Combining the Elicitor Up-Regulated Production of Unusual Linear Diterpene-Derived Variants for an In-Depth Assessment of the Application Value and Risk of the Medicinal and Edible Basidiomycete Schizophyllum commune.

To better assess the practical value and avoid potential risks of the traditionally medicinal and edible basidiomycete Schizophyllum commune, which may arise from undescribed metabolites, a combination of elicitors was introduced for the first time to discover products from cryptic and low-expressed gene clusters under laboratory cultivation. Treating S. commune NJFU21 with the combination of five elicitors led to the upregulated production of a class of unusual linear diterpene-derived variants, including eleven new ones (1-11), along with three known ones (12-14). The structures and stereochemistry were determined by 1D and 2D NMR, HRESIMS, ECD, OR and VCD calculations. Notably, the elongation terminus of all the diterpenes was decorated by an unusual butenedioic acid moiety. Compound 1 was a rare monocyclic diterpene, while 2-6 possessed a tetrahydrofuran moiety. The truncated metabolites 4, 5 and 13 belong to the trinorditerpenes. All the diterpenes displayed approximately 70% scavenging of hydroxyl radicals at 50 µM and null cytotoxic activity at 10 µM. In addition, compound 1 exhibited potent antifungal activity against the plant pathogenic fungi Colletotrichum camelliae, with MIC values of 8 µg/mL. Our findings indicated that this class of diterpenes could provide valuable protectants for cosmetic ingredients and the lead compounds for agricultural fungicide development.

Assessment of the cardioprotective effect of liraglutide on methotrexate induced cardiac dysfunction through suppression of inflammation and enhancement of angiogenesis in rats.

OBJECTIVE: Methotrexate (MTX) is one of the most commonly used anti-cancer drugs for various types of neoplasms. It is associated with multiple cytotoxic effects including nephrotoxicity, hepatotoxicity and cardiotoxicity. Liraglutide (LIR) is a potent anti-diabetic drug and also has antioxidant and anti-inflammatory properties. In this study, we tried to investigate the protective effect of LIR on MTX induced cardiotoxicity and to identify the molecular mechanisms for this protection. MATERIALS AND METHODS:  Rats were divided into 4 groups, including control group, LIR group, MTX group and LIR + MTX group. ECG was measured then blood samples were taken, and hearts were excised for biochemical and histological investigations. RESULTS: MTX group exhibited a mild non-significant irregular bradycardia, an increase of CK-MB besides a decrease of total antioxidant capacity. MTX administration also resulted in downregulation of vascular endothelial growth factor (VEGF), while caused upregulation of interleukin 1 beta (IL-1B) and interleukin 6 (IL-6) in comparison to the control group. Also, MTX group showed histological abnormalities besides negative VEGF and positive iNOS as detected by immunohistochemical staining compared to the control group. LIR administration could reverse these results. CONCLUSIONS: LIR prevented MTX induced cardiotoxicity through its antioxidant and anti-inflammatory properties.

Assessment of Thiosemicarbazone-Containing Compounds as Potential Antileukemia Agents against P-gp Overexpressing Drug Resistant K562/A02 Cells.

P-Glycoprotein (P-gp) overexpression is considered to be the leading cause of multidrug resistance (MDR) and failure of chemotherapy for leukemia. In this study, seventeen thiosemicarbazone-containing compounds were prepared and evaluated as potential antileukemia agents against drug resistant K562/A02 cell overexpressing P-gp. Among them, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could significantly inhibit K562/A02 cells proliferation with an IC50 value of 0.96 µM. Interestingly, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could dose-dependently increase ROS levels of drug resistant K562/A02 cells, thus displaying a potential collateral sensitivity (CS)-inducing effect and selectively killing K562/A02 cells. Furthermore, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide possessed potent inhibitory effect on HDAC1 and HDAC6, and could promote K562/A02 cells apoptosis via dose-dependently increasing Bax expression, reducing Bcl-2 protein level, and inducing the cleavage of PARP and caspase3. These present findings suggest that N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide might be a promising lead to discover novel antileukemia agents against P-gp overexpressing leukemic cells.

MicroRNA-31-5p Exacerbates Lipopolysaccharide-Induced Acute Lung Injury via Inactivating Cab39/AMPKα Pathway.

