Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

Insights of ethyl acetate fraction from Vassobia breviflora in multidrug-resistant bacteria and cancer cells: from biological to therapeutic.

Cancer and infectious diseases are among the leading causes of death in the world. Despite the diverse array of treatments available, challenges posed by resistance, side effects, high costs, and inaccessibility persist. In the Solanaceae plant family, few studies with Vassobia breviflora species relating to biological activity are known, but promising results have emerged. The phytochemicals present in the ethyl acetate fraction were obtained using ESI-MS-QTOF, and the antioxidants assays 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical capture (ABTS), plasma ferric reduction capacity (FRAP), and total antioxidant capacity (TAC). Cytotoxic activity was evaluated by MTT, Neutral Red, and lactate dehydrogenase (LDH) released. The production of reactive oxygen species, nitric oxide, and purinergic enzymes was also investigated. Antibacterial activity was measured through minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antibiofilm activity, in addition to genotoxicity in plasmid DNA. Five major masses were identified D-glucopyranose II, allyl disulfide, γ-lactones, pharbilignoside, and one mass was not identified. V. breviflora exhibited relevant antioxidant and cytotoxic activity against the HeLa cell line and enhanced expression effect in modulation of purinergic signaling. Antibacterial activities in the assays in 7 ATCC strains and 8 multidrug-resistant clinical isolates were found. V. breviflora blocked biofilm formation in producing bacteria at the highest concentrations tested. However, there was no plasmid DNA cleavage at the concentrations tested. Data demonstrated that V. breviflora exhibited an antioxidant effect through several methods and proved to be a promising therapeutic alternative for use against tumor cells via purinergic signaling and multidrug-resistant microorganisms, presenting an anti-biofilm effect.

New insights into the antiviral activity of nordihydroguaiaretic acid: Inhibition of dengue virus serotype 1 replication.

BACKGROUND: Dengue virus (DENV) is considered one of the most important pathogens in the world causing 390 million infections each year. Currently, the development of vaccines against DENV presents some shortcomings and there is no antiviral therapy available for its infection. An important challenge is that both treatments and vaccines must be effective against all four DENV serotypes. Nordihydroguaiaretic acid (NDGA), isolated from Larrea divaricata Cav. (Zygophyllaceae) has shown a significant inhibitory effect on a broad spectrum of viruses, including DENV serotypes 2 and 4. PURPOSE: We evaluated the in vitro virucidal and antiviral activity of NDGA on DENV serotype 1 (DENV1), including the study of its mechanism of action, to provide more evidence on its antiviral activity. METHODS: The viability of viral particles was quantified by the plaque-forming unit reduction method. NDGA effects on DENV1 genome and viral proteins were evaluated by qPCR and immunofluorescence, respectively. Lysosomotropic activity was assayed using acridine orange and neutral red dyes. RESULTS: NDGA showed in vitro virucidal and antiviral activity against DENV1. The antiviral effect would be effective within the first 2 h after viral internalization, when the uncoating process takes place. In addition, we determined by qPCR that NDGA decreases the amount of intracellular RNA of DENV1 and, by immunofluorescence, the number of cells infected. These results indicate that the antiviral effect of NDGA would have an intracellular mechanism of action, which is consistent with its ability to be incorporated into host cells. Considering the inhibitory activity of NDGA on the cellular lipid metabolism, we compared the antiviral effect of two inhibitors acting on two different pathways of this type of metabolism: 1) resveratrol that inhibits the sterol regulatory element of binding proteins, and 2) caffeic acid that inhibits the 5-lipoxygenase (5-LOX) enzyme. Only caffeic acid produced an inhibitory effect on DENV1 infection. We studied the lysosomotropic activity of NDGA on host cells and found, for the first time, that this compound inhibited the acidification of cell vesicles which would prevent DENV1 uncoating process. CONCLUSION: The present work contributes to the knowledge of NDGA activity on DENV. We describe its activity on DENV1, a serotype different to those that have been already reported. Moreover, we provide evidence on which stage/s of the viral replication cycle NDGA exerts its effects. We suggest that the mechanism of action of NDGA on DENV1 is related to its lysosomotropic effect, which inhibits the viral uncoating process.

