Hinokiflavone induces apoptosis via activating mitochondrial ROS/JNK/caspase pathway and inhibiting NF-κB activity in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common malignancy with limited treatment options. Hinokiflavone (HF), a natural biflavonoid, has shown to inhibit the proliferation of melanoma, whereas its antitumour effect against HCC and the underlying mechanisms remain elusive. Here, we aimed at evaluating its antitumour effect against HCC in both in vitro and in vivo. Cell counting kit 8, colony formation assay, PI/RNase staining and Western blotting revealed that HF inhibited the proliferation of HCC cells via G0/G1 cell cycle arrest with p21/p53 up-regulation. DAPI staining, Annexin V-FITC/PI staining and Western blotting confirmed that HF triggered caspase-dependent apoptosis. Moreover, HF increased the levels of mitochondrial reactive oxygen species (mtROS) and activated c-Jun N-terminal kinase (JNK) pathway, as measured by MitoSOX Red staining and Western blotting. After respectively inhibiting mtROS (Mito-TEMPO) and JNK (SP600125), HF-induced apoptosis was reversed. Additionally, Western blotting documented that HF suppressed nuclear factor kappa B (NF-κB) activity and the anti-apoptotic genes downstream, contributing to cell apoptosis. Finally, in vivo studies demonstrated that HF significantly impaired tumour growth in HCC xenograft. Collectively, these findings suggested that HF induced apoptosis through activating mtROS/JNK/caspase pathway and inhibiting NF-κB signalling, which may represent a novel therapeutic agent for treating HCC.

Enhanced glycometabolism as a mechanism of NQO1 potentiated growth of NSCLC revealed by metabolomic profiling.

NAD(P)H:quinone oxidoreductase 1 (NQO1), a cytoplasmic 2-electron reductase, has been considered as a potential poor prognostic biomarker and a promising therapeutic target for patients with non-small cell lung cancer (NSCLC) due to its frequent overexpression and significantly increased activity in NSCLC. Previous studies have shown that depleting tumor-NQO1 potentiates anoikis and inhibits growth of NSCLC. However, the underlying mechanisms whereby NQO1 potentiates proliferation have not been fully understood. In this study, based on a metabolomics analysis, we found that cell proliferation inhibition upon NQO1 depletion was accompanied by suppressed glycometabolism in NQO1 high expression human NSCLC A549 cells. Also we found that NQO1 depletion significantly decreased the gene expression levels of hexokinase II (HKII), a key mediator of aerobic glycolysis responsible for the transformation of glucose into glucose-6-phosphate. Taken together, we proposed that NQO1 could potentiate NSCLC cell proliferation by enhancing cellular glycometabolism, and HKII is a key mediator of this mechanism.

Glycyrrhizin Protects against Acetaminophen-Induced Acute Liver Injury via Alleviating Tumor Necrosis Factor α-Mediated Apoptosis.

Acetaminophen (APAP) overdose is the leading cause of drug-induced acute liver failure in Western countries. Glycyrrhizin (GL), a potent hepatoprotective constituent extracted from the traditional Chinese medicine liquorice, has potential clinical use in treating APAP-induced liver failure. The present study determined the hepatoprotective effects and underlying mechanisms of action of GL and its active metabolite glycyrrhetinic acid (GA). Various administration routes and pharmacokinetics-pharmacodynamics analyses were used to differentiate the effects of GL and GA on APAP toxicity in mice. Mice deficient in cytochrome P450 2E1 enzyme (CYP2E1) or receptor interacting protein 3 (RIPK3) and their relative wild-type littermates were subjected to histologic and biochemical analyses to determine the potential mechanisms. Hepatocyte death mediated by tumor necrosis factorα(TNFα)/caspase was analyzed by use of human liver-derived LO2 cells. The pharmacokinetics-pharmacodynamics analysis using various administration routes revealed that GL but not GA potently attenuated APAP-induced liver injury. The protective effect of GL was found only with intraperitoneal and intravenous administration and not with gastric administration. CYP2E1-mediated metabolic activation and RIPK3-mediated necroptosis were unrelated to GL's protective effect. However, GL inhibited hepatocyte apoptosis via interference with TNFα-induced apoptotic hepatocyte death. These results demonstrate that GL rapidly attenuates APAP-induced liver injury by directly inhibiting TNFα-induced hepatocyte apoptosis. The protective effect against APAP-induced liver toxicity by GL in mice suggests the therapeutic potential of GL for the treatment of APAP overdose.

