Neutral Red Uptake Research Articles

Polarization of avian macrophages upon avian flavivirus infection

Avian Tembusu virus (TMUV) is a newly emerging avian pathogenic flavivirus that spreads rapidly, has an expanding host range and undergoes cross-species transmission. Our previous study identified avian monocytes/macrophages as the key targets of TMUV infection, since the infection of host monocytes/macrophages was crucial for the replication, transmission, and pathogenesis of TMUV. The polarization of host macrophages determines the functional phenotypes of macrophages; however, the effect of TMUV infection on macrophage polarization remains unclear. Here, we analysed the expression spectra of the marker genes of macrophage polarization upon TMUV infection in the HD11 chicken macrophage cell line and primary monocytes/macrophages isolated from the peripheral blood of specific pathogen-free (SPF) chickens and ducks. We found that viral replication mainly induced M1 marker genes and triggered nitric oxide (NO) release at different levels, suggesting that TMUV infection led mainly to host macrophages polarizing into the classically activated (M1) type. The NO that was increased upon infection did not function as an antiviral agent against TMUV, since the replication of TMUV in HD11 cells was not affected by the addition of an organic NO donor. Furthermore, upon TMUV infection, polarized HD11 cells exhibited increased migration but reduced phagocytosis, as evidenced by scratch assay and neutral red uptake assay, respectively. Our present study characterized the polarization of host monocytes/macrophages upon TMUV infection, which may lay a foundation for further research on the immune escape mechanism and pathogenic mechanism of TMUV.

New Class of Anti-Inflammatory Therapeutics Based on Gold (III) Complexes in Intestinal Inflammation-Proof of Concept Based on In Vitro and In Vivo Studies.

Inflammatory bowel diseases (IBD) are at the top of the worldwide rankings for gastrointestinal diseases as regards occurrence, yet efficient and side-effect-free treatments are currently unavailable. In the current study, we proposed a new concept for anti-inflammatory treatment based on gold (III) complexes. A new gold (III) complex TGS 121 was designed and screened in the in vitro studies using a mouse macrophage cell line, RAW264.7, and in vivo, in the dextran sulphate sodium (DSS)-induced mouse model of colitis. Physicochemical studies showed that TGS 121 was highly water-soluble; it was stable in water, blood, and lymph, and impervious to sunlight. In lipopolysaccharide (LPS)-stimulated RAW264.7 cells, the complex showed a potent anti-inflammatory profile, as evidenced in neutral red uptake and Griess tests. In the DSS-induced mouse model of colitis, the complex administered in two doses (1.68 μg/kg, intragastrically, and 16.8 μg/kg, intragastrically, once daily) produced a significant (* p < 0.05) anti-inflammatory effect, as shown by macroscopic score. The mechanism of action of TGS 121 was related to the enzymatic and non-enzymatic antioxidant system; moreover, TGS 121 induced changes in the tight junction complexes expression in the intestinal wall. This is the first study proving that gold (III) complexes may have therapeutic potential in the treatment of IBD.

Nitric-Acid Oxidized Single-Walled Carbon Nanohorns as a Potential Material for Bio-Applications-Toxicity and Hemocompatibility Studies.

The results of in vitro studies of single-walled carbon nanohorn (SWCNH) oxidized materials’ cytotoxicity obtained by the cell membrane integrity (Neutral Red Uptake (NRU)) and metabolic activity (by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) on A549 and human dermal fibroblasts (HDF) cell lines are presented. We also present hemocompatibility studies on human and porcine blood, and an erythrocyte concentrate to prove that the obtained samples will not interfere with blood components. Characterization of the materials is supplemented by ζ-potential measurements, Transmission Electron Microscope (TEM) imaging, and thermogravimetric studies (TG). The presented results show the correlation between the specific surface area of materials and the platelet aggregation, when the ID/IG ratio determined from Raman spectra correlates with hemoglobin release from the erythrocytes (in whole blood testing). A plausible mechanism explaining the observed correlations is given. The cytotoxicity and hemocompatibility studies prove that the studied materials are acceptable for use in biomedical applications, especially a sample SWCNH-ox-1.5 with the best application potential.

Effects of UV-filter Photostabilizers in the Photostability and Phototoxicity of Vitamin A Palmitate Combined with Avobenzone and Octyl Methoxycinnamate.

