Immunochemical and LC-MS/MS Detection of Protein Adduction Resulting from Metabolic Activation of Columbin In Vitro and In Vivo.

Columbin (CLB) is a diterpenoid furanolactone compound occurring in some herbal medicines. Administration of CLB has been reported to induce liver injury. The reported CLB hepatotoxicity is suggested to require metabolism to a cis-enedial intermediate. We successfully detected hepatic protein adduction resulting from the metabolic activation of CLB and found that the intermediate reacted with lysine residues or lysine/cysteine residues to produce the corresponding pyrroline derivative or pyrrole derivative. The detection was achieved by proteolysis- and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods. Furthermore, we prepared a polyclonal antibody approach which allowed us to detect the protein adduction in the forms of protein immunoblot as well as tissue- and cell-based immunostaining. The antibody technique verified the protein adduction detected by LC-MS/MS.

Evaluating and predicting the correlations of hepatic concentration and pyrrole-protein adduction with hepatotoxicity induced by retrorsine based on pharmacokinetic/pharmacodynamic model.

Retrosine (RTS) is a pyrrolozidine alkaloid and a known hepatotoxin that widely exist in nature. The mechanisms involved in toxic action of pyrrolizidine alkaloids need further investigation. The objective of the present study was to evaluate the correlation of RTS hepatotoxicity with hepatic RTS concentration and pyrrole-protein adduction. Mice were intragastrically treated with RTS alone or RTS and ketoconazole (KTZ) simultaneously. Sera and liver tissues were collected at various time points after administration, followed by the determination of changes in serum transaminase activity, hepatic RTS concentration and pyrrole-protein adduction. The correlation of RTS hepatotoxicity with hepatic RTS concentration and hepatic pyrrole-protein adduction were examined by use of Sigmoid-Emax PK/PD models. Dose-dependent hepatotoxicity, hepatic RTS concentration and pyrrole-protein adduction were observed in the animals, which could be modulated by co-treatment with KTZ. The fit parameters indicated pyrrole-protein adduction was more closely related with liver injury than hepatic RTS concentration. Similar correlation was observed in mice given low-dose of RTS for 4 consecutive days. RTS hepatotoxicity is correlated with hepatic pyrrole-protein adduction derived from RTS rather than hepatic RTS concentration. The observed protein modification would be a good indicator to predict the hepatoxicity of RTS at low dose.

The SWGEDWGEIW from Soybean Peptides Reduce Oxidative Damage-Mediated Apoptosis in PC-12 Cells by Activating SIRT3/FOXO3a Signaling Pathway.

The goal of the investigation was to study the protective effects of the SWGEDWGEIW (the single peptide, TSP) from soybean peptides (SBP) on hydrogen peroxide (H2O2)-induced apoptosis together with mitochondrial dysfunction in PC-12 cells and their possible implications to protection mechanism. Meanwhile, the SBP was used as a control experiment. The results suggested that SBP and TSP significantly (p < 0.05) inhibited cellular oxidative damage and ROS-mediated apoptosis. In addition, SBP and TSP also enhanced multiple mitochondrial biological activities, decreased mitochondrial ROS levels, amplified mitochondrial respiration, increased cellular maximal respiration, spare respiration capacity, and ATP production. In addition, SBP and TSP significantly (p < 0.05) raised the SIRT3 protein expression and the downstream functional gene FOXO3a. In the above activity tests, the activity of TSP was slightly higher than that of SBP. Taken together, our findings suggested that SBP and TSP can be used as promising nutrients for oxidative damage reduction in neurons, and TSP is more effective than SBP. Therefore, TSP has the potential to replace SBP and reduce neuronal oxidative damage.

Hepatic RNA adduction derived from metabolic activation of retrorsine in vitro and in vivo.

Pyrrolizidine alkaloids (PAs) are among the most significant hepatotoxins widely distributed in plant species. Incidence of liver injuries caused by PAs has been reported worldwide, and the reactive metabolites of PAs are known to play a critical role in causing the hepatotoxicity. To better understand the toxicity-induction mechanisms, we explored the interactions of PA metabolites with cellular RNA molecules, and examined their effects on the biochemical and metabolic properties of hepatic RNAs. After exposure to retrorsine, adduction on adenosine and guanosine were detected in mouse liver microsomal incubations, cultured mouse primary hepatocytes, and mouse liver tissues. NMR analysis showed that the exocyclic amino group participated in the adduction. We found drastically altered properties and metabolism of the adducted RNA such as reverse-transcriptability, translatability, and RNase-susceptibility. In addition, endogenous modification of N6-methyladenosine (m6A) was remarkably reduced.