Acute lung injury (ALI) and the subsequent acute respiratory distress syndrome remain devastating diseases with high mortality rates and poor prognoses among patients in intensive care units. The present study is aimed at investigating the role and underlying mechanisms of microRNA-31-5p (miR-31-5p) on lipopolysaccharide- (LPS-) induced ALI. Mice were pretreated with miR-31-5p agomir, antagomir, and their negative controls at indicated doses for 3 consecutive days, and then they received a single intratracheal injection of LPS (5 mg/kg) for 12 h to induce ALI. MH-S murine alveolar macrophage cell lines were cultured to further verify the role of miR-31-5p in vitro. For AMP-activated protein kinase α (AMPKα) and calcium-binding protein 39 (Cab39) inhibition, compound C or lentiviral vectors were used in vivo and in vitro. We observed an upregulation of miR-31-5p in lung tissue upon LPS injection. miR-31-5p antagomir alleviated, while miR-31-5p agomir exacerbated LPS-induced inflammation, oxidative damage, and pulmonary dysfunction in vivo and in vitro. Mechanistically, miR-31-5p antagomir activated AMPKα to exert the protective effects that were abrogated by AMPKα inhibition. Further studies revealed that Cab39 was required for AMPKα activation and pulmonary protection by miR-31-5p antagomir. We provide the evidence that endogenous miR-31-5p is a key pathogenic factor for inflammation and oxidative damage during LPS-induced ALI, which is related to Cab39-dependent inhibition of AMPKα.

A toxicogenomic approach for the risk assessment of the food contaminant acetamide.

Acetamide (CAS 60-35-5) is detected in common foods. Chronic rodent bioassays led to its classification as a group 2B possible human carcinogen due to the induction of liver tumors in rats. We used a toxicogenomics approach in Wistar rats gavaged daily for 7 or 28 days at doses of 300 to 1500 mg/kg/day (mkd) to determine a point of departure (POD) and investigate its mode of action (MoA). Ki67 labeling was increased at doses ≥750 mkd up to 3.3-fold representing the most sensitive apical endpoint. Differential gene expression analysis by RNA-Seq identified 1110 and 1814 differentially expressed genes in male and female rats, respectively, following 28 days of treatment. Down-regulated genes were associated with lipid metabolism while up-regulated genes included cell signaling, immune response, and cell cycle functions. Benchmark dose (BMD) modeling of the Ki67 labeling index determined the BMD10 lower confidence limit (BMDL10) as 190 mkd. Transcriptional BMD modeling revealed excellent concordance between transcriptional POD and apical endpoints. Collectively, these results indicate that acetamide is most likely acting through a mitogenic MoA, though specific key initiating molecular events could not be elucidated. A POD value of 190 mkd determined for cell proliferation is suggested for risk assessment purposes.

Assessment of doxorubicin-induced remodeling of Ca2+ signaling and associated Ca2+ regulating proteins in MDA-MB-231 breast cancer cells.

Triple-negative breast cancers (TNBC) are often associated with high relapse rates, despite treatment with chemotherapy agents such as doxorubicin. A better understanding of the signaling and molecular changes associated with doxorubicin may provide novel insights into strategies to enhance treatment efficacy. Calcium signaling is involved in many pathways influencing the efficacy of chemotherapy agents such as proliferation and cell death. However, there are a limited number of studies exploring the effect of doxorubicin on calcium signaling in TNBC. In this study, MDA-MB-231 triple-negative, basal breast cancer cells stably expressing the genetically-encoded calcium indicator GCaMP6m (GCaMP6m-MDA-MB-231) were used to define alterations in calcium signaling. The effects of doxorubicin in GCaMP6m-MDA-MB-231 cells were determined using live cell imaging and fluorescence microscopy. Changes in mRNA levels of specific calcium regulating proteins as a result of doxorubicin treatment were also assessed using real time qPCR. Doxorubicin (1 µM) produced alterations in intracellular calcium signaling, including enhancing the sensitivity of MDA-MB-231 cells to ATP stimulation and prolonging the recovery time after store-operated calcium entry. Upregulation in mRNA levels of ORAI1, TRPC1, SERCA1, IP3R2 and PMCA2 with doxorubicin 1 µM treatment was also observed. Doxorubicin treatment is associated with specific remodeling in calcium signaling in MDA-MB-231 cells, with associated changes in mRNA levels of specific calcium-regulating proteins.