A challenge for cosmetic and dermatologic products is to develop new high-performance and safer anti-aging products based on new compounds to enhance the stability of retinyl palmitate combined with broad-spectrum UV-filters. Consequently, the aim of this work was to evaluate the effects of three often used avobenzone photostabilizers-ethylhexyl methoxycrylene (EHMCR), tris(tetramethylhydroxypiperidinol) citrate (TTMHP) and tris-biphenyl triazine (TBPT)-on the photostability and phototoxicity of the combination of avobenzone (AVO), octyl methoxycinnamate (OMC) and retinyl palmitate (RP). The photostability studies were performed by the exposure of formulations to UVA radiation. The phototoxicity was evaluated by the 3T3 neutral red uptake phototoxic assay (OECD TG 432). The addition of EHMCR, TBPT, and TTMHP in the formulations, with/or without RP, improved the photostability of AVO and RP, but EHMCR was the most effective in stabilizing RP. In the phototoxicity assay, the combinations AVO-OMC containing or not RP showed phototoxic potential. EHMCR and TTMHP reduced the phototoxicity of the combination AVO-OMC, whereas EHMCR also decreased the phototoxicity of the combination containing RP. Therefore, EHMCR might be used to the photostabilization of formulations of AVO-OMC with/or not RP, while TTMHP can be added to this photounstable UV-filter combination.

Co-electrospun-electrosprayed PVA/folic acid nanofibers for transdermal drug delivery: Preparation, characterization, and in vitro cytocompatibility

In this study, hydrophilic based bioactive nanofibers were produced via an electrospinning and electrospraying simultaneous process. Poly(vinyl alcohol) (PVA), poly(vinyl alcohol)-gelatin (PVA-Gel), and poly(vinyl alcohol)-alginate (PVA-Alg) polymers were used as the matrix material and folic acid (FA) particles were dispersed simultaneously on the surface of the nanofibers. The morphology of the nanofibers (NFs) was uniform and confirmed by scanning electron microscopy. Thermal behavior, chemical structure of the composite nanofibers were investigated by thermogravimetric analysis, and Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy which showed that no chemical bonding between vitamin and polymers. A fast release of FA-loaded electrospun fibers was carried out by UV-Vis in vitro study within the 8 hour-period in artificial sweat solutions (pH 5.44). The obtained PVA/FA, PVA-Gel/FA, and PVA-Alg/FA fibers released 49.6%, 69.55%, and 50.88% of the sprayed FA in 8 h, indicating the influence of polymer matrix and polymer-drug interactions, on its release from the polymer matrix. Moreover, biocompatibility of all developed novel NFs was assessed by two different cytotoxicity tests, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and neutral red uptake (NRU) assay in L929 (mouse fibroblasts) cell lines. In all cases, it is concluded that these new electrospun fibers had fast-release of the vitamin and the hybrid process is suitable for transdermal patch applications, especially for skin-care products. The results of cytocompatibility assays on L929 reveal that all prepared NFs have no or slight cell toxicity. PVA and PVA-Gel with/without FA nanofibers seems more biocompatible than PVA-Alg nanofibers.

Protective effect of Phyllanthus niruri leaf extracts on Saccharomyces cerevisiae cells subjected to oxidative stress

Phyllanthus niruri has been widely used to treat a number of ailments in traditional medicine. The pharmacological and preventive potential of Phyllanthus niruri, such as in urolithiasis, hyperglycemia, hypertension, pain and mild cases of malaria, are well known. The present investigation was designed to study the protective effects of aqueous, methanol and chloroform extract of Phyllanthus niruri against oxidative stress and cytotoxicity induced by hydrogen peroxide (H2O2) in Saccharomyces cerevisiae cells. Cytotoxicity of leaf extracts and H2O2 was identified by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sulforhodamine B assay (SRB) and neutral red uptake (NRU) assays. All the three extracts showed protection against H2O2 induced toxicity and reduced the loss of cell viability proving the cytoprotective effect of the extracts against H2O2 induced toxicity in Saccharomyces cerevisiae cells. The aqueous extract rendered maximum protection when compared to that of methanolic and chloroform extract.

Comparative sensitivity of proliferative and differentiated intestinal epithelial cells to the food contaminant, deoxynivalenol.

