Cd Accumulation and Cd–Metallothionein as a Biomarker in <Emphasis Type="Italic">Cepaea hortensis</Emphasis> (Helicidae,Pulmonata) from Laboratory Exposure and Metal-polluted Habitats

Cepaea hortensis is a widespread terrestrial pulmonate, contributing significantly to element fluxes in soil ecosystems. Due to its capacity of accumulating certain trace elements in its tissues, Cepaea hortensis can serve as a biological indicator of metal accumulation in contaminated areas. In response to Cd exposure this species and related helicid pulmonates are also able to synthesize an inducible, Cd-binding metallothionein (MT) isoform specifically serving in binding and detoxification of this metal. As shown by field-collected garden-snails from a metal-contaminated site near a zinc smelting works in Avonmouth (UK) and an unpolluted reference site in Reutte (Tyrol, Austria), Cd and Cd–MT concentrations in midgut gland of C. hortensisfrom these sites increased with rising Cd concentrations in the soil substrate from the same contaminated sites. By combining the results of these field data with laboratory experiments it appears that midgut gland Cd–MT of Cepaea hortensis seems to fulfil the criteria of a successful biomarker in many respects. First, the synthesis of the protein can rapidly be induced by Cd exposure. Second, the level of Cd–MT induction in C. hortensis directly reflects the intensity of metal exposure. Third, the induced signal of increased Cd–MT concentration in C. hortensis is persistent over extended periods of time. Fourth, the Cd–MT signal in C. hortensis seems to be very specific for Cd exposure. Regression analyses demonstrate that tissue levels of Cd and Cd–MT in C. hortensis depend on Cd concentrations in the substrate which is represented by either soil or plant material on which snails normally feed. In both cases the best fit for this dependence is exhibited by a semi-logarithmic relationship, with substrate (soil or plant feed) concentrations expressed on a logarithmic scale. It is concluded that C. hortensis and other related pulmonates can successfully be used either as biological indicators of Cd accumulation, or as key species in biomonitoring studies focusing on Cd–MT induction as a biomarker for Cd exposure.     

环境镉接触人群尿金属硫蛋白排泄与镉致骨损伤效应

目的研究环境镉接触人群尿金属硫蛋白(UMT)的排泄与镉致骨损伤效应的关系。方法镉污染区居民为环境镉接触人群,非污染区居民为对照人群。人群尿镉(UCd)与血镉(BCd)用原子吸收分光光度法测定,用环境接触量估测总镉摄入量(TCd)。UMT、尿β2微球蛋白(UB2M)、尿视黄醇结合蛋白(URBP)及尿白蛋白(UALB)用ELISA法测定,尿N-乙酰-β-D-氨基葡萄糖苷酶(UNAG)和UNAG同工酶B(UNAGB)用荧光分析法测定,均用尿肌酐校正。单光子骨密度仪测定人群前臂骨密度。结果UMT能反映机体接触镉时镉负荷的变化。而高剂量接触镉可先后导致肾功能障碍及骨质疏松。人群UMT排泄量与骨密度的关系同UMT排泄量本身是否异常有关。根据各指标与尿镉的剂量-反应曲线计算得到的基准剂量95%低限水平BMDL值,从小到大排列为UNAGB、UNAG、UB2M、UMT、URBP、T评分及UALB。结论镉致骨质疏松的发生与镉致肾功能障碍有关并迟于后者。UMT不仅能特异而敏感的反映镉致肾的毒性,而且能在一定程度上反映镉对骨骼的损伤。     

Characterisation of Two Metallothionein cDNAs from the Shore Crab for use as Biomarkers of Heavy Metal Pollution

Molecular biological procedures open up possibilities for the development of new biomarker assays for use in environmental monitoring studies. Metallothionein (MT) is a useful biomarker for monitoring pollution by heavy metals and since very little information is available on the genes for MT in marine invertebrates, studies have been initiated in order to develop probes for use in biomarker assays for MT in the shore crab (Carcinus maenas). RNA isolated from the gills of shore crabs was used to produce complementary DNA (cDNA) from which two incomplete and two complete MT cDNAs have been isolated and characterised. The first complete cDNA (cDNA-4) encodes for a protein of 58 amino acids with a predicted molecular mass of 6151 Da; the predicted amino acid sequence of this protein is identical to that determined earlier for MT-Ib isolated from cadmium-exposed crabs. The second complete cDNA (cDNA-3) encodes a protein of 41 amino acids with a predicted molecular mass of 4484 Da; only the 5 C-terminal residues of this truncated MT differ from those at the corresponding positions of MT-Ib and this may correspond to a 4100 Da MT also reported previously. The implications of these findings and the use of these cDNAs as biomarkers in ecotoxicological studies are discussed.     

