The protective mechanism of antioxidants in cadmium-induced ototoxicity in vitro and in vivo

BACKGROUND: Several heavy metals have been shown to have toxic effects on the peripheral and central auditory system. Cadmium (Cd2+) is an environmental contaminant showing a variety of adverse effects. Given the current rate of release into the environment, the amount of Cd2+ present in the human body and the incidence of Cd2+-related diseases are expected to increase. OBJECTIVE: The overall aim of this study was to gain further insights into the mechanism of Cd2+-induced ototoxicity. METHODS: Cell viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cytochrome c (cyt c), phosphorylated extracellular signal-regulated protein kinase (p-ERK), caspases, morphologic change, and functional changes in HEI-OC1 cells, rat cochlear explants, and mouse cochlea after Cd2+ exposure were measured by flow cytometry, immunohistochemical staining, Western blot analysis, and auditory brainstem response (ABR) recording. Mechanisms underlying Cd2+ototoxicity were studied using inhibitors of different signaling pathways, caspases, and antioxidants. RESULTS: Cd2+ exposure caused cell death, ROS generation, MMP loss, cyt c release, activation of caspases, ERK activation, apoptosis, and finally auditory threshold shift. Cd2+ toxicity interfered with inhibitors of cellular signaling pathways, such as ERK and c-jun N-terminal kinase, and with caspase inhibitors, especially inhibitors of caspase-9 and caspase-3. The antioxidants N-acetyl-l-cysteine and ebselen showed a significant protective effect on the Cd2+ toxicity. CONCLUSIONS: Cd2+ is ototoxic with a complex underlying mechanism. However, ROS generation may be the cause of the toxicity, and application of antioxidants can prevent the toxic effect.     

Effects of heavy metals on mitogen-activated protein kinase pathways

The signaling pathways leading to cellular protection or cell death following exposure to heavy metals have not been fully clarified. Mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated protein kinase (ERK), c-Jun NH₂-terminal kinase (JNK) and p38 MAPK transmit extracellular signals into the nucleus, and have been shown to participate in a diverse array of cellular functions such as cell growth, differentiation and apoptosis. Treatment with cadmium, inorganic mercury or tributyltin can activate ERK, JNK and p38 MAPK, and induces the expression of c-fos and c-jun genes prior to the development of apoptosis. However, the members of the MAPK family appear to be differentially activated depending on the heavy metal and the cell type exposed. Consequently, various cellular responses may be caused by the distinct pattern of MAPKs activation. MAPKs may be one of the important cellular signal transduction pathways affected by various environmental pollutants, including heavy metals.     

Distribution and binding of intratracheally instilled cadmium in rat lung

Retention, subcellular distribution, and the nature of binding of cadmium in lungs were studied following a single intratracheal instillation of cadmium. The clearance of intratracheally instilled cadmium from the lungs was very rapid during the initial periods of exposure and liver accumulated a maximum amount within 1 day. A low-molecular-weight cadmium-binding protein was isolated and characterized from lung cytosol. Cadmium administration increased metallothionein-like protein content but not sulfhydryls and zinc. The decrease in metallothionein at subsequent days of the exposure indicates the possibility of transport of this protein to some other target organs or the destruction of few cell types involved in providing protection against cadmium toxicity.     

Effect of the chelating agent sodium tripolyphosphate on cadmium toxicity in mice

The mechanism by which Sodium tripolyphosphate (STPP) increases cadmium toxicity after sc administration was investigated in mice after a dose of Cd (30 μmole/kg), alone or with STPP (90 μmole/kg). The effects of STPP on acute Cd toxicity, development of Cd-induced liver necrosis, rate of Cd transport, distribution of Cd among organs, and Cd binding to metallothionein (MT) was followed. Histological liver changes were not observed during the first 12 hr after injection of Cd, but already 6–8 hr after injection of Cd + STPP early centrilobular necroses and blood stasis appeared. At 12 hr more advanced necroses were present. STPP administered alone was nontoxic and did not change the liver morphology, when compared to animals killed immediately after injection. During the first 12 hr after administration of Cd with STPP there was a much faster transport of Cd giving rise to higher liver and kidney concentrations of Cd and partial inhibition of Cd-MT binding, compared with animals receiving the same dose of Cd without STPP.     

