镉中毒大鼠睾丸与肝脏金属硫蛋白表达的时相研究

啮齿目动物睾丸对镉毒性较肝脏更敏感.为阐明睾丸的镉毒性分子机制,比较了肝脏与睾丸金属硫蛋白（MT）表达的时相变化.mRNA采用RT-PCR技术分析并用光密度扫描定量;蛋白质定量用ELISA方法.结果显示,睾丸中存在MT,镉中毒后MT1与MT2 mRNA明显升高,但MT没有相应增加;肝脏镉中毒后MTmRNA与MT均明显升高.结果提示：镉虽然能诱导睾丸MTmRNA的转录,但没有促进其MT的合成,这可能是睾丸对镉毒性与致癌作用较肝脏更敏感的重要原因.     

Effects of Chronic Cadmium Poisoning on Zn, Cu, Fe, Ca, and Metallothionein in Liver and Kidney of Rats

An experiment was conducted to invest effects of chronic cadmium poisoning on Zn, Cu, Fe, Ca, and metallothionein gene expression and protein synthesis in liver and kidney in rats. Forty rats, 6?weeks old, were randomly allocated into two groups. A group was given CdCl(2) (1?mg/KgCd(2+)) by intraperitoneal injection once a day. The other group was treated with normal saline in the same way. Liver and kidney were collected for analysis at the end of the third week. Results showed that Cd exposure increased Cd (P?相似文献   

The binding of gold(I) to metallothionein

The binding of gold(I) to metallothionein, MT, has been unambiguously established by the reaction of Na₂AuTM with purified horse kidney MT. Zinc was displaced more readily than cadmium although the latter could be displaced using large Au/Cd ratios. The metal exchange reactions were complete within 2 hr of mixing. Further evidence that such reactions might be physiologically significant were obtained by studying in vitro metal displacements in the liver cytosol of in vivo metal treated rats: When Na₂AuTM was added to the cytosol of rats administered CdCl₂ in vivo, zinc, copper and cadmium were displaced in 2/1/1 ratios from the metallothionein fraction. The zinc and cadmium displacement provide direct evidence that the gold was binding to MT. Addition of Cd⁺² to liver cytosol of gold-treated rats resulted in displacement of copper and zinc, but not gold, from the MT fractions. When liver MT is prepared from rats exposed to Au or Cd, the Cd/protein ratio increased during the preparation, but the Au/protein ratio decreased. The Mt-bound metals account for 95% of the cytosolic Cd but only 15%–30% of the cytosolic gold in these studies. Thus, the nonspecific binding of gold to MT in vivo should be considered as one aspect in its equilibration among protein binding sites, which include, inter alia, metallothionein. Gold was found to coelute with zinc and cadmium in the MT fraction of rat kidney cytosol, when both Cd and Na₂AuTM were administered to the rats. The possible significance of gold binding to MT in the treatment of rheumatoid arthritis-chrysotherapy-is briefly discussed.     

Biological function of metallothionein. VI. Metabolic interaction of cadmium and zinc in rats

The accumulation and depletion of cadmium in liver and kidney metallothionein (MT) and the effects of dietary zinc deficiency on cadmium metabolism were studied in rats. The accumulation of cadmium in liver MT started to plateau after 80 days, but there was a linear accumulation of this element in kidney MT over the entire 300-day experiment. Cadmium in MT fractions was depleted very slowly when rats were changed to a diet without cadmium. The accumulation of cadmium in MT also caused zinc to accumulate in this protein, even in rats fed zinc-deficient diets. However, the reverse situation was found not to be true; zinc did not cause cadmium to accumulate in MT. Dietary zinc deficiency limited the binding of injected¹⁰⁹Cd to MT of liver, but not of kidneys or testes. However, zinc-deficient rats fed cadmium in their diets metabolized cadmium similarly to zinc-supplemented rats, suggesting that zinc deficiency does not limit the ability of cadmium to stimulate MT synthesis.     

Protective effects of zinc on cadmium toxicity in rodents

A study of acute and subacute toxicity of cadmium ions [Cd(II)] was carried out on male Swiss mice and Sprague-Dawley rats with and without previous administration of zinc chloride. The LD₅₀ of Cd(II) as cadmium sulfate (ip) was lower in animals previously given 10 mg/kg of zinc(II) chloride (sc). Factors such as animal weight variations, biochemical parameters, and accumulation patterns of Cd(II) and Zn(II) were taken into consideration when the subacute toxicity was evaluated. Alteration of the activities of glutamic pyruvic transaminase (GPT) and of glutamic oxaloacetic transaminase (GOT) was observed in short-term-exposure (<6 h) cases. These alterations reverted to normal after 1 wk. The activity of alkaline phosphatase (ALP) and the concentrations of cholesterol and triglycerides in serum are also changed, especially so in the groups given CdSO₄ alone. In the experimental groups treated with ZnCl₂ prior to administration of cadmium, proteinuria was detected 5 wk after the treatment. Also at 5 wk, both Zn-treated and nontreated groups showed an abnormally low liver mass with respect to total body mass. Both Cd and Zn are retained preferentially in the liver but show also in the kidneys. If CdSO₄ and ZnCl₂ are given simultaneously, especially after 1 wk of treatment, Cd is accumulated in greater amounts in these organs when compared to the groups given only cadmium sulfate.     

Metallothionein levels in willow ptarmigan (<Emphasis Type="Italic">Lagopus lagopus</Emphasis>) populations with different natural loads of cadmium

The tissues of willow ptarmigan in some Norwegian mountain areas contain elevated concentrations of cadmium (Cd). It is not known whether such high Cd levels would have negative impacts in otherwise healthy populations of this species. The aim of the current study was to clarify relationships between hepatic and renal metallothionein (MT) and Cd concentrations in willow ptarmigan to assess effects from this metal. The study reported here was undertaken on willow ptarmigan from the Kongsvoll area, with a naturally high Cd load, and the Essand area, with a naturally low Cd load. Cd values in liver and kidney in willow ptarmigan from Kongsvoll were significantly higher than in willow ptarmigan from Essand. The MT content in both tissues was also highest in willow ptarmigan from Kongsvoll. The MT concentration in kidney was twice that in liver for ptarmigan from both areas and at all times of the year. The MT level in both liver and kidney varied greatly throughout the season, with the highest content in spring (May). The variation was greatest in liver. The total material showed a significant linear relationship between Cd and MT levels in both liver and kidney, but a breakdown of the material into seasons and areas gave a varying degree of significance. MT in willow ptarmigan may be an important mechanism for detoxifying Cd, and populations exposed to high load may “respond” to the loads by increasing MT synthesis in tissues such as liver and kidney. There is no evidence that willow ptarmigan from areas with high natural Cd loads have reached a limit for MT synthesis in either liver or kidney.     

Kidney dysfunction and cadmium exposure--factors influencing dose-response relationships

Our early toxicological studies showed that metallothionein (MT) is a protein that carries cadmium (Cd) to the kidney, explaining why Cd exposures during long time periods may give rise to kidney dysfunction. This dysfunction is usually considered to be the critical effect, i.e. the adverse effect that occurs at the lowest exposure level. MT also provides intracellular protection against cadmium toxicity. In studies of population groups in cadmium contaminated areas in China, we investigated factors that affected the relationship between internal dose of Cd, as indicated by blood Cd (BCd) or urinary Cd (UCd), and the prevalence of kidney dysfunction. We found dose-response relationships between UCd and the prevalence of increased levels of biomarkers of renal tubular dysfunction (urinary beta-2-microglobulin, B2M, or N-acetyl-beta-d-glucosaminidase - NAG) or urinary albumin (UAlb), a biomarker of glomerular kidney dysfunction. Two years after Cd intake from contaminated rice was diminished, renal tubular dysfunction appeared unchanged or aggravated among those with higher UCd; Another 8 years later, i.e. 10 years after Cd intake was decreased, the prevalence of renal tubular dysfunction was still increased but UAlb had returned to normal. Factors that influenced the dose-response relationships were: (1) time after maximum exposure. (2) Concomitant exposure to other nephrotoxic agents such as inorganic arsenic. (3) Cd induced metallothionein mRNA levels in peripheral blood lymphocytes, used as a biomarker of the ability of each person, to synthesize MT. (4) The occurrence of increased levels in blood plasma of autoantibodies against MT. The two last points further support a role in humans of MT as a protective protein against tissue damage from cadmium and gives support to previous ideas developed partly in experimental systems.     

