Study On The Interaction Between Haloacetonitrile And Nucleophilic Biomolecules Based On Computational Simulation And In Vitro Experiments

With the popularization of chloramine disinfection technology and the increase of pollutants and natural organic matter(NOM)in natural water bodies,nitrogen-containing disinfection by-products(N-DBPs)have gradually become a major category of disinfection by-products after drinking water disinfection.Studies have shown that N-DBPs has a stronger toxic mechanism in organisms,and the cytotoxicity and genotoxicity induced by N-DBPs are 2 to 3 orders of magnitude higher than that of C-DBPs.Haloacetonitriles(HANs)are a typical type of N-DBPs.Although their concentration in water is lower than that of controlled C-DBPs such as THMs and HAAs,they are more toxic.In recent years,domestic and foreign research on HANs has mainly focused on exploring its formation mechanism and controlling its production.There are relatively few studies on its toxicology,and its toxicity mechanism is not clear,and further exploration is needed.HANs are electrophilic and toxic substances that can covalently bond with the nucleophilic groups of biomolecules.The common nucleophilic biomolecules in organisms include glutathione(Glutathione,GSH),N-acetyl-L-Cysteine(NAC),cysteine(Cysteine,Cys)and DNA.Studying the interaction between HANs and nucleophilic biomolecules will help understand their toxicological mechanism,correctly assess their health risks,and clarify the HANs that need priority control.This thesis systematically studied the interaction of 9 kinds of HANs and three different kinds of nucleophilic substances(GSH,NAC,Cys)and four kinds of DNA bases(guanine,adenine,thymine,cytosine)through quantum chemical calculations.At the same time,6 kinds of HANs and GSH reacted products under physiological conditions were detected by liquid chromatography-triple quadrupole mass spectrometer and the possible reaction sites were estimated.Mainly draw the following conclusions:(1)The reaction between HANs and nucleophilic biomolecules is a bimolecular nucleophilic substitution reaction(S_N2 reaction),that is,nucleophilic biomolecules attack theα-C of electrophilic HANs,so that the halogen atom onα-C is replaced,the reaction is an exothermic process;HANs with different types and numbers of halogen atoms have different reactivity.The overall reactivity is:monohaloacetonitrile>dihaloacetonitrile>trihaloacetonitrile,iodoacetonitrile>bromoacetonitrile>chloroacetonitrile;for the sulfhydryl end reaction site,the reactivity of HANs and three nucleophilic substances is:NAC>Cys>GSH;for the amino terminal reaction site,the reactivity of the two nucleophilic substances is:Cys>GSH;for the amino terminal and sulfhydryl terminal of the same substance,the reactivity with HANs is sulfhydryl>amino.(2)In vitro experiments,the detected products are mainly mono-substituted products,and di-substituted products are almost undetectable.This indicates that the reaction between HANs and GSH mainly takes place in a substitution reaction;and the relative molecular weight of the detected products can determine the reaction.The type of reaction is S_N2;the secondary mass spectrum combining GSH and an adduct of GSH and HANs can determine the site of the addition reaction,and finally determine that the reaction site of HANs and GSH is mainly located on the sulfhydryl group of GSH,that is,HANs preferentially Reacts with the sulfhydryl group on GSH.These conclusions drawn from in vitro experiments are consistent with the results of quantum chemistry calculations.(3)The reaction between HANs and DNA bases is a S_N2 reaction which is an exothermic process;HANs with different types and numbers of halogen atoms have different reactivity.The overall reactivity is:monohaloacetonitrile>dihaloacetonitrile>trihaloacetonitrile,iodoacetonitrile>bromoacetonitrile>chloroacetonitrile;the selected12 base positions,the most easily reacted with HANs are guanine N7,cytosine O2,adenine N1,and the most difficult position to react is thymine O4,N3,O2 site.The main factors affecting the reactivity of different sites are electronegativity and steric hindrance.In summary,HANs have different reactivity with different nucleophilic substances,resulting in different biological toxicity and health risks.In addition,this study revealed the influence of the type and number of halogen substitutions of HANs on the reactivity.The above research helps to understand the toxic mechanism of HANs,correctly assess their health risks,and clarify the priority control of HANs.