Human AKR1A1 involves in metabolic activation of carcinogenic aristolochic acidⅠ

OBJECTIVE To investigate whether aldo-keto reductases(AKRs)can act as a nitrore⁃ductase(NR)and bioactivate aristolochic acidⅠ(AA-Ⅰ)to produce AA-Ⅰ-DNA adducts.METHODS①Human-induced hepatocytes(hiHeps)and human bladder RT4 cells were used as tool cells and treated with AA-Ⅰ0,0.5,1.0 and 2μmol·L^(-1)for 24 h.Cell viability was detected using the CCK-8 method,and the half maximal inhibition concentration(IC_(50))was calculated using the CCK-8 method and the level of DNA adduct production was calculated.②hiHeps and RT4 cells were treated with AKR inhibitor luteotin(0,5,10 and 25μmol·L^(-1))+AA-Ⅰ0.2 and 1.0μmol·L^(-1)for 24 h,respectively,and the levels of DNA adducts were detected by a liquid chromatography-tandem mass spectrometer(LC-MS/MS).③hiHeps cells were incubated with 80 nmol·L^(-1)small interfering RNAs(si-AKRs)for 48 h and treated with AA-Ⅰ1.0μmol·L^(-1)for 24 h.Real-time qualitative PCR(RT-qPCR)method was used to detect the mRNA expression of AKRs gene and LC-MS/MS technology was used to investigate the effect of specific AKR gene knockdown on DNA adduct levels.④500 nmol·L^(-1)human AKR recombinant proteins AKR1A1 and AA-Ⅰwere incubated in vitro under anaerobic conditions and the formation of AA-Ⅰ-DNA adducts was detected.RESULTS①The IC_(50)of AA-Ⅰto hiHeps and RT4 cells was 1.9 and 0.42μmol·L^(-1),respec⁃tively.The level of DNA adduct production of the two cell lines was significantly different(P<0.01).②Luteolin≥5μmol·L^(-1)significantly inhibited the production of AA-Ⅰ-DNA adducts in both cells(P<0.05),and there was a concentration-dependent effect in hiHeps cells(P<0.01,R=0.84).③In the AKR family,the knockdown of AKR1A1 gene up to 80%inhibited the generation of AA-Ⅰ-DNA adducts by 30%-40%.④The AA-Ⅰ-DNA adducts were detected in the incubation of recombinant protein AKR1A1 and AA-Ⅰunder anaerobic conditions in vitro,approximately 1 adduct per 107 nucleotides.CONCLU⁃SION AKR1A1 is involved in AA-Ⅰbioactivation,providing a reference for elucidation of the carcino⁃genic mechanism of AA-Ⅰ.

EFFECTS OF PROSTAGLANDIN E1 ON THE PROGRESSION OF ARISTOLOCHIC ACID NEPHROPATHY

Objective To investigate the effects of prostaglandin E1 (PGE1) on the progression of aristolochic acid nephropathy (AAN). Methods Twenty-four patients diagnosed as AAN with serum creatinine (Scr) between 1.5 mg/dL and 4 mg/dL during September 2001 to August 2003 were randomly divided into 2 groups. All patients had ingested long dan xie gan wan con-taining aristolochic acid (0.219 mg/g) for at least 3 months. Twelve patients were injected with Alprostadil (10 μg/d for 10 days in one month, summing up to 6 months). Except for PGE1, the other therapy was same in both groups. Renal function was assessed using reciprocal serum creatinine levels (1/Scr). Results The level of Scr and serum hemoglobin (Hgb) was similar in both groups prior to therapy. During follow-up, 1/Scr levels in PGE1 group were significantly higher than control group (P < 0.01), and Hgb levels in PGE1 group were sig-nificantly increased compared with control (P < 0.05).Conclusion PGE1 can slow the progression of renal failure and increase Hgb level of AAN patient.

