The clastogenic effects of chronic exposure to particulate and soluble Cr(VI) in human lung cells

Hexavalent chromium (Cr(VI)) is a well-designated human lung carcinogen, with solubility playing an important role in its carcinogenic potential. Although it is known that particulate or water-insoluble Cr(VI) compounds are more potent than the soluble species of this metal, the mechanisms of action are not fully elucidated. In this study, we investigated the hypothesis that the difference in potency between particulate and soluble Cr(VI) is due to more chronic exposures with particulate chromate because it can deposit and persist in the lungs while soluble chromate is rapidly cleared. Chronic exposure to both insoluble lead chromate and soluble sodium chromate induced a concentration and time-dependent increase in intracellular Cr ion concentrations in cultured human lung fibroblasts. Intracellular Pb levels after chronic exposure to lead chromate increased in a concentration-dependent manner but did not increase with longer exposure times up to 72 h. We also investigated the effects of chronic exposure to Cr(VI) on clastogenicity and found that chronic exposure to lead chromate induces persistent or increasing chromosome damage. Specifically, exposure to 0.5 μg/cm² lead chromate for 24, 48 and 72 h induced 23, 23 and 27% damaged metaphases, respectively. Contrary to lead chromate, the amount of chromosome damage after chronic exposure to sodium chromate decreased with time. For example, cells exposed to 1 μM sodium chromate for 24, 48 and 72 h induced 23, 13 and 17% damaged metaphases, respectively. Our data suggest a possible mechanism for the observed potency difference between soluble and insoluble Cr(VI) compounds is that chronic exposure to particulate Cr(VI) induces persistent chromosome damage and chromosome instability while chromosome damage is repaired with chronic exposure to soluble Cr(VI).     

The cytotoxicity and genotoxicity of hexavalent chromium in Steller sea lion lung fibroblasts compared to human lung fibroblasts

In this study we directly compared soluble and particulate chromate cytotoxicity and genotoxicity in human (Homo sapiens) and sea lion (Eumetopias jubatus) lung fibroblasts. Our results show that hexavalent chromium induces increased cell death and chromosome damage in both human and sea lion cells with increasing intracellular chromium ion levels. The data further indicate that both sodium chromate and lead chromate are less cytotoxic and genotoxic to sea lion cells than human cells, based on an administered dose. Differences in chromium ion uptake explained some but not all of the reduced amounts of sodium chromate-induced cell death. By contrast, uptake differences could explain the differences in sodium chromate-induced chromosome damage and particulate chromate-induced toxicity. Altogether they indicate that while hexavalent chromium induces similar toxic effects in sea lion and human cells, there are different mechanisms underlying the toxic outcomes.     

The effects of hexavalent chromium on thioredoxin reductase and peroxiredoxins in human bronchial epithelial cells

Inhalational exposure to hexavalent chromium (Cr(VI)) compounds (e.g., chromates) is of concern in many Cr-related industries and their surrounding environments. The bronchial epithelium is directly exposed to inhaled Cr(VI). Cr(VI) species gain easy access inside cells, where they are reduced to reactive Cr species, which may also contribute to the generation of reactive oxygen species. The thioredoxin (Trx) system promotes cell survival and has a major role in maintaining intracellular thiol redox balance. Previous studies with normal human bronchial epithelial cells (BEAS-2B) demonstrated that chromates cause dose- and time-dependent oxidation of Trx1 and Trx2. The Trx’s keep many intracellular proteins reduced, including the peroxiredoxins (Prx’s). Prx1 (cytosolic) and Prx3 (mitochondrial) were oxidized by Cr(VI) treatments that oxidized all, or nearly all, of the respective Trx’s. Prx oxidation is therefore probably the result of a lack of reducing equivalents from Trx. Trx reductases (TrxR’s) keep the Trx’s largely in the reduced state. Cr(VI) caused pronounced inhibition of TrxR, but the levels of TrxR protein remained unchanged. The inhibition of TrxR was not reversed by removal of residual Cr(VI) or by NADPH, the endogenous electron donor for TrxR. In contrast, the oxidation of Trx1, Trx2, and Prx3 was reversible by disulfide reductants. Prolonged inhibition of TrxR in Cr(VI)-treated cells might contribute to the sustained oxidation of Trx’s and Prx’s. Reduced Trx binds to an N-terminal domain of apoptosis signaling kinase (ASK1), keeping ASK1 inactive. Cr(VI) treatments that significantly oxidized Trx1 resulted in pronounced dissociation of Trx1 from ASK1. Overall, the effects of Cr(VI) on the redox state and function of the Trx’s, Prx’s, and TrxR in the bronchial epithelium could have important implications for redox-sensitive cell signaling and tolerance of oxidant insults.     