Deacetylepoxyazadiradione Derived from Epoxyazadiradione of Neem (Azadirachta indica A. Juss) Fruits Mitigates LPS-Induced Oxidative Stress and Inflammation in Zebrafish Larvae.

Reactive oxygen species (ROS) produced by cell metabolism have a duplex role in oxidation and inflammation reactions which involve cell damage or repair responses. Excess ROS production has detrimental effects on the survival of cells. We examined the protective effect of a semi-natural compound NF2 (deacetylepoxyazadiradione), for its protective activity against free radical-mediated stress and inflammatory response to lipopolysaccharide (LPS) using zebrafish larvae. Preliminary antioxidant assays indicated an increase in scavenging of free radicals from NF2 than NF1 (Epoxyazadiradione) in a concentration-dependent manner. Cell cytotoxicity was determined using rat myoblast cell lines (L6), and more than 95 % of cell viability was obtained. Zebrafish developmental toxicity test indicated that NF2 is not toxic even at 150 µM. The percentage of ROS, lipid peroxidation, nitric oxide and apoptosis were reduced significantly in NF2 treated LPS-stressed zebrafish larvae. The reduced number of employed macrophages on NF2 treatment was observed in neutral red dye-marked macrophage localization images. Relative expression of antioxidant genes in zebrafish larvae after treatment with NF2 is significantly increased. The RT-PCR quantification of antioxidant and anti-inflammatory gene expression indicated decreased relative folds of pro-inflammatory cytokines, iNOS and increased relative folds of mitochondrial antioxidant genes (GR, GST and GPx) in LPS stressed zebrafish larvae after treatment with NF2. From the overall obtained results, it can be concluded that NF2 reduced the oxidative stress and inflammatory response by scavenging free radicals caused by LPS.

Protective role of Portuguese natural mineral waters on skin aging: in vitro evaluation of anti-senescence and anti-oxidant properties.

Natural mineral waters (NMWs) emerge from the earth as springs and their beneficial therapeutic effect has been empirically recognized in different countries. Portugal has diverse NMW resources that are sought for the relief of different afflictions including dermatological complications. However, there is a lack of scientific validation supporting this empiric knowledge. In this study, we aimed to screen the in vitro bioactivity of Portuguese NMWs with different chemical profiles, namely sulfurous/bicarbonate/sodic (SBS), bicarbonate/magnesium, sulfated/calcic, sulfurous/chlorinated/sodic, sulfurous/bicarbonate/fluoridated/sodic, and chlorinated/sodic, focusing on aging-related skin alterations. Mouse skin fibroblasts and macrophages were exposed to culture medium prepared in different NMWs. Cellular viability was evaluated by MTT assay and etoposide-induced senescence was analyzed through the beta-galactosidase staining kit. Wound healing was investigated by the scratch assay, and phototoxicity/photoprotection after UVA irradiation was evaluated using a neutral red solution. ROS production was quantified using the 2'7'-dichlorofluorescin diacetate dye, and the activity of superoxide dismutase (SOD) was analyzed by a commercial kit after lipopolysaccharide exposure. NMWs within the SBS profile demonstrated anti-senescence activity in skin fibroblasts, along with a variable effect on cellular viability. Among the tested NMWs, two decreased cellular senescence and preserved cell viability and were therefore selected for subsequent studies, together with a SBS NMW with therapeutic indications for dermatologic diseases. Overall, the selected NMW promoted wound healing in skin fibroblasts and activated SOD in macrophages, thus suggesting an anti-oxidant effect. None of the NMWs prevented phototoxicity after UV irradiation. Our results shed a light on the anti-aging potential of Portuguese NMW, supporting their putative application in cosmetic or medical products.

Preventive potential of Bacillus sonorensis exopolysaccharide upon hepatocellular carcinoma and quantitation of tumor suppressor protein p53.

BACKGROUND: Exopolysaccharide, a carbohydrate polymer, is known to possess several biological activities. This approach was designed to clarify the cytotoxic mechanism of Bacillus sonorensis exopolysaccharide (EPS-1) on Huh7, HepG2 and BNL cells besides exploring its influence on the expression of the tumor suppressor protein p53. p53 is the biomarker of the prognosis and occurrence of severe stages of the tumor and activation of both cell-cycle arrest and apoptosis in cancer cells which are the most targeted cellular processes for the therapy of tumor patients. METHODS: The cytotoxic impact of EPS-1 was quantified via neutral red uptake assay and the results were confirmed by a morphology study. The expression level of p53 was analyzed using quantitative real-time PCR. RESULTS: The outcomes of the present study explicated that EPS-1 with IC 50 = 164 and 398 µg ml -1 exhibited an inhibitory influence on Huh7 and HepG2 cells growth after 48 h incubation time respectively. EPS-1 showed no influence on normal BNL cells. Furthermore, the molecular genetic analysis revealed that EPS-1 provoked significant upregulation in the expression level of the p53 gene in the treated Huh7 cell line more than that in HepG2, whereas no significant gene expression was noticed in BNL cells ( P = 0.006, 0.65 and 0.83), respectively. CONCLUSION: The antitumor activity displayed by this compound may be of interest for further studies of its structure-activity relationship. Before application in phase 1 of the clinical study, in-vivo studies would be needed to confirm the results obtained in the hope of finding more active and selective anticancer agents for drug development in the future.

Rhein induces changes in the lysosomal compartment of HeLa cells.

Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal compartment, which indirectly influences cell death. The experiment was performed on HeLa cells by treating them with rhein at concentrations of 100-300 µM. LC3-II protein and caspase 3/7 activity, level of apoptosis, the concentration of reactive oxide species (ROS), and mitochondrial potential (Δψm) were evaluated by the cytometric method. To evaluate the permeability of the lysosomal membrane (LMP), staining with acridine orange and the assessment of activity of cathepsin D and L in the lysosomal and extralysosomal fractions were used. Cell viability was assessed by -(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) and neutral red (NR) assays. Changes in cells were also demonstrated at the level of electron, optical, confocal, and fluorescence microscopy. Inhibition of autophagy was done using chloroquine. Rhein-induced degradation processes were confirmed by an increase in the number of primary lysosomes, autophagosomes, and autolysosomes. At high concentrations, rhein caused the generation of ROS, which induced LMP expressed by quenching of acridine orange fluorescence. These results correlated with a reduction of lysosomes, as visualized in graphical modeling, with the decreased uptake of NR by lysosomes, and increased activity of cathepsin D and L in the extralysosomal fraction. The studies also showed an increase in the activity of caspase 3/7 and a decrease in the expression of Bcl-2 protein, indicative of rhein-stimulated apoptosis. At the same time, we demonstrated that preincubation of cells with chloroquine inhibited rhein-induced autophagy and contributed to increased cytotoxicity to HeLa cells. Rhein also induced DNA damage and led to cycle arrest in the S phase. Our results indicate that rhein, by inducing changes in the lysosomal compartment, indirectly affects apoptosis of HeLa cells and in combination with autophagy inhibitors may be an effective form of anticancer therapy.

S-allylmercapto-N-acetylcysteine protects bone cells from oxidation and improves femur microarchitecture in healthy and diabetic mice.

Oxidative stress is involved in the deterioration of bone quality and mechanical strength in both diabetic and aging adults. Therefore, we studied the ability of the antioxidant compound, S-allylmercapto-N-acetylcysteine (ASSNAC) to protect bone marrow stromal cells (BMSCs) from advanced glycation end-products (AGEs) cytotoxicity and improve bone microarchitecture of adult healthy and obese/diabetic (db/db) female mice. ASSNAC effect on AGEs-treated cultured rat BMSCs was evaluated by Neutral Red and XTT cell survival and reactive oxygen species (ROS) level assays. Its effect on healthy (C57BL/6) and obese/diabetic (C57BLKS/J Leprdb+/+; db/db) female mice femur parameters, such as (1) number of adherent BMSCs, (2) percentage of CD73+/CD45- cells in bone marrow (BM), (3) glutathione level in BM cells, and (4) femur microarchitecture parameters by microcomputed tomography, was studied. ASSNAC treatment protected BMSCs by significantly decreasing AGEs-induced ROS production and increasing their cellular resistance to the cytotoxic effect of AGEs. ASSNAC treatment of healthy female mice (50 mg/kg/day; i.p.; age 12-20 weeks) significantly increased the number of BMSCs (+60%), CD73+/CD45- cells (+134%), and glutathione level (+110%) in the femur bone marrow. Furthermore, it increased the femur length (+3%), cortical diameter (+3%), and cortical areal moment of inertia (Ct.MOI; +10%) a surrogate for biomechanical strength. In db/db mice that demonstrated a compromised trabecular bone and growth plate microarchitecture, ASSNAC treatment restored the trabecular number (Tb.N, +29%), bone volume fraction (Tb.BV/TV, +130%), and growth plate primary spongiosa volumetric bone mineral density (PS-vBMD, +7%) and thickness (PS-Th, +18%). In conclusion, this study demonstrates that ASSNAC protects bone marrow cells from oxidative stress and may improve bone microarchitecture in adult healthy and diabetic female mice.

