Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models.

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation.

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

In vitro activity of essential oils of Lippia sidoides and Lippia gracilis and their major chemical components against Thielaviopsis paradoxa, causal agent of stem bleeding in coconut palms

Essential oils of Lippia sidoides, Lippia gracilis and their main chemical components were investigated for in vitro control of Thielaviopsis paradoxa. Mycelial growth and a number of pathogen conidia were inhibited by the essential oil of L. sidoides at all concentrations tested (0.2; 0.5; 1.0; 3.0 µL mL-1). L. sidoides oil contained 42.33% thymol and 4.56% carvacrol, while L. gracilis oil contained 10% thymol and 41.7% carvacrol. Mycelial growth and conidial production of T. paradoxa were completely inhibited by thymol at a 0.3 µL m-1 concentration. The results suggest that thymol could potentially be used for controlling coconut stem bleeding.

In vitro radical-scavenging activity, toxicity against A. salina, and nmr profiles of extracts of lichens collected from Brazil and Antarctica

Extracts of six lichen species collected from Brazil and Antarctica were investigated for their potential toxicity and radical-scavenging properties. The composition of the extracts was investigated using TLC and NMR, leading to identification of atranorin (1), along with salazinic (2), barbatic (3), α-alectoronic (4), α-collatolic (5), cryptochlorophaeic (6), caperatic (7), lobaric (8), and protolichesterinic (9) acids. All acetone extracts were evaluated for their 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and subjected to Artemia salina bioassay. The free-radical-scavenging activities of each extract (100 µg) ranged from 8.9 ± 0.1% to 38.7 ± 2.5% and the EC50 values ranged from 0.24 ± 2.10 to 3.54 ± 0.28 mg mL-1, while the toxicity of the extracts against A. salina were low (151.0 to >600 µg mL-1).

Characterization of nitrate reductase activity in vitro in Gracilaria caudata J. Agardh (Rhodophyta, Gracilariales)

The marine red alga Gracilaria caudata J. Agardh has been used in Brazil for agar extraction, mainly in the northeast region of the country. Nitrogen availability is the most important abiotic factor in seawater that limits the growth of seaweeds. The enzyme nitrate reductase (NR) is the key regulatory point in the nitrogen assimilation in photosynthetic organisms. This study describes an in vitro assay, characterizing the enzymatic activity of NR in terms of kinetic constants and stability, its oscillation during the day and glucose effect on NR modulation. Maximal peaks of NR activity were recorded at 20 ºC and pH 8.0. The enzymatic stability in crude extracts stored at 3 ± 1 ºC decreased significantly after 48 hours. Apparent Michaelis-Menten constants (K M) for NADH and nitrate were 22 µM and 3.95 mM, respectively. Gracilaria caudata NR activity showed an oscillation under light:dark photoperiod (14:10 hours LD) with 3-fold higher activity during the light phase, peaking after 10 hours of light. Under optimal assay conditions, the maximal activity was 92.9 10-3 U g-1. The addition of glucose induced the enzymatic activity during the light and dark phase, evidencing a possible modulation of this enzyme by the photosynthesis. This relationship can be explained by the need of carbon skeletons, produced by the photosynthetic process, to incorporate the intermediary metabolites of nitrate assimilatory pathway, avoiding the toxic intracellular accumulation of nitrite and ammonium. The optimization of enzymatic assay protocols for NR is essential to establish appropriate conditions to study nutritional behaviour, compare different taxonomic groups and to understand its regulatory mechanism.

In vivo and in vitro effect of imipramine and fluoxetine on Na+,K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM) from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10%) in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27%) in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group). When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80%) occurring at 0.5 mM. We suggest that a) imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b) stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients.

