【作者】 叶新；

【导师】 杨旭；

【作者基本信息】 华中师范大学 ， 动物学， 2013， 博士

【摘要】 甲醛,是一种主要的化工产品,广泛的存在于环境中,是…种常见的空气污染物。近年来甲醛已经被WHO (World Health Organization)组织划分为人类A1级别致癌物,已经明确指出甲醛可以导致鼻咽癌,引起哮喘等。并且美国NTP (National Toxicology Program)组织也已经将其划分为导致白血病的元凶,但是其导致远距离器官产生的毒性机理仍然不清楚,特别是其如何导致骨髓细胞中产生毒性机理的研究还值得去解决。本研究通过氧化应激反应平衡的变化来揭示这个问题。一、内环境甲醛浓度变化对BALB/c小鼠的毒性影响将不同浓度的甲醛溶液依据小鼠体重(6,12,18,30和60μg/g)通过尾静脉注射入小鼠体内,实验发现当BALB/c小鼠内的甲醛浓度平衡受到影响时,将会引起组织器官发生一系列的氧化应激反应,在肺、肝、脑、肾脏和骨髓细胞等器官都检测到氧化损伤情况的发生。其中活性氧自由基(Reactive oxygen species, ROS),除了肺组织外,在其他组织器官中均随着注射甲醛浓度的上升ROS值在不断增大。肺组织中没有检测到ROS增加,可能与其是呼吸的重要器官,组织结构有所不同,对ROS耐受性较强有关：对于膜脂过氧化重要的产物丙二醛(Malondialdehyde, MDA),发现组织器官随着注射甲醛浓度的上升MDA值在不断增大,并且在注射甲醛浓度为12μg/g时候,除脑组织以外肝,肺,肾和骨髓细胞中MDA值都是最大值;并且在反应细胞中DNA-蛋白质交联(DNA protein crosslinks, DPC)指标中,肝,肺,脑,肾和骨髓细胞,在浓度6～18μg/g时(在12μg/g时DPC值最高),DPC系数都随之增大,但在注射高浓度组30-60μg/g时反而减少。这些结果说明：1)微量的甲醛就能够导致机体内产生氧化应激反应从而导致机体器官的损伤,这是一个新发现。2)而且在微量甲醛的影响下,体内应该存在一个甲醛应答的阈值；从本实验来看,体内甲醛浓度达到12μg/g时似乎达到了这个闽值,引发连锁反应改变体内甲醛浓度的平衡。当注入体内甲醛浓度增大时,机体通过缓冲反应又可以清除氧化应激分子,使体内甲醛重新达到平衡。3)进而推测,短时间急性甲醛染毒应该能导致相关毒理反应。二、甲醛口鼻式气态染毒对小鼠和大鼠的影响对与甲醛的气态染毒而言,无论啮齿类动物如大小鼠,或灵长类动物恒河猴等等,都是可以用来做实验对象的,目前还没有论断到底那种动物是最好的动物模型,而且也没有专门的用于甲醛气态染毒的模式实验动物。目前国际上研究气态甲醛暴露问题的实验动物,大多数都选择的是大鼠(如Wistar大鼠)来进行实验的,用小鼠来做为动物模型进行甲醛毒性研究还比较少。本实验研究显示,在气态甲醛浓度为3.0mg/m3条件下,Wistar大鼠的体内多个器官特别是鼻腔和骨髓中明显可以检测出(与实验对照组相比)生物标志物的改变。活性氧自由基(ROS)水平上升,并且与之对应的DNA-蛋白质交联水平(DPC)也显著上升；并且抗氧化性化合物还原性谷胱甘肽(Glutathione, GSH)水平在骨髓,肺和脾脏组织中都发生显著下降。这些结果说明大鼠在3.0mg/m3浓度下组织器官还是受到了氧化损伤的影响,其中在骨髓和鼻腔中比较明显。在肺和脾脏中没有直接检测到ROS,DPC产生变化,但仍然能够发现在抗氧化系统中发现GSH水平产生了变化。在气态甲醛3.0mg/m3的暴露条件下,同样的状况也可以在BALB/c小鼠的试验中发现：在鼻腔和骨髓组织中(与对照组相比),ROS和DPC指标显著上升而抗氧化损伤指标GSH也与之对应的显著下降；在肺和脾脏组织中,ROS和DPC未检测出明显变化,但也仍然检测出GSH指标出现下降趋势。通过对比Wistar大鼠和BALB/c小鼠的ROS, GSH和DPC,我们可以推断在甲醛气态染毒实验中,大鼠和小鼠产生的由甲醛刺激造成氧化损伤情况大致相同。虽然以前甲醛气态染毒所用的实验对象大部分是在鼠,但本实验证明用BALB/c小鼠所获得的毒理特征儿乎与大鼠一致,可以很好的替代大鼠用于染毒实验。三、甲醛口鼻式气态染毒致小鼠的骨髓及远距离毒性影响本实验通过甲醛对BALB/c鼠在短期且模拟职业暴露条件(8h/d,7day)吸入染毒,检测了甲醛对小鼠骨髓细胞的毒性。通过ROS, MDA,GSH,DPC和甲醛浓度(FA)等指标检测了甲醛对小鼠骨髓细胞的氧化损伤,通过给小鼠依据100mg/kg体重灌胃注入GSH溶液,建立了甲醛毒性抑制组；利用RT-PCR检测了其对在骨髓细胞中的造血细胞生长分化起重要作用的转化生长因子(TGF-p)和造血生长因子(GM-Csf)的表达的影响；然后义检测了骨髓细胞周期变化,并且做了骨髓病理切片验证实验结果。发现随甲醛染毒浓度升高(0,0.5,1.0,3.0mg/m3),肺、肝和骨髓细胞中的ROS,MDA和DPC值极显著升高(P<0.01),并致GSH值下降(P<0.01)；肝和骨髓组织中FA值也随之升高,肺组织中FA值没有变化。而外周血淋巴单核细胞中这些指标儿乎都没有变化。而且加入GSH抑制组后,能显著的降低甲醛暴露导致的机体氧化损伤。通过RT-PCR结果也显示随吸入甲醛浓度的升高,TGF-P和GM-Csf的表达量也随之升高。并且我们通过对骨髓细胞周期的检测,骨髓病理切片的检测也印证了吸入甲醛确实能导致骨髓细胞损伤。总体来说以上结果可以证明甲醛能致使小鼠骨髓细胞产生损伤,以及其能致使远距离器官产生毒性作用。从基因以及细胞周期等方面向我们提示,高浓度的甲醛暴露有增加患白血病的风险。当然我们需要更多更充分的证据来证明甲醛暴露和白血病的关系。更多还原

【Abstract】 Formaldehyde is a major chemical product, widely existence in the environment, and is a common air pollutant. In recent years, formaldehyde has been classified by WHO as carcinogen (A1level), and has made clear conclusion that formaldehyde can cause nasopharyngeal cancer, asthma and other diseases. US NTP organization has also considered formaldehyde leads to leukemia, but the mechanism how it causes toxicity of non-respiratory organs remains is unclear, particularly how it causes the toxicity of bone marrow cell is also worth to mark clear. In this study, the change of oxidative stress balance has been found related to the problem.1. The formaldehyde concentration in body influenced the toxicity of the Balb/c mice.Different concentrations formaldehyde solution which based the mouse body weight (6,12,18,30and60μg/g) was injected into the