NMR Studies on the Subacute Biochemical Effects of Aristolochic Acid on Rat Serum

The subacute effect of aristolochic acid （AA） on rat serum was studied by NMR method. The biochemical effects induced by AA were characterized by an increase in the amounts of creatinine, trimethylamine N-oxide, acetoacetate, acetate and 3-D-hydroxybutyrate and lactate in serum from ^1H NMR spectra. Principal component analysis was used for further comparing the similarities of ^1H NMR spectral profiles of serum from rats treated with AA and model toxins.

Nephrotoxicity and carcinogenesis of aristolochic acids and their derivates

Aristolochic acids （AAs）, a natural mixture of 8-methoxy-6-nitro-phenanthro-（3,4-d）-1,3-dioxolo-5-carboxylic acid （AAI）and 6-nitro-phenanthro-（3,4-d）-1,3-dioxolo-5-carboxylic acid （AAII）, derived from aristolochiaceae species, has beenreported to cause AAS-induced nephropathy and upper urothelial cancer. In this review, we summarize the informationon the nephrotoxicity and carcinogenesis of AAs and their derivatives. AAs nephrotoxicity can lead to apoptosis andoxidative stress of renal tubular cells, and inhibition of the expression of aquaporins. AAs can also reduce the capabilityfor renal tubular epithelial cell repair after acute injury and further produce renal fibrosis by activating TGF-β-Smadsignaling and promoting the migration of macrophages. Moreover, AAs-induced carcinogenesis may be due to theformation of covalent adducts with DNA which can lead to the mutation in certain tumor suppressor genes orproto-oncogenes and the different catalyzing capacity of the microsomal cytochrome P450 of individuals in AAImetabolism.

Improved preparation and identification of aristolochic acid-DNA adducts by solid-phase extraction with liquid chromatography-tandem mass spectrometry

Aristolochic acid （AA） is a known nephrotoxin and potential carcinogen, which can form covalent DNA adducts after metabolic activation in vivo and in vitro. A simple method for preparation and characterization of aristolochic acid-DNA adducts was developed. Four AA-adducts were synthesized by a direct reaction of AAI/AAII with 2′-deoxynucleosides. The reaction mixture was first cleaned-up and pre-concentrated using solid phase extraction （SPE）, and further purified by a reversed-phase high performance liquid chromatography （HPLC）. By the application of developed SPE procedure, matrices and byproducts in reaction mixture could be greatly reduced and adducts of high purity （more than 94% as indicated by HPLC） were obtained. The purified AA-DNA adducts were identified and characterized with liquid-electrospray ionization-quadrupole-time of flight-mass spectrometry （LC-ESI-Q-TOF-MS/MS） and LC-Diode array detector-fluorescence （LC-DAD-FL） analysis. This work provides a robust tool for possible large-scale preparation of AA-DNA adduct standards, which can promote the further studies on carcinogenic and mutagenic mechanism of aristolochic acids.

A NEW ARISTOLOCHIC ACID FROM ARISTOLOCHIA CINNABARINA

A new compound, aristolochoc acid Ⅲ a- 6-0-β-D-glucoside, was isolated along with four known compounds from Aristolochia cinnabarina. The structure of the new,compound was elucidated on the basis of spectral evidences

Electrochemistry of aristolochic acid interacting with guanine

Interaction of Aristolochic acid （AA） and guanine （G） was studied by electrochemical techniques in this paper. When AA was added into the guanine solution, the oxidation peak currents of mixture solution decreased, while the peak potential and the electrochemical kinetic parameters remained the same as when AA was absent, except that the electrode process of guanine that involved two protons and two electrons changed from adsorption controlled to diffusion controlled. It is suggested that an electrochemical inactive supramolecular adduct AA-Gua （1：1） was formed in the system. The adduct cannot be oxidized on the glassy carbon electrode, which indirectly results in the decrease of the free concentration of guanine in the reaction solution and the decrease of peak currents. The binding constant （13） of this adduct is calculated as 7.14× 10^3 mol/L. The possible mechanism for the interaction of Aristolochic acid and DNA was proposed, that may provide a possible pathway for the nosogenesis research of aristolochic acid.

