Perturbation of copper (Cu) homeostasis and expression of Cu-binding proteins in cadmium-resistant lung fibroblasts.

To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.     

The influence of metallothionein on exposure to metals: An in vitro study on cellular models

In the present study, the interactions between zinc (Zn) and copper (Cu) or iron (Fe) have been examined. Rat hepatoma cell line H4-II-E-C3, fibroblast cell line mutant MT−/−, and wild-type MT+/+ cells treated with ZnSO₄ or CuSO₄ or FeSO₄ or CuSO₄ + ZnSO₄ or ZnSO₄ + FeSO₄ for different times have been employed to study the effect of metallothionein (MT), glutathione (GSH) and metal (Cu, Fe and Zn) accumulation during cellular adaptation to supraphysiological metal concentrations. To investigate the different biological functions in the processes of metal homeostasis and detoxification, the levels of both MT-1 and MT-2 mRNAs have been evaluated. The three cell lines responded differently to metal treatments suggesting that the uptake and storage of these metals are affected by the specific cellular model and MT presence. In particular, Zn treatment significantly decreased Fe accumulation (p < 0.05), whereas MT induced by Zn increased intracellular Cu content (p < 0.05). Moreover, in H4-II-E-C3 cells administration of metals resulted in a rapid and transient induction of MT (p < 0.05) and in GSH accumulation (p < 0.05) suggesting synergistic interactions in which both appear essential for a protective regulatory function against the redox activity of metals. Taken together these results demonstrate that Zn affects the cellular levels of Cu and Fe by competition with the same ligand sites and/or by coordinate regulation of MT and GSH content.     

Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR

Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles and oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv(BAS)) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv(BAS) increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.     

Metallothionein induction in aquatic oligochaete tubifex tubifex exposed to herbicide isoproturon

Metallothioneins (MTs) are low-molecular-weight proteins mainly involved in metal ion detoxification. Recently it has been demonstrated that MTs participate in several cellular functions such as regulation of growth and antioxidative defenses. Moreover, pesticides can induce their synthesis. The aim of the current work was to determine the effects of isoproturon, either pure or formulated as Matin (suspension containing an isoproturon concentration of 500 g. L(-1)), on the metallothionein and total protein contents of the aquatic worm Tubifex tubifex. MT levels in exposed worms increased significantly after 7 and 15 days of exposure to a concentration of the herbicide of 50 mg. L(-1). Isoproturon reduced the metal (Cu, Zn, and Cd) content of metallothioneins, and it also increased the total protein content of the worms. These results suggest that MT induction may not be considered a specific biomarker of metal exposure but that it can be used as a nonspecific biomarker of the effect of isoproturon effect in aquatic worms.     

Arsenic-copper interaction in the kidney of the rat.

1. The interaction between As and three toxic metals (Cd, Ni and Pb) and Cu (an essential trace metal) in the kidney was investigated in the rat by feeding diets containing various concentrations of As whilst maintaining constant concentrations of the other elements. After 1, 3, 7 and 15 weeks of feeding, metal contents in the renal cortex and medulla, red blood cells and plasma were determined by atomic emission spectrometry (ICP-AES). 2. As accumulated in the whole kidney, whereas Cu accumulated only in the cortex. Accumulation of Cu was found to depend on the feeding period and dietary As concentration. 3. As was also accumulated in red blood cells, where saturation was found at 550 micrograms As g-1 cells. 4. Although Cd was also accumulated in the cortex, its accumulation was independent of the dietary As concentration. Ni and Pb were not detected by ICP-AES. 5. Chromatography of the supernatants from cortical homogenates of control and As-treated rat kidney suggested that Cu accumulated in renal metallothionein (MT). Its accumulation in this fraction was independent of that of Cd, indicating that the As-Cu interaction was not a result of MT induction, but rather that it might result from altered renal handling of Cu with subsequent incorporation into MT.     

Involvement of cytokines in the hepatic expression of metallothionein by ursolic acid

Ursolic acid (UA), a pentacyclic triterpene acid, is reported to have inducing activity of hepatic metallothionein (MT) which responsible for the detoxification of heavy metals; however, the mechanism underlying its effects is poorly understood. To further determine the underlying mechanism of UA, this study investigated the effects of UA on the induction of hepatic MT expression in an in vitro model, using murine hepatoma cell line Hepa-1c1c7 and murine macrophage cell line RAW 264.7 cell cultures. The UA added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels were markedly increased when Hepa-1c1c7 cells were cultured with UA-treated conditioned media from RAW 264.7. Concomitant treatment with UA and pentoxifylline, a TNF-alpha synthesis inhibitor, to RAW 264.7 cells decreased the effects of UA on the MT induction. In UA-exposed RAW 264.7 cell cultures, TNF-alpha and IL-6 production and TNF-alpha and IL-6 mRNA levels increased. When antibodies to TNF-alpha or/and IL-6 were added to UA-treated conditioned media from RAW 264.7, the MT induction activity was inhibited. These results demonstrate that UA induces hepatic MT expression through TNF-alpha and IL-6 released from UA-activated macrophages, which may be the mechanism, whereby UA elicits its biological effects.     

Metallothionein and apoptosis during differentiation of myoblasts to myotubes: protection against free radical toxicity.

The changes in subcellular localization of metallothionein (MT) during differentiation were studied in two muscle cell lines, L6 and H9C2, myoblasts in order to understand the nuclear presence of MT and its antiapoptotic property. In myoblasts, MT and zinc were localized mainly in the cytoplasm but were translocated into the nucleus of newly formed myotubes during early stage of differentiation, which was initiated by lowering FBS from 10% to 1%. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. These changes in subcellular localization of MT and Zn were accompanied by increased apoptosis in myotubes. The changes in the apoptosis at different stages of differentiation were measured by both DNA ladder formation and TUNEL technique. The results also show that the apoptosis may be initiated by free radical generation and may be accompanied by p53 expression. The H9C2 cells contained high levels of MT, differentiated slowly, and had low incidence of apoptotic bodies compared to L6 cell line.     

The protective effect of metallothionein on the toxicity of various metals in rat primary hepatocyte culture

Metallothionein (MT), a low-molecular-weight, cysteine-rich, metal-binding protein, has been implicated in the detoxification of Cd. However, whether MT protects against the cellular toxicity of other metals has not been examined thoroughly. This study was therefore designed to determine the effects of Zn-induced MT on the toxicity of seven metals in rat primary hepatocyte cultures. Hepatocytes were grown in monolayer culture for 22 hr and subsequently treated with ZnCl2 (100 microM) for 24 hr which produced a 15-fold increase in MT concentration. Following Zn pretreatment, hepatocytes were exposed to various concentrations of Ag, Co, Cu, Hg, Ni, Pb, or Zn for 24 hr. Cytotoxicity was assessed by enzyme leakage and loss of intracellular K+. The toxicity of all seven metals was significantly less in the Zn-pretreated cells. Zn pretreatment had no appreciable effect on the hepatocellular uptake (1-24 hr) of 110Ag or 203Hg, but markedly altered their subcellular distribution, with metals accumulating more in the cytosol and less in the nuclear, mitochondrial, and microsomal fractions. In the cytosol of control cells, the metals were bound mainly to high-molecular-weight proteins whereas in the Zn-pretreated cells, the metals were mainly associated with MT. In summary, Zn-induced MT in rat primary hepatocyte cultures protects against Ag-, Co-, Cu-, Hg-, Ni-, Pb-, and Zn-induced cytotoxicity. This protection appears to be due to the binding of metals to MT with a concomitant reduction of metal content in critical organelles and proteins.     

Effects of copper on cortisol receptor and metallothionein expression in gills of Oncorhynchus mykiss

Effects of waterborne Cu (2.4 microM) on the expression of glucocorticoid receptor (GR) and metallothionein (MT) in the branchial epithelium of freshwater rainbow trout (Oncorhynchus mykiss) was studied by immunocytochemistry. After 5 days of Cu exposure, the number of GR-immunoreactive (GR-ir) cells in the gill epithelium had decreased, whereas the number of MT-ir cells had increased. Localization of GR in chloride cells was achieved by double staining for Na(+)/K(+)-ATPase; other cell types were identified on the basis of their topology. GRs were present in the chloride cells in both the filaments and lamellae, in respiratory cells in the lamellae, in pavement cells, basal layer cells and undifferentiated cells in the filaments. Co-localization of Na(+)/K(+)-ATPase and MT revealed chat MT was expressed in chloride cells, both in filaments and lamellae. Occasionally, MT immunoreactivity was found in pavement cells and in undifferentiated cells. By double staining for Na(+)/K(+)-ATPase and GR, for Na(+)/K(+)-ATPase and MT and for GR and MT, we can conclude that after 5 days of Cu stress there are chloride cells that express GR and MT, GR or MT alone or neither of the two proteins. This apparent functional heterogeneity of branchial chloride cells may reflect a limited window when chloride cell subpopulations show an adaptive response to Cu.     

