| BackgroundAcrylonitrile is an important raw material and product in the petrochemical and chemical fiber industry,as well as an important harmful substance in cigarette smoke,plastic packaging,and medical products.Acrylonitrile is highly toxic.It is a recognized animal carcinogen and easily invades the human body through the respiratory tract,skin and gastrointestinal tract.Until now,the prevention and therapy of acrylonitrile-induced toxicity,especially neurotoxicity,is still a serious public and clinical problem.Acrylonitrile primarily attacks the nervous system,where astrocytes are the target cells.The metabolism of acrylonitrile in vivo is closely related to sulfur,and H2S is an important functional molecule in the sulfur-metabolizing system.H2S is the third gasotransmitter along with carbon monoxide and nitric oxide,which participates in the regulation of multiple physiological systems.Previously,we have found that exogenous H2S attenuates the acute toxicity induced by acrylonitrile in primary rat astrocytes,but the physiological basis for such function exerted by exogenous H2S remains unclear,that is,whether it is due to that endogenous H2S balance is impaired by acrylonitrile.We speculate that acrylonitrile disrupts H2S biosynthesis,leading to a decrease in endogenous H2S contents,which is involved in acrylonitrile-induced acute toxicity.ObjectivesThis study uses various biological models,including Caenorhabditis elegans（C.elegans）,primary rat astrocytes and rats,to evaluate the role of gasotransmitter H2S in acrylonitrile-induced toxicity both in vivo and in vitro.This study aims to clarify the toxicity mechanism of acrylonitrile and discovering new targets for clinical therapy for acrylonitrile poisoning,and to provide preliminary evidence for exploring the health-monitoring indicators for chronically acrylonitrile-exposed populations.Methods（1）Toxicity screening of acute acrylonitrile exposure in C.elegans:C.elegans were maintained and used to observe the effects of acrylonitrile（1,2.5,5,10,25,50,100 m M,1 h）on the survival rate of C.elegans.Then,the effects of acrylonitrile（5,10,25 m M,1 h）on the movement ability,including moving speed and body bends,and the morphology of dopaminergic neurons of C.elegans were observed.In addition,after treatment with acrylonitrile（10 m M）for 1 h,the lifespan,the content of reduced glutathione（GSH）and the ratio of GSH/oxidized glutathione（GSSG）of C.elegans were detected.（2）The effects of acute acrylonitrile exposure on the endogenous H2S content and H2S-producing enzymes in C.elegans:Wild type worms were treated with acrylonitrile（5,10,25 m M）for 1 h,H2S contents were detected by methylene blue spectrophotometry method and lead acetate test paper;H2S-producing activity was detected by methylene blue spectrophotometry method;the m RNA levels of H2S-producing enzymes and H2S-metabolizing enzymes were detected by q RT-PCR.（3）The effects of regulating H2S biosynthesis on the acrylonitrile-induced acute toxicity in C.elegans:The gene mutant and knockout strains which encode H2S-producing enzymes,including cth-1 knockout,cth-2 mutant and mpst-1 knockout strains were used.After acrylonitrile treatment（10 m M,1 h）,q RT-PCR was used to detect the m RNA level of H2S-producing enzymes;lead acetate test paper was used to detect the H2S contents;methylene blue spectrophotometry method was used to detect H2S-producing activity;the changes in the survival rate and lifespan of C.elegans were observed.（4）The effects of acute acrylonitrile exposure on the endogenous H2S contents and H2S-producing enzymes in primary rat astrocytes:The primary rat astrocytes were isolated and cultured.After treatment with different concentrations of acrylonitrile（1,2.5,5,10 m M）for 12 h,cell viability and cytotoxicity were detected by MTT reduction and LDH leakage assay respectively;Az Mc fluorescent probe was used to detect H2S contents;western blotting was used to detect the protein level of H2S-producing enzymes,including cystathionine-β-synthase（CBS）and 3-merecaptopyruvate sulfurtransferase（3-MPST）.（5）The effects of regulating H2S biosynthesis on the acrylonitrile-induced acute toxicity in primary rat astrocytes:Pharmacological and genetic regulation manners targeting H2S-producing enzymes were used,including CBS inhibitor aminooxyacetate（AOAA,50 and 100μM）treatment for 12 h,3-MPST-targeted si RNA transfection,CBS activator S-adenosyl-L-methionine（SAM,25 and 50μM）pretreatment for 3 h,adenovirus carrying3-MPST transfection and Na HS（400μM）pretreatment for 1 h.Then,MTT method was used to detect the changes in cell viability caused by acrylonitrile in primary rat astrocytes.