Ginsenosides Regulate PXR/NF-κB Signaling and Attenuate Dextran Sulfate Sodium-Induced Colitis.

Pregnane X receptor (PXR) activation exhibits anti-inflammatory effects via repressing nuclear factor-κB (NF-κB); however, its overactivation may disrupt homeostasis of various enzymes and transporters. Here we found that ginsenosides restore PXR/NF-κB signaling in inflamed conditions without disrupting PXR function in normal conditions. The effects and mechanisms of ginsenosides in regulating PXR/NF-κB signals were determined both in vitro and in vivo. Ginsenosides significantly inhibited NF-κB activation and restored the expression of PXR target genes in tumor necrosis factor-α-stimulated LS174T cells. Despite not being PXR agonists, ginsenosides repressed NF-κB activation in a PXR-dependent manner. Ginsenosides significantly increased the physical association between PXR and the NF-κB p65 subunit and thereby decreased the nuclear translocation of p65. Ginsenoside Rb1 and compound K (CK) were major bioactive compounds in the regulating PXR/NF-κB signaling. Consistently, ginsenosides significantly attenuated dextran sulfate sodium-induced experimental colitis, which was associated with restored PXR/NF-κB signaling. This study indicates that ginsenosides may elicit anti-inflammatory effects via targeting PXR/NF-κB interaction without disrupting PXR function in healthy conditions. Ginsenoside Rb1 and CK may serve as leading compounds in the discovery of new drugs that target PXR/NF-κB interaction in therapy for inflammatory bowel disease.

Mechanism-based inhibitory and peroxisome proliferator-activated receptor α-dependent modulating effects of silybin on principal hepatic drug-metabolizing enzymes.

Silybin, a major pharmacologically active compound in silymarin, has been widely used in combination with other prescriptions in the clinic to treat hepatitis and a host of other diseases. Previous studies suggested that silybin is a potential inhibitor of multiple drug-metabolizing enzymes (DMEs); however, the in vitro to in vivo translation and the mechanisms involved remain established. The aim of this study was to provide a mechanistic understanding of the regulatory effects of silybin on principal DMEs. Silybin (50 or 150 mg/kg/d) was administered to mice for a consecutive 14 days. The plasma and hepatic exposure of silybin were detected; the mRNA, protein levels, and enzyme activities of principal DMEs were determined. The results demonstrated that the enzyme activities of CYP1A2, CYP2C, CYP3A11, and UGT1A1 were significantly repressed, whereas little alteration of the mRNA and protein levels was observed. Silybin inhibits these DMEs in a mechanism-based and/or substrate-competitive manner. More importantly, silybin was found to be a weak agonist of peroxisome proliferator-activated receptor (PPAR)α, as evidenced from the molecular docking, reporter gene assay, and the targeting gene expression analysis. However, silybin could significantly compromise the activation of PPARα by fenofibrate, characterized with significantly repressed expression of PPARα targeting genes, including L-FABP, ACOX1, and UGT1A6. This study suggests that silybin, despite its low bioavailability, may inhibit enzyme activities of multiple DMEs in a mechanism-based mode, and more importantly, may confer significant drug-drug interaction with PPARα agonists via the repression of PPARα activation in a competitive mode.

Glucuronidation of edaravone by human liver and kidney microsomes: biphasic kinetics and identification of UGT1A9 as the major UDP-glucuronosyltransferase isoform.

