Hepatic Metallothionein Research Articles

Metallothionein gene expression under different time in testicular Sertoli and spermatogenic cells of rats treated with cadmium

The rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than the liver. To clarify the molecular mechanism underlying tissue and cell differences in Cd sensitivity, we compared metallothionein (MT) gene expression, MT protein accumulation, and Cd retention under different times in freshly isolated testicular Sertoli and spermatogenic cells and liver of rats treated with Cd. Adult male Sprague–Dawley rats received a s.c. injection of 4.0 μmol Cd/kg and 1, 3, 6, or 24 h later and untreated animals (0 h) tissue were sampled and testicular Sertoli and spermatogenic cells isolated. MT1 and MT2 mRNA levels were determined by semi-quantitative RT-PCR analysis followed by densitometry scanning, and MT was estimated by the enzyme-linked immunosorbent assay (ELISA) method. Cadmium content was determined by atomic absorption spectrophotometry. Testicular lesions were not grossly or histologically observed in rats treated with 4.0 μmol Cd/kg. In the present study, we demonstrated that the rat testis indeed expressed MT1 and MT2, the major isoforms. We also found that untreated animals contained relatively high basal levels of both isoform mRNA, which were increased after Cd treatment in liver and peaked at 3 h, followed by a decline, in contrast, the mRNA levels in Sertoli cells peaked at 6 h. Interestingly, the induction of MT1 mRNA was lower than MT2 mRNA in Sertoli cells and liver of rats treated with Cd. However, the MT1 mRNA levels of spermatogenic cells decreased 0–3 h after Cd treatment, followed by an increase; in contrast, MT2 mRNA levels increased 0–3 h after Cd treatment, followed by a reduction, but induced extents of them are lower than those of Sertoli cells and liver. Cd exposure substantially increased hepatic MT, but did not increase MT translation in Sertoli and spermatogenic cells. These results indicate: (1) that Cd-induced MT mRNA expression is cell- and time-dependent; (2) that the inability to induce the metal-detoxicating MT protein in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative to liver.

Effect of lead on ALA-D activity, metallothionein levels, and lipid peroxidation in blood, kidney, and liver of the toadfish Halobatrachus didactylus

The effects of lead (Pb) on ALA-D activity, metallothionein (MT) levels, and lipid peroxidation in liver, kidney, and blood of the toadfish Halobatrachus didactylus were investigated. A time-course experiment was performed with sampling on days 0, 2, 5, and 7 following intraperitoneal Pb injection. This indicated a rank order for lead concentration of kidney>liver>blood in fish exposed to Pb. No significant variation of ALA-D activity was observed in liver and kidney while in blood, a slight decrease of ALA-D activity was found but this was not attributed to acute metal stress. Hepatic and renal MT levels were both affected in different ways by metal uptake. The progressive decrease of MDA concentration in the liver and the lack of a clear induction in kidney suggested the hypothesis that Pb is not a good inductor of lipid peroxidation. The histological and histochemical results demonstrated degenerative effects of lead accumulation on the tissues and the activation of lysosomal responses to induced stress.

Property of metallothionein as a Zn pool differs depending on the induced condition of metallothionein

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, heavy metal-binding protein with several putative physiological functions as a radical scavenger and a regulator of metabolism of Zn. Although the induction of hepatic MT by a wide range of stressors is a well-known phenomenon, little is known about the role of MT in stressful situations. Since MT regulates Zn metabolism, we investigated the differences between affinities of MT for Zn in various stressful conditions in this study. Divalent cadmium ions are capable of displacement of Zn from MT in vitro. Therefore, we assayed the binding of Zn to MT induced by various stimuli using in vivo administration of Cd. MT was induced by paraquat (PQ), a reactive oxygen generator, fasting stress and restraint stress. Apo-MT induced by them bound to Zn in vivo. Zn, which bound to MT induced by PQ, was displaced by the administration of Cd. However, Zn that had bound to MT induced by fasting stress and restraint stress was not displaced by Cd. Moreover, we assessed the in vivo affinity of Zn to MT induced by fasting stress under the condition of subchronic Cd exposure. Cd was administered to mice by subcutaneously implanted Alzet osmotic minipumps, which released constant amounts of Cd over a 14-day period. After 4 days, mice were fasted for 24 h and hepatic MT was examined. Interestingly, it was found that Zn had displaced Cd bound to MT. These findings indicate that the affinity of MT for Zn differs depending on the stimulus by which MT was induced. This is the first report on differences in the properties of MT depending on the stimulus used to induce MT.