Diosbulbin B: An important component responsible for hepatotoxicity and protein covalent binding induced by Dioscorea bulbifera L.

BACKGROUND: Dioscorea bulbifera L. (DBL) is an herbal medicine used for the treatment of thyroid diseases and tumors in China. However, the hepatotoxicity of DBL limits its wide safe use. Diosbulbin B (DSB) is the most abundant diterpene lactone occurring in DBL. Numbers of studies showed that this furanoterpenoid plays an important role in DBL-induced liver injury and that DSB is metabolized to a cis-enedial intermediate which reacts with protein to form protein covalent binding and induces hepatotoxicity. PURPOSE: The present study aimed to define the association of DSB content in DBL with the severity of DBL hepatotoxicity to ensure the safe use of the herbal medicine in clinical practice and to determine the role of DSB in DBL-induced liver injury. METHODS: Chemical chromatographic fingerprints of DBL were established by UPLC-MS/MS. Their hepatotoxicity potencies were evaluated in vitro and in vivo. Metabolic activation of DSB was evaluated by liver microsomal incubation. Protein modification was assessed by mass spectrometry and immunostaining. RESULTS: The contents of DSB in DBL herbs collected from 11 locations in China varied dramatically with as much as 47-fold difference. The hepatotoxicity potencies of DBL herbs were found to be proportional to the contents of DSB. Intensified protein adduction derived from the reactive metabolite of DSB was observed in mice administered DBL with high contents of DSB. CONCLUSION: The findings not only demonstrated that contents of DSB can be quite different depending on harvest location and special attention needs to pay for quality control of DBL but also suggest DSB is a key contributor for DBL-induced hepatotoxicity.

From part to whole, operative link on to endoscopic grading of gastric intestinal metaplasia, pathology to endoscopy: gastric intestinal metaplasia graded by endoscopy.

Objective: To conduct a systematic review and meta-analysis on the prediction of severity of gastric intestinal metaplasia (GIM) in localized and entire gastric mucosa using endoscopy. Methods: The authors searched Web of Science, PubMed, Embase and Cochrane Central Register of Controlled Trials and performed systematic searches on endoscopic grading of GIM of the entire stomach using Meta-DiSc and Stata. Results: Sensitivity and specificity for the stratified prediction of overall GIM were 0.91 (95% CI: 0.85-0.95) and 0.91 (95% CI: 0.88-0.93), respectively. Sensitivity in predicting the different grades of GIM was higher in operative link on GIM assessment grades 0, III and IV but lower in grades I and II. Conclusion: Digital chromoendoscopy is well suited to predicting the severity of localized and overall GIM.

2-Naphthalenemethanol participates in metabolic activation of 2-methylnaphthalene.

2-Methylnaphthalene (2-MN) is an environmental pollutant. Studies have shown that 2-MN is teratogenic, carcinogenic, and cytotoxic. However, the mechanisms of 2-MN induced toxicities remain unclear.This study aimed to characterise reactive metabolites of 2-MN, to define the metabolic pathway, and to determine the enzymes participating in the metabolic activation.A hydroxylation metabolite of 2-MN, 2-naphthalenemethanol (2-NM), was observed in 2-MN-containing mouse liver microsomes.A glutathione (GSH) conjugate was detected in mouse S9 incubations fortified with 2-MN and GSH. A GSH conjugate and an NAC conjugate were found in mouse liver and urine, respectively, in animals given 2-MN. Hepatic protein covalent binding derived from 2-NM was observed in animals administered 2-MN.Cytochrome P450 enzymes and sulfotransferases participated in the metabolic activation of 2-MN.

Metabolic Activation of Militarine In Vitro and In Vivo.

Bletilla striata is consumed as food and herbal medicine. Militarine (MLT) is a major ingredient in B. striata. Previous studies demonstrated that MLT showed teratogenic toxicity to zebrafish embryos. The present study aimed to identify reactive metabolites possibly involved in the cytotoxicity of MLT and determine the metabolic pathways involved. MLT was found to be hydrolyzed to p-hydroxybenzyl alcohol (HBA) by ß-glucosidase and esterases. The resulting HBA further underwent spontaneous dehydration to form quinone methide. HBA was also metabolized to the corresponding sulfate, followed by departure of the sulfate to generate a quinone methide. The resultant quinone methide reacted with hepatic glutathione (GSH) and protein to form the corresponding GSH conjugate and protein adduction. Additionally, inhibition of sulfotransferases (SULTs) attenuated the susceptibility of hepatocytes to the toxicity of MLT. This study provides that the hydrolytic enzymes ß-glucosidase, esterases, and SULTs participate in the metabolic activation of MLT.

Metabolic activation of 3-aminodibenzofuran mediated by P450 enzymes and sulfotransferases.

3-Aminodibenzofuran (3-ADBF) is a potent bladder carcinogen. This study aimed to identify reactive metabolites and the metabolic pathways of 3-ADBF. The in vitro and in vivo studies demonstrated that 3-ADBF was oxidized to the corresponding hydroxylamine by cytochrome P450 enzymes, followed by sulfation of the hydroxyl group mediated by sulfotransferases. The resulting sulfate conjugate was chemically reactive to GSH and cysteine residues of hepatic protein to form the corresponding GSH conjugate and protein adduction. Exposure of 3-ADBF to primary hepatocytes caused protein covalent binding and decreased cell viability. The resultant protein adduction was found to correlate the observed cytotoxicity of 3-ADBF.

Toxicokinetic and bioavailability studies on retrorsine in mice, and ketoconazole-induced alteration in toxicokinetic properties.

Retrorsine (RTS) is a toxic retronecine-type pyrrolizidine alkaloid, which is widely distributed. The purpose of this study was to develop a high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum RTS determination in mice. Serum samples were deproteinated by acetonitrile, separated on a C18 -PFP column and delivered at 0.8 ml/min with an eluting system composed of water containing 0.1% (v/v) formic acid and acetonitrile containing 0.1% (v/v) formic acid as mobile phases. RTS and the internal standard S-hexylglutathione (H-GSH) were quantitatively monitored with precursor-to-product transitions of m/z 352.1 → 120.1 and m/z 392.2 → 246.3, respectively. The method showed excellent linearity over the concentration range 0.05-50 µg/ml, with correlation coefficient r2 = 0.9992. The extraction recovery was >86.34%, and the matrix effect was not significant. Inter- and intra-day precisions (RSD) were <4.99%. The validated LC-MS/MS method was successfully applied to study the toxicokinetic profiles of serum RTS in mice after intravenous, oral administration and co-treated with ketoconazole, which showed that RTS displayed a long half-life (~11.05 h) and good bioavailability (81.80%). Co-administration of ketoconazole (KTZ) increased the peak serum concentration and area under the concentration-time curve and decreased the clearance and mean residence time. Summing up, a new standardized method was established for quantitative determination of RTS in sera.

RNA adduction derived from electrophilic species in vitro and in vivo.

RNA molecules are essential for cell function by not only serving as genetic materials, but also providing cells with structural support and catalytic functions. Due to nucleophilicity of nucleobases, RNA molecules can react with electrophilic species thus to be "adducted". The electron-deficient agents potentially inducing adduction exist in a variety of natural sources including metabolic products of biomolecules. Although evident and readily detected in human tissue, RNA adduction remains poorly understood for their physiological and pathological function. In this article, we review a collection of exogenous and endogenous molecular species that participate in RNA adduction and elaborates on the chemical nature of their RNA adduction sites. Furthermore, we provide perspectives on the potential of RNA adducts as biomarkers of environmental insults. Finally, we project future investigations that are necessary for understanding the mechanisms of cellular toxicity of RNA adduction.

Application of DNA barcoding and metabarcoding for species identification in salmon products.