The intestinal epithelium is a functional and physical barrier formed by a cell monolayer that constantly differentiates from a stem cell in the crypt. This is the first target for food contaminants, especially mycotoxins. Deoxynivalenol (DON) is one of the most prevalent mycotoxins. This study compared the effects of DON (0-100μM) on proliferative and differentiated intestinal epithelial cells. Three cell viability assays (LDH release, ATP content and neutral red uptake) indicated that proliferative Caco-2cells are more sensitive to DON than differentiated ones. The establishment of transepithelial electrical resistance (TEER), as a read out of the differentiation process, was delayed in proliferative cells after exposure to 1μM DON. Transcriptome analysis of proliferative and differentiated exposure to 0-3μM DON for 24h revealed 4862 differentially expressed genes (DEG) and indicated an effect of both the differentiation status and the DON treatment. KEGG enrichment analysis indicated involvement of metabolism, ECM receptors and tight junctions in the differentiation process, while ribosome biogenesis, mRNA surveillance, and the MAPK pathway were involved in the response to DON. The number of differentially expressed genes and the amplitude of the effect were higher in proliferative cells exposed to DON than that in differentiated cells. In conclusion, our study shows that proliferative cells are more susceptible than differentiated ones to DON and that the mycotoxin delays the differentiation process.

Carbofuran cytotoxicity, DNA damage, oxidative stress, and cell death in human umbilical vein endothelial cells: Evidence of vascular toxicity.

Carbofuran is a broad-spectrum carbamate insecticide, which principally inhibits the acetylcholinesterase (AChE) enzyme in the nervous system. Nonetheless, their selective action is not restricted to a single species and expanded to humans. No studies are available on the toxicological effects of carbofuran in the endothelial cells (ECs), which first confronts the toxicants in blood vessels. Hence, we have exposed the human umbilical vein ECs (HUVECs) with carbofuran for 24 h, which significantly reduced the cell survival to 25.16% and 33.48% at 500 and 1,000 μM analyzed by MTT assay. In the neutral red uptake (NRU) assay, 16.68%, 30.99%, and 58.11% survival decline was found at 250, 500, and 1,000 μM of carbofuran. HUVECs exposed to carbofuran showed significant increase in the intracellular reactive oxygen species (ROS), indicating oxidative stress at low concentrations. In parallel, HUVECs showed hyperpolarization effects in the mitochondrial membrane potential (ΔΨm) upon carbofuran exposure. Carbofuran induced DNA damage in HUVECs measured as 8.80, 11.82, 35.56, and 79.69 Olive tail moment (OTM) in 100-, 250-, 500-, and 1,000-μM exposure groups. Flow cytometric analysis showed apoptotic peak (SubG1) and G2M arrest in the HUVECs exposed to carbofuran. Overall, our novel data confirm that carbofuran is toxic for the EC cells, especially at the higher concentrations, which may affect the vascular functions and possibly angiogenesis. Hence, carbofuran should be applied judiciously, and detailed vascular studies are warranted to gain an in-depth information focusing the transcriptomic and translation changes employing suitable in vivo and in vitro test models.

Inactivation of Herpes Simplex Virus by Photosensitizing Anthraquinones Isolated from Heterophyllaea pustulata.

Heterophyllaea pustulata is a phototoxic plant from Argentina. Aerial parts extracts, high in photosensitizing anthraquinones, have shown in vitro antiviral activity. The purpose of this study was to study the antiherpetic activity of the main purified anthraquinones, even evaluating their competence as photodynamic sensitizers to photo-stimulate the antiviral effect. In vitro antiviral activity against Herpes Simplex virus type I and the photo-inactivation of viral particle were studied by the Neutral Red uptake test and observation of the cytopathic effect. Rubiadin 1-methyl ether and 5,5'-bisoranjidiol produced a significant effect (≥ 80% inhibition) with minimal damage to host cells (subtoxic concentration). Anthraquinones with poor antiherpetic activity at its maximum noncytotoxic concentration showed an important photo-stimulated effect, such is the case of soranjidiol and 5,5'-bisoranjidiol (28.0 ± 6.3 vs. 81.8 ± 2.1% and 15.5 ± 0.3 vs. 89.8 ± 1.7%, respectively). The study also proved the decrease of viral particles, necessary to reduce infection. Therefore, photosensitizing anthraquinones from natural resources could be proposed to develop new treatments for localized viral lesions with antimicrobial photodynamic therapy.

