Chronic Effect Of Methylamine On Endothelium Of Rats And Rabbits

Semicarbazide-sensitive amine oxidase (SSAO) is a group of enzymes containing copperand quinine and sensitive to semicarbazide. Increased serum SSAO activities were found inpatients with diabetic complications, vascular disorder and heart disease. Methylamine (MA) isboth an exogenous (present in cigarette smoke, wine and foods) and an endogenous amine. It is ametabolite of diverse compounds, including epinephrine, creatine, nicotine, and sarcosine. MA isa physiological substrate for SSAO and can be deaminated to formaldehyde (FA), hydrogenperoxide (H₂O₂) and ammonia. FA is an extremely reactive chemical. It can interact withmonoamines or amides to form methylene bridges and with proteins and single-strand DNAproduce irreversible covalent cross-linking complexes. H₂O₂ is a major reactive oxygen species,which is potentially cytotoxic. FA and H₂O₂ may act synergistically in causing cellular damage.Although MA itself is relatively nontoxic to cultured endothelial cells, it can lead to endothelialinjury in the presence of SSAO. However, SSAO inhibitor (MDL-72974A) can protect the cells.Therefore, chronic effect of MA in vivo may be a potential risk factor for human vasculardisease. Endothelium is a thin layer of epithelium that lines the heart, blood vessels, lymphvessels, and the serous cavities of the body. In addition, endothelium is a reactive endocrineorgan. It can secret various vasoactive agents and participate in the regulation of cell function.Endothelial dysfunction is one of the initial steps of atherosclerosis. Therefore, in order toexplore the effect of MA and its metabolite on endothelium, MA was chronically administered toWistar rats and New Zealand rabbits.Methods:1. Wistar rats and New Zealand white rabbits (half male and half female) were randomlydivided into two groups, 6 rabbits and 10 rats as control (CON), and 12 rabbits and 20 ratsfor methylamine hydrochloride (MA)-treated groups. The MA group received a low dose of100 mg/kg body weight (rabbit) and 50 mg/kg of body weight (rat) of MA, respectively. Thetime of experiment was 6 months. The rabbits were weighed every other week; the rats wereweighed every two weeks. All dosage of the ig was adjusted by the body weight of animals.Samples were collected during the period of the ig.2. SSAO enzyme activity and FA concentration in the plasma of rabbits and rats were measuredby high performance liquid chromatography (HPLC).3. Endothelial cells in the arterial blood of rabbits were detected by FCM.4. Nitric oxide concentration in the serum of rabbits and in the plasma of rats was measured by Nitrate Reductase Method.5. The expression of v Wf in the thoracic aorta of rats was detected by immunohistochemistry.6. Ultrastructure of endothelial cells in the ascending aorta and endocardium of rabbits and ratswere observed by transmission electron microscopy.Result:1. The weight of rats (MA group) in 1, 2, 3, 4, 5 month compared witti the CON group, whichwas not statistically significant. The weight of rabbits (MA group) in 1, 2, 3, 4, 5, 6 monthscomparied with the CON group, which was not statistically significant.2. SSAO activities of the rabbits (MA group) at 1, 2, 3, 4, 5, 6 month compared with the CONgroup, it was statistically significant (P＜0.05). In rats, after treatment for 6 months, SSAOactivity in MA group was 1.85±0.29 nmol·h^-1·ml^-1, SSAO activity in CON group was 1.69±0.17 nmol·h^-1·ml^-1. There was no statistically significant between the two groups.3. FA concentration of rabbits (MA group) at 1, 2, 3, 4, 5, 6 month were higher compared withCON group. How.ever, it is statistically significant only at 1, 2, 3 month (P＜0.05). In rats,after treatment for 6 months, FA concentration in MA group compared with CON group, itwas no statistically significant.4. The NO cocentrations in the serum of rabbits (MA group) at 3, 4, 5, 6 month compared withCON group were higher; however, it is statistically significant only at 3 and 4 months (P＜0.05). In rats, after treatment for 6 months, NO concentration in the plasma of MA groupcompared with CON group, it was no statistically significant.5. 23 weeks after rabbits were intragastricly administered. Blood samples from CON group(n=5) had a lower number of CECs (17±10/150,000 cells) compared with MA group(n=6)(67±37/150,000 cells)(P＜0.05)6. The expression of vWf was relatively weak in the thoracic aorta of rats (CON group).However in the rats (MA group), strongly positive expression of vWf in endothelium,subendothelium and perivascular matrix was always observed.7. As to the ultrastructure of endothelial cells, the endothelial cells in the ascending aortas ofrabbits (MA group) contained multiple intranuclear inclusions. Karyopyknosis andkaryorrhexis could be seen in the endothelial cells in the endocardium of rabbits (MA group).However, the endothelial cells in the CON group showed normal endothelial morphology.The endothelial cells in the ascendingaortas of MA group (rats) contained intracytoplasmicextensive vacuolization. Endothelial cells in the endotcardium of MA group (rats) showedkaryopyknosis and irregular nucleus. However, the endothelial cells in the CON group showed normal endothelial morphology.Conclusion and significance:1. Chronic supply of MA of low dose in the rats and rabbits has no effect on their weight.2. Chronic supply of MA of low dose in the rabbits can elevate plasma SSAO activity of rabbits;however, chronic supply of MA of low dose in the rats has no significantly effect on theirSSAO activity.3. Formaldehyde concentration increased significantly at first three months in MA group ofrabbits compared with CON group, it shows that chronic supply of MA to rabbits can affectthe metabolism of FA. This phenomenon might resulted from that FA concentration might beelevated for increasing of MA metabolism; and then, returned to normal level for theelevation of formaldehyde dehydrogenase and/or interation of formaldehyde with tissue. Thelater reaction can induce injury of tissue and cause the happening of diseases. The same levelof formaldehyde in the MA and CON group in rats might result from the relative low level ofSSAO activity in rats.4. Chronic supplement of MA to the rats and rabbits might result in the endothelial injury.