epub 2015 Aug 10.

The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing 100730, China.

Aim: To determine the genetic basis of the low warfarin dose requirement in a Chinese patient.

Materials & Methods: Bi-directional sequencing of CYP2C9, VKORC1 and CYP4F2 genes was performed. CYP2C9 variants were highly expressed in yeast and insect-cell microsomes. Three typical CYP2C9 probe drugs were used to evaluate the catalytic activity.

Results: A novel missense mutation (1400 T>C) was identified in CYP2C9 and had been named as new allele *60. When expressed in yeast and insect cells, compared with wild-type enzyme, variant CYP2C9.60 exhibited lower protein expression capacity and showed significantly decreased metabolic activities for the hydroxylation of S-warfarin, tolbutamide and diclofenac.

Conclusion: The novel mutation can greatly decrease the enzymatic activity of the CYP2C9 enzyme both in vitro and in vivo.
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http://dx.doi.org/10.2217/pgs.15.89DOI Listing
June 2016

In vitro functional analysis of 24 novel CYP2C19 variants recently found in the Chinese Han population.

Xenobiotica 2015 7;45(11):1030-5. Epub 2015 Jul 7.

a The Key Laboratory of Geriatrics , Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health , Beijing , China .

1. CYP2C19 is a highly polymorphic enzyme responsible for the metabolism of a wide range of clinical drugs. Alterations to the CYP2C19 gene contribute to the variability of CYP2C19 enzyme activity, which causes pharmacokinetics and drug efficacies to vary and adverse drug reactions to occur in different persons. Recently, we identified 24 novel CYP2C19 allelic variants in the Chinese Han population. The purpose of present study is to assess the impact of these newly found nucleotide mutations on the enzymatic activity of the CYP2C19 protein. 2. Dual-expression vectors were constructed and transiently transfected into 293FT cells. Forty-eight hours after transfection, cells were re-suspended and incubated with two typical probe substrates, omeprazole and S-mephenytoin, to determine the activities of each variant relative to the wild-type protein. 3. Immunoblotting results showed that the protein expression levels of the CYP2C19 variants were diverse. Enzymatic ability analysis showed that the variant 35FS exhibited no functional activity, and most of the other variants showed significantly decreased metabolic activities toward both omeprazole and S-mephenytoin compared with wild-type. 4. These findings greatly enrich the knowledge of biological effects of these newly found CYP2C19 mutations and aid the application of this knowledge to future individualized drug therapy in clinic.
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http://dx.doi.org/10.3109/00498254.2015.1028512DOI Listing
June 2016

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C9*59.

Drug Metab Dispos 2015 Aug 20;43(8):1246-9. Epub 2015 May 20.

The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing, China (D.-P.D., J. C., J.-P.C.); The Clinical Laboratory of Beijing Hospital, Ministry of Health, Beijing, China (C.-B.L.); Department of Pharmacology, School of Pharmacy, Wenzhou Medical University, Wenzhou, Zhejiang, China (J.C., H.W., G.-X.H.); and The Laboratory of Clinical Pharmacy, The People's Hospital of Lishui, Lishui, Zhejiang, China (S.-H.W., P.-W.G.)

CYP2C9, one of the most important drug-metabolizing enzymes, is responsible for metabolizing approximately 15% of clinically important drugs, including warfarin, diclofenac, and losartan. Similar to other CYP members, human CYP2C9 exhibits marked genetic polymorphisms among individuals of different ethnicities. In this study, a novel missense mutation (1300A>T) was identified in a warfarin-sensitive patient after a genetic screen of three candidate genes related to high variability in response to warfarin doses. This base transversion leads to an Ile-to-Phe amino acid substitution at codon 434 within the CYP2C9 protein, and this new variant has been named a novel allele, CYP2C9*59, by the Human CYP Allele Nomenclature Committee (http://www.cypalleles.ki.se/cyp2c9.htm). The exogenous expression of CYP2C9.59 in insect cell microsomes revealed that, despite a similar protein expression level as wild-type CYP2C9, variant CYP2C9.59 exhibited significantly reduced maximal velocity, Vmax, and/or increased Michaelis constant, Km, values toward three CYP2C9-specific substrates. Our data suggest that the 1300A>T mutation can greatly decrease the enzymatic activity of the CYP2C9 protein both in vitro and in vivo.
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http://dx.doi.org/10.1124/dmd.115.063412DOI Listing
August 2015

Analysis of the oxidative damage of DNA, RNA, and their metabolites induced by hyperglycemia and related nephropathy in Sprague Dawley rats.

Free Radic Res 2015 Oct;49(10):1199-209

a Graduate School, Chinese Academy of Medical Sciences and Peking Union Medical College , Dongdan, Beijing , China.

We used a sensitive and accurate method based on isotope dilution high-performance liquid chromatography-triple quadrupole mass spectrometry (ID-LC-MS/MS) to determine the levels of 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosin (8-oxo-Gsn) in various tissue specimens, plasma, and urine of hyperglycemic Sprague Dawley rats induced by streptozotocin (STZ). The oxidative DNA and RNA damages were observed in various organs and the amounts of 8-oxo-dGsn and 8-oxo-Gsn derived from DNA and RNA were increased with hyperglycemic status. In contrast to the results of the nucleic acid samples derived from tissues, the levels of 8-oxo-Gsn in urine and plasma were significantly higher compared with that of 8-oxo-dGsn, which most likely reflected the RNA damage that occurs more frequently compared with DNA damage. For the oxidative stress induced by hyperglycemia, 8-oxo-Gsn in urine may be a sensitive biomarker on the basis of the results in urine, plasma, and tissues. In addition, high levels of urinary 8-oxo-Gsn were observed before diabetic microvascular complications. Based on that the 8-oxo-dGsn was associated with diabetic nephropathy and RNA was more vulnerable to oxidative stress compared with DNA. We also propose that 8-oxo-Gsn is correlated with diabetic nephropathy and that 8-oxo-Gsn in urine could be a useful and sensitive marker of diabetic nephropathy.
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http://dx.doi.org/10.3109/10715762.2015.1033416DOI Listing
October 2015
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