Metallothionein as Potential Biomarker of Cadmium Exposure in Persian Sturgeon (<Emphasis Type="Italic">Acipenser persicus</Emphasis>)

Metallothionein (MT) concentration in gills, liver, and kidney tissues of Persian sturgeon (Acipenser persicus) were determined following exposure to sublethal levels of waterborne cadmium (Cd) (50, 400, and 1,000 μg l⁻¹) after 1, 2, 4, and 14 days. The increases of MT from background levels were 4.6-, 3-, and 2.8-fold for kidney, liver, and gills, respectively. The results showed that MT level change in the kidney is time and concentration dependent. Also, cortisol measurement revealed elevation at the day 1 of exposure and followed by MT increase in the liver. Cd concentrations in the cytosol of experimental tissues were measured, and the results indicated that Cd levels in the cytosol of liver, kidney, and gills increased 240.71-, 32.05-, and 40.16-fold, respectively, 14 days after exposure to 1,000 μg l⁻¹ Cd. The accumulation of Cd in cytosol of tissues is in the order of liver > gills > kidney. Pearson correlation coefficients showed that the MT content in kidney is correlated with Cd concentration, the value of which is more than in liver and gills. Thus, kidney can be considered as a tissue indicator in A. persicus for waterborne Cd contamination.     

Metallothionein response to cadmium and zinc exposures compared in two freshwater bivalves, Dreissena polymorpha and Corbicula fluminea

Metallothionein (MT) response to cadmium (Cd) and zinc (Zn) bioaccumulation after single or combined direct exposure was compared in two freshwater bivalves, Dreissena polymorpha (zebra mussel) and Corbicula fluminea (Asiatic clam). Bivalves were exposed to 0.133 μM Cd and/or 15.3 μM Zn, with metal and MT concentrations analysed in the whole soft body after 1, 3, 10 and 24 days of exposure and compared with controls. Results showed significant increase in MT concentrations in both species exposed to Cd and Cd+Zn with a higher accumulation of the protein compared to the control in D. polymorpha for nevertheless similar Cd levels accumulated with time. Exposure to Zn alone led to a significant increase in MT concentrations only in C. fluminea, whereas there was a lack of MT gene induction in the zebra mussels which was confirmed by MT mRNA quantification in gills (RT-PCR). Mussel mortality after 10 days of exposure to Zn and Cd + Zn is discussed with regard to detoxification mechanisms, which include metallothioneins.     

Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn

In this work we have studied the accumulation of heavy metals in two brown trout (Salmo trutta) populations in their natural environment and the participation of metal binding to metallothionein (MT) in this process. Cd, Cu and Zn concentrations, total MT (including Cu MT) and Cd/Zn MT were measured in the gills, liver and kidney of trout inhabiting two rivers, one Cu-contaminated and the other Cd/Zn-contaminated, located at Røros, Central Norway. In both populations, high levels of Cu were found in the liver, whereas Cd was accumulated in liver and particularly in the kidney. The proportions of Cd/Zn MT and Cu MT in liver and kidney, but not in gills, reflected the accumulated and the environmental concentrations of these metals. The total Cu MT concentrations in the investigated tissues, however, were highest in trout from the river with the lowest ambient Cu concentration. It is suggested that MTs are of less importance in Cu-acclimated trout. The data also suggest that acclimation to a Cu-rich environment involves reduced Cu accumulation or increased Cu elimination. In trout from the Cd-rich environment, this metal was mainly bound to MT, whereas in trout from the Cu-rich environment Cd was also associated with non-MT proteins. These findings emphasize the importance to determine both Cd/Zn MT and Cu MT levels, when the participation of this protein in metal handling in trout tissues is investigated.     

Cadmium uptake in rat hepatocytes in relation to speciation and to complexation with metallothionein and albumin

Cadmium (Cd) uptake has been studied in primary cultures of rat hepatocytes focusing on the impact of inorganic and organic speciation. Uptake time-course studies over a 60-min exposure to 0.3 microM (109)Cd revealed a zero-time uptake and a slower process of accumulation which proceeds within minutes. (109)Cd uptake showed saturation kinetics (K(m) = 3.5 +/- 0.8 microM), and was highly sensitive to inhibition by Zn and Hg. There was no evidence for sensitivity to the external pH nor for any preferential transport of the free cation Cd(2+) over CdCl(n) (2-n) chloro-complexes. According to the assumption that only inorganic metal species are available, metal uptake decreased upon albumin (BSA) addition to the exposure media. In contrast, higher levels of (109)Cd accumulation were obtained under optimal conditions for Cd complexation by MT. Comparison among uptake data obtained under inorganic and organic conditions revealed that Cd-MT would be taken up 0.4 times as rapidly as Cd(inorg). We conclude that uptake of Cd in rat hepatocytes involves specific transport mechanism(s) subjected to Zn or Hg interactions. Uptake of inorganic Cd is not proportional to the levels of free Cd(2+) and does not involve the divalent cation transporter DCT1 nor the co-transporter Fe(2+)-H(+) NRAMP2. We found Cd-MT but not Cd-BSA to be available for the liver cells, and have estimated a binding affinity four orders of magnitude higher for Cd complexation with MT compared to BSA; MT may have a significant role in Cd delivery to the liver.     