Reductive activation of hexavalent chromium by human lung epithelial cells: generation of Cr(V) and Cr(V)-thiol species

Chromium(VI) compounds (e.g. chromates) are cytotoxic, mutagenic, and potentially carcinogenic. The reduction of Cr(VI) can yield reactive intermediates such as Cr(V) and reactive oxygen species. Bronchial epithelial cells are the primary site of pulmonary exposure to inhaled Cr(VI) and are the primary cells from which Cr(VI)-associated human cancers arise. BEAS-2B cells were used here as a model of normal human bronchial epithelium for studies on the reductive activation of Cr(VI). Cells incubated with Na₂CrO₄ exhibited two Cr(V) ESR signals, g = 1.979 and 1.985, which persisted for at least 1 h. The g = 1.979 signal is similar to that generated in vitro by human microsomes and by proteoliposomes containing P450 reductase and cytochrome b₅. Unlike many cells in culture, these cells continued to express P450 reductase and cytochrome b₅. Studies with the non-selective thiol oxidant diamide indicated that the g = 1.985 signal was thiol-dependent whereas the g = 1.979 signal was not. Pretreatment with phenazine methosulfate eliminated both Cr(V) signals suggesting that Cr(V) generation is largely NAD(P)H-dependent. ESR spectra indicated that a portion of the Cr(VI) was rapidly reduced to Cr(III). Cells incubated with an insoluble chromate, ZnCrO₄, also generated both Cr(V) signals, whereas Cr(V) was not detected with insoluble PbCrO₄. In clonogenic assays, the cells were very sensitive to Na₂CrO₄ and ZnCrO₄, but considerably less sensitive to PbCrO₄.     

Proteomic analysis of chromium cytotoxicity in cultured rat lung epithelial cells

Chromium (Cr) has been widely used in industry for more than one century. Exposure to hexavalent Cr compounds is strongly associated with increasing risk of lung cancer. Extensive researches at DNA level indicated that generation of ROS from the reduction of Cr(VI) leading to DNA damage is the major cause of the toxicity and carcinogenicity of Cr(VI). The present study in cellular and protein levels confirmed that Cr(VI) induced apoptosis of lung epithelial cells (LEC) via ROS generation. To view the differentially expressed proteins in the process of Cr(VI) reduction, subcellular proteomics was applied and allowed the identification of more than 30 proteins with expression alteration. Most of those proteins are correlated with ROS-elicited responses, which were further validated by Western blotting analysis, induction of p53 pathway and antioxidative treatment. The current findings provided additional evidence in protein level to support the claim that ROS generated during the process of Cr(VI) reduction are involved in the Cr(VI)-induced toxicity and carcinogenesis.     

The cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human lung cells

Hexavalent chromium (Cr(VI)) is a human lung carcinogen. Cr(VI) is a particularly important and dangerous carcinogen, because there is widespread exposure to it both occupationally and to the general public. However, despite the potential for widespread exposure and the fact that the lung is its target organ, there are few reports of the genotoxicity of Cr(VI) in human lung cells. Clearly, in order to better understand this carcinogen, its effects in its target cells need to be evaluated. Accordingly, we determined the cytotoxicity and clastogenicity of both particulate (water-insoluble) and soluble Cr(VI) in primary human bronchial fibroblasts (PHBFs). We used lead chromate (PbCrO(4)) and sodium chromate (Na(2)CrO(4)) as prototypical particulate and soluble Cr(VI) salts, respectively. Both compounds induced concentration-dependent cytotoxicity after a 24h exposure in PHBFs. The relative survival was 87, 46, 26 and 2% after exposure to 0.1, 0.5, 1 and 5 microg/cm(2) PbCrO(4), respectively, and 74, 57, 13 and 0% after exposure to 1, 2.5, 5 and 10 microM Na(2)CrO(4), respectively. Similarly, the amount of chromosome damage increased with concentration after 24h exposure to both compounds. Specifically, 0.1, 0.5 and 1 microg/cm(2) PbCrO(4) damaged 15, 34 and 42% of metaphase cells with the total amount of damage reaching 18, 40 and 66 aberrations per 100 metaphases, respectively. PbCrO(4) (5 microg/cm(2)) induced such profound cell cycle delay that no metaphases were found. Na(2)CrO(4) (1 and 2.5 microM) damaged 18 and 33% of metaphase cells with the total amount of damage reaching 19 and 43 aberrations per 100 metaphases, respectively. Na(2)CrO(4) (5 and 10 microM) induced such profound cell cycle delay that no metaphases were found. Overall the data clearly indicate that Cr(VI) compounds are cytotoxic and genotoxic to human lung cells.     