Toxicity assessment of green synthesized Cu nanoparticles by cell-free extract of Pseudomonas silesiensis as antitumor cancer and antimicrobial

Spherical homogeneous 32 nm, protein coated Pseudomonas silesiensis strain A3 CuNPs was investigated for their cytotoxicity effect as well as antimicrobial and antitumor activity. CuNPs cytotoxicity was estimated using human normal lung cell lines (Wi38) against CuNPs with concentrations ranging from 25 to 150 μg/mL using neutral red uptake assay. The cytotoxicity study revealed that the bacterial CuNPs had an impact on Wi38 cell viability at concentrations of 25, 50, 100 and 150 μg/mL CuNPs were 95.8, 91.1, 89.2 and 82.3%, respectively, with a strong correlation coefficient (r = 0.94) and a CuNPs IC50 value of 1057.0 μg/mL. CuNPs exhibit a broad-spectrum antimicrobial activity against various microorganism species, including fungi and Gram positive and negative bacteria using the agar-well diffusion method. The findings revealed that the most sensitive pathogens were Staphylococcus aureus ATCC5638 and Aspergillus flavus ATCC 9643 which tended to have a high inhibition zone diameter (50 and 47 mm, respectively). The minimum inhibitory concentration (MIC) of CuNPs was 50 μg/mL. The minimum lethal concentration (MLC) values were 50 and 75 μg/mL for S. aureus ATCC 5638 and A. flavus ATCC 9643, respectively. MLC/MIC ratio was ≤2, suggesting the CuNPs had a bactericidal or fungicidal effect on both pathogenic strains. Results also indicated that bacterial CuNPs at varying concentrations of 25, 50, 100 and 150 μg/mL were such a good antitumor agent against A549 lung carcinoma cell lines with an IC50 value of 137.5 μg/mL and a cell viability of 89.3, 79.6, 64.9 and 44.1%, respectively. The results also suggested that the biosynthesized-CuNPs were an antimicrobial and anticancer agent that could be used in future in food preservation, biomedicine and pharmaceutical fields.

Physicochemical characterization of a DMPC-based nanoemulsion for dry eye and compatibility test with soft contact lenses in vitro

IntroductionThe use of contact lenses (CL) is often associated with hallmark symptoms of dry eye disease (DED) such as red eye and dryness. Even though lipid-based artificial tears are already marketed for DED, there is little evidence that supports their use while wearing soft CL. MethodsAn oil in water (O/W) nanoemulsion was formulated with a highly-stable oily phase composed of castor oil and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Physicochemical characterization of the DMPC-based nanoemulsion (DMPC-NE) was performed using the Zetasizer Nano ZSP, and its long-term stability was evaluated over 24 months; in addition, the in vitro cytotoxicity of DMPC-NE was determined by Neutral Red Uptake (NRU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Finally, the physical compatibility of the DMPC-NE with soft CL was tested by measuring the power, diameter, thickness and UV transmittance of two different types of CL. ResultsDMPC-NE had a mean particle size of 86.48 ± 4.22 nm, a polydispersity index of 0.22 ± 0.01 and a zeta potential of -33.23 ± 0.93 mV that remained with no changes after 24 months. DMPC-NE formulation, free of preservatives, showed no cytotoxicity and it was compatible with the physical properties of FDA-class II and -class IV CL. ConclusionDMPC-NE is a highly stable formulation for dry eye that is safe to be used while wearing soft CL.

Investigations on the impact of the introduction of the Aloe vera into the hydrogel matrix on cytotoxic and hydrophilic properties of these systems considered as potential wound dressings

In the paper, synthesis of chitosan-based hydrogels modified with Aloe vera juice is presented. The novelty of the research was a combination of hydrogel materials with properties beneficial in viewpoint of their use as modern wound dressings and Aloe vera juice supporting the wound healing process. Hydrogels have been obtained via UV radiation. The impact of the amount of the crosslinking agent as well as the introduction of the Aloe vera juice into the hydrogel matrix has been determined. Performed measurements involved analysis of the swelling ability, characteristics of the surface roughness, determining the release profile of Aloe vera and the contact angles of hydrogels. Furthermore, the analysis of the dehydration process of the polymer membrane, investigations on the cytotoxicity of hydrogels via MTT reduction assay and the neutral red uptake assay as well as the studies on the pro-inflammatory activity have also been performed. It was proved that the addition of Aloe vera juice improves the hydrophilic properties of the materials (e.g. contact angle changed from 82.5° to 73.0°). Next, the use of 25% more of the crosslinker resulted even in the increase of the contact angle by 86%. Modified hydrogels showed higher swelling properties even by 15% than unmodified materials. Furthermore, obtained hydrogels show an ability to release Aloe vera – after 5 h approx. 80% of this additive has been released in an acidic environment. Tested materials do not exhibit cytotoxic properties, the addition of Aloe vera results in an improvement of the viability of L929 murine fibroblasts and, importantly, these materials show lower pro-inflammatory activity than the positive control. Performed investigations allow to state that obtained materials show a great application potential.

