Effects of polycyclic aromatic hydrocarbons on the proliferation and differentiation of placental cells

Background The purpose of this study was to investigate the effects of polycyclic aromatic hydrocarbons (PAHs) other than bisphenol A (BPA) and BPA substitutes on placental cells. Methods HTR-8/SVneo cells were treated with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, endocrine disruptors, and 4,4-(9-fluorenylidene)diphenol, which is used as a substitute for BPA-free products. After confirming the dose response for each reagent using the prepared cells, the cells were incubated for 24, 48, and 72 h. Cell viability was confirmed using the XTT assay. Each experiment was performed with the minimum number of samples (n = 3) required for statistical analysis. The results were analyzed using t-tests; p < 0.05 was considered statistically significant. Results After treatment with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, endocrine disruptors, and 4,4-(9-fluorenylidene)diphenol, the absorbance measured using the XTT assay decreased significantly with increasing concentration. The absorbance decreased significantly over time following treatment with each endocrine disruptor at the concentration confirmed by the dose–response analysis. Conclusions This study showed that anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4-(9-fluorenylidene)diphenol—a BPA substitute—affect cell viability and necrosis in the placental cell line. The study indicates the serious effects of PAHs that negatively affect pregnancy but were previously unknown. Further, this study would serve as a reference for the identification of harmful PAHs during pregnancy prognosis in women who are more susceptible to PAH exposure.

Toxic Potentials of Methamphetamine in Neuroblastoma Cell Line and its Effects on the Mitochondrial Activity

The cytotoxicity potentials of methamphetamine (METH) is presumably associated with oxidative stress induced apoptosis, this study therefore, investigated the toxic potentials of METH in neuroblastoma cells and further determined it effects on the mitochondrial activity. Human neuroblastoma SK-N-BE (2) cells cultured in DMEM/F12 were used in this study. The cells were treated acutely with methamphetamine (1, 5, 10, 20, and 50 µg/mL) over 24, and were allowed to recover from METH treatment over 48, 72, and 96 h. Cell viability study was done with Trypanblue exclusion assay. The cell proliferative characteristics of the neuroblastoma cell line were investigated by constructinga cell proliferation curve. Mitochondrial activity was assessed using the XTT Assay. Statistical analysis were done with Graph Pad prism and significant difference were considered at p<0.001, 0.01 and 0.05. The result showed normal growth in the untreated neuroblastoma cell over the 96 h of monitoring. Following treatment with METH, significant decrease in cell growth was observed when treated acutely with 5 and 10 µg/mL METH and allowed till 72 and 96 h recovery period. The SK-N-BE (2) treated with increasing concentration of METH showed no significant difference in cell viability over the recovery period from METH exposure. Toxicity of SK-N-BE (2) cells was only observed when treated with 10 µg/mL of METH. Significant decrease in mitochondria activity was observed when the cells were treated with 5, 10, 20, and 50 µg/mL METH and allowed till 72 h recovery. The result showed that METH is cytotoxic to the SK-N-BE (2) cells and the mechanism of toxicity might be associated with inhibition of mitochondrial activity.

Methods for Candida albicans biofilm formation on temporary soft liner/ Métodos para a formação de biofilme de Candida albicans em reembasador macio temporário

This study evaluated methods for the contamination of a soft liner material (Softone™) with Candida albicans biofilm. Specimens were either submitted or not to pretreatment in artificial saliva in an orbital incubator, and then held suspended in different positions (horizontal or vertical) and different storage conditions (bacteriological incubator or orbital incubator) during biofilm formation. Eight conditions were tested. All specimens were immersed in C. albicans inoculum and stored in an orbital incubator at 75 rpm or in a bacteriological incubator, both at 37ºC for 90 min. Then, they were washed in PBS, and maintained in RPMI-1640 medium under the same conditions for 48 h. The degree of contamination was determined by the XTT assay. Data were submitted to ANOVA 1-factor/Tukey HSD test (α=0.05). Specimens held horizontally in an orbital incubator showed the highest cell viability, while the ones kept vertically in a bacteriological incubator had the lowest viability (p0.0001). The best condition for C. albicans biofilm formation was obtained when specimens were not submitted to pretreatment in saliva and were held horizontally in an orbital incubator.

Lichen Polyphenolic Compounds for the Eradication of Candida albicans Biofilms

Lichens, due to their symbiotic nature (association between fungi and algae), constitute a chemical factory of original compounds. Polyphenolic compounds (depsides and depsidones) are the main constituents of lichens and are exclusively biosynthesized by these organisms. A panel of 11 polyphenols was evaluated for their anti-biofilm activity against Candida albicans biofilms on the maturation phase (anti-maturation) (MMIC50) as well as on preformed 24-h-old biofilm (anti-biofilm) (MBIC50) using the XTT assay. Minimum inhibitory concentrations of compounds (MICs) against C. albicans planktonic yeast were also determined using a broth microdilution method. While none of the tested compounds were active against planktonic cells (IC50 &gt; 100 µg/ml), three depsides slowed the biofilm maturation (MMIC50 ≤12.5 µg/ml after 48 h of contact with Candida cells). Evernic acid was able to both slow the maturation and reduce the already formed biofilms with MBIC50 ≤12.5 µg/ml after 48 h of contact with the biofilm. This compound shows a weak toxicity against HeLa cells (22%) at the minimal active concentration and no hemolytic activity at 100 µg/ml. Microscopic observations of evernic acid and optimization of its solubility were performed to further study this compound. This work confirmed the anti-biofilm potential of depsides, especially evernic acid, and allows to establish the structure–activity relationships to better explain the anti-biofilm potential of these compounds.