Prolonged exposure to particulate Cr(VI) is cytotoxic and genotoxic to fin whale cells

BackgroundHexavalent chromium [Cr(VI)] is a human lung carcinogen and global marine pollutant. High Cr concentrations, resembling the ones observed in occupationally exposed workers, have been observed in fin whales (Balaenoptera physalus) in the Gulf of Maine. This outcome suggests Cr might be disrupting the health of fin whale populations. Indeed, Cr in acute (24 h) exposure does cause toxicity in fin whale cells. However, human cell culture data indicate prolonged exposures (120 h) induce a higher amount of toxicity compared to 24 h exposure due to an inhibition of homologous recombination repair. However, whether prolonged exposure causes similar outcomes in fin whale cells is unknown.ObjectiveDue to the importance of assessing prolonged exposure toxicity, this study focuses on characterizing acute and prolonged exposure of Cr(VI) in male and female fin whale cells.MethodsCytotoxicity was measured by the clonogenic assay, also known as colony forming assay, which measures the ability of cells to proliferate and form colonies after the treatment. DNA double strand breaks were analyzed by neutral comet assay. Clastogenicity was measured using the chromosome aberration assay. Intracellular Cr levels were measured with Graphite Furnace Atomic Absorption Spectrometry (GFAAS) with Syngistix Software.ResultsIn this study, we demonstrate that particulate Cr(VI) induces cytotoxicity and genotoxicity in a treatment-dependent manner after 24 h and 120 h exposures. Cytotoxicity levels were generally low with relative survival above 64 %. DNA double strand break data and chromosome aberration data were elevated after a 24 h exposure, but decreased after a 120 h exposure. While cytotoxicity was similar after 24 h and 120 h exposures, less DNA double strand breaks and chromosomal instability occurred with prolonged exposure.ConclusionParticulate Cr(VI) is cytotoxic and genotoxic to fin whale cells after acute and prolonged exposures. The reduction of genotoxicity we have observed after 120 h exposure may be partly explained by lower intracellular Cr levels after 120 h. However, the decrease in intracellular levels is not reflected by a similar decrease in chromosome aberrations suggesting other mechanisms may be at play. Male fin whale cells appear to be more susceptible to the genotoxic effects of particulate Cr(VI) while female cells are less susceptible possibly due to increased cell death of damaged cells, but more work is needed to clarify if this outcome reflects a sex difference or interindividual variability. Overall, the study shows particulate Cr(VI) does induce toxicity at both acute and prolonged exposures in fin whales cells indicating Cr(VI) exposure is a health risk for this species.     

Cytotoxicity and genome-wide microarray analysis of intestinal smooth muscle cells in response to hexavalent chromium induction