Edaravone was launched in Japan in 2001 and was the first neuroprotectant developed for the treatment of acute cerebral infarction. Edaravone is mainly eliminated as glucuronide conjugate in human urine (approximately 70%), but the mechanism involved in the elimination pathway remains unidentified. We investigated the glucuronidation of edaravone in human liver microsomes (HLM) and human kidney microsomes (HKM) and identified the major hepatic and renal UDP-glucuronosyltransferases (UGTs) involved. As we observed, edaravone glucuronidation in HLM and HKM exhibited biphasic kinetics. The intrinsic clearance of glucuronidation at high-affinity phase (CL(int1)) and low-affinity phase (CL(int2)) were 8.4 ± 3.3 and 1.3 ± 0.2 µl · min(-1) · mg(-1), respectively, for HLM and were 45.3 ± 8.2 and 1.8 ± 0.1 µl · min(-1) · mg(-1), respectively, for HKM. However, in microsomal incubations contained with 2% bovine serum albumin, CL(int1) and CL(int2) were 16.4 ± 1.2 and 3.7 ± 0.3 µl · min(-1) · mg(-1), respectively, for HLM and were 78.5 ± 3.9 and 3.6 ± 0.5 µl · min(-1) · mg(-1), respectively, for HKM. Screening with 12 recombinant UGTs indicated that eight UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B17) produced a significant amount of glucuronide metabolite. Thus, six UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A9, UGT2B7, and UGT2B17) expressed in human liver or kidney were selected for kinetic studies. Among them, UGT1A9 exhibited the highest activity (CL(int1) = 42.4 ± 9.5 µl · min(-1) · mg(-1)), followed by UGT2B17 (CL(int) = 3.3 ± 0.4 µl · min(-1) · mg(-1)) and UGT1A7 (CL(int) = 1.7 ± 0.2 µl · min(-1) · mg(-1)). Inhibition study found that inhibitor of UGT1A9 (propofol) attenuated edaravone glucuronidation in HLM and HKM. In addition, edaravone glucuronidation in a panel of seven HLM was significantly correlated (r = 0.9340, p = 0.0021) with propofol glucuronidation. Results indicated that UGT1A9 was the main UGT isoform involved in edaravone glucuronidation in HLM and HKM.

Metabolic profile, enzyme kinetics, and reaction phenotyping of ß-lapachone metabolism in human liver and intestine in vitro.

ß-Lapachone (ß-Lap) is an NAD(P)H: quinone oxidoreductase 1 (NQO1) target antitumor drug candidate in phase II clinical trials. The present study aimed to uncover the metabolic profile, enzyme kinetics, and enzyme isoforms for the metabolism of ß-Lap in human liver and intestine in vitro. NQO1-mediated quinone reduction and subsequent glucuronidation is the predominant metabolic pathway for ß-Lap in humans; a pair of regioisomers (M1 and M2) of reduced ß-Lap glucuronides were the major metabolites found from human S9 incubations. The overall glucuronidation clearance of ß-Lap in human liver S9 was 4754.90 µL/min/mg of protein and was 8.1-fold of that in human intestinal S9. Recombinant UDP-glucuronosyltransferase (UGT) screening, correlation analysis, enzyme kinetics, and chemical inhibition study were performed to determine the UGT isoforms involved in ß-Lap metabolism. UGT1A7, UGT1A8, and UGT1A9 are the predominant isoforms responsible for the formation of M2 while UGT2B7 is the main isoform for M1, suggesting a regioselective glucuronidation of reduced quinone by UGTs. It was of interest to find that ß-Lap underwent nonenzymatic two-electron reduction, providing a novel explanation for the toxicities of ß-Lap to NQO1-negative cells at high concentration and with long-time incubation. In conclusion, this study contributes to a better understanding of not only ß-Lap metabolism but its antitumor property as well.

Therapeutic drug monitoring of amikacin: quantification in plasma by liquid chromatography-tandem mass spectrometry and work experience of clinical pharmacists.

OBJECTIVES: As part of the service provided by clinical pharmacists in our hospital, an assay for plasma amikacin quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been established for clinical use since 2018. This study was undertaken to describe: (1) the establishment of this assay; (2) the application and results of the testing; and (3) the analysis and impact for patients. METHODS: The amikacin quantification assay was validated and the plasma amikacin concentration data were extracted and analysed. The clinical data for related patients were collected from electronic health and medical records. RESULTS: 121 plasma samples from 53 patients were included in this statistical analysis. The use of amikacin was mostly monitored in the intensive care unit and the haematology department, and the monitoring range of amikacin concentrations were about 0.1-57µg/mL. The main indications for amikacin concentration detection were combined medications, impaired renal function, or people over 65 years old, which may increase the incidence of adverse reactions. Amikacin prescribing decisions were diversified due to the combination of assay results and clinical disease progression, and the effective rate of amikacin administration was about 52.8% (28/53). CONCLUSIONS: The assay for plasma amikacin concentration has been successfully established to monitor the clinical use of amikacin, and the assay results served as one of the references for amikacin prescribing decisions.

Potential Nexus of Non-alcoholic Fatty Liver Disease and Type 2 Diabetes Mellitus: Insulin Resistance Between Hepatic and Peripheral Tissues.

The liver is the central metabolic organ and plays a pivotal role in regulating homeostasis of glucose and lipid metabolism. Aberrant liver metabolism promotes insulin resistance, which is reported to be a common characteristic of metabolic diseases such as non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). There is a complex and bidirectional relationship between NAFLD and T2DM. NAFLD patients with hepatic insulin resistance generally share a high risk of impaired fasting glucose associated with early diabetes; most patients with T2DM experience non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and other more severe liver complications such as cirrhosis and hepatocellular carcinoma (HCC). Additionally, hepatic insulin resistance, which is caused by diacylglycerol-mediated activation of protein kinase C epsilon (PKC𝜀), may be the critical pathological link between NAFLD and T2DM. Therefore, this review aims to illuminate current insights regarding the complex and strong association between NAFLD and T2DM and summarize novel and emerging targets for the treatment of hepatic insulin resistance based on established mechanistic knowledge.

The complete mitochondrial genome of Brachythemis contaminata (Odonata: Libellulidae).

In this study, we reported the complete mitochondrial genome of the dragonfly Brachythemis contaminate (Odonata: Libellulidae). The entire circular genome is 15,056 bp in length and represents the smallest in presently known odonatan mitogenomes. The DNA molecule contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding control region of 323 bp. There were a total of 137 bp short intergenic spacers and 89 bp overlaps in the genome. The gene arrangement is similar to other dragonflies. The base composition of the genome is A (40.2%), T (32.8%), C (15.6%) and G (11.4%) with an AT content of 73.0%. Four start codons (ATA, ATT, ATC and ATG) and two stop codons (TAG and TAA/TA) were found in 13 protein-coding genes. The length of 22 tRNA genes ranged from 63 (trnP) to 72 bp (trnK).

The complete mitochondrial genomes of four cockroaches (Insecta: Blattodea) and phylogenetic analyses within cockroaches.

Three complete mitochondrial genomes of Blaberidae (Insecta: Blattodea) (Gromphadorhina portentosa, Panchlora nivea, Blaptica dubia) and one complete mt genome of Blattidae (Insecta: Blattodea) (Shelfordella lateralis) were sequenced to further understand the characteristics of cockroach mitogenomes and reconstruct the phylogenetic relationship of Blattodea. The gene order and orientation of these four cockroach genomes were similar to known cockroach mt genomes, and contained 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and one control region. The mt genomes of Blattodea exhibited a characteristics of a high A+T composition (70.7%-74.3%) and dominant usage of the TAA stop codon. The AT content of the whole mt genome, PCGs and total tRNAs in G. portentosa was the lowest in known cockroaches. The presence of a 71-bp intergenic spacer region between trnQ and trnM was a unique feature in B. dubia, but absent in other cockroaches, which can be explained by the duplication/random loss model. Based on the nucleotide and amino acid datasets of the 13 PCGs genes, neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and bayesian inference (BI) analyses were used to rebuild the phylogenetic relationship of cockroaches. All phylogenetic analyses consistently placed Isoptera as the sister cluster to Cryptocercidae of Blattodea. Ectobiidae and Blaberidae (Blaberoidea) formed a sister clade to Blattidae. Corydiidae is a sister clade of all the remaining cockroach species with a high value in NJ and MP analyses of nucleotide and amino acid datasets, and ML and BI analyses of the amino acid dataset.

