Toxicogenetic effects of low concentrations of the pesticides imidacloprid and sulfentrazone individually and in combination in in vitro tests with HepG2 cells and Salmonella typhimurium

The insecticide imidacloprid and the herbicide sulfentrazone are two different classes of pesticides that are used for pest control in sugarcane agriculture. To evaluate the genotoxic potential of low concentrations of these two pesticides alone and in mixture, the comet assay and the micronucleus (MN) test employing fluorescence in situ hybridization (FISH) with a centromeric probe were applied in human hepatoma cell lines (HepG2), in a 24-h assay. Mutagenicity was assessed by Salmonella/microsome assay with TA98 and TA100 strains in the absence and presence of an exogenous metabolizing system (S9). The results showed significant inductions of MN in HepG2 cells by both pesticides, for all the tested concentrations. As evidenced in the comet assay, only the imidacloprid presented significant responses. When the two pesticides were associated, a significant induction of damage was observed in the HepG2 cells by the comet assay, but not by the MN test. Moreover, the MN induced by the mixtures of the pesticides appeared at lower levels than those induced by sulfentrazone and imidacloprid when tested alone. According to the FISH results, the damage induced by imidacloprid in the HepG2 cells resulted from a clastogenic action of this insecticide (76.6% of the MN did not present a centromeric signal). For the herbicide sulfentrazone and for the mixture of the pesticides, a similar frequency of MN with and without the presence of the centromeric signal (herbicide: 52.45% of the MN without centromeric signal and 47.54% of the MN with centromeric signal; mixture: 48.71% of the MN without centromeric signal and 51.42% of the MN with centromeric signal) was verified. Based on these results, it was concluded that each one of the pesticides evaluated interacts with the DNA of HepG2 cells and causes irreparable alterations in the cells. However, the combination of the pesticides showed an antagonistic effect on the cells and the damage induced was milder and not persistent in HepG2 cells. The results obtained by the Ames test did not point out significant results.

Genome-wide scan for visceral leishmaniasis in mixed-breed dogs identifies candidate genes involved in T helper cells and macrophage signaling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mechanisms involved in reproductive damage caused by gossypol in rats and protective effects of vitamin E

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of the genotoxic potential of low concentrations of Malathion on the Allium cepa cells and rat hepatoma tissue culture

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The role of qualea multiflora on mammary tumour cells and immune system

Species of the genus Qualea are used by the Brazilian public as a natural anti-inflammatory. Based on this evidence, we evaluated the effects of terpene fractions (βF and TF) obtained from Qualea multiflora on nitric oxide production (Griess assay), cytokines (IL-1, IL-10, IL-12, and TNF-a) and the transcription factor NF-κB by peritoneal macrophages. Since there is a relationship between inflammation and cancer, the cytotoxicity of βF and TF against mammary tumoural cell lineage, and macrophages was evaluated. Inhibition levels close to 90% of the production of NO, IL-1, IL-12 and TNF-a; about 32% of NF-κB; and a large stimulation of IL-10 production (close to the positive control) by peritoneal macrophages were observed in response to βF and TF which are correlated with anti-inflammatory activity. Additionally, the samples showed exceptional cytotoxic activity against tumoural cells but not against macrophages. Since anti-inflammatory activity is important in tumour inhibition, further examination of potential anti-cancerous activity of Qualea multiflora is warranted.

IGFBP7 participates in the reciprocal interaction between acute lymphoblastic leukemia and BM stromal cells and in leukemia resistance to asparaginase

The interaction of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells (BMSCs) has a positive impact on ALL resistance to chemotherapy. We investigated the modulation of a series of putative asparaginase-resistance/sensitivity genes in B-precursor ALL cells upon coculture with BMSCs. Coculture with stromal cells resulted in increased insulin-like growth factor (IGF)-binding protein 7 (IGFBP7) expression by ALL cells. Assays with IGFBP7 knockdown ALL and stromal cell lines, or with addition of recombinant rIGFBP7 (rIGFBP7) to the culture medium, showed that IGFBP7 acts as a positive regulator of ALL and stromal cells growth, and significantly enhances in-vitro resistance of ALL to asparaginase. In these assays, IGFBP7 function occurred mainly in an insulin-and stromal-dependent manner. ALL cells were found to contribute substantially to extracellular IGFBP7 levels in the conditioned coculture medium. Diagnostic BM plasma from children with ALL had higher levels of IGFBP7 than controls. IGFBP7, in an insulin/IGF-dependent manner, enhanced asparagine synthetase expression and asparagine secretion by BMSCs, thus providing a stromal-dependent mechanism by which IGFBP7 protects ALL cells against asparaginase in this coculture system. Importantly, higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (Cox regression model, P = 0.003) in precursor B-cell Ph(-) ALL patients (n = 147) treated with a contemporary polychemotherapy protocol.