Chronic ingestion of high concentrations of hexavalent chromium Cr(VI) in drinking water induces intestinal tumors in mice; however, information on its toxicity on intestinal smooth muscle cells is limited. The present study aimed to assess the in vitro and in vivo toxicological effects of Cr(VI) on intestinal smooth muscle cells. Human intestinal smooth muscle cells (HISM cells) were cultured with different concentrations of Cr(VI) to evaluate effects on cell proliferation ability, oxidative stress levels, and antioxidant system. Furthermore, tissue sections in Cr(VI) exposed rabbits were analyzed to evaluate toxicity on intestinal muscle cells in vivo. Gene chips were utilized to assess differential gene expression profiles at the genome-wide level in 1 μmol/L Cr(VI) treated cells. Intestinal tissue biopsy results showed that Cr(VI) increased the incidences of diffuse epithelial hyperplasia in intestinal jejunum but caused no obvious damage to the structure of the muscularis. Cell proliferation analysis revealed that high concentrations (≥64 μmol/L) but not low concentrations of Cr(VI) (≤16 μmol/L) significantly inhibited the growth of HISM cells. For oxidative stress levels, the expression of reactive oxygen species (ROS) and nitric oxide (NO) was elevated at high concentrations (≥64 μmol/L) but not at low concentrations of Cr(VI) (≤16 μmol/L). In addition, dose-dependent increases in the activity of oxidized glutathione (GSSH)/total-glutathione (T-GSH) were also observed. Gene chip screened 491 differentially expressed genes including genes associated with cell apoptosis, oxidations, and cytoskeletons. Some of these differentially expressed genes may be unique to smooth muscle cells in response to Cr(VI) induction.     

Transformed human bronchial epithelial cells (beas-2b) alter the growth and morphology of normal human bronchial epithelial cells in vitro

Normal human bronchial epithelial cells (BE) and adenovirus-12 SV40 hybrid virus transformed, non-tumorigenic human bronchial epithelial cells (BEAS-2B) were cultured for 7 days in a serum free hormone supplemented medium. BE cells after 3 days in culture were exposed to conditioned medium (CM_t) from confluent BEAS-2B cells. By day 7, CM_t-treated BE cells exhibited a lower colony forming efficiency (CFE), fewer cells per colony, and a reduced mitotic index (MI) and BrdU (bromodeoxyuridine) labeling index. CM_t also enhanced the expression of a terminally differentiated squamous phenotype in BE cells. Cell free lysates from BEAS-2B cells (CFL_t) had effects similar to CM_t on the MI and morphology of BE cells. In contrast, CM_t and CFL_t did not inhibit the growth, or alter the morphology of BEAS-2B cells. Conditioned medium from BE cells (CM_n) did not reduce the growth of BEAS-2B cells, and had little effect on the morphology of BE cells. In co-culturesAbbreviations BE normal bronchial epithelial cells - BEAS-2B adenovirus-12 SV40 hybrid virus transformed bronchial epithelial cells - CM_n conditioned medium from BE cells - CM_t conditioned medium from BEAS-2B cells - CF_n cell free lysate from BE cells - CFL_t cell free lysate from BEAS-2B cells - BrdU bromodeoxyuridine - KGM keratinocyte growth medium - TGF- transforming growth factor type - NCI-LHC National Cancer Institute-Laboratory of Human Carcinogenesis Contribution No. 2801 from the Pathobiology Laboratory, University of Maryland.     

Various cells of the immune system and intestine differ in their capacity to reduce hexavalent chromium

The cells of the immune system form a strong line of defence against foreign substances. The present study was undertaken to investigate the capacity of different cells of Wistar rats to reduce potentially carcinogenic hexavalent chromium (Cr-VI) into less toxic trivalent chromium in vitro. 5 x 10(6) cells were incubated with 10 or 25 microg ml(-1) of Cr (VI) in the form of K2Cr2O7 at 37 degrees C in the presence of 5% CO2 in air. At various time periods the remaining amount of Cr (VI) was measured and the percentage of Cr (VI) reduced was calculated. Among the single cell suspensions from the splenic cells a peak reduction of 55% was observed with the total spleen cells, 40% with the B-lymphocyte-enriched subpopulation, 10% with T-lymphocytes and 24% with the macrophages. The reduction by splenic and peritoneal macrophages was similar. Total thymocytes reduced 54% of the Cr (VI). Since the most common route of entry of chromium is through drinking water and food, intestinal cells were also investigated. Among the intestinal cells the maximum reduction of 100% (of 10 microg ml(-1)) was observed with the upper villus cells and 72% with the middle villus cells while reduction was the least (4%) with the crypt cells. The reduction in the intestinal loop in situ was 100%. The time taken by each cell type for the peak reduction to Cr (VI) was markedly different. The findings thus show that the capacity of different cells in the body differs vastly in their capacity and time taken to reduce hexavalent chromium. The most efficient handling of Cr (VI) by the intestine, due to the presence of a variety of cells and bacteria, protects the body from its adverse effects.     

