Metabolic Activation of Chrysene by Human Hepatic and Pulmonary Cytochrome P450 Enzymes

The metabolic activation of chrysene, a weak carcinogen found in tobacco smoke, gasoline engine exhaust, and other environmental sources was analyzed in 18 human hepatic and 11 pulmonary microsomal samples. The major metabolites formed were 3,4-dihydroxy-3,4-dihydrochrysene (chrysene-3,4-diol), chrysene-1,2-diol and to lower extents phenols. Chrysene-5,6-diol was found in trace amounts only. All human liver samples formed the proximate carcinogen chrysene-1,2-diol (1.3–5.8 pmol/mg protein/min). Comparable results were seen with pulmonary microsomes, but metabolites were formed to about 10 fold lower extents. Here the most prominent metabolite was chrysene-1,2-diol. Catalytic activities known to be associated with specific cytochrome P450s were determined and correlated with the levels of metabolites formed in each sample. The results of the correlation analysis as well as studies with the inhibitor α-naphthoflavone indicated that hepatic cytochrome P450 1A2 plays a major role in the formation of the proximate carcinogen chrysene-1,2-diol and most of the other metabolites. The formation of metabolites in human lung seems mainly to be due to cytochrome P450 1A1 activity. These results demonstrate that cytochrome-P450 1A mediated metabolic activation processes occur for chrysene in human liver and lung.     

Substrate Enantioselective and Product Stereoselective Metabolism of Racemic 2-Hydroxy-3-methylcholanthrene by Rat Liver Microsomes

Racemic 2-hydroxy-3-methylcholanthrene (rac-2-OH-3MC) is a potent carcinogen and 2S-OH-3MC is the most abundant metabolite formed in the metabolism of 3-methylcholanthrene (3MC) by rat liver microsomes. Eighteen identifiable metabolites were formed in the metabolism of rac-2-OH-3MC by liver microsomes prepared from phenobarbital-treated rats. Metabolites were separated by sequential use of reversed-phase and normal-phase high performance liquid chromatography (HPLC) and characterized by ultraviolet-visible absorption, mass, and circular dichroism spectral, and chiral stationary phase (CSP) HPLC analyses. Identified metabolites were: 2-OH-3MC 7,8-dihydrodiol (enriched in 7R,8R enantiomer), 2-OH-3MC 9,10-dihydrodiol (2S,9R,10R:2R,9S,10S ≈ 27:73 and 2R,9R,10R:2S,9S,10S ≈ 81:19), 2-OH-3MC 11,12-dihydrodiol (2R,11R,12R:2S,11S,12S ≈ 5:95 and an essentially optically pure 2S,11R,12R enantiomer), 3MC trans-1,2-diol (1S,2S:1R,2R ≈ 68:32), 3MC cis-1,2-diol (1S,2R:1R,2S ≈ 18:82), 2-OH-3-hydroxymethyl-cholanthrene (2-OH-3-OHMC, 2R:2S ≈ 19:81), 2-OH-3-OHMC 9,10-dihydrodiol (9R,10R:9S,10S ≈ 62:38), 3MC-2-one 9,10-dihydrodiol (9R,10R:9S,10S ≈ 7:3), 3MC cis-1,2-diol:trans-9,10-dihydrodiol, 8-hydroxy-2-OH-3-OHMC, 9-hydroxy-2-OH-3MC, 10-hydroxy-2-OH-3MC, and 3MC-2-one. The enantiomer ratios of some metabolites formed were determined by circular dichroism spectra and/or CSP-HPLC. Six 9,10-dihydrodiols formed in the metabolism of 2-OH-3MC may be further converted to potentially reactive 9,10-diol-7,8-epoxides and may contribute to the overall carcinogenicity exhibited by 3MC.     

Identification of Human UDP-Glucuronosyltransferase 1A4 as the Major Isozyme Responsible for the Glucuronidation of 20(S)-Protopanaxadiol in Human Liver Microsomes

20(S)-protopanaxadiol (PPD), one of the representative aglycones of ginsenosides, has a broad spectrum of pharmacological activities. Although phase I metabolism has been investigated extensively, information regarding phase II metabolism of this compound remains to be elucidated. Here, a glucuronidated metabolite of PPD in human liver microsomes (HLMs) and rat liver microsomes (RLMs) was unambiguously identified as PPD-3-O-β-d-glucuronide by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry. The chemical inhibition and recombinant human UDP-Glucuronosyltransferase (UGT) isoforms assay showed that the PPD glucuronidation was mainly catalyzed by UGT1A4 in HLM, whereas UGT1A3 showed weak catalytic activity. In conclusion, PPD-3-O-β-d-glucuronide was first identified as the principal glucuronidation metabolite of PPD in HLMs, which was catalyzed by UGT1A4.     