Chemical profiling of principle active and toxic constituents in herbs containing aristolochic acids

Objective:To clear the amounts of the principal active/toxic components in herbs containing aristolochic acids(HCAAs),which are still used as medicine and/or seasoning in many ethnic minority areas of China.Methods:In this study,six major active and toxic components in HCAAs were extracted with ultrasonic extraction.With 6-O-methyl guanosine as internal standard,the target compounds were analyzed qualitatively and quantitatively by using ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry(UPLC-ESI-MS/MS)with multiple reaction monitoringinformation dependent acquisition-enhanced production ion scanning mode(MRM-IDA-EPI)combined with dynamic background subtraction(DBS)function.Results:The method showed good linearity in the linear range of the six analytes.The limit range of detection was from 0.01 ng/mL to 0.27 ng/mL.All of the detection repeatability,extraction repeatability and accuracy of the method were good.After extraction,the samples remained stable at 15℃ within 24 h.Six analytes were all found in samples except aristolactam(AL)in sample 2,and the contents varied greatly.The contents of these compounds decreased in fruits,leaves and stems of Aristolochia delavayi successively.Conclusion:This method has the advantages of less sample dosage,simple operation,short analysis cycle,high sensitivity,specificity and accuracy.It laid a good foundation for guiding the safety of HCAAs,the indepth study of pharmacological and toxicological effects and the scientific and standardized processing and compatibility of HCAAs.

No Incidence of Liver Cancer Was Observed in A Retrospective Studyof Patients with Aristolochic Acid Nephropathy

Objective:To assess the risk of aristolochic acid(AA)-associated cancer in patients with AA nephropathy(AAN).Methods:A retrospective study was conducted on patients diagnosed with AAN at Peking University First Hospital from January 1997 to December 2014.Long-term surveillance and follow-up data were analyzed to investigate the influence of different factors on the prevalence of cancer.The primary endpoint was the incidence of liver cancer,and the secondary endpoint was the incidence of urinary cancer during 1 year after taking AA-containing medication to 2014.Results:A total of 337 patients diagnosed with AAN were included in this study.From the initiation of taking AA to the termination of follow-up,39 patients were diagnosed with cancer.No cases of liver cancer were observed throughout the entire follow-up period,with urinary cancer being the predominant type(34/39,87.17%).Logistic regression analysis showed that age,follow-up period,and diabetes were potential risk factors,however,the dosage of the drug was not significantly associated with urinary cancer.Conclusions:No cases of liver cancer were observed at the end of follow-up.However,a high prevalence of urinary cancer was observed in AAN patients.Establishing a direct causality between AA and HCC is challenging.

Activation of LONP1 by 84-B10 alleviates aristolochic acid nephropathy via re-establishing mitochondrial and peroxisomal homeostasis

Pharmaceutical formulations derived from Aristolochiaceae herbs, which contain aristolochic acids(AAs), are widely used for medicinal purposes. However, exposure to these plants and isolated AAs is linked to renal toxicity, known as AA nephropathy(AAN). Currently, the mechanisms underlying AAN are not fully understood, leading to unsatisfactory treatment strategies. In this study, we explored the protective role of 84-B10(5-[[2-(4-methoxyphenoxy)-5-(trifluoromethyl) phenyl] amino]-5-oxo-3-phenylpentanoic acid) against AAN. RNA-seq analysis revealed that the mitochondrion and peroxisome were the most affected cellular components following 84-B10 treatment in AAN mice. Consistently, 84-B10 treatment preserved mitochondrial ultrastructure, restored mitochondrial respiration, enhanced the expression of key transporters(carnitine palmitoyltransferase 2) and enzymes(acylCoenzyme A dehydrogenase, medium chain) involved in mitochondrial fatty acid β-oxidation, and reduced mitochondrial ROS generation in both aristolochic acid I(AAI)-challenged mice kidneys and cultured proximal tubular epithelial cells. Additionally, 84-B10 treatment increased the expression of key transporters(ATP binding cassette subfamily D) and rate-limiting enzymes(acyl-CoA oxidase 1) involved in peroxisomal fatty acid β-oxidation and restored peroxisomal redox balance. Knocking down LONP1 expression diminished the protective effects of 84-B10 against AAN, suggesting LONP1-dependent protection. In conclusion, our study provides evidence that AAN is associated with significant disturbances in both mitochondrial and peroxisomal functions. The LONP1 activator 84-B10 demonstrates therapeutic potential against AAN, likely by maintaining homeostasis in both mitochondria and peroxisomes.