NAMPT inhibition synergizes with NQO1-targeting agents in inducing apoptotic cell death in non-small cell lung cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD(+), essential for a number of enzymes and regulatory proteins involved in a variety of cellular processes, including deacetylation enzyme SIRT1 which modulates several tumor suppressors such as p53 and FOXO. Herein we report that NQO1 substrates Tanshione IIA (TSA) and ß-lapachone (ß-lap) induced a rapid depletion of NAD(+) pool but adaptively a significant upregulation of NAMPT. NAMPT inhibition by FK866 at a nontoxic dose significantly enhanced NQO1-targeting agent-induced apoptotic cell death. Compared with TSA or ß-lap treatment alone, co-treatment with FK866 induced a more dramatic depletion of NAD(+), repression of SIRT1 activity, and thereby the increased accumulation of acetylated FOXO1 and the activation of apoptotic pathway. In conclusion, the results from the present study support that NAMPT inhibition can synergize with NQO1 activation to induce apoptotic cell death, thereby providing a new rationale for the development of combinative therapeutic drugs in combating non-small lung cancer.

Farnesoid X receptor activation promotes cell proliferation via PDK4-controlled metabolic reprogramming.

Farnesoid X receptor (FXR) plays a pivotal role in the regulation of various metabolic pathways as well as liver regeneration. However, the casual link between cell proliferative effects during liver regeneration and metabolic regulation of FXR was elusive. In this study, we found that FXR activation significantly promotes HepG2 cell proliferation accompanied with metabolic switch towards the excessive accumulation of aerobic glycolytic intermediates including lactic acid, pyruvate and the subsequently increased biosynthesis of glycine. This FXR-induced metabolic switch was found dependent on an up-regulation of pyruvate dehydrogenate kinase 4 (PDK4), a FXR target gene. FXR agonists were found to promote liver regeneration in the murine model of APAP induced liver injury, which was associated with a metabolic switch favoring the accumulation of glycolytic intermediates as precursors for generation of biomass. However, FXR activation has little effect on the glycolytic metabolism in healthy primary hepatocytes in vitro and the liver of healthy mice in vivo. Therefore, we conclude that FXR may promote the proliferation of tumor cells and the hepatocytes in the process of liver regeneration by activating the PDK4-mediated metabolic reprogramming to generate glycolytic intermediates essential for rapid biomass generation, establishing a mechanistic link between cell proliferation and metabolic switch.

De-novo NAD+ synthesis regulates SIRT1-FOXO1 apoptotic pathway in response to NQO1 substrates in lung cancer cells.

Tryptophan metabolism is essential in diverse kinds of tumors via regulating tumor immunology. However, the direct role of tryptophan metabolism and its signaling pathway in cancer cells remain largely elusive. Here, we establish a mechanistic link from L-type amino acid transporter 1 (LAT1) mediated transport of tryptophan and the subsequent de-novo NAD+ synthesis to SIRT1-FOXO1 regulated apoptotic signaling in A549 cells in response to NQO1 activation. In response to NQO1 activation, SIRT1 is repressed leading to the increased cellular accumulation of acetylated FOXO1 that transcriptionally activates apoptotic signaling. Decreased uptake of tryptophan due to the downregulation of LAT1 coordinates with PARP-1 hyperactivation to induce rapid depletion of NAD+ pool. Particularly, the LAT1-NAD+-SIRT1 signaling is activated in tumor tissues of patients with non-small cell lung cancer. Because NQO1 activation is characterized with oxidative challenge induced DNA damage, these results suggest that LAT1 and de-novo NAD+ synthesis in NSCLC cells may play essential roles in sensing excessive oxidative stress.

UDP-glucuronosyltransferase 1A determinates intracellular accumulation and anti-cancer effect of ß-lapachone in human colon cancer cells.

ß-lapachone (ß-lap), an NAD(P)H: quinone oxidoreductase 1 (NQO1) targeting antitumor drug candidate in phase II clinical trials, is metabolically eliminated via NQO1 mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation. This study intends to explore the inner link between the cellular glucuronidation and pharmacokinetics of ß-lap and its apoptotic effect in human colon cancer cells. HT29 cells S9 fractions exhibited high glucuronidation activity towards ß-lap, which can be inhibited by UGT1A9 competitive inhibitor propofol. UGT1A siRNA treated HT29 cells S9 fractions displayed an apparent low glucuronidation activity. Intracellular accumulation of ß-lap in HCT116 cells was much higher than that in HT29 cells, correlated with the absence of UGT1A in HCT116 cells. The cytotoxic and apoptotic effect of ß-lap in HT29 cells were much lower than that in HCT116 cells; moreover, ß-lap triggered activation of SIRT1-FOXO1 apoptotic pathway was observed in HCT116 cells but not in HT29 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol significantly decreased ß-lap's cytotoxic and apoptotic effects, due to the repression of glucuronidation and the resultant intracellular accumulation. In conclusion, UGT1A is an important determinant, via switching NQO1-triggered redox cycle to metabolic elimination, in the intracellular accumulation of ß-lap and thereafter its cytotoxicity in human colon cancer cells. Together with our previous works, we propose that UGTs determined cellular pharmacokinetics is an important determinant in the apoptotic effects of NQO1 targeting substrates serving as chemotherapeutic drugs.

Pharmacodynamics and potential synergistic effects of Mai-Luo-Ning injection on cardiovascular protection, based on molecular docking.

As a computer-assisted approach, molecular docking has been universally applied in drug research and development and plays an important role in the investigation and evaluation of herbal medicines. Herein, the method was used to estimate the pharmacodynamics of Mai-Luo-Ning injection, a traditional Chinese compound herbal prescription. Through investigating the interactions between several important proteins in cardiovascular system and characteristic components of the formula, its effect on cardiovascular protection was evaluated. Results showed the differences in the interactions between each component and the selected target proteins and revealed the possible mechanisms for synergistic effects of various characteristic components on cardiovascular protection. The study provided scientific evidence supporting the mechanistic study of the interactions among multi-components and targets, offering a general approach to investigating the pharmacodynamics of complicated materials in compound herbal prescriptions.

PPARα-UGT axis activation represses intestinal FXR-FGF15 feedback signalling and exacerbates experimental colitis.

Bile acids play a pivotal role in the pathological development of inflammatory bowel disease (IBD). However, the mechanism of bile acid dysregulation in IBD remains unanswered. Here we show that intestinal peroxisome proliferator-activated receptor α (PPARα)-UDP-glucuronosyltransferases (UGTs) signalling is an important determinant of bile acid homeostasis. Dextran sulphate sodium (DSS)-induced colitis leads to accumulation of bile acids in inflamed colon tissues via activation of the intestinal peroxisome PPARα-UGTs pathway. UGTs accelerate the metabolic elimination of bile acids, and thereby decrease their intracellular levels in the small intestine. Reduced intracellular bile acids results in repressed farnesoid X receptor (FXR)-FGF15 signalling, leading to upregulation of hepatic CYP7A1, thus promoting the de novo bile acid synthesis. Both knockout of PPARα and treatment with recombinant FGF19 markedly attenuate DSS-induced colitis. Thus, we propose that intestinal PPARα-UGTs and downstream FXR-FGF15 signalling play vital roles in control of bile acid homeostasis and the pathological development of colitis.

Disturbance of hepatic and intestinal UDP-glucuronosyltransferase in rats with trinitrobenzene sulfonic acid-induced colitis.