Lipidomic Profile and Enzymes Activity in Hepatic Microsomes of Rats in Physiological and Pathological Conditions

Among the risk factors affecting the development of cancer, nutritional factors occupy a significant place. Pomegranate seed oil (PSO) and bitter melon extract (BME), used for ages in folk medicine, are nowadays used in the prevention of many diseases and as ingredients of dietary supplements. Despite numerous publications on these raw materials or their active substances, their mechanism of action in various pathological states has not been recognized yet, nor has the safety of their simultaneous use been evaluated. The study aimed to assess how dietary supplementation with either PSO, with BME, or both, affects fatty acids’ profiles and their metabolism in hepatic microsomes, as well as the activity of selected microsomal enzymes (COX-2 and CYP1B1). Experimental animals (Sprague-Dawley rats) were divided into eight parallel experimental groups, differing in applied dietary modifications (control, PSO, BME and both PSO and BME) and introduction of chemical carcinogen—7,12-dimethylbenz[a]nthracene. Obtained results indicated the pronounced effect of the cancerous process on lipid metabolism and demonstrated the antagonistic effect of applied dietary supplements on the content of individual fatty acids and the activity of CYP1B1 and COX-2. The applied broad analytical approach and chemometric data analysis confirmed that raw materials, for which potential cancer prevention has been previously demonstrated, may differ in effects depending on the coexisting pathological state.     

Differential Effects of Estrogen and Progestin on Apolipoprotein B100 and B48 Kinetics in Postmenopausal Women

The distinct effects of the estrogen and progestin components of hormonal therapy on the metabolism of apolipoprotein (apo) B‐containing lipoproteins have not been studied. We enrolled eight healthy postmenopausal women in a placebo‐controlled, randomized, double‐blind crossover study. Each subject received placebo, conjugated equine estrogen (CEE, 0.625 mg/day) and CEE plus medroxyprogesterone acetate (MPA, 2.5 mg/day) for 8 weeks in a randomized order, with a 4‐week washout between phases. Main outcomes were the fractional catabolic rate (FCR) and production rate (PR) of apo B100 in triglyceride‐rich lipoproteins (TRL), intermediate‐density lipoproteins (IDL) and low ‐density lipoprotein (LDL) and of apo B48 in TRL. Compared to placebo, CEE increased TRL apo B100 PR (p = 0.04). CEE also increased LDL apo B100 FCR (p = 0.02), but this effect was offset by a significant increase in LDL apo B100 PR (p = 0.04). Adding MPA to CEE negated the CEE effects resulting in no significant changes in TRL apo B100 PR and LDL apo B100 FCR and PR relative to placebo. Relative to placebo, during CEE there was a trend toward a reduction in plasma apo B48 concentrations and PR (p = 0.07 and p = 0.12, respectively). Compared with CEE, CEE + MPA significantly increased TRL apo B48 FCR (p = 0.02) as well as apo B48 PR (p = 0.01), resulting in no significant changes in apo B48 concentration. Estrogen and progestin have independent and opposing effects on the metabolism of the atherogenic apo B100‐ and apo B48‐containing lipoproteins.     

Biotransformation and DNA Adduct Formation of Trans-8,9-Dihydroxy-8,9-Dihydrodibenzo[a,l]Pyrene by Induced Rat Liver and Human CYP1A1 Microsomes

In order to explain the adduct patterns observed from the human CYP1A1-mediated binding of dibenzo[a, l]pyrene (DB[a, l]P) to DNA, we have investigated the further metabolism and DNA adduct activity of trans-DB[a, l]P-8,9-diol by induced rat liver and human CYP1A1 microsomes. trans-DB[a, l]P-8,9-diol was synthesized and metabolic studies with β-naphthoflavone-induced rat liver microsomes indicated three major metabolites: 2 diastereomers of trans,trans-8,9,11,12-tetrahydro-8,9,11,12-tetrahydroxy-DB[a, l]P and 8,9,13,14-tetrahydro-8,9,13,14-tetrahydroxy-DB[a, l]P. DB[a, l]P when activated by CYP1A1/epoxide hydrase (EH) and calf thymus DNA gave a complex pattern of DNA adducts most of which cochromatograph with syn- and anti-DB[a, l]P fjord region diol epoxide-DNA standards. Two highly polar eluting adducts were also observed, one which cochromatographs with the single major DNA adduct obtained from the CYP1A1/EH activation of trans-DB[a, l]P-8,9-diol. The relative retention time of this adduct suggests either a bis-diol epoxide adduct or a more polar diol epoxide adduct.     