Fatty acid lithium salts from Cunninghamella echinulata have cytotoxic and genotoxic effects on HL‐60 human leukemia cells

Polyunsaturated fatty acids, especially gamma linolenic acid (GLA), are potentially useful agents in the treatment of cancer. Cunninghamella echinulata, a fungus species that is able to synthesize GLA, when cultivated under nitrogen‐limited conditions in a medium having glucose as carbon and energy source, accumulated 32–35% of lipids containing 11–18% GLA. The conversion yield of glucose to lipid was around 0.11 g per gram of glucose consumed while the lipid production was 5 g/L. Fatty acid lithium salts (FALS) were prepared from the total Cunninghamella lipids and studied for their effects on HL‐60 human leukemic cells. Cytotoxicity of FALS on HL‐60 leukemic cells was linearly related to the FALS concentration. High FALS concentration (i.e. 15 and 20 μg/mL) induced DNA fragmentation, while concurrent treatment of cells with H₂O₂ (at 100 μM) and FALS resulted in enhanced cytotoxicity of H₂O₂. However, when FALS were employed at low concentrations (i.e. 5 and 10 μg/mL), they demonstrated a protective effect on HL‐60 cells against H₂O₂ genotoxicity, whereas at 20 μg/mL FALS enhanced the ability of H₂O₂ to induce DNA fragmentation. It is concluded that FALS derived from C. echinulata lipids could be an effective preparation against HL‐60 human leukemic cells.     

An in vitro test for measuring cytotoxicity of mercuric chloride to human kidney epithelial cells by specific enzyme release

Mercuric compound toxicity is well documented in animals and man for practically all organs. The recent development of cell culture techniques appeared as a novel fruitful tool in toxicology, especially in renal toxicology. Heavy metal induced renal cell alterations can be evaluated by membrane permeability damages.The present study evaluates mercuric chloride nephrotoxic effect in human kidney epithelial cells by measuring the release of two specific nephrotoxicity marker enzymes, Gamma Glutamyl Transferase (GGT) and Alkaline Phosphatase (ALP) in the culture medium. Cultured kidney epithelial cells were exposed to different HgCl₂ concentrations (5, 10, 20, and 50 g). Cultures were examined after 6 and 24 hours exposure. A good correlation between mercury dose and toxic effect, and exposure time and toxic effect was found. Enzymes were significantly released into the culture medium for 5 g and 10 g HgCl₂/ml after 6 hours exposure; and after 24 hours exposure, enzymes were released for 5 g/ml only.It appears that the specific tubular enzyme release in the culture medium is a good in vitro test for quantification of specific tubular damage.     

Comparative cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human and sperm whale (Physeter macrocephalus) skin cells

Chromium (Cr) is a global marine pollutant, present in marine mammal tissues. Hexavalent chromium [Cr(VI)] is a known human carcinogen. In this study, we compare the cytotoxic and clastogenic effects of Cr(VI) in human (Homo sapiens) and sperm whale (Physeter macrocephalus) skin fibroblasts. Our data show that increasing concentrations of both particulate and soluble Cr(VI) induce increasing amounts of cytotoxicity and clastogenicity in human and sperm whale skin cells. Furthermore, the data show that sperm whale cells are resistant to these effects exhibiting less cytotoxicity and genotoxicity than the human cells. Differences in Cr uptake accounted for some but not all of the differences in particulate and soluble Cr(VI) genotoxicity, although it did explain the differences in particulate Cr(VI) cytotoxicity. Altogether, the data indicate that Cr(VI) is a genotoxic threat to whales, but also suggest that whales have evolved cellular mechanisms to protect them against the genotoxicity of environmental agents such as Cr(VI).     