Effects of low-level cadmium in rats: influence of pretreatment with thiol-modulating agents

The effects of low-level cadmium (Cd) administration to rats on animal health, liver and kidney thiols, metallothionein, and glutathione reductase (GSSG reductase) and their modulation by cysteine (as a possible protector) and diethyl maleate (as a possible potentiator) have been investigated. Male Sprague-Dawley rats were treated with sodium or Cd acetate (25 micrograms Cd/kg) orally five times a week for 6 weeks. A second group of animals received cysteine (500 mg/kg; po) before each gavage while a third group received diethyl maleate (DEM) (0.85 mg/kg; ip) in addition to sodium or Cd acetate. When rats were treated with cadmium alone neither weight gain nor serum parameters indicative of hepato- or nephro-toxicity were affected. However, acid-soluble thiols, primarily glutathione, were decreased by about 25% in liver only. A tendency to a decrease in hepatic protein thiols was also noted. No changes were observed for hepatic or renal metallothionein in response to this low level of cadmium administration alone or in combination with the other treatments. Animals receiving cysteine, either alone or with cadmium, showed decreased body weight gain, but no change in serum parameters. Acid-soluble thiols in liver were lower in cysteine-treated rats (24%) and cysteine + Cd (33%) while kidney thiols were unaffected. Administration of DEM alone or with Cd did not cause any alteration in body weight gain. When given DEM + Cd, however, an increase in serum bilirubin was observed, which suggests interference with hepatobiliary function. Acid-soluble thiols were decreased by DEM alone (45%) and DEM + Cd (51%) in liver while renal thiols showed no change. Our data indicate that low-level Cd gavage decreases hepatic cellular thiols but not those of kidney. Cysteine gavage does not protect from the Cd-related effect. Indeed, cysteine itself was found to reduce acid-soluble thiols under the experimental conditions. This was observed only in liver, as was the decrease in thiols due to DEM treatment. DEM administration together with Cd resulted in signs of liver toxicity. There is no indication that inhibition of GSSG reductase by Cd might be involved in the thiol-decreasing effect of short-term repeated low-level gavage of Cd to rats.     

Effects of subchronic digestive exposure to organic or inorganic cadmium on biomarkers in rat tissues

In an experimental food chain, Wistar rats were fed cadmium (Cd) in an inorganic (CdCl₂) or organic (mainly associated with metallothionein from Helix aspersa snail viscera) form. After 1 month of exposure to 100 μg inorganic Cd g⁻¹ in food, an induction of metallothionein was observed in all target tissues. In liver, glutathione peroxidase (GSH-Px) activity decreased and alanine aminotransferase (ALAT) activity increased, suggesting that Cd causes hepatotoxicity. However, lipid peroxidation as well as catalase and caspase 3 (a marker of apoptosis) activities were not modified. At a rather low exposure (2.5 μg Cd g⁻¹), metallothionein level in the kidney was found to be the most sensitive biomarker of exposure for both Cd forms. In the small intestine of rats ingesting inorganic Cd, metallothionein expression was significantly higher than that observed for rats fed organic Cd. Present results allowed proposing a simple design to assess the effect of a chemical in a trophic transfer approach.     

Cadmium, zinc, and copper in rabbit kidney metallothionein--relation to kidney toxicity

Twenty-two rabbits were given repeated subcutaneous injection of cadmium chloride. The cumulative cadmium dose given ranged from 13 to 214 mumole/kg body weight. Five rabbits served as controls. The treatment resulted in cadmium concentrations in kidney cortex that ranged from 0.3 to 3.2 mmole Cd/kg and a subsequent production of metallothionein. The molar ratio of cadmium, zinc, and copper in metallothionein fractions from kidneys with different concentrations of cadmium was determined. At low concentrations of cadmium in rabbit kidneys, zinc was the dominating metal bound to metallothionein (70-90%). At high concentrations of cadmium in kidneys, cadmium was the dominating metal in metallothionein. Evidence of kidney toxicity, in the form of beta2-microglobulinuria, was seen when cadmium constituted 85% of the metal ions recovered from metallothionein fractions. The remaining 15% was zinc. This indicates that at most six of the seven metal-binding sites in mammalian metallothionein are occupied by cadmium and that the remaining site is occupied by zinc. Our data provide further support for the hypothesis that chronic cadmium nephrotoxicity develops when there is a lack of metal-binding sites available for cadmium in metallothionein.     