Protective role of metallothionein (I/II) against pathological damage and apoptosis induced by dimethylarsinic acid.

To better clarify the main target organs of dimethylarsinic acid toxicity and the role of metallothionein (MTs) in modifying dimethylarsinic acid (DMAA) toxicity. MT-I/II null (MT(-/-)) mice and the corresponding wild-type mice (MT(+/+)), six in each group, were exposed to DMAA (0-750 mg/kg body weight) by a single oral injection. Twenty four hours later, the lungs, livers and kidneys were collected and undergone pathological analysis, induction of apoptotic cells as determined by TUNEL and MT concentration was detected by radio-immunoassay. Remarkable pathological lesions were observed at the doses ranging from 350 to 750 mg/kg body weight in the lungs, livers and kidneys and MT(+/+) mice exhibited a relatively slight destruction when compared with that in dose matched MT(-/-) mice. The number of apoptotic cells was increased in a dose dependent manner in the lungs and livers in both types of mice. DMAA produced more necrotic cells rather than apoptotic cells at the highest dose of 750 mg/kg, however, no significant increase was observed in the kidney. Hepatic MT level in MT(+/+) mice was significantly increased by DMAA in a dose-dependent manner and there was no detectable amount of hepatic MT in untreated MT(-/-) mice. DMAA treatment can lead to the induction of apoptosis and pathological damage in both types of mice. MT exhibits a protective effect against DMAA toxicity.

Use of Biochemical Endpoints to Determine Relationships between Contaminants and Impaired Fish Health in a Freshwater Stream

Bayou Bartholomew is a low gradient stream predominantly influenced by agricultural inputs and stormwater discharge from the urban areas of Pine Bluff, Arkansas. Preliminary studies indicated induction of hepatic cytochrome P450 1A protein (CYP1A) and CYP1A-catalyzed enzyme activity (ethoxyresorufin O-deethylase-EROD) in several fish species collected in the waterway, which meanders through residential areas of Pine Bluff, and forested regions in the outskirts of the city. Largemouth bass (Micropterus salmoides) showed CYP1A repression and a lack of correlation between CYP1A and EROD activity. In addition, hepatic heme oxygenase activity was elevated in several species and demonstrated an inverse relationship with hepatic CYP1A. Hepatic metallothionein was unchanged in any species collected from these locations. Four years after this evaluation, sediments were collected and analyzed for agents that could be mechanistically consistent with the biochemical responses observed. Utilizing seven criteria for causality, these data indicated that PCBs appear to be at least one group of agents responsible for the biological effects observed in fish species from the upper Bayou Bartholomew. While this supports causality under the criteria, further studies in this waterway would strengthen the relationship between this class of compound and the effects observed in resident fish species.

Copper kinetics and hepatic metallothionein levels in the frog Rana ridibunda, after exposure to CuCl2.

Adult female frogs Rana ridibunda were exposed to 50 and 100 ppm of Cu (as CuCl2) dissolved in water for 5, 15 and 30 days. We measured the Cu content in the liver, kidneys, ventral skin, and large intestine. Hepatic metallothionein (MT) was also measured and we identified by elution the type of proteins bound to copper. Gross morphological characteristics of the frogs were not affected by Cu accumulation. Cu uptake took place first across the skin, then accumulated first in the large intestine, and then in the liver which was continuously accumulating Cu at all exposure concentrations and times. The highest concentration of the metal was recorded in the kidneys at 30 days and 100 ppm exposure. It appears that the kidneys act as the secondary route of Cu detoxification, probably after a Cu overload of liver. The concentration of hepatic MT increased with the increase of Cu concentration in liver at the 5th and 15th day of exposure but we observed a decrease by the end of the experiment. Cu was observed in the MT-fraction, and in the high-molecular weight protein fraction.