Myocardial hypertrophy is improved with berberine treatment via long non-coding RNA MIAT-mediated autophagy.

OBJECTIVES: This study aimed to evaluate berberine (BBR) effects on myocardial hypertrophy (MH) and associated mechanisms. METHODS: BBR effects on MH were evaluated in rats with constriction of abdominal aorta (CAA). qRT-PCR assay was used to measure MH-related genes, long non-coding RNAs (lncRNAs) and autophagy-related genes expressions. Western blot was performed to detect autophagy markers expression. Filamentous actin and phalloidin expressions were detected using immunofluorescence assay. KEY FINDINGS: BBR significantly attenuated CAA-induced MH and cardiomyocyte enlargement. CAA upregulated ß myosin heavy chain and atrial natriuretic peptide expressions in heart tissues, which was attenuated by BBR. BBR suppressed myocardial infarction associated transcript (MIAT) expression in rats with CAA. p62 mRNA expression was upregulated and beclin1 and autophagy related 5 were downregulated in CAA versus control groups. The effects were abolished by BBR. In vitro studies showed that BBR ameliorated angiotensin II-induced MH and attenuated Ang II-induced MIAT expression in H9C2 cells. Expressions of phosphorylated mTOR, phosphorylated AMPK and LC3 were upregulated in H9C2 cells after Ang II stimulation, and the effects were abolished by BBR. CONCLUSIONS: BBR exerted beneficial effects on MH induced by CCA, and the mechanisms were associated with decreased MIAT expression and enhanced autophagy.

Effect of suboptimal temperature on the regulation of endogenous antigen presentation in a rainbow trout hypodermal fibroblast cell line.

Rainbow trout (Oncorhynchus mykiss) face low environmental temperatures over winter months and during extreme low temperature events. Suboptimal temperatures are known to negatively impact the teleost immune system, although there is mixed evidence in rainbow trout as to the effect on the endogenous antigen processing and presentation pathway (EAPP). The EAPP is an important pathway for antiviral defense that involves the presentation of endogenous peptides on the cell surface for recognition by cytotoxic T cells. Using a rainbow trout hypodermal fibroblast (RTHDF) cell line as an in vitro model, we determined that constitutive EAPP transcript levels are not impaired at low temperature, but induction of up-regulation of these transcripts is delayed at the suboptimal temperature following exposure to poly(I:C) or viral haemorrhagic septicaemia virus IVb, which was still able to enter and replicate in the cell line at 4 °C, albeit with reduced efficiency. The delay in the induction of EAPP mRNA level up-regulation following poly(I:C) stimulation coincided with a delay in ifn1 transcript levels and secretion, which is important since interferon-stimulated response elements were identified in the promoter regions of the EAPP-specific members of the pathway, implying that IFN1 is involved in the regulation of these genes. Our results suggest that the ability of rainbow trout to mount an effective immune response to viral pathogens may be lessened at suboptimal temperatures.

Assessment of the anti-inflammatory, analgesic and sedative effects of oleuropein from Olea europaea L.

More and more studies show that inflammation, pain and insomnia have become the main common diseases. Effective treatments of inflammation, pain and insomnia have become an issue of primary concern in clinical practice. Oleuropein (OLE), the main phenolic component of Mediterranean extra virgin olive oil, has shown many pharmacological properties. In the present study, the anti-inflammatory effect of OLE was firstly evaluated using RAW264.7 macrophages subjected to stimulation with lipopolysaccharide (LPSC). The results obtained revealed that OLE caused significant and dose-dependent downregulation of nitric (NO), COX-2, inducible NO synthase iNOS, and the inflammation-associated cytokines IL-6 and TNF-α. From the mechanism, the expression of COX-2, cytokines IL-6 and TNF-α OLE is closely related to analgesic and sedation effect. Further evaluations showed significant analgesic and sedative effects of OLE in tail-flick test and sedation test conducted in SD rats in vivo. All these results indicate that OLE has anti-inflammatory, analgesic and sedative effects both in vitro and in vivo.

Assessment of Feng-Liao-Chang-Wei-Kang as a potential inducer of cytochrome P450 3A4 and pregnane X receptors.

Feng-Liao-Chang-Wei-Kang (FLCWK), a traditional Chinese patent medicine, consists primarily of Polygonum hydropiper and Daphniphyllum calycinum roots. As a complex containing several kinds of flavonoids, FLCWK has the potential to impact the drug metabolism enzyme P450 3A4 (CYP3A4) and nuclear receptors. The purpose of this research was to probe the effects of FLCWK on CYP3A1, the homolog of CYP3A4 in rats, and to confirm whether FLCWK interferes with PXR and CAR-mediated transactivation of CYP3A4. The effects of FLCWK on Cyp3a1 mRNA, catalytic activity levels, and protein expression in Sprague-Dawley (SD) rat liver tissues were examined using real-time PCR, western blotting, and high-performance liquid chromatography (HPLC) assays, respectively. The efficacy of PXR and CAR on CYP3A4 transcriptional activity were detected using luciferase reporter assays and further research of the impact of FLCWK on CYP3A4 gene expression mediated by the PXR pathway was examined by transient transfection of PXR siRNA. FLCWK significantly increased Cyp3a1 mRNA, CYP3A1 activity, and protein expression levels in SD rats. FLCWK highly induced CYP3A4 luciferase activity mediated by PXR in PXRCYP3A4 co-transfected cells. A siRNA-mediated drop-off in PXR expression greatly cut the effect of FLCWK on CYP3A4 mRNA expression in HepG2 cells. These findings show that FLCWK up-regulates CYP3A4 levels via the PXR pathway. This effect should be considered being applied in clinical use as FLCWK has the potential to interact with other drugs.

Knockdown of LncRNA MALAT1 contributes to the suppression of inflammatory responses by up-regulating miR-146a in LPS-induced acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a type of severe pulmonary inflammatory disease with high rates of morbidity and mortality. Now, an increasing number of studies suggest that lncRNAs may act as key regulators of the inflammatory response and play a crucial role in the pathogenesis of many inflammatory diseases. Our study firstly explored the function and underlying mechanism of lncRNA metastasis-associated lung adenocarcinoma transcription 1 (MALAT1) in regulating the inflammatory response of lipopolysaccharide (LPS)-induced ALI in rats. METHODS: The ALI rats were constructed by intratracheal instillation with LPS. Hematoxylin and eosin (HE) for histological examination were performed to detect histopathological changes in the lung tissues. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of cytokines TNF-α, IL-6, and IL-1ß in the supernatants of the bronchoalveolar lavage fluid (BALF). Quantitative real-time PCR (qRT-PCR) analysis was employed to assess the expression of MALAT1, miR-146a, TNF-α, IL-6, and IL-1ß in lung tissues. Luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to detect the relationship between MALAT1 and miR-146a. RESULTS: The results revealed that MALAT1 knockdown played a protective role in the LPS-induced ALI rat model. In addition, knockdown of MALAT1 in vitro inhibited LPS-induced inflammatory response in murine alveolar macrophages cell line MH-S and murine alveolar epithelial cell line MLE-12. This study found that MALAT1 acts as a molecular sponge for miR-146a and MALAT1 negatively regulated miR-146a expression. Mechanistically, MALAT1 overexpression alleviated the inhibitory effect of miR-146a on LPS-induced inflammatory response in MH-S. CONCLUSIONS: Together, our study provided the first evidence that MALAT1 knockdown could suppress inflammatory response by up-regulating miR-146a in LPS-induced ALI, which provided a potential therapeutic target for the treatment of ALI.

TRPM8 activation improves energy expenditure in skeletal muscle and exercise endurance in mice.

Skeletal muscle serving as the major organ is responsible for energy expenditure and exercise endurance, which directly influence cardiometabolic risk factors. Transient receptor potential melastatin 8 (TRPM8), a Ca2+-permeable non-selective cation channel, plays vital roles in the regulation of various cellular functions. It has been reported that TRPM8 activation enhanced the energy metabolism of adipocytes. However, the involvement of TRPM8 in the energy metabolism of skeletal muscle remains unexplored. Our data revealed that TRPM8 was expressed in cultured C2C12 myocytes. Menthol treatment increased uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) expression in C2C12 myotubes through TRPM8 activation. Moreover, dietary menthol upregulated the expression of UCP1 and PGC1α in skeletal muscle of mice. In addition, dietary menthol enhanced exercise endurance and reduced blood lactic acid and triglycerides through TRPM8 activation. It is concluded that dietary menthol improves energy metabolism and exercise endurance by increasing UCP1 and PGC1α in skeletal muscles, suggesting dietary menthol might be a novel therapeutic approach for cardiometabolic diseases management and prevention.