Synthesis, isolation and characterization of methyl levulinate from cellulose catalyzed by extremely low concentration acid

A direct synthesis of methyl levulinate from cellulose alcoholysis in methanol medium under mild condition(180 210 C)catalyzed by extremely low concentration sulfuric acid(0.01 mol/L)and the product isolation were developed in this study.Effects of different process variables towards the catalytic performance were performed as a function of reaction time.The results indicated that sulfuric acid concentration,temperature and initial cellulose concentration had significant effects on the synthesis of methyl levulinate.An optimized yield of around 50%was achieved at 210 C for 120 min with sulfuric acid concentration of 0.01 mol/L and initial cellulose concentration below 100 g/L.The resulting product mixture was isolated by a distillation technique that combines an atmospheric distillation with a vacuum distillation where n-dodecane was added to help distill the heavy fraction.The light fraction including mainly methanol could be reused as the reaction medium without any substantial change in the yield of methyl levulinate.The chemical composition and structural of lower heavy fraction were characterized by GC/MS,FTIR,1H-NMR and13C-NMR techniques.Methyl levulinate was found to be a major ingredient of lower heavy fraction with the content over 96%.This pathway is efficient,environmentally benign and economical for the production of pure levulinate esters from cellulose.

Effect of Lanthanum on Acid Secretion from Isolated Mouse Stomach in Vitro

To explore the effect and the mechanism of La^(3+) on gastric acid secretion (GAS) of isolated mouse stomach with perfused lumen, 12 cm H_2O column intragastric pressure-provided, whole stomach preparations from mice were incubated in buffer at 37 ℃ in vitro, and perfusate was measured for pH with a pHS-3 type pH meter. The results show that La^(3+) (0.41～820×10^(-6) mol·L^(-1)) significantly promotes GAS in a concentration-dependant manner. Proglutamine, a blocker of gastrin receptor, potently inhibits GAS, and it may block the promotive effect of La^(3+) on GAS, and this effect increases with the increase of proglutamin concentration. Cimetidine, a blocker of histamine H_2 receptor, also potently inhibits GAS, and blocks the promotive effect of La^(3+) on GAS in the same manner with proglutamine. These results suggest that La^(3+) promotes GAS in isolated stomach possibly by stimulating the releases of gastrin from G cell and Histamine from ECL cell or by activating the gastrin receptors and Histamine H_2 receptors on the parietal cell, thereby accelerating the acid secretion of parietal cells in stomach.

Isolation and Identification of High-Quality Lactic Acid Bacteria in Forage Corn

[ Objective] To screen suitable lactic acid bacterium strains from forage corn which can be used as silage additives. [ Method] The lactic acid bacterium strains were isolated by inoculation on MRS solid medium containing calcium carbonate, and they were preliminarily identified through morphological, physiological and biochemical experiments. The acid production efficiency was determined. Twelve strains having strong acid-pro- duction ability were selected, and their salt tolerance and acid tolerance were detected. The sequences of their 16 S rDNA were also analyzed. [ Result] A total of 44 lactic acid bacterium strains were isolated from the forage com. As evidenced by the physiological and biochemical experi- ments, the twelve strains having strong acid-production ability belonged to Leuconostoc, Lactobacillus and Enterococcus, respectively, and they had strong salt tolerance and acid tolerance. According to the sequences of 16 S rDNA, A4, B9, B11, B12 and B14 were Lactobacillus plantarum; A1, A2., A7, A11 and B8 were Leuconostoc mesenteroides dextran subspecies; and AB and A9 were Enterococcus hirae. [ Conclusion ] The lactic acid bacterium strains with strong acid-production ability isolated from forage corn can be developed into silage additives.

