| ObjectiveCisplatin (cis - diamine - dichloroplatinum, CDDP) is a potent antitumor compound that is widely used for the treatment of a large number of tumors, especially useful in the treatment of epithelial malignancies, such as head and neck cancers. However, the treatment effects are dose - limited by those side effects such as ototoxicity, nephrotoxicity, haematological toxicity and bone marrow suppression. Until now the mechanisms of side effects induced by cisplatin are not clearly understood. Recently, evidence has been accumulated to demonstrate that these side effects are closely related to oxidative stress. There are a number of studies concerning the role of reactive oxygen radical species in the pathophysiology of CDDP - induced side effects. Several antioxidant agents have been reported to prevent these side - effects but there is no study regarding the combinative protective actions of protective agents administered simultaneously. Now researches are focused on the potential mechanisms underlying cisplatin toxicity and protective agents against the toxic effects. The data of studies suggested that the cisplatin toxicity could be induced by multi - ways and ameliorated by some protective agents. As the toxic effects of cisplatin are induced by multi - ways, the best protective effects could be achieved by combined use of multi - protective agents with different anti - ways. As the antitumor and toxic effects are mediated in part by different mechanisms, thus, permitting a selective inhibition of these side effects while the antitumor effect is not altered..In present study, we attempted to suppress CDDP - induced ototoxicity, hepatic toxicity and nephrotoxicity by exploring the combinative effects of nutri-ents against the toxicity of cisplatin.MethodsIn the experiment 1: Male mice weighted from 28 to 30g, were divided into five groups, with 10 mice in each group. Before cisplatin administration, auditory brainstem response (ABR) threshold of mice were determined, then cisplatin solution were injected intraperitoneally (i. p.) to mice in doses of 2. 0, 3. 0, 3.5 and 4. Omg/kg body weight (b. w. ) for five days, the mice in control group received i. p 0. 1ml isotonic saline solution at meantime. Three days after the last administration, ABR was checked again and mice were sacrificed by cervical dislocation. Blood, kidney and liver were immediately taken and removed. Indicators of kidney ratio (Kr) , liver ratio (Lr) , ABR, levels of blood urea nitrogen (BUN) , activities of serum alanine aminotransferase (ALT) and the levels of glutathione (GSH) and malondialdehyde (MDA) , activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH - Px) in liver and kidney were examined with spectrophotometric method.In the experiment 2: The mice in the three experimental groups received i. p. 3. 5mg/kg b. w. of cisplatin consecutively for five days, and were sacrificed by cervical dislocation three, five and ten days after the last injection. The other procedure and the indicators examined were the same as above.In the experimental 3: Orthogonal design was used, 7 drugs with two levels were tested by animal experiments. At first the ABR of all mice were examined as the base levels, then eight experimental groups with six mice in each group and one control group with 8 mice were divided randomly, the protective agents of zinc (Zn), selenium (Se) , fosfomycin (Fos), sodium thiosulfate (STS) ,N - acetyl - cysteine ( NAC) , methionine ( Met) , taurine (Tau) , which have been shown to reduce the side effects of cisplatin effectively, with the doses of 25 mg/kg b. w. of Zn, 1. 5mg/kg b. w. of Se, 300mg/kg b. w. of Fos, 500mg/ kg b. w. of NAC, 1000mg/kg b. w. of STS, 200mg/kg b. w. of Met or 400mg/ kg b. w. of Tau, were given to mice by lavage in different combinations as arranged in the orthogonal table, respectively. Two days later, the mice receivedi. p. 3.5mg/kg b. w of cisplatin for five days simultaneously, after cisplatin administration the protective agents were given continuously for two days. Mice were sacrificed and the indicators were determined as shown above.In the experimental 4: Ten ICR mice were divided into two groups with 5 mice in each group. 3.5mg/kg b. w. of cisplatin were given to the mice in experimental group for consecutive five days, and mice in control group received i-sotonic saline solution. Three days after the last administration, mice were sacrificed and brain and kidney were taken immediately. Brain and kidney were embedded in paraffin and sectioned at 4 μm, after deparaffinization with xylene and ethanol, 0.1% trypsin treatment and denaturing of nuclear DNA, the sections were incubated with 10% normal rabbit serum, then incubated with monoclonal antibody for 8 - OHdG (5μg/ml; Japan Institute for the Control of Aging, Shi-zuoka, Japan) for one hour at room temperature, followed by biotin - labeled rabbit anti — mouse IgG serum, and finally streptavidin - biotin - alkaline phos-phatase complex (Histofine SAB - AP(M) , Nichirei, Japan). Substrate for alkaline phosphatase was obtained from Nichirei. As negative controls, we omitted the first antibody from the staining procedure.ResultsAmong the administered dose ranges of 3.0 to 4. 0mg/kg b. w, mice treated with cisplatin were found to have ABR threshold shifts of 10 to 40 dB in all frequency checked, levels of Lr, GSH in liver and body weight of experimental mice were decreased significantly; the levels of Kr, BUN, MDA in liver and GSH in kidney, activities of serum ALT were increased significantly along with the doses of cisplatin increased. Furthermore, activities of GSH - Px and SOD in liver were decreased, and activities of GSH - Px and SOD in kidney were increased induced by cisplatin administration as compared with those of mice in control group. The changes of these indicators were most obviously at the three or four days after the last administration of cisplatin, then they were alleviated gradually.Mice treated with protective agents plus cisplatin all had ABR thresholdsshifts of less than 20dB. The decreased activities of GSH - Px and increased levels of MDA in liver of animals administered with cisplatin were significantly improved with Zn; The increased Kr and levels of MDA, activities of serum ALT were significantly improved with Se; The changed indicators were all improved significantly with Fos, excluding the increased Kr and activities of GSH - Px and SOD in kidney; The activities of serum ALT and indicators of kidney were significantly improved with STS; Reduced body weight, increased ABR threshold, LR, Kr, levels of BUN and activities of GSH - Px in kidney and decreased levels of GSH in liver were significantly improved with Met; only the increased levels of MDA in liver were significantly improved with Tau; No changed indicators were significantly improved with NAC. On the other hand, nearly all the changed indicators examined in these study were significantly improved by combinative administration of Se, Fos, Met and Tau.Positive staining of 8 - OHdG were shown in the neuron of the cerebral cortex and hippocampus, in the endothelia cell of capillary of hippocampus, and in the endothelial cells of glomerular capillary in kidney.ConclusionIn conclusion, these data demonstrated that the obvious toxic effects on hearing loss, kidney and liver could be induced by cisplatin administered in doses among 3.0 to 4. 0mg/kg b. w. Oxidative damage was the one mechanism of toxic effects on liver induced by cisplatin, while in the kidney, the compensative reaction against the oxidative stress was induced by the cisplatin administration, and kidney was protected from oxidative damage. Though the oxidative damage was not the main mechanism of toxic effects on kidney induced by the cisplatin, the results suggested that overproduction of free radicals might be induced by cisplatin administration. These results showed that oxidative damage is not the only mechanism by which cisplatin induced nephrotoxicity and hepatic toxicity. Toxic effects were the worst at the three or four days after the last injection of 3. 5mg/kg b. w. cisplatin in a consecutive five days administration. After then, the mice were recovered gradually from the toxic effects of cisplatin. |
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