Site-Specific Induction of Metallothionein in N-Nitrosodimethylamine-Treated Rat Liver.

The effects of dietary protein and sulfur-containing amino acids on oxidative responses against N-nitrosodimethylamine (NDMA) treatment were investigated in rat liver, focusing on the mechanism of metallothionein (MT) induction. Rats were fed a 10% soybean protein isolate (SPI) diet or SPI supplemented with 0.3% L-methionine (SPI-Met) for 3 weeks after weaning. Rats fed on SPI showed lower glutathione (GSH) levels and higher MT levels in the liver than those fed on SPI-Met. After treatment with NDMA (20 mg/kg, i.p.), MT levels were elevated significantly in both groups, whereas GSH levels were only elevated in the SPI group. Pathologically, necrosis and hemorrhage were seen in the livers of NDMA-treated rats of both dietary groups, suggesting oxidative damage caused by NDMA treatment. Immunological histochemistry using anti-MT antibody showed that most of the MT was distributed in the cytosol region of all the rat groups. The ratios of the MT-positive cells were correlated to MT levels that were chemically analyzed. It was notable that the localization of MT staining in the hepatic lobules was altered after NDMA injection. The MT staining was principally observed around the central vein before injection, whereas it was more markedly observed at the peripheral region after NDMA treatment. These results indicate that the synthesis of hepatic MT basically occurs around the central vein with oxygen stress caused by insufficient oxygen supplement and/or stimulated oxygen consumption. NDMA could be oxidatively metabolized at the peripheral region with sufficient supplemental oxygen from the artery, resulting in oxidative stress and stimulation of MT synthesis there. Thus, distribution of tissue MT might vary depending on the site of oxidative stress.

Multi-element speciation of metalloproteins in fish tissue using size-exclusion chromatography coupled "on-line" with ICP-isotope dilution-time-of-flight-mass spectrometry.

An on-line isotope dilution (ID) method in combination with the coupling of size-exclusion high-performance liquid chromatography (SE-HPLC) to an axial inductively coupled plasma time-of-flight mass spectrometer (ICP-TOFMS) was applied to the quantitative speciation of Cu, Zn and Cd in carp and eel cytosols. Quantitative information on the distribution of essential and toxic metals between the different molecular weight fractions in kidney cytosols of control and Cd exposed carp was obtained with species-unspecific spiking by post-column addition of the enriched isotopes 65Cu, 67Zn and 106Cd. The isotope ratios 63Cu/65Cu, 64Zn/67Zn and 114Cd/106Cd, were monitored by ICP-TOFMS in transient signal mode. The determination of the total cytosolic element concentration was performed by flow injection conventional nebulization ICP-TOFMS. Speciation differences were encountered between control and Cd-stressed carp, indicating that quantitative speciation studies might be useful for ecotoxicological and biomonitoring purposes. Application of this methodology to quantitation of metals associated with metallothioneins (MT) in European eel, sampled at three different sites in Flanders, Belgium was also addressed. A significant increase of the amount of Cd, Cu and Zn bound to hepatic MT was observed with the increase of the total metal and MT concentrations of hepatic eel tissue.

Protective Effect of Zinc against Lipopolysaccharide/D-Galactosamine-Induced Lethality.

Zinc is an essential and multifunctional element for all cells. Metallothionein (MT) is a low molecular weight, cystein-rich, metal-binding protein that is involved in zinc homeostasis. During the acute-phase reaction, hepatic MT production is induced and hepatic zinc accumulation is stimulated. We previously reported that MT-I and II-deficient (MT-null) mice are highly sensitive to the lethal effects of lipopolysaccharide (LPS) plus D-galactosamine (GalN). The sensitization may relate to attenuatoin of α1-acid glycoprotein (AGP) expression in MT-null mice. In the present study, we hypothesized that MT-induced hepatic zinc accumulation promotes AGP expression and prevents LPS/GalN-induced lethality. To determine whether zinc reduces LPS/GalN toxicity, zinc was administered to mice. Simultaneous administration of zinc and LPS/GalN showed no effect on the lethality of LPS/GalN in mice. Zinc administration at 3 hr prior to LPS/GalN challenge reduced LPS/GalN-induced death. However, zinc pre-administration at 24 hr before LPS/GalN challenge did not reduce LPS/GalN-induced death. The expression of AGP mRNA was elevated at 3 hr after zinc administration, 24-hr pretreatment was ineffective. The protective effect of zinc was observed in both wild-type and MT-null mice. These results show that the protective effects of zinc were not caused by MT induction, but by AGP expression. We suggest that MT-induced hepatic zinc accumulation may promote AGP expression and thus prevent LPS/GalN-induced lethality.