Risk assessment of a formamidine pesticide, Amitraz, focusing on thyroid hormone receptors (TRs) in rainbow trout, Oncorhynchus mykiss.

Amitraz, a formamidine pesticide, and their metabolites have the potential to disrupt endocrine homeostasis in a variety of organisms, nevertheless there is a lack of information concerning such effects and underlying mechanisms in any fish species.To evaluate the potential impacts of Trasil (EC; active constituent 200 g amitraz/L), a commercial product of amitraz, on thyroid hormone (TH) homeostasis of rainbow trout (Oncorhynchus mykiss); mRNA levels of thyroid hormone receptors (TRs), TRα and TRß, were determined by RT-PCR soon after sub-lethal administration in a static bio-assay system. The sub-lethal exposure of 0.84 mg/L amitraz resulted in upregulation of both TRαand TRßgenes for muscle and liver, respectively in a tissue-manner, though the differences were found statistically insignificant (P>0.05). The present results emerged an endocrine interaction between amitraz based formulation and TH homeostasis, but still needs further detail studies to a better understanding of TH mechanism in teleosts in response to environmental compounds.

Modulation of macrophage phagocytosis in vitro-A role for cholinergic stimulation?

Acetylcholine is synthetized and released from neural cells, but also by non-neuronal cells such as epithelial cells or keratinocytes. Cholinergic agonists enhance the phagocytosis of zymosan particles in primary peritoneal macrophages. The aim of this study was to investigate the effect of carbachol stimulation on phagocytosis in a macrophage cell line using microspheres. The murine cell line MH-S was used in a phagocytosis assay with fluorescent latex beads. The amount of the ingested beads was determined using flow cytometry. Gene expression was investigated using polymerase chain reaction. Gene expression of the muscarinic receptors M1, M3, M4 and M5 but not M2 was found. Carbachol slightly increased the phagocytosis of microspheres in the macrophages. A co-stimulation using lipopolysaccharide and carbachol did not increase the effect of lipopolysaccharide alone. In conclusion, cholinergic stimulation in vitro only moderately modulates the phagocytosis of microspheres. M2 might have a role in stimulation of macrophage phagocytosis.

Stimulation of the histamine 4 receptor upregulates thymic stromal lymphopoietin (TSLP) in human and murine keratinocytes.

The cytokine thymic stromal lymphopoietin (TSLP) is involved in the development and the progression of allergic diseases. It is mainly released by epithelial cells at barriers such as skin and gut in response to danger signals. Overexpression of TSLP in keratinocytes (KC) can provoke the development of a type 2 inflammatory response. Additionally, TSLP directly acts on sensory neurons and thereby triggers itch. Since histamine is also increased in lesions of inflammatory skin diseases, the aim of this study was to investigate possible effects of histamine as well as different histamine receptor subtype agonists and antagonists on TSLP production in KC. We therefore stimulated human KC with histamine in the presence or absence of the known TSLP-inductor poly I:C and measured TSLP production at protein as well as mRNA level. Histamine alone did not induce TSLP production in human KC, but pre-incubation with histamine prior to challenge with poly I:C resulted in a significant increase of TSLP production compared to stimulation with poly I:C alone. Experiments with different histamine receptor agonists (H1R: 2-pyridylethylamine; H2R: amthamine; H2R/H4R: 4-methylhistamine (4MH)) revealed a dominant role for the H4R receptor, as 4-MH in combination with poly I:C displayed a significant increase of TSLP secretion, while the other agonists did not show any effect. The increase in TSLP production by 4MH was blocked with the H4R antagonist JNJ7777120. This effect was reproducible also in the murine KC cell line MSC. Taken together, our study indicates a new role for the H4 receptor in the regulation of TSLP in keratinocytes. Therefore, blocking of the H4R receptor in allergic diseases might be promising to alleviate inflammation and pruritus via TSLP.