Soy Protein Isolate Film by Incorporating Mandelic Acid as Well as Through Fermentation Mediated by Bacillus Subtilis

Soy protein isolate(SPI)biopolymeric films were prepared by adding different contents of mandelic acid(1 to 5%wrt SPI)to glycerol plasticized SPI by solution casting method.Also,SPI was fermented by Bacillus subtilis to get fermented SPI films by solution casting.Molecular mass determination of mandelic acid incorporated and fermented SPI films was carried out by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE).Mandelic acid incorporated and fermented SPI films were characterized by Fourier-transform infrared spectroscopy(FT-IR),dynamic mechanical analysis(DMA),tensile strength,water uptake and optical transmittance studies.Results indicated that incorporation of mandelic acid in SPI resulted in high tensile strength(8.03 MPa)and highα-relaxation(Tα)as well as low water uptake.On the other hand,films cannot be prepared from fermented SPI with SPI contents of 8%and 12%.However,film from fermented SPI with 16%SPI content could be prepared but it exhibited low tensile strength(3.18 MPa)and low Tαas well as high water uptake.The resulting mandelic acid incorporated SPI films were also subjected to antimicrobial studies.At all the concentration of mandelic acid,we can easily observe the antimicrobial effect in mandelic acid incorporated SPI films unlike fermented SPI films.This work will be helpful in fabricating antimicrobial SPI film from renewable resources.

ISOLATION AND IDENTIFICATION OF β-N-OXALO-L-α,β-DIAMINOPROPIONIC ACID AND ITS ISOMER α-N-OXALO-L-α,β-DIAMINOPROPIONIC ACID FROM PANAX SPECIES

The neuroexcitotoxic nonprotein amino acid β-N-oxalo-L-α, β-diaminopropionic acid (β-N-ODAP)and its isomer a-N-oxalo-L-α, β-diaminopropionic acid (α-N-ODAP)in Panax ginseng C. A. Meyer(cultivated ih Northeastern China), Panax quinquefolius L., Panax notoginseng F.H. Chen (cultivated in Southwestern China), Korean red ginseng and Jilin red ginseng were isolated and identified. Efficient separation and purification methods for β-N-ODAP and α-N-ODAP were developed.

Antimicrobial Profile of Lactic Acid Bacteria Isolated from Vegetables and Indigenous Fermented Foods of India against Clinical Pathogens Using Microdilution Method

In dairy and food industries lactic acid bacteria （LAB） have been used in form of starter culture that plays vital role in fermentation; as flavouring and texturizing or as preservative agents. There is increasing evidence that lactobacilli which inhabit the gastrointestinal tract develop antimicrobial activities and participate in the host＇s defence system[1]. During fermentation, most of the LAB produces a number of different compounds like organic acids, hydrogen peroxide, diacetyl, acetaldehyde, carbon dioxide, polysaccharides, and proteinaceous compounds called bacteriocins or bacteriocinogenic peptides.

Isolation and Characterization of Acid-soluble Collagen and Pepsin-soluble Collagen from the Skin of Hybrid Sturgeon

By using the wastes fish skin of sturgeon processed as a raw material, a macromolecule biomaterial of collagen was extracted. Acid-soluble collagen(ASC) and pepsin-soluble collagen(PSC) were successfully isolated from the skin of hybrid sturgeon with two extraction methods. The yields of ASC and PSC based on the wet weight of skin were 5.73 ± 0.11% and 10.26 ± 0.39%, respectively. The denaturation and melting points of ASC(26.83 ℃ and 110.49 ℃) and PSC(26.54 ℃ and 102.99 ℃) were assessed by Circular dichroism(CD) and Differential scanning calorimetry(DSC). ASC and PSC appeared to be dense sheet-like film linked by random-coiled filaments under scanning electron microscopy(SEM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Fourier transform infrared spectroscopy(FTIR) confirmed that both the ASC and PSC were Type I collagen and maintained a complete triple helix structure. These results indicated that both ASC and PSC possessed good biological activity and could be widely used in medical biomaterials and other fields.

