Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007.

Sixteen animals affected in 2 outbreaks of pet food-associated renal failure (2 dogs in 2004; 10 cats and 4 dogs in 2007) were evaluated for histopathologic, toxicologic, and clinicopathologic changes. All 16 animals had clinical and laboratory evidence of uremia, including anorexia, vomiting, lethargy, polyuria, azotemia, and hyperphosphatemia. Where measured, serum hepatic enzyme concentrations were normal in animals from both outbreaks. All animals died or were euthanized because of severe uremia. Distal tubular lesions were present in all 16 animals, and unique polarizable crystals with striations were present in distal tubules or collecting ducts in all animals. The proximal tubules were largely unaffected. Crystals and histologic appearance were identical in both outbreaks. A chronic pattern of histologic change, characterized by interstitial fibrosis and inflammation, was observed in some affected animals. Melamine and cyanuric acid were present in renal tissue from both outbreaks. These results indicate that the pet food-associated renal failure outbreaks in 2004 and 2007 share identical clinical, histologic, and toxicologic findings, providing compelling evidence that they share the same causation.     

三聚氰胺与三聚氰酸混合物对小鼠肾脏的影响

40只21日龄SPF雄性昆明小鼠随机分为4组,三聚氰胺(MA)和三聚氰酸(CA)按1∶1比例混合染毒,分别给予低剂量(0.6 mg.kg-1)、中剂量(3 mg.kg-1)和高剂量(15 mg.kg-1),连续30 d灌胃,玉米油作溶剂,对照组灌胃等量的溶剂玉米油。最后一次染毒后24 h,眼静脉采集小鼠血液,无痛处死小鼠。分离小鼠血清,测定血清肌酐(Cr)及尿素氮(BUN)含量,对肾脏组织进行光镜和电镜观察。肾脏功能指标测定结果表明:与对照组相比,中、高剂量组小鼠血清肌酐、尿素氮含量显著升高(P<0.05或P<0.01);HE及PAS法观察结果显示:低、中剂量组小鼠肾脏肾小管管腔轻微扩张,中剂量组小鼠肾脏局部出现溶解的结晶,高剂量组小鼠肾脏肾小管中出现晶体,肾小管管腔极度扩张,小管上皮细胞扁平化,刷状缘破损,间质不同程度炎性细胞浸润,纤维结缔组织增生等病理改变。     

Clinicopathologic, histologic, and toxicologic findings in 70 cats inadvertently exposed to pet food contaminated with melamine and cyanuric acid

OBJECTIVE: To document clinicopathologic, histologic, and toxicologic findings in cats inadvertently exposed to pet food contaminated with melamine and cyanuric acid. DESIGN: Case series. ANIMALS: 70 cats from a single cattery inadvertently fed contaminated food that was the subject of a March 2007 recall. PROCEDURES: Clinical signs, clinicopathologic and histopathologic findings, and results of toxicologic analyses were recorded. RESULTS: Clinical signs were identified in 43 cats and included inappetence, vomiting, polyuria, polydipsia, and lethargy. Azotemia was documented in 38 of the 68 cats for which serum biochemical analyses were performed 7 to 11 days after consumption of the contaminated food. One cat died, and 13 were euthanized. Histologic examination of kidney specimens from 13 cats revealed intratubular crystalluria, tubular necrosis with regeneration, and subcapsular perivascular inflammation characterized by perivascular fibroplasia or fibrosis and inflammation with intravascular fibrin thrombi. Toxicologic analyses revealed melamine and cyanuric acid in samples of cat food, vomitus, urine, and kidneys. CONCLUSIONS AND CLINICAL RELEVANCE: In cats unintentionally fed pet food contaminated with melamine and cyanuric acid, the most consistent clinical and pathologic abnormalities were associated with the urinary tract, specifically tubular necrosis and crystalluria.     