Effect of zinc deficiency on the accumulation of metallothionein and cadmium in the rat liver and kidney

The effects of zinc (Zn) deficiency and repeated exposure to cadmium (Cd) on the accumulation and distribution of metallothionein (MT), Cd and Zn in the liver and kidney were studied. Male Sprague-Dawley rats were fed either a Zn-deficient (1 ppm) or a Zn-adequate (40 ppm) diet during the experiment, and the rats were injected subcutaneously with a cadmium chloride solution (1.0 mg Cd/kg of body weight, 5 days a week) for 4 weeks. Cadmium, Zn, and Cd-induced MT concentrations in the liver and kidney were lower in the Zn-deficient rats (–Zn + Cd) than in the Zn-adequate rats (+ Zn + Cd), while the content of Cd bound to high molecular weight proteins (HMWP) was greater in the Zn-deficient rats (–Zn + Cd). The Zn bound to Cd-induced MT was reduced to 30% in the liver and to 60% in the kidney of the Zn-deficient rats (–Zn + Cd) as compared with that of the Zn-adequate rats (+ Zn + Cd). In the kidney of Zn-deficient rats, exposure to Cd caused a decrease in essential Zn associated with HMWP as compared with that of Zn-adequate rats (+ Zn + Cd). Thus, Zn-deficiency affected the distribution of Cd in tissues, MT and HMWP and accelerated substantially Cd-induced Zn-deficiency in the kidney. Although the renal Cd concentration was lower in the Zn-deficient rats (–Zn + Cd) than in the Zn-adequate rats (+ Zn + Cd), exposure to Cd for four weeks resulted in glucosuria and an increase in liver and kidney weights in the Zn-deficient rats (–Zn + Cd), but not in the Zn-adequate rats (+ Zn + Cd). These results suggest that development of Cd toxicity is related to the Zn status of the body, to the accumulation of Cd in HMWP and to the amount of essential Zn associated with HMWP.     

Mechanisms of cadmium toxicity in terrestrial pulmonates: programmed cell death and metallothionein overload

A sublethal dose of cadmium (Cd2+) administered via the diet during short-term exposure over 10 d induced programmed cell death in the hepatopancreas of the terrestrial pulmonate snail Helix pomatia. Condensed cell residues were predominantly phagocytosed by calcium cells, suggesting a specific function of these epithelial cells in metal detoxification or in clearing the organ of cellular debris from cell death. The considerable cell loss recorded by histological analysis was accompanied by enhanced cell proliferation. Intoxication with Cd was further associated with the pronounced abundance of residual bodies, predominantly recorded in excretory cells, and with pathological changes in the endoplasmic reticulum. During long-term Cd exposure, mortality increased with increasing Cd concentrations in the diet, as demonstrated by feeding experiments in the laboratory. Lethal effects of Cd appeared to be correlated with Cd overloading of the Cd-specific metallothionein isoform (Cd-MT), isolated and characterized previously from the animal's hepatopancreas. Stoichiometric analysis shows that the capacity of Cd-MT to bind six molar equivalents of Cd corresponds to a tissue Cd concentration of approximately 4 micromol/g dry weight. At this tissue concentration, all high-affinity metal-binding sites of Cd-MT are occupied by Cd2+. Cadmium exposure beyond this level gives rise to progressive destabilization of Cd-MT cluster structure in vitro, resulting in increasing proportions of weakly bound, or even unbound, Cd2+ ions. Our results suggest that in vivo, the observed overburdening of Cd-MT with Cd2+ reduces the viability of affected animals.     

Cadmium uptake and defense mechanism in insect cells

The uptake of cadmium and the defense mechanism against this heavy metal were studied in the Aedes albopictus C6/36 cell line. The internalization of cadmium was a very quick process and exhibited saturation kinetics over the metal concentration gradient (1.37 to 131 micromol/L). Cd toxicity and influx were both shown to be temperature dependent. The uptake was not influenced by a 2, 4-dinitrophenol pretreatment but was significantly decreased by the Ca2+ antagonist verapamil. These data suggest that cadmium is readily taken up through mediated transport, not requiring metabolic energy. A considerable amount of the metal passes through the Ca2+ channels, but probably (an)other transporting molecule(s) also play(s) an important role in the uptake process. The remarkable, nonsigmoid viability pattern of Cd-treated cultures suggests that CdCl2 concentrations above 33 micromol/L induce a cellular defense system. This phenomenon went together with increased protein synthesis. We found a major induction of a group consisting of 71-, 75-, and 78-kDa proteins, probably belonging to the HSP70 family, as similar proteins were induced by heat shock. A slight induction of a 120-kDa protein also occurred. At the highest Cd concentrations 98-, 108-, and 110-kDa proteins were induced. These data suggest that heat shock proteins may play an important role in the Aedes cell protection against Cd insult.     

