Induction Of Hepatic Metallothionein Research Articles

Biliary and hepatic metallothionein, metals and trace elements in environmentally exposed neotropical cichlids Geophagus brasiliensis

One of the metal detoxifying mechanisms that occurs in fish is metallothionein (MT) induction and metal binding. Hepatic MT induction has been well described, but biliary MT metal detoxification has only recently been described in fish. In this scenario, metal-metal interactions have been increasingly evaluated to further understand the behavior of these contaminants regarding homeostasis and biological functions, as well as their toxic effects. Studies, however, have been mainly conducted concerning the elemental pair Se-Hg, and scarce reports are available concerning other metal pairs. Therefore, this study aimed to evaluate biliary and hepatic MT metal detoxification mechanisms in a territorial neotropical cichlid, Geophagus brasiliensis. Fish were sampled from the anthropogenically impacted estuarine Rodrigo de Freitas Lagoon, located in Southern Rio de Janeiro, and trace elements and MT were determined by inductively coupled plasma mass spectrometry (ICP-MS) and UV-Vis spectrophotometry, respectively, in fish liver and bile. MT in bile were significantly lower than in liver. Significant differences between bile and liver were observed for many trace elements, and, although most were higher in liver, Cd and Ni were significantly higher in bile, indicating efficient excretion from the body via the biliary route. A significant correlation was observed between MT and Fe in bile, and between MT in liver and Cu and Zn in bile. Molar ratio calculations demonstrated protective elements effects against Al, As, Cd, Hg, Pb and V in both bile and liver, as well as some novel interrelationships, indicating the importance of these investigations regarding the elucidation of element detoxifying mechanisms. Furthermore, investigation of other elemental associations may aid in decision-making processes regarding environmental contamination scenarios linked to public health.

Immunolocalization of metallothionein in hepatocytes of guppy fish (Poecilia reticulata) exposed to tannery effluent: A biomarker study

Composite tannery effluent (TE) contaminated with a load of metals, especially chromium, is used in East Calcutta Wetland Ecosystem (ECW; a Ramsar site of West Bengal; No 1208) after natural stabilization for pisciculture and agriculture. Quantity and synergistic and antagonistic activities of both essential and nonessential metals in the effluents can induce a variety of complex changes in animal physiology. High fish yield and no apparent adverse influence on the fish biosystems thriving under pollutant stress at ECW allowed the practice to thrive sustainably over the last nearly 100 years. Thus, the present study was conducted to investigate the role of metallothionein (MT) as a potential biomarker in a fish biosystem following acute and chronic exposure to sublethal concentrations of tannery wastewater. MT localization in the liver tissue of guppy fish (Poecilia reticulata) was studied by the immuno gold-labeled electron microscopic technique. The data demonstrated increased metal content in fish body upon exposure to TE. Electron micrographs showed significant induction of hepatic MT in exposed P. reticulata over control specimens. Liver, being the detoxification site of an organism, plays a crucial role in the synthesis of MT and arrest of excess metal. The method employed in the present study for demonstrating the induction and localization of MT is innovative and could be used as a biomarker in fish exposed to metal stress. Our study also indicated that the metalloprotein complexes in fish tissue sequestered excess load of metals and thereby arrested unwanted interferences of excess metal loads in metabolic processes.

Zinc and liver disease.

Zinc is an essential trace element required for normal cell growth, development, and differentiation. It is involved in DNA synthesis, RNA transcription, and cell division and activation. It is a critical component in many zinc protein/enzymes, including critical zinc transcription factors. Zinc deficiency/altered metabolism is observed in many types of liver disease, including alcoholic liver disease (ALD) and viral liver disease. Some of the mechanisms for zinc deficiency/altered metabolism include decreased dietary intake, increased urinary excretion, activation of certain zinc transporters, and induction of hepatic metallothionein. Zinc deficiency may manifest itself in many ways in liver disease, including skin lesions, poor wound healing/liver regeneration, altered mental status, or altered immune function. Zinc supplementation has been documented to block/attenuate experimental ALD through multiple processes, including stabilization of gut-barrier function, decreasing endotoxemia, decreasing proinflammatory cytokine production, decreasing oxidative stress, and attenuating apoptotic hepatocyte death. Clinical trials in human liver disease are limited in size and quality, but it is clear that zinc supplementation reverses clinical signs of zinc deficiency in patients with liver disease. Some studies suggest improvement in liver function in both ALD and hepatitis C following zinc supplementation, and 1 study suggested improved fibrosis markers in hepatitis C patients. The dose of zinc used for treatment of liver disease is usually 50 mg of elemental zinc taken with a meal to decrease the potential side effect of nausea.