Increased Antioxidant Content in Juice Enriched with Dried Extract of Pomegranate (Punica granatum) Peel

Antioxidants are compounds responsible for free radical scavenging in the body. They protect the organism from oxidative modification of cells and tissues. These modifications have been associated with degenerative diseases, atherosclerosis and carcinogenesis. Punica granatum displays high antioxidant potential due to the presence of phenolic compounds, which are capable of disease prevention. The present study showed the highest antioxidant activity in pomegranate peel than in seeds and pulp. Based on these results, pomegranate peel was used to produce dried extract that was added to commercial tomato juice and orange juice with strawberries. Analysis to determine the content of phenolic compounds and antioxidant activity was performed on pomegranate pulp, seeds and peel and in juices enriched with dried extract of pomegranate peel. The dried extract was responsible for a significant increase in antioxidant activity of the juices, proportional to the concentrations added. However, although both flavors of enriched juices displayed high antioxidant levels, the samples with higher dried extract concentrations received the lowest scores from sensory analysis participants due to the characteristic astringent flavor of pomegranate peels. Therefore, to obtain greater acceptance in the consumer market, we concluded that the maximum addition of dried pomegranate peel extract is 0.5% in tomato juice and orange juice with strawberries.

Anti-CD25 Treatment Depletes Treg Cells and Decreases Disease Severity in Susceptible and Resistant Mice Infected with Paracoccidioides brasiliensis

Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4(+)CD25(+)Foxp3(+) Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-beta. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4(+)CD25(+)Foxp3(+) Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4(+) and CD8(+) T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25(+) cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.

Antiproliferative Effects of Fluoxetine on Colon Cancer Cells and in a Colonic Carcinogen Mouse Model

The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G(0)/G(1) phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.

Increased Expression of Regulatory T Cells and Down-Regulatory Molecules in Lepromatous Leprosy

T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-gamma (IFN-gamma) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-beta (TGF-beta), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-beta. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth.

Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model

The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4+ and CD8+ immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207+, CD11c+, CD4+, CD8+, iNOS+ cellular densities. Cytokine (IFN-?, IL-4 and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207+ and CD11c+ were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4+ and CD8+ were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS+ was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-? was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response.

The Aurora A and B kinases are up-regulated in bone marrow-derived chronic lymphocytic leukemia cells and represent potential therapeutic targets

Background The malignant B cells in chronic lymphocytic leukemia receive signals from the bone marrow and lymph node microenvironments which regulate their survival and proliferation. Characterization of these signals and the pathways that propagate them to the interior of the cell is important for the identification of novel potential targets for therapeutic intervention. Design and Methods We compared the gene expression profiles of chronic lymphocytic leukemia B cells purified from bone marrow and peripheral blood to identify genes that are induced by the bone marrow microenvironment. Two of the differentially expressed genes were further studied in cell culture experiments and in an animal model to determine whether they could represent appropriate therapeutic targets in chronic lymphocytic leukemia. Results Functional classification analysis revealed that the majority of differentially expressed genes belong to gene ontology categories related to cell cycle and mitosis. Significantly up-regulated genes in bone marrow-derived tumor cells included important cell cycle regulators, such as Aurora A and B, survivin and CDK6. Down-regulation of Aurora A and B by RNA interference inhibited proliferation of chronic lymphocytic leukemia-derived cell lines and induced low levels of apoptosis. A similar effect was observed with the Aurora kinase inhibitor VX-680 in primary chronic lymphocytic leukemia cells that were induced to proliferate by CpG-oligonucleotides and interleukin-2. Moreover, VX-680 significantly blocked leukemia growth in a mouse model of chronic lymphocytic leukemia. Conclusions Aurora A and B are up-regulated in proliferating chronic lymphocytic leukemia cells and represent potential therapeutic targets in this disease.

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 mu M tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.

SK-HEP cells and lentiviral vector for production of human recombinant factor VIII

Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs.