Metallothionein-bound Cadmium Research Articles

Elevated metallothionein-bound cadmium concentrations in urine from bladder carcinoma patients, investigated by size exclusion chromatography-inductively coupled plasma mass spectrometry

Cadmium is discussed as being involved in the development of transitional cell carcinoma (TCC) of the bladder and can be observed in urine of these patients. Investigations of urinary samples from bladder cancer patients and normal controls were carried out with special emphasis on metallothionein (MT)-bound cadmium. Compounds that are constituents of urine were separated in urine samples by means of size exclusion chromatography and cadmium was monitored continuously with a hyphenated inductively coupled plasma mass spectrometry (ICP-MS) system. MT-bound cadmium was quantified by peak area integration, taking into account the intensity of the rhodium signal which was added continuously before ICP-MS detection. The obtained results show that urinary cadmium is predominantly bound to the observed MT-fraction. The median of the MT-bound cadmium concentration in the control group was found to be 0.8 μg L −1 whereas the cancer group has a median of 1.8 μg L −1. The variance of the data in the cancer group is much higher than in the controls. However, the urinary MT-bound cadmium is significantly elevated in the cancer group; odds-ratio test: 7.11 (95% C.I.: 1.89–26.80), taking into account the total protein content. Due to the fact that only one main cadmium-containing fraction was observed, there is no necessity to separate the MT-fraction before cadmium determination in urine samples in future studies.

Development of a method for the determination of inorganic cadmium and cadmium metallothioneins in fish liver by continuous preconcentration on fullerene and flame atomic absorption spectrometry

The adsorptive potential of C60 fullerene for the preconcentration of metallothionein-bound cadmium (Cd-MT) traces was assessed for the first time in this work. A simple flow injection system for the on-line preconcentration of Cd-MT was developed. The compounds were directly retained on the sorbent column and subsequently eluted, by hydrolysis of the complexes with diluted HNO3, and the cadmium was transferred to a flame atomic absorption spectrometer. Total cadmium (inorganic and Cd-MT) was also determined by preparing the sample in acid medium and complexing total cadmium with Na-DDC. The neutral chelates of cadmium, retained also on C60, were eluted with IBMK and transferred to the atomic absorption instrument. By the difference between both determinations, inorganic and organometallic forms of cadmium could be distinguished. The analytical figures of merit of the proposed method are: linear range, 0.3–12 ng ml−1 (using 10 ml of sample); limit of detection, 0.1 ng ml−1 and precision (RSD), ca. 4%. No interferences from metals such as Zn, Fe, Pb, Cu and Hg, as inorganic salts, were observed in the determination of total Cd. The method was applied to the determination of inorganic and organometallic cadmium compounds in spiked fish liver samples, with acceptable recoveries.

Metallothionein-bound cadmium in the gut of the insect Orchesella cincta (Collembola) in relation to dietary cadmium exposure

Metallothionein is considered to be a potential biomarker for heavy metal exposure in the terrestrial environment. However, limited information is available on metallothioneins from insects, a major class of terrestrial invertebrates. In this study we have quantified metallothioneins in the springtail Orchesella cincta by determining metallothionein-bound cadmium after separation of these proteins using gel filtration and reversed phase chromatography from total body homogenates of animals dietary exposed to different concentrations of cadmium. Furthermore, we have studied in more detail where cadmium and metallothionein-bound cadmium is located within this animal. The concentration of metallothionein-bound cadmium increases with the exposure concentration in the same way as the total internal concentration. Both reach a plateau at an exposure concentration of approximately 1.0 μmol Cd/dry food. Cadmium is primarily located within the gut of O. cincta and isolation of metallothionein from this organ gives results identical to isolations from total bodies. Based on this results an estimation of the metallothionein level at the highest exposure concentration results in a concentration of about 115 μg metallothionein/g fresh gut. The O. cincta metallothionein gives the possibility of using this protein as a biomarker for heavy metal exposure in soil insects.

Speciation of metal complexes with biomolecules by reversed-phase HPLC with ion-spray and inductively coupled plasma mass spectrometric detection

Complementarity of ion-spray MS and ICP-MS as detection techniques in reversed-phase HPLC for the characterization of metals complexed by biomacromolecules is discussed by the example of the speciation of metallothionein-bound cadmium. The commercially purified rabbit liver MT-2 isoform is eluted from a microbore C8 column with a gradient of up to 50% methanol in acetate buffer (pH 6.0) to give one major and three minor peaks detected at 254 nm. The preparation is further characterized by using an ICP mass spectrometer interfaced with HPLC via a direct injection nebulizer which allows for the specific detection of cadmium down to the 10 ng mL–1 level. On-line detection by mass spectrometry with an ion-spray (pneumatically-assisted electrospray) ion source further allows the determination of the molecular masses of the eluted compounds.

Failure of Metallothionein-Bound Cadmium to Undergo Apoptosis in Porcine Renal LLC-PK<SUB>1</SUB> Cells (Proceedings of the 22nd Symposium on Toxicology and Environmental Health)

Failure of Metallothionein-Bound Cadmium to Undergo Apoptosis in Porcine Renal LLC-PK<SUB>1</SUB> Cells (Proceedings of the 22nd Symposium on Toxicology and Environmental Health)

Further observations on the etiology of preeclampsia: Mobilization of toxic cadmium-metallothionein into the serum during pregnancy

Cadmium-metal loth ionein, mobilized from the liver, might be the toxic serum factor associated with pre-eclampsia. We base this on four documented concepts. First, during pregnancy, maternal physiology adjusts to assure the fetus of the proper amounts of nutrients necessary for growth. Our focus is on zinc and progesterone. Second, because zine and cadmium are similar, they compete for binding sites. Our focus is on the storage protein metallothionein. Third, the manifestations of cadmium toxicity closely mimic the manifestations of toxemia (i.e. hypertension, proteinuria, edema). Our focus is on cadmium-induced indovasculitis. Fourth is the concept that metalloth ionein-bound cadmium can be mobilized from the liver into the serum during pregnancy as it follows the mobilization of metallothionein-bound zinc. Our focus is on the extreme toxicity of extracellular cadmium-metallothionein. We correlate these four concepts into a rational theory on the etiology of toxemia, and we suggest a method of proof.

Possible mechanism for zinc protection against cadmium cytotoxicity in cultured vascular endothelial cells

We investigated the effect of zinc on cadmium cytotoxicity in vascular endothelial cells in a culture system. The cytotoxicity was evaluated by [ 3H]adenine release assay. Cadmium at 2 μM concentration and above significantly increased the [ 3H]adenine release, but zinc at 80 μM and below did not induce such a response after a 24-h incubation. Metallothionein was induced by cadmium at 0.1 μM and above but not by zinc at 300 μM and below; however, zinc at 10 μM and above significantly decreased cadmium-(2 and 5 μM) induced cytotoxicity. Zinc protection against cadmium cytotoxicity was also observed in the presence of 1 μM cycloheximide. Zinc caused significanlty less accumulation of cadmium in the cell layer accompanied with a significant accumulation of zinc. The distribution (%) of cadmium in the particulate fraction of the cells was significantly decreased by zinc. In contrast, cadmium in the cytosol fraction was increased in the cells treated with both cadmium and zinc. Gel filtration chromatography of the cytosol showed that cadmium was capable of being bound to high-molecular-weight proteins and metallothionein. The metallothionein-bound cadmium was not increased by zinc; however, the relative distribution of cadmium in the high-molecular-weight fraction in the cytosol was decreased in cadmium plus zinc-treated cells. From these results, it was suggested that the mechanism by which zinc protects endothelial cells from cadmium cytotoxicity was decreased accumulation of cadmium in the particulate fraction and in the high-molecular-weight fraction in the cytosol of the cells. This alteration is postulated to be caused by both zinc-induced decrease in the intracellular cadmium accumulation and the sequestration of cadmium by cadmium-induced metallothionein.

Schedule dependency of a cadmium-complexing dithiocarbamate analog in mice

A number of dosing regimens was assessed to determine the optimum schedule of administration of N-(4-methoxybenzyl)- N-dithiocarboxy- d-glucamine (MeOBDCG) in depleting whole-body, renal and hepatic levels of metallothionein-bound cadmium (Cd) in mice. A comparison of 4.0 mmol/kg given as a single injection versus 0.5 mmol/kg given as 8 hourly injections revealed the latter regimen to be superior in reducing renal Cd levels, but less effective than a bolus dose in lowering hepatic Cd concentrations. Administration of 1.33 mmol/kg for 3 consecutive days or 0.8 mmol/kg for 5 days effected a more extensive depletion of renal Cd concentrations than did a single injection of 4.0 mmol/kg. Three injections of 1.0 mmol/kg given at 4- to 7-day intervals were generally more effective in reducing renal Cd concentrations than were 3 consecutive daily injections in mice which had low or moderately high total Cd burdens. The lowest effective dose of MeOBDCG in lowering whole-body, liver and kidney Cd levels when given repetitively was about 0.2 mmol/kg. While schedule variations did not alter appreciably the whole-body Cd reductions at any given total dose of MeOBDCG, repetitive dosing schedules in which injections were given at intervals of several days rather than daily were typically more effective in reducing renal Cd levels. Based upon consideration of pharmacological response as influenced by body surface area, it was calculated that doses of MeOBDCG of the order of 2.0 g/d may be effective in reducing renal Cd levels in individuals with chronic renal dysfunction secondary to chronic Cd intoxication.

The teratogenicity of cadmium-metallothionein in the rat.

A single dose in the range 0.25-1.0 mg metallothionein-bound cadmium (MT-Cd)/kg body weight, when administered parenterally to the rat between day 8 and day 14 of gestation (gd 8-gd 14), is teratogenic. In vitro, the development of the isolated rat conceptus, explanted at 8.5 days of gestation, is unaffected by the addition of 1.5 microM MT-Cd to the culture medium, whereas the same concentration of ionic Cd (as CdCl2) is lethal. The incorporation of appreciable amounts of Cd into the embryo (860 pg), placenta (970 pg) and yolk sac (65.4 ng) without toxic manifestations under the former conditions suggests that the metalloprotein is incorporated pinocytotically, but without degradation, by the conceptus in vitro. It does not follow, therefore, that MT-Cd is without embryo/foetotoxicity in the pregnant rat since, in-vivo, liberation of some of the protein-bound Cd is known to occur in the blood. At short times after injection of 0.25 mg MT-Cd/kg body weight on gd 12, however, the maximal foetal and placental contents of Cd (less than 25 pg and 2 ng, respectively) are low in comparison with those after a teratogenic dose of CdCl2 and are of the same order as those in the embryo (46 pg) and placenta (100 pg) + yolk sac (3.8 ng) of the rat conceptus, cultured in the presence of the highest no-effect concentration of CdCl2 (0.065 microM). From this evidence, therefore, it is concluded that the uptake by the conceptus in vivo of either CdMT, or of Cd liberated therefrom, is unlikely to contribute to the teratogenic response. In the pregnant, as in the non-pregnant rat, the kidney appears to be the only organ that is affected directly by the metalloprotein. All doses in the range 0.25-1.0 mg MT-Cd/kg body weight are nephrotoxic and, because of this, result in prolonged anorexia in the pregnant animal. While some of the foetal deformities that occur in the CdMT-dosed animal seem to be direct consequences of the renal dysfunction, others apparently are secondary to the maternal anorexia, since they are induced in pregnant, normal rats by appropriate reductions in food intake. In rats that are injected i.p. on gd 12 with 0.25 mg MT-Cd/kg body weight, renal uptake of Cd is slower, but the final concentration is higher than in animals that are given the same dose i.v.(ABSTRACT TRUNCATED AT 400 WORDS)

On the role of metallothionein in cadmium induced renal toxicity.

Metallothionein plays an important role in the metabolism and toxicity of cadmium. Human beings are exposed to increasing amounts of cadmium. The critical organ in long term cadmium exposure is the kidney. Some groups in the population fall within the risk zone for developing renal tubular dysfunction from cadmium exposure. After absorption, cadmium is to a large extent distributed to the liver where it is bound to and induces the synthesis of metallothionein. Subsequently metallothionein-bound cadmium is slowly released into blood and efficiently taken up by renal tubular cells after glomerular filtration. The biological half-time of cadmium in the human kidney is considered to be very long: 10-15 years and this explains the life-long accumulation of cadmium, reaching a toxic concentration of approximately 200 ug/g in excessively exposed individuals. Human adult renal metallothionein has a natural content of cadmium which also induces the resynthesis of this protein upon its catabolism. This could be one explanation of the long biological half-time which has been reported for cadmium. Injection of metallothionein-bound cadmium can induce renal tubular kidney dysfunction at lower renal cadmium concentrations (10-20 ug/g), probably because of the fast catabolism and relatively slow synthesis of metallothionein in renal cells. A model of these and other mechanisms causing tubular kidney damage in cadmium exposure is presented.

General aspects of cadmium: transport, uptake and metabolism by the kidney.

Cadmium taken up from lung and gastrointestinal tract is transported via blood to liver and kidney. On long-term exposure to cadmium, renal tubular dysfunction develops in humans and experimental animals. Data from animal experiments demonstrate that initially after exposure cadmium in blood is bound to albumin and proteins with higher molecular weight. Such cadmium is mainly taken up in liver. For a few days after exposure cadmium exists as metallothionein in plasma and blood cells. After both single and long-term administration of cadmium bound to metallothionein, cadmium is taken up by the kidney. The concentration of metallothionein-bound cadmium in plasma is quite low due to continuous renal clearance. Cadmium from metallothionein is taken up in renal tubules by pinocytosis and subsequently degraded in lysosomes, thereby releasing cadmium which stimulates de novo synthesis of metallothionein but also binds to reabsorbed metallothionein. Catabolizing and rebinding are continuous and prevent excretion of cadmium. Because of differences in transport, renal metabolic handling forms of cadmium are also different for different forms of cadmium administered and rate of administration. A single dose of metallothionein-bound cadmium given intravenously is almost immediately and completely taken up in the renal tubule. Under such conditions, resynthesis and rebinding processes are insufficient to sequester cadmium from sensitive tissue receptors, and renal damage occurs at total tissue concentrations much lower than when renal cadmium concentrations rise slowly. This explains the wide range (10-200 micrograms Cd/g wet weight) of cadmium in the renal cortex that associated with renal tubular dysfunction in experimental animals.

Distribution of metallothionein-bound cadmium and cadmium chloride in mice: preliminary studies.

Metallothionein from livers of mice was isolated by gel chromatography and isoelectric focusing. One of two forms thus obtained contained 32 percent cysteine. This form, labeled in vitro with 109Cd, was injected intravenously in mice, and the distribution of 109Cd was studied. Animals killed after 4 hrs had over 80 percent of the injected dose in the kidneys. Protein obtained after gel chromatography, containing both forms of cadmium-binding protein, was also labeled in vitro with 109Cd and injected intravenously. Animals killed 4 hrs after injection had 50 percent of the injected dose in the kidneys. Whole-body measurements and wholebody autoradiography demonstrated that approximately 40-60 percent of the injected dose had been excreted in urine. The results show a selective accumulation of metallothionein-bound cadmium in the kidney and indicate possible differences in distribution and excretion of cadmium depending on binding to different forms of low molecular weight cadmium-binding proteins.