Data cleaning method for the process of acid production with flue gas based on improved random forest

Acid production with flue gas is a complex nonlinear process with multiple variables and strong coupling.The operation data is an important basis for state monitoring,optimal control,and fault diagnosis.However,the operating environment of acid production with flue gas is complex and there is much equipment.The data obtained by the detection equipment is seriously polluted and prone to abnormal phenomena such as data loss and outliers.Therefore,to solve the problem of abnormal data in the process of acid production with flue gas,a data cleaning method based on improved random forest is proposed.Firstly,an outlier data recognition model based on isolation forest is designed to identify and eliminate the outliers in the dataset.Secondly,an improved random forest regression model is established.Genetic algorithm is used to optimize the hyperparameters of the random forest regression model.Then the optimal parameter combination is found in the search space and the trend of data is predicted.Finally,the improved random forest data cleaning method is used to compensate for the missing data after eliminating abnormal data and the data cleaning is realized.Results show that the proposed method can accurately eliminate and compensate for the abnormal data in the process of acid production with flue gas.The method improves the accuracy of compensation for missing data.With the data after cleaning,a more accurate model can be established,which is significant to the subsequent temperature control.The conversion rate of SO_(2) can be further improved,thereby improving the yield of sulfuric acid and economic benefits.

Isolation and Identification of High-Quality Lactic Acid Bacteria from Wheat Haulm

[Objective] The aim of this study was to isolate and identify lactic acid bacteria （LAB） from wheat haulm and to select efficient strains for silage fermentation. [ Method] From 78 LAB strains isolated on the MRS solid medium containing calcium carbonate, we selected 43 strains having better acid-production ability through morphological observation, Gram staining, physiological and biochemical tests, acid production test, acid tolerance test and salt tolerance test. These strains were finally identified by sequencing 16 S rDNA. [ Result] Of the 43 LAB strains having better acid-production ability, 37 belonged to Lactobacillus paracasei subsp., one belonged to Lactobacillus rhamnosus and five belonged to Enterococcus faecium, as shown by the sequences of 16 S rDNA. [ Conclusion ] A total of 43 LAB strains having better acid-production ability were selected, which may be developed as high-quality silage additives.

Fabrication of a solid superacid with temperature-regulated silica-isolated biochar nanosheets

This paper reports a new strategy for the structural reconstruction of biomass carbon sulfonic acid(BCSA)to its solid superacid counterpart.In this approach,a cheap layered biomass carbon(BC)source is chemically exfoliated by cetyltrimethyl ammonium bromide and then converted to silica-isolated carbon nanosheets(CNSs)by a series of conversion steps.The state of the silica-isolated CNSs and the stacking density of their nanoparticles are regulated by the dehydration temperature.Only the highly isolated and non-crosslinked CNSs with loose particle stacking structures obtained upon dehydration at 250℃ can be turned into superacid sites(with stronger acidity than that of 100%H2 SO4)after sulfonation.This is accompanied by the creation of abundant hierarchical slit pores with high external surface area,mainly driven by the strong hydrogen bonding interactions between the introduced sulfonic acid groups.In typical acid-catalyzed esterification,etherification,and hydrolysis reactions,the newly formed superacid exhibits superior catalytic activity and stability compared to those of common BCSA and commercial Amberlyst-15 catalysts,owing to its good structural stability,highly exposed stable superacidic sites,and abundance of mesoporous/macroporous channels with excellent mass transfer rate.This groundbreaking work not only provides a novel strategy for fabricating bio-based solid superacids,but also overcomes the drawbacks of BCSA,i.e.,unsatisfactory structural stability,acidity,and porosity.