ZnO particulate matter requires cell contact for toxicity in human colon cancer cells.

There is ongoing concern regarding the toxicity of nanoparticles with sizes less than 100 nm as compared to larger particles of the same nominal substance. Two commercial ZnO types, one sold as a 8-10 nm powder and the other described as -325 mesh (<44 mum) powder, were evaluated in human colon-derived RKO cells. The powders had a volume-to-surface area ratio equivalent to 40 and 330 nm spheres, respectively. Both materials formed micrometer-sized agglomerates in cell culture media. The nanosized ZnO was more cytotoxic than the micrometer-sized ZnO with LC(50) values of 15 +/- 1 and 29 +/- 4 mug/cm(2), respectively. Transfer of Zn from the solid phase to the cell culture media in the presence of RKO cells was time- and concentration-dependent. However, direct particle-cell contact was required for RKO cell cytotoxicity, and the toxicity of particles was independent of the amount of soluble Zn in the cell culture media. The mechanism of cell death includes the disruption of mitochondrial function. Robust markers of apoptosis, Annexin V staining, loss of mitochondrial potential, and increased generation of superoxide were observed when cells were treated with ZnO particulate matter but not when treated with comparable concentration of a soluble Zn salt. Both ZnO samples induced similar mechanisms of toxicity, but there was a statistically significant increase in potency per unit mass with the smaller particles.

Effects of cell type and culture media on Interleukin-6 secretion in response to environmental particles.

Cultured lung cells provide an alternative to animal exposures for comparing the effects of different types of air pollution particles. Studies of particulate matter in vitro have reported proinflammatory cytokine signaling in response to many types of environmental particles, but there have been few studies comparing identical treatments in multiple cell types or identical cells with alternative cell culture protocols. We compared soil-derived, diesel, coal fly ash, titanium dioxide, and kaolin particles along with soluble vanadium and lipopolysaccharide, applied to airway-derived cells grown in submerged culture. Cell types included A549, BEAS-2B, RAW 264.7, and primary macrophages. The cell culture models (specific combinations of cell types and culture conditions) were reproducibly different in the cytokine signaling responses to the suite of treatments. Further, Interleukin-6 (IL-6) response to the treatments changed when the same cells, BEAS-2B, were grown in KGM versus LHC-9 media or in media containing bovine serum. The effect of changing media composition was reversible over multiple changes of media type. Other variables tested included culture well size and degree of confluence. The observation that sensitivity of a cell type to environmental agonists can be manipulated by modifying culture conditions suggests a novel approach for studying biochemical mechanisms of particle toxicity.

TOR controls transcriptional and translational programs via Sap-Sit4 protein phosphatase signaling effectors.

The Tor kinases are the targets of the immunosuppressive drug rapamycin and couple nutrient availability to cell growth. In the budding yeast Saccharomyces cerevisiae, the PP2A-related phosphatase Sit4 together with its regulatory subunit Tap42 mediates several Tor signaling events. Sit4 interacts with other potential regulatory proteins known as the Saps. Deletion of the SAP or SIT4 genes confers increased sensitivity to rapamycin and defects in expression of subsets of Tor-regulated genes. Sap155, Sap185, or Sap190 can restore these responses. Strains lacking Sap185 and Sap190 are hypersensitive to rapamycin, and this sensitivity is Gcn2 dependent and correlated with a defect in translation, constitutive eukaryotic initiation factor 2alpha hyperphosphorylation, induction of GCN4 translation, and hypersensitivity to amino acid starvation. We conclude that Tor signals via Sap-Sit4 complexes to control both transcriptional and translational programs that couple cell growth to amino acid availability.

Stromal production of prostacyclin confers an antiapoptotic effect to colonic epithelial cells.

The mechanism whereby cyclooxygenase-2 and its prostaglandin (PG) products are involved in colonic carcinogenesis is not fully understood. Prostacyclin (PGI(2)) is a major PG with antiapoptotic activity and is produced in the gastrointestinal tract. We reported previously that a human colorectal cancer (CRC) cell line, HCA-7, produces significant levels of PGE(2), PGD(2), thromboxane, and PGF(2alpha), but not PGI(2). We now report that human colonic fibroblast cell lines produce significant amounts of PGI(2) and that fibroblast lines derived from normal-appearing colonic mucosa of hereditary nonpolyposis CRC individuals produce 50-fold more PGI(2) than normal fibroblast lines derived from individuals with nonhereditary CRC. Coculture of HCA-7 cells with hereditary nonpolyposis CRC fibroblasts, but not normal fibroblasts, markedly reduced butyrate-induced apoptosis of HCA-7 cells. This antiapoptotic effect was inhibited by the cyclooxygenase-2 inhibitor rofecoxib and was restored by the stable PGI(2) analogue carbaprostacyclin. PGI(2) binds either G protein-coupled cell surface PGI(2) receptor or the nuclear peroxisome proliferator-activated receptor (PPAR) delta. PPAR delta likely mediates this antiapoptotic effect because HCA-7 cells express this receptor, and another PPAR delta agonist, docosahexaenoic acid, mimics the effect. We propose a novel mechanism by which stromal production of PGI(2) promotes survival of colonocytes through PPAR delta activation. This mechanism may have relevance to maintenance of cells in the normal crypt and to clonal expansion of mutant colonocytes during tumorigenesis.

Responses of human cells to ZnO nanoparticles: a gene transcription study.

The gene transcript profile responses to metal oxide nanoparticles was studied using human cell lines derived from the colon and skin tumors. Much of the research on nanoparticle toxicology has focused on models of inhalation and intact skin exposure, and effects of ingestion exposure and application to diseased skin are relatively unknown. Powders of nominally nanosized SiO2, TiO2, ZnO and Fe2O3 were chosen because these substances are widely used in consumer products. The four oxides were evaluated using colon-derived cell lines, RKO and CaCo-2, and ZnO and TiO2 were evaluated further using skin-derived cell lines HaCaT and SK Mel-28. ZnO induced the most notable gene transcription changes, even though this material was applied at the lowest concentration. Nano-sized and conventional ZnO induced similar responses suggesting common mechanisms of action. The results showed neither a non-specific response pattern common to all substances nor synergy of the particles with TNF-α cotreatment. The response to ZnO was not consistent with a pronounced proinflammatory signature, but involved changes in metal metabolism, chaperonin proteins, and protein folding genes. This response was observed in all cell lines when ZnO was in contact with the human cells. When the cells were exposed to soluble Zn, the genes involved in metal metabolism were induced but the genes involved in protein refoldling were unaffected. This provides some of the first data on the effects of commercial metal oxide nanoparticles on human colon-derived and skin-derived cells.

Transcription factor binding to a putative double E-box motif represses CYP3A4 expression in human lung cells.

Two vital enzymes of the CYP3A subfamily, CYP3A4 and CYP3A5, are differentially expressed in the human lung. However, the molecular mechanisms that regulate tissue-selective expression of the genes are poorly understood. The ability of the 5' upstream promoter region of these two genes to drive luciferase reporter activities in human lung A549 cells was dramatically different. The CYP3A5 promoter region activated luciferase gene expression by 10-fold over the promoterless construct, whereas the CYP3A4 promoter did not drive expression. Sequence comparisons of the promoters identified a 57-base pair insertion in the CYP3A4 promoter region (-71 to -127) that was absent in the CYP3A5 promoter. Deletion of the 57-bp motif from CYP3A4 or insertion into the CYP3A5 promoter, showed that this motif represses CYP3A4 expression in lung. EMSA analysis using nuclear extracts from either A549 cells or human lung tissues showed two specific protein/DNA complexes formed with the (32)P-labeled CYP3A4 57-bp oligonucleotide. EMSA analyses identified two E-box motifs as the minimal specific cis-elements. Supershift assays with antibodies directed against known double- or single-E-box binding factors (TAL1, deltaEF1, E2A, HEB, etc.) failed to identify this factor as a previously characterized trans-acting double E-box binding protein. These results demonstrated that the 5'-upstream region of CYP3A4 contains an active putative double E-box repressor motif, not present in the 5'-upstream region of the CYP3A5 gene, that attenuates CYP3A4 expression in the human lung. We believe that this is the first documented case in which a cytochrome P450 gene is actively repressed in a tissue-specific manner.

Molecular mechanisms regulating human CYP4B1 lung-selective expression.

Lung-selective cytochrome P450 expression is well recognized; however, little is known regarding regulatory mechanisms. To address this knowledge gap, transient expression of CYP4B1/luciferase constructs was used to identify a proximal, positively acting regulatory element, position -139 to -45, that functioned in all cells examined; a negatively acting element, position -457 to -216, that only functioned in HepG2 hepatoblastoma cells; and a distal, positively acting element, position -1087 to -1008, that functioned in A549 or BEAS-2B lung-derived cells but not HepG2 cells or 293 kidney-derived cells. Competitive electrophoretic mobility shift assays further localized specific A549, but not HepG2, nuclear protein binding to two sites within the distal element, CYP4B1 position -1052 to -1042 and -1026 to -1008. Several potential lung-selective transcription factor recognition sequences were identified within these elements. However, attempts to identify specific factor(s) were unsuccessful. In contrast, in vitro DNA/protein binding assays combined with transient expression and mutagenesis studies identified two functional Sephadex protein/Krüppel-like factor families of transcription factor sites within the proximal element (position -118 to -114 and position -77 to -73) that bound both Sephadex protein 1 (Sp1) and Sephadex protein 3 (Sp3) in vitro. Furthermore, Sp1-dependent synergistic regulation was observed in A549 cells involving the proximal and distal regulatory elements. Chromatin immunoprecipitation assays demonstrated binding of neither Sp1 nor Sp3 to the CYP4B1 proximal element in human liver tissue, whereas selective Sp1 binding was observed in human lung tissue. Thus, the composite findings are consistent with both the proximal Sp1 elements and the distal regulatory element acting to synergistically control CYP4B1 lung-selective expression.

Sp1 and Sp3 regulate basal transcription of the human CYP2F1 gene.

Selective transcription of the human CYP2F1 gene in lung tissues may control the susceptibilities of this organ to diverse pneumotoxicants and lung carcinogens. However, the mechanisms responsible for CYP2F1 organ-selective transcription have not been elucidated. The objectives of the current studies were to identify and characterize basal transcription elements within the TATA-less promoter region of CYP2F1. Four putative Sp1-like sites were identified in the CYP2F1 promoter. Competitive electrophoretic mobility shift assay analysis with mutated oligonucleotide probes and lung A549 cell nuclear extract, along with supershift studies using antibodies to either Sp1 or Sp3 proteins, demonstrated that all four sites formed three specific protein-DNA complexes. Mutations in any of the four core Sp1-like motifs abolished protein-DNA binding. Western blot analysis of both human tissues and cells showed that Sp1 was considerably higher in lung than liver and that Sp3 was much higher in liver than lung. Promoter activation of a luciferase reporter construct was sequentially increased by addition of each of the four Sp1-like motifs in lung A549 cells but not in liver HepG2 cells. Cotransfection of a Sp1 expression vector with the reporter construct dramatically increased luciferase activity in either A549 cells or Sp1-deficient Drosophila Schneider line 2 (SL-2) cells. However, similar cotransfections with an Sp3 expression vector failed to increase activity. Cotransfection of both the Sp1 and Sp3 expression vectors considerably decreased Sp1-mediated activity in A549 cells and abolished activity in SL-2 cells. Thus, these studies demonstrated that four Sp1-dependent proximal promoter elements drive organ-selective CYP2F1 gene transcription, and that Sp1 and Sp3 factors interact to modulate constitutive CYP2F1 transcription in lung cells.