648 Utilizing primary human tissue/cell models to assess for pesticide-induced placental toxicity

Chorionic villi serve as the primary interface between mother and fetus. Dysfunction at this interface is implicated in pregnancy complications. Pesticides are widely used in agriculture and vector control, but the full impact on human pregnancy is unknown. Specific pesticides such as organophosphate pesticides (OPs) are suspected to target the developing central nervous system and placenta. We aimed to leverage ex vivo models of the human placenta (primary chorionic villous explants and isolated cytotrophoblasts (CTBs)) for in vitro studies of toxicological agents. Isolated CTBs from 2nd trimester placentas were exposed to 9 diverse pesticides at 5 concentrations and compared to vehicle (0.1% DMSO) and media-only controls. At 24h, viability and cell death were assayed by neutral red lysosomal uptake or lactate dehydrogenase activity, respectively. Benchmark concentrations (BMC10) of a 10% change in cytotoxicity were identified to assess relative potencies. Human chorionic villi explants dissected from placentas were exposed to naled or vehicle for 24h. After exposure, 1st trimester villi were evaluated by gross morphology and 2nd trimester villi by histopathology and immunofluorescence. Sections were scored by 2 blind evaluators for syncytiotrophoblast (STB) denudation, quantified by ImageJ analysis. We used ANOVA and Tukey’s test to determine significant changes between groups. We observed concentration-dependent cytotoxicity with the three OPs (naled, dichlorovos and malathion) in CTBs (p<0.05; Figure 1). Naled was the most toxic pesticide. Gross morphologic evaluation of naled-exposed villi showed frayed appearance and impaired vascularity compared to the control groups. Naled-exposed explants displayed a ∼4-fold increase in STB denudation compared with media-only or vehicle controls (p<0.001, Figure 2). Our results suggest that naled, an OP commonly-used in agriculture and mosquito control is particularly toxic in two human placental models. We demonstrate that primary cell/tissue models can be used to evaluate placental toxicity of pesticide compounds.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Preventive effects of the antioxidant and antigenotoxicAchyrocline satureioides extract against zearalenone-induced mammal cytogenotoxicity and histological damage

Zearalenone (ZEN), aFusarium’s mycotoxin, is immunotoxic, genotoxic, hepatonephrotoxic and, affects the reproductive system. ZEN induces toxic and genotoxic effects on humans and other animals.Achyrocline satureioides has several medicinal properties. Moreover, the aqueous extract ofA. satureioides is a safe agent that exerts low cytotoxicity and no genotoxicity. This extract is a promissory candidate to counteract ZEN effects. The present study aimed to investigate the capacity of cold aqueous extract fromA. satureioides to protect against ZEN multi-target toxicity in different experimental mammal models. Anticytotoxicity was evaluated by neutral red uptake and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium reduction assays. Comet assay and micronuclei test, oxidative stress (TBARs), and histopathological damage were evaluated in Balb/C mice. Anticytotoxic studies indicated that cold aqueous extract (100 and 300 μg/ml) protected from damage induced by ZEN (50 μg/ml) on Vero cells.In vivo studies indicated that ZEN (40 mg/kg body weight) induced an increase of genotoxicity: micronuclei (34 MNPCE/1000 PCE) and increase of damage (tail moment) in blood cells. Also, it increased lipid peroxidation in liver and kidneys and generated several histopathological alterations in both organs. Cold aqueous extract (100 mg/kg body weight) protected from genotoxicity induced by ZEN in both tests. Cold aqueous extract, also, reduced the lipid peroxidation and histopathological damage in liver and kidneys. In conclusion, the cold aqueous extract ofA. satureioides that contains bioactive flavonoids prevents the multi-target toxicity induced by ZEN improving all the parameters evaluatedin vitro andin vivo, which is a valuable and original finding in order to develop future treatments for human and veterinary medicine.

