Bile and liver metallothionein behavior in copper-exposed fish

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2 mg L⁻¹) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC–ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.     

Early effects of surgery on zinc and metallothionein levels in female rats

Time-response effects of experimental surgery on zinc (Zn) and metallothionein (MT) homeostasis were investigated in female rats up to 24 h. Hepatic Zn content increased at 20 and 24 h postsurgery, whereas serum Zn levels decreased. Hepatic MT increased significantly by 9 h postsurgery and peaked at up to twofold of control at 12 h after surgery. Following the peak at 12 h, hepatic MT content decreased with time but did not reach control levels at the end of this study. When MT isoforms were evaluated, MT-II levels were elevated to the highest extent by 12 h after surgery, whereas MT-I levels started to decrease after 3 h postsurgery but then increased by 20 h. The early increases in MT content are probably mediated by nonmetallic mediators released during the postsurgical inflammatory process, favoring the plasma/tissue mobilization of Zn. This process might be part of the overall mechanisms occurring in the inflammation.     

Renal metallothionein and platinum levels in diabetic and nondiabetic rats injected with cisplatin

This study was designed to investigate the relationship between the attenuation of cisplatin-induced nephrotoxicity in experimental diabetes and the increased level of renal metallothionein (MT) reported to occur in this condition. Two groups of male Sprague-Dawley rats were used: 42-day streptozotocin diabetics and age-matched nondiabetics. Half of each group was injected with a nephrotoxic dose of cisplatin (5 mg/kg, ip) and half with vehicle. Four hours after injection, renal MT and platinum (Pt) content were quantified. Mean renal MT concentration in vehicle-injected diabetics was about triple that found in nondiabetics. Comparison of renal MT concentrations in cisplatin-injected diabetics and nondiabetics with their vehicle-injected counterparts suggested an inducing effect of the drug. In contrast to the marked elevations of MT in diabetic kidney, mean renal Pt concentration in the cisplatin-injected diabetic group was only about one-fourth that of the nondiabetic group. No difference was evident in the intracellular distribution Pt between cytosolic and particulate fractions from diabetic and nondiabetic kidneys. It was concluded that: (i) Sequestration of Pt by MT cannot account for the resistance of diabetic kidney to cisplatin toxicity. (ii) Rather, the resistance is due to a significant decrease in renal uptake/retention of cisplatin or derivatives during the critical first few hours after injection.     

Antioxidants and metallothionein levels in mercury-treated mice

Acute effects of mercury on mouse blood, kidneys, and liver were evaluated. Mice received a single dose of mercuric chloride (HgCl₂, 4.6 mg/kg, subcutaneously) for three consecutive days. We investigated the possible beneficial effects of antioxidant therapy (N-acetylcysteine (NAC) and diphenyl diselenide (PhSe)₂) compared with the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS), an effective chelating agent in HgCl₂ exposure in mice. We also verified whether metallothionein (MT) induction might be involved in a possible mechanism of protection against HgCl₂ poisoning and whether different treatments would modify MT levels and other toxicological parameters. The results demonstrated that HgCl₂ exposure significantly inhibited δ-aminolevulinate dehydratase (δ-ALA-D) activity in liver and only DMPS treatment prevented the inhibitory effect. Mercuric chloride caused an increase in renal non-protein thiol groups (NPSH) and none of the treatments modified renal NPSH levels. Urea concentration was increased after HgCl₂ exposure. NAC plus (PhSe)₂ was partially effective in protecting against the effects of mercury. DMPS and (PhSe)₂ were effective in restoring the increment in urea concentration caused by mercury. Thiobarbituric acid-reactive substances (TBARS), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities and ascorbic acid levels were not modified after mercury exposure. Mercuric chloride poisoning caused an increase in hepatic and renal MT levels and antioxidant treatments did not modify this parameter. Our data indicated a lack of therapeutic effect of the antioxidants tested.     

Immunohistochemical localization of metallothionein in developing rat tissues

Metallothionein (MT) is a cysteine-rich, low molecular weight protein inducible by heavy metal ions and various endogenous factors. Using an indirect immunofluorescent technique, we studied the localization of MT in developing rat tissues (kidney, small intestine, and liver). In kidney of the neonate and fetus, MT was found in both the cytoplasm and the nucleus of renal tubular epithelia. Localization of MT changed with shift of zonation in the renal cortex during development. Metallothionein was found mainly in the inner zone of the cortex but not in tubules of the neogenic zone on Day 4. Until Day 18, tubular cells containing MT were observed in a part of the cortex adjacent to the medulla, followed by a significant decrease in immunostaining by Day 27. In small intestine of the neonate, MT was localized predominantly in Paneth and goblet cells which play secretory roles. The number of goblet cells with strong immunostaining for MT was maximal on Day 27. In liver of 20-day fetuses and of 4-day-old neonates, both the cytoplasm and the nucleus of hepatocytes exhibited strong immunofluorescence. The intensity of MT staining diminished with development, and by 18-27 days after birth no immunofluorescence was observed in the nucleus. We further studied a possible association of MT with development by localizing MT in livers obtained from partially hepatectomized and laparotomized rats. Hepatectomy led to the appearance of MT not only in the nucleus and cytoplasm of hepatocytes but also in sinusoids and bile canaliculi. After laparotomy, MT immunofluorescence was observed only in the cytoplasm. The present results suggest a possible involvement of MT in cell proliferation and differentiation, as well as in transport and secretion of this metal-binding protein.     