Copper Oxide Nanoparticles Exhibit Cell Death Through Oxidative Stress Responses in Human Airway Epithelial Cells: a Mechanistic Study.

Copper oxide nanoparticles (CuONPs) are purposefully used to inhibit the growth of bacteria, algae, and fungi. Several studies on the beneficial and harmful effects of CuONPs have been conducted in vivo and in vitro, but there are a few studies that explain the toxicity of CuONPs in human airway epithelial cells (HEp-2). As a result, the purpose of this study is to look into the dose-dependent toxicity of CuONPs in HEp-2 cells. After 24 h of exposure to 1-40 µg/ml CuONPs, the MTT and neutral red assays were used to test for cytotoxicity. To determine the mechanism(s) of cytotoxicity in HEp-2 cells, additional oxidative stress assays (LPO and GSH), the amount of ROS produced, the loss of MMP, caspase enzyme activities, and apoptosis-related genes were performed using qRT-PCR. CuONPs exhibited dose-dependent cytotoxicity in HEp-2 cells, with an IC50 value of ~ 10 µg/ml. The morphology of HEp-2 cells was also altered in a dose-dependent manner. The involvement of oxidative stress in CuONP-induced cytotoxicity was demonstrated by increased LPO levels and ROS generation, as well as decreased levels of GSH and MMP. Furthermore, activated caspase enzymes and altered apoptotic genes support CuONPs' ability to induce apoptosis in HEp-2 cells. Overall, this study demonstrated that CuONPs can cause apoptosis in HEp-2 cells via oxidative stress; therefore, CuONPs may pose a risk to human health and should be handled and used with caution.

The neutral red assay can be used to evaluate cell viability during autophagy or in an acidic microenvironment in vitro.

Harsh conditions within the tumor microenvironment, such as hypoxia and extracellular acidic pH (pHe), inactivate some chemotherapies, which results in limited or no cytotoxicity. Standard MTT, ATPlite and protease assays that are used to determine the potency of newly developed drugs often give erroneous results when applied under hypoxic or acidic conditions. Therefore, development of a cytotoxicity assay that does not yield false positive or false negative results under circumstances of both hypoxia and acidic pHe is needed. We evaluated currently used cell viability assays as well as neutral red staining to assess viability of ovarian and pancreatic cancer cells grown in an acidic pHe microenvironment after treatment with carboplatin, gemcitabine or chloroquine. We validated cell viability using western blotting of pro-caspase-9 and cleaved-caspase-9, and LC3-I and - II. Standard cell viability assays indicated cell viability accurately at pHe 7.4, but was not correlated with induction of apoptosis or autophagy at acidic pHe. By contrast, our modified neutral red assay detected cell viability accurately over a range of pHe as demonstrated by its correlation with induction of apoptosis and autophagy. Neutral red staining is effective for evaluating the effect of chemotherapeutic agents on cell viability under acidic pHe or hypoxic conditions.

Mechanistic Insight into the Photodynamic Effect Mediated by Neutral Red and a New Azine Compound in Staphylococcus aureus Cells.

The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25 µm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers.

Tracking the evolution of CNS remyelinating lesion in mice with neutral red dye.

