Purine Metabolism In Cells Research Articles

Electrochemical voltammetry assessment of cellular DNA damage caused by drugs with different mechanisms: A comparative analysis with the γH2AX focus and alkaline comet assays

As industrialization accelerates, there is an increasing need to find a quick, easy, and affordable method to assess the potential DNA damage caused by newly developed chemicals. Electrochemical voltammetry using intracellular purine bases as markers shows promise for detecting DNA damage. However, it is unclear whether drugs with different DNA damage mechanisms will impact purine metabolism in cells and consequently disturb the electrochemical indicators of DNA damage. In this paper, the electrochemical behaviors of HepG2 cells exposed to the direct DNA-damaging agent (MNNG), the indirect DNA-damaging agent (CPT11), and the non-DNA-damaging agent (KCl) were studied. The findings showed that the electrochemical signals of cells increased with the rising concentration of DNA-damaging drugs. A time-dependent effect was observed, with a lower threshold dose for 4 h compared to 1 h. However, the non-DNA-damaging agent did not induce an increase in the cellular electrochemical signal. Despite the higher cytotoxicity of CPT11, MNNG triggered DNA damage at a lower dose, as detected by electrochemical indicators. The results of the electrochemical indicators assessment of DNA damage correlated significantly with those of the γH2AX focus and alkaline comet assays, with r > 0.98 and P < 0.01. Particularly, there was a stronger correlation with the alkaline comet assay. Furthermore, electrochemical voltammetry detected DNA damage at a lower threshold compared to other assessment methods, demonstrating a higher sensitivity to DNA damage. These findings suggest that by using purine bases as markers, electrochemical voltammetry enables precise assessment of DNA damage caused by various mechanisms.

Simultaneous determination of canonical purine metabolism using a newly developed HILIC-MS/MS in cultured cells

Purine metabolism acts as the core role in human metabolic network. It offers purine metabolites as raw material for building blocks in cell survival and proliferation. Purine metabolites are the most abundant metabolic substrates in organisms. There are few reports to simultaneously quantify canonical purine metabolism in cells. A novel hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS/MS) method was developed to simultaneously determine purines profile in biological samples. Chromatographic separation was achieved using a HILIC (Waters Xbridge™ Amide) column. Different optimizing chromatographic conditions and mass spectrometric parameters were tested in order to provide the best separation and the lowest limit of quantification (LLOQ) values for targeted metabolites. The validation was evaluated according to the Food and Drug Administration guidelines. The limit of determination (LOD) and the LOQ values were in the range of 0.02–8.33 ng mL−1 and 0.1–24.5 ng mL−1, respectively. All calibration curves displayed good linear relationship of with excellent correlation coefficient (r) ranging from 0.9943 to 0.9999. Both intra-day and inter-day variability were below 15 %, respectively. Trueness, expressed as relative error, was always within ±15 %. In addition, no derivatization procedure and ion-pair reagents are in need. The innovated approach demonstrates high sensitivity, strong specificity, and good repeatability, making it suitable for absolute quantitative studies of canonical purine metabolism in cultured cells.

Evaluation on purine metabolism in human skin fibroblast cells exposed to oxygenated polycyclic aromatic hydrocarbons.

A rapid and sensitive assessment of the toxicity of oxygenated polycyclic aromatic hydrocarbons (OPAHs), widely distributed persistent organic pollutants in the environment, is crucial for human health. In this study, using high-performance liquid chromatography, the separation and detection of four purines, xanthine (X), guanine (G), adenine (A), and hypoxanthine (HX) in cells were performed. The aim was to evaluate the cytotoxicity of three OPAHs, namely 1,4-benzoquinone (1,4-BQ), 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone (9,10-PQ), with higher environmental concentrations, from the perspective of purine nucleotide metabolism in human skin fibroblast cells (HFF-1). The results revealed that the levels of G and A were low in HFF-1 cells, while the levels of HX and X showed a dose-response relationship with persistent organic pollutants concentration. With increased concentration of the three persistent organic pollutants, the purine metabolism in HFF-1 cells weakened, and the impact of the three persistent organic pollutants on purine metabolism in cells was in the order of 9,10-PQ > 1,4-BQ > 1,2-NQ. This study provided valuable insights into the toxic mechanisms of 1,4-BQ, 1,2-NQ and 9,10-PQ, contributing to the formulation of relevant protective measures and the safeguarding of human health.

Purine auxotrophy: Possible applications beyond genetic marker.

Exploring new drug candidates or drug targets against many illnesses is necessary as "traditional" treatments lose their effectivity. Cancer and sicknesses caused by protozoan parasites are among these diseases. Cell purine metabolism is an important drug target. Theoretically, inhibiting purine metabolism could stop the proliferation of unwanted cells. Purine metabolism is similar across all eukaryotes. However, some medically important organisms or cell lines rely on their host purine metabolism. Protozoans causing malaria, leishmaniasis, or toxoplasmosis are purine auxotrophs. Some cancer forms have also lost the ability to synthesize purines de novo. Budding yeast can serve as an effective model for eukaryotic purine metabolism, and thus, purine auxotrophic strains could be an important tool. In this review, we present the common principles of purine metabolism in eukaryotes, effects of purine starvation in eukaryotic cells, and purine-starved Saccharomyces cerevisiae as a model for purine depletion-elicited metabolic states with applications in evolution studies and pharmacology. Purine auxotrophic yeast strains behave differently when growing in media with sufficient supplementation with adenine or in media depleted of adenine (starvation). In the latter, they undergo cell cycle arrest at G1/G0 and become stress resistant. Importantly, similar effects have also been observed among parasitic protozoans or cancer cells. We consider that studies on metabolic changes caused by purine auxotrophy could reveal new options for parasite or cancer therapy. Further, knowledge on phenotypic changes will improve the use of auxotrophic strains in high-throughput screening for primary drug candidates.

An Enzyme Assisted Electrochemical Detection System of Purine Intracellular Utilizing MWCNTs-IL Modified Glassy Carbon Electrode

Monitoring purines intracellular quickly and accurately is critically important for revealing physiological processes relate to purine metabolism. However, the overlapped signals of guanine (G) or xanthine (X) and adenine (A) or hypoxanthine (H) hinder the accurate detection of individual purine change in cells. In this paper, a novel electrochemical detecting system has been constructed by introducing xanthine oxidase, which can catalyze H or X to uric acid during the detection process. Using this enzyme-assisted electrochemical detecting system, the individual concentrations of G, X, A and H in the Human breast cancer (MCF-7) cells can be detected simultaneously without any pretreatment. As a result, more information about the purine metabolism in cells can be provided. The cytotoxicity of cyclophosphamide on MCF-7 cell activity was also evaluated by this system, and the results are consistent with that of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which paves a new way to the accurate detection of purines intracellular with the assistance of xanthine oxidase.

Purine metabolism in cells of a mangrove plant, Sonneratia alba, in tissue culture

Summary A tissue culture system was established from pistils of flower buds of a mangrove plant, Sonneratia alba . The optimum growth rate of callus was observed in Murashige and Skoog's medium that contained 50 mM NaCl. In this tissue culture system, we examined the effects of short-term salt stress on the size of the pool of purine nucleotides, as well as the capacity for the salvage and the degradation of purines. At 100 mM NaCl, the size of the total adenylate pool was reduced, but the «adenylate energy charge» was, by contrast, increased. No marked change in the size of guanylate pool occurred in response to NaCl. The rate of conversion of [8- 14 C] adenosine to adenylate was decreased by NaCl, but that to nucleic acids was not significandy affected. In contrast to the results obtained with maize root tips (Peterson et al., 1988), salt stress did not increase the catabolism of [8- 14 C] hypoxanthine. Most of the radioactivity from [8- 14 C] adenosine was accumulated in adenine while that in [8- 14 C] hypoxanthine remained unaffected in Sonneratia alba . The low capacity of the purine-degradation system might ensure a supply of purine bases and nucleosides that can be utilized as substrates for the rapid and efficient biosynthesis of nucleotides by the salvage pathways.