Decreases of metallothionein and aminopeptidase N in renal cancer tissues

Good molecular markers for investigating the biochemical differences between renal cancer and surrounding tissues have not yet been developed. Sixteen kidney samples (clear cell RCC) were investigated to determine the differences in the protein components between renal cancer and surrounding tissues, using HPLC analysis. The metallothionein (MT) and zinc levels were consistently lower in renal cancer tissues compared with in surrounding tissues. The mean concentration of MT in normal tissues surrounding renal tumors was about 15 times higher than that in cancer tissues. An immunohistochemical study confirmed that the expression of MT in renal cancer tissues was lower than that in adjacent normal tissues. The activities of aminopeptidases (APs) were significantly decreased in renal cancer tissues compared with in adjacent normal tissues. An immunohistochemical study and Western blot analysis confirmed that the expression of AP-N in renal cancer tissues was also lower than in adjacent normal tissues. These results suggest that the immunohistochemical detection of MT and AP-N could provide useful information as a pathological diagnostic tool for classifying renal cancer and surrounding tissues.     

Effect of zinc supplementation on metallothionein,copper, and zinc concentration in various tissues of copper-loaded rats

The present study was designed to investigate the effects of Zn administration on metallothionein concentrations in the liver, kidney, and intestine of copper-loaded rats. Male CD rats were fed a diet containing 12 mg Cu and 67 mg Zn/kg body wt. They were divided into either acute or chronic experimental protocols. Rats undergoing acute experiments received daily ip injections of either Cu (3 mg/kg body wt) or Zn (10 mg/kg body wt) for 3 d. Chronic experiments were carried out on rats receiving Cu ip injections on d 1, 2, 3, 10, 17, and 24, Cu injections plus a Zn-supplemented diet containing 5 g Zn/kg solid diet, or a Zn-supplemented diet alone. Rats injected Zn or Cu had increased MT concentrations in liver and kidney. Zn produced the most important effects and the liver was the most responsive organ. Rats fed a Zn-supplemented diet had significantly higher MT concentrations in liver and intestine with respect to controls. Increased MT synthesis in the liver may contribute to copper detoxification; the hypothesis of copper entrapment in enterocytes cannot be confirmed.     

Turnover rate of metallothionein and cadmium in Mytilus edulis

The results demonstrate the first attempt to determine metallothionein turnover in the whole soft tissues of mussels Mytilus edulis exposed to cadmium. Half-lives for metallothionein and cadmium are 25 and 300 days, respectively. As metallothionein degrades the released cadmium induces further synthesis of the protein, to which the metal becomes resequestered. The slow metallothionein turnover rates (compared with mammals) and the lack of significant cadmium excretion testify to the relatively stable nature of the cadmium-metallothionein complex in these invertebrates and supports the view of a detoxifying role for metallothionein in the mussels.     

Immunohistochemical localization of metallothionein in the eye of rats

In order to elucidate possible physiological roles of metallothionein (MT), we have studied immunohistological localization of MT in the eye of the rat, using an avidin-biotin peroxidase complex method. As a result, strong MT immunostaining was observed in the epithelium of the lens and cornea. In the retina, considerably strong MT immunostaining was observed in the pigment cell layer while the nerve fiber layer and inner plexiform layer showed weak MT staining. Glial cells in the optic nerve were found to have marked MT staining. The present result is consistent with the hypothesis that MT may be involved not only in activation of zinc enzymes and cell proliferation through supply of zinc ions, but also in a protective mechanism in the blood-retina barrier.     

Immunohistochemical localization of metallothionein in the eye of rats

Summary In order to elucidate possible physiological roles of metallothionein (MT), we have studied immunohistological localization of MT in the eye of the rat, using an avidin-biotin peroxidase complex method. As a result, strong MT immunostaining was observed in the epithelium of the lens and cornea. In the retina, considerably strong MT immunostaining was observed in the pigment cell layer while the nerve fiber layer and inner plexiform layer showed weak MT staining. Glial cells in the optic nerve were found to have marked MT staining. The present result is consistent with the hypothesis that MT may be involved not only in activation of zinc enzymes and cell proliferation through supply of zinc ions, but also in a protective mechanism in the blood-retina barrier.     

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.     