Animal models of central nervous system (CNS) demyelination, including toxin-induced focal demyelination and immune-mediated demyelination through experimental autoimmune encephalomyelitis (EAE), have provided valuable insights into the mechanisms of neuroinflammation and CNS remyelination. However, the ability to track changes in transcripts, proteins, and metabolites, as well as cellular populations during the evolution of a focal lesion, has remained challenging. Here, we developed a method to label CNS demyelinating lesions by the intraperitoneal injection of a vital dye, neutral red (NR), into mice before killing. We demonstrate that NR-labeled lesions can be easily identified on the intact spinal cord in both lysolecithin- and EAE-mediated demyelination models. Using fluorescence microscopy, we detected NR in activated macrophages/microglia and astrocytes, but not in oligodendrocytes present in lesions. Importantly, we successfully performed RT-qPCR, Western blot, flow cytometry, and mass spectrometry analysis of precisely dissected NR-labeled lesions at 5, 10, and 20 d postlesion (dpl) and found differential changes in transcripts, proteins, cell populations, and metabolites in lesions over the course of remyelination. Therefore, NR administration is a simple and powerful method to track and analyze the detailed molecular, cellular, and metabolic changes that occur within the lesion microenvironment over time following CNS injury. Furthermore, this method can be used to identify molecular and metabolic pathways that regulate neuroinflammation and remyelination and facilitate the development of therapies to promote repair in demyelinating disorders such as multiple sclerosis.

A Selective Inhibitor of Ubiquitin-Specific Protease 4 Suppresses Colorectal Cancer Progression by Regulating ß-Catenin Signaling.

BACKGROUND/AIMS: Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of ß-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities. METHODS: By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of ß-catenin, as shown by immunoblotting, and it affected the target gene expression of ß-catenin, as shown by quantitative real-time PCR. NR's potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model. RESULTS: We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased ß-catenin stability and reduced expression of ß-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume. CONCLUSION: The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents.

Predicting Cytotoxicity and Enzymatic Activity of Diverse Chemicals Using Goldfish Scale Tissue and Topminnow Hepatoma Cell Line-based Data.

Quantitative structure-toxicity relationship (QSTR) models were built for two in vitro endpoints: cytotoxicity and enzymatic activity of diverse chemicals to goldfish (Crassius auratus) scale tissue (GFS) and topminnow (Poeciliopsis lucida) hepatoma cell line (PLHC-1), respectively. The data sets were based on experimental cytotoxicity measured with uptake of 3-amino-7-dimethylamino-2-methylphenazine hydrochloride dye (Neutral Red assay) representing lysosomal damage and enzymatic activity measured with Ethoxyresorufin-O-deethylase (EROD) induction potency. The descriptors were calculated with DRAGON 6 and SPARTAN 10 software packages. Descriptor selection was made by 'All Subset' and Genetic Algorithm-based features implemented in QSARINS software. The proposed QSTR models validated both internally and externally. Additionally, the QSTR models generated for cytotoxicity and EROD induction potency were used to predict the relevant endpoint values for external set chemicals with structural coverage of 95.0 % and 92.1 %, respectively. A strong correlation of experimental in vivo fish lethality data with predicted in vitro cytotoxicity and EROD induction potency values for external set chemicals was found. It was concluded that the proposed QSTR models might be useful to provide an initial screening and prioritization for these diverse chemicals. Also, regarding the strong correlations between predicted in vitro and experimental in vivo data, the use of QSTR predictions as an alternative to the acute fish toxicity assessment can be claimed.

Development of macaque secondary follicles exposed to neutral red prior to 3-dimensional culture.

