DNA microarray analysis of human gene expression induced by a non-lethal dose of cadmium

Cadmium (Cd) is a hazardous heavy metal present in working and living environments. Cd affects many cellular functions, but little is known about the mechanisms of its toxicity and cellular defense against it. Recently, advanced gene expression analysis employing DNA microarrays provided us the means to profile the expression of thousands of genes simultaneously. We describe here a study of Cd-induced gene expression profile. Messenger RNA was prepared from HeLa cells exposed to a non-lethal dose of CdSO4, and analyzed by the use of an array consisting of 7,075 human cDNAs. Many stress response genes including those coding for metallothioneins and heat shock proteins were observed to be induced by Cd. The cellular metabolism inclined toward the synthesis of cysteine and glutathione after Cd exposure. Anti-oxidant genes also appeared to be induced to protect cell components and to quench reactive oxygen species. Ubiquitin pathway was activated as well probably to degrade proteins which might not be renatured. These data suggest that human cells mobilize every genomic resource (induction of some genes and repression of others) to overcome cytotoxicity caused by Cd.     

DNA microarray analysis of altered gene expression in cadmium-exposed human cells

Cadmium (Cd) is a heavy metal known to be toxic and carcinogenic, but its mechanism of action remains to be elucidated. Development of the DNA microarray technology has recently made the comprehensive analysis of gene expression possible, and it could be a powerful tool also in toxicological studies. With microarray slides containing 7,000-9,000 genes, we have been studying the gene expression profiles of a human cell line exposed to Cd. By exposure to a non-lethal concentration of Cd, 46 upregulated and 10 downregulated genes whose expression levels changed twofold or greater were observed. The expression of genes related to cellular protection and damage control mechanisms such as those encoding metallothioneins, anti-oxidant proteins and heat shock proteins was simultaneously induced. In addition, altered expression of many genes involved in signaling, metabolism and so on was newly observed. As a whole, a number of genes appear to be coordinately regulated toward survival from Cd toxicity. When cells were exposed to a higher concentration of Cd, more remarkable effects were observed both in the number of affected genes and in the extent of altered expression. These findings will contribute to the understanding of the complicated biological effects of Cd.     

Marginal zinc deficiency exacerbates experimental colitis induced by dextran sulfate sodium in rats

We investigated the impact of Zn status on the maintenance of mucosal homeostasis. Rats were fed diets containing different amounts of Zn (30, 10, 5, <1 mg Zn/kg diet) for 21 d. Serum Zn concentrations were lower in rats fed marginally Zn-deficient (MZD; 5 mg Zn/kg diet) and severely Zn-deficient (<1 mg/kg) diets but not in those fed the marginally Zn-adequate diet (10 mg/kg) or the Zn-adequate (ZA; 30 mg/kg) group (P < 0.05). However, organ weights, colonic epithelial cell proliferation, and crypt fission did not differ between the MZD and ZA groups. We then evaluated whether MZD modulated dextran sulfate sodium (DSS)-induced colonic inflammation by administering 2% DSS to the MZD and ZA groups for 7 d. Myeloperoxidase activity and TNFα production increased in response to DSS in the MZD group (P < 0.03). Colonic permeability in the 2 groups did not differ after DSS administration. In a culture experiment using isolated mesenteric leukocytes, TNFα production was higher (P < 0.05) and TNF receptor type I (TNFR1) expression was detected in culture medium containing 20 and 30 μmol/L of Zn compared with culture medium lacking Zn supplementation. These results suggest that MZD exacerbated colitis by modulating the immune response through the impairment of TNFα production and TNFR1 expression rather than through the impairment of epithelial barrier function.     

缺锌对生长期大鼠肾脏维生素D受体及钙结合蛋白基因表达的影响

【目的】研究锌缺乏对生长期大鼠肾脏维生素D受体(vitamin Dreceptor,VDR)和钙结合蛋白(CaBP)基因表达水平的影响。【方法】新生大鼠随机分为缺锌组(ZD)、配饲组(PF)、对照组(ZA)3窝,每窝10只;21 d断奶后每只分笼饲养,喂养至第60 d处死;取肾脏,用试剂盒抽提组织RNA,用实时荧光定量PCR检测肾脏组织VDR mRNA及CaBP mRNA的表达。【结果】大鼠肾脏VDR基因表达ZD组较PF组下调34.78%,PF组较ZA组下调25.81%,ZD组较ZA组下调51.61%;CaBP基因表达ZD组较PF组下调23.19%,PF组较ZA组下调13.75%,ZD组较ZA组下调33.75%。【结论】锌缺乏通过改变生长期大鼠肾脏VDR的活性而影响VDR mRNA,从而影响靶基因CaBP转录,进而影响肾脏钙重吸收,可能是导致骨生成障碍的机制之一。     

Vitamin A repletion in rats with concurrent vitamin A and iodine deficiency affects pituitary TSHbeta gene expression and reduces thyroid hyperstimulation and thyroid size

Concurrent vitamin A (VA) deficiency (VAD) and iodine deficiency (ID) are common in developing countries. VAD has effects on thyroid metabolism that may be dependent on iodine status. The aim of this study was to investigate the effect of VA supplementation (VAS) and/or dietary iodine repletion, alone and in combination, on the thyroid-pituitary axis in rats with concurrent VAD and ID. Weanling rats (n = 96) were fed diets deficient in VA and iodine or sufficient in both (control), for 30 d. Subsequently, deficient rats were repleted with iodine and/or single VAS or remained deficient for 10 d. Serum retinol (SR), thyroid hormones, serum thyrotropin (TSH), pituitary TSHbeta mRNA expression level, and thyroid weight were measured. High-dose VAS restored SR concentrations to normal in both iodine-deficient and iodine-sufficient rats. Despite continuing VAD, provision of the iodine-sufficient diet entirely reversed the abnormalities of the pituitary-thyroid axis produced by VAD and ID. In iodine-sufficient rats, VAS had no discernible effects on the pituitary-thyroid axis; in iodine-deficient rats, VAS reduced pituitary production of TSH and thyroid stimulation but had no discernible effects on circulating thyroid hormone concentrations. Primary hypothyroidism in rats with concurrent VAD and ID does not reduce the efficacy of VAS, nor does VAD reduce the efficacy of dietary iodine to correct pituitary-thyroid axis dysfunction due to ID. In concurrent VAD and ID, VAS, independent of iodine repletion, reduces thyroid hyperstimulation and size, an effect likely mediated through the effects of VA on pituitary TSHbeta gene expression.     

Differences in muscular changes in rats with vitamin E and with selenium deficiency

The recti muscles of rats with vitamin E deficiency and with selenium deficiency were examined by electron microscopy and compared. In vitamin E deficiency, the first changes occurred in blood vessels and in the extracellular components. The endothelium contained many vacuoles and less cytoplasmic organelles. The basal lamina of the endothelium appeared thicker. As it progressed, vitamin E deficiency caused abnormal pigment granules, mitochondrial degeneration, decreased numbers and irregular arrangement of muscle fibers, and irregular arrangement of z bands. On the other hand, selenium deficiency caused no abnormal changes in the recti muscles. These results suggest that selenium plays a different role from that of vitamin E which is an antioxidant.     

Kidney injury induced by lipid peroxide produced by vitamin E deficiency and GSH depletion in rats

Four-week-old Wistar male rats were fed a vitamin E (VE)-deficient (0E) or a VE-sufficient (10E) diet for 6 weeks and then intraperitoneally treated with buthionine sulfoximine (BSO) at 1 mmol/kg body weight once a day for 3 days. Glutathione (GSH) depletion by BSO treatment caused injuries especially in the kidneys of VE-deficient rats. The kidney weight increased in the VE-deficient rats after BSO treatment (0E-BSO). It was observed that the epithelial cells of the renal tubules in this group were strongly impaired and the injuries were necrosis and desquamation. No injury was observed in the kidneys of the BSO-untreated 0E group and the 10E groups. The TBA value of the kidney of 0E-BSO group was lower than that of the BSO-untreated 0E group, but the lipofuscin content of the kidney of the 0E-BSO group was 10 times higher than that of the BSO-untreated 0E group. These results suggest that the kidney injuries in rats may be caused by lipid peroxidation induced by vitamin E deficiency and glutathione depletion.     