Sharing the Roles: An Assessment of Japanese Medaka Estrogen Receptors in Vitellogenin Induction.

Teleost fish express at least three estrogen receptor (ER) subtypes. To date, however, the individual role of these ER subtypes in regulating expression of estrogen responsive genes remains ambiguous. Here, we investigate putative roles of three ER subtypes in Japanese medaka (Oryzias latipes), using vitellogenin (VTG) I and II as model genes. We identify specific ligand/receptor/promoter dynamics, using transient transactivation assays that incorporate luciferase reporters comprising 3kb promoter/enhancer regions of medaka VTGI and VTGII genes. Four steroidal estrogens (17ß-estradiol, estrone, estriol, and 17α-estradiol) were tested in these assays. Results indicate that all three medaka ERs (mERs) are capable of initiating transactivation of both VTG I and II, with ERß2 exhibiting greatest activity. Promoter deletion analysis suggests that ligand-specific receptor transactivation and utilization of regional-specific estrogen response elements may be associated with differential activities of each medaka ER. Further, cluster analysis of in vivo gene expression and in vitro transactivation suggests that all three ER subtypes putatively play a role in up-regulation of VTG. Results illustrate that preferential ligand/receptor/promoter interactions may have direct implications for VTG gene expression and other ER-mediated regulatory functions that are relevant to the risk assessment of estrogenic compounds.

Effects of elevated pCO2 and feeding on net calcification and energy budget of the Mediterranean cold-water coral Madrepora oculata.

Ocean acidification is a major threat to calcifying marine organisms such as deep-sea cold-water corals (CWCs), but related knowledge is scarce. The aragonite saturation threshold (Ωa) for calcification, respiration and organic matter fluxes were investigated experimentally in the Mediterranean Madrepora oculata Over 10 weeks, colonies were maintained under two feeding regimes (uptake of 36.75 and 7.46 µmol C polyp-1 week-1) and exposed in 2 week intervals to a consecutively changing air-CO2 mix (pCO2) of 400, 1600, 800, 2000 and 400 ppm. There was a significant effect of feeding on calcification at initial ambient pCO2, while with consecutive pCO2 treatments, feeding had no effect on calcification. Respiration was not significantly affected by feeding or pCO2 levels. Coral skeletons started to dissolve at an average Ωa threshold of 0.92, but recovered and started to calcify again at Ωa≥1. The surplus energy required to counteract dissolution at elevated pCO2 (≥1600 µatm) was twice that at ambient pCO2 Yet, feeding had no mitigating effect at increasing pCO2 levels. This could be due to the fact that the energy required for calcification is a small fraction (1-3%) of the total metabolic energy demand and corals even under low food conditions might therefore still be able to allocate this small portion of energy to calcification. The response and resistance to ocean acidification are consequently not controlled by feeding in this species, but more likely by chemical reactions at the site of calcification and exchange processes between the calicoblastic layer and ambient seawater.

Comparative assessment of HIF-1α and Akt responses in human lung and skin cells exposed to benzo[α]pyrene: Effect of conditioned medium from pre-exposed primary fibroblasts.

Exposure to atmospheric pollutants has been accused for many adverse health effects. Benzo[α]pyrene (Β[α]Ρ) in particular, the most extensively studied member of pollutants, is implicated in both cancer initiation and promotion. In the present study, we compared the effects of noncytotoxic doses of Β[α]Ρ, between human skin and lung epithelial cells A431 and A549, respectively, focusing on Akt kinase and HIF-1α, as it is well known that these proteins are upregulated in various human cancers promoting survival, angiogenesis and metastasis of tumor cells. Also, taking into consideration that fibroblasts are involved in cancer progression, we tested the possible modulation of epithelial cell response by paracrine factors secreted by Β[α]Ρ-treated fibroblasts. Low doses of Β[α]Ρ were found to enhance epithelial cell proliferation and upregulate both Akt kinase and HIF-1α, with A549 cells exhibiting a more sustained profile of upregulation. It is to notice that, the response of HIF-1α was remarkably early, acting as a sensitive marker in response to airborne pollutants. Also, HIF-1α was induced by Β[α]Ρ in both lung and skin fibroblasts indicating that this effect may be conserved throughout different cell types and tissues. Interestingly however, the response of both proteins was differentially modified upon treatment with conditioned medium from Β[α]Ρ-exposed fibroblasts. This is particularly evident in A459 cells and confirms the critical role of intercellular and paracrine factors in the modulation of the final response to an extracellular signal. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1103-1112, 2016.