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.     

Isoform‐specific responses of metallothioneins in a marine pollution biomonitor,the green‐lipped mussel Perna viridis,towards different stress stimulations

Metallothioneins (MTs) are commonly used as biomarker for metal pollution assessment in marine ecosystems. Using integrated genomic and proteomic analyses, this study characterized two types of MT isoform in the digestive gland of a common biomonitor, the green‐lipped mussel Perna viridis, towards the challenges of a metal (cadmium; Cd) and a non‐metal oxidant (hydrogen peroxide; H₂O₂) respectively. The two isoforms differed in their deduced protein sequences, with 73 amino acids for MT10‐I and 72 for MT10‐II (a novel type), but both consisted of a high percentage (27.4 to 29.2%) of cysteine. Two‐dimensional gel and Western blot showed that the MT proteins were present in multiple isoform spots, and they were further validated to be MT10‐I and MT10‐II using MS analysis coupled with unrestricted modifications searching. Expression of mRNA revealed that MT10‐I responded promptly to Cd but had a lagged induction to H₂O₂ treatments, while MT10‐II was exclusively induced by Cd treatment over the course of exposure. Expression of the MT proteins also showed a delayed response to H₂O₂, compared to Cd treatments. This study uncovered the potential different functional roles of various MTs isoforms in P. viridis and thus advances the resolution of using MTs as biomarkers in future applications.     

Age-dependent changes in metallothionein levels in liver and kidney of the Japanese

Samples of liver, renal cortex, and medulla were obtained from 55 forensic autopsies (0- to 95-yr-old Japanese). Metallothionein (MT) was determined by the Ag-hem or Cd-hem method. Zinc (Zn), copper (Cu), and cadmium (Cd) were determined by atomic absorption spectrophotometry. The mean levels of MT were 250 μg/g in the liver, and 394 μg/g (cortex) and 191 μg/g (medulla) in the kidney. Age-dependent changes were observed in both the liver and kidney. In the liver, MT level decreased during infancy and increased thereafter with age. Similar age-dependent changes in the levels of Zn and Cu were observed. In the kidney cortex, MT level increased with age, although no correlation was found after middle age. The levels of Cd and Zn also increased with age until middle age; however, they decreased thereafter. These results suggest that age-dependent changes in renal MT levels are associated with accumulation of Cd.     

Age- and tissue-dependent metallothionein and cytosolic metal distribution in a native Mediterranean fish, Mullus barbatus, from the Eastern Adriatic Sea

The levels of metallothionein (MT), a biomarker of metal exposure, and of cytosolic metals (Zn, Cu, Cd), known as MT inducers, were investigated as variables of age (1 to 8 years) and tissue mass (liver, kidney, brain) of red mullet (Mullus barbatus). Within the age from 1 to 8 years the most significant increase is evident for cytosolic Cd in liver (43-fold) and in kidney (5-fold). MT and essential metals are constant with age or slightly increased. Over the growth period, statistically significant MT and metal increase is evident only between 1 and 6-8 years old specimens, while for Cd in liver and kidney cytosol significant increase already exists at 4 years old specimens. Metal distribution in all tissues follows the order: Zn>Cu>Cd, with even 500-800 times lower Cd levels than essential metal levels. Consequently, MTs follow the levels of essential metals, Zn and Cu, indicating MT involvement in homeostasis of essential metals. In contrast to kidney and brain, hepatic MT levels are not age-dependent. Inclusion of hepatic MT measurements and the associated cytosolic metals will be useful in the assessment of long-term metal effects in demersal fish M. barbatus.     

