Silver nanoparticles (AgNPs) is known as one of the most valuable metal nanoparticles in antibacterial and anticancer application. AgNPs-resistant bacteria has been documented, but it is unclear whether cancer cells can also escape the anti-cancer effect of AgNPs. In this study, we aimed to investigate this phenomenon and its underlying mechanism. The antibacterial activity and cytotoxicity of AgNPs were measured in the presence of HeLa cell metabolites. The status of AgNPs in the system associated with metabolites were characterized by UV-Vis, Zetasizer Nano ZS, and transmission electron microscopy. Non-targeted metabolomics was used to reveal the metabolites components that bind with AgNPs. HeLa cells were injected intraperitoneally to establish the tumor-bearing mice model, and the stability of AgNPs in mice serum was analyzed. The results manifested that HeLa cell metabolites inhibited the anticancer and antibacterial effects of AgNPs in a dose-dependent manner by causing AgNPs aggregation. Effective metabolites that inhibited the biological activity of AgNPs were stable in 100 ℃, insoluble in chloroform, containing sulfur elements, and had a molecular weight less than 1 kDa in molecular weight. There were 115 compounds bound with AgNPs. In vitro experiments showed that AgNPs aggregation occurred only when the concentration of α-ketoglutarate (AKG) and glutathione (GSH) together reached a certain threshold. Interestingly, the concentration of AKG and GSH in HeLa cellular metabolites was 10 and 6 times higher than that in normal cervical epithelial cells, respectively, which explained why the threshold was reached. Furthermore, the stability of AgNPs in the serum of tumor-bearing mice decreased by 20% (P < 0.05) compared with the healthy mice. In conclusion, our study demonstrates that HeLa cells escaped the anti-cancer effect of AgNPs through the synergistic effect of AKG and GSH, suggesting the need to develop strategies to overcome this limitation.
Humans ; Animals ; Mice ; HeLa Cells ; Silver/pharmacology* ; Ketoglutaric Acids/pharmacology* ; Metal Nanoparticles ; Anti-Bacterial Agents/pharmacology* ; Glutathione ; Microbial Sensitivity Tests

A large amount of alpha-ketoglutarate (alpha-KG) (6.8 g/L) was accumulated in flask culture when CaCO3 was used as a buffering agent in the production of pyruvate by multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019. In a 5 L jar-fermentor, less alpha-KG (1.3 g/L) was produced when NaOH was used to adjust the pH, while more alpha-KG (11.5 g/L) detected when CaCO3 was used as the buffer. In the latter case, the molar carbon ratio of pyruvate to alpha-KG (C(PYR)/ CalphaKG) was similar to that obtained in flask culture, suggesting the accumulation of alpha-ketoglutarate was related to the addition of CaCO3. Furthermore, it was found that: (1) delaying the addition time of CaCO3 decreased the a-ketoglutarate formation but increased C(PYR)/ C(alphaKG); and (2) under vitamin limitation conditions increasing the concentration of CaCO3 led to an increased a-KG accumulation at the expenses of pyruvate. To study which ions in CaCO3 was responsible for the accumulation of alpha-KG, the effects of different pH buffers on the a-KG accumulation were studied. The level of alpha-KG was found to correlate with the levels of both Ca2+ and CO3(2-), with Ca2+ played a dominant role and CO3(2-) played a minor role. To find out which pathway was responsible for the accumulation of alpha-KG, the effects of biotin and thiamine on alpha-KG accumulation was investigated. The increase in biotin concentration led to an increase in alpha-KG accumulation and a decrease in C(PYR)/ C(alpha-KG), while the levels of alpha-KG and C(PYR)/C(alphaKG) were not affected by thiamine concentration. The activity of pyruvate carboxylase was increased as much as 40% when the medium was supplemented with Ca2+ . On the other hand, the activity of the pyruvate dehydrogenase complex was unaffected by the presence of Ca2+. To conclude, the higher level of a-KG was caused by higher activity of pyruvate carboxylase stimulated by Ca2+, with CO3(2-) served as the substrate of the reaction.
Biotechnology ; methods ; Calcium Carbonate ; pharmacology ; Candida glabrata ; drug effects ; metabolism ; Fermentation ; drug effects ; Ketoglutaric Acids ; metabolism ; Models, Biological ; Pyruvic Acid ; metabolism

OBJECTIVETo investigate the biochemical changes in rat brain and liver following acute exposure to a lethal dose of cyanide, and its response to treatment of alpha-ketoglutarate (alpha-KG) in the absence or presence of sodium thiosulfate (STS).