PURPOSE: Neutral red (NR) may assist identification of preantral follicles in pieces of cortical tissue prior to cryopreservation in cancer patients requesting fertility preservation. This study is the first to analyze this effect by follicle growth rate after long-term culture in primates. METHODS: Ovarian cortex was obtained from adult rhesus macaques, was cut into fragments, and was incubated with NR. Secondary follicles were readily visualized following NR staining and then were encapsulated into alginate beads and cultured individually for 4 weeks in αMEM media supplemented with 10 ng/ml FSH at 5% O2. RESULTS: The survival rates of secondary follicles during culture were similar between those derived from control tissue (71 ± 13%) and those treated with NR (68 ± 9%). The proportion of surviving follicles that formed an antrum were also similar in both groups (70 ± 17% control; 48 ± 24% NR-treated). Follicle diameters were not different between control follicles (184 ± 5µm) and those stained with NR (181 ± 7 µm) on the day of isolation. The percentages of surviving follicles within three cohorts based on their diameters at week 4 of culture were similar between the control group and NR-stained tissue group, fast-grow follicles (24 ± 6% vs. 13 ± 10%), slow-grow follicles (66 ± 5% vs. 60 ± 9%), or no-grow (10 ± 9% vs. 27 ± 6%), respectively. There were no differences in follicle diameters between groups during the culture period. Pre-exposure of secondary follicles to NR diminished their capacity to produce both estradiol and androstenedione by week 4 of culture, when follicles are exhibiting an antrum. Inhibitory effects of NR on steroid production by slow-grow follicles was less pronounced. CONCLUSIONS: NR does not affect secondary follicle survival, growth, and antrum formation during long-term culture, but steroid hormone production by fast-grow follicles is compromised. NR can be used as a non-invasive tool for in situ identification of viable secondary follicles in ovarian cortex before tissue cryopreservation without affecting follicle survival and growth in vitro. Whether maturation or developmental competence of oocytes derived from antral follicles in 3D culture that were previously isolated from NR-stained tissue is normal or compromised remains to be determined. Likewise, the functional consequences of pre-exposure to NR prior to ovarian cortical tissue cryopreservation and transplantation are unknown.

Individual marking of soft-bodied subtidal invertebrates in situ - A novel staining technique applied to the giant plumose anemone Metridium farcimen (Tilesius, 1809).

The ability to recognize individuals and track growth over time is crucial to population dynamics research as well as studies of animal behavior. Invertebrates are particularly difficult to track as they often molt, have regenerative capabilities, or lack hard parts to attach markers. We tested, in laboratory and field studies, a new way of marking sea anemones (order Actiniaria) by injection of three vital stains (i.e., neutral red, methylene blue, and fluorescein). Neutral red and methylene blue did not affect growth or survival, but fluorescein was lethal at high concentrations. Marked individuals could be identified up to seven months after injection with neutral red, six weeks with methylene blue, and three days with low concentrations of fluorescein. Neutral red could be used for long-term monitoring of growth and survival in the field, and in combination with methylene blue could be used to mark individuals in distinguishable patterns for short-term studies such as examining predator-prey interactions, movement of individuals, and recruitment survival.

The monoamine oxidase inhibition properties of selected structural analogues of methylene blue.

The thionine dye, methylene blue (MB), is a potent inhibitor of monoamine oxidase (MAO) A, a property that may, at least in part, mediate its antidepressant effects in humans and animals. The central inhibition of MAO-A by MB has also been linked to serotonin toxicity (ST) which may arise when MB is used in combination with serotonergic drugs. Structural analogues and the principal metabolite of MB, azure B, have also been reported to inhibit the MAO enzymes, with all compounds exhibiting specificity for the MAO-A isoform. To expand on the structure-activity relationships (SARs) of MAO inhibition by MB analogues, the present study investigates the human MAO inhibition properties of five MB analogues: neutral red, Nile blue, new methylene blue, cresyl violet and 1,9-dimethyl methylene blue. Similar to MB, these analogues also are specific MAO-A inhibitors with cresyl violet (IC50=0.0037µM), Nile blue (IC50=0.0077µM) and 1,9-dimethyl methylene blue (IC50=0.018µM) exhibiting higher potency inhibition compared to MB (IC50=0.07µM). Nile blue also represents a potent MAO-B inhibitor with an IC50 value of 0.012µM. From the results it may be concluded that non-thionine MB analogues (e.g. cresyl violet and Nile blue) also may exhibit potent MAO inhibition, a property which should be considered when using these compounds in pharmacological studies. Benzophenoxazines such as cresyl violet and Nile blue are, similar to phenothiazines (e.g. MB), representative of high potency MAO-A inhibitors with a potential risk of ST.

Enhanced Butanol Production Through Adding Organic Acids and Neutral Red by Newly Isolated Butanol-Tolerant Bacteria.