Cold Atmospheric Plasma Jet as a Possible Adjuvant Therapy for Periodontal Disease

Due to the limitations of traditional periodontal therapies, and reported cold atmospheric plasma anti-inflammatory/antimicrobial activities, plasma could be an adjuvant therapy to periodontitis. Porphyromonas gingivalis was grown in blood agar. Standardized suspensions were plated on blood agar and plasma-treated for planktonic growth. For biofilm, dual-species Streptococcus gordonii + P. gingivalis biofilm grew for 48 h and then was plasma-treated. XTT assay and CFU counting were performed. Cytotoxicity was accessed immediately or after 24 h. Plasma was applied for 1, 3, 5 or 7 min. In vivo: Thirty C57BI/6 mice were subject to experimental periodontitis for 11 days. Immediately after ligature removal, animals were plasma-treated for 5 min once—Group P1 (n = 10); twice (Day 11 and 13)—Group P2 (n = 10); or not treated—Group S (n = 10). Mice were euthanized on day 15. Histological and microtomography analyses were performed. Significance level was 5%. Halo diameter increased proportionally to time of exposure contrary to CFU/mL counting. Mean/SD of fibroblasts viability did not vary among the groups. Plasma was able to inhibit P. gingivalis in planktonic culture and biofilm in a cell-safe manner. Moreover, plasma treatment in vivo, for 5 min, tends to improve periodontal tissue recovery, proportionally to the number of plasma applications.

Δ9–Tetrahydrocannabinol (THC)-Rich Compositions from Cannabis Have Cytotoxic Activity Against Ovarian Cancer Cells and Act Synergistically With Niraparib in vitro

Ovarian cancer (OC) is the most lethal gynecologic malignancy. Cannabis sativa is being used to treat different medical conditions. We sought to examine the effectiveness of combinations of cannabis compounds against OC. Cytotoxic activity was determined by XTT assay on HTB75 and HTB161 cell lines. Apoptosis and cell cycle were determined by fluorescence-activated cell sorting (FACS). Gene expression was determined by quantitative PCR. The two most active fractions, F5 and F7, from a high &Delta;9&ndash;tetrahydrocannabinol (THC) cannabis strain extract and their standard mix (SM) showed cytotoxic activity against OC cells. The most effective phytocannabinoid combination was THC+cannabichromene (CBC)+cannabigerol (CBG). F5, F7 and SM affected cell cycle, led to cell apoptosis and to a marked reduction in cell migration. Moreover, these fractions act in synergy with niraparib, and were ~50 fold more cytotoxic to OC cells than to normal keratenocytes. Niraparib+F7 treatment was effective on OC patient's cells. F7 and the niraparin+fraction (F5 and F7) treatments reduced Mitogen-Activated Protein Kinase 4 (MAPK4) gene expression; this reduction may act in synergy with the niraparib inhibition of Poly (ADP-ribose) polymerase 1 (PARP1) activity. Combinations of cannabis compounds and niraparib should be examined for efficacy in pre-clinical studies and clinical trials.

Size Attenuated Copper Doped Zirconia Nanoparticles Enhances in Vitro Antimicrobial Properties

Biofilm formation hinders the activity of antimicrobial drugs at the site of infections and any agent that can act on both Gram- positive and Gram- bacteria by inhibiting the bacterial growth and rupture the biofilm is needed for the management of infection. In the present study, we have synthesized ZrO2 NPs and copper doped zirconia nanoparticles (Cu-ZrO2 NPs) and characterized them using dynamic light scattering, X-ray diffractometry and scanning electron microscopy (SEM). The size of the Cu-ZrO2 NPs drastically reduced compared to ZrO2 NPs and the antimicrobial activity was studied against Gram- positive bacteria (Lactobacillus sp.) and Gram- negative bacteria (Pseudomonas aeruginosa), respectively. The synthesized Cu-ZrO2 NPs showed superior inhibitory action against Lactobacillus sp. compared to ZrO2 NPs, due to the negatively charged cell wall of Lactobacillus sp. which could attract readily the positively charged Cu-ZrO2 NPs, thereby inhibiting its activity. The biocompatibility was tested using XTT assay in FL cells and the results demonstrated that Cu-ZrO2 NPs were non- toxic to mammalian cells. Hence, it could be proposed that the synthesized Cu-ZrO2 NPs possess possible biomedical applications and can be used as antibacterial agents without causing toxicity in mammalian cells.

