Non-exposed Cells Research Articles

Enhancing pulmonary delivery and immunomodulation of respiratory diseases through virus-mimicking nanoparticles

This study introduces the nanobromhexine lipid particle (NBL) platform designed for effective pulmonary drug delivery. Inspired by respiratory virus transport mechanisms, NBL address challenges associated with mucus permeation and inflammation in pulmonary diseases. Composed of low molecular weight polyethylene glycol-coated lipid nanoparticles with bromhexine hydrochloride, NBL exhibit a size of 118 ± 24 nm, a neutral zeta potential, osmolarity of 358 ± 28 mOsmol/kg, and a pH of 6.5. Nebulizing without leakage and showing no toxicity to epithelial cells, NBL display mucoadhesive properties with a 60% mucin-binding efficiency. They effectively traverse the dense mucus layer of Calu-3 cultures in an air-liquid interface, as supported by a 55% decrease in MUC5AC density and a 29% increase in nanoparticles internalization compared to non-exposed cells. In assessing immunomodulatory effects, NBL treatment in SARS-CoV-2-infected lung cells leads to a 40-fold increase in anti-inflammatory MUC1 gene expression, a proportional reduction in pro-inflammatory IL-6 expression, and elevated anti-inflammatory IL-10 expression. These findings suggest a potential mechanism to regulate the excessive IL-6 expression triggered by virus infection. Therefore, the NBL platform demonstrates promising potential for efficient pulmonary drug delivery and immunomodulation, offering a novel approach to addressing mucus permeation and inflammation in pulmonary diseases.

Exploring the structure-activity relationship (SAR) of Schiff bases as effective compounds in scavenging free radicals

Naphthol-imine compounds containing N-phenylethylenediamine (1) and quinoline amine (2) moieties were synthesized and characterized. Density Functional Theory (DFT) calculations were performed aiming to obtain information on the chemical properties of these molecules. In silico ADME/Tox profile analyses were performed on molecules 1 and 2 using the web tools pkCSM-pharmacokinetics, Molinspiration, and Osiris Property Explorer. Drug-likeness levels of 1 and 2 were predicted according to the Lipinski rules. The radical scavenger activity of compounds 1 and 2 was determined by in vitro assays such as 1,1-diphenyl-2-picrylhydrazyl free radicals (DPPH.) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals (ABTS.+). Molecules 1 and 2 were ineffective as radical scavengers in the DPPH radical capture assay at the test doses. However, 1 showed activity in eliminating ABTS+ radical with IC50 = 17.4 ± 0.9 µM. The neutral red assay determined the cytotoxicity of 1 and 2 against the V79 cell line. Molecule 1 was more cytotoxic than 2, with IC50 values ​​of 36.67 µM and 51.26 µM, respectively. The alkaline comet assay was used to investigate the in vitro genotoxicity of 1 and 2. After 24 h of exposure to a dose of 20 μM, 1 did not cause significant DNA damage compared to non-exposed cells. The compounds were tested for toxicity using Artemia saline assay.

#2928 Fabry disease may imbalance integrin α3 related to endothelial dysfunction

Abstract Background and Aims Fabry Disease (FD) is a rare disease characterized by a mutation in the GLA gene and consequently by the functional anomaly of the α-Galactosidase A (α-Gal) enzyme. The lack of this functional enzyme leads to lysosomal accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different cell types, causing several symptoms throughout the body, which aggravate during the patient's longevity. We already demonstrated that exposure of endothelial cells to chloroquine, to mimic FD effects, leads to lysosomal accumulation in human endothelial cells. In the present study, we analyzed the endothelial changes in ultrastructural morphology and cell adhesion under evaluation of Integrin α3 expression, an important integrin that stabilizes cell adhesion and is involved in several signaling pathways in endothelial cells. Method Human endothelial cells (EA.hy926, ATCC CRL-2922) were exposed to chloroquine diphosphate (0.5 µg/mL) for 72 hours. The ultrastructural morphology was analyzed through scanning electron microscopy (SEM) and the Integrin α3 expression was analyzed through Western Blot and RT-qPCR. Results Through SEM (Fig. 1C-F) it was observed that after the exposure to chloroquine diphosphate, there was a decrease in cell expansion and adhesion and an increase in vesicular content. Curiously, the analysis of the Integrin α3 gene (ITGA3) and protein expression (Fig. 1A-B) demonstrated a significant increase (P < 0.05) in cells that were exposed to chloroquine diphosphate compared to non-exposed cells (control cells). Conclusion The in vitro condition of the FD in human endothelial cells demonstrated an increase in Integrin α3; however, there was a decrease in cell adhesion and expansion. The evaluation of the Integrin α3 expression could be important for monitoring the endothelial dysfunction in FD, as it is intrinsically involved in cell adhesion, remodeling, and fibrosis. These important changes can lead to renal impairment, proteinuria, and decreased glomerular filtration rate.

Glycocalyx transduces membrane leak in brain tumor cells exposed to sharp magnetic pulsing

Mechanisms by which electric (E) or magnetic (B) fields might be harnessed to affect tumor cell behavior remain poorly defined, presenting a barrier to translation. We hypothesized in early studies that the glycocalyx of lung cancer cells might play a role in mediating plasma membrane leak by low-frequency pulsed magnetic fields (Lf-PMF) generated on a low-energy solenoid platform. In testing glioblastoma and neuroblastoma cells known to overexpress glycoproteins rich in modifications by the anionic glycan sialic acid (Sia), exposure of brain tumor cells on the same platform to a pulse train that included a 5 min 50Hz Lf-PMF (dB/dt ∼ 2 T/s at 10 ms pulse widths) induced a very modest but significant protease leak above that of control nonexposed cells (with modest but significant reductions in long-term tumor cell viability after the 5 min exposure). Using a markedly higher dB/dt system (80 T/s pulses, 70 μs pulse-width at 5.9 cm from a MagVenture coil source) induced markedly greater leak by the same cells, and eliminating Sia by treating cells with AUS sialidase immediately preexposure abrogated the effect entirely in SH-SY5Y neuroblastoma cells, and partially in T98G glioblastoma cells. The system demonstrated significant leak (including inward leak of propidium iodide), with reduced leak at lower dB/dt in a variety of tumor cells. The ability to abrogate Lf-PMF protease leak by pretreatment with sialidase in SH-SY5Y brain tumor cells or with heparin lyase in A549 lung tumor cells indicated the importance of heavy Sia or heparan sulfate glycosaminoglycan glycocalyx modifications as dominant glycan species mediating Lf-PMF membrane leak in respective tumor cells. This “first-physical” Lf-PMF tumor glycocalyx event, with downstream cell stress, may represent a critical and “tunable” transduction mechanism that depends on characteristic anionic glycans overexpressed by distinct malignant tumors.

Analyses of cellular responses of phorate-incubated Lactiplantibacillus plantarum by transcriptomics and proteomics

Lactiplantibacillus plantarum (L. plantarum) is a versatile species that can resist multiple environmental stresses, including pesticides. Our previous work even observed that the L. plantarum P9 could detoxify phorate and other pesticide-induced toxicity in rats and humans. However, the cellular responses of L. plantarum P9 on phorate exposure have not been described. Therefore, this study applied transcriptomics and proteomics to investigate the cellular responses of L. plantarum P9 on phorate exposure. After 8-h incubation of L. plantarum P9 in 10 mg/L phorate, 149 differentially expressed genes and 92 differentially expressed proteins were detected between phorate-exposed and non-exposed cells, suggesting phorate exposure could induce differential gene regulation and metabolism in these bacteria. We observed up-regulation in genes/proteins associated with carbohydrate metabolism, fatty acid biosynthesis, amino acid biosynthesis and purine metabolism. Moreover, phorate stress also resulted in differential expression in some transcriptional/translational regulators, and heat shock proteins. These transcriptomics- and proteomics-level modulation in phorate-exposed cells might together optimize the energy metabolism, cell membrane configuration, and defense mechanisms against phorate-associated toxicity/oxidative stress to enhance cell survival. Our study provides novel insights into the cellular adaptation of L. plantarum to survive in phorate.

Inflammatory Genes Associated with Pristine Multi-Walled Carbon Nanotubes-Induced Toxicity in Ocular Cells.

The wide application of multi-walled carbon nanotubes (MWCNTs) in various fields has raised enormous concerns regarding their safety for humans. However, studies on the toxicity of MWCNTs to the eye are rare and potential molecular mechanisms are completely lacking. This study was to evaluate the adverse effects and toxic mechanisms of MWCNTs on human ocular cells. Human retinal pigment epithelial cells (ARPE-19) were treated with pristine MWCNTs (7-11 nm) (0, 25, 50, 100 or 200 μg/mL) for 24 hours. MWCNTs uptake into ARPE-19 cells was examined using transmission electron microscopy (TEM). The cytotoxicity was evaluated by CCK-8 assay. The death cells were detected by Annexin V-FITC/PI assay. RNA profiles in MWCNT-exposed and non-exposed cells (n = 3) were analyzed using RNA-sequencing. The differentially expressed genes (DEGs) were identified through the DESeq2 method and hub of which were filtered by weighted gene co-expression, protein-protein interaction (PPI) and lncRNA-mRNA co-expression network analyses. The mRNA and protein expression levels of crucial genes were verified using quantitative polymerase chain reaction (qPCR), colorimetric analysis, ELISA and Western blotting. The toxicity and mechanisms of MWCNTs were also validated in human corneal epithelial cells (HCE-T). TEM analysis indicated the internalization of MWCNTs into ARPE-19 cells to cause cell damage. Compared with untreated ARPE-19 cells, those exposed to MWCNTs exhibited significantly decreased cell viabilities in a dose-dependent manner. The percentages of apoptotic (early, Annexin V positive; late, Annexin V and PI positive) and necrotic (PI positive) cells were significantly increased after exposure to IC50 concentration (100 μg/mL). A total of 703 genes were identified as DEGs; 254 and 56 of them were, respectively, included in darkorange2 and brown1 modules that were significantly associated with MWCNT exposure. Inflammation-related genes (including CXCL8, MMP1, CASP3, FOS, CXCL2 and IL11) were screened as hub genes by calculating the topological characteristics of genes in the PPI network. Two dysregulated long non-coding RNAs (LUCAT1 and SCAT8) were shown to regulate these inflammation-related genes in the co-expression network. The mRNA levels of all eight genes were confirmed to be upregulated, while caspase-3 activity and the release of CXCL8, MMP1, CXCL2, IL11 and FOS proteins were demonstrated to be increased in MWCNT-treated ARPE-19 cells. MWCNTs exposure also can induce cytotoxicity and increase the caspase-3 activity and the expression of LUCAT1, MMP1, CXCL2, and IL11 mRNA and protein in HCE-T cells. Our study provides promising biomarkers for monitoring MWCNT-induced eye disorders and targets for developing preventive and therapeutic strategies.

TNF-α Preconditioning Promotes a Proangiogenic Phenotype in hiPSC-Derived Vascular Smooth Muscle Cells.

hiPSC-VSMCs have been suggested as therapeutic agents for wound healing and revascularization through the secretion of proangiogenic factors. However, methods of increasing cell paracrine secretion and survivability have thus far yielded inconsistent results. This study investigates the effect of pre-conditioning of hiPSC-VSMCs with TNF-α and their integration into 3D collagen scaffolds on cellular viability and secretome. hiPSC-VSMCs were dual-plated in a 2D environment. TNF-α was introduced to one plate. Following incubation, cells from each plate were divided and added to type-I collagen scaffolds. TNF-α was introduced to two sets of scaffolds, one from each 2D plate. Following incubation, scaffolds were harvested for their media, tested for cell survivability, cytotoxicity, and imaged. Intra-media VEGF and bFGF levels were evaluated using ELISA testing. hiPSC-VSMCs exposed to TNF-α during collagen scaffold proliferation and preconditioning showed an increase in cell viability and less cytotoxicity compared to non-exposed cells and solely-preconditioned cells. Significant increases in bFGF expression were found in pre-conditioned cell groups with further increases found in cells subsequently exposed during intra-scaffold conditioning. A significant increase in VEGF expression was found in cell groups exposed during both pre-conditioning and intra-scaffold conditioning. Fibroblasts treated with any conditioned media demonstrated increased migration potential. Conditioning hiPSC-VSMCs embedded in scaffolds with TNF-α improves cellular viability and increases the secretion of paracrine factors necessary for wound healing mechanisms such as migration. The online version contains supplementary material available at 10.1007/s12195-023-00764-0.

Metabolic signature of HepaRG cells exposed to ethanol and tumor necrosis factor alpha to study alcoholic steatohepatitis by LC-MS-based untargeted metabolomics.

