Randomized phase II study of loratadine for the prevention of bone pain caused by pegfilgrastim.

PURPOSE: Bone pain is a common side effect of pegfilgrastim and can interfere with quality of life and treatment adherence. This study investigated the impact of antihistamine prophylaxis on pegfilgrastim-induced bone pain. METHODS: This is a two-stage enrichment trial design. Patients receiving an initial dose of pegfilgrastim after chemotherapy were enrolled into the observation (OBS) stage. Those who developed significant back or leg bone pain (SP) were enrolled into the treatment (TRT) stage and randomized to daily loratadine 10 mg or placebo for 7 days. SP was defined by Brief Pain Inventory as back or leg pain score ≥5 and a 2-point increase after pegfilgrastim. The primary end point of TRT was reduction of worst back or leg bone pain with loratadine, defined as a 2-point decrease after treatment compared to OBS. RESULTS: Two hundred thirteen patients were included in the final analysis. Incidence of SP was 30.5 %. The SP subset had a worse overall Functional Assessment of Cancer Therapy-Bone Pain score (33.9 vs. 51.7, p < 0.001) and a higher mean white blood cell count (15.4 vs. 8.4 K/cm(3), p = 0.013) following pegfilgrastim than those without SP. Forty-six patients were randomized in the TRT. Benefit was 77.3 % with loratadine and 62.5 % with placebo (p = 0.35). Baseline NSAID use was documented in four patients (18.2 %) in loratadine arm and two patients (8.3 %) in placebo arm, with baseline non-NSAID use documented in five (22.7 %) and six (25 %) patients, respectively. Eight additional patients used NSAIDS by day 8 compared to day 1 (six in the loratadine and two in the placebo arm). A total of six additional patients used non-NSAIDS by day 8 compared to day 1 (four in the loratadine and two in the placebo arm). CONCLUSIONS: Administration of prophylactic loratadine does not decrease the incidence of severe bone pain or improve quality of life in a high-risk patient population. ClinicalTrials.gov identifier: NCT01311336.

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.

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).

Pulmonary metallothionein expression in rats following single and repeated exposure to cadmium aerosols.

This study examined the expression of metallothionein (MT) isoforms in the lungs of Lewis rats exposed to Cadmium (Cd) aerosols. With the use of isoform-specific oligonucleotide probes and Northern hybridization analysis, we demonstrated that a dramatic, rapid, and coordinate increase occurred in pulmonary MT-1 mRNA and MT-2 mRNA following Cd inhalation exposure. MT mRNAs levels reached a maximum at 2 h post-exposure and remained above control levels at 96 h after exposure. A considerable lag between the time of maximal elevations in MT mRNAs and MT protein accumulation was observed and suggested that regulatory mechanisms in addition to transcriptional control could be involved. MT expression (protein and mRNA) and Cd lung burden were directly related to aerosol Cd concentration. In situ hybridization and immunohistochemistry studies showed good correlation between the localization of MT protein and MT mRNAs. However, staining for MT protein and MT mRNA was not uniformly distributed in the lung. MT was particularly prominent within the alveolar compartment. Even within this area, however, heterogeneity of MT expression was evident. Experiments were subsequently conducted to determine whether prior exposure to Cd modulates the transcriptional activity of MT genes such that there is a greater elevation in gene expression upon reexposure to Cd. Surprisingly, animals pretreated with Cd exhibited a smaller incremental increase in MT mRNA levels in response to subsequent Cd exposure than controls with no prior treatment. Moreover, MT mRNA levels were elevated to a similar extent regardless of whether animals were exposed to Cd aerosols for 1, 2, or 3 weeks (3 h/day; 5 days/week). MT protein and lung Cd burden, on the other hand, exhibited an increasing linear trend as a function of exposure number. In summary, this study has demonstrated that: (1) the lung responds to Cd inhalation exposure by increasing MT mRNA and MT protein levels; (2) MT expression is prominent within alveolar cells but not all cells are MT positive; and (3) Cd-pretreatment does not increase the transcriptional potential of MT genes when the animal is subsequently reexposed to Cd.

Increased oxidant resistance of alveolar macrophages isolated from rats repeatedly exposed to cadmium aerosols.

This study investigated potential mechanisms of oxidant resistance in alveolar macrophages (AM) isolated from Lewis rats exposed repeatedly to cadmium aerosols. Macrophages from Cd-adapted animals significantly greater resistance to oxidant-induced cytotoxicity than control cells when challenged with hydrogen peroxide in vitro. Elevations in glutathione peroxidase and glutathione reductase activities were associated with increased oxidant tolerance but catalase activity was unchanged. Metallothionein (MT) expression (protein and mRNA) was dramatically up-regulated in response to in vivo Cd exposure. A study using immunocytochemistry and in situ hybridization techniques revealed significantly heterogeneity in the expression of metallothionein by AMs. The percentage of AMs positive for MT (protein and mRNA) and the degree of MT expression within individual cells increased in response to additional Cd exposures. A putative state of activation was suggested by differences in size and number of inclusion bodies in macrophages from Cd-adapted animals and by secretion of a cytokine with interleukin-1-like characteristics. In summary, AMs from Cd-adapted animals are distinguished from control cells with respect to: (1) increased oxidant resistance, (2) secretion of cytokines, (3) elevations in enzymes associated with glutathione metabolism, and (4) up-regulation in metallothionein expression.