三聚氰胺及其三聚氰酸混合物对小鼠肝脏的损伤及损伤机理

试验采取检测各攻毒组小鼠肝脏受损情况、肝脏细胞凋亡及相关凋亡机制。结果显示,三聚氰胺及其三聚氰酸混合物对小鼠肝脏的损伤随着混合物剂量的上升呈量效关系,剂量越高损伤作用越严重,而三聚氰胺单独组和三聚氰酸单独组对小鼠肝脏无明显损伤作用。三聚氰胺与三聚氰酸混合物主要是通过诱导小鼠肝脏细胞凋亡而致肝组织损伤,Bax和caspase-3基因参与了三聚氰胺和三聚氰酸诱导小鼠肝细胞凋亡的调控。     

Residue depletion of melamine and cyanuric acid in catfish and rainbow trout following oral administration

Reimschuessel, R., Evans, E., Andersen, W. C., Turnipseed, S. B., Karbiwnyk, C. M., Mayer, T. D., Nochetto, C., Rummel, N. G., Gieseker, C. M. Residue depletion of melamine and cyanuric acid in catfish and rainbow trout following oral administration. J. vet. Pharmacol. Therap. 33 , 172–182. The intentional addition of triazines such as melamine to animal feeds and the lack of information about residue accumulation in food animals caused global concerns for food safety during 2007 and 2008. We report the results of a good laboratory practices (GLP) study to determine melamine and cyanuric acid residues in catfish and trout filets harvested at 1, 3, 7, 14, 28, and 42 days after a single oral dose of 20 mg/kg body weight of melamine, cyanuric acid, or melamine and cyanuric acid together. Peak melamine concentrations were 12.73 mg/kg (ppm) in catfish (mean = 9.98), 12.26 mg/kg in trout (mean = 7.89) on day 1. Within 7 days (catfish) or 14 days (trout) residues were <2.5 mg/kg, a level in foods accepted by many risk assessors worldwide to be unlikely to pose health risks to consumers. Peak cyanuric acid residues also occurred on day 1, 0.68 mg/kg in catfish (mean = 0.46), 2.59 mg/kg in trout (mean = 0.86). Cyanuric acid muscle residues were <2.5 mg/kg by day 3. The half‐lives for melamine and cyanuric acid ranged between 1 and 4 days. Renal crystals formed in fish given both melamine and cyanuric acid, persisting for weeks after the single dose.     

Depletion of melamine and cyanuric acid in kidney of catfish Ictalurus punctatus and trout Oncorhynchus mykiss

A risk assessment conducted in 2007 identified significant knowledge gaps about tissue residues of melamine and related triazine analogs such as cyanuric acid in animals that had eaten contaminated food. The USFDA subsequently designed studies to determine residue levels in muscle, serum, and kidneys of catfish and trout given a single gavage dose of 20 mg/kg body weight (BW) of melamine, cyanuric acid, or 20 mg/kg BW of both compounds simultaneously. Renal triazines were determined by LC‐MS/MS at postdose days 1, 3, 7, 14, 28 (and day 42 for trout). When dosed individually, melamine and cyanuric acid kidney residues depleted much faster than those in fish given both compounds together. Combined dose residue depletion was punctuated by extreme outliers due to the formation of persistent renal melamine cyanurate crystals.     

Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs

OBJECTIVE: To determine whether renal crystals can be experimentally induced in animals fed melamine or the related triazine compound cyanuric acid, separately or in combination, and to compare experimentally induced crystals with those from a cat with triazine-related renal failure. ANIMALS: 75 fish (21 tilapia, 24 rainbow trout, 15 channel catfish, and 15 Atlantic salmon), 4 pigs, and 1 cat that was euthanatized because of renal failure. PROCEDURES: Fish and pigs were fed a target dosage of melamine (400 mg/kg), cyanuric acid (400 mg/kg), or melamine and cyanuric acid (400 mg of each compound/kg) daily for 3 days and were euthanatized 1, 3, 6, 10, or 14 days after administration ceased. Fresh, frozen, and formalin-fixed kidneys were examined for crystals. Edible tissues were collected for residue analysis. Crystals were examined for composition via Raman spectroscopy and hydrophilic-interaction liquid chromatography-tandem mass spectrometry. RESULTS: All animals fed the combination of melamine and cyanuric acid developed goldbrown renal crystals arranged in radial spheres (spherulites), similar to those detected in the cat. Spectral analyses of crystals from the cat, pigs, and fish were consistent with melamine-cyanurate complex crystals. Melamine and cyanuric acid residues were identified in edible tissues of fish. CONCLUSIONS AND CLINICAL RELEVANCE: Although melamine and cyanuric acid appeared to have low toxicity when administered separately, they induced extensive renal crystal formation when administered together. The subsequent renal failure may be similar to acute uric acid nephropathy in humans, in which crystal spherulites obstruct renal tubules.     