Liver Bile Acid Changes in Mouse Models of Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive cognitive impairment. It is hypothesized to develop due to the dysfunction of two major proteins, amyloid-β (Aβ) and microtubule-associated protein, tau. Evidence supports the involvement of cholesterol changes in both the generation and deposition of Aβ. This study was performed to better understand the role of liver cholesterol and bile acid metabolism in the pathophysiology of AD. We used male and female wild-type control (C57BL/6J) mice to compare to two well-characterized amyloidosis models of AD, APP/PS1, and App^NL-G-F. Both conjugated and unconjugated primary and secondary bile acids were quantified using UPLC-MS/MS from livers of control and AD mice. We also measured cholesterol and its metabolites and identified changes in levels of proteins associated with bile acid synthesis and signaling. We observed sex differences in liver cholesterol levels accompanied by differences in levels of synthesis intermediates and conjugated and unconjugated liver primary bile acids in both APP/PS1 and App^NL-G-F mice when compared to controls. Our data revealed fundamental deficiencies in cholesterol metabolism and bile acid synthesis in the livers of two different AD mouse lines. These findings strengthen the involvement of liver metabolism in the pathophysiology of AD.     

Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis,Characterization and Comparison

Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.     

Metabolic Instability of Cyanothiazolidine‐Based Prolyl Oligopeptidase Inhibitors: a Structural Assignment Challenge and Potential Medicinal Chemistry Implications

As part of the development of cyanothiazolidine‐based prolyl oligopeptidase inhibitors, initial metabolism studies suggested multiple sites of oxidation by P450 enzymes. Surprisingly, in‐depth investigations revealed that epimerization at multiple stereogenic centers was responsible for the conversion of the single primary metabolite into a panel of secondary metabolites. The rapid isomerization of all seven detected molecules precluded the use of NMR spectroscopy or X‐ray crystallography for complete structural determination, presenting an interesting structure elucidation challenge. Through a combination of LC–MS analysis, synthetic work, deuterium exchange studies, and computational predictions, we were able to characterize all metabolites and to elucidate their dynamic behavior in solution. In the context of drug development, this study reveals that cyanothiazolidine moieties are problematic due to their rapid P450‐mediated oxidation and the unpredictable stability of the corresponding metabolites.     

Synthesis of Fe-MCM-48 and its catalytic performance in phenol hydroxylation

Highly ordered Fe-MCM-48 was synthesized by a mixed templation method under low molar ratio (0.17:1) of mixed surfactants to silica and characterized by XRD, ²⁹Si-MAS NMR, ESR, and UV-Visible. Its catalytic activity and selectivity was studied for phenol hydroxylation using H₂O₂ (30%). The substituting element (Fe³⁺) is partially incorporated into the framework position forming a new type of active site which raises the phenol conversion to 43.6% and the diphenol (the mixture of catechol (CAT) and hydroquinone (HQ)) selectivity to 97.7%.     

硅基介孔材料MCM-48的合成、改性及催化领域应用的研究进展

概述了硅基介孔材料MCM-48的发展、合成及应用,介绍了具有三维螺旋孔道结构的MCM-48的主要合成方法及各个方法的优缺点,分析了MCM-48合成过程中原料,反应条件对合成产物的影响.总结了金属改性MCM-48的两种方法,以及改性后在吸附和催化领域的应用,指出了未来MCM-48的研究方向.     

Effects of brown rice and white rice on expression of xenobiotic metabolism genes in type 2 diabetic rats

Xenobiotics constantly influence biological systems through several means of interaction. These interactions are disturbed in type 2 diabetes, with implications for disease outcome. We aimed to study the implications of such disturbances on type 2 diabetes and rice consumption, the results of which could affect management of the disease in developing countries. In a type 2 diabetic rat model induced through a combination of high fat diet and low dose streptozotocin injection, up-regulation of xenobiotic metabolism genes in the diabetic untreated group was observed. Xenobiotic metabolism genes were upregulated more in the white rice (WR) group than the diabetic untreated group while the brown rice (BR) group showed significantly lower expression values, though not as effective as metformin, which gave values closer to the normal non-diabetic group. The fold changes in expression in the WR group compared to the BR group for Cyp2D4, Cyp3A1, Cyp4A1, Cyp2B1, Cyp2E1, Cyp2C11, UGT2B1, ALDH1A1 and Cyp2C6 were 2.6, 2, 1.5, 4, 2.8, 1.5, 1.8, 3 and 5, respectively. Our results suggest that WR may upregulate these genes in type 2 diabetes more than BR, potentially causing faster drug metabolism, less drug efficacy and more toxicity. These results may have profound implications for rice eating populations, constituting half the world's population.     