Bioavailability indicators of inhaled cadmium compounds

In a thirty-day inhalation study male Wistar rats were continuously exposed to submicron aerosols of three different cadmium compounds. The cadmium chloride (CdCl2) and cadmium oxide (CdO) aerosol concentrations were 0.1 mg/m3 Cd. Because of its lower solubility the cadmium sulfide (CdS) level was 1 mg/m3 Cd. For CdCl2 and CdO, most of the cadmium was found in the lung cytosolic compartment, but for CdS only 30% of the cadmium was retrieved from the lung cytosols. This was observed both at the end of the inhalation and also after an additional 2-month period in fresh air. The cadmium contents of the lung homogenates, cytosols, and the lung cytosolic metallothionein were found to be twice as much for exposure to CdO than for exposure to CdCl2. For exposure to CdS at cadmium concentrations 10 times higher the same cadmium levels were found as for CdO. These results are confirmed by results from alveolar lavage analysis indicating that in the lung-inhaled CdO is even more available to lung tissue than the very soluble CdCl2, and CdO has an availability 10 times as much as CdS. This study proved that lung compartmental cadmium and metallothionein contents as well as lung lavage analysis can describe the bioavailability of inhaled cadmium.     

Cadmium-induced antioxidant defense mechanism in freshwater teleost Oreochromis mossambicus (Tilapia)

Antioxidant defenses consisting of catalase, superoxide dismutase (SOD), xanthine oxidase (XOD), glutathione peroxidase (GPX), and glutathione S-transferase were estimated in liver and kidney of freshwater fish subjected to a sublethal concentration of cadmium chloride (Cd2+), i.e., 5 ppm. The aim of the study was to evaluate the role of antioxidant defenses during cadmium-induced oxidative stress. Significant elevations in liver and kidney of all of the above detoxification enzymes were evident from the 7th day onward, were maintained until the 15th day, and then decreased slightly on the 30th day of exposure to cadmium stress. Between the two tissues studied, liver recorded higher activity for all enzymes except GPX, which was elevated significantly in kidney (82.85%). Both liver and kidney recorded more or less similar increases of SOD (86.61% and 86.32%, respectively), and XOD (86.41% and 84.19%, respectively). The findings indicate that tissue glutathione-dependent enzymes as well as other antioxidant enzymes function in protection against Cd2+ toxicity and that these antioxidants provide a first line of defense against Cd2+ before the induction of any metallothionein synthesis occurs.     

Bioaccumulation and Toxicodynamics of Cadmium to Freshwater Planarian and the Protective Effect of N-Acetylcysteine

Although toxic responses of freshwater planarians after exposure to environmental toxicants can be observed through external toxicological end points, physiological responses inside the bodies of treated planarians have rarely been investigated. The present study was designed, using cadmium (Cd) as a reference toxicant, to determine its bioaccumulation and toxicodynamics in the freshwater planarian, Dugesia japonica, after acute toxicity was obtained. Accumulated Cd concentrations, metallothionein levels, and the oxidative status in planarians were determined after exposure to Cd. Furthermore, we hypothesized that the acute death of Cd-treated planarians was associated with increased oxidative stress. After Cd-treated planarians were coexposed to antioxidant, N-acetylcysteine (NAC), we found that NAC protected planarians from Cd lethality by maintaining the oxidative status and decreasing the bioaccumulation of Cd. The results of the present study support planarians being used as a practical model for toxicological studies of environmental contaminants in the future.     

