{"title":"Heavy metals altered the xenobiotic metabolism of rats by targeting the GST enzyme: An in vitro and in silico study","authors":"","doi":"10.1016/j.tox.2024.153946","DOIUrl":null,"url":null,"abstract":"<div><p>Among all the heavy metals, Pb, Cd, and As are the most harmful pollutants in the environment. They reach into the organisms via various levels of food chains i.e. air and water. Glutathione-s-transferase (GST, E.C. 2.5.1.18), a key enzyme of xenobiotics metabolism, plays an important role in the removal of several toxicants. The present study aimed to evaluate any inhibitory action of these heavy metals on the GST enzyme isolated from the hepatic tissues of rats. A 10 % (w/v) homogenate of rat liver was prepared in cold and centrifuged at 4 °C at 9000xg for 30 min. The supernatant was collected and kept frozen at −20 °C or used fresh for carrying out different experiments. The activity of GST was monitored spectrophotometrically at 340 nm using 220 μg of soluble protein with varying equal substrate concentrations (0.125–2 mM) in phosphate buffer (50 mM, pH 6.5). To assess the impact of heavy metals on the enzyme activity, different concentrations of Cd (0–0.6 mM) and Pb (0–2 mM) were added to the reaction mixture followed by monitoring the residual activity. The optimum temperature and pH of rat liver GST were found to be 37 °C and 6.5, respectively. The K<sub>m</sub> value for GST was 0.69 mM and the V<sub>max</sub> was found to be 78.67 U/mg. The Cd and Pb significantly altered the kinetic behaviour of the enzyme. The V<sub>max</sub> and K<sub>cat</sub>/K<sub>m</sub> parameters of GST were recorded to be decreased after interaction with Cd and Pb individually and showed a mixed type of inhibition pattern suggesting that these inhibitors may have a greater binding affinity either for the free enzyme or the substrate-enzyme complex. These metals showed a time-dependent enzyme inhibition profile. Cd was found to be the most potent inhibitor when compared to other treated metals; the order of inhibitory effect of metal ions was Cd>Pb>As. The <em>in silico</em> ion docking analysis for determining the probable interactions of Cd and Pb with fragmented GST validated that Cd exhibited higher inhibition potential for the enzyme as compared to Pb. The results of the present study indicated that exposure of both the Cd and Pb may cause significant inhibition of hepatic GST; the former with higher inhibitory potential than the later. However, As proved to be least effective against the enzyme under the aforesaid experimental conditions.</p></div>","PeriodicalId":23159,"journal":{"name":"Toxicology","volume":null,"pages":null},"PeriodicalIF":4.8000,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Toxicology","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0300483X24002270","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PHARMACOLOGY & PHARMACY","Score":null,"Total":0}
引用次数: 0
Abstract
Among all the heavy metals, Pb, Cd, and As are the most harmful pollutants in the environment. They reach into the organisms via various levels of food chains i.e. air and water. Glutathione-s-transferase (GST, E.C. 2.5.1.18), a key enzyme of xenobiotics metabolism, plays an important role in the removal of several toxicants. The present study aimed to evaluate any inhibitory action of these heavy metals on the GST enzyme isolated from the hepatic tissues of rats. A 10 % (w/v) homogenate of rat liver was prepared in cold and centrifuged at 4 °C at 9000xg for 30 min. The supernatant was collected and kept frozen at −20 °C or used fresh for carrying out different experiments. The activity of GST was monitored spectrophotometrically at 340 nm using 220 μg of soluble protein with varying equal substrate concentrations (0.125–2 mM) in phosphate buffer (50 mM, pH 6.5). To assess the impact of heavy metals on the enzyme activity, different concentrations of Cd (0–0.6 mM) and Pb (0–2 mM) were added to the reaction mixture followed by monitoring the residual activity. The optimum temperature and pH of rat liver GST were found to be 37 °C and 6.5, respectively. The Km value for GST was 0.69 mM and the Vmax was found to be 78.67 U/mg. The Cd and Pb significantly altered the kinetic behaviour of the enzyme. The Vmax and Kcat/Km parameters of GST were recorded to be decreased after interaction with Cd and Pb individually and showed a mixed type of inhibition pattern suggesting that these inhibitors may have a greater binding affinity either for the free enzyme or the substrate-enzyme complex. These metals showed a time-dependent enzyme inhibition profile. Cd was found to be the most potent inhibitor when compared to other treated metals; the order of inhibitory effect of metal ions was Cd>Pb>As. The in silico ion docking analysis for determining the probable interactions of Cd and Pb with fragmented GST validated that Cd exhibited higher inhibition potential for the enzyme as compared to Pb. The results of the present study indicated that exposure of both the Cd and Pb may cause significant inhibition of hepatic GST; the former with higher inhibitory potential than the later. However, As proved to be least effective against the enzyme under the aforesaid experimental conditions.
期刊介绍:
Toxicology is an international, peer-reviewed journal that publishes only the highest quality original scientific research and critical reviews describing hypothesis-based investigations into mechanisms of toxicity associated with exposures to xenobiotic chemicals, particularly as it relates to human health. In this respect "mechanisms" is defined on both the macro (e.g. physiological, biological, kinetic, species, sex, etc.) and molecular (genomic, transcriptomic, metabolic, etc.) scale. Emphasis is placed on findings that identify novel hazards and that can be extrapolated to exposures and mechanisms that are relevant to estimating human risk. Toxicology also publishes brief communications, personal commentaries and opinion articles, as well as concise expert reviews on contemporary topics. All research and review articles published in Toxicology are subject to rigorous peer review. Authors are asked to contact the Editor-in-Chief prior to submitting review articles or commentaries for consideration for publication in Toxicology.