tail vein of mice, and then it was found that along with the formaldehyde concentration in the Balb/c mice is increased, the cells of organs can be found with a series of oxidative stress, while the oxidative injury was detected in the lung, liver, brain, kidneys, and bone marrow. The ROS (reactive oxygen species, ROS) level and oxidative damage were found increasing along with the rising in the concentration of formaldehyde in these organs except the lung. This phenomenon may relate to the fact that lung is a vital organ of the respiratory, organizational structure is different from others, so it has stronger tolerance against ROS. The malondialdehyde (malondialdehyde, MDA), is an important peroxidation product for membrane lipid, was found increased in the tissues and organs of the mice along with the injection of formaldehyde. When the injection concentration of formaldehyde reached at12μg/g, the MDA values of liver, lung, kidney and bone marrow cells reached to maximum. The DNA-protein cross-linking (DPC) in liver, lung, brain, kidney and bone marrow cells was also found increased at the formaldehyde levels of6～18μg/g (the highest DPC was at12μg/g), but decreased in the higher concentrations at30～60μg/g.These results indicate that:1) the trace quantities of formaldehyde generated in the body can lead to oxidative stress in body organs, which is a new discovery.2) Under the influence of trace formaldehyde, the body should have a formaldehyde response threshold. From our experimental results, the concentration of formaldehyde at12μg/g of the body may close to the threshold, triggering a chain reaction to change the balance of formaldehyde concentration in body. When injected formaldehyde concentration is increasing, the body can clear the adverse effects through the buffer and oxidative stress response elements, and then the body keeps formaldehyde rebalance.3) Further speculated that acute formaldehyde exposure can cause acute toxicity related reactions.2. The affect between Mice to Rats under the nose-mouth formaldehyde exposed. Exposed to the gaseous formaldehyde, whether rodents such as rats and mice, or primate rhesus monkey, etc., are all used to do the experiment, there is no confirmed which is the best kind of animal in toxicological experiments, and then no special model animals for gaseous formaldehyde exposured. The current international studies for gaseous formaldehyde exposure experiments, are most chosing the rat (for example Wistar rats) to carry out experiments, whereas use mice as animal models of formaldehyde toxicity studies is still relatively less. This study showed that using rats as animal model we have found the toxicity effects under exposure of3.0mg/m3gaseous formaldehyde. The biomarks in various organs of Wistar rats, particularly evident in the nasal cavity and the bone marrow can be positive detected compared with the control groups. The levels of reactive oxygen species (ROS) significantly increased, and the DPC also increased. As one of the biomarker of the antioxidant system, the reduced of glutathione (GSH) levels in the bone marrow, lung and spleen tissues were significantly decreased. These results indicated that3.0mg/m3formaldehyde exposure could induce oxidative damage on rat tissues and