Mislabelling is a significant manifestation of food fraud. Traditional Sanger sequencing technology is the gold standard for seafood species identification. However, this method is not suitable for analysing processed samples that may contain more than one species. This study tested the feasibility of next-generation sequencing in identifying mixed salmon products. Salmon samples containing up to eight species were amplified using 16S rRNA mini-barcode primers, and sequenced on an Illumina HiSeq2500 platform. All species were accurately identified, and mixtures as low as 1% (w/w) could be detected. Furthermore, this study conducted a market survey of 32 products labelled as salmon. For pure and mixed fish products, Sanger and next-generation sequencing techniques were respectively used for species identification, and for NGS results, we also used real-time PCR method to cross-validate the mixed products to further verify the accuracy of the DNA metabarcoding technology established in this study. DNA barcoding and metabarcoding of commercial salmon food products revealed the presence of mislabelling in 16 of 32 (50%) samples. The developed DNA barcoding and metabarcoding methods are useful for the identification of salmon species in food and can be used for quality control of various types of salmon products.

A meta-analysis and systematic review on subtypes of gastric intestinal metaplasia and neoplasia risk.

BACKGROUND: Gastric intestinal metaplasia (GIM) is a significant risk factor for gastric cancer. Risk of gastric cancer/dysplasia between complete intestinal metaplasia (CIM) and incomplete intestinal metaplasia (IIM) was controversial. Our study aimed to pool relative risk (RR) of cancer/dysplasia of IIM compared with CIM in GIM patients. METHODS: PubMed, EMBASE, Cochrane Library and Web of Science were searched for studies concerning cancer/dysplasia in GIM patients. Random-effects or fixed-effects model was utilized for pooling RR. Sensitivity and publication bias analyses were conducted. Stability of results would be evaluated in case of publication bias. RESULTS: 12 studies were included. Compared with CIM, pooled RR of cancer/dysplasia in IIM patients was 4.48 (95% CI 2.50-8.03), and the RR was 4.96 (95% CI 2.72-9.04) for cancer, and 4.82 (95% CI 1.45-16.0) for dysplasia. The pooled RR for cancer/dysplasia in type III IM was 6.27 (95% CI 1.89-20.77) compared with type II + I IM, while it was 5.55 (95% CI 2.07-14.92) compared with type II IM. Pooled RR between type II IM and type I IM was 1.62 (95% CI 1.16-2.27). Subgroup analyses showed that IIM was associated with a higher risk of gastric cancer/dysplasia in Western population (pooled RR = 4.65 95% CI 2.30-9.42), but not in East Asian population (pooled RR = 4.01 95% CI 0.82-19.61). CONCLUSIONS: IIM was related to a higher risk of cancer/dysplasia compared with CIM. Risk of developing cancer/dysplasia from type I, II, and III intestinal metaplasia increased gradually.

Atomic-Scale Structural Modification of 2D Materials.

2D materials have attracted much attention since the discovery of graphene in 2004. Due to their unique electrical, optical, and magnetic properties, they have potential for various applications such as electronics and optoelectronics. Owing to thermal motion and lattice growth kinetics, different atomic-scale structures (ASSs) can originate from natural or intentional regulation of 2D material atomic configurations. The transformations of ASSs can result in the variation of the charge density, electronic density of state and lattice symmetry so that the property tuning of 2D materials can be achieved and the functional devices can be constructed. Here, several kinds of ASSs of 2D materials are introduced, including grain boundaries, atomic defects, edge structures, and stacking arrangements. The design strategies of these structures are also summarized, especially for atomic defects and edge structures. Moreover, toward multifunctional integration of applications, the modulation of electrical, optical, and magnetic properties based on atomic-scale structural modification are presented. Finally, challenges and outlooks are featured in the aspects of controllable structure design and accurate property tuning for 2D materials with ASSs. This work may promote research on the atomic-scale structural modification of 2D materials toward functional applications.

Disassembly of 2D Vertical Heterostructures.

As one of the most widely discussed fields, the assembly of nanomaterials has always been extensively studied. However, its inverse process, namely disassembly, is still limited in the ambit of biomolecules. Specifically, in the emerging 2D research field, disassembly still remains unexplored. Inspired by the disassembly of DNA molecules via breaking intermolecular hydrogen bonds, the disassembly of 2D vertical heterostructures (2DVHs) is first achieved through the weakening of the interlayer van der Waals interactions. As a demonstration, ReS2 /WS2 VHs is successfully disassembled into individual building blocks. Density functional theory calculations are performed to study the disassembly of the 2DVHs, which simulate that 2DVHs are first activated by the disassembly promoters and then disassembled with weakened interlayer van der Waals interactions. Such a disassembly process demonstrates that it has great potential to be expanded as a general strategy to achieve the disassembly of other 2D superstructures.