Joint effects of dietary cadmium and polychlorinated biphenyls on metallothionein induction, lipid peroxidation and histopathology in the kidneys and liver of bank voles

Free-living bank voles have been shown to be more sensitive to cadmium (Cd) toxicity than the rodents exposed to Cd under laboratory conditions. The present study was designed to find out whether polychlorinated biphenyls (PCBs), common environmental co-contaminants, increase susceptibility to Cd toxicity through inhibition of metallothionein (MT) synthesis-a low molecular weight protein that is considered to be a primary intracellular component of the protective mechanism. For 12 weeks, the male bank voles were provided with diets containing Cd (0.05 microg/g (control) and 10 microg/g dry wt) and PCBs (0, 10 and 50 microg/g dry wt) alone or in combination under long (16 h) and short (8 h) photoperiods. At the end of exposure period, histological examinations and analyses of MT, Cd, Fe and lipid peroxidation in the kidneys and liver were carried out. Dietary PCBs did not affect Cd inducibility of renal MT, but decreased it significantly in the liver; however, no signs of Cd toxicity (measured by histopathology) occurred in both organs. On the contrary, PCBs at the highest dose increased significantly lipid peroxidation in the kidneys and liver (4-fold) only in the bank voles raised under a long photoperiod; the PCB-induced hepatic lipid peroxidation was accompanied by extensive histopathological changes including hepatocyte enlargement, necrosis and steatosis. Co-treatment with dietary Cd significantly suppressed the increase in lipid peroxidation and apparently reduced hepatic damage. These data indicate that (1) dietary PCBs do not enhance Cd toxicity in the kidneys and liver of bank voles and (2) dietary Cd suppresses PCB-induced hepatotoxicity that appears to be photoperiod-dependent.     

Effect of pH on cadmium toxicity,speciation, and accumulation during naphthalene biodegradation

Lowering pH of a microbiological medium from 7 to 4 decreased cadmium toxicity during naphthalene biodegradation by a Burkholderia sp. Cadmium speciation and cadmium accumulation in the system were studied to explain this effect. Cadmium speciation was determined by direct measurement and by geochemical modeling. Previous studies have implicated the monovalent hydroxylated cadmium (CdOH+) species in the effect of pH on cadmium toxicity. Modeling analysis predicted CdOH+ formation only at very low concentrations (< or = 0.0128 microM), while the measured concentration of divalent ionic cadmium (Cd2+) was at least three orders of magnitude greater, suggesting that Cd2+ is the more significant metal form. With respect to cadmium accumulation, cells contained in media adjusted to pH 4 accumulated only 2.76 +/- 0.76 mg Cd/g cells, whereas cells in media adjusted to pH 7 accumulated 8.52 +/- 0.71 mg Cd/g cells. These data suggest that cadmium toxicity is correlated with increased cadmium accumulation rather than the formation of CdOH as pH is increased. At low pH, the decrease in cadmium accumulation may be caused by increased competition between hydrogen and cadmium ions for binding sites on the cell surface or by an increase in metal efflux pump activity due to an increase in the proton gradient that drives the efflux pump.     

Assessment of stress-related gene expression in the heavy metal-exposed nematode Caenorhabditis elegans: a potential biomarker for metal-induced toxicity monitoring and environmental risk assessment

The toxicity of cadmium, lead, chromium, and arsenite on Caenorhabditis elegans was investigated to identify sensitive biomarkers for environmental monitoring and risk assessment. Effects of these metals on stress-related gene expression, growth, reproduction, and mortality of C. elegans were investigated under laboratory conditions. The possibility of using C. elegans as a biosensor for environmental toxicity monitoring was also tested using a green fluorescent protein transgenic nematode. The 24-h median lethal concentrations of cadmium, lead, chromium, and arsenite in C. elegans were 846, 34, 115, and 92 mg/L, respectively. Cadmium exposure led to an increase in the expression of most of the genes tested. The degree of increase was more than threefold compared to control in heat shock protein 16.2, heat shock protein 70, metallothionein 2, cytochrome P450 family protein 35A2, glutathione-S-transferase 4, superoxide dismutase 1, catalase 2, C. elegans p53-like protein 1, and apoptosis enhancer 1 genes. The lead-, chromium-, and arsenite-exposed nematode, on the other hand, showed little change in gene expression. Alterations in growth and reproduction were observed in cadmium- and chromium-exposed worms. To consider a transgenic nematode as a biosensor for toxicity monitoring, the responses of stress-related gene promoters need to be tested with a variety of metals. The overall results suggest that cadmium exhibits a high level of tolerance compared to the other metals tested. Use of the responses of stress-related gene expression therefore has considerable potential as a sensitive biomarker for the diagnosis of cadmium contamination, and C. elegans seems to be a good biological model for this approach.