Bio-Morphological Reaction of Human Periodontal Ligament Fibroblasts to Different Types of Dentinal Derivates: In Vitro Study

Understanding the biological and morphological reactions of human cells towards different dentinal derivate grafting materials is fundamental for choosing the type of dentin for specific clinical situations. This study aimed to evaluate human periodontal ligament fibroblasts (hPLF) cells exposed to different dentinal derivates particles. The study design included the in vitro evaluation of mineralized dentine (SG), deproteinized and demineralized dentine (DDP), and demineralized dentine (TT) as test materials and of deproteinized bovine bone (BIOS) as the positive control material. The materials were kept with the hPLF cell line, and the evaluations were made after 24 h, 72 h, and 7 days of in vitro culture. The evaluated outcomes were proliferation by using XTT assays, the morphological characteristics by light microscopy (LM) and by the use of scanning electron microscopy (SEM), and adhesion by using confocal microscopy (CLSM). Overall, the experimental materials induced a positive response of the hPLFs in terms of proliferation and adhesion. The XTT assay showed the TT, and the SG induced significant growth compared to the negative control at 7 days follow-up. The morphological data supported the XTT assay: the LM observations showed the presence of densely packed cells with a modified shape; the SEM observations allowed the assessment of how fibroblasts exposed to DDP and TT presented cytoplasmatic extensions; and SG and BIOS also presented the thickening of the cellular membrane. The CLMS observations showed the expression of the proliferative marker, as well as and the expression of cytoskeletal elements involved in the adhesion process. In particular, the vinculin and integrin signals were stronger at 72 h, while the actin signal remained constantly expressed in all the follow-up of the sample exposed to SG material. The integrin signal was stronger at 72 h, and the vinculin and actin signals were stronger at 7 days follow-up in the sample exposed to DDP material. The vinculin and integrin signals were stronger at 72 h follow-up in the sample exposed to TT material; vinculin and integrin signals appear stronger at 24 h follow-up in the sample exposed to BIOS material. These data confirmed how dentinal derivates present satisfying biocompatibility and high conductivity and inductivity properties fundamental in the regenerative processes. Furthermore, the knowledge of the effects of the dentin’s degree of mineralization on cellular behavior will help clinicians choose the type of dentine derivates material according to the required clinical situation.

Biofilm Formation of Multidrug-Resistant MRSA Strains Isolated from Different Types of Human Infections

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens causing chronic infections, mainly due to its capacity to form biofilms. However, the mechanisms underlying the biofilm formation of MRSA strains from different types of human infections are not fully understood. MRSA strains isolated from distinct human infections were characterized aiming to determine their biofilm-forming capacity, the biofilm resistance to conventional antibiotics and the prevalence of biofilm-related genes, including, icaA, icaB, icaC, icaD, fnbA, fnbB, clfA, clfB, cna, eno, ebpS, fib and bbp. Eighty-three clinical MRSA strains recovered from bacteremia episodes, osteomyelitis and diabetic foot ulcers were used. The biofilm-forming capacity was evaluated by the microtiter biofilm assay and the biofilm structure was analyzed via confocal scanning laser microscopy. The antimicrobial susceptibility of 24-h-old biofilms was assessed against three antibiotics and the biomass reduction was measured. The metabolic activity of biofilms was evaluated by the XTT assay. The presence of biofilm-related genes was investigated by whole-genome sequencing and by PCR. Despite different intensities, all strains showed the capacity to form biofilms. Most strains had also a large number of biofilm-related genes. However, strains isolated from osteomyelitis showed a lower capacity to form biofilms and also a lower prevalence of biofilm-associated genes. There was a significant reduction in the biofilm biomass of some strains tested against antibiotics. Our results provide important information on the biofilm-forming capacity of clinical MRSA strains, which may be essential to understand the influence of different types of infections on biofilm production and chronic infections.

Evaluation of Cocaine Effect on Endogenous Metabolites of HepG2 Cells Using Targeted Metabolomics

Cocaine toxicity has been a subject of study because cocaine is one of the most common and potent drugs of abuse. In the current study the effect of cocaine on human liver cancer cell line (HepG2) was assessed. Cocaine toxicity (IC50) on HepG2 cells was experimentally calculated using an XTT assay at 2.428 mM. The metabolic profile of HepG2 cells was further evaluated to investigate the cytotoxic activity of cocaine at 2 mM at three different time points. Cell medium and intracellular material samples were analyzed with a validated HILIC-MS/MS method for targeted metabolomics on an ACQUITY Amide column in gradient mode with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring. About 106 hydrophilic metabolites from different metabolic pathways were monitored. Multivariate analysis clearly separated the studied groups (cocaine-treated and control samples) and revealed potential biomarkers in the extracellular and intracellular samples. A predominant effect of cocaine administration on alanine, aspartate, and glutamate metabolic pathway was observed. Moreover, taurine and hypotaurine metabolism were found to be affected in cocaine-treated cells. Targeted metabolomics managed to reveal metabolic changes upon cocaine administration, however deciphering the exact cocaine cytotoxic mechanism is still challenging.