Experimental Study On Mechanism Of Toxic Effects Of Melamine On Infant Urinary System And Potential Risk Prevention

Chapter 1 InductionThe outbreak of infant disease induced by melamine-tainted formula in the summer of 2008 is a food disaster in China. By the means of effective treatments, at present the melamine-related urinary stones in most infants have been successfully passed and renal function in a few of infants with acute renal injury has returned to normal. However, nobody could know what the degree of risk of occurrence of renal and bladder disorders in the future for these hundreds of thousands of infants who were affected by high-does melamine and whose organ systems were still developing would be. In addition, the study of the mechanism of infant disease induced by melamine-tainted formula is useful to further understand the toxicity of melamine for humans. Therefore, my doctoral program was performed to study the mechanism of infant nephrolithiasis and renal injury, their long-term influences, and corresponding preventive measures.The ceftriaxone-associated nephrolithiasis is rarely observed in the world. However, within the recent one month two children ceftriaxone-associated urinary stone samples were received in our hospital. Then worrying about a possibility of a drag safety accident of ceftriaxone sodium occurring prompted us to identify these stone compositions and investigate risk factors of this stone formation as early as possible. My doctoral program also enclosed this study.Chapter 2 Compositional analysis method of melamine-related urinary stoneSection 1 Identification of melamine-related stone by Infrared spectroscopyObjective:To identify stone compositions of urinary stone induced by melamine-tainted formula in a child by infrared spectroscopy.Methods:The melamine-related mixtures were respectively prepared by mixing powders of melamine with cyanuric acid or uric acid in equimolar ratio. The melamine-related precipitates mimicking its related stone formation were respectively prepared by mixing melamine with cyanuric acid or uric acid in water at the given conditions. Subsequently, the melamine-related mixtures and precipitates, and a child urinary stone induced by melamine-tainted formula were analyzed by infrared spectroscopy.Results:The wave-number positions of powder mixtures of melamine-cyanuric acid and melamine-uric acid were a combination of these of their individual ingredients. The typical wave-number positions of melamine were showed in two melamine-contained mixtures. In contrast, these positions were disappeared or shifted greatly in the two melamine-related precipitates. In total, the spectrum of precipitates of melamine with cyanuric acid and uric acid had significantly differences with their powder mixtures. The child urinary stone induced by melamine-tainted formula showed the nearly same of the wave-number positions as melamine-uric acid precipitate.Conclusion:Our results indicated the compositions of urinary stone from a child affected by melamine-tainted formula were melamine and uric acid, in a form of melamine-uric complex.Section 2 Quantitative analysis of melamine-related urinary stone compositions by High Performance Liquid ChromatographyObjective:To establish a method to quantitatively analyze melamine-related stone compositions and analyze urinary stone from a child affected by melamine-tainted formula.Methods:Separation and quantification of melamine, cyanuric acid, and uric acid from urine or urinary stone was performed using a Agilent 1100 HPLC system, a Agilent HC-C18 (4.6x250 mm,5 μm) column, and a Agilent Zorbax Entend-C18 (4.6x12.5 mm, 5 μm) guard column.100% phosphate buffer (10 mmol/1, pH 7.5) was used as mobile phase, the wavelength was 200 nm, and flow rate was 1.0 min/min. The stone from a child affected by melamine-tainted formula after sample preparation was analyzed by this method.Results:The Limit of detection (LOD), linear range, Relative Standard Deviation (RSD), and average recover rate of melamine were 0.005 μg/ml,0.05-50 μg/ml,< 10%,> 90%. The LOD, linear range, RSD, and average recover rate of cyanuric acid were 0.025 μg/ml, 0.1-50 μg/ml,< 10%, and> 90%. The LOD, linear range, RSD, and average recover rate of uric acid were 0.025 μg/ml,0.1-50 μg/ml,< 10%, and> 90%. The stone from a child affected by melamine tainted formula consisted of melamine and uric acid (molar ratio= 2.1:1), they accounted for 89.6% of whole stone weight.Conclusion:The High Performance Liquid Chromatography we established could be used to simultaneously quantitatively analyze melamine, cyanuric acid, and uric acid from urine or urinary stone. This method was convenient, fast, and accurate. The melamine-related stone from a child consisted of melamine and uric acid with a molar ratio of 2.1:1.Chapter 3 Study injury toxicity of melamine to renal tubular and bladder epithelial cell linesObjective:To study injury toxicity of melamine to epithelial cell of renal proximal tubule, renal distal tubule, and bladder, and its preventive measures.Methods:Human renal proximal tubular epithelial cell line (HK-2), Canine renal distal tubular epithelial cell line (MDCK), and human bladder epithelial cell line were (SV-HUC-1) respectively treated with 625、1250、2500, and 5000 μg/ml melamine. After 12 h culture malondialdehyde (MDA) content in culture solution, reactive oxygen species (ROS) content within cell, and cell apoptosis rate of these treated cells were measured by TBA assay, DCFH-DA assay, and Annexin V-FITC/PI assay respectively. After 24 h culture the cell proliferation rates were measured by CCK-8 assay. When Hk-2, MDCK, and SV-HUC-1 cells were co-treated by 2500 μg/ml melamine with super oxygen dehydrogenises (SOD) at 6.25-400 U/ml, catalase (CAT) at 6.25-400 U/ml, and mannitol at 500-4000 μg/ml respectively for 24 h. The cell proliferation rates were measured by CCK-8 assay.Results:The LD50 of Hk-2, MDCK, and SV-HUC-1 cells treated by melamine for 24 h were 3817、3118, and 4101 μg/ml. After 12 h culture, the MDA concentrations in culture solution, the contents of ROS within cell, and cell apoptosis rates of Hk-2, MDCK, and SV-HUC-1 cells treated by 2500 and 5000 μg/ml melamine were significantly higher than these treated without melamine (P< 0.05). The proliferation rates of Hk-2, MDCK, and SV-HUC-1 cells treated by 2500μg/ml melamine with CAT and mannitol could be , obviously improved than cells treated with melamine along (P< 0.05). The SOD had no such a protective effect.Conclusion:The results showed melamine at high-dose could injury epithelial, cell of renal proximal tubule, renal distal tubule, and bladder. Its toxicity was associated with cell oxidative stress and apoptosis. The cell injury caused by a high-dose melamine could be alleviated by CAT and mannitol.Chapter 4 Study mechanism of melamine-induced toxic effects in urinary systemObjective:To study mechanism of toxic effects in urinary system induced by melamine-tainted formula in infants.Methods:Sprague-Dawley male rats were fed with diets containing 0.3%,1%, and 3% melamine for 4 weeks. Sprague-Dawley male rat with hyperuricosuria were established by intraperitoneal injection of potassium oxonate and uric acid, this model rats was fed with diets containing 1% melamine for 4 weeks. Sprague-Dawley male rats were fed with half protein-contained diets containing 1% melamine for 4 weeks. Sprague-Dawley male rats were fed with diets containing 1% melamine and 0.01% cyanuric acid for 4 weeks. Before or after melamine feeding was stopped,24 h urinary lithogenic factors, blood biochemical indexes, stone incidence, stone compositions, and histomorphology in all rats were respectively measured.Results:Compared with rats fed with normal diets, in rats fed with 3% melamine 24 h urinary volume and calcium concentration were significantly increased, urinary oxalate and citrate concentration were significantly decreased, urinary pH, urate, and phosphate concentration remained unchanged, and injury degree of renal function and parenchyma was significantly aggregated. In hyperuricosuria rats fed with 1% melamine, crystals were found within the renal parenchyma, and injury degree of renal function and parenchyma was significantly aggregated compared with normal rats fed with the same diets. In rats fed with a half protein-contained diet containing 1% melamine, the injury degree of renal parenchyma but renal function was slightly aggregated. In rats fed 1% melamine and 0.01% cyanuric acid, crystals were also found within the renal parenchyma, and injury degree of renal function and parenchyma was significantly aggregated compared with normal rats fed with the same diet. The stone incidences in rats fed with 0.3%,1%,3% melamine were 0,20%, and 50%, and all stone consisted of melamine and a trace of uric acid. The stone incidence in hyperuricosuria rats fed with 1% melamine was 30%. The stone incidence of rats fed with a half protein-contained diet containing 1% melamine was 10%. And the stone incidence of rats fed with 1% melamine and 0.01% cyanuric acid was 20%. All stones consisted with melamine and a trace of uric acid except of stones in rats fed with melamine and cyanuric acid which contained a trace of cyanuric acid..Conclusion:The, urinary stone and renal injury could be induced in male rats fed with diets containing high-dose melamine for 4 weeks, and 24 h urinary concentration of some lithogenic factors but urate and pH in rats could be changed. The degree of renal injury in rats fed with diets containing high-dose melamine could be significantly aggregated and crystals could be formed in renal parenchyma on conditions of hyperuricosuria and melamine adulterated with a few of cyanuric acid other than low-protein diet. It seems stone incidence of rats fed with diets containing high-does melamine could not influenced by conditions of hyperuricosuria, low-protein diet, and melamine adulterated with a few of cyanuric acid but the melamine content in diets.Charpter 5 Study of potential kidney injury and bladder cancer induced by melamine and investigating corresponding preventive measuresObjective:To study toxicity in kidney and bladder after a long-term exposure of melamine and investigate corresponding preventive measures.Methods:130 weaning male F344 rats were randomly dividend into five groups,20 rats in group A were fed with normal diets for 48 weeks,20 rats in group B were fed with diets containing 3% melamine for 24 week and then with normal diets for 24 weeks,30 rats in group C were fed with diets containing 3% melamine for 48 weeks,30 rats in group D were fed with diets containing 3% melamine and 3% green tea powers for 48 weeks, and 30 rats in group E were fed with diets containing 3% melamine and 0.02% genistein for 48 weeks. At 24th week,10 rats in A, C, D, E groups were executed. At 48th week, remained rats in all groups were executed. After execution, blood was collected to test renal function, the weights of urinary stones, both kidney organs, and bladder organs were measured, morphologies of kidney and bladder organs were examined by direct observation and histological sections. The compositions of urinary stones were analyzed by FTIR and HPLC.Results:At 24th week, plasma creatinine in group C, D, and E were significantly increased compared with group A (p< 0.05), and plasma creatinine in group D was significantly reduced compared group C (p< 0.05). Dilated renal distal tubules with or without crystals, lymphocytic infiltration and fibrosis surrounding affected tubules were observed. The stone incidences in group C, D, and E rats were 100%,90% and 100%, average stone weight in these groups was> 0.1 g, and stone compositions in these groups all consisted of melamine and a trace of uric acid. The coefficients of bladder weight/body weight in group C, D, and E were increased compared with group A (p< 0.05), obvious proliferations of bladder epithelial cells in all rats in group C, D, and E were observed, but no neoplasm was observed in lumens. At 48th week, plasma creatinine and coefficients of both kidney weight/body weight in group B, C, D, and E were significantly increased compared with group A except of plasma creatinine in group B (P< 0.05), and plasma creatinine in group D was significantly reduced compared with group C (p< 0.05). Dilated renal distal tubules, lymphocytic infiltration and fibrosis in renal interstitium which were obviously aggregated than observation at 24th week, and sclerosed glomerruli were observed. The stone incidences in group B, C, D, and E rats were 5%,44.4%, 33.3% and 52.6%, average stone weight in these groups was< 0.01 g. The coefficients of bladder weight/body weight in group C, D, and E were increased compared with group A (P< 0.05). The incidences of bladder cancer in group B, C, D, and E were 22.2%,72.3%,22.2%, and 31.6%, and the incidences of bladder cancer in group B, D, and E were significantly decreased than group C (P< 0.05).Conclusion:Urinary stones, injures of renal function and parenchyma, and carcinogenesis of bladder epithelial cells could be induced in weaning F344 male rats after feeding diets containing high-does melamine for a year, of which urinary stones were decreased or disappeared, injures of renal function and parenchyma were aggregated, and bladder proliferating epithelial cells became carcinogenesis at a period of last half year. The injures of renal function and parenchyma carcinogenesis of bladder epithelial cells were remained in rats fed firstly with diets containing high-does melamine and then fed with normal diet respectively for half a year, although the degree of renal injures were alleviated and incidence of bladder cancer were decreased. Diets containing high-does tea powers could alleviate renal injures induced by high-does melamine, and Diets containing high-does tea powers or a trace of genistein could reduce incidences of bladder cancer induced by 3% melamine.Chapter 6 Characterizing ceftriaxone-associated urinary stone and in-vitro investigating its possible predisposing factorsSection 1 Characterizing ceftriaxone-associated urinary stone inChildrenObjective:To investigate the physiochemical characteristics of urinary stone induced by ceftriaxone.Methods:Two children ceftriaxoen-associated urinary stone samples were received for compositional analysis in our hospital in April 2012, of which one was from a boy whose clinical data was not available, and the other was from a body who suffered acute lower abdominal pain and vomiting after treatment with ceftriaxone for 5 d in nearly April. Ultrasound demonstrated a stone in right upper ureter. Computed tomography showed right upper ureteral stone, which was radiolucent on plain abdominal radiography. After a conservative treatment for 3 d, the stone in right upper ureter was spontaneously passed, which was confirmed by ultrasound and intravenous urography. The two received stone samples were analyzed by infrared spectroscopy, and scanning electron microscopy and energy disperse spectroscopy for component analysis.Results:The compositions of two stone samples were free ceftriaxone and calcium ions combined with a molar ratio of 1:1.Conclusion:The ceftriaxone could induce urinary stone in children. This special stone was radiolucent imaging, and it is composed of calcium ceftriaxone salt.Section 2 Investigating risk factors of ceftriaxone-associated stone formation using an in vitro methodObjective:To Investigate risk factors of ceftriaxone-associated stone formation using an in vitro method.Methods:The experiment was designed based on stone compositions and reference values of some urinary electrolytes and ceftriaxone. The effects of crystallized time, ceftriaxone, calcium, citrate, and pH on degree of calcium ceftriaxone crystallization in water solution with 0.16 mol/1 ionic strength were investigated. The degree of calcium ceftriaxone crystallization was defined as the depletion of calcium/total calcium in solution × 100%.Results:The degree of calcium ceftriaxone crystallization was crystallized time-, ceftriaxone-, calcium-, citrate-, and pH dependent, of which crystallized time, ceftriaxone and calcium were promoting factors while citrate and acidified pH were inhibiting factors.Conclusion:Urinary obstruction, high does of ceftriaxone, hypercalcinuria, hypocitraturia, and higher urine pH are possible predisposing factors of ceftriaxone-associated stone formation.