Effect of ascorbic acid and vitamin E on biochemical changes associated with vitamin E deficiency in rats

Weanling male Sprague Dawley rats were fed a vitamin E and C-free basal diet with or without supplementation of 100 IU vitamin E per kg diet. After 20 weeks, the vitamin E-deficient rats were divided into four groups, six in each group, and received supplemental ascorbic acid and/or vitamin E by tube feeding daily for 7 days: Group I, 30 mg ascorbic acid/100 g body wt.; Group II, 0.03 mg RRR-alpha-tocopheryl acetate/100 g body wt.; Group III, 30 mg ascorbic acid and 0.03 mg RRR-alpha-tocopheryl acetate/100 g body wt.; and Group IV, placebo. The six control rats (Group V) received placebo. The rats were sacrificed, blood and liver samples were collected for biochemical determinations. Vitamin E deficiency significantly increased erythrocyte (RBC) spontaneous hemolysis, liver thiobarbituric acid (TBA) value, activities of glutamateoxaloacetate transaminase (GOT), pyruvate kinase (PK), and creatine phosphokinase (CPK) in plasma, and significantly lowered plasma vitamin E levels and glutathione peroxidase (GPX) activities. Tube-feeding ascorbic acid for 7 days produced partial reversal effect on liver TBA values, activities of plasma PK, GOT, CPK, and plasma vitamin E levels but not on RBC hemolysis and plasma GPX activity. Tube feeding both ascorbic acid and vitamin E showed similar partial reversal effect as feeding vitamin E alone on all the parameters stated above. The results suggest that ascorbic acid may spare the metabolism of vitamin E and partially reverse the changes in some of the biochemical parameters characteristic of vitamin E deficiency.     

运用基因芯片技术筛选亚慢性汞中毒大鼠肾脏差异表达基因

目的 运用基因芯片技术分析亚慢性汞中毒大鼠肾脏基因表达语的变化.方法 健康雄性SD大鼠20只随机分成染毒组和对照组,每组10只.染毒组皮下注射0.2 mg/kg氛化汞溶液2个月,建立亚慢性汞中毒肾脏损伤大鼠模型,用基因芯片方法筛选大鼠肾脏差异表达基因,并用逆转录-聚合酶链反应(RT-PCR)方法验证部分差异表达基因厂结果筛选出亚慢性汞中毒肾脏差异表达基因385个,其中上调139个,下调246个.上调基因主要步及毒物异物应答、炎症应答、神经递质代谢、溶质体转运等功能;下调基因主要涉及基础物质代谢、细胞信号传导、免疫调节、细胞增殖、转录调节等功能基因.RT-PCR方法验证结果与芯片结果一致.结论　汞中毒肾脏损伤是多墓因参与的结果;筛选出的明显差异表达基因可能在肾脏损伤机制中发挥重要作用,可能是氛化汞肾脏毒性作用的敏感基因或靶基因.     

生长期缺锌对大鼠海马Egr家族基因表达影响

目的 研究锌缺乏对生长期大鼠海马Egr家族(Egr1、Egr2、Egr3、Egr4)基因表达的影响,探讨缺锌影响学习记忆的基因转录调节机制.方法 选取刚出生的Sprague-Dawley大鼠3窝,每窝1只母鼠、10只仔鼠,3窝母鼠与仔鼠体重相近,仔鼠雌雄各半;随机分为3组:缺锌组(ZD)、配饲组(PF)、对照组(ZA),实验期60 d;用实时荧光定量PCR检测大鼠海马Egr家族(Egr1、Egr2、Egr3、Egr4)mRNA的表达.结果 大鼠海马Egr1、Egr3、Egr4基因表达在3组大鼠间差异有统计学意义(F值分别为13.631,24.435,21.825,),ZD组(2.44×10~(-3),8.72×10~(-3),1.88×10~(-2))表达量低于PF组(3.13×10~(-3),1.60×10~(-2),2.68×10~(-2))、ZA组(3.96×10~(-3),1.92×10~(-2),3.53×10~(-2)),且PF组低于ZA组;海马Egr2基因表达在各组间差异无统计学意义.结论 缺锌使海马锌指蛋白Egr1、Egr3、Egr4基因表达下调,是影响海马学习记忆的机制之一.     

