B.A. Hart

Characterization of cadmium-induced apoptosis in rat lung epithelial cells : evidence for the participation of oxidant stress

The mode of cadmium-induced cell death was investigated in a rat lung epithelial cell line. Cells, grown to near confluence, were exposed to 0-30 microM CdCl2 for 0-72 h. Phase contrast microscopy and fluorescent nuclear staining showed that Cd caused morphological alterations in lung epithelial cells that are characteristic of apoptosis. These changes included cell shrinkage, detachment of the cell from its neighbors, cytoplasmic and chromatin condensation, and fragmentation of the nucleus into multiple chromatin bodies surrounded by remnants of the nuclear envelope. Apoptotic DNA degradation was validated and quantitated using a sensitive enzyme-linked immunosorbent assay (ELISA) which measures the amount of histone-bound DNA fragments in the cytosol. Using this technique, a maximum level of apoptosis (5-fold higher than control) was observed in cultures exposed for 48 h to 20 microM CdCl2. The terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling method (TUNEL) was subsequently used to determine the percentage of cells that contained Cd-induced DNA strand breaks. After 48 h, approximately 54% of the cells exposed to 20 microM Cd were TUNEL positive compared to less than 2% for control cells. Although the mechanisms by which Cd initiates apoptosis in these cells are presently not known, reactive oxygen species are likely to play a role. This possibility is supported by the finding that the first morphological features indicative of apoptosis were preceded by the up-regulation of oxidant stress genes (glutathione S-transferase-alpha, gamma-glutamylcysteine synthetase, and metallothionein-1), activation of redox sensitive transcription factors (AP-1 and NF-kappaB), and changes in various forms of glutathione (reduced, oxidized, and protein-bound).

Toxicity and bioaccumulation of cadmium in Chlorella pyrenoidosa

Abstract Chlorella pyrenoidosa cultures grown at pH 7 in the presence of 0, 0.25, 0.50, and 1.00 mg of Cd/liter had doubling times of 11, 21, 22, and 35 hours, respectively, whereas similarly exposed cultures grown at pH 8 had doubling times of 11, 16, 17, and 25 hours, respectively. C. pyrenoidosa is capable of concentrating cadmium, and the amount accumulated is directly proportional to the concentration of metal present initially and is dependent upon the pH of the medium. No accumulation occurs in the dark, at 4°C, or in dead cells. Cadmium accumulation is not affected by the concentration of calcium, magnesium, molybdenum, copper, zinc, or cobalt in the growth medium, whereas a level of manganese equal to 0.20 mg/liter completely blocks cadmium accumulation; iron may also play a role in regulating cadmium accumulation. Cells which had accumulated cadmium could still fix atmospheric CO2, albeit at reduced rates; O2 evolution was also inhibited, but to a lesser extent. The ability of C. pyrenoidosa to accumulate large concentrations of cadmium before showing adverse effects may be related to the presence of cadmium-sequestering agent(s) within the cell. The concentration of cadmium by C. pyrenoidosa could pose a hazard to the freshwater food chain.

Suppressed oxidant-induced apoptosis in cadmium adapted alveolar epithelial cells and its potential involvement in cadmium carcinogenesis.

Apoptosis involves a series of genetically programmed events associated with endonucleolytic cleavage of DNA. This process is triggered by a variety of agents, including oxidants such as hydrogen peroxide (H(2)O(2)) and it plays a key role in eliminating pre-neoplastic cells from the lung. Failure to do so could favor tumor promotion. The current study demonstrated that alveolar epithelial cells, adapted to cadmium (CdCl(2)) by repeated in vitro exposure, exhibit lower levels of H(2)O(2)-induced apoptosis than similarly challenged non-adapted cells. An immunologic assay, measuring cytoplasmic histone-associated DNA fragments, indicated maximal apoptosis 24 h after exposure to 400 microM H(2)O(2). Non-adapted cells showed a 13-fold increase in oxidant-induced apoptosis while Cd-adapted cells had only a 4-fold elevation. A terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to assess the percentage of cells with DNA breaks consistent with apoptosis. Cd-adapted and non-adapted cells that were not exposed to H(2)O(2) did not differ in TUNEL positivity. However, after H(2)O(2) treatment, the percentage of TUNEL positive cells was 4-fold higher in non-adapted cultures than in adapted ones. Suppression of oxidant-induced apoptosis is due, in part, to up-regulation in the gene expression of several resistance factors including metallothioneins (MT-1 and MT-2), glutathione S-transferases (GST-alpha and GST-pi), and gamma-glutamylcysteine synthetase catalytic subunit (gamma-GCS). These steady-state mRNA changes, determined by Northern blotting, were accompanied by increased levels of MT and gamma-GCS protein, GST activity, and glutathione (GSH). Suppressed oxidant-induced apoptosis, resulting at least in part from these response modifications, could leave pre-neoplastic or neoplastic cells alive, favor clonal expansion, and ultimately lead to cancer development.

Mechanisms regulating the cadmium-mediated suppression of Sp1 transcription factor activity in alveolar epithelial cells

This study demonstrates that in vitro exposure of adult rat alveolar epithelial cells to CdCl(2) decreases DNA binding activity of specificity protein 1 (Sp1), a zinc-finger transcription factor known to play a key role in eukaryotic gene expression, maintenance of homeostasis, cell cycle control, terminal differentiation, and apoptosis. Suppression of Sp1 function, as assessed by electrophoretic mobility shift assays (EMSAs), is dependent upon cadmium (Cd) dose and duration of exposure. A 45% decrease of Sp1 activity occurs as early as 30 min after Cd addition. By 2 h, Sp1 activity is reduced even further with no loss of cell viability, suggesting that Sp1 inactivation precedes cell death. If Cd is removed from cultures during these early periods of exposure, inhibition of Sp1 binding activity is reversed. Sp1 inactivation does not appear to be a generalized, non-selective response to Cd as other transcription factors are up-regulated under the same conditions. Phosphorylation is involved in Sp1 down-regulation, as evidenced by the finding that alkaline phosphatase treatment of nuclear extracts from cells exposed to Cd for 2 h helps restore Sp1 binding activity. A broad spectrum Protein Kinase C (PKC) inhibitor, GF109203X, substantially reduces the Cd-mediated effect on Sp1 suggesting that a member of the PKC family is required for Sp1 phosphorylation. More prolonged Cd exposure promotes Sp1 degradation with the appearance of cleavage products (40 and 50 kDa), as detected by Western blotting. Changes in the integrity of the Sp1 protein are accompanied by a corresponding decline in cell survival. Cd-induced cell death is substantially attenuated if cells are pretreated with antagonists of PKC activity which implies that a PKC isoform is also a participant in this process.

Effect of thiols on cadmium-induced expression of metallothionein and other oxidant stress genes in rat lung epithelial cells

This study examined cadmium-induced alterations in metallothionein-1 (MT), glutathione-S-transferase Ya (GST), and heme oxygenase-1 (HO) gene expression in an adult rat lung epithelial cell line. Elevations in MT mRNA and HO mRNA occurred as early as 1 h after exposure to a sub-toxic concentration of CdCl(2) (10 microM) whereas GST expression did not increase significantly until 4 h after Cd addition. At t = 8 h, levels of GST, MT, and HO mRNA were elevated 9-fold, 27-fold, and 44-fold, respectively, over basal expression. By 24 h, MT expression was almost back to baseline levels. GST mRNA and HO mRNA were also reduced, compared to 8 h, but to a lesser extent than MT expression. The MT gene was more responsive to low Cd concentrations (5 microM) than the genes for HO or GST whereas HO was induced more than the others at higher Cd doses (10-20 microM). Pro-oxidant conditions play a role in Cd-induced gene expression, as suggested by the rapid decline (15-30 min) in glutathione (GSH), amounting to 25-30% of baseline, that occurred after exposure to 10 microM CdCl(2). This was followed by resynthesis of GSH to a concentration higher than the initial. Depleting GSH by treatment of cells with buthionine sulfoximine (BSO) enhanced Cd-induced expression of MT, GST, and HO whereas thiol supplementation, by treatment with N-acetyl cysteine (NAC), had an attenuating effect. BSO and NAC pretreatment had no effect on basal gene expression or Cd uptake. In summary, this study has shown that: (1) Cd increases MT, GST, and HO gene expression in a time- and dose-dependent fashion: (2) MT gene expression appears to be most sensitive to Cd whereas the HO gene is most inducible at higher Cd concentrations; (3) Cd-induced expression is enhanced by GSH depletion and suppressed by thiol supplementation.

