Differentiation of action mechanisms between natural and synthetic repellents through neuronal electroantennogram and proteomic in Aedes aegypti (Diptera: Culicidae).

Natural-based compounds with repellent activity arise nowadays with the possibility to replace commercial synthetic repellents wholly or partially, such as N,N-Diethyl-m-toluamide (DEET). It is due to DEET's demonstrated toxicity and cutaneous irritation for human beings. Besides, research recommends avoiding using it with kids and pregnant women. The search for a repellent product implies early stages of detailed research that resolve the modes of action against the target insect. Therefore the objective of the current study was to analyze neuronal electrophysiological signals and olfactory system protein expression when the Aedes aegypti mosquito with exposition to natural-based repellents. Adult females of Ae. aegypti of Rockefeller strain were exposed to specific concentrations of repellent compounds like geranyl acetate, α-bisabolol, nerolidol, and DEET. The neuronal effect was measured by electroantennography technique, and the effect of exposure to either DEET or a mixture of natural molecules on protein expression was determined with 2D-PAGE followed by MALDI-TOF-mass spectrometry (MS). This approach revealed that DEET affected proteins related to synapses and ATP production, whereas natural-based repellents increased transport, signaling, and detoxification proteins. The proteomic and electrophysiology experiments demonstrated that repellent exposure disrupts ionic channel activity and modifies neuronal synapse and energy production processes.

Impact of Cymbopogon flexuosus (Poaceae) essential oil and primary components on the eclosion and larval development of Aedes aegypti.

The current study describes the effects of sub-lethal concentrations and constituent compounds (citral and geranyl acetate) of Cymbopogon flexuosus essential oil (EO) on the development of Aedes aegypti. We treated eggs with 6, 18, or 30 mg L-1 and larvae with 3 or 6 mg L-1 of EO and its major compounds (citral and geranyl acetate). Citral and geranyl acetate were evaluated at 18, 30, and 42 mg L-1 and compared with commercial growth inhibitors (diflubenzuron and methoprene). We measured larval head diameter, siphon length, and larval length. Finally, we examined concentrations of molt hormone (MH) and juvenile hormone III (JH III) using high-performance liquid chromatography coupled to mass spectrometry. All geranyl acetate concentrations decreased egg hatching, while EO altered molting among larval instars and between larvae and pupae, with an increase in the larval length (3 mg L-1: 6 ± 0.0 mm; 6 mg L-1: 6 ± 0.7 mm) and head width (3 mg L-1: 0.8 ± 0 mm; 6 mg L-1: 0.8 ± 0.0 mm) compared with the control group. We did not detect chromatographic signals of MH and JH III in larvae treated with C. flexuosus EO or their major compounds. The sub-lethal concentrations C. flexuosus EO caused a similar effect to diflubenzuron, namely decreased hormone concentrations, an extended larval period, and death.

Laser photo-thermal therapy of epithelial carcinoma using pterin-6-carboxylic acid conjugated gold nanoparticles.

Gold nanoparticles functionalized with folic acid toward the internalization into cancer cells have received considerable attention recently. Folic acid is recognized by folate receptors, which are overexpressed in several cancer cells; it is limited in normal cells. In this work, pterin-6-carboxylic acid is proposed as an agonist of folic acid since the pterin-6-carboxylic acid structure has a pterin moiety, the same as folic acid that is recognized by the folate receptor. Here a simple photochemical synthesis of gold nanoparticles functionalized with pterin-6-carboxylic acid is studied. These conjugates were used to cause photothermal damage of HeLa cells irradiating with a diode laser of 808 nm. Pterin-6-carboxylic acid-conjugated gold nanoparticles caused the death of the cell after near-infrared irradiation, dose-dependently. These results indicate a possible internalization of AuNPs via folate receptor-mediated endocytosis due to the recognition or interaction between the folate receptors of HeLa cells and pterin, P6CA.

Tetrahydroquinoline/4,5-Dihydroisoxazole Molecular Hybrids as Inhibitors of Breast Cancer Resistance Protein (BCRP/ABCG2).

Multidrug resistance (MDR) is one of the major factors in the failure of many chemotherapy approaches. In cancer cells, MDR is mainly associated with the expression of ABC transporters such as P-glycoprotein, MRP1 and ABCG2. Despite major efforts to develop new selective and potent inhibitors of ABC drug transporters, no ABCