Effect of oral zinc supplementation on metallothionein and superoxide dismutase concentrations in patients with inflammatory bowel disease

Abstract Oxygen-derived free radicals may contribute to intestinal tissue damage in inflammatory bowel disease. The concentrations of metallothionein and superoxide dismutase, two copper and zinc containing proteins involved in the scavenging of free radicals, were previously found to be decreased in the intestinal mucosa of patients with this disorder. The plasma zinc concentration is often decreased also in these patients. Since zinc is reported to be an efficient inducer of metallothionein synthesis, and probably of superoxide dismutase, we evaluated the effect of oral zinc supplementation on metallothionein and superoxide dismutase levels in patients with inflammatory bowel disease. Fourteen patients with inactive to moderately active inflammatory bowel disease received oral zinc supplementation (300 mg zinc aspartate, equal to 60 mg elemental zinc per day) for 4 weeks in a placebo-controlled double-blind cross-over trial. The plasma zinc concentration of these patients was low at the start of the study (12.2 ± 1.7 μmol/L, P <0.05), when compared to that of 22 healthy controls (13.6 ± 2.3 μmol/L), but increased (P <0.05) towards the levels of controls during the supplementation period (13.3 ± 2.5 μmol/L). The concentrations of metallothionein and superoxide dismutase in plasma and in erythrocytes did not change in relation to the supplementation. The metallothionein concentration in both inflamed and non-inflamed intestinal mucosa was slightly higher after zinc supplementation but the superoxide dismutase concentration in the tissue was not altered. The histological inflammation score of intestinal biopsies, plasma albumin levels, and the disease activity index of the patients did not change during the study. Thus, although zinc supplementation therapy increased plasma zinc concentrations, there was no effect on the plasma, erythrocyte and mucosal metalloprotein levels in inactive to moderately active patients with inflammatory bowel disease.     

锌对小鼠肝金属硫蛋白合成的影响

本文研究了小鼠在宽的剂量范用内（１０，２０，４０，８０，１００，１２０，１４０，１６０，１８０，２００ｍｇ／ｋｇｂｗ）口服锌（Ｚｎ）盐时，对肝金属硫蛋白（ＭＴ）合成的影响。结果显示：因服用过量Ｚｎ（２＋），按剂量──效应关系发生肝ＭＴ的诱导合成，而且在肝Ｚｎ浓度和肝ＭＴ浓度之间存在正向关系。表明Ｚｎ是一个ＭＴ的诱导剂。在口服Ｚｎ８０～１００ｍｇ／ｋｇ的剂量范围时，无论是肝ＭＴ水平或肝Ｚｎ水平均有明显的改变。提示：在Ｚｎ诱导ＭＴ合成中，存在一个敏感的剂量范围。     

PP2A B56α亚基介导镉诱导的细胞毒性

目的： 探讨蛋白磷酸酶2A(PP2A)B56α亚基在调控CdCl2诱导的细胞毒性中的作用及其机制。方法：利用病毒感染法在人胚肾上皮细胞HEK上构建PP2A B56α表达降低的细胞株模型(HEK-SHB56α-1和HEK-SHB56α-2),采用改良四甲基偶氮唑盐(MTT)法检测CdCl2诱导的细胞毒性。用不同浓度(0、10、20、40和80 μmol/L)CdCl2联合或不联合c-Jun氨基末端激酶(JNK)抑制剂SP600125染毒细胞不同时间(0、2、6、12和24 h),采用免疫印迹检测B56α亚基、金属硫蛋白(MT)的表达和JNK的磷酸化水平。结果：免疫印迹结果证实细胞株HEK-SHB56α-1和HEK-SHB56α-2构建成功。与对照组比较,PP2A B56α表达抑制导致镉诱导的细胞毒性减小,JNK的磷酸化水平和MT的表达水平均显著增加。用不同剂量CdCl2处理细胞株12 h,发现B56α表达抑制导致JNK磷酸化(p-JNK)分别增加了2.78和1.26倍(P<0.05),MT的表达也分别增加了1.36和1.19倍(P<0.05)。用p-JNK抑制剂SP600125作用时,p-JNK的表达降低了35%~38%(P<0.05),MT的表达下降了13%~35%(P<0.05), CdCl2诱导的细胞毒性显著增加(P<0.05)。此外,用CdCl2处理细胞不同时间点,B56α的表达逐渐降低(P<0.05),而p-JNK和MT的表达水平呈逐渐增加趋势(P<0.05)。结论：PP2A B56α在调控CdCl2诱导的细胞毒性中起着重要作用,其作用机制可能通过介导JNK的去磷酸化调控MT的表达而发挥作用。本研究揭示了PP2A参与重金属应激的关键靶点和信号通路的调控。     

Immunohistochemical metallothionein expression in hepatocellular carcinoma: relation to tumor progression and chemoresistance to platinum agents

Background Metallothionein (MT), which is known to detoxify heavy metal ions, is considered to serve as a mechanism of resistance to platinum complex compounds. In the present study, MT expression in hepatocellular carcinoma (HCC) was immunohistologically investigated to clarify its relationship to clinical background factors and responsiveness to anticancer drugs.Methods Specimens from 117 patients with HCC were immunohistologically studied, using a monoclonal anti-MT antibody. the percentage of MT-positive HCC (MT ratio) cells was determined, to evaluate the extent of staining with anti-MT antibody. Staining with an MT ratio of more than 50% was categorized as diffusely positive; an MT ratio of 5% to less than 50% was focally positive; and an MT ratio of less than 5% was negative. Twenty-two patients received repeated arterial infusion chemotherapy with carboplatin (CBDCA), a platinum-containing compound, and the MT expression was analyzed in relation to their chemotherapeutic response.Results The ratio of MT-positive cells in HCC decreased with the degree of histological differentiation and also decreased with higher tumor stage. In patients treated with CBDCA, the ratio of MT-positive cells in responders was significantly lower than that in non-responders.Conclusions MT expression decreases with the degree of histological differentiation and decreases with increasing tumor stage in HCC. In addition, MT expression may lower the antitumor effect of CBDCA.     

Gene expression in gastric biopsies from patients infected with Helicobacter pylori

Background: Helicobacter pylori infection has protean effects on gene expression in the host gastric mucosa, which have been investigated by gene chip analysis in vitro. In this study the effects of H. pylori infection on host gene expression in the gastric antral mucosa in patients were examined. Methods: One gastric antral biopsy was obtained from a total of 18 untreated patients undergoing routine endoscopic evaluation of chronic abdominal complaints. Nine patients had histologic evidence of H. pylori infection and 9 age- and sex-matched patients had no histologic evidence of H. pylori infection. A microarray analysis was performed using a gene chip containing 35,000 human expressed sequence tags on RNA extracted from endoscopic, gastric antral biopsies, and average gene expression among infected and uninfected patients was compared. Results: Underexpressed genes in infected patients' mucosa included gastric intrinsic factor and several metallothionein isoforms. Overexpressed genes in infected patients' mucosa comprised MHC Class II molecules, immunoglobulin and B-cell activation genes, as well as genes known to induce apoptosis. Changes in expression were confirmed for a subset of genes by SYBR green real-time PCR. Conclusions: Microarray analysis of antral biopsies from patients with and without H. pylori infection revealed differential expression of metal regulatory, immunity and inflammation-related genes.     

金属硫蛋白在前列腺癌中的表达及临床意义

目的探讨金属硫蛋白（Metallothionein，MT）在前列腺癌组织中的表达及临床意义。方法应用免疫组织化学方法观察不同分级的66例前列腺癌、20例前列腺增生组织MT的表达情况，分析MT与临床病理因素及预后之间的关系。结果20例前列腺增生组织MT的表达率为100％（20／20），66例前列腺癌组织中MT的表达率为59．1％（39／66），两者比较差异有统计学意义（P〈0．05）。MT的表达随着前列腺癌组织学分级、临床分期的增加而增加（P〈0．05）；与患者血清PSA水平、年龄无相关性（P〉0．05）；MT阳性表达组与阴性表达组之间的生存率差异有统计学意义（P〈0．05）。结论MT的表达与前列腺癌发生、发展及预后有一定的关系。     

金属硫蛋白对培养神经细胞迟发性损伤的作用和一氧化氮的表达

本文以新生大鼠原代培养皮质神经元为实验材料，造成迟发性神经元损伤模型．在不同时程内，测试培养液中的细胞乳酸脱氢酶漏出量和用还原型尼克酰胺腺嘌呤二核着苷酸脱氢酶染色反应，观察培养细胞中一氧化氮合酶的表达水平．结果表明，缺血组在缺血与再灌流中，细胞乳酸脱氢酶漏出量明显高于对照组，差异显著（Ｐ＜０．００１）．一氧化氮合酶阳性神经元数量在缺血组的缺血时即明显高于对照组（Ｐ＜０．０１），再灌流后一氧化氮合酶表达仍然强烈，与对照组相比有显著差异（Ｐ＜０．０１）．当损伤细胞再灌流时，同时加入金属硫蛋白后，显示一氧化氮合酶表达减弱，和缺血组相比有显著差异（Ｐ＜０．０５～０．００１），细胞乳酸脱氢酶漏出量在再灌流后的早期近似于对照组．结合文献和本实验结果提示：在迟发性神经元损伤形成过程中一氧化氮起着重要作用；金属硫蛋白对脑缺血后迟发性神经元损伤有一定保护作用，是一种细胞保护剂，可望用于临床，控制缺血再灌流损伤。     

含金属硫蛋白蛋奶粉对H-22小鼠肝癌的预防作用

目的：观察含金属硫蛋白（MT）蛋奶粉对BALB/c小鼠H-22肝癌的预防作用。方法：雌性BALB/c纯系小鼠70只随机分为正常对照组(10只)、荷瘤鼠对照组(20只)、含MT蛋奶粉预防肿瘤低剂量及高剂量组(各20只)。将MT低和高剂量蛋奶粉给小鼠灌胃2周后,皮下接种H-22细胞,建立小鼠移植肿瘤模型,观察MT蛋奶粉对肿瘤出现时间及肿瘤大小和生长的影响;观察1个月后处死小鼠,MTT法检测脾脏淋巴细胞转化功能,流式细胞术检测脾脏CD4⁺、CD8⁺和CD25⁺ T细胞百分率以及细胞周期进程和凋亡的变化,³H-TdR掺入法检测CTL和NK细胞杀伤活性。结果：与荷瘤对照组相比,含MT蛋奶粉组小鼠其肿瘤生长速率显著降低（P<0.05）,淋巴细胞增殖率显著增加 （P<0.01）,脾脏CD4⁺和CD8⁺ T细胞百分率及CD4⁺/CD8⁺比值显著升高（P<0.05或P<0.01）, CD25⁺ T细胞百分率和淋巴细胞凋亡百分率均显著降低（P<0.05或P<0.01）, NK细胞毒性显著提高 （P<0.05或P<0.01）,CTL细胞毒性亦显著增高（P<0.01）。结论：含MT蛋奶粉可明显增强荷瘤小鼠的免疫功能,对小鼠移植性肿瘤的生长起到一定程度的抑制作用。     

Immunohistochemical expression of metallothionein in normal human colorectal mucosa, in adenomas and in adenocarcinomas and their associated metastases

The immunohistochemial distribution pattern of metallothionein, a low molecular weight protein with strong affinity for divalent heavy metal ions, has been investigated in normal and neoplastic conditions of the large bowel. Utilizing a monoclonal mouse antibody the following formalin-fixed paraffin-embedded surgical or biopsy samples were studied: tubulo-villous adenomas (8 cases); adenocarcinomas with various degree of differentiation (85), nine of which were mucinous-type; synchronous tubular or tubulo-villous adenomas separate from carcinomas (30); transitional mucosa (45); metastases in lymph nodes (43); and distant metastases (45). Twenty biopsies from the right and left colon of 10 patients affected by irritable bowel syndrome were also analyzed. Normal colonic mucosa as well as transitional mucosa showed metallothionein immunopositivity in enterocytes at the luminal surface and crypts. Evident nuclear and cytoplasmic staining was encountered in tubulo-villous adenomas; the same reactivity was noted in the basal glandular component of colorectal carcinomas-synchronous adenomas, while less intense staining was noted in the apical villous portions. A variable metallothionein immunostaining was observed in adenocarcinomas (62.3%), in lymph node (55.8%) and distant hepatic (17.2%) and omental (43.8%) metastases, although it was not always concordant with that reported in the corresponding primary tumour. Whether the metallothionein positivity observed in normal and neoplastic cells is the result of expression of a stable form of the protein or an accumulation in the nucleus and cytoplasm remains to be clarified.     

Effect of maternal diabetes and dietary copper on fetal development in rats

To test whether diabetes associated alterations in copper metabolism contribute to diabetes-induced teratogenicity in rats, pregnancy outcome was compared between diabetic and nondiabetic rats fed either a copper adequate (12 μg/g diet) or low copper diet (1 μg/g diet). The dietary regimen was begun two weeks prior to mating and continued throughout pregnancy. To facilitate the reduction of maternal copper stores in the low copper groups, the low copper diet was supplemented with a copper chelator, triethylenetetraamine, at 1% for one week; the chelator was removed from the diet one week prior to mating. Pregnancy was terminated on gestation day 20. Maternal and fetal tissues were assessed for copper concentrations, the activities of the cuproenzymes copper, zinc superoxide dismutase and ceruloplasmin, and the copper binding protein metallothionein. Dams fed the low copper diet had low tissue copper concentrations, and low plasma ceruloplasmin and erythrocyte superoxide dismutase activities compared to copper-adequate dams. Fetuses in the low copper groups were characterized by low liver copper concentrations. Gross structural and skeletal anomalies were only observed in the diabetic groups; maternal copper intake did not influence the frequency of these anomalies. However, fetuses in the low-copper nondiabetic group, and both diabetic groups, were characterized by low liver copper, zinc superoxide dismutase activity suggesting that fetal copper metabolism was influenced by both copper intake and diabetes.