【作者】 于光艳；

【导师】 孙志伟；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2008， 博士

【摘要】 甲醛、苯是主要的室内空气污染物,具有来源广、毒性大、污染水平高、污染时间长等特点。近年的研究表明装饰装修材料中释放的甲醛、苯等挥发性有机物可以引起不良建筑物综合征,高污染水平的甲醛和苯很可能是儿童过敏性哮喘、白血病等重大疾病发病率升高的诱因,但具体机制还不清楚。目前对甲醛和苯的研究多局限于高浓度,单一污染物,而对于二者联合毒性及其机制,尤其是幼鼠骨髓细胞毒作用机制的研究还没有相关报道。本研究在建立气态甲醛/苯单独和混合后吸入染毒小鼠模型基础上,采用细胞生物学、细胞遗传学、分子生物学和免疫学等实验方法,检测幼鼠骨髓细胞酶活性、CFU-F、膜电位、DNA损伤、细胞周期、细胞凋亡、以及凋亡相关蛋白表达。结果显示:一定浓度下,甲醛和苯可以引起小鼠外周血WBC、PLT数量降低;骨髓有核细胞及CFU-F数量降低;骨髓细胞SOD、GSH-Px活性降低,MDA含量增加;可增强MPO活性及促进其蛋白表达;引起骨髓细胞DNA损伤;线粒体膜电位下降;骨髓细胞S期阻滞;诱导细胞凋亡及凋亡相关蛋白CytC、Bax、Caspases-9、Caspases-3的表达增强,Bcl-2蛋白表达减弱;且一定浓度的甲醛和苯联合染毒时具有协同作用。结果提示甲醛和苯可以造成幼鼠骨髓细胞氧化损伤及DNA损伤,影响细胞周期进程,诱导细胞凋亡。本研究为探讨甲醛联合苯对幼鼠骨髓细胞毒性及其相关机制,筛选室内甲醛、苯暴露人群的早期生物学标志,进行甲醛、苯暴露人群的健康监护、慢性中毒的危险度评价提供实验依据。更多还原

【Abstract】 Formaldehyde and benzene have become the two most important indoor air pollutants released by decoration. High level concentration of formaldehyde and benzene may cause severely diseases, such as asthma ,leukemia, et al.However, the mechanisms of pathogenic associated with them are not well-known. Presently, some researches about formaldehyde and benzene are focused on high level concentration of them , or a single pollantant of them. The reports of combining toxic mechanism of them are not enough, especially researches about toxic effect of neonatal mice bone marrow cells and the relative mechanism are not reported.This paper was designed to study damage mechanisms of combined toxicity of formaldehyde and benzene on bone marrow cell (BMC) of neonatal mice.The experiments were performed ICR male mice, weighted 13±1 g. Both formaldehyde and benzene were given to ICR male mice by static inhalation for 15 days and 2 h/d. Changes of peripheral blood were detected by blood analysis technology. CFU-F countings was measured by using cuture method in vitro and detected numbers of bone marrow karyote. The oxidative damage including SOD activity, MDA content and GSH-Px activity was examined by biochemical method.The activity of metabolic enzyme: myeloperoxidase (MPO) was detected by biochemical method. Expression of MPO protein was measured by immunocytochemical method. DNA damage was examined by Micronucleus method and SCGE method. The change of mitochondrion membrane potential (MMP) was detected by Flow cytometry (FCM). Changes of cell cycle and apoptosis were measured by FCM. The expression of apoptotic related proteins in mitochondrial pathway was observed by immunocytochemical method. Results as follows:1. Effect of combined toxicity of formaldehyde and benzene on mice spleen coefficient and liver coefficient.Spleen coefficient of each group was decreased with dose increasing; liver coefficient of each group was increased with dose increasing. But both were no obvious change compared with control group and among different dose groups.2. Effect of combined toxicity of formaldehyde and benzene on peripherial blood.With the dose increased, WBC countings in the groups treated with formaldehyde and benzene were decreased. WBC countings in median and high dose formaldehyde groups, benzene groups and combined groups had obvious change compared with control group (P<0.05);and which in high dose formaldehyde groups, benzene groups and combined groups had obvious change compared with low and median dose groups(P<0.05). PLT countings in the groups treated with formaldehyde and benzene were decreased as the dose increased. PLT countings in median and high dose formalhehyde groups, median and high dose benzene groups and combined groups decreased significantly compared with those in negative controls(P<0.05); and which in high dose combined groups had obvious change compared with low and median dose groups(P<0.05). WBC and PLT countings in combined groups decreased significantly compared with those in benzene groups (P<0.05). Three doses level of low, median and high have not interactions by combined effect analysis. RBC counting and hemoglobin content were also decreased with dose increasing, but there both were no obvious change compared with control group. RBC counting and hemoglobin content have not significantly difference with formaldehyde group and benzene group.3. Effect of combined toxicity of formaldehyde and benzene on numbers of bone marrow karyote and CFU-F countings.With the dose increased, numbers of bone marrow karyote were decreased. Numbers of bone marrow karyote in high dose formaldehyde group, median and high dose benzene group and combined group