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium

Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186%, L-leucine _ 155% and L-phenylalanine - 168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

In vitro activity of hypnophilin from Lentinus strigosus: a potential prototype for Chagas disease and leishmaniasis chemotherapy

Hypnophilin and panepoxydone, terpenoids isolated from Lentinus strigosus, have significant inhibitory activity onTrypanosoma cruzi trypanothione reductase (TR). Although they have similar TR inhibitory activity at 10 μg/mL (40.3 μM and 47.6 μM for hypnophilin and panepoxydone, respectively; ~100%), hypnophilin has a slightly greater inhibitory activity (~71%) on T. cruzi amastigote (AMA) growth in vitro as well as on in vitro phytohemagglutinin (PHA)-induced peripheral blood mononuclear (PBMC) proliferation (~70%) compared to panepoxydone (69% AMA inhibition and 91% PBMC inhibition). Hypnophilin and panepoxydone at 1.25 μg/mL had 67% inhibitory activity onLeishmania (Leishmania) amazonensis amastigote-like (AMA-like) growth in vitro. The panepoxydone activity was accompanied by a significant inhibitory effect on PHA-induced PBMC proliferation, suggesting a cytotoxic action. Moreover, incubation of human PBMC with panepoxydone reduced the percentage of CD16+ and CD14+ cells and down-regulated CD19+, CD4+ and CD8+ cells, while hypnophilin did not alter any of the phenotypes analyzed. These data indicate that hypnophilin may be considered to be a prototype for the design of drugs for the chemotherapy of diseases caused by Trypanosomatidae.

Esculetin, a coumarin derivative, exerts in vitro and in vivo antiproliferative activity against hepatocellular carcinoma by initiating a mitochondrial-dependent apoptosis pathway

This study investigated the in vitro and in vivo antiproliferative activity of esculetin against hepatocellular carcinoma, and clarified its potential molecular mechanisms. Cell viability was determined by the MTT (tetrazolium) colorimetric assay. In vivoantitumor activity of esculetin was evaluated in a hepatocellular carcinoma mouse model. Seventy-five C57BL/6J mice were implanted with Hepa1-6 cells and randomized into five groups (n=15 each) given daily intraperitoneal injections of vehicle (physiological saline), esculetin (200, 400, or 700 mg·kg-1·day-1), or 5-Fu (200 mg·kg-1·day-1) for 15 days. Esculetin significantly decreased tumor growth in mice bearing Hepa1-6 cells. Tumor weight was decreased by 20.33, 40.37, and 55.42% with increasing doses of esculetin. Esculetin significantly inhibited proliferation of HCC cells in a concentration- and time-dependent manner and with an IC50 value of 2.24 mM. It blocked the cell cycle at S phase and induced apoptosis in SMMC-7721 cells with significant elevation of caspase-3 and caspase-9 activity, but did not affect caspase-8 activity. Moreover, esculetin treatment resulted in the collapse of mitochondrial membrane potential in vitro and in vivo accompanied by increased Bax expression and decreased Bcl-2 expression at both transcriptional and translational levels. Thus, esculetin exerted in vitro and in vivo antiproliferative activity in hepatocellular carcinoma, and its mechanisms involved initiation of a mitochondrial-mediated, caspase-dependent apoptosis pathway.

Antioxidant activity in vivo and in vitro of Halimeda incrassata aqueous extracts

The aim of the present paper was to provide the evidences for the antioxidant activity in Halimeda incrassata (Ellis) Lamouroux aqueous extracts obtained after simple water extraction of the fresh algae at room temperature (23°C). Previously in the literature, only antioxidant activity associated to carotenoids fractions of seaweeds has been reported. From different species of seaweeds, Halimeda incrassata aqueous extract exhibited the highest antioxidant activity on the inhibition of TBARS formed during the spontaneous lipid peroxidation of rat brain homogenates with an IC50 of 0.340mg.mL-1. Halimeda incrassata aqueous extract (0.5mg.mL-1), was also capable of decreasing the in vitro generation of hydrogen peroxide by two distinct metabolic pathways involving glutamic and malonic acids. Also, Halimeda incrassata (at doses of 50, 100 and 200mg.Kg-1) showed a neuroprotective effect in vivo on the gerbil model of bilateral carotid occlusion because of decreasing the locomotor and exploratory activity induced by ischemia. In summary, Halimeda incrassata aqueous extracts exhibit antioxidant properties in different in vitro as well as in vivo models which could be explained by the presence of several hydrosoluble compounds. Further studies on this way are necessary to elucidate the precise structure of these compounds. Low toxicity of most seaweeds to humans, but particularly of Halimeda genus may favor its use as functional food.