Analysis of stress-induced gene expression in fish cell lines exposed to heavy meals and heat shock

We have examined the effect of heavy metals on the expression of two major groups of stress-induced proteins in fish cell lines: the 70 kDa heat-shock proteins (hsp70) and metallothioneins (MTs). The rainbow trout hepatoma (RTH) cell line synthesized the hsp70 protein in response to zinc and heat shock, while chinook salmon embryonic (CHSE) cells synthesized this protein in response to these inducers, as well as cadmium. The synthesis of this 70 kDa protein was correlated with the accumulation of hsp70 mRNA as measured by hybridization to a trout hsp70 gene probe. Heavy metals also induced the synthesis of MT in RTH cells. However, heat shock did not result in induction of MT and its mRNA. Unlike RTH cells, CHSE cells did not synthesize MT following exposure to cadmium or zinc. When these cells were treated with 5-azacytidine prior to heavy metal treatment, accumulation of MT mRNA was observed. Northern blot analysis of total RNA from 5-azacytidine treated CHSE cells, using a trout MT (tMT-B) cDNA probe, indicated that the time-course of induction and the maximal level of MT mRNA accumulation in response to cadmium and zinc paralleled that observed in RTH cells. Copper and dexamethasone were ineffective in inducing MT mRNA in 5-azacytidine-treated CHSE cells. These results indicate that MT is specifically induced in response to heavy metal treatment, whereas the synthesis of hsp70 appears to be a general stress response. Furthermore, MT is differentially regulated by heavy metals and dexamethasone in these cell lines and the expression of MT is cell-type-specific.     

Analysis of cadmium and lead: their immunosuppressive effects and distribution in various organs of mice

Chronic exposure (3.5 mo) of mice to cadmium (Cd), lead (Pb), or a cadmium-lead mixture at a concentration of 1 ppm in drinking water induced a highly significant inhibition of antibody response to human serum. The highest immunosuppression (84.4%) was induced by the Cd-Pb mixture, whereas Cd caused the lowest immunosuppression (53.6%). The body burden of Cd and Pb in various organs was investigated in the four groups of mice by atomic absorption spectrometry. The highest level of Cd was found in the kidney of the Cd-treated group, and the highest level of Pb was found in the liver of the Pb- and Cd-Pb-treated groups. It is concluded that when mice are exposed concurrently to Cd and Pb, they develop synergistic immunosuppression. Analysis of Cd levels using atomic absorption spectrometry revealed that it was distributed in the following order: kidney > liver > spleen > heart, whereas Pb was distributed in the following order: liver > kidney > spleen > heart.     

Alterations in isoforms of glutathione S-transferase in liver and kidney of cadmium exposed rhesus monkeys: Purification and kinetic characterization

Exposure of animals to cadmium (Cd) (25 mg kg-1 body wt day-1) for 10 weeks resulted in preferential accumulation of the metal in liver and kidney. Cd accumulation concomitantly increased zinc (Zn) concentration in both the organs. However, significant decrease in copper level was observed in liver, whereas kidney showed increase in copper (Cu) level. Cd exposure resulted in decreased total GST activity in liver (63%) and kidney (41%) as compared to control group monkeys on normal diet (group I). On isoelectric focusing (IFP) control liver GST segregated into thirteen isoenzymes, while in Cd-treated experimental animals (group II) liver GST resolved into nine isoenzymes. Similarly kidney GST from control animals separated into seven isoenzymes as compared to four isoenzymes from Cd-treated animals. Kinetic analysis showed that Cd exposure did not alter the affinity constant (Km) of GST for GSH and CDNB whereas maximal velocity (Vmax) for these substrates decreased as compared to controls in both the organs, indicating inhibition in GST synthesis by Cd. Cd resulted in a noncompetitive type of inhibition with respect to GSH in vitro. On isoelectric focussing GST of liver and kidney in group II resolved into nine and four isoenzymes as compared to thirteen and seven in group I, showing loss of four basic isoenzymes in case of liver and three isoenzymes in case of kidney. Monkey liver and kidney expressed all the three classes of GST isoenzymes i.e. , µ and , which were serologically identical to human , µ and GSTs. (Mol Cell Biochem 166: 55-63, 1997)     

Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay

A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1–100 ng for MT-2 and 10–1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1% Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our anti-serum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%) for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2. Therefore, the MT-2 specific antibodies may be useful for the study of the functions of MT isoforms. Levels of total MT in tissues and biological fluids of rats injected with CdCl₂ (0.3 mg Cd/kg) and Cd-MT (0.3 mg Cd/kg) were estimated by ELISA. The results suggest urinary MT levels may be related to kidney damage.