Gamma-melanocyte stimulating hormone regulates the expression and cellular localization of epithelial sodium channel in inner medullary collecting duct cells

Gamma₂-melanocyte-stimulating hormone (γ₂MSH) is a peptide hormone released by the pituitary gland which is thought to act directly on the renal inner medulla to promote increased sodium excretion into urine (natriuresis). The aim of this study was to determine if a stable analog, [Nle³, D-Phe⁶]-γ₂MSH (NDP-γ₂MSH), of the native peptide regulated the activity, expression and cellular localization of epithelial sodium channel (ENaC) in a murine inner medullary collecting duct (mIMCD-3) cell line. Our results indicate that expression of the γ₂MSH receptor, melanocortin receptor 3 receptor (MC3R), is up-regulated by culturing the cells in media with an increased osmolality (∼400 mOsm/kg). Furthermore, stimulation of cAMP signaling and sodium transport by 1 nM NDP-γ₂MSH occurs only in cells cultured in the high osmolality media. Finally, treatment of mIMCD-3 cells cultured in high osmolality medium for 1 h with 1 nM NDP-γ₂MSH causes a reduction in expression of serum- and glucocorticoid-induced kinase (sgk1) and a reduction in expression and cell surface abundance of the alpha subunit of ENaC. Collectively, this data suggest that γ₂MSH directly regulates both ENaC expression and cellular localization in the inner medulla to exert its natriuretic effect.     

Isolation,culture, and verification of human sweat gland epithelial cells

Human sweat gland epithelial cells (SGECs) have been isolated and grown in vitro, However, slow proliferation makes the culture of these cells extremely difficult. The present study was carried out to explore the modified culture medium for SGECs in vitro. Full-thickness skin samples were minced (1 mm³) and digested overnight with type II collagenase. The gland coils were removed under an inverted phase-contrast microscope. An adherent culture method was used to isolate and culture SGECs. Staining with hematoxylin and eosin was performed, followed by observation of the morphologic features of these cells. Immunofluorescence staining with antibodies to cytokeratins CK7, CK18, and CK19 and carcinoembryonic antigen (CEA) was performed to verify the presence of SGECs. Growth curves by MTT were created for cells grown in serum-free keratinocyte medium and in modified keratinocyte medium containing 2.5% fetal bovine serum (FBS). One week after culturing, the cells grew well and were polygonal or irregular in shape by inverted phase contrast microscopy. Cell fusion, with a characteristic paving-stone arrangement, reached 100% after approximately 3 weeks in culture. Immunofluorescence staining indicated expression of CK7, CK18, CK19, and CEA. Compared with SGECs grown in serum-free keratinocyte medium, the proliferation of SGECs grown in modified culture medium with low concentration of FBS at days 6, 9, and 12 was significantly accelerated (p < 0.05). This study suggests that keratinocyte medium supplemented with 2.5% FBS is effective and suitable for the culture of SGECs.     

Human mesenchymal stem cells are resistant to cytotoxic and genotoxic effects of cisplatin in vitro

Mesenchymal stem cells (MSCs) are known for their important properties involving multilineage differentiation potential., trophic factor secretion and localization along various organs and tissues. On the dark side, MSCs play a distinguished role in tumor microenvironments by differentiating into tumor-associated fibroblasts or supporting tumor growth via distinct mechanisms. Cisplatin (CIS) is a drug widely applied in the treatment of a large number of cancers and is known for its cytotoxic and genotoxic effects, both in vitro and in vivo. Here we assessed the effects of CIS on MSCs and the ovarian cancer cell line OVCAR-3, by MTT and comet assays. Our results demonstrated the resistance of MSCs to cell death and DNA damage induction by CIS, which was not observed when OVCAR-3 cells were exposed to this drug.     

Lysophosphatidic acid increases soluble ST2 expression in mouse lung and human bronchial epithelial cells

Lysophosphatidic acid (LPA), a naturally occurring bioactive lysophospholipid increases the expression of both pro-inflammatory and anti-inflammatory mediators in airway epithelial cells. Soluble ST2 (sST2), an anti-inflammatory mediator, has been known to function as a decoy receptor of interleukin (IL)-33 and attenuates endotoxin-induced inflammatory responses. Here, we show that LPA increased sST2 mRNA expression and protein release in a dose and time dependent manner in human bronchial epithelial cells (HBEpCs). LPA receptors antagonist and Gαi inhibitor, pertussis toxin, attenuated LPA-induced sST2 release. Inhibition of NF-κB or JNK pathway reduced LPA-induced sST2 release. LPA treatment decreased histone deacetylase 3 (HDAC3) expression and enhanced acetylation of histone H3 at lysine 9 that binds to the sST2 promoter region. Furthermore, limitation of intracellular LPA generation by the down-regulation of acetyl glycerol kinase attenuated exogenous LPA-induced histone H3 acetylation on sST2 promoter region, as well as sST2 gene expression. Treatment of HBEpCs with recombinant sST2 protein or sST2-rich cell culture media attenuated endotoxin-induced phosphorylation of PKC and airway epithelial barrier disruption. These results unravel a novel sST2 mediated signaling pathway that has physiological relevance to airway inflammation and remodeling.     

