苯暴露小鼠呼出气中生物标志物的筛查

Screening of biomarkers in exhaled breath of mice exposed to benzene

目的基于呼出气在线分析系统筛查苯暴露小鼠呼出气中的生物标志物。方法30只8周龄的雄性C57BL/6小鼠随机分为对照组(0 mg/m^(3))、3、32、324、648、1296 mg/m^(3)六个组,采用静式吸入法进行苯染毒28 d。染毒结束后检测小鼠外周血血常规和全血还原型谷胱甘肽(GSH)含量,并利用体外细胞实验检测小鼠血浆染毒HL60细胞的丙二醛(MDA)含量;采用二次电喷雾电离源高分辨质谱(SESI-HRMS)收集小鼠呼出气数据,进行苯代谢产物和氧化应激小分子代谢物的靶向分析,并筛查呼出气中苯暴露及毒效应的生物标志物。结果苯暴露小鼠的外周血细胞数目出现了不同程度的降低,其中白细胞(WBC)的下降最为显著,32和324 mg/m^(3)组的WBC与对照组相比分别下降了27.76%和52.87%(P<0.05)。同时,与对照组相比,324 mg/m^(3)组中小鼠外周血细胞的GSH含量降低了13.16%(P<0.05),324 mg/m^(3)组小鼠的血浆处理HL60细胞后MDA含量增加了18.11%(P<0.05)。小鼠呼出气中的苯酚、氢醌/儿茶酚、苯三酚和反-反式粘康酸(t,t-MA)4种苯代谢产物,可作为苯暴露生物标志物(R^(2)>0.8,P<0.001);5种氧化应激相关的小分子代谢产物(ω-羧脂肪酸C_(5)H_(10)O_(3)、ω-羧脂肪酸C_(6)H_(12)O_(3)、谷氨酸、半胱氨酸和MDA)的峰值强度随着苯浓度的增加而增加(P<0.05),且与WBC数量降低呈负相关(P<0.001),可作为苯毒效应的生物标志物。结论呼出气中的苯酚、氢醌/儿茶酚、苯三酚和t,t-MA可作为苯暴露标志物;ω-羧脂肪酸C_(5)H_(10)O_(3)、ω-羧脂肪酸C_(6)H_(12)O_(3)、谷氨酸、半胱氨酸和MDA可能作为苯效应标志物。

Objective To screen the biomarkers in the exhaled breath of mice exposed to benzene by using exhaled breath online analysis system.Methods Thirty 8-week-old male C57BL/6 mice were randomly divided into six groups(0,3,32,324,648,and 1296 mg/m^(3))and treated with benzene vapour for 28 days.At the end of the exposure,the peripheral blood cell counts and blood glutathione(GSH)were detected.The content of malondialdehyde(MDA)in HL60 cells treated by mice plasma was examined.Exhaled breath data from mice were collected by Secondary electrospray ionization source high resolution mass spectrometry(SESI-HRMS).Targeted analysis underlying benzene metabolites and oxidative stress metabolites was performed to screen the biomarkers in exhaled breath.Results After benzene exposure,the number of peripheral blood cells was decreased in different degrees,particularly in the white blood cells(WBC)number.The WBC in 32 and 324 mg/m^(3) groups was declined by 27.76%and 52.87%,respectively compared to that in control group(P<0.05).Meanwhile,compared with the control group,the GSH content of peripheral blood cells from 324 mg/m^(3) group decreased by 13.16%(P<0.05).In addition,MDA content was increased by 18.11%in HL60 cells treated with plasma from 324 mg/m^(3) group mice(P<0.05).The phenol,hydroquinone/catechol,benzenetriol and trans,trans-Muconic acid(t,t-MA)in the exhaled gas of mice could be used as biomarkers for benzene exposure(R^(2)>0.8,P<0.001).The peak intensity of five small molecular metabolites related to oxidative stress(ω-carboxylic fatty acid C_(5)H_(10)O_(3),ω-carboxylic fatty acid C_(6)H_(12)O_(3),glutamate,cysteine and MDA)increased with the increase of benzene concentration(P<0.05),which was negatively correlated with WBC decline(P<0.001),suggesting that these molecules mignt be used as biomarkers of benzene-induced toxicity.Conclusions Phenol,hydroquinone/catechol,benzenetriol and trans,trans-Muconic acid(t,t-MA)in exhaled breath of mice could be used as biomarkers for benzene exposure;ω-carboxylic fatty acid C_(5)H_(10)O_(3),ω-carboxylic fatty acid C_(6)H_(12)O_(3),glutamate,cysteine and MDA might be used as markers of benzene-induced toxicity.