METHODSFemale rats were administered 2.0 LD50 potassium cyanide (KCN; oral) in the absence or presence of pre-treatment (-10 min), simultaneous treatment (0 min) or post-treatment (+2-3 min) of alpha-KG (2.0 g/kg, oral) and/or STS (1.0 g/kg, intraperitoneal, -15 min, 0 min or + 2-3 min). At the time of onset of signs and symptoms of KCN toxicity (2-4 min) and at the time of death (5-15 min), various parameters particularly akin to oxidative stress viz. cytochrome oxidase (CYTOX), superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH) and oxidized glutathione (GSSG) in brain, and CYTOX, sorbitol dehydrogenase (SDH), alkaline phosphatase (ALP), GSH and GSSG in liver homogenate were measured.

RESULTSAt both time intervals brain CYTOX, SOD, GPx, and GSH significantly reduced (percent inhibition compared to control) to 24%, 56%, 77%, and 65%, and 44%, 46%, 78%, and 57%, respectively. At the corresponding time points liver CYTOX and GSH reduced to 74% and 63%, and 44% and 68%, respectively. The levels of GSSG in the brain and liver, and hepatic ALP and SDH were unchanged. Pre-treatment and simultaneous treatment of a-KG alone or with STS conferred significant protection on above variables. Post-treatment was effective in restoring the changes in liver but failed to normalize the changes in the brain.

CONCLUSIONSOral treatment with alpha-KG alone or in combination with STS has protective effects on cyanide-induced biochemical alterations in rat brain and liver.

Animals ; Antidotes ; pharmacology ; Brain ; drug effects ; metabolism ; Electron Transport Complex IV ; metabolism ; Female ; Glutathione Peroxidase ; metabolism ; Glutathione Reductase ; metabolism ; Ketoglutaric Acids ; pharmacology ; Liver ; drug effects ; metabolism ; Oxidative Stress ; Poisoning ; prevention & control ; Potassium Cyanide ; poisoning ; Rats ; Rats, Wistar ; Superoxide Dismutase ; metabolism ; Thiosulfates ; pharmacology

To investigate the intervention effects of Morinda officinalis How. on 'Kidney-yang deficiency syndrome' induced by hydrocortisone in rats, the metabolic profiles of rat urine were characterized using proton nuclear magnetic resonance and principal component analysis (PCA) was applied to study the trajectory of urinary metabolic phenotype of rats with 'Kidney-yang deficiency syndrome' under administration of M. officinalis at different time points. Meanwhile, the intervention effects of M. officinalis on urinary metabolic potential biomarkers associated with 'Kidney-yang deficiency syndrome' were also discussed. The experimental results showed that in accordance to the increased time of administration, an obvious tendency was observed that clustering of the treatment group moved gradually closed to that of the control group. Eight potential biomarkers including citrate, succinate, alpha-ketoglutarate, lactate, betaine, sarcosine, alanine and taurine were definitely up- or down-regulated. In conclusion, the effectiveness of M. oficinalis on 'Kidney-yang deficiency syndrome' is proved using the established metabonomic method and the regulated metabolic pathways involve energy metabolism, transmethylation and transportation of amine. Meanwhile, the administration of M. officinalis can alleviate the kidney impairment induced by 'Kidney-yang deficiency syndrome'.
Alanine ; urine ; Animals ; Betaine ; urine ; Biomarkers ; urine ; Citric Acid ; urine ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Hydrocortisone ; Ketoglutaric Acids ; urine ; Kidney Diseases ; chemically induced ; urine ; Lactic Acid ; urine ; Magnetic Resonance Spectroscopy ; Male ; Metabolomics ; methods ; Morinda ; chemistry ; Plants, Medicinal ; chemistry ; Principal Component Analysis ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sarcosine ; urine ; Succinic Acid ; urine ; Taurine ; urine ; Yang Deficiency ; chemically induced ; urine