In vitro and in vivo antifungal activity of natural inhibitors against Penicillium expansum

Penicillium expansum is the causative agent of apple blue mold. The inhibitory effects of the capsaicin derived from Capsicum spp. fruits and yeast Hansenula wingei against P. expansum were evaluated in an in vitro and in in vivo assay using Fuji apples. The minimum inhibitory concentration of capsaicin determined using the broth micro-dilution method was 122.16 µg mL-1. Capsaicin did not reduce blue mold incidence in apples. However, it was able to delay fungal growth in the first 14 days of the in vivo assay. The in vivo effect of the yeast Hansenula wingei AM2(-2), alone and combined with thiabendazole at low dosage (40 µg mL-1), on the incidence of apple diseases caused by P. expansum was also described. H. wingei AM2(-2) combined with a low fungicide dosage (10% of the dosage recommended by the manufacturer) showed the best efficacy (100%) up to 7 days of storage at 21 ºC, later showing a non-statistically different decrease (p > 0.05) after 14 (80.45%) and 21 days (72.13%), respectively. These results contribute providing new options for using antifungal agents against Penicillium expansum.

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite: Chitin synthase, the enzyme responsible for the synthesis of chitin in fungal cell wall was extracted from young hyphae of Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts, respectively, to the mycoparasite, Piptocephalis virginiana. Crude enzyme was identified and characterized by measuring the incorporation of the substrate [14C]-UDP-N-acetylglucosamine, into chitin. Most activity occurred in mixed membrane fraction. Inhibition of activity with Polyoxin D and activation with proteases, N-acetyl-glucosamine and magnesium and other ions was observed. Properties of the crude enzyme preparation such as cofactor requirement, Vmax , apparent Km value for UDP-GlcNAc, inhibition by Polyoxin D, response to pH and to temperature, and stability at 4°C were determined. Enzyme activity from both fungi displayed basically the same features as the corresponding enzymes reported from other mucoraceous fungi. However, the two preparations from P. articulosus and C. cucurbitarum differed from each other in their expressed activity (i.e., the preparations from ~ articulosus exhibited higher latency and higher specific chitin synthase activity than the corresponding preparations from ~ cucurbitarum). Trypsin was effective in activation only over a narrow concentration range. Acid protease was the most effec.tive activator. En.dogenous protease estimation indicated higher protease activity in C. cucurbitarum than in P. articulosus. The suggestion is made that regulation of chitin synthase activities may be related to host resistance in the mycoparasitic system.

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growthmedium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett–Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150 ng ml-1, respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53 % metabolic activity and 24.8 % DNA synthesis could be obtained, which suggested that IGF-I and IGFII had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells

Senescence-associated β-galactosidase activity in the in vitro ovarian stromal fibroblasts

Introduction: A growing biological research field is the cellular senescence, a mechanism that has been associated, under certain circumstances, with malignant transformation. Given the high incidence of ovarian cancer and its main origin from the ovarian surface epithelium, as well as the possibility that an epithelial-mesenchymal transition occurs, we evaluated both the in vitro growth of stromal fibroblasts from the ovarian cortex and their β-galactosidase activity at pH 6, enzyme whose expression is considered as a marker of replicative senescence. Methods: 48 samples of ovarian cortical fibroblasts from donors without a history of cancer were serially cultured until the end of their replicative life. β-galactosidase activity at pH 6 was quantified in each passage by the chemiluminiscent method. As control, we used ovarian epithelial cell cultures from the same donors. The enzyme activity was also evaluated in fibroblasts previously induced to senescence by exposure to hydrogen peroxide. Results: The analysis of the enzyme activity and the replicative capacity taken together showed that the fibroblast cultures reached the senescent state at passages 4-5, as what happened with the control epithelial cells. Fibroblasts induced to senescence showed high variability in the values of enzymatic activity. Conclusions: The similarity between both types of cells in reaching the senescent state deserves to be taken into account in relation to the epithelialmesenchymal transition that has been proposed to explain their behavior in the genesis of cancer arising from ovarian surface epithelium. Low β-galactosidase activity values at pH 6 would suggest possible inactivation of the response pathways to oxidative stress.