（6）The effect of subchronic acrylonitrile exposure on the endogenous H2S contents and H2S-producing enzymes in rats:50 rats were randomly divided into five groups,including control group（normal saline）,6.25,12.5,25,and 50 mg/kg acrylonitrile groups.Saline and acrylonitrile were injected intraperitoneally（one time/day,five days/week,for four weeks）.Rat body weight,and rat brain and liver organ coefficients were observed.Methylene blue spectrophotometry method was used to detect H2S contents in serum and plasma,cerebral cortex and liver of rats.Western blotting was used to detect the protein level of H2S-producing enzymes in rat cerebral cortex and liver.Results（1）Toxicity screening of acute acrylonitrile exposure in C.elegans:Acute acrylonitrile exposure（5,10,25 m M for 1 h）decreased survival rate,moving speed and body bends,and impaired dopaminergic neurons morphology of C.elegans in a concentration-dependent manner.Acrylonitrile（10 m M）treatment for 1 h had no effects on the lifespan of C.elegans,while significantly decreased GSH contents and the ratio of GSH/GSSG.（2）The effects of acute acrylonitrile exposure on the endogenous H2S contents and H2S-producing enzymes in C.elegans:Acute acrylonitrile exposure（5,10,25 m M for 1 h）significantly decreased the H2S contents and inhibited the H2S-producing activity of 3-MPST,yet had no significant effects on the H2S-producing activity of total CBS and CSE activity.Acute acrylonitrile exposure significantly increased the m RNA level of cth-2 and decreased that of mpst-1,while had no effects on the other H2S-producing and H2S-metabolising genes.（3）The effects of regulating endogenous H2S biosynthesis on the acrylonitrile-induced acute toxicity in C.elegans:cth-1 knockout didn’t affect the endogenous H2S contents and survival rate induced by acute acrylonitrile exposure in C.elegans.cth-2 mutation and mpst-1 knockout significantly decreased the H2S contents and corresponding H2S-producing activity,and significantly decreased the survival rate and lifespan of C.elegans induced by acrylonitrile.（4）The effects of acute acrylonitrile exposure on the endogenous H2S contents and H2S-producing enzymes in primary rat astrocytes:Acrylonitrile treatment（1,2.5,5 and 10 m M for 12 h）decreased cell viability and increased LDH leakage in a concentration-dependent manner in astrocytes.Notably,acrylonitrile treatment significantly decreased the H2S contents and inhibited the expression of CBS and 3-MPST.（5）The effects of regulating endogenous H2S biosynthesis on the acrylonitrile-induced astrocytic toxicity:CBS inhibitor AOAA and 3-MPST knockdown significantly decreased the acrylonitrile-induced cell viability in astrocytes.In contrast,CBS activator SAM and 3-MPST overexpression,as well as Na HS pretreatment significantly increased the acrylonitrile-induced cell viability in astrocytes.（6）The effects of subchronic acrylonitrile exposure on the endogenous H2S contents and H2S-producing enzymes in rats:Subchronic acrylonitrile exposure significantly inhibited the body weight growth of rats and decreased the organ coefficients of rat liver.Subchronic acrylonitrile exposure significantly increased H2S levels in rat plasma and serum,but not in liver.The endogenous H2S level in rat cerebral cortex was also significantly increased upon acrylonitrile treatment,paralleled by the expression of the major H2S-generating enzymes（CBS and 3-MPST）in brain.Protein levels of CBS and cystathionine-γ-lyase（CSE）were significantly increased,whereas levels of 3-MPST were significantly decreased in rat liver.ConclusionsAcute acrylonitrile exposure impaired the endogenous H2S biosynthesis,thus decreasing H2S contents,which contributes to the acute acrylonitrile toxicity,especially acute neurotoxicity.H2S may be a novel chemopreventive and therapeutic target for acute acrylonitrile poisoning.Acute and subchronic exposure to acrylonitrile have differential effects on endogenous H2S.Subchronic acrylonitrile exposure increased the H2S contents in rat blood and cerebral cortex,but not in the liver,which paralleled by the expression profile of H2S-producing enzymes.These results suggest that H2S may serve as a novel biomarker for chronically acrylonitrile-exposed populations. |
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