The Study Of Acute And Chronic Toxicity Of Methamphetamine On Rat

BACKGROUNDMethamphetamine (MA) is known as methylamphetamine, which belongs to the amphetamine-type stimulants (ATS). This drug is one of psychotropic substances which are controlled by our country. Because it has strong excitement actions and is easy to produce, it was abused rapidly after being applied to clinic. Though methamphetamine is a new drug, it was widely abused for its instant and long time effect. The most important feature of it is that people will be addicted for the first use. So it is called the Drug Lord. Recently the drug abuse gets more and more serious in the word and many major cities in China. Experts agreed that this drug will spread to all round the world and will become to one of the main drugs in 21st century. The global investigation of United Nations Office on Drugs and Crime (UNODC) showed that by 2004, there were 30 million people abuse in the world. The drugger's age become lower ages and the sex to trend feminize, even to rich. Recently the drugs of usurpation get more and more serious. We can know a lot of druggers from the magazines. It brings the disasters not only to the person in the physiology and the mind but also to the family and the society.The drug was found to have much psychic dependence, stimulant action on the central nervous system and sympathomimetic action. Some researches show that METH has severe injury to central nervous system, heart, kidney and skeletal muscle. And METH related heart diseases may be one of most important causes which induce druggers' sudden death. MTHE can cause vasoconstriction, vasospasm, tachycardia and high blood pressure through stimulating the peripheral nervous system to release huge amounts of catecholamines. This paper will carry out studies aimed at the toxicity of METH on heart and heart function.The METH-induced neurotoxicity is a complicated process that via many ways and mechanisms. The definite mechanism has not been known, so there is no effective method to prevent it. Our form discussing group found that the increased DDAH1 (Nω,Nω-diMAyl-L-arginine diMAylaminohydrolase, DDAH) in cerebral striatum and cortex which is a new regulatory enzyme to help the synthesis of NO. DDAH takes part in the catabolism of ADMA(asymmetric diMAylated L-arginine, ADMA), which is an endogenic inhibitor of NOS and can inhibit three subtypes of NOS. So DDAH regulates the activity of NOS and the synthesis of NO via adjusting the degradation of ADMA. The increase of the activity of NOS and the synthesis of NO can produce a great amount of Reactive Nitrogen Species (RNS), which will cause nitrosation, nitration and oxidation of proteins. And tyrosine nitration of proteins is an important posttranslational modification, leading to the gain or lose of protein functions. Protein is the final executant of life functions and the damage of the key proteins will bring about the lose of cell functions and the death of cells. Protein tyrosine nitration can be detected through identify 3-nitrotyrosine with special antibody. Many researches show that the nitration of key proteins plays an important role in many neurodegenerative diseases such as amyotrophic lateral sclerosis, Parkinson disease and Alzheimer's disease.On the basis of the previous two studies, we tried to utilize serum proteomicson to explore the METH toxicity on the whole organism. Serum proteomics is a new method, which is based on the normal protein expression profiles, to study the expression of serum total protein of the target population, look for differences between the proteins, and identify disease-related proteins, and then study the structures and functions of the proteins. It will help to study the pathophysiology mechanism of major diseases, find specific markers for early diagnosis, and research drug targets. Acute METH toxicity may lead to the changes in quantity and quality of body's protein. Which may reveal the mechanism of METH addiction at the molecular level. Besides, serum proteins may contain biological markers to distinguish physiological or pathological state of the body.OBJECTIVETry to study the toxicity of METH on rats and mechanism by histopathological, behavioral and neurochemical methods. On the bases of these results, proteomic method was used to screen serum proteins which may be biological markers to show the body damage associated with METH toxicity.METHODS1. Observation on heart toxicity of acute and chronic METH abuseWistar rats, weighting from 180g to 220g (the Laboratory Animal Center of Southern Medical University, China) were made two kinds of METH toxicity models-the acute one and chronic one. And the control group was set up respectively. The control group was injected with the identical volume of saline. The behavior changes, heart rate and blood pressure were observed. The rats were anaesthetized and killed and trunk blood was collected in serum tubes for the determination of serum Tn-T and BNP. Rapidly, the hearts were removed, weighed and gross observed. The changes of heart were observed by HE staining.2. The neurotoxicity in striatum of acute METH exposed ratsWistar rats were made acute METH toxicity models.And set up the control group which was injected with the identical volume of saline. The rats were anaesthetized and killed. Rapidly, the striatum were removed on ice. To detect the concentration of NO in striatum homogenate by nitrate reductase method; To detect the activity of NOS with colorimetry; To detect the contents of 3-nitrotyrosine with western blot.3. Proteomic profiling of serum proteins associated with METH-induced toxicityProteins of serum were separated by two-dimensional gel electrophoresis. The different expressed proteins were identified by tandem mass spectrum and data base search.RESULTS1. In acute METH toxicity models, the times of stereotyped behavior were increased obviously, mainly including quest, shinny, nasil and so on; The grades of behavior were increased obviously (P<0.001); The heart rate and mean arterial pressure(MAP) increased significantly than the control groups; The gross observation of hearts were normal; local myofibril fracture and dissolution, as well as contraction band necrosis were found in HE staining section; The content of Tn-T in serum rose obviously (P<0.05), but that of BNP increased without statistical significance.2. In chronic METH toxicity models, the times of stereotyped behavior were increased obviously, mainly including quest, shinny, nasil and so on; The grades of behavior were increased obviously (P<0.001); The heart rate and MAP increased significantly than the control groups; The gross observation of hearts were normal; acidophilic degeneration, vacuolar degeneration, local myofibril fracture and dissolution, as well as fibrosis were found in HE staining section; The content of Tn-T and BNP in serum increased significantly(P<0.05).3. In the striatum of acute METH exposed rats, the content of NO and the activity of NOS are increased, and the content of protein with 3-nitrotyrosine was increased significantly (P<0.001). 4. There are 16 spots expressed differently in serum of rats, and ten of them are significant via tandem mass spectrum and data base search. There are three increased spots, and they are Glial fibrillary acidic protein, haptoglobin (Hp) and Carcinoembryonic antigen-related cell adhesion molecule 3 precursor. And there are six decreased spots, which are GTP-binding protein Rab-3D, Testis-specific serine kinase substrate, Ras-related protein Rab-3A, Sodium/hydrogen exchanger 3, Advanced glycosylation end product -specific receptor precursor and Alpha-1-inhibitor 3 precursor.CONCLUSION1. Both acute and chronic METH toxicity can cause obvious damage on heart, inducing the increase in heart rate and blood pressure, as well as the content of serum Tn-T. The long-time use of METH can increase the content of BNP, that means the heart function has been effected. While the short-time ones can not influence the heart function.2. METH can increase the content of NO and the activity of NOS in the striatum, then increase the content of protein with 3-nitrotyrosine, which may cause the damage or lose of protein functions. It may be one of the main mechanisms in METH-induced neurotoxicity.3. There are 16 spots expressed differently in serum of acute METH exposed rats, and ten of them are significant via tandem mass spectrum and data base search. These proteins mainly participate in cytoskeleton reconsitution, signal transduction pathway /transport/binding, and regulation of enzyme activity. The injury of these proteins may be the main mechanism of METH-induced toxicity. Additionally, the change of proteins in serum may be one of index to indicate METH-induced toxicity.