Metal accumulation and metallothionein in two populations of brown trout, Salmo trutta, exposed to different natural water environments during a run-off episode

Cd, Cu and Zn concentrations were measured in ambient water as well as in gills, liver and kidney tissues of two natural populations of brown trout (Salmo trutta) during a run-off episode in two rivers with different metal compositions due to mining pollution. Metallothionein (MT) was also measured in these tissues. The two rivers, Rugla (Cu contaminated) and Naustebekken (Cd and Zn contaminated), are located in two neighboring drainage basins separated by the topographic divide near the city of Røros in the County of Sør-Trøndelag, Norway. In Rugla, the Cu concentration increased from 15 μg/l at the low water level to 41 μg/l during the run-off episode. In Naustebekken, corresponding values for Cd were 90–170 ng/l and those for Zn were 49–91 μg/l. Gill concentrations of Cu and Cd/Zn MT in both populations of native trout clearly reflect the presence of these metals in the rivers during the run-off, in accordance with the hypothesis of protection caused by MT induction. When Rugla trout were transferred to Naustebekken and vice versa, both the amounts of MT itself and the Cu contents reflected the concentration of this metal in the new environment, indicating that MT induction also protects against acutely increased metal levels. The measured levels of MT in both native and transferred trout can account for all the Cd present in the tissues, but not for all of the Cu and Zn. The capacity of MT to regulate Cd and Cu in the trout populations in their natural habitat therefore seems clearly present. Our data also indicate that the MT I and II isoforms may bind metals selectively.     

Tissue‐specific copper accumulation,zinc levels,induction, and purification of metallothionein in freshwater crab Sinopotamon henanense exposed to subacute waterborne copper

Copper (Cu) is one of the most important essential metals for crustaceans, buttoxic in excess. Metallothioneins (MT) are a family of low molecular weight, cysteine‐rich, metal‐binding proteins, which play important roles in metal homeostasis, detoxification, and cytoprotection. In the present study, Sinopotamon henanense were exposed to 0 (controls), 2.86, and 14.3 mg L^?1 waterborne Cu, Cu accumulation, zinc (Zn) levels and MT induction in gills and hepatopancreas were determined with Cd/Hemoglobin saturation assay and atomic absorption spectrophotometry method. Results showed that Cu accumulation and MT levels were both tissue‐specific and revealed some time‐dependent and dose‐dependent, respectively. The highest Cu accumulations of 82.10 ± 16.38 μg g^?1 w wt were observed in the gill after 15 days of 14.3 mg L^?1 Cu exposure, the peak MT induction of 136.16 ± 19.39 μg g^?1 w wt were observed in the hepatopancreas after 3 day of 14.3 mg L^?1 Cu exposure.In addition, the essential metal homeostasis of Zn was disturbed in some ways by subacute Cu exposure. The calculated ratios of actual Cu to theoretical maximum metal bound by MT indicating that the hepatopancreas had much greater Cu‐binding potentials than the gills. Positive correlation were shown between MT induction and Cu accumulation both in hepatopancreas and gills, indicating that MT induction in S. henanense can be considered as a biomarker for subacute waterborne Cu pollution. Furthermore, the Cu induced MT (CuMT) from S. henanense was purified using acetone precipitation (50–80%), followed by gel filtration chromatography and anion exchange chromatography. SDS‐PAGE and time‐of‐flight mass spectrometry analysis showed that S. henanense CuMT possess two isoforms and both mainly existed as monomer and dimmer forms. These present studies will be helpful to increase the database information of heavy metal‐induced MT in terms of crustaceans. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 407–417, 2014.     

Developmental and sex differences in cadmium distribution and metallothionein induction and localization

Age- and sex-related differences in hepatic and renal distribution of cadmium (Cd) and the effect of Cd injection (10 mumol/kg) on tissue zinc (Zn), copper (Cu) and metallothionein (MT) levels were investigated in 2- to 84-day old rats. Renal Cd accumulation increased with age of the animal. Sex differences in renal Cd accumulation were noted in young animals where the 2- and 8-day old males had significantly greater concentration than the females. There were no clear effects of Cd on renal Zn. Renal Cu levels, however, were elevated in the adults. The adult females contained about twice as much MT as the adult males. Cd treatment had no effect on renal MT levels of 8- to 84-day old animals but depressed the levels in 2-day old. Age-related increase in hepatic Cd accumulation was also found; the pattern was more clear cut in females than in males. In addition, in the females the hepatic Cd concentration was significantly higher than in the males. Cd-injection significantly increased hepatic Zn and MT concentrations only in weaned animals. While there were no sex differences in MT levels in the young animals, the weaned females had significantly more MT than the corresponding males. Immunohistochemical staining for MT showed positive staining in both cytoplasm and nuclei of the parenchymal cells. The number of MT-positive nuclei was dependent on the relative MT concentration of the liver. In spite of the intense nuclear staining in 2-day old controls and 84-day old Cd-injected rats, less than 1% of the hepatic MT was present in the nuclear fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

厄贝沙坦对高糖刺激的大鼠肾小球系膜细胞结缔组织生长因子及膜型基质金属蛋白酶-1表达的影响

目的研究血管紧张素Ⅱ受体1拮抗剂厄贝沙坦对高糖刺激的大鼠肾小球系膜细胞结缔组织生长因子(CTGF)和膜型基质金属蛋白酶-1(MT1-MMP)表达的影响。方法体外培养的大鼠肾小球系膜细胞,分别给予高糖和厄贝沙坦干预,采用RT-PCR及Western blot法分别检测CTGF和MT1-MMP的mRNA及蛋白的表达,用酶联免疫吸附法(ELISA)检测培养上清中Ⅳ型胶原的含量。结果与对照组相比,高糖组各时间点系膜细胞CTGF表达明显上调,Ⅳ型胶原的分泌增加,且二者随时间持续增高;而MT1-MMP的表达则随时间呈明显下降趋势。厄贝沙坦能够抑制高糖引起的上述变化。结论高糖可诱导肾小球系膜细胞CTGF表达增加,抑制MT1-MMP的表达。厄贝沙坦抑制肾小球系膜细胞基质分泌的作用可能部分通过抑制CTGF,增加MT1-MMP的表达而实现。     

Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers

The literature on metallothioneins (MT) and metallothionein-like proteins (MTLP) in aquatic invertebrates is large and increasing, and yet inconsistencies and confusion remain, not least over the physiological role of MT and their use as biomarkers in environmental monitoring programmes. We have collated published information on MT in three important groups of aquatic invertebrates-the molluscs, crustaceans and annelid worms, and attempted to seek explanations for some of the apparent inconsistencies present in the dataset. MTs can be induced by the essential metals Cu and Zn and the non-essential metals Cd, Ag and Hg in both vertebrates and invertebrates, but their induction is variable. Such variation is intraspecific and interspecific, and is down to a variety of reasons environmental and physiological explored here. Against this background of variability MTs do appear to play roles both in the routine metabolic handling of essential Cu and Zn, but also in the detoxification of excess amounts intracellularly of these metals and of non-essential Cd, Ag and Hg. Different isoforms of MT probably play different physiological roles, and the dependence on MT in detoxification processes varies environmentally and between zoological groups. MTs can be used as biomarkers if used wisely in well-designed environmental monitoring programmes.     

Localization and quantification of Cd- and Cu-specific metallothionein isoform mRNA in cells and organs of the terrestrial gastropod Helix pomatia

A quantitative assay based on real-time detection polymerase chain reaction (rtdPCR) was applied to analyze basal and metal-induced mRNA levels of two metallothionein (MT) isoforms (Cd-MT and Cu-MT) in organs of the terrestrial gastropod Helix pomatia. The results show that specific Cd-MT mRNA levels increase with Cd tissue burden, identifying hepatopancreas and gut as the main organs of Cd accumulation and, accordingly, the predominant organs of Cd-MT mRNA expression. In situ hybridization localized this isoform in epithelial cells of hepatopancreas, gut, and kidney. In contrast to the observed Cd-dependent inducibility of the Cd-binding MT isoform, gene expression of the Cu-binding MT could not be induced by either Cd or Cu exposure. Only very low mRNA amounts of the Cu-MT isoform were found in snail hepatopancreas and kidney, whereas the mantle exhibited high basal mRNA levels of this isoform. In situ localization revealed that the Cu-MT gene expression was restricted to one cell type, the so-called rhogocytes, which are present to various extents in the different organs examined. These results suggest a metal-specific sharing of functions between the two MT isoforms. The Cd-MT isoform apparently plays a crucial role in Cd detoxification, as demonstrated by the inducibility of this isoform, as well as its specific localization in the main metabolic and Cd storing organs. The predominant presence of Cu-MT in rhogocytes of snail mantle strengthens the hypothesis that this isoform may regulate Cu availability in hemocyanin synthesis.     