Expression of metallothionein gene at different time in testicular interstitial cells and liver of rats treated with cadmium.

Rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than liver. To clarify the molecular mechanism of Cd-induced toxicity in testes, we compared metallothionein (MT) gene expression, MT protein accumulation, and Cd retention at different time in freshly isolated testicular interstitial cells and liver of rats treated with Cd. Adult male Sprague-Dawley rats weighing 250-280 g received a s.c injection of 4.0 micromol Cd/kg and were euthanized by CO(2) asphyxiation 1 h, 3 h, 6 h, or 24 h later. Tissue was sampled and testicular interstitial cells were isolated. There were three replicates per treatment and 3 animals per replicate for RNA analyses, others, three replicates per treatment and one animal per replicate. MT1 and MT2 mRNA levels were determined by semi-quantitative RT-PCR analysis followed by densitometry scanning, and MT was estimated by the enzyme-linked immunosorbent assay (ELISA) method. Cadmium content was determined by atomic absorption spectrophotometry. The same parametersd were also analyzed in the liver, since this tissue unquestionably accumulate MT. The rat testis expressed MT1 and MT2, the major isoforms. We also found that untreated animals contained relatively high basal levels of both isoform mRNA, which were increased after Cd treatment in liver and peaked at 3 h, followed by a decline. In contrast, the mRNA levels in interstitial cells peaked at 6 h. Interestingly, the induction of MT1 mRNA was lower than MT2 mRNA in liver of rat treated with Cd, but it was opposite to interstitial cells. Cd exposure substantially increased hepatic MT (3.9-fold increase), but did not increase MT translation in interstitial cells. Cd-induced expression of MT isoforms is not only tissue dependent but also time-dependent. The inability to induce the metal-detoxicating MT-protein in response to Cd, may account for a higher susceptibility of testes to Cd toxicity and carcinogenesis compared to liver.

Metallothionein induction related to hepatic structural perturbations and antioxidative defences in roach (Rutilus rutilus) exposed to the fungicide procymidone

A variety of stresses, hormones, glucocorticoids and cytokines are known to induce metallothioneins (MTs) in animals. The aim of this study was to investigate the effects of chemical stress induced by the dicarboximide fungicide procymidone on hepatic structure, MT content and antioxidative defences (catalase and glutathione reductase activities and glutathione content) in the common fish Rutilus rutilus. Catalase and glutathione reductase activities remained stable throughout the experiment. Four days of exposure to 0.2 or 0.4 mg l−1 of procymidone induced an obvious increase in liver MT content, perturbation of metal MT contents, and an increase in hepatic glutathione content. After 14 days' exposure, obvious and large structural alterations of the hepatic parenchyma occurred simultaneously with a decrease in MT and glutathione content. These events were interpreted as degeneration of the liver. Fish exposed for 14 days to procymidone and then placed for 14 days in clean water showed nearly complete decontamination of the liver, but MT concentrations remained high. The toxicological significance of these events is discussed.

Stress-induced Hepatic Metallothionein Engages in Radioprotective Control of Organic Radicals

Stress-induced Hepatic Metallothionein Engages in Radioprotective Control of Organic Radicals

Jejunal transfer rates of 109cadmium chloride increase in rats in vitro and in vivo after oral pretreatment with cadmium or zinc chloride

An increased body retention of Cd in rats orally pretreated with Cd or Zn is explained by induction of hepatic and renal metallothionein. Whether intestinal absorption of Cd increases after such treatments is not clear yet. To approach this problem we measured jejunal transfer rates of 109Cd in vitro and in vivo in pretreated rats (0.44 mmol Cd/l or 4.6 mmol Zn/l in the drinking water for 10 days) and compared them with those of untreated controls. Isolated jejunal segments were used for in vitro perfusion. In vivo perfusion was performed in anaesthetized rats with blood collected from mesenteric venules substituting corresponding losses by reinfusion of rat blood. Water and glucose transfer did not differ between controls and pretreated rats. At a luminal concentration of 5 μmol 109CdCl 2/l, Cd and Zn pretreatment significantly increased the transfer rate of 109Cd in vitro and in vivo similarly. The 109Cd transfer rates in controls in the final perfusion intervals (80–120 min) were 0.06 (pmol/cm/min) in vivo and 0.05 in vitro; the corresponding rates in Cd or Zn pretreated rats were significantly higher ( P<0.05) and amounted to 0.11 and 0.18 or 0.15 and 0.23, respectively. Mucosal concentrations of 109Cd measured at the end of the perfusion period tended to be lower in the pretreated animals than in the controls. This suggests that pretreatment with Cd or Zn reduces the amount of 109Cd bound to the tissue leaving more 109Cd for the transfer step. As compared to a level of mucosal metallothionein of 8 μg/g wet weight in controls, increased amounts of 67 or 52 μg/g wet weight in the Cd or Zn pretreated rats, respectively, thus did not decrease but increased transfer rates of 109Cd. Therefore, increased small intestinal transfer rates of Cd can contribute to increase the body retention of Cd seen after oral pretreatment with Cd or Zn.

Sexual maturation and reproductive zinc physiology in the female squirrelfish.

Female squirrelfish (Holocentridae) accumulate higher concentrations of hepatic zinc than any other known organism. In the liver cells, up to 70% of zinc is bound to metallothionein (MT), which is expressed at extremely high levels. These attributes are related to reproduction in ways that have not been fully characterized. In the present study, we have demonstrated that female-specific zinc and MT accumulation and distribution are strongly correlated to the onset of sexual maturity in Holocentrus adsenscionis. Sexual maturation not only resulted in increased concentrations of zinc in the liver and plasma, but also increased levels of hepatic MT mRNA. Furthermore, mature female squirrelfish exhibited greater proportions of MT protein in the nuclear liver cell fractions. To characterize the physiology further, we have examined the influence of the female sex hormone 17 beta-estradiol (E(2)). E(2) was not sufficient to elicit an increase in hepatic zinc concentrations or MT mRNA levels. E(2) administration did, however, result in increased levels of MT in the nuclear fraction as well as overall hepatic MT protein. E(2) also increased concentrations of zinc in the plasma. The changes in zinc concentration in the bloodstream followed the same time course as vitellogenin (VTG) transport from the liver. However, the high ratio of molar concentrations of zinc to VTG in the bloodstream suggest that VTG may not be the primary vehicle for hepato-ovarian zinc transport in squirrelfish.

Baseline measurements of indicators for sublethal effects of metals in lake whitefish (Coregonus clupeaformis).

Histological and metallothionein (MT) analyses of liver and kidney and measurements of serum electrolytes and hematocrit from lake whitefish (Coregonus clupeaformis), 3.5 years of age, were conducted over a 90-day period that included maximal gonadal maturation in females. Condition factors were higher in females at day 1 and when all data were combined over the 90-day sampling period. Significant differences between females and males of 1.8-fold in hepatic MT concentrations were observed at day 20 when ovaries appeared to be in the most advanced state of maturation, although this parameter was not quantified. Hematocrits were slightly but significantly lower at day 90. No sex differences were observed in the quantitative assessments of epithelium cell height, lumen diameter (LD), tubule diameter (TD), and ratio of LD:TD in the second segment of the proximal tubule (P2); and of hepatocyte area, hepatocyte nuclear diameters, and nucleus area:cytoplasmic area (N:C) ratio. Positive correlations were observed between N:C ratios and fish weight, fork length, and liver weight. No significant correlations between these histological morphometrics and MT concentrations were observed in liver or kidney. The results of this study provide baseline information for investigations designed to determine sublethal effects of metals in lake whitefish.

Enhancement of MT synthesis by leptin in fasted mice

It is known that metallothionein (MT) synthesis occurs in the liver in various stressful situations such as immobilization and fasting. However, the mechanism of MT synthesis in stressful situations is not fully understood. In this study, we examined the involvement of leptin, the obese gene product, in MT synthesis induced by fasting stress. Despite an increase in hepatic MT levels induced by 24-hr fasting in wild-type mice, both wild-type and MT-null mice showed decreases in plasma leptin levels after 24 hr of fasting. Hepatic MT levels increased to levels comparable with that in control mice in ICR, C3H, 129Sv and Balb/c mice fasted for 24 hr, and plasma leptin levels decreased significantly. Repetition of fasting and feeding in turn every 24 hr caused a gradual decrease in hepatic MT levels after the fasting period. In contrast, the reduced plasma leptin levels increased after the fasting period with repetition of fasting-feeding cycles. The findings indicate that there is an adaptation to starvation. On the other hand, subcutaneous leptin infusion in fasted mice via an osmotic pump resulted in increases in hepatic MT levels compared to the levels in vehicle-treated mice after 24 hr of fasting. Only leptin infusion had no effect on hepatic MT levels. These results suggest that MT synthesis in fasting stress is not correlated with decrease in plasma leptin levels but that leptin itself is a potent inducer of MT in a fasting situation.

Metallothionein and antioxidant enzymes in Long-Evans Cinnamon rats treated with zinc.

The Long-Evans Cinnamon (LEC) rat is a mutant animal model for Wilson's disease. It is known that an abnormal accumulation of Cu and Fe in the liver and low concentrations of both ceruloplasmin and Cu in the serum occur in these rats. The accumulation of Cu is explained by the defective expression of the Cu-transporting P-type ATPase gene, homologous to the gene for Wilson's disease (ATP7B). The aim of this work was to clarify the action mechanism of Zn, and to verify the role that this metal plays in LEC rats in short-term treatment experiments (1 and 2 weeks) on concentrations of Cu, Zn, Fe, metallothionein (MT), 8-hydroxy-2'-deoxyguanosine (oh(8)dG) and on the activity of antioxidant enzymes. It is well known that Zn induces MT and has the ability to prevent redox-active metals, Cu and Fe, binding to and causing oxidative damage at active sites of Zn metalloenzymes and nonspecific binding sites on proteins. Zn administration reduces Cu and Fe transport from mucosal to serosal intestinal sides through competitive mechanisms. Our findings show that treatment with zinc acetate increases tissue Zn and MT contents and decreases Cu and Fe concentrations in the liver and kidneys, even if hepatic Zn and MT concentrations decrease with treatment period. Induction of MT synthesis by Zn contributes to the reduction in free radicals produced by Cu and Fe. We also observed that the superoxide dismutase (SOD)activity in liver decreases with treatment duration in association with the Cu and Fe liver decrease. However, the SOD activity in kidney increases in untreated rats at 2 weeks relative to those untreated for 1 week.

Metallothionein-Independent Zinc Protection from Alcoholic Liver Injury

Previous studies using metallothionein (MT)-overexpressing transgenic mice have demonstrated that MT protects the liver from oxidative injury induced by alcohol. The mechanism of action of MT is unknown. Because MT primarily binds to zinc under physiological conditions and releases zinc under oxidative stress and zinc is an antioxidant element, it is likely that zinc mediates the protective action of MT. The present study was undertaken to determine the distinct role of zinc in hepatic protection from alcoholic injury. MT I/II-knockout (MT-KO) mice along with their wild-type controls were treated with three gastric doses of ethanol at 5 g/kg at 12-hour intervals. Zinc sulfate was injected intraperitoneally in a dosage of 5 mg/kg/day for 3 days before ethanol treatment. MT concentrations in MT-KO mice were very low and zinc concentrations in MT-KO mice were lower than in wild-type mice. Zinc treatment significantly elevated hepatic MT concentrations only in wild-type mice and increased zinc concentrations in both MT-KO and wild-type mice. Ethanol treatment caused degenerative morphological changes and necrotic appearance in the livers of MT-KO mice. Microvesicular steatosis was the only ethanol-induced change in the liver of wild-type mice. Ethanol treatment decreased hepatic glutathione concentrations and increased hepatic lipid peroxidation, and the concentrations of lipid peroxide products in the wild-type mice were lower than in the MT-KO mice. All of these alcohol-induced toxic responses were significantly suppressed by zinc treatment in both MT-KO and wild-type mouse livers. These results demonstrate that zinc, independent of MT, plays an important role in protection from alcoholic liver injury. However, MT is required to maintain high levels of zinc in the liver, suggesting that the protective action of MT in the liver is likely mediated by zinc.