Combined Effect of Dietary Cadmium and Benzo(a)pyrene on Metallothionein Induction and Apoptosis in the Liver and Kidneys of Bank Voles

Bank voles free living in a contaminated environment have been shown to be more sensitive to cadmium (Cd) toxicity than the rodents exposed to Cd under laboratory conditions. The objective of this study was to find out whether benzo(a)pyrene (BaP), a common environmental co-contaminant, increases Cd toxicity through inhibition of metallothionein (MT) synthesis—a low molecular weight protein that is considered to be primary intracellular component of the protective mechanism. For 6 weeks, the female bank voles were provided with diet containing Cd [less than 0.1 μg/g (control) and 60 μg/g dry wt.] and BaP (0, 5, and 10 μg/g dry wt.) alone or in combination. At the end of exposure period, apoptosis and analyses of MT, Cd, and zinc (Zn) in the liver and kidneys were carried out. Dietary BaP 5 μg/g did not affect but BaP 10 μg/g potentiated rather than inhibited induction of hepatic and renal MT by Cd, and diminished Cd-induced apoptosis in both organs. The hepatic and renal Zn followed a pattern similar to that of MT, attaining the highest level in the Cd + BaP 10-μg/g group. These data indicate that dietary BaP attenuates rather than exacerbates Cd toxicity in bank voles, probably by potentiating MT synthesis and increasing Zn concentration in the liver and kidneys.

Assessment of tissue-specific accumulation and effects of cadmium in a marine fish fed contaminated commercially produced diet

Commercially produced fish diet is now widely used in fish farming but it often contains elevated levels of cadmium (Cd). However, the adverse effects on fish are poorly understood. In this study, farm-raised marine grunts, Terapon jarbua, were fed Cd-contaminated diet or exposed to waterborne Cd for 4 weeks. Tissue-specific Cd bioaccumulation and its effects were subsequently examined. We found that Cd was accumulated in different fish tissues (digestive tracts, gills or livers). At the end of the exposure, Cd accumulation peaked in the fishes’ livers (5.0–6.3 μg g −1), followed by the digestive tracts (0.83–3.16 μg g −1) and gills (0.27–2.74 μg g −1). Endpoints such as the survival rate, specific growth rate, condition factor, and superoxide dismutase activity were not significantly affected by Cd exposure. In contrast, metallothionein (MT) induction and subcellular Cd distribution indicated that there were possible sublethal effects of Cd exposure. MT was induced in response to Cd accumulation, but it returned to the control levels after a longer exposure period, except for hepatic MT induction resulting from waterborne or low dietary Cd exposure. The Cd percentage in the metallothionein-like protein (MTLP) fraction increased over exposure time, and it accounted for more than 57% Cd in the fishes’ livers and 80% Cd in their digestive tracts by the end of the exposure period. Overall, although Cd in commercial fish diet did not have significant lethality to T. jarbua, sensitive responses such as hepatic MT induction and subcellular Cd distribution revealed that the Cd-induced storage and detoxification in T. jarbua may increase fish's tolerance to toxic metals.

Chemical and ecotoxicological analyses of sediments and elutriates of contaminated rivers due to e-waste recycling activities using a diverse battery of bioassays

A multi-trophic, multi-exposure phase assessment approach was applied to characterize the toxicity of sediments collected from two rivers in Guiyu, China, an e-waste recycling centre. Elutriate toxicity tests (bacterium Vibrio fischeri and microalga Selenastrum capricornutum) and whole sediment toxicity test (crustacean Heterocypris incongruens) showed that most sediments exhibited acute toxicity, due to elevated heavy metals and PAHs levels, and low pH caused by uncontrolled acid discharge. The survival rates of crustaceans were negatively ( p < 0.05) correlated with total PAHs in sediments (411–1755 mg kg −1); EC50s of V. fischeri on the elutriates were significantly correlated with elutriate pH ( p < 0.01). Significant ( p < 0.05) correlations between the induction of hepatic metallothionein in tilapia ( Oreochromis mossambicus) and metal concentrations (Cu, Zn, Pb) in sediments were also observed, when fish were fed with diets containing sediment. The results showed that uncontrolled e-waste recycling activities may bring adverse effects to local aquatic ecosystem.

Induction of Metallothionein by Manganese Is Completely Dependent on Interleukin-6 Production

Metallothionein (MT) is a cysteine-rich protein that binds to and is inducible by heavy metals such as cadmium and zinc. However, the precise mechanism of MT induction by other metals remains unclear. In the present study, we investigated the mechanism of MT induction by manganese, focusing on the involvement of cytokine production. Administration of MnCl(2) to mice resulted in the induction of MT dose-dependently in the liver with little accumulation of manganese. Speciation analysis of metals in the liver cytosol showed that the major metal bound to the induced MT was zinc. Administration of MnCl(2) caused an increase in mRNA levels of interleukin-6 (IL-6) in the liver as well as an increase in serum levels of IL-6 but not those of other inflammatory cytokines. Subsequently, serum levels of serum amyloid A (SAA), an acute-phase protein induced by IL-6, increased with a peak at 24 h. However, no increase in serum alanine aminotransferase activity was observed, suggesting that manganese enhanced the production of IL-6 and SAA without causing liver injury. In response to IL-6, the expression of a zinc transporter, ZIP14, was enhanced in the liver, possibly contributing to the synthesis of hepatic zinc-MT. In IL-6-null mice, the induction of hepatic MT by treatment with MnCl(2) was completely suppressed to the control level. These results suggest that manganese is a unique metal that induces the synthesis of hepatic MT completely depending on the production of IL-6 without accompanying liver injury.

Pentavalent vanadium induces hepatic metallothionein through interleukin-6-dependent and -independent mechanisms

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein which has a high affinity for metals. The synthesis of MT is induced by heavy metals such as cadmium and zinc. However, little is known about the induction of MT by tetravalent or pentavalent metals. We investigated the induction of MT synthesis by a pentavalent vanadium compound in mice. Hepatic MT concentrations were increased by subcutaneous injection of ammonium metavanadate (AMV) dose-dependently, and to the similar levels as those induced by zinc chloride. However, accumulation of vanadium in the liver was very low, while high concentrations of vanadium were detected in the kidney. High performance liquid chromatography/inductively coupled argon plasma-mass spectrometry (HPLC/ICP-MS) chromatogram of the liver cytosol of AMV-treated mice revealed that the major metal bound to MT was not vanadium, but zinc. The chromatogram of the liver cytosol of MT null mice demonstrated the existence of a low-molecular-weight vanadium-binding protein that is different from MT. A time-course study showed that concentrations of serum interleukin-6 (IL-6) and serum amyloid A (SAA), an acute-phase protein, increased after the AMV injection. To confirm the involvement of IL-6 in MT induction by AMV administration, IL-6 null and wild-type mice were injected with AMV. In IL-6 null mice, hepatic MT induction by AMV administration decreased significantly to about a half of wild-type mice. These data suggest that both IL-6-dependent and -independent mechanisms are involved in MT induction by vanadium compounds in mice.

Induction of Hepatic Metallothionein by Trivalent Cerium: Role of Interleukin 6

Metallothionein (MT) is a small sulfydryl-rich protein that binds to and is inducible by heavy metals such as mercury, cadmium, zinc, and copper. However, little is known about the induction of MT by trivalent metals except for bismuth. In this study, we examined the induction of MT synthesis by cerium, a trivalent lanthanoid metal. Administration of cerium chloride (CeCl3) to mice resulted in accumulation of cerium and induction of MT in the liver in a dose-dependent manner. Distribution profiles of metals in the soluble fraction of the liver of CeCl3-treated mice analyzed by high performance liquid chromatography/inductively coupled argon plasma-mass spectrometry (HPLC/ICP-MS) demonstrated that the metal bound to MT-I and MT-II was zinc, but not cerium. Administration of CeCl3 caused increases in the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the levels of serum amyloid A (SAA), an acute phase protein. Among inflammatory cytokines examined, interleukin 6 (IL-6) exhibited a marked increase in the serum at 3 h after the CeCl3 administration. In order to evaluate the involvement of IL-6 in the induction of MT by cerium, we examined MT induction by CeCl3 in IL-6 null mice. Both the induction of hepatic MT and the increases in SAA levels were markedly suppressed in IL-6 null mice. These results suggest that IL-6 plays an important role in the induction of hepatic MT by cerium.

