Proteomics Study Of Rat Lung Damage Induced By Formaldehyde Inhalation

Formaldehyde (FA) is a ubiquitous chemical found in garments, food, indoor air, gasoline and diesel exhausts, etc. It belongs to volatile organic chemicals with a boiling point at -19℃ and is easy to volatilize at indoor temperature. Therefore gaseous FA is more dangerous to health compared with liquid FA. Its critical health effects on health include sensory irritation, allergenic reaction and a variety of genotoxic actions. Long-term inhalation experiments in rats have confirmed that FA is a nasal carcinogen. Formaldehyde is classified as a "identified human carcinogen". The WHO identifies it as a class 1A carcinogen at 2004. At present, the toxic effects of FA are clear, however, the underlying mechanism is little understood. We speculated that protein molecules were involved in harmful effects of FA, and used proteomics technique to explore the toxicity mechanism of FA and to find indicative protein markers for its toxicity.Along with the successful completion of the human genome project (HGP) on April 14,2003 and the comimg of post genomic era, proteomics has become an important area of research in life science. Coined by Wilkins and Williams in 1995, the word "proteome" indicates "the entire proteins expressed by a genome". The conception of "functional proteome" is a basis of proteome study[7]. At present, researches concerning FA are mainly focused on environment monitoring, epidemiological investigating and DNA damage at molecular level, while studies at proteome level have not been reported.Respiratory system is the major target of FA. It was reported that after rats inhale FA, the volume of FA is higher in the lung than in the blood, brain, liver and kidney[8]. Much attention to the effects of FA is paid on the upper airway, in particular the nose, much less concern is focused on the pulmonary toxicity. The aim of this study was to apply the method of proteomics to analyze protein changes of lung of rats in response to FA inhalation and probe into the mechanism and function of protein alteration. The acquired results as follows:1. The technique of two-dimensional electrophoresis(2-DE) for proteome analysis of normal rat lung was established.By control and optimizition of many experimental conditions in protein determination, samplepreparation, first dimension and second dimension electrophoresis, the 2-DE patters with good resolution and reproducibility were obtained. The technique could detect about 720 spots on 18cm IPG strip (pH3-10L) for normal rat lung, and the mean match rate of protein spots was 72.2%. The technique of 2-DE which has been established is the basis for later work.2. The protein alteration patterns in rat lung induced by FA exposure were obtained.Firstly we established an inhalation model of rat exposed to gaseous FA in different concentrations (0> 15-20> 32-37 mg/m3 ;4h/d,15d).Secondly we obtained protein alteration patterns in rat lung induced by FA exposure. Lung samples were solubilized and separated via two-dimensional electrophoresis (2-DE), and gel patterns were scanned and analyzed for detection of differently expressed protein spots. 13 proteins were altered significantly in FA exposure group. Of the altered proteins, 4 proteins were up-regulated significantly in 32-37 mg/ro3 FA group, 2 proteins was down-regulated significantly in 32-37 mg/m3 FA group, 4 proteins were expressed only in 32-37 mg/m3 FA group; 1 protein was all up-regulated significantly in two FA exposure groups and presented dose-reponse relationship, 2 proteins were all up-regulated significantly in two FA exposure groups, but didn't have dose-reponse relationship.3. The altered proteins on 2-DE patterns were identified by mass spectrometry, and relationship between these proteins and FA toxicity was approached.6 protein spots were succeedly identified by in-gel digestion, MALDI-TOF-MS detection and NCBInr protein database searching. They were aldose reductase, LIM protein, glyceraldehyde-3-phosphate dehydrogenase, chloride intracellular channel 3 and cyt p-450, two of them are chloride intracellular channel 3. The result of AR activity test was in accordance with its alteration on 2-DE pattern. Pathology study showed that expression variety of the glyceraldehyde-3-phosphate dehydrogenase and cyt p-450 on 2-DE pattern was involved in ultrastructural damage of rat lung.4. Further study on the mechanism and function of aldose reductase (AR) increase were carried out.Rats which were exposed to FA could produce the reaction of oxidative stress because of the formation of reactive oxygen species (ROS) and the falling of anti-oxidative ability, so significantly up-regulated AR could reduce the rat lung damage induced by oxidative stress. By cell culture,...