Time-dependent uptake, distribution and biotransformation of chromium(VI) in individual and bulk human lung cells: application of synchrotron radiation techniques

Chromium(VI) is a human carcinogen, primarily affecting the respiratory tract probably via active transport into cells, followed by the reduction to Cr(III) with the formation of DNA-damaging intermediates. Distribution of Cr and endogenous elements within A549 human lung adenocarcinoma epithelial cells, following treatment with Cr(VI) (100 M, 20 min or 4 h) were studied by synchrotron-radiation-induced X-ray emission (SRIXE) of single freeze-dried cells. After the 20-min treatment, Cr was confined to a small area of the cytoplasm and strongly co-localized with S, Cl, K, and Ca. After the 4-h treatment, Cr was distributed throughout the cell, with higher concentrations in the nucleus and the cytoplasmic membrane. This time-dependence corresponded to ~100% or 0% clonogenic survival of the cells following the 20-min or 4-h treatments, respectively, and could potentially be explained by a new cellular protective mechanism. Such processes may also be important in reducing the potential hazards of Cr(III) dietary supplements, for which there is emerging evidence that they exert their anti-diabetic effects via biological oxidation to Cr(VI). The predominance of Cr(III) was confirmed by micro-XANES spectroscopy of intracellular Cr hotspots. X-ray absorption spectroscopy (XANES and EXAFS, using freeze-dried cells after the 0–4-h treatments) was used to gain insight into the chemical structures of Cr(III) complexes formed during the intracellular reduction of Cr(VI). The polynuclear nature of such complexes (probably with a combination of carboxylato and hydroxo bridging groups and O-donor atoms of small peptides or proteins) was established by XAFS data analyses.Electronic Supplementary Material Supplementary material is available for this article at     

Appearance and endocytic activity of epithelial cells from human efferent ducts in primary monolayer culture

Summary The culture of epithelial cells lining human efferent ducts, obtained from prostatic carcinoma patients, is described. Ciliated cells were observed to beat for at least one month on plastic. On pervious filters low cuboidal cells characterized the monolayers. Cells comprising monolayers over the filter were 5 to 9 m in height whereas taller cells were found over the original fragments (14 m). Some non-ciliated cells contained dark and light vacuoles, others were found to lack them. Both non-ciliated and ciliated cells maintained tight junctional complexes restricting the paracellular movement of horseradish peroxidase. Both types of cultured cells exhibited fluid-phase and adsorptive endocytosis from both apical and basal surfaces. It is reported for the first time that the monolayers form high resistance barriers (150 cm²) that prevent the apical medium from draining to the basal compartment over 24 h.     

TWEAK/Fn14 interaction stimulates human bronchial epithelial cells to produce IL-8 and GM-CSF

TNF-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) family, is a multifunctional cytokine that regulates cellular proliferation, angiogenesis, inflammation, and apoptosis. In this study, we investigated the effect of TWEAK on human bronchial epithelial cells. A human bronchial epithelial cell line, BEAS2B, expressed a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14), and produced IL-8 and GM-CSF upon TWEAK stimulation in a dose-dependent manner, which was abrogated by anti-Fn14 blocking antibody. TWEAK induced phosphorylation of IkappaBalpha and BAY11-7082, a selective inhibitor of IkappaBalpha phosphorylation, inhibited the TWEAK-induced IL-8 and GM-CSF production by BEAS2B cells. Moreover, primary cultured human bronchial epithelial cells also expressed Fn14 and produced IL-8 and GM-CSF upon TWEAK stimulation. Collectively, TWEAK stimulated human bronchial epithelial cells to produce IL-8 and GM-CSF through Fn14. Because IL-8 and GM-CSF are associated with inflammatory conditions, these results suggest that TWEAK/Fn14 interaction may play some roles in airway inflammatory responses.