巨噬细胞移动抑制因子在大鼠实验性结肠炎肠组织中的表达

目的观察巨噬细胞移动抑制因子（MIF）在三硝基苯磺酸（TNBS）诱发的大鼠结肠炎肠组织中的表达及其与疾病严重程度的关系。方法采用TNBS／乙醇溶液灌肠制备大鼠结肠炎模型,24只动物分为正常组、轻、重模型组共3组。每天观察各组大鼠疾病活动指数（DAI）;生化法检测髓过氧化物酶（MPO）活性,采用逆转录,聚合酶链反应（RT-PCR）技术,对肠组织MIF的表达水平进行半定量测定。结果正常组DAI、MPO活性及MIFmRNA的表达水平分别为0．51±0．28、0．38±0．18、0．11±0．03;模型Ⅰ组5．04±0．73、1．68±0．45、0．65±0．04;模型Ⅱ组8．13±0．71、2．70±0．35、0．81±0．05。与正常组相比,模型组肠组织中MIFmR-NA的表达显著增强（P〈0．05）,MPO活性及DAI也显著增高（P〈0．05）,且在病情严重组增高尤为明显。结论大鼠结肠炎肠组织中MIF的表达升高,并与疾病严重程度正相关,提示其可能与大鼠结肠炎的发病有关。     

Vitamin a deficiency induced histological and histochemical alterations in the testis of rats

In order to seek some explanatory evidence for the possible endocrine and/or biochemical correlation for vitamin A deficiency induced testicular lesion sin rats, detailed histopathological chanes and histochemical alterations in the localisation of lipids and distribution of steroid dehydrogenases and phosphomonoestrases have been studied in Charles Foster strain of male rats maintained on a vitamin A deficient diet for 5 weeks. Experimental rats depicted significant testicular size reduction and extensive pathological alterations affecting both the shape and size of the seminiferous tubules as well as degenerative changes involving germ cells. Alterations in sertoli cell cytology and hyperamia and oedema were also noted. Histochemically the tubules were marked by increased lipid accumulation and intense 17-ß-HSDH activity with reduced phosphomonoestrase activity. The intensity and activity pattern of phosphomonoestrases were both altered in vitamin A deficient rats. Another noticeable change was the loss of 3-ß-HSDH activity from the tubules. Both histologically and histochemically, the interstitial cells did not show any noticeable alterations. These changes have been taken to indicate the possibility of increased steroid catabolism and reduced androgen retention within the tubules alongwith functional break down of the blood-testis barrier, all of which together could provide the causal explanation for the testicular dysfunction associated with vitamin A deficiency.     

Neonatal-age treatment with vitamin A delays postweaning vitamin A deficiency and increases the antibody response to T-cell dependent antigens in young adult rats fed a vitamin A-deficient diet

Vitamin A supplementation for infants and young children is recommended by WHO/UNICEF for countries with a high prevalence of vitamin A deficiency, and vitamin A is often administered at immunization contacts. Using a rat model, we tested whether supplementation with vitamin A or other retinoids at the time of neonatal immunization has prospective benefit in terms of preventing postweaning vitamin A deficiency and promoting antibody responses to T-cell dependent (TD) antigens administered at the neonatal stage and at the young adult stage. Rats were treated orally on postnatal d 6-8 with oil (placebo control), vitamin A, retinoic acid, or a combination of both (VARA) (n > or = 12/group), and immunized with tetanus toxoid (TT) on d 7. The primary anti-TT response was measured on d 21, after which weanling rats were fed the vitamin A-deficient diet until approximately 10 wk. At 8 wk, rats were immunized again with TT to determine the recall response, and with a novel TD antigen, keyhole limpet hemocyanin (KLH), to assess the adult primary response. None of the supplements affected the plasma titer of anti-TT immunoglobulin G (IgG) on d 21 (P = 0.25). However, neonatal-age supplementation with vitamin A or VARA at the young adult stage resulted in: >5 times higher anti-TT IgG recall response (P < 0.01); 5- and 9-times higher anti-KLH primary IgM and IgG responses, respectively (P < 0.05), and plasma retinol in the normal range (approximately 1.0 micromol/L vs. approximately 0.35 micromol/L in retinoic acid-treated and control groups, P < 0.0001). We conclude that early-life supplementation with vitamin A or VARA can prospectively benefit the primary and recall antibody responses to TD antigens administered at the young adult stage, which may involve the maintenance of normal plasma retinol levels.