Cadmium transport by Chlorella pyrenoidosa.

Abstract Chlorella pyrenoidosa , incubated at 31°C in a medium containing CdCl 2 concentrations ranging from 0.025 to 0.500 μg/ml, incorporated cadmium in a linear fashion for 60 min. The rate of uptake was dependent upon the exogenous cadmium concentration; 2.98 μ M CdCl 2 was required to half-saturate the transport system. Cd uptake is reduced by lower temperatures and when cultures are not illuminated. Both manganese and iron inhibited cadmium uptake but zinc and cobalt had no effect. The inhibition of cadmium uptake by manganese could be correlated with the concentration of manganese present; kinetic studies showed manganese to be a competitive inhibitor. Similar kinetic studies revealed that Mn transport is competitively inhibited by cadmium. The ability of C. pyrenoidosa to incorporate cadmium and manganese is determined, in large part, by the composition of the growth medium. Cells, grown in a medium containing low levels of Mn, incorporate both Cd and Mn at a faster rate than cells grown in a medium containing high levels of Mn. The rate of Cd and Mn incorporation is also faster in cells that have been previously exposed to Cd. The data support the conclusion that manganese and cadmium share a common transport system in C. pyrenoidosa .

Cadmium adaptation in the lung : a double-edged sword?

This review article discusses the major cellular and molecular responses characterizing pulmonary adaptation to cadmium (Cd) that may ultimately contribute to Cd carcinogenesis. Hallmarks of Cd adaptation include hyperplasia and hypertrophy of type II alveolar epithelial stem cells, an inflammatory response involving polymorphonuclear leukocytes, and the increased gene and protein expression of several resistance factors. The most prominent biochemical change is associated with Cd-induced up-regulation of metallothionein, a cysteine-rich, metal-binding protein that sequesters Cd and also possesses considerable free radical scavenging ability. Increased levels of glutathione (GSH) and induction of enzymes involved with both the synthesis of GSH (gamma-glutamylcysteine synthetase regulatory and catalytic subunits) and its metabolism (GSH S-transferases) also constitute important components of the pulmonary adaptive response. Enhancement of several important cellular defense systems in response to Cd exposure may, at first, appear to be beneficial. However, recent evidence suggests that the Cd-adaptive phenotype could have deleterious consequences and may represent a double-edged sword. It has been discovered that Cd-adapted alveolar epithelial cells have a reduced ability to repair DNA damage due, in part, to the inhibition of two base excision repair enzymes (8-oxoguanine-DNA glycosylase and endonuclease III). Cells with genetic aberrations resulting from unrepaired DNA lesions would normally be removed from the lung by apoptosis. However, another study has demonstrated that apoptotic cell death, following an oxidant challenge, is significantly attenuated in Cd-adapted cells compared to non-adapted counterparts. Suppressed apoptosis could leave pre-neoplastic or neoplastic cells alive, favor their clonal expansion, and ultimately promote tumor development. The presence of superior antioxidant defenses would also be expected to increase the resistance of these tumors to chemotherapeutic agents.

Cadmium-mediated oxidative stress in alveolar epithelial cells induces the expression of γ-glutamylcysteine synthetase catalytic subunit and glutathione S-transferase α and π isoforms: Potential role of activator protein-1