Thermococcus kodakarensis-derived L-asparaginase: a candidate for the treatment of glioblastoma

L-Asparaginase is a chemotherapeutic agent against various types of cancer due to its asparagine depleting ability. Previously, we have characterized a highly active and thermostable L-Asparaginase (TK1656) from Thermococcus kodakarensis and reported its anti-cancerous effects against acute lymphoblastic leukemia. In the current study, we report anti-proliferative and cytotoxic effects of TK1656 on glioblastoma cell line SF767. Our results showed that TK1656 reduced the proliferation of glioblastoma cells after 72 h in a dose-dependent manner while leaving Vero cells unaffected. The IC50 of TK1656 against SF767 cells was calculated as 18 µg/mL by neutral red uptake assay. Phase-contrast microscopy showed pronounced morphological changes in TK1656 treated SF767 cells. Likewise, DAPI staining and DNA laddering showed that TK1656 has strong genotoxic effects on glioblastoma cells. The AO/EB dual staining pointed out the induction of apoptosis in TK1656 treated cells. Activation of Caspases 3, 6, and 8, after TK1656 treatment, further substantiated the notion of apoptosis mediated death in these cells. Furthermore, flow cytometry data showed that TK1656 treatment induced cell cycle arrest with an increased sub-G1 cell population and downregulation of CDK2 and CDK4. Altogether, our data revealed that TK1656 induces apoptosis and cell cycle arrest in glioblastoma cells. Our findings prompt further investigations to approve the use of TK1656 for the effective treatment of glioblastoma.

Cytotoxicity and genotoxicity of methomyl, carbaryl, metalaxyl, and pendimethalin in human umbilical vein endothelial cells

AbstractPesticides have adverse effects on the cellular functionality, which may trigger myriad of health consequences. However, pesticides‐mediated toxicity in the endothelial cells (ECs) is still elusive. Hence, in this study, we have used human umbilical vein endothelial cells (HUVECs) as a model to quantify the cytotoxicity and genotoxicity of four pesticides (methomyl, carbaryl, metalaxyl, and pendimethalin). In the MTT assay, HUVECs exposed to methomyl, carbaryl, metalaxyl, and pendimethalin demonstrated significant proliferation inhibition only at higher concentrations (500 and 1000 μM). Likewise, neutral red uptake (NRU) assay also showed proliferation inhibition of HUVECs at 500 and 1000 μM by the four pesticides, confirming lysosomal fragility. HUVECs exposed to the four pesticides significantly increased the level of intracellular reactive oxygen species (ROS). Comet assay and flow cytometric data exhibited DNA damage and apoptotic cell death in HUVECs after 24 h of exposure with methomyl, metalaxyl, carbaryl, and pendimethalin. This is a first study on HUVECs signifying the cytotoxic–genotoxic and apoptotic potential of carbamate insecticides (methomyl and carbaryl), fungicide (metalaxyl), and herbicide (pendimethalin). Overall, these pesticides may affect ECs functions and angiogenesis; nonetheless, mechanistic studies are warranted from the perspective of vascular biology using in vivo test models.

Efficacy and safety evaluation of alcohol-containing and alcohol-free mouth rinses: A clinicocytological study.

Context:Whether the alcohol-based mouth rinses are as good as nonalcoholic mouth rinses as far as oral mucosal safety is concerned?Aims:The aim of the study was to investigate the oral mucosal safety of widely used alcohol- and nonalcohol-based mouth rinses at their recommended doses.Settings and Design:The clinical and cytological investigations were carried out by enrolling 120 systemically healthy volunteers fulfilling the inclusion criteria. The volunteers were subjected to a repeated mouth rinse for 60 days to either alcohol-based or alcohol-free mouth rinses at their recommended dosages. A comparative analysis for any clinical adverse response on the oral mucosa and efficacy, i.e., reduction of plaque and gingival index was done at the terminal of the exposure. The studies were also carried out to investigate the cytotoxicity and genotoxicity potential of alcohol-based and alcohol-free mouth rinses in the exposed mucosal cells.Subjects and Methods:The data have been presented in comparative account between alcohol-based and alcohol-free mouth rinses in the volunteers at day 0 and day 60. The cytotoxicity and genotoxicity potential of prescribed doses of alcohol- and alcohol-free mouth rinses have also been evaluated using tetrazolium bromide salt 3-(4, 5-dimethylthiazol-2-yl)-2,-diphenyl tetrazolium bromide, neutral red uptake, and trypan blue dye, micronucleus and chromosomal aberrations.Results:The study findings reveal no statistically as well as biologically significant adverse responses of both alcohol-based and alcohol-free mouth rinses at clinical and cytological level.Conclusions:Under cytological observation, repeated dose exposure up to 60 days of the mouth rinses (alcohol-based and alcohol-free) used in the study was found to be effective and safe at their prescribed dosages.