Induction of Hepatic Metallothionein by Mitochondrial Oxidative Stress in Mice

or malonic acid and were analyzed for the hepatic levels of thiobarbituric acid-reactive substances (TBARS) and MT. Sodium azide and atractyloside increased the hepatic MT level transiently around 6 and 12 hr, respectively, after the injection. Malonic acid significantly increased the MT level even at 3 hr and the increase continued until at least 24 hr after the injection. In all three cases, the changes in hepatic MT level showed almost the same patterns as those of TBARS with respect to both kinetics and magnitude. The increases in both TBARS and MT in the liver due to malonic acid were inhibited by about 90% by preinjection of succinic acid. These results suggest that the inhibitors of mitochondrial respiratory functions directly affect the mitochondria of hepatic cells, causing the production of reactive oxygen species, and consequently resulting in the induction of MT in mice.

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.

Toxicological effects of dietary arsenic exposure in lake whitefish ( Coregonus clupeaformis)

Adult lake whitefish were fed As contaminated diets at nominal concentrations of 0, 1, 10, and 100 μg As/g food (dry weight) for 10, 30, and 64 days. Reduced feed consumption was observed in lake whitefish fed the 100 μg As/g food, beginning on day 45 of exposure. The accumulation and distribution of As in these fish are described in the previous manuscript [Pedlar, R.M., Klaverkamp, J.F., 2001. The accumulation and distribution of dietary arsenic in lake whitefish ( Coregonus clupeaformis). Aquat. Toxicol., in press]. At the molecular level of organization, metallothionein (MT) induction occurred in lake whitefish fed the 100 μg As/g food after 10 and 30 days, and in fish fed the 1 and 10 μg As/g diets for 64 days. Dietary As exposure did not have a significant effect on plasma lipid peroxide (LPO) concentrations. At the tissue and organ level, mean liver somatic index decreased significantly in lake whitefish fed the 100 μg As/g food for 64 days. Blood parameters (hematocrit, hemoglobin concentration, red blood cell count, mean cell volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration) were not affected by exposure to As contaminated diets. Liver and gallbladder histopathologies were observed in lake whitefish fed all As contaminated diets after each duration of exposure. Histopathology observed in liver included nuclear, architectural and structural alterations, areas of inflammation, and focal necrosis. Sloughing of the epithelium, dilation of vascular elements, inflammation, edema, fibrosis, and increased width of the submucosa were some of the alterations observed in gallbladders of lake whitefish fed As contaminated diets. Both organs were sensitive to As exposure, as damage occurred with exposure to dietary concentrations of As as low as 1 μg/g. Whole organism parameters were unaltered by dietary As exposure. Based on the results of this study, histopathological alterations in liver and gallbladder, and hepatic MT induction may be useful indicators of As toxicity in environmental monitoring programs that also measure As concentrations in those tissues.

Hepatic and renal metallothionein induction by an oral equimolar dose of zinc, cadmium or mercury in mice