As alternative microorganisms for butanol production with high butanol tolerant and productivity are in high demand, one excellent butanol-tolerant bacterium, S10, was isolated and identified as Clostridium acetobutylicum S10. In order to enhance the performance of butanol production, organic acids and neutral red were added during butanol fermentation. Synergistic effects were exhibited in the combinations of organic acids and neutral red to promote butanol production. Consequently, the optimal concentrations of combined acetate, butyrate, and neutral red were determined at sodium acetate 1.61 g/L, sodium butyrate 1.88 g/L, and neutral red 0.79 g/L, respectively, with the butanol yield of 6.09 g/L which was 20.89 % higher than that in control. These results indicated that combination of adding organic acid and neutral red is a potential effective measure to improve butanol production.

In vitro photosafety and efficacy screening of apigenin, chrysin and beta-carotene for UVA and VIS protection.

Currently most of sunscreens provide effective protection in the full UV range but lack VIS protection. The addition of effective antioxidants to sunscreens might afford suitable UV-VIS protection. Apigenin (API), chrysin (CRI) and beta-carotene (BTC) have shown potential for UV-VIS protection. This paper reports a photosafety and efficacy screening of such antioxidants through evaluation of the photostability, photoreactivity and phototoxicity as well as UVA/UVB ratio and critical wavelength. The assessment of the photostability, photoreactivity and phototoxicity of API, CRI and BTC, isolated and combined (CMB) was performed by HPLC, ROS assay and 3T3 NRU phototoxicity test, respectively. The phototoxicity test was also performed for CMB plus bemotrizinol (BMZ). The in vitro evaluation of the UVA protection was assessed by the determination of the UVA/UVB ratio and the critical wavelength. The antioxidants API, CRI, BTC and CMB were stable under UVA/VIS and VIS light. However weak photoreactivity after UVA/VIS irradiation was observed for API, CRI and CMB in the ROS assay. In the 3T3 NRU phototoxicity test, phototoxic potential was observed for CRI, BTC, CMB and CMB+BMZ after UVA/VIS exposure, and for BTC and CMB after VIS exposure. BMZ reduced the phototoxic potential of CMB in the VIS range. In the in vitro evaluation of UVA protection API, CRI, BTC, CMB and CMB+BMZ presented ultra UVA protection (UVA/UVB ratio>0.9) and exhibited critical wavelength close to or above 370nm. In conclusion, the use of API, CRI, BTC and their CMB aiming skin photoprotection could be considered safer in the VIS range. Furthermore, API presented the best performance in the photosafety screening among the studied antioxidants, since it was photostable and non-phototoxic in UVA/VIS and photostable, non-photoreactive and non-phototoxic in VIS range.

Chronic toxicity of commercial chlorpyrifos to earthworm Pheretima peguana.

A chronic toxicity study was conducted in earthworms (Pheretima peguana) exposed to soil spiked with chlorpyrifos at concentrations of 0, 0.1, 1, 10, and 100 mg/kg soil dry matter for 7, 14, and 28 days. The integrity of the coelomocyte lysosomal membrane, nervous system, and male reproductive tissue was monitored using, respectively, the neutral-red retention assay, acetylcholinesterase (AChE) enzyme assay, and histomorphology of spermatogenic cells in the seminal vesicles and cocoon production (at 28 days after 28 days' exposure). Chlorpyrifos decreased the coelomocyte neutral-red retention time (NRRT) significantly (p < 0.05) at concentrations > 0.1 mg/kg soil as early as day 7 of exposure and was dose- and time-dependent. Chlorpyrifos inhibition of AChE activity was greater at day 7 than at day14 (p < 0.05) indicating possibly nerve recovery. Chlorpyrifos induced concentration-dependent damage to spermatogenic cells and cytophores in premature stages. The number and size of premature, maturing, and fully mature spermatogenic stages were increased at low concentrations (<1 mg/kg) but a number of these maturation stages declined at higher concentrations (10 and100 mg/kg) on day 28. The most severe effects were observed in the maturing and fully mature stages at the highest chlorpyrifos concentration, and this had an adverse impact on cocoon production and cocoon viability. Collectively, the results suggest induction of widespread effects on multiple organ systems in P. peguana exposed to chlorpyrifos. Although NRRT and AChE activity were the most sensitive of the biomarkers, cocoon production and cocoon viability could still be considered as diagnostic tools for monitoring effects from low-dose long-term chlorpyrifos toxicity and for evaluating population effects. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1450-1459, 2016.