Increased oxidant resistance of alveolar epithelial type II cells. Isolated from rats following repeated exposure to cadmium aerosols.

Alveolar epithelial type II cells (AEIIC) were isolated from male Lewis rats following repeated in vivo cadmium aerosol exposure and were subsequently evaluated for their oxidant resistance in vitro. AEIIC from Cd-adapted animals removed a greater proportion of hydrogen peroxide from the extracellular milieu and incurred less oxidant-induced cytotoxicity than AEIIC from air controls. This altered response to oxidants occurred coincident with changes in cellular resistance factors. A two-fold increase in glutathione peroxidase activity and a 1.5-fold increase in the activities of glutathione reductase and catalase were observed in Cd-adapted AEIIC compared to control cells. These cells also exhibited a dramatic induction of metallothionein (MT), a thiol-rich protein known to scavenge free radicals in vitro. MT concentration increased as a function of exposure number. MT was localized within the nucleus and cytoplasm of AEIIC by immunocytochemical techniques. MT positive cells showed a wide variation of MT content, particularly in the nucleus. The biochemical and physiological features of these AEIIC may explain, in part, why animals pretreated with Cd aerosols develop cross-tolerance to hyperoxia.

In vivo expression of metallothionein in rat alveolar macrophages and type II epithelial cells following repeated cadmium aerosol exposures.

This study examined the ability of alveolar macrophages and alveolar type II epithelial cells to accumulate cadmium (Cd) and to express metallothionein (MT). Lung cells were isolated from Lewis rats repeatedly exposed to a Cd aerosol (1.6 mg Cd/m3). Intracellular Cd concentration rose following Cd exposure and showed an increasing rend as a function of exposure number. Alveolar macrophages accrued approximately four times more Cd than type II epithelial cells similarly exposed. Macrophages and type II cells responded to the presence of intracellular Cd by increasing MT protein levels. MT concentration was highly correlated with intracellular Cd. Immunocytochemical studies revealed that not all macrophages and type II cells from Cd-exposed animals were immunopositive for MT and that the intensity of immunostaining varied within each cell population. Although a greater percentage of macrophages were immunopositive for MT than type II cells, a greater proportion of type II cells showed moderate and dark MT staining patterns. Oligonucleotide probes, shown to distinguish between MT-1 and MT-2 mRNA isoforms, were used to test for cell-specific differences in MT isoform gene expression. The basal level of MT-1 mRNA was greater in macrophages than in type II cells. Following Cd administration, the level of MT-1 mRNA and MT-2 mRNA increased in each cell class but the response to Cd was three times greater in alveolar macrophages. Neither macrophages nor type II cells expressed MT mRNA isoforms in equal proportions. Macrophages expressed more MT-1 mRNA when exposed to air and more MT-2 mRNA in response to Cd exposure. Type II cells, on the other hand, expressed more MT-2 mRNA than MT-1 mRNA regardless of whether the cells were exposed to air or Cd. In conclusion, the present study has demonstrated that (1) alveolar macrophages and type II cells respond to in vivo Cd exposure by increasing MT protein and mRNA levels; (2) MT expression is greater in macrophages than in type II cells and correlates well with intracellular Cd concentration; and (3) the MT-2 mRNA to MT-1 mRNA ratio is cell and treatment specific.

Oxidant resistance of cadmium-adapted human lung fibroblasts.

Metallothionein (MT) is a metal and thiol-rich protein readily induced by cadmium (Cd) exposure. In vitro experiments have demonstrated that MT is able to serve as a scavenger of hydroxyl radicals as well as superoxide anions, albeit to a lesser extent. The role of MT as a mediator in Cd induced oxidant resistance was investigated in a nontransformed human lung fibroblast cell line (IMR-90). Cells were passaged three times either in a Cd-containing medium (8.9 microM CdCl2) or in a medium which lacked Cd. Cellular MT content, as quantitated by a modification of the heme/109Cd binding assay, increased significantly with each passage in Cd. Immunocytochemistry studies revealed that all Cd-pretreated cells contained MT and that MT was localized in both cytoplasmic and nuclear compartments. Immunolabeling was more intense in some cells compared to others. Very slight immunolabeling was observed in physiological control cells, grown in the absence of Cd, and virtually no staining was observed in Cd-adapted or non-adapted cells when primary antibody was omitted. Using the xanthine/xanthine oxidase system as a generating system for active oxygen species, we found that the magnitude of cell injury for Cd-adapted and non-adapted fibroblasts was dependent upon oxidant concentration and duration of oxidant exposure. Cd-adapted fibroblasts, which were characterized by over-expression of MT, were significantly more resistant to injury by active oxygen species and also exhibited a greater ability to scavenge extracellular hydrogen peroxide compared to cells with no previous history of Cd exposure. Experiments with aminotriazole demonstrated that catalase was not a major contributor to the additional hydrogen peroxide scavenging capacity of Cd-adapted cells. The data presented in this report are consistent with involvement of MT in protecting critical cellular targets from reactive oxygen species.