The hepatic and the renal subcellular distribution of zinc, cadmium or mercury and induction of tissue metallothionein (MT) at 24, 48 and 72 h following an oral equimolar dose (15 μmol metal/kg) of zinc (II) chloride, cadmium (II) chloride or mercury (II) chloride in male albino mice were investigated. There was a moderate increase in hepatic and renal zinc levels mainly in their nuclear mitochondrial fraction (NMF) 24 h post zinc chloride administration. Subsequently, the hepatic zinc increased and the renal zinc declined with time. The zinc-induced hepatic MT level was maximum at 48 h, which decreased slightly thereafter, while there was no marked increase in renal MT level at any time interval. The cadmium was equally distributed in liver and kidney more in their supernatant cytosol fraction (SCF) than in their NMF at 24 h after a dose of cadmium chloride. The cadmium levels showed a decreasing trend in hepatic fractions and an increasing trend in renal fractions with time. The cadmium-induced hepatic and renal MT were substantial at 24 h post cadmium administration, the former decreased thereafter while the latter enhanced at 48 h before declining. The accumulation of mercury in kidney was 1.5 times that in liver, which was localised more in their SCF than in their NMF at 24 h in response to a dose of mercuric chloride. The mercury levels of hepatic and renal subcellular fractions started declining after 24 h and at 72 h they were significantly lower. The induction of hepatic and renal MT was maximum at 24 h after mercuric chloride administration, which declined thereafter concomitant with the decrease in their mercury levels. However, the MT levels in both the organs remained considerably higher than in normal animals at 72 h post exposure. The results show that the accumulation of metal in liver and kidney follows the order: Hg>Cd>Zn and the induction of MT follows Hg>Cd>Zn in liver and Cd>Hg>Zn in kidney. The alterations in zinc and copper homeostasis were more marked in liver than in kidney and follows the order: Hg>Cd>Zn.

Induction of hepatic metallothioneins in tumor-bearing IL-6-null mice.

Interleukin-6 (IL-6) has been thought to play a key role in the induction of hepatic metallothionein (MT) synthesis in tumor-bearing animals. In order to clarify the role of IL-6 and to distinguish its effect from those of other cytokines, we inoculated IL-6-null and B6J129Sv (wild-type control) mice SC with 1 x 10(7) Ehrlich carcinoma cells and examined tumor growth, hepatic MT, and serum cytokines. We have found that tumor growth was followed by an increase of hepatic MT in both IL-6-null and wild-type mice and that the two strains of mice had a similar hepatic MT induction followed by zinc accumulation in the liver. These results could be explained by our finding that tumor-bearing IL-6-null mice had a high level of tumor-secreted IL-6 in the blood. In conclusion, by utilizing tumor-bearing IL-6-null mice, we have demonstrated that IL-6 secreted from the tumor cells is responsible for the tumor-evoked increase of hepatic MT level.

Arctic char (Salvelinus alpinus) metallothionein: cDNA sequence, expression, and tissue‐specific inhibition of cadmium‐mediated metallothionein induction by 17β‐estradiol, 4‐OH‐PCB 30, and PCB 104

AbstractIn this study, we haveexamined the basallevel expression and tissue‐specificexpressionpatternsof metallothionein (MT) in Arctic char following metal and E2 (17β‐estradiol) treatment. To study the gene regulation in Arctic char, the two MT isoforms were isolated from a lambda‐ZAP hepatic cDNA library and characterized. Determination of basal MT mRNA and MT expression for 10 different tissues revealed a lack of correlation between MT mRNA and MT levels. The inducibility of MT mRNA and the correlation to resulting MT levels were then determined for liver and kidney. We found a more rapid and stronger induction of MT mRNA in liver than in kidney at day 1 and 3 postinjection, whereas the MT protein quantification showed higher MT levels in kidney than in liver at days 3 and 7 postinjection. These discrepancies indicate that differences in metal handling or posttran‐scriptional regulation of MT exists between tissues. Whereas metals induce MT synthesis, E2 inhibit the hepatic MT expression. To examine the tissue specificity of this inhibition, we determined the effect of 17pj‐estradiol (E2) and two estrogenic PCBs (4′‐OH‐PCB 30 and PCB 104) on Cd‐mediated MT induction in liver and kidney. Although E2 and the estrogenic PCBs inhibited cadmium‐mediated hepatic MT induction, these compounds did not interfere with renal MT induction.