had obvious change compared with control group(P<0.05); and which in high dose formaldehyde groups and combined groups had obvious change compared with low and median dose groups(P<0.05), which in median dose combined groups had obvious change compared with low dose groups(P<0.05), and which in high dose benzene groups had also obvious change compared with low dose groups(P<0.05). CFU-F countings were decreased as the dose increased. And CFU-F countings in high dose formalhehyde groups, high dose benzene groups and combined groups decreased significantly compared with those in negative controls(P<0.05);and which in high dose formaldehyde groups, benzene groups and combined groups had obvious change compared with low and median dose groups(P<0.05), which in median dose combined groups had obvious change compared with low dose groups,too(P<0.05). Numbers of bone marrow karyote and CFU-F countings in median and high dose combined groups decreased significantly compared with those in formaldehyde group and benzene group(P<0.05). Three doses level of low, median and high have not interactions by combined effect analysis.4. Oxidative damage of BMC of neonatal mice induced by combined toxicity of formaldehyde and benzeneWith the increase of exposure dosage, SOD activity decreased and has significantly differences with control group (P<0.05); and which in high dose formaldehyde groups had obvious change compared with low dose groups(P<0.05), which in median and high dose benzene groups had obvious change compared with low dose groups(P<0.05), and which in high dose combined groups had also obvious change compared with low and median dose groups(P<0.05). SOD activity in combined groups decreased significantly compared with those in formaldehyde group and benzene group (P<0.05). Low and median doses have not interactions ,but high doses has interactions(P<0.05)by combined effect analysis. It was suggested that formaldehyde and benzene in high dose has synergistic effect. MDA content increased in all dose groups compared with control group (P<0.05); and which in high dose formaldehyde groups, benzene groups and combined groups had obvious change compared with low and median dose groups(P<0.05), which in median dose combined groups had also obvious change compared with low dose groups(P<0.05). MDA content in combined groups increased significantly compared with those in formaldehyde group and median and high dose benzene group(P<0.05). GSH-Px activity also decreased in each other groups except for low-dose formaldehyde group compared with control group(P<0.05); which in high dose benzene groups had also obvious change compared with low dose groups(P<0.05). And GSH-Px activity in combined groups had obvious change compared with those in formaldehyde and benzene groups (P<0.05).Three doses level of low, median and high have not interactions by combined effect analysis.5. Changes of MPO activity and expression of BMC of neonatal mice induced by combined toxicity of formaldehyde and benzeneWith the increase of exposure dosage, MPO activity in all dose groups increased compared with control group (P<0.05); and which in high dose benzene groups had obvious change compared with low dose groups(P<0.05), which in median dose combined groups had also obvious change compared with low dose groups(P<0.05),and which in high dose combined groups had also obvious change compared to low and median dose groups(P<0.05). MPO activity in combined groups increased significantly compared with those in formaldehyde group and median and high benzene group (P<0.05). Low and median doses have not interactions ,but high doses has interactions(P<0.05)by combined effect analysis. It was suggested that formaldehyde and benzene in high dose has synergistic effect.Positive cell rate in all dose groups increased compared with control group (P<0.05); and which in median and high dose formaldehyde groups ,benzene groups and combined groups had obvious change compared with low dose groups(P<0.05), which in high dose groups had also obvious change compared with median dose groups(P<0.05). Positive cell rate in combined groups increased significantly compared with those in formaldehyde group and median and high benzene group (P<0.05). Three doses level of low, median and high have interactions by combined effect analysis.It shows that formaldehyde and benzene has synergistic effect.6. Effect of combined toxicity of formaldehyde