| Background:Melamine also called cyanurimide is used widely as basic organic chemical intermediate inseveral products. At present its most important role is as a raw material for melamine-formaldehyde resin (MF).Melamine has been a cause for food adulteration concern. One example is in the United Statespet food contamination in2007nother unforgettable incident was in2008China’s milkpowder incident occurred resulting in more than290,000sick children of whom6died due tourinary tract (UTI) and kidney complications and it caused serious poisoning incidents. As aresult it has attracted worldwide attention. When the State Food and Drug Administrationissued the results of dairy products melamine contamination in China it caused panic amongthe general public, consequently Chinese dairy industry suffered heavy losses. U.S. FDApublished a report on melamine tolerable dietary intake value (TDI) and pointed out thataccording to the current studies it is hard to establish the standard of the infant formula whichcan help gain public confidence on the safety of milk products in China. Studying biological andtoxicological effects of melamine is not merely for the purposes of research, but also for thesocial stability needed. However, there is no detailed or more systematic report yet, becausethe horizon of the already published body of literature concerning melamine spans differentresearch objectives and targets.Aim:Although a lot has been discovered about the characteristics of this compound, but there arestill some problems some of which are; the mechanisms of kidney stone pathogenesis is notfully understood, the effect of this compound on other organ systems, the synergistic effect ofmelamine and its homologues is not well explained. Thus, this study was aimed to determine the toxicity of melamine and one of its homologues cyanuric in vitro in cell culture and in orderto determine IC50values at cellular level. And then later, based on the in-vitro studies,investigate the dual and individual toxicity of these two compounds in various organs of wistarrats to gain information on the toxicity of these substances in tissues other than kidney andurinary system related organs.Methodology:In this project laboratory animals (wistar rats) are studied for potential effects of melamine andboth melamine-cyanuric acid administration through oral means. In this study, initially themedian lethal doses (IC50) of melamine and cyanuric acid were determined using the MTTcolorimetric assay, after first adding different concentrations of melamine and/or cyanuric acidin NRK-52E AND293T cell lines in vitro and then calculating the cell proliferation activity. Laterthese doses were used to administer to lab rats, one month old wistar rats in this case, for aperiod of4weeks. Then according to the cell toxicity of melamine and cyanuric acid in vivo.Melamine and cyanuric acid were adminstered to experimental animals. One month old wistarrats were randomLy divided into four groups, each comprising four rats (2male and2female).And adminstration was every24hours for a period of four weeks through oral gavage.Adminstration per group proceeded as；Group1:3mL normal saline; Group2: the3mLmelamine (180mg/Kg bwt.,); Group3:3mL cyanuric acid (150mg/Kg bwt.,) and, Group4: was1.5mL melamine (180mg/Kg bwt.)+1.5mL cyanuric acid (150mg/Kg bwt.). A month later, thegroup of experimental animals were sacrificed, weighed, and the rat organs such as: kidney,bladder, liver, stomach, heart, spleen, ovaries, uterus and testis were collected and their weightwas recorded. Also, each organ was divided in to three parts and three methods (hematoxylinand eosin staining, ELISA method, and HPLC-MS method) were used to study the pathologicaland toxicological effects of melamine, cyanuric acid and the combination of the two. One partwas preserved in4%paraformaldehyde and the other two parts in-80oC refrigerator. Thenkidney, stomach, ovary and testes were stained with HE stain for histo-pathology. The secondpart of the kidney, stomach, uterus and testes were homogenized and concentration ofmelamine and cyanuric acid was determined in each organ using competitive binding ELISA. The third part of each preserved organ was homogenized too and the specimen was analyzed usingHPLC-MS method and the concentration of melamine was determined in each group and finallythe concentration of melamine was expressed in ng per g of rat organ in each organ.Results:1. The NRK cell toxicity studies of both Mel and Cya revealed that the IC50values of thesecompounds according to cell culture studies on NRK-52E cells were1.892mg/mL for melamineand4.2mg/mL for cyanuric acid. And the293T cell toxicity studies of both Mel and Cyaresulted in IC50values of2.069mg/mL for melamine and3.7mg/mL for cyanuric acid.2. The histopathologic studies revealed the formation of yellowish brown melamine-cyanuratecrystals formation in the medulla-cortex area in the kidneys and loss of chief cells in the liningof the stomach in the group administered with melamine plus cyanuric acid. And melamine andcyanuric acid alone showed no significant morphological changes in various organs.3. Organ homogenates tested with ELISA method showed that the melamine concentration,four groups of melamine content is57.30±4.44ng/g, and63.02±7.10ng/g, and55.23±6.27ng/g and59.23±4.60ng/g, and the cyanuric acid content is59.79±5.24ng/g, and58.44±1.58ng/g, and56.74±1.29ng/g and64.14±1.10ng/g, inthe kidney, stomach, uterus and testis respectively.4. Organ homogenates tested by HPLC-MS method r showed that the various organs ofmelamine in the group of melamine, cyanuric acid group and melamine and cyanuric acidcombined group were higher than those in the control group. In the group adminstered withmelamine and cyanuric acid the kidney showed the highest content of melamine (296ng/g),and renal involvement in the melamine group was most obvious (11.83ng/g), followed by theuterus (9.6ng/g) and liver (6.8ng/g). Conclusion:1. Histological study and HPLC-MS experiments showed that the combined effects of melamineand cyanuric acid resulted in the formation of melamine-cyanurate complexes, and that thetoxicity of this substance is far greater than the individual toxicity of melamine or cyanuric acidalone. And the kidney is the most affected organ.2. HPLC-MS detection results showed that melamine is present not only in the kidneys, but theuterus and liver too will have a large number of melamine deposition.3. Cell culture experimental results showed that, melamine is more toxic than its homologuecyanuric acid. However, at lower doses both have very low toxicity.4. The enzyme-linked immunosorbent assay is not a reliable method for the detection of thecontent of melamine and its analogues in tissues, hence high performance liquidchromatography-mass spectrometry is a better choice. |
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