Research On The Accumulative Toxicity And Subchronic Toxicity Of Trimethytin Chloride In Rats

Objective To Study the accumulative effect of trimethytin chloride (TMT). Observe the no observed adverse effect level (NOAEL) of the sub-chronic oral toxicity, the target organs and the influence on the neurobehavior in rats; Give a preliminary discussion about the metabolic distribution in the rats' blood and urine samples.Methods (1) Cumulative toxicity test:SD Rats and KM mice were given increasing doses of TMT in gavage, respectively. The accumulative coefficients in rats and mice were calculated. The pathological changes of main target organs from the deceased rats were detected. (2) Sub-chronic toxicity (90 days) test; 55 female SD rats of 6 weekly were divided in to five groups at random, called blank control (C) low dosage (L) medium dosage (M) high dosage (H) super high dosage(S) respectively. They were treated at a oral dose of 0,8.20,32.81,131.25,262.50μg·kg-1·d-1. Drug concentration was adjusted according to the average water consumption and animal weight weekly. Animals' behaviors were observed everyday and the consumption of water and food and body weights were measured weekly. In the middle and final of the tests, blood and urine were collected respectively. in the end, some tests about the neuroethology were conducted including the open-field test, Morris water maze and the step-down test. At the end of the experiment (at day 90th), we collected the urine and the blood to determine the biochemistry, electrolyte and TMT concentration. we also collected the main organs to measure the visceral ratios, pathological changes and the activity of hydrogen potassium ATPase and natrium potassium ATPase. We made use of Western Blotting to measure the protein expression of H+-K+-ATPase and real-time RT-PCR to measure the mRNA expression level of H+-K+-ATPase in kidney.Results (1) Cumulative toxicity test:The accumulative coefficients of TMT in rats and mice were 1.7 and 3.8, respectively. Diffuse fatty vacuolization in cerebella cortex, swelling and necrosis of neuron in brainstem and diffuse fibrosis in spleen were observed in rats which were treated with TMT. (2) Sub-chronic toxicity(90 days) test:①control (C) low dosage(L) group: There were no obvious abnormal performance in the control (C) and the low dosage(L) group.②Medium dosage (M) group:One animal was died because of the tumor and bladder calculi in the medium dosage (M) group. The renal surfaces were asystematic granular pathological changes. Alkaline phosphatase (AKP), uric acid (UA), triglyceride (TG) decreased.③High dosage (H) group:Most animals in high dosage (H) group became nervous and easy to be scared in the end of the test, even shrieked sometimes. We could see renal tubular sedimentary calcium salt and the renal tubular epithelial cells necrosis. There was obvious hyperplasia in the epithelial cells of the renal pelvis and renal papilla. ALT (ALT), alkaline phosphatase (AKP), uric acid (UA), triglyceride (TG), alpha hydroxyl butyrate dehydrogenase (HBDH) and lactate dehydrogenase (LDH) were reduced.④Super high dosage(S) group:the poisoning performance became obvious from the 11th week. Two rats died of urinary calculus formation in urinary system. The pathological changes were the same as high dosage (H) group. We could also see renal pelvis expansion and the tubules lumens protein deposition. The amount of rats drinking water and urine increased. Rats' body weight decreased distinctly. Rats' visceral indexes of kidney, adrenals and spleen were greater than the control's obviously. The urine creatinine (CREA) decreased in 14th weeks and the RBC in Urine (BLD) was positive.The concentrations of K+, ALT, UA, TG, HBDH and LDH in serum reduced and Glucose (GLU), alkaline phosphatase (AKP), urea nitrogen (BUN) increased of Super high dosage(S) group (P<0.05). The H+-K+-ATPase enzyme activity of kidney decreased drastically. The test results of WB (Western Blotting) and the real-time RT-PCR (real-time reverse transcription-polymerase chain reaction) showed that there were no obvious expression level differences of protein and mRNA of kidney H+-K+-ATPase enzyme. The Na+-K+ATP enzyme activity of kidney had a descending tendency.There were no obvious differences between the five groups in neuroethology test including open-field test, Morris water maze and the step-down test. The concentration of TMT in RBC is so far higher than plasma concentrations, about 195,204,121,63times in group of S H, M, L, respectively. In the urine, for the low concentration of plasma 44%,27%,31% and 16%.Conclusion TMT was obvious accumulation for rats and medium accumulation for mice. Main pathological damaged organs were cerebellum, the brainstem and spleen. For the TMT sub-chronic toxicity, the NOAEL was 7.74μg·kg-1·d-1. The central nervous system was not the main damaged organs. The main target organ was the kidney. TMT can inhibit renal H+-K+-ATPase activity and lead to hypokalemia and the renal tubule stones. Red cell was one of the main cumulative places.