and benzene on the level of DNA.With the increase of exposure dosage, micronuclear cell rate in all dose groups increased compared with control group (P<0.05); and which in high dose formaldehyde groups and combined groups had obvious change compared with low dose groups(P<0.05). Micronuclear cell rate in combined groups increased significantly compared with those in formaldehyde group and benzene group (P<0.05). Low and median doses have not interactions ,but high doses has interactions(P<0.05)by combined effect analysis. It shows that formaldehyde and benzene has synergistic effect.DNA damage rate in each group except all formaldehyde groups and low-dose benzene group also increased compared with control group (P<0.05); and which in high dose benzene groups and combined groups had obvious change compared with low dose groups(P<0.05). DNA damage rate in combined groups increased significantly compared with those in formaldehyde group (P<0.05).7. Effect of combined toxicity of formaldehyde and benzene on mitocho- ndrion membrane potential.With the increased of exposure dosage, MMP in high-dose formaldehyde group, median and high-dose benzene groups and combined groups were decreased compared with control group(P<0.05); and which in high dose formaldehyde groups and combined groups had obvious change compared with low dose groups(P<0.05), which in median dose combined groups had also obvious change compared with low dose groups(P<0.05). MMP in combined groups decreased significantly compared with those in formaldehyde and benzene group (P<0.05). Three doses level of low, median and high have not interactions by combined effect analysis.8. Changes of cell cycle of BMC of neonatal mice induced by combined toxicity of formaldehyde and benzene.The rate of cell in G0/G1 and G2/M phase in all dose groups decreased as the dose increased. The rate of cell in G0/G1 phase in combined groups has significantly difference with control groups (P<0.05).The number of S cells in high-dose formaldehyde group, benzene groups and combined groups increased compared with control group (P<0.05); and which in high dose formaldehyde groups had obvious change compared with low dose groups.The number of S cells in combined groups has no obvious change compared with those in formaldehyde and benzene group. Three doses level of low, median and high have not interactions by combined effect analysis.9. Apoptotic rate of neonatal mice induced by combined toxicity of form- aldehyde and benzeneApoptotic rate in all dose groups increased as the dose increased. Apoptotic rate in high-dose formaldehyde, high-dose benzene and combined groups increased compared with control group (P<0.05); and which in median dose benzene groups and combined groups had obvious change compared with low dose groups(P<0.05), which in high dose groups had also obvious change compared with low and median dose groups(P<0.05).Apoptotic rate in combined groups increased significantly compared with those in formaldehyde and high-dose benzene group (P<0.05). Three doses level of low, median and high have not interactions by combined effect analysis.Apoptotic index increased as the dose increased, but only which in high dose combined groups had obvious change compared with control groups(P<0.05).10. Expression of apoptosis related proteins in mitochondrial pathway induced by combined toxicity of formaldehyde and benzeneExpressions of CytC, Bax, Caspase-9 and Caspase-3 in high-dose formaldehyde, benzene and combined groups increased significantly compared with control group (P<0.05) , but expression of Bcl-2 decreased;and which in median and high dose formaldehyde groups, benzene groups and combined groups had obvious change compared with low dose groups (P<0.05),which in high dose groups had also different degree change compared with low dose groups (P<0.05).Expression of CytC, Bax, Caspase-3 and Bcl-2 in three doses level of low, median and high have interactions by combined effect analysis. It shows that formaldehyde and benzene has synergistic effect.It was suggested that formaldehyde and benzene at certain doses could induce neonatal mice bone marrow Oxidative damage, DNA damage, apoptosis and changes of cell cycle. A certain concentration of combined formaldehyde and benzne can cause interactions on SOD activity, MPO activity and protein expression, micronulear cell rate, and expression of CytC, Bax, Bcl-2 and Caspase-3 protein.更多还原