Gadolinium chloride pretreatment prevents cadmium chloride-induced liver damage in both wild-type and MT-null mice.

The heavy metal cadmium (Cd) causes hepatotoxicity upon acute administration. Kupffer cells, the resident macrophages of the liver, have been suggested to play a role in Cd-induced hepatotoxicity. Gadolinium chloride (GdCl3) may prevent Cd-induced hepatotoxicity by suppressing Kupffer cells. However, GdCl3 also induces the Cd-binding protein, metallothionein (MT). Therefore, this study was conducted to determine whether GdCl3 prevents Cd-induced hepatotoxicity via the induction of MT. Hepatic MT and Kupffer cell counts were analyzed 24 h after wild-type (WT) mice were administered saline or 10, 30, or 60 mg GdCl3/kg. GdCl3 induced MT in a dose-dependent manner without affecting nonprotein sulfhydryl content. All examined doses of GdCl3 were effective at eliminating Kupffer cells from the liver. To examine the hepatoprotective effects of GdCl3, WT and MT-null mice were pretreated with saline or 10, 30, or 60 mg GdCl3 24 h prior to a hepatotoxic dose of Cd (2.5 mg Cd/kg). Blood and livers were removed 16 h later and analyzed for hepatotoxicity as well as MT, Cd, and Kupffer cell content. Hepatotoxicity was alleviated in both WT and MT-null mice that were pretreated with 30 or 60 mg GdCl3/kg, indicating that MT induction is not required for the hepatoprotective effects of GdCl3. Hepatic Cd content was not decreased by GdCl3, demonstrating that GdCl3 does not negatively affect Cd distribution to the liver. Kupffer cells were depleted at all three doses of GdCl3, whereas hepatoprotection was only observed at doses of 30 and 60 mg GdCl3/kg. This does not rule out Kupffer cells in the mechanism of Cd-induced hepatotoxicity, but it does suggest that GdCl3 exerts hepatoprotective effects on the liver aside from depleting Kupffer cells. In summary, these data substantially rule out MT induction and decrease the importance of Kupffer cells as mechanisms of GdCl3-induced protection from Cd-induced hepatotoxicity.

Relative bioavailability of organic zinc sources based on tissue zinc and metallothionein in chicks fed conventional dietary zinc concentrations

An experiment was conducted to estimate relative bioavailability (RBV) of Zn in Zinc proteinate and Zinc methionine compared to Zinc acetate as the standard. A total of 432, 1-day-old male broiler chicks was assigned randomly to dietary treatments which included the basal maize–soyabean meal diet (24 mg Zn kg −1 DM) supplemented with 0, 30, 60, or 90 mg kg −1 added Zn as reagent grade Zinc acetate or 30 or 60 mg kg −1 added Zn from Zinc methionine or Zinc proteinate fed for 3, 6, or 9 days. Accumulation of Zn in bone increased ( P<0.001) with supplemental Zn and age. Bone Zn concentration was greater ( P<0.001) in chicks given Zinc proteinate. Hepatic and mucosal metallothionein (MT) increased ( P<0.001) with supplemented Zn and age. Using Zinc acetate as a standard (1.00) in multiple linear regressions gave estimated RBV values of 1.10, 1.24, and 1.16 for Zinc proteinate and 0.88, 0.91 and 0.78 for Zinc methionine at 3, 6, and 9 days, respectively, based on bone Zn concentrations. Similar estimates were calculated using mucosal MT. The coefficient of determination was greater for the mucosal MT regression at 3 days compared to that for bone Zn. Mucosal MT appears to be a suitable criterion to estimate bioavailability of Zn sources during short term trials.