Despite the high prevalence of alcoholic liver disease, its identification and characterization remain poor, especially in early stages such as alcoholic fatty liver disease and alcoholic steatohepatitis. This latter implies diagnostic difficulties, few therapeutic options and unclear mechanisms of action. To elucidate the metabolic alterations and pinpoint affected biochemical pathways, alcoholic steatohepatitis was simulated in vitro by exposing HepaRG cells to ethanol (IC10, 368mM) and tumor necrosis factor alpha (TNF-α, 50ng/mL) for 24h. This combined exposure was compared to solely ethanol-exposed as well as -nonexposed cells. Four different metabolomics platforms were used combining liquid chromatography, high-resolution mass spectrometry and drift tube ion mobility to elucidate both intracellular and extracellular metabolic alterations. Some of the key findings include the influence of TNF-α in the upregulation of hepatic triglycerides and the downregulation of hepatic phosphatidylethanolamines and phosphatidylcholines. S-Adenosylmethionine showed to play a central role in the progression of alcoholic steatohepatitis. In addition, fatty acyl esters of hydroxy fatty acid (FAHFA)-containing triglycerides were detected for the first time in human hepatocytes and their alterations showed a potentially important role during the progression of alcoholic steatohepatitis. Ethoxylated phosphorylcholine was identified as a potential new biomarker of ethanol exposure.

Characterization of the genotoxic profile of antineoplastic drugs using the cytokinesis-block micronucleus cytome assay

Since antineoplastic agents are frequently used in cancer therapy and able to affect the patient’s DNA, it is important to know the genotoxic consequences on non-cancerous tissue. Therefore, we aimed to characterize the genotoxic profile of antineoplastic drugs belonging to different classes, using the cytokinesis-block micronucleus cytome assay in a human monocytic cell line (THP-1). All tested antineoplastic agents resulted in increased micronucleus formation. Exposure to anthracyclines led to an increased number of vacuolated cells and cell death, while for mitotic spindle inhibitors, (different stages of) cell death and an increased nuclear bud formation was observed. Alkylating agents induce a high proportion of vacuolated cells and increased nuclear bud formation. No striking differences of nuclear division index or nucleoplasmic bridge formation were observed between exposed and non-exposed cells. The here presented class-specific aberrations may facilitate interpretation of genotoxic aberrations when evaluating clinical samples from patients treated with these antineoplastic agents.

Ganoderma lucidum polysaccharide inhibits the proliferation of leukemic cells through apoptosis.

To investigate the cytotoxic effect of polysaccharides derived from Ganoderma lucidum on T lymphocyte leukemia cells. Water-soluble polysaccharides were extracted from the fruit bodies of G. lucidum, purified, and characterized using HPGPC-MALLS and NMR. The cytotoxicity of G. lucidum polysaccharide fraction 5 (GLP5) to T lymphocyte leukemia cell line Jurkatand human immortalized epidermal cell line HaCat was assessed using MTT assay. Apoptosis was assessed using flow cytometry. Expressions of apoptosis-related genes in the cells after being exposed to GLP5 were detected using Western blot assay. GLP5 was a β-(1→3) and β-(1→6) linked glucan. It inhibited the proliferation of Jurkat cells in a concentration-dependent manner and the half-maximal inhibitory concentration (IC50) was 34.5 mg/L but did not suppress the growth of HaCat cells. Apoptotic cells in Jurkatcells were detected to increase with increasing GLP5 concentrations. The expression levels of cleaved caspase-3 were significantly higher after the cells were exposed to 25 and 50 mg/L GLP5 when compared to non-exposed cells (Control). In addition, the expression levels of BAX and Bcl2 were significantly up- and down-regulated after treatment with GLP5 at 25 and 50 mg/L when compared with control (P<0.05), respectively. GLP5 has antiproliferative activity against Jurkat cells and the activity is likely mediated through the activation of apoptosis pathways.

Evaluation of Pulmonary Effects of 3-D Printer Emissions From Acrylonitrile Butadiene Styrene Using an Air-Liquid Interface Model of Primary Normal Human-Derived Bronchial Epithelial Cells.

This study investigated the inhalation toxicity of the emissions from 3-D printing with acrylonitrile butadiene styrene (ABS) filament using an air-liquid interface (ALI) in vitro model. Primary normal human-derived bronchial epithelial cells (NHBEs) were exposed to ABS filament emissions in an ALI for 4hours. The mean and mode diameters of ABS emitted particles in the medium were 175 ± 24 and 153 ± 15nm, respectively. The average particle deposition per surface area of the epithelium was 2.29 × 107 ± 1.47 × 107 particle/cm2, equivalent to an estimated average particle mass of 0.144 ± 0.042μg/cm2. Results showed exposure of NHBEs to ABS emissions did not significantly affect epithelium integrity, ciliation, mucus production, nor induce cytotoxicity. At 24hours after the exposure, significant increases in the pro-inflammatory markers IL-12p70, IL-13, IL-15, IFN-γ, TNF-α, IL-17A, VEGF, MCP-1, and MIP-1α were noted in the basolateral cell culture medium of ABS-exposed cells compared to non-exposed chamber control cells. Results obtained from this study correspond with those from our previous in vivo studies, indicating that the increase in inflammatory mediators occur without associated membrane damage. The combination of the exposure chamber and the ALI-based model is promising for assessing 3-D printer emission-induced toxicity.

Single Cell Transcriptomes of In Vitro Bradyzoite Infected Cells Reveals Toxoplasma gondii Stage Dependent Host Cell Alterations.

Toxoplasma gondii bradyzoites establish chronic infections within their host cells. Recent studies have demonstrated that several parasite effector proteins are translocated to host cells during the bradyzoite stage of chronic infection. To understand the interaction between host cells and bradyzoites at the transcriptomic landscape level, we utilized single-cell RNA-sequencing (scRNA-Seq) to characterize the bradyzoite-induced host cell response. Distinct gene expression profiles were observed in infected host, cells with low parasite mapped reads, and mock (non-exposed) control cells. Gene set enrichment analysis showed that c-Myc and NF-κB signaling and energy metabolic pathways were upregulated by infection. Type I and II interferon response pathways were upregulated in cells with low parasite mapped reads compared to the non-exposed host control cells, and this upregulation effect was reversed in infected cells. Differences were observed in the host cells depending on the differentiation status of the parasites, as determined by BAG1 and SAG1 expression. NF-κB, inflammatory response pathways, and IFN-γ response pathways were downregulated in host cells containing T. gondii BAG1+/SAG1−, whereas this downregulation effect was reversed in case of T. gondii BAG1−/SAG1+. We also identified two distinct host cell subsets that contained T. gondii BAG1+/SAG1−, one of which displayed distinct transcriptomes with upregulated c-Myc expression. Overall, these data clearly demonstrate that host cell transcriptional alteration by bradyzoite infection is different from that of tachyzoite infection, indicating fine-tuning of the host immune response.

Suppression of Cholangiocarcinoma Cell Growth and Proliferation by Atractylodes lancea (Thunb) DC. through ERK-Signaling Cascade.

Objective:The study aimed to investigate the inhibitory effects of AL on the ERK signaling molecules (ERK, p-ERK, cyclin D, and eIF4B) and the growth and proliferation of CCA cells. Materials and methods:The viability of the three CCA cell lines CL-6, HuCCT1, and HuH28 was determined using MTT assay. The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was investigated. The protein extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed by using RT-PCR. Results:The potency of cytotoxic activity of AL (by MTT assay) was about three times higher than the standard drug 5-fluorouracil. The IC50 (concentration that inhibits cell growth by 50%) of AL for the CL-6, HuCCT-1 and HuH28 cell lines were 29.77±6.64, 35.45±4.96, and 35.32±6.69 µg/mL (mean+SD), respectively. The cells were exposed to AL extract at the IC50 for 0, 12, 24, 48, and 72 hours in the absence and presence of Ras/ERK inhibitors (salirasib and XMD8-92). Protein expression was determined by Western blot analysis. The results suggested the lack of significant inhibitory effect of AL on ERK at 48 and 72 hours of exposure in all CCA cell types. On the other hand, a significant inhibitory effect was observed with p-ERK expression in all CCA cell types. Cyclin D was significantly down-regulated at 72 hours of exposure in all cell types with different potencies. The expression of eIF4B was markedly inhibited in HuCCT-1 but slightly inhibited in CL-6 and HuH28 cells. Real-time PCR analysis revealed significant down-regulation of ERK following 72 hours of AL exposure in the HuCCT1 and HuH28, but not CL-6 cell. Conclusion:The ERK signaling cascade and downstream molecules are potential targets of action of AL in CCA.

Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing.

Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as “hyper-down” genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

Lupin γ-conglutin protects against cell death induced by oxidative stress and lipotoxicity, but transiently inhibits in vitro insulin secretion by increasing KATP channel currents

Lupin γ-conglutin beneficially modulates glycemia, but whether it protects against oxidative and lipotoxic damage remains unknown. Here, we studied the effects of γ-conglutin on cell death provoked by hydrogen peroxide and palmitate in HepG2 hepatocytes and insulin-producing MIN6 cells, and if a modulation of mitochondrial potential and reactive oxygen species (ROS) levels was involved. We also investigated how γ-conglutin influences insulin secretion and electrical activity of β-cells. The increased apoptosis of HepG2 cells exposed to hydrogen peroxide was prevented by γ-conglutin, and the viability and ROS content in γ-conglutin-treated cells was similar to that of non-exposed cells. Additionally, γ-conglutin partially protected MIN6 cells against hydrogen peroxide-induced death. This was associated with a marked reduction in ROS. No significant changes were found in the mitochondrial potential of γ-conglutin-treated cells. Besides, we observed a partial protection against lipotoxicity only in hepatocytes. Unexpectedly, we found a transient inhibition of insulin secretion, plasma membrane hyperpolarization, and higher KATP channel currents in β-cells treated with γ-conglutin. Our data show that γ-conglutin protects against cell death induced by oxidative stress or lipotoxicity by decreasing ROS and might also indicate that γ-conglutin promotes a β-cell rest, which could be useful for preventing β-cell exhaustion in chronic hyperglycemia.

Evidence of bystander effect induced by radiofrequency radiation in a human neuroblastoma cell line

In previous studies we demonstrated that radiofrequency (RF) electromagnetic fields (EMF) is able to reduce DNA damage induced by a subsequent treatment with genotoxic agents, resembling the adaptive response, a phenomenon well known in radiobiology. In this study we report on the capability of the culture medium from SH-SY5Y neuroblastoma cells exposed to 1950 MHz to elicit, in recipient non-exposed cells, a reduction of menadione-induced DNA damage (P < 0.05; comet assay), indicating the capability of non-ionizing radiation to elicit a bystander effect. A comparable reduction was also detected in cultures directly exposed to the same EMF conditions (P < 0.05), confirming the adaptive response. In the same exposure conditions, we also evidenced an increase of heat shock protein 70 (hsp70) in culture medium of cells exposed to RF with respect to sham exposed ones (P < 0.05; western blot analysis), while no differences were detected in the intracellular content of hsp70. On the whole, our results evidence a protective effect of RF against menadione-induced DNA damage in directly and non-directly exposed cells, and suggest hsp70 pathway to be investigated as one of the potential candidate underpinning the interaction between RF exposure and biological systems.

Cytotoxicity assessment of Aflatoxin B1 after high voltage atmospheric cold plasma treatment

Aflatoxin B1 (AFB1) is a secondary metabolite produced by Aspergillus flavus and A. parasiticus, and is a known carcinogen in humans and animals. High voltage atmospheric cold plasma (HVACP) technology has already shown promise to decontaminate AFB1 in food and feed. This study aimed to investigate the cytotoxicity of AFB1 after HVACP treatment. AFB1 (100 μM) was treated at 85 kV with HVACP for 0, 2, 5, 10, and 20 min. HepG2 cells were exposed to HVACP-treated AFB1 for 72 h and assessed for cell viability, caspase-3 activity, DNA fragmentation, and protein carbonyls for each treatment time. Cell viability, caspase-3 activity, DNA fragmentation levels, and protein carbonyls contents of HepG2 cells exposed to HVACP-treated AFB1 after 20 min was not significantly different compared to non-exposed HepG2 cells (P > 0.05). However, their contents were significantly higher in non-exposed cells compared to the other HVACP treatment times (P < 0.01). Twenty minutes of HVACP treatment for AFB1 significantly reduced AFB1 cytotoxicity and oxidative damage and showed potential as a safe aflatoxin decontamination technology.

UV disinfection studies on chlorine tolerant bacteria recovered from treated sewage

Chlorine tolerance is a major concern during disinfection of treated wastewater. Here, the UV disinfection efficacy was assessed for controlling two previously reported chlorine tolerant bacteria, Stenotrophomonas maltophilia and Klebsiella sp. along with a standard E.coli strain using a collimated beam apparatus. UV exposure was applied in two sets - short exposure (0–60 s) and long exposure (0–60 min). Bacterial counts immediately after, and 24 h post irradiation (regrowth) were analysed using media plating. Morphological characteristics and intracellular Mn/Fe ratio were examined by SEM and ICP-OES respectively, to understand the possible reasons for sensitivity/ resistance to irradiation. E.coli and Klebsiella sp. were effectively reduced (> 4 log) but S. maltophilia showed low Effective Reduction (ER < 4) at short exposures. S. maltophilia also showed ER < 4 at long UV exposure due to a high regrowth potential (1.25–3.95). Membrane damage was apparent in SEM analysis in Klebsiella sp. whereas cellular aggregation was evident in S. maltophilia upon UV exposure. Intracellular Mn/Fe ratio was found to be low in both sensitive (Klebsiella sp.) and resistant (S. maltophilia) strains, but the Mn concentration in UV irradiated S. maltophilia cells was not reduced as compared to the non-exposed cells indicating a possible accumulation of Mn released by the damaged cells in the surviving cells. The results indicate that chlorine tolerant S. maltophilia is also resistant to UV due to cellular aggregation and possible Mn accumulation. However, further studies are needed to prove this mechanism of UV resistance.

Electronic cigarette aerosols alter the expression of cisplatin transporters and increase drug resistance in oral cancer cells

Tobacco smoking is the leading preventable cause of cancer. Moreover, continued smoking during cancer therapy reduces overall survival. Aware of the negative consequences of tobacco smoking and the challenges of smoking cessation, cancer patients are inquiring whether they should switch to electronic cigarettes (e-cigarettes). To obtain evidence-based data to inform this decision, we examined the effects of e-cigarette aerosol exposure on cisplatin resistance in head and neck cancer cells. Our results show that cancer cells exposed to e-cigarette aerosol extracts and treated with cisplatin have a significant decrease in cell death, increase in viability, and increase in clonogenic survival when compared to non-exposed cells. Moreover, exposure to e-cigarette aerosol extracts increased the concentration of cisplatin needed to induce a 50% reduction in cell growth (IC50) in a nicotine-independent manner. Tobacco smoke extracts induced similar increases in cisplatin resistance. Changes in the expression of drug influx and efflux transporters, rather than activation of cell growth-promoting pathways or DNA damage repair, contribute to e-cigarette induced cisplatin resistance. These results suggest that like combustible tobacco, e-cigarette use might increase chemotherapy resistance, and emphasize the urgent need for rigorous evaluation of e-cigarettes health effects to ensure evidence-based public health policies.

Cytotoxicity and dentin composition alterations promoted by different chemomechanical caries removal agents: A preliminary in vitro study.

BackgroundThe use of chemomechanical agents for caries removal has been indicated as a non-invasive treatment option; however, their possible deleterious effects on the dental-pulp complex have been insufficiently studied. This study assessed the direct cytotoxicity of two chemomechanical caries removal agents (Brix 3000™ - BX and Papacarie Duo™ - PD) on pulp cells from deciduous teeth, as well as to assess the morphology and chemical compositions of the dentin surface after the application of these materials. Material and MethodsThe cells were seeded (50,000 cells/cm²) in a culture medium (DMEM with 10% fetal bovine serum - FBS). After 24 hours, the BX and PD materials were added to 1:20, 1:100, and 1:1000 dilutions. Non-exposed cells were considered as the control group. The viability test (MTT), Trypan Blue assay (TB), and cell morphology (Scanning Electron Microscopy - SEM) were performed after 24 hours of agent application. For the SEM and chemical (energy-dispersive X-ray spectrometry - EDS) dentin evaluation, 0.3-mm-thick dentin discs were obtained and divided into control group (no treatment) and surfaces covered with 37% phosphoric acid, BX, or PD. Data were compared by one-way ANOVA and Tukey’s test (p<0.05). ResultsDecreases in cell viability and numbers of viable cells were observed for both materials, at all dilutions, when compared with the control group (p<0.05). The BX and PD materials did not cause visually perceptible changes, according to SEM, on the surfaces of dentin discs. The EDS analysis did not indicate a statistically significant difference in the levels of calcium (Ca) and phosphorus (P) between the materials and the control group (p>0.05). ConclusionsBoth materials showed cytotoxicity when in direct contact with the pulp cells from deciduous teeth, and the BX material presented lower cytotoxicity than the PD material. Moreover, both materials did not significantly change the dentin composition. Key words:Cell culture, cytotoxicity, dental pulp, papacarie, primary teeth.