Effect of dietary iron deficiency on mineral levels in tissues of rats

To clarify the influence of iron deficiency on mineral status, the following two synthetic diets were fed to male Wistar rats: a control diet containing 128 micrograms iron/g, and an iron-deficient diet containing 5.9 micrograms iron/g. The rats fed the iron-deficient diet showed pale red conjunctiva and less reactiveness than the rats fed the control diet. The hemoglobin concentration and hematocrit of the rats fed the iron-deficient diet were markedly less than the rats fed the control diet. The changes of mineral concentrations observed in tissues of the rats fed the iron-deficient diet, as compared with the rats fed the control diet, are summarized as follows: . Iron concentrations in blood, brain, lung, heart, liver, spleen, kidney, testis, femoral muscle, and tibia decreased; . Calcium concentrations in blood and liver increased; calcium concentration in lung decreased; . Magnesium concentration in blood increased; . Copper concentrations in blood, liver, spleen and tibia increased; copper concentration in femoral muscle decreased; . Zinc concentration in blood decreased; . Manganese concentrations in brain, heart, kidney, testis, femoral muscle and tibia increased. These results suggest that iron deficiency affects mineral status (iron, calcium, magnesium, copper, zinc, and manganese) in rats.     

Determination of metallothionein in tissues by radioimmunoassay and by cadmium saturation method

The diversity of reported basal metallothionein (MT) values in animal tissues has made it necessary that the presently available methods be further developed and compared for their accuracy, reproducibility, sensitivity, and specificity. A radioimmunoassay (RIA) to quantitate MT in animal tissues was developed and its performance compared to that of a cadmium saturation method. The procedure is more accurate and reliable but no more time-consuming than other techniques in current use. It offers the advantages of greater specificity and sensitivity thus enabling the determination of basal levels of MT in tissues and the analysis of small samples, for example, biopsies, cultured cells, in vitro protein synthesis, etc. The use of a polyclonal antiserum is advantageous in that total MT can be determined in any tissue from a variety of animal species. Both nonspecific and specific interference in the assay can be eliminated by heat treatment of the sample followed by a short preincubation with cadmium. The sensitivity of the RIA is 10 ng MT/g tissue. The cadmium saturation assay is unsuitable for the measurement of low levels of MT due to its nonspecific nature and its detection limit (10 micrograms MT/g tissue) but it is useful where large amounts of the protein are present in a tissue. Difficulties arising in the analysis of MT by both methods are discussed and solutions are offered. The basal levels of MT in the brain, kidney, liver, lung, muscle, pancreas, small intestine, and spleen of rats are determined by RIA and are shown to be generally lower than the currently accepted values measured by other techniques.     

Endogenous and heavy-metal-ion-induced metallothionein gene expression in salmonid tissues and cell lines

Endogenous levels of metallothionein (MT) mRNA were detected by RNA probes in several somatic and germ-line tissues of rainbow trout, such as eggs, ovaries and immature testis. These levels may be related to metal-ion homeostasis in the observed tissues. The induction kinetics of trout MT isoform B (MT-B) mRNA were studied after single intraperitoneal injections of CdCl2, CuCl2 and ZnCl2. MT-B mRNA was induced within 12 h in liver, kidney, spleen and gills. However, over the 48-h experimental period, the kinetics of MT-B mRNA accumulation differed in response to the three metal salts, possibly due to differential handling of the salts by these tissues. Multiple metal-salt injections induced high levels of MT-B mRNA in the four tissues studied. In the rainbow trout hepatoma cell line, ZnCl2 was a better inducer of the MT-B gene, as compared to CdCl2 and CuCl2. The expression of the exogenous trout MT-B promoter in Chinook salmon embryonic cell line indicates the presence of MT regulatory factors. In contrast, the endogenous MT genes in these cells are quiescent, possibly due to the methylation of their promoter region.     

Role of metallothionein in regulating the abundance of histochemically reactive zinc in rat tissues

The objectives of this study were (i) to investigate the modulating effects of zinc nutrition on histochemically reactive zinc in the rat intestine and liver and (ii) to assess the relationship between histochemically reactive zinc and metallothionein-bound zinc in these tissues under varying zinc nutrition. Male Wistar rats were fed a zinc-deficient (3 mg zinc/kg), adequate-zinc (30 mg zinc/kg, ad libitum or pair-fed), or zinc-supplemented (155 mg zinc/kg) diet for 2 or 6 weeks. Plasma N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide-reactive zinc reflected dietary zinc intake. Abundance of the intestine histochemically reactive zinc was correlated with dietary zinc intake after 2 weeks of dietary treatment. Dietary zinc intake had no effect on the abundance of the intestine histochemically reactive zinc after 6 weeks of dietary treatment and the hepatic histochemically reactive zinc after both 2 and 6 weeks of dietary treatment. This lack of effect of dietary zinc intake on the abundance of histochemically reactive zinc was associated with a higher level of metallothionein. The molecular-mass distribution profile revealed that N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide-reactive zinc and metallothionein-bound zinc represented two different, but interrelated, pools of zinc. Overall, these results suggested that the abundance of histochemically reactive zinc was homeostatically regulated, which was partially achieved through the regulation of metallothionein levels in rats.