Role of IL-6 in the induction of hepatic metallothionein in mice after partial hepatectomy

Metallothioneins (MT) are induced upon partial hepatectomy (PH), possibly mediated by various cytokines. In the present study, we studied cytokine-dependent MT synthesis in partially hepatectomized IL-6 gene knock-out (GKO) mice in the remaining lobe of the liver. We focused on IL-6, TNFα and IL-1β, the major cytokines thought to be involved in MT synthesis. The IL-6 GKO mice and B6J129Sv (wild-type control) mice were subjected to 70% PH or laparotomy. We found that MT was significantly decreased in IL-6 GKO mice, although PH induced hepatic MT in both strains of mice. Laparotomy induced MT in the liver of wild-type mice but not in IL-6 GKO mice. Pretreatment of IL-6 GKO mice with rIL-6 (5 μg/mouse) restored hepatic MT synthesis. Serum IL-6 level in wild-type mice was maximal at 6 h after surgery and decreased thereafter. Serum IL-1β was the same in both strains of mice. Serum TNFα basal level in IL-6 GKO mice was higher than in wild-type mice. PH caused an increase in serum TNFα level in both strains of mice, and it was two times higher in IL-6 GKO mice than in wild-type mice at 18 h after surgery. We conclude that IL-6 plays a predominant role in hepatic MT synthesis after PH, but that IL-6 GKO mice still reserve the capacity to synthesize MT by an as yet unidentified mechanism.

Induction of metallothionein as an adaptive mechanism affecting the magnitude and progression of toxicological injury.

Pretreatment of rats with low doses of Cd produces adaptive tolerance to a subsequent high dose of Cd-induced lethality, thus shifting the dose-response curve to the right. Cd pretreatment of animals also protects against the hepatotoxicity produced by high doses of Cd. This protection is attributable to the 10- to 50-fold induction of hepatic metallothionein (MT) by Cd pretreatment. As a result hepatic subcellular distribution of Cd is significantly altered, with more Cd bound to MT in the cytosol and a concomitant reduction of Cd in other critical organelles. In addition MT-transgenic animals are more resistant, whereas MT-null mice are more sensitive than controls to Cd-induced lethality and hepatotoxicity. This further demonstrates that MT is important in Cd detoxication. Induction of hepatic MT by zinc also protects mice from carbon tetrachloride (CCl4)-induced liver injury, with more 14C-CCl4 bound to MT in the cytosol. MT-null mice are more sensitive to CCl4-induced hepatotoxicity, which supports the hypothesis that induction of MT also plays a protective role for nonmetallic chemicals. These results indicate that MT is a part of cellular adaptive mechanisms affecting the magnitude and progression of toxic insults from metals such as Cd as well as from organic chemicals such as CCl4.

Suppression of Kupffer cell function prevents cadmium induced hepatocellular necrosis in the male Sprague-Dawley rat

Exposure of humans to toxic metals and metalloids is a major environmental problem. Many metals, such as cadmium, can be hepatotoxic. However, the mechanisms by which metals cause acute hepatic injury are in many cases unknown. Previous reports suggest a major role for inflammation in acute cadmium induced hepatotoxicity. In initial experiments we found that a non-hepatotoxic dose of cadmium chloride (CdCl2; 2.0 mg/kg, i.v.) markedly increased the clearance rate of colloidal carbon from the blood, which is indicative of enhanced phagocytic activity by Kupffer cells (resident hepatic macrophages). Thus, the objective these studies was to determine the involvement of Kupffer cells in cadmium induced liver injury by inhibiting their function with gadolinium chloride (GdCl3). Male Sprague-Dawley rats were administered GdCl3 (10 mg/kg, i.v.) followed 24 h later by a single dose of CdCl2 (3.0 and 4.0 mg/kg, i.v.). Twenty four hours after CdCl2 administration animals were killed and the degree of liver toxicity was assessed using plasma alanine aminotransferase (ALT), as well as light microscopy. Cadmium chloride administration produced multifocal hepatocellular necrosis and increased plasma ALT activity. Pretreatment with GdCl3 significantly reduced both the morphological changes and hepatic ALT release caused by CdCl2. However, the protection was specific to the liver, and did not alter CdCl2 induced testicular injury, as determined by histopathological damage. In many cases, the inducible cadmium-binding protein, metallothionein (MT) is often an essential aspect of the acquisition of cadmium tolerance in the liver. Although cadmium caused a dramatic induction of hepatic MT (32-fold), GdCl3 caused only a minor increase (2-fold). Combined CdCl2 and GdCl3 treatment did not induce levels to an extent greater than CdCl2 alone. As expected, GdCl3 also caused a slight increase in the amount of cadmium associated with the liver. In cultured hepatocytes isolated from GdCl3 pretreated rats, CdCl2 induced cytotoxicity was not significantly altered compared to control hepatocytes, indicating that the mechanism of tolerance required the presence of other cell types. Thus, GdCl3 attenuation of CdCl2 induced hepatotoxicity does not appear to be caused by increased tissue MT content or a decreased susceptibility of hepatocytes to cadmium. From these data, we concluded that tolerance to cadmium induced hepatotoxicity involves the inhibition of Kupffer cell function which results in a decreased inflammatory response and an altered progression of hepatic injury. These data further indicate that Kupffer cell function is critical to cadmium induced hepatocellular necrosis.

Tolerance Induced by All-trans-Retinol to the Hepatotoxic Effects of Cadmium in Rats: Role of Metallothionein Expression

Recently, it has been shown that large doses of all-trans-retinol (vitamin A) can potentiate the hepatotoxicity of several organic chemicals in the rat. Whether retinol pretreatment can alter the acute hepatotoxicity of an inorganic chemical, such as cadmium, is unknown. Therefore, the objective of this study was to determine how retinol might affect the acute toxicity of cadmium chloride (CdCl2) and to elucidate possible mechanisms. Cadmium exposure can induce acute, lethal hepatocellular necrosis in rodents, as well as lesions in the lung, kidney, testis, and gastrointestinal tract. In the present studies, male Sprague–Dawley rats were pretreated with retinol (75 mg/kg/day, po) for 7 consecutive days. One day after the last dose of retinol, animals were given a single injection of CdCl2(2.5 to 4.0 mg/kg, iv). Cadmium chloride administration to unpretreated control rats caused extensive hepatic, renal, pulmonary, and testicular toxicity at 6, 24, and 48 hr postdosing as evaluated by plasma enzymes and/or histopathology. In retinol-pretreated rats, a significant attenuation of CdCl2-induced tissue injury was observed. Since the inducible cadmium-binding protein metallothionein (MT) is often an essential aspect of cadmium tolerance, its content in tissue was assessed using the cadmium–hemoglobin assay. Interestingly, retinol pretreatment significantly increased MT in the liver by sevenfold, but had no effect on lung, kidney, testicular, or pancreatic MT content. Although this increase in hepatic MT was much less than that induced by CdCl2, it was additive to the induction of CdCl2. Furthermore, the tissue distribution of cadmium was significantly altered by retinol pretreatment. The liver accumulated more cadmium, while less cadmium was found in the lung, kidney, and testis in retinol-pretreated rats than in controls. In monolayers of primary isolated hepatocytes, CdCl2-induced toxicity was significantly reduced in cells isolated from retinol-pretreated rats compared to those isolated from control rats. The dose response was shifted to the right and thein vitrocadmium LC50 was increased byin vivoretinol exposure from 1.1 ± 0.1 to 2.4 ± 0.04 μm. From these data it is concluded that the induction of hepatic MT is an essential aspect of retinol-induced tolerance to CdCl2hepatotoxicity, as well as toxicity in other tissues.

Metallothionein Induction and Hepatoprotection by Echinoside A and Sakuraso-saponin

Metallothionein-inducing activities of 11 saponins were investigated in mice. Of the saponins investigated, echinoside A and sakuraso-saponin were highly effective. Sakuraso-saponin showed dose-dependent and time-dependent induction of hepatic metallothionein. The isoforms of the induced metallothionein in the liver were determined to be metallothionein 1 and 2. Induction of metallothionein was observed specifically in the liver and heart. Echinoside A showed similar effects to sakuraso-saponin except that no induction was observed in the heart. Pretreatment of mice with these saponins blocked CCl4-induced hepatic injury, such as the elevation of plasma transaminase activity and centrilobular necrosis in the liver. CCl4-induced elevation of lipid peroxide level in the liver was also blocked by injection of sakuraso-saponin. The hepatoprotective activities of the saponins found in this study may have been due to their MT-inducing activity. © 1997 by John Wiley & Sons, Ltd.