An improved diagnostic method for chronic hepatic disorder: analyses of metallothionein isoforms and trace metals in the liver of patients with hepatocellular carcinoma as determined by capillary zone electrophoresis and inductively coupled plasma-mass spectrometry

It is desirable to diagnose hepatocellular carcinoma (HCC) in the early stages during its development since its treatment is usually difficult. We previously proposed a new diagnostic method that made use of the total metallothionein (MT), zinc (Zn), and copper (Cu) concentrations in the liver of the HCC patients. We recently found that MT-1 is involved in the metabolism or detoxification of toxic metals, such as cadmium; on the other hand, MT-2 is responsible for the homeostasis of essential metals such as copper, in experimental models such as Long Evans Cinnamon (LEC) rats. In order to device a better diagnostic method than the one we proposed previously, in this study, we newly propose an improved method that includes the discriminative determination data regarding the MT isomers, namely, MT-1 and MT-2, in the liver of patients with or without HCC as compared with the total MT level. The total MT and Zn concentrations in the HCC patients were confirmed to be significantly lower than those in patients without hepatic disorders (Ctrl). In contrast, Cu concentrations of the HCC patients were higher than those of the Ctrl patients. In addition, in the juxta-tumor portion with HCC, MT-1 concentrations were significantly higher than those of MT-2. In contrast, the MT-1 concentrations in the tumor portion were significantly lower than that in the juxta-tumor portion. In addition, MT-1/MT-2 ratio in the tumor portion was significantly lower than that of the juxta-tumor portion. By using parameters such as concentrations of Cu, Zn, total MT, and MT isomers, we performed the multivariate discriminative analysis (MDA). The results suggest that the concentrations of MT isomers change depending on the progress of the tumor, and information on MT isomers and trace elements is very useful in determining the stage of the chronic hepatic disorder.     

Steady-state distribution of metals among metallothionein and other cytosolic ligands and links to cytotoxicity in bivalves living along a polymetallic gradient

The present study was designed to assess the environmental effects of metals in a field setting. We explored exposure-->bioaccumulation-->effects relationships in freshwater molluscs exposed to metals in their natural habitat. Indigenous floater mussels (Pyganodon grandis) were collected from ten limnologically similar lakes located along a Cd, Cu and Zn gradient. Ambient free-metal ion concentrations were estimated as a measure of metal exposure. Metallothionein (MT) was measured in mussel gills and metal partitioning among the various cytosolic protein pools was determined by size exclusion chromatography. Various biomarkers were also measured, including malondialdehyde (MDA) concentrations in the gills and in the digestive gland, glutathione-peroxidase and glutathione-reductase activities in the digestive gland, and lipid concentrations in the gonad. Cadmium and MT concentrations in the gill cytosol increased along the contamination gradient, but Cu and Zn levels were independent of the ambient free-metal ion concentrations. The distribution of Cd among the various cytosolic complexes remained quite constant: 80% in the MT-like pool, 7% in the low molecular weight pool (LMW<1.8 kDa) and 13% in the high molecular weight pool (HMW>18 kDa). For these chronically exposed molluscs there was thus no threshold exposure concentration above which spillover of Cd occurred from the MT pool to other cytosolic ligands. However, the presence of Cd in the LMW and HMW fractions suggests that metal detoxification was imperfect, i.e. that P. grandis was subject to some Cd-related stress at low chronic exposure concentrations. Consistent with this suggestion, MDA concentrations, an indicator of oxidative stress, increased with gill cytosolic Cd. In the digestive gland, MDA concentrations were unrelated to any of the measured metals, but glutathione-peroxidase and glutathione-reductase activities increased with gill cytosolic copper. We speculate that cytosolic Cu catalyses the production of reactive oxygen species, to which the organism reacts by increasing activities of the two enzymes, thus preventing the accumulation of reactive oxygen species. Lipid concentrations in the gonad did not decrease with any of the measured toxicological parameters, suggesting that energy reserves for reproduction were not compromised in the metal-contaminated mussels. The results of the present study, where chronically exposed bivalves were collected from their natural habitat along a metal contamination gradient, contrast markedly with what would have been predicted on the basis of experimental metal exposures, and clearly demonstrate the need to study metal exposure-->bioaccumulation-->effects relationships in natural populations.     

Contribution of glutathione and metallothioneins to protection against copper toxicity and redox cycling: quantitative analysis using MT+/+ and MT-/- mouse lung fibroblast cells

Glutathione (GSH) and metallothioneins (MT) are believed to play important roles in protecting cells against high copper (Cu) concentrations. Little is known, however, about their specific intracellular interactions and the coordination of protective functions. We investigated contributions of GSH and MT to protection against Cu toxicity in fibroblasts derived from wild-type (MT+/+) and knockout (MT-/-) mice that were challenged with cupric nitrilotriacetate (Cu-NTA). Endogenous levels of GSH and MT were manipulated using an inhibitor of gamma-glutamylcysteine synthetase, buthionine sulfoximine (BSO, 5 microM), as GSH depletor and ZnCl(2) (100 microM) as inducer of MT expression. BSO pretreatment markedly decreased cellular GSH levels in MT+/+ and MT-/- cells, by 65% and 70%, respectively, which resulted in Cu cytotoxicity accompanied by its elevated redox-cycling activity and enhanced Cu-induced membrane phospholipid peroxidation. BSO-pretreated MT-/- cells were markedly more sensitive to Cu despite the fact that the residual levels of GSH were similar in both BSO-pretreated MT+/+ and MT-/- cells. Zn pretreatment resulted in more than 10-fold induction of MT in MT+/+ cells but not in MT-/- cells. Accordingly, Zn pretreatment afforded significant protection of MT+/+ cells against Cu cytotoxicity, likely associated with MT-dependent suppression of Cu redox-cycling activity and phospholipid peroxidation, but it exerted no protection in MT-/- cells (as compared to naive cells). To determine whether MT functions specifically in Cu regulation or rather acts as a nonspecific Cu-binding cysteine-rich nucleophile, experiments were performed using MT+/+ and MT-/- cells pretreated with both BSO and Zn. BSO pretreatment did not affect Zn-induced MT expression in MT+/+ cells. As compared with BSO pretreatment alone, exposure to Cu of MT+/+ cells after Zn/BSO pretreatment resulted in the following: (i) a significantly higher viability; (ii) attenuated Cu-dependent redox-cycling activity; and (iii) a lower level of phospholipid peroxidation. In BSO/Zn-pretreated MT-/- cells, the redox-cycling activity of Cu and the level of phospholipid peroxidation remained remarkably higher than in naive cells and were not significantly different from those in cells pretreated with BSO alone. Cu-induced toxicity was remarkably higher in BSO/Zn-pretreated MT-/- cells than in naive or Zn-pretreated cells, although slightly lower than in the MT-/- cells pretreated with BSO alone.     

Biochemical changes in the rat lung and liver following intratracheal instillation of cadmium oxide

Acute biochemical changes in the rat lung and liver following intratracheal instillation of cadmium oxide (CdO) were observed at a dose of 5 micrograms Cd/rat to investigate the defense mechanism to Cd intoxication via airway. In the lung metallothionein (MT) was induced, reaching a maximum at 2 days. A slight increase in reduced glutathione (GSH) concentration was observed at 4 days. The activity of glucose-6-phosphate dehydrogenase (G6PDH) was increased and superoxide dismutase (SOD) activity was slightly decreased, but glutathione peroxidase (GPx) and glutathione reductase (GR) activities were not changed. These observations suggested that MT played a key role in detoxification of instilled CdO, but that the antioxidant enzymes had a minimal role. In the liver MT and GSH concentrations were diminished 7 h after instillation and returned to their control levels. Hepatic GPx activity was increased 1 day after instillation and the significantly elevated level lasted up to 7 days, while hepatic GR activity was decreased. These hepatic biochemical changes are suggested to be due to the secondary effects of the lung injury.     

成人肝癌组织褪黑素受体的表达和结合特性

目的检测成人肝癌组织是否表达褪黑素受体(me-latonin receptor,MR),并探讨其结合特性。方法免疫组化法检测肝癌组织MR两种亚型(MT1、MT2)的表达和分布。采用放射配体结合法检测肝癌组织MR特异性结合和动力学特性。结果免疫组化结果显示,成人肝癌组织MT1和MT2均呈聚集型粽黄色颗粒阳性染色,主要分布在细胞膜和细胞质,细胞核中表达最少。放射配体结合结果表明,成人肝癌组织存有125I标记的褪黑素(125I-Mel)特异结合位点,最大结合容量(Bmax)值为(0.29±0.07)pmol.g-1 protein,平衡解离常数(Kd)值为(48.7±6.5)pmol·L-1,动力学分析表明该特异性结合特点为可饱和及可逆性结合。结论成人肝癌组织表达MR,包括MT1和MT2,其结合特性为低结合容量、高亲和力、可饱和性及可逆性。