organs. Comparing to the nasal cavity, the bone marrow, lungs and spleen were not very sensitive (for example ROS, DPC changes did not obvious), but the changes of GSH could still be found. The same situations could be also found when Balb/c mice exposed to gaseous formaldehyde3.0mg/m3:in the nasal tissue and bone marrow (compared with the control group), the ROS and DPC index increased significantly and oxidative damage indicators corresponding GSH also decreased significantly; in lung and spleen tissues, the changes of ROS and DPC did not detected, but the GSH could still be detected a downward trend.By comparation of Wistar rats with Balb/c mice’s ROS, GSH and DPC, we can infer that, for gaseous formaldehyde exposure experiments, the rats and mice may have similar oxdative effects. During previous gaseous formaldehyde exposure experiments the rats were wildly used as subjects, with the present experiment that Balb/c mice had similar oxdative effects with rats’toxicological studies, we think mice can be a good alternative to rats for exposure experiments.3. Mouse bone marrow and other organ toxicity induced by formaldehyde exposure via nose-mouth inhalationIn this study, the experimental BALB/c mice were undertaken a short term and simulated occupational formaldehyde inhalation exposure (8h/d,7day), formaldehyde toxicity effects were detected on mouse bone marrow cells. ROS, MDA, GSH, DPC and other biomarkers have been used for testing formaldehyde induced oxidative damage on mouse bone marrow cells, according to100mg/kg body weight via gavage to mice injected, GSH was used for the establishment of formaldehyde toxicity suppression group. By RT-PCR detection, the formalydehy exposure to hematopoietic cells in the bone marrow had been found to play an important role in growth and differentiation of transforming growth factor (TGF-β) and hematopoietic growth factors (GM-Csf) during transcription; then the cell cycle changes were detected for bone marrow cells, and then bone marrow biopsy was undertaken to verify the pathological changes.Along with formaldehyde exposure concentration (0,0.5,1.0,3.0mg/m3) incresing, the biomarkers ROS, MDA and DPC values in lung, liver and bone marrow cells were significantly increased (P<0.01), and the value of GSH decreased (P<0.01); while the formaldehyde values of liver and bone marrow tissue were also increased (but lung tissue FA value does not change). The peripheral blood mononuclear cells in lymphoid almost no change for these biomakers. In GSH suppression group, the formaldehyde induced oxidative effects significantly reduced.The RT-PCR results also showed that, along with the formaldehyde concentration increasing, TGF-β and GM-Csf transcription also increased. The cell cycle detection of bone marrow cells and bone marrow biopsy also confirmed that the inhaled formaldehyde can really cause damage to bone marrow cells.Overall, these results suggst that formaldehyde can cause damage of bone marrow cells, as well as other organs cells. It can produce toxic effects via gene and cell cycle negative changes, suggesting high concentrations of formaldehyde exposure may increase the risk of leukemia. Of course, we still need more evidences to prove the relationship between formaldehyde and leukemia.更多还原