UDP-glucuronosyltransferase (UGT) is an important class of phase II metabolizing enzymes, playing a pivotal role in detoxifying various substances and in the pathological procedures of some diseases. The present study aims to uncover the potential dysregulation pattern of UGTs in trinitrobenzene sulfonic acid (TNBS)-induced colitis. Colitis was induced by intra-rectally administering a single dose of TNBS (100 mg/kg). The expression and enzyme activity of hepatic UGTs of colitis rats were all down-regulated significantly except UGT1A7, for which the mRNA level was up-regulated. In contrast, UGT isoforms in the small intestine were relatively unaffected. In the colon, where the inflammation occurs, the mRNA level and enzyme activity of UGT1A1 and 1A6 were down-regulated, but those of UGT1A7 and 2B1 up-regulated. The mRNA levels of various transcription factors, including AhR, CAR, PXR, PPARγ, and FXR were all decreased, except for AhR and CAR in the small intestine and colon. Our data suggests that colitis induces an isoform-dependent and tissue-specific dysregulation of UGTs and their related transcription factors.

Dysregulations of intestinal and colonic UDP-glucuronosyltransferases in rats with type 2 diabetes.

Diabetes mellitus is a chronic disease of complex metabolic disorder associated with various types of complications. UDP-glucuronosyltransferases (UGTs), the major phase II conjugation enzymes, mediate the metabolism of both drugs and endogenous metabolites that may raise great concerns in the condition of diabetes. The aim of this study was to determine whether diabetes could affect UGTs in the intestinal and colonic tract. A high-fat diet combined with low-dose streptozotocin was used to induce a type 2 diabetic model in rats. The mRNA levels and enzymatic activities of UGT1A1, -1A6, and -1A7 in the diabetic intestine and colon were higher than those in nondiabetic rats. In contrast, both the activity and mRNA level of UGT2B1 in diabetic rats were lower than those in nondiabetic rats. Notably, the diabetic intestine and colon exhibited an inflammatory state with increased pro-inflammatory cytokines. Various transcriptional factors involved in UGT regulation were unanimously upregulated in the diabetic intestine and colon. These findings strongly suggest that the regulating pathways of the UGT1 family are adaptively upregulated in the diabetic gastrointestinal tract. Given the essential regulatory role of the gastrointestinal site in drug disposition, such changes in UGTs may have a dynamic and complex impact on therapeutic drugs and endogenous metabolomes.

Absolute quantification of NAD(P)H:quinone oxidoreductase 1 in human tumor cell lines and tissues by liquid chromatography-mass spectrometry/mass spectrometry using both isotopic and non-isotopic internal standards.

NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase) is a prognostic biomarker and a potential therapeutic target for various tumors. Therefore, it is of significance to develop a robust method for the absolute quantification of NQO1. This study aimed to develop and validate a LC-MS/MS based method and to test the appropriateness of using non-isotopic analog peptide as the internal standard (IS) by comparing with a stable isotope labeled (SIL) peptide. The chromatographic performance and mass spectra between the selected signature peptide of NQO1 and the non-isotopic peptide were observed to be very similar. The use of the two internal standards was validated appropriate for the absolute quantification of NQO1, as evidenced by satisfactory validation results over a concentration range of 1.62-162 fmol µL(-1). This method has been successfully applied to the absolute quantification of NQO1 expression in various tumor cell lines and tissues. NQO1 expression in human tumor tissues is much higher than that in the neighboring normal tissues in both the cases of lung and colon cancer. The quantitative results obtained from the isotopic and non-isotopic methods are quite similar, further supporting that the use of non-isotopic analog peptide as internal standard is appropriate and feasible for the quantification of NQO1. By comparing with a classical isotopic IS, the present study indicates that the use of a non-isotopic peptide analog to the proteotypic peptide as the internal standard can get equal accuracy and preciseness in measuring NQO1. The universal applicability of the non-isotopic IS approach for the quantification of proteins warrants further research.