Current Issues in Photosafety Evaluation

Phototoxicity is a toxic response elicited by topically applied or systemically administered photoreactive chemicals after exposure to light and can be broadly categorized into photoirritation, photoallergy, photogenotoxicity, and photocarcinogenicity. The need in the 21st century for accurate evaluation of photosafety has led to the publication of a number of guidelines from government agencies in Europe and the U.S.A. as well as the Organisation for Economic Co-operation and Development (OECD). In this review, we first discuss the mechanisms of phototoxicity and how they can be evaluated. We then discuss the state of the art and challenges now faced in photosafety evaluation of pharmaceuticals and cosmetics. Additionally, we describe the latest developments in OECD test guidelines (TG) for assessing photosafety, including revisions to the in vitro 3T3 neutral red uptake (NRU) phototoxicity test (TG 432) and the newly adopted reactive oxigen species (ROS) assay (TG 495). We will emphasize the importance of selecting the most appropriate means of evaluation with reference to the latest guidelines and other legal criteria for conducting photosafety evaluation.

Assessment of the toxicity and antiproliferative activity of hemocyanins from Helix lucorum, Helix aspersa and Rapana venosa

Hemocyanins (Hcs) are respiratory, oxygen-carrying metalloproteins that are freely dissolved in the hemolymph of many molluscs and arthropods. The interest in hemocyanins has grown significantly since it was found that they can be successfully used in immunotherapy of neoplastic diseases as non-specific or active stimulators of the immune system. The present study aims to assess the cytotoxicity, in vivo toxicity and antiproliferative activity of hemocyanins isolated from marine snail Rapana venosa (RvH), garden snails Helix lucorum (HlH) and Helix aspersa (HaH). For in vitro safety testing, 3T3 Neutral Red Uptake (NRU) test was used. The experiments for antiproliferative activity of the hemocyanins were performed by MTT assay on a panel of cell lines - a model of breast cancer. The in vivo toxicological assessment was performed by regular clinical examinations of hemocyanin-treated laboratory mice and histopathological analysis of hematoxylin/eosin stained preparations of parenchymal organs. The evaluation of the in vitro cytotoxicity showed that the tested hemocyanins does not induce toxic effects in nontumorigenic epithelial cell lines. In contrast, significant reduction of the viability of human breast carcinoma cell lines was found after treatment with high concentrations of hemocyanins. The in vivo experiments showed no signs of organ and systemic toxicity in the hemocyanin-treated animals. The presented data indicate that Hcs show a potential for development of novel anticancer therapeutics due to their beneficial properties, biosafety and lack of toxicity or side effects. Key words: hemocyanins (Hcs); cytotoxicity; antitumor activity; in vivo biosafety testing.

Thiourethane-functionalized fillers: biological properties and degradation resistance.

This study determined the effect of thiourethane-functionalized fillers (TU) on the antimicrobial properties, cytotoxicity, degree of conversion (DC), water sorption (Wsp) and solubility (Wsl) of experimental composites. TU-modified fillers were added at different ratios in experimental composites: 0 (Control-TU0), 25% (TU25), 50% (TU50), 75% (TU75) and 100wt% (TU100). The antimicrobial properties were detected through the exhaustion test and counting of Streptococus mutans colonies for biofilm formation. Cytotoxicity to human gingival fibroblasts was evaluated in three different parameters: XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide), NRU (Neutral Red Uptake assay) and CVDE (Crystal Violet Dye Exclusion test)) at the same cells. ELISA was used to measure the IL-6 and b-FGF biomarkers. DC was determined by Fourier-transformed infrared spectroscopy, while Wsp and Wsl by mass variations. Inhibitory capacity of biofilm formation was not observed for any material. All groups presented at least 70% of cell survival within the observed periods (24h and 7 days). Positive control (toxic) had high IL-6 values and low b-FGF values. No significant variations in DC, Wsp, and Wsl were observed among the experimental groups. The use of thiourethane did not present antimicrobial and cytotoxic activity and the tested materials presented equivalent properties to those conventionally used in dentistry.