Exposure of rat alveolar epithelial cells to 10 μmol/L CdCl2 causes time-dependent increases in steady-state mRNA levels of the γ-glutamylcysteine synthetase catalytic (heavy) subunit (γ-GCS) and of glutathione S-transferase isoforms (GST-α and GST-π). The expression of γ-GCS was significantly increased as early as 2 h after addition of cadmium. Maximal induction of γ-GCS mRNA (∼4-fold), at 8 h, was subsequently followed by increases in γ-GCS activity/protein and glutathione (GSH) levels. Maximal elevations in GST-π (∼2-fold) and GST-α (∼10-fold) transcripts, at 8 and 24 h, respectively, were also accompanied by enhanced GST activity. Cadmium-induced oxidative stress, assessed by alterations in GSH homeostasis and an accelerated rate of intracellular oxidant production, could constitute early events in the signal transduction pathway mediating these responses. The dimeric transcription factor, activator protein-1 (AP-1), may also play a regulatory role in this process. This association is suggested by transcriptional activation of the immediate-early response genes, c-fos and c-jun, within 15 min after exposure to cadmium and by the enhancement of AP-1 DNA binding activity, involving a c-Jun protein complex, which is maximally induced (∼4-fold) by 2 h. These molecular changes likely function together to protect alveolar epithelial cells against cadmium toxicity.

Cellular and biochemical response of the rat lung to repeated inhalation of cadmium

Male Lewis rats were exposed from 1 to 6 weeks (3 hr/day, 5 days/week) to a Cd aerosol (1.6 mg Cd/m3). After the first week, there were significant elevations in airway amounts of lactic dehydrogenase, alkaline and acid phosphatase, protein, and polymorphonuclear leucocytes. After 2 weeks of exposures, airway cytological and biochemical alterations intensified and pulmonary histopathology was observed. The severity of pulmonary injury did not progress beyond this point, although Cd continued to accumulate in the lung in a linear fashion. During the next 3 weeks of exposures, airway alterations diminished and lung histology became normal, suggesting that pulmonary adaptation to Cd might have occurred. Cd-binding proteins, with properties similar to hepatic metallothionein (MT), were isolated from the lungs of Cd-exposed animals. Pulmonary MT quantities increased significantly with repeated exposure to Cd. Sequestration of Cd by MT may be involved in the partial resolution of the lung injury. Translocation of Cd to the liver and kidney also occurred following inhalation exposure. Prior Cd inhalation exposure increased Cd translocation to the kidney, but not to the liver. Liver and kidney Cd burdens increased during the 6 weeks of Cd exposure. MT values also rose but hepatic MT quantities increased faster and to a greater extent than renal MT quantities.

Inhibition of oxidative DNA repair in cadmium-adapted alveolar epithelial cells and the potential involvement of metallothionein.

This study evaluated the effects of cadmium (Cd) adaptation in cultured alveolar epithelial cells on oxidant-induced DNA damage and its subsequent repair. Using the comet assay, we determined that lower levels of DNA damage occurred in Cd-adapted cells compared with non-adapted cells following treatment of cells with hydrogen peroxide (H(2)O(2)). This may be a consequence of increased thiol-containing antioxidants that were observed in adapted cells, including metallothionein and glutathione. Cd-adapted cells were, however, less efficient at repairing total oxidative DNA damage compared with non-adapted cells. Subsequently, we investigated the effect of Cd adaptation on the repair of particular oxidized DNA lesions by employing lesion-specific enzymes in the comet assay, namely formamidopyrimidine DNA glycosylase (Fpg), an enzyme that predominantly repairs 8-oxoguanine (8-oxoG), and endonuclease III, that is capable of repairing oxidized pyrimidines. The data demonstrated that adaptation to Cd results in significantly impaired repair of both Fpg- and endonuclease III-sensitive lesions. In addition, in situ detection of 8-oxoG using a recombinant monoclonal antibody showed that Cd-adaptation reduces the repair of this oxidative lesion after exposure of cells to H(2)O(2). Activities of 8-oxoG-DNA glycosylase and endonuclease III were determined in whole cell extracts using 32P-labeled synthetic oligonucleotides containing 8-oxoG and dihydrouracil sites, respectively. Cd adaptation was associated with an inhibition of 8-oxoG-DNA glycosylase and endonuclease III enzyme activity compared with non-adapted cells. In summary, this study has shown that Cd adaptation: (1) reduces oxidant-induced DNA damage; (2) increases the levels of key intracellular antioxidants; (3) inhibits the repair of oxidative DNA damage.