The Role of LGR4 (GPR48) in Normal and Cancer Processes

Leucine-rich repeats containing G protein-coupled receptor 4 (LGR4) is a receptor that belongs to the superfamily of G protein-coupled receptors that can be activated by R-spondins (RSPOs), Norrin, circLGR4, and the ligand of the receptor activator of nuclear factor kappa-B (RANKL) ligands to regulate signaling pathways in normal and pathological processes. LGR4 is widely expressed in different tissues where it has multiple functions such as tissue development and maintenance. LGR4 mainly acts through the Wnt/β-catenin pathway to regulate proliferation, survival, and differentiation. In cancer, LGR4 participates in tumor progression, invasion, and metastasis. Furthermore, recent evidence reveals that LGR4 is essential for the regulation of the cancer stem cell population by controlling self-renewal and regulating stem cell properties. This review summarizes the function of LGR4 and its ligands in normal and malignant processes.     

Lack of TRPV1 Channel Modulates Mouse Gene Expression and Liver Proteome with Glucose Metabolism Changes

To investigate the role of the transient receptor potential channel vanilloid type 1 (TRPV1) in hepatic glucose metabolism, we analyzed genes related to the clock system and glucose/lipid metabolism and performed glycogen measurements at ZT8 and ZT20 in the liver of C57Bl/6J (WT) and Trpv1 KO mice. To identify molecular clues associated with metabolic changes, we performed proteomics analysis at ZT8. Liver from Trpv1 KO mice exhibited reduced Per1 expression and increased Pparα, Pparγ, Glut2, G6pc1 (G6pase), Pck1 (Pepck), Akt, and Gsk3b expression at ZT8. Liver from Trpv1 KO mice also showed reduced glycogen storage at ZT8 but not at ZT20 and significant proteomics changes consistent with enhanced glycogenolysis, as well as increased gluconeogenesis and inflammatory features. The network propagation approach evidenced that the TRPV1 channel is an intrinsic component of the glucagon signaling pathway, and its loss seems to be associated with increased gluconeogenesis through PKA signaling. In this sense, the differentially identified kinases and phosphatases in WT and Trpv1 KO liver proteomes show that the PP2A phosphatase complex and PKA may be major players in glycogenolysis in Trpv1 KO mice.     

Establishment of Metabolism and Transport Pathways in the Rodent and Human Fetal Liver

The ultimate fate of drugs and chemicals in the body is largely regulated by hepatic uptake, metabolism, and excretion. The liver acquires the functional ability to metabolize and transport chemicals during the perinatal period of development. Research using livers from fetal and juvenile rodents and humans has begun to reveal the timing, key enzymes and transporters, and regulatory factors that are responsible for the establishment of hepatic phase I and II metabolism as well as transport. The majority of this research has been limited to relative mRNA and protein quantification. However, the recent utilization of novel technology, such as RNA-Sequencing, and the improved availability and refinement of functional activity assays, has begun to provide more definitive information regarding the extent of hepatic drug disposition in the developing fetus. The goals of this review are to provide an overview of the early regulation of the major phase I and II enzymes and transporters in rodent and human livers and to highlight potential mechanisms that control the ontogeny of chemical metabolism and excretion pathways.     

除草剂在植物体内的代谢与选择性及使用

简要论述了除草剂在植物体内的代谢反应及其调控。     

Simple Analysis of Primary and Secondary Bile Salt Hydrolysis in Mouse and Human Gut Microbiome Samples by Using Fluorogenic Substrates

Animals produce bile to act as an antibacterial agent and to maximize the absorption of lipophilic nutrients in the gut. The physical properties of bile are largely dictated by amphipathic bile salt molecules, which also participate in signaling pathways by modulating physiological processes upon binding host receptors. Upon excretion of bile salts from the gall bladder into the intestine, the gut microbiota can create metabolites with modified signaling capabilities. The category and magnitude of bile salt metabolism can have positive or negative effects on the host. A key modification is bile salt hydrolysis, which is a prerequisite for all additional microbial transformations. We have synthesized five different fluorogenic bile salts for simple and continuous reporting of hydrolysis in both murine and human fecal samples. Our data demonstrate that most gut microbiomes have the highest capacity for hydrolysis of host-produced primary bile salts, but some microbially modified secondary bile salts also display significant turnover.