Multilevel assessment of Cry1Ab Bt-maize straw return affecting the earthworm Eisenia fetida.

Non-target effects of two varieties of Bacillus thuringiensis (Bt)-maize straw (5422Bt1 [event Bt11] and 5422CBCL [MON810]) return on the Eisenia fetida were investigated by using multilevel assessments, compared to near-isogenic non-Bt-maize (5422). 5422Bt1 straw return had no deleterious effects on adult earthworms and had significantly positive effects on juveniles over three generations. Negative, no, and positive effects on adults treated with 5422CBCL straw were observed in the 1st, 2nd and 3rd generation, respectively. Negative and positive effects were observed on juveniles produced from the 1st- and 2nd-generation adults treated with 5422CBCL straw, respectively. Glutathione peroxidase activity of earthworms from Bt-maize treatments was significantly higher than that of control on the 90th d. Translationally controlled tumour protein (TCTP) and superoxide dismutase (SOD) genes were down-regulated, while annetocin (ANN) expression was up-regulated in 5422Bt1 treatments. TCTP and SOD genes were up-regulated, while ANN and heat shock protein 70 were down-regulated in E. fetida from 5422CBCL treatments. Enzyme-linked immunosorbent assay revealed that Cry1Ab released from 5422Bt1 and 5422CBCL straw degraded rapidly on the 15th and 30th d and had a slow decline in the rest testing time. Cry1Ab concentrations in the soil, casts and guts of earthworm significantly decreased over the course of the experiment. This study was the first to evaluate generational effects of Bt-maize straw return on earthworms under laboratory conditions. The responses of enzymes activity and genes expression may contribute to better understand above different effects of Bt-maize straw return on earthworms from the 1st generation.

High-throughput, quantitative assessment of the effects of low-dose silica nanoparticles on lung cells: grasping complex toxicity with a great depth of field.

BACKGROUND: The toxicity of manufactured fumed silica nanoparticles (NPs) remains poorly investigated compared to that of crystalline silica NPs, which have been associated with lung diseases after inhalation. Amorphous silica NPs are a raw material for manufactured nanocomposites, such as cosmetics, foods, and drugs, raising concerns about their potential toxicity. RESULTS: The size of the NPs was determined by dynamic light scattering and their shape was visualized by atomic force microscopy (10 ± 4 nm). The pertinent toxicological concentration and dynamic ranges were determined using viability tests and cellular impedance. We combined transcriptomics and proteomics to assess the cellular and molecular effects of fumed silica in A549 human alveolar epithelial cells. The "no observed transcriptomic adverse effect level" (NOTEL) was set to 1.0 µg/cm(2), and the "lowest observed adverse transcriptional effect level" (LOTEL) was set at 1.5 µg/cm(2). We carried out genome-wide expression profiles with microarrays and identified, by shotgun proteomics, the exoproteome changes in lung cells after exposure to NP doses (0.1, 1.0, 1.5, 3.0, and 6.0 µg/cm(2)) at two time points (24 h and 72 h). The data revealed a hierarchical, dose-dependent cellular response to silica NPs. At 1.5 µg/cm(2), the Rho signaling cascade, actin cytoskeleton remodeling, and clathrin-mediated endocytosis were induced. At 3.0 µg/cm(2), many inflammatory mediators were upregulated and the coagulation system pathway was triggered. Lastly, at 6.0 µg/cm(2), oxidative stress was initiated. The proteins identified in the extracellular compartment were consistent with these findings. CONCLUSIONS: The alliance of two high-throughput technologies allowed the quantitative assessment of the cellular effects and molecular consequences of exposure of lung cells to low doses of NPs. These results were obtained using a pathway-driven analysis instead of isolated genes. As in photography, toxicogenomics allows, at the same time, the visualization of a wide spectrum of biological responses and a "zoom in" to the details with a great depth of field. This study illustrates how such an approach based on human cell culture models is a valuable predictive screening tool to evaluate the toxicity of many potentially harmful emerging substances, alone or in mixtures, in the framework of future regulatory reinforcements.