Oxidative stress and hypoxia observed in the kidneys of mice after a 13-week oral administration of melamine and cyanuric acid combination

Both melamine and cyanuric acid have low toxicity, but together they may cause serious lesions to the kidney, via an unknown mechanism. This study was aimed to estimate whether lesions to the kidney were relative to oxidative damage and hypoxia in the kidney after mice exposed to 1 mg/kg/day, 5 mg/kg/day or 25 mg/kg/day of a mixture of melamine and cyanuric acid for 13 weeks. Pathological changes to the kidneys, oxidative stress and energy parameters and hypoxia-inducible factor-1α (HIF-1α) change in the kidneys were evaluated. Pathological changes were found in the distal tubules of kidneys, such as crystals, proteinaceous casts and compensatory expansion, indicating that the mixture induced toxicity to the kidney. The activities of total antioxidant capacity (TAC) and superoxide dismutase (SOD) and the concentration of glutathione (GSH) decreased, while the concentrations of lipid peroxidation (MDA) and protein carbonyl groups (PC) increased after exposure to the mixture, demonstrating that the mixture resulted in imbalance of antioxidant and reactive oxygen species (ROS) and excessive ROS induced oxidant damage to lipid and proteins in kidneys. The activities of malate dehydrogenase (MDH) and succinate dehyrogenase (SDH) decreased, however, the activity of lactic dehydrogenase (LDH) and the concentration of HIF-1α increased after exposure to the mixture. Accordingly, it was concluded that the mixture resulted in a hypoxic state in kidneys and that both oxidative stress and hypoxia contributed to the lesion of kidneys. The exact cause of oxidative damage and hypoxia is not clear, it might be caused by either a direct effect or by an indirect effect, which is secondary to substantial renal damage caused by tubular obstruction due to crystal formation.     

Effect of melamine-contaminated diet on tissue distribution of melamine and cyanuric acid,blood variables,and egg quality in laying hens

1. The aim of the study was to investigate the effect of melamine-contaminated feed (100 mg/kg) on the distribution of melamine and cyanuric acid in the tissues of laying hens. The effect of a 5-week melamine administration in feed on the egg quality and blood variables of layers was also investigated.

2. A total of twenty 36-week-old ISA Brown layers were used in the experiment. The layers were equally divided into an experimental melamine group (n = 10) and a control group without melamine (n = 10). At the end of the experiment, samples of liver, kidney, breast and thigh muscles were collected from all hens and analysed for the presence of melamine and cyanuric acid by gas chromatography and tandem mass spectrometry.

3. Layers receiving the melamine-contaminated diet laid eggs with decreased eggshell strength. The decrease in shell strength was found in weeks 3 and 4 compared to the initial state (week 0) and week 1.

4. Hens receiving the melamine-contaminated diet also exhibited a higher total red blood cell count and lower mean corpuscular haemoglobin compared to the control group. However, melamine at 100 mg/kg feed had no effects on the blood variables of layers.

5. Melamine was detected in all analysed tissues of layers fed on the melamine-contaminated diet, with its mean concentrations decreasing in the following order: kidney (7.43 mg/kg) > breast muscle (3.88 mg/kg) > liver (3.11 mg/kg) > thigh muscle (1.91 mg/kg). The kidney and liver of layers fed on the melamine-contaminated diet also exhibited the presence of cyanuric acid.

6. On the basis of our results, it can be concluded that the biotransformation of melamine into cyanuric acid proceeded mainly in the liver, and cyanuric acid was eliminated in urine.     

Erythrocyte protoporphyrin concentrations in clinically normal cats and cats with lead toxicity.

Erythrocyte protoporphyrin (EPP) and blood lead concentrations were determined in 91 clinically healthy cats living in the inner suburban area of Sydney, Australia. The mean EPP concentration was 223.4 +/- 186.1 micrograms litre-1 whole blood and the mean blood lead concentration 0.62 +/- 0.25 mumol litre-1. EPP concentrations were also monitored in three cats with confirmed lead toxicity--at the time of diagnosis and one week and one month after chelation therapy with calcium EDTA. EPP concentrations were elevated in two cats and within the normal range in the third cat at the time of diagnosis. EPP concentration were higher in two cats one week after treatment than at the time of diagnosis. One month after chelation therapy, EPP concentrations were normal in two cats but still substantially elevated in the third cat although its blood lead concentration had returned to normal and all clinical signs of lead toxicity had resolved. It was determined that the predominant form of protoporphyrin present in cats with lead toxicity was zinc protoporphyrin. The EPP assay may have limited value in the diagnosis of acute lead toxicity and in monitoring the success of chelation therapy in cats.     

Pharmacokinetics and toxicity of zonisamide in cats

With the eventual goal of making zonisamide (ZNS), a relatively new antiepileptic drug, available for the treatment of epilepsy in cats, the pharmacokinetics after a single oral administration at 10mg/kg and the toxicity after 9-week daily administration of 20mg/kg/day of ZNS were studied in healthy cats. Pharmacokinetic parameters obtained with a single administration of ZNS at 10mg/day were as follows: Cmax=13.1microg/ml; Tmax=4.0h; T(1/2)=33.0h; areas under the curves (AUCs)=720.3microg/mlh (values represent the medians). The study with daily administrations revealed that the toxicity of ZNS was comparatively low in cats, suggesting that it may be an available drug for cats. However, half of the cats that were administered 20mg/kg/day daily showed adverse reactions such as anorexia, diarrhoea, vomiting, somnolence and locomotor ataxia.     

三聚氰胺对小白鼠亚慢性毒性

将80只昆明小白鼠随机分为4组（每组20只）：空白对照组（Ⅰ组）、三聚氰胺高剂量组（300mg／kg）（Ⅱ组）、三聚氰胺中剂量组（150mg／kg）（Ⅲ组）和三聚氰胺低刺量组（37．5mg／kg）（Ⅳ组）,试验周期为30d。试验期内观察小白鼠一般情况、体质量变化及食物利用率。结果显示：三聚氰胺染毒组小鼠体增重缓慢,食物利用率极显著降低（P〈0．01）;Ⅱ、11组小鼠血清ALT、AST、BUN、Cr和UA值显著高于Ⅰ组（P〈O．05）,Ca和P显著低于Ⅰ组（P〈0．05）;Ⅱ组小鼠肝、肾指数显著高于I组（P〈0．05）,Ⅱ、Ⅲ组脾指数显著低于Ⅰ组（P〈o．05）;Ⅱ、Ⅲ组小鼠肺、肝、脾、肾及膀胱有病理变化。结果表明,三聚氰胺亚慢性毒性会影响动物生长,造成肺、肝、肾及脾损害。     

In vitro toxicity of melamine against Tetrahymena pyriformis cells

This study assessed the toxicity of melamine against the unicellular eukaryotic system of Tetrahymena (T.) pyriformis exposed to 0, 0.05, 0.25, 0.5, 2.5, and 5 mg/mL of melamine. Cell growth curves of different cultures, the half maximum inhibition concentration (IC₅₀) value of melamine, and morphological changes in cells were obtained via optical and transmission electron microscopic observation. The effects of eleven melamine concentrations, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 mg/mL, on protein expression levels of T. pyriformis were examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results showed an obvious inhibitory effect of melamine on the growth of eukaryotic cells. Cell growth dynamics indicated that the IC₅₀ value of melamine on T. pyriformis was 0.82 mg/mL. The cellular morphology was also affected in a concentration-dependent manner, with characteristics of atrophy or cell damage developing in the presence of melamine. The relative contents of the top four main proteins corresponding to peak mass-to-charge ratios (m/z) of 4466, m/z 6455, m/z 6514, and m/z 7772 in the MALDI-TOF-MS spectra were all found to be closely correlated with the melamine concentrations. In conclusion, exposure of eukaryotic cells to melamine could inhibit cell growth, cause changes in cytomorphology and even disturb the expression of proteins in a concentration-dependent manner. The described method of examining four sensitive proteins affected by melamine was also proposed to be used in a preliminary study to identify protein biomarkers in T. pyriformis.     

GC-MS法检测包装材料中的三聚氰酸残留

<正>三聚氰胺在一般情况下较稳定, 但在强酸、强碱条件下能发生水解,氨基逐步被羟基取代,生成三聚氰酸二酰胺(ammeline)、三聚氰酸一酰胺(ammelide)