外源金属硫蛋白基因表达对NIH3T3细胞耐寒力的影响

目的：研究金属硫蛋白基因对细胞耐寒力的影响．方法：通过将金属硫蛋白基因（ｍｅｔａｌｌｏｔｈｉｏｎｅｉｎ,ＭＴ）的表达载体ｐＢＡｃＮｅｏ－ＭＴⅡＡ转染ＮＩＨ３Ｔ３细胞（小鼠胚胎成纤维细胞系）,筛选获得了高表达ＭＴ的细胞克隆ＭＴ１（ＭＴ阳性细胞百分比为２９．４％）,用四唑盐（ＭＴＴ）比色实验观察了该细胞的生长曲线以及不同低温条件下的存活能力,并通过电镜观察超微结构以进一步证实．结果：与转染ｐＢＡｃＮｅｏ空载体的细胞克隆Ｎｅｏ相比,常温下二者的增殖能力无显著差别．在不同的低温条件下,存活能力都受到一定程度的影响,细胞克隆ＭＴ１的存活细胞数明显高于阴性对照（Ｐ＜０．０１）,超微结构改变不明显．结论：上调金属硫蛋白基因表达可能具有增强耐寒的能力．     

有机镉染毒对小鼠免疫球蛋白及其亚类水平的影响

为研究有机镉化合物对体液免疫功能的影响。有机镉组采用分离纯化的镉金属硫蛋白 (CdMT)作为受试物给予低、中、高剂量 (Cd2 +0 2、0 5、1 0mg kg) ,无机镉组给予CdCl2 (Cd2 +0 5mg kg) ,BALB c小鼠经灌胃染毒 2周和 4周 ,检测血清中免疫球蛋白 (Ig)及其亚类的水平。结果显示染毒 4周后与对照组和脱金属硫蛋白 (Apo MT)组比较 ,CdCl2 组血清IgG1、IgG2a、IgG2b、IgG3、IgA、IgM和IgLλ各指标均显著性降低 (P <0 0 5 ) ;CdMT组IgG2a和IgG2b明显降低 (P <0 0 5 ) ,但无剂量 反应关系 ,其余指标未见显著性差异 (P >0 0 5 )。结果表明有机镉CdMT可诱导体液免疫功能抑制 ,但明显轻于无机镉作用 ,提示不同形态镉的体液免疫毒性不同并与金属硫蛋白的作用有关     

Cd modifies hepatic Zn deposition and modulates δ-ALA-D activity and MT levels by distinct mechanisms

Cadmium (Cd) is a pollutant that is harmful to human and animals. The liver is a target for Cd accumulation and it can disrupt Zn homeostasis. Here we examined the interaction of Zn and Cd to determine how these two metals could affect δ-aminolevulinate-dehydratase (δ-ALA-D) and metallothionein (MT), two potential molecular endpoints for Cd hepatotoxicity. Cd exposure (0.25 and 1 mg kg1 body weight, i.p., for 10 days) caused a marked increase in hepatic Zn deposition, which was not modified by treatment with Zn (2 mg kg1 , i.p.). Cd caused a dose-dependent increase in hepatic Cd content that was not modified by Zn. Zn and/or Cd treatment increased hepatic δ-ALA-D activity, although the increase caused by Cd was less marked. Reactivation index of δ-ALA-D by DTT was decreased by Zn and Cd exposure, which indicates that Zn protects enzyme from oxidation. Hepatic MT was increased only after exposure to 1 mg kg(-1) Cd and Zn reduced the stimulation of MT synthesis. The results presented here indicate that Cd can redistribute Zn from non-hepatic tissues to liver and the increase in hepatic Zn deposition can account for the increase in hepatic δ-ALA-D activity after Cd exposure. However, Zn blocked the increase in hepatic MT levels caused by Cd. Thus, the modulation of the two molecular endpoints of Cd toxicity used here was distinct, which indicates that the mechanism(s) involved in Zn and Cd distribution, δ-ALA-D and MT regulation are not coincident.     

Meta-analysis of 12 genomic studies in bipolar disorder

Multiple genome-wide expression studies of bipolar disorder have been published. However, a unified picture of the genomic basis for the disease has not yet emerged. Genes identified in one study often fail to be identified in other studies, prompting the question of whether microarray studies in the brain are inherently unreliable. To answer this question, we performed a meta-analysis of 12 microarray studies of bipolar disorder. These studies included >500 individual array samples, on a range of microarray platforms and brain regions. Although we confirmed that individual studies showed some differences in results, clear and striking regulation patterns emerged across the studies. These patterns were found at the individual gene level, at the functional level, and at the broader pathway level. The patterns were generally found to be reproducible across platform and region, and were highly statistically significant. We show that the seeming discordance between the studies was primarily a result of the following factors, which are also typical for other brain array studies: (1) Sample sizes were, in retrospect, too small; (2) criteria were at once too restrictive (generally focusing on fold changes >1.5) and too broad (generally using p<0.05 or p<0.01 as criteria for significance); and (3) statistical adjustments were not consistently applied for confounders. In addition to these general conclusions, we also summarize the primary biological findings of the meta-analysis, focusing on areas that confirm previous research and also on novel findings.     

Effect of Chlorella intake on Cadmium metabolism in rats

This study was performed to investigate the effect of chlorella on cadmium (Cd) toxicity in Cd- administered rats. Sixty male Sprague-Dawley rats (14 week-old) were blocked into 6 groups. Cadmium chloride was given at levels of 0 or 325 mg (Cd: 0, 160 ppm), and chlorella powder at levels of 0, 3 and 5%. Cadmium was accumulated in blood and tissues (liver, kidney and small intestine) in the Cd-exposed groups, while the accumulation of Cd was decreased in the Cd-exposed chlorella groups. Fecal and urinary Cd excretions were remarkably increased in Cd-exposed chlorella groups. Thus, cadmium retention ratio and absorption rate were decreased in the Cd exposed chlorella groups. Urinary and serum creatinine, and creatinine clearance were not changed in experimental animals. In addition, metallothionein (MT) synthesis in tissues was increased by Cd administration. The Cd-exposed chlorella groups indicated lower MT concentration compared to the Cd-exposed groups. Moreover, glomerular filtration rate (GFR) was not changed by dietary chlorella and Cd administration. According to the results above, this study could suggest that Cd toxicity can be alleviated by increasing Cd excretion through feces. Therefore, when exposed to Cd, chlorella is an appropriate source which counteracts heavy metal poisoning, to decrease the damage of tissues by decreasing cadmium absorption.     

The role of ZIP8 down‐regulation in cadmium‐resistant metallothionein‐null cells

The mechanisms of cellular cadmium uptake in mammalian cells remain obscure. To solve this problem, we established cadmium‐resistant cells (A7 and B5) from metallothionein‐null mouse cells, and found that cadmium accumulation was markedly suppressed in these cells. DNA microarray and real‐time PCR analyses revealed that expressions of ZIP (Zrt‐, Irt‐related protein) 8 and ZIP14 were down‐regulated in A7 and B5 cells. In particular, both mRNA and protein levels of ZIP8 were markedly suppressed in A7 and B5 cells. Introduction of short hairpin RNA (shRNA) of ZIP8 into parental cells reduced the accumulation of cadmium to about 35% of that of mock‐transfected cells, whereas the introduction of shRNA of divalent metal transporter 1 hardly changed cadmium accumulation. Thus, the cadmium resistance in A7 and B5 cells may be conferred primarily by the down‐regulation of ZIP8. In mouse tissues, high expression of ZIP8 was noted in the liver, kidney, lung and testis. These data suggest that ZIP8 plays an important role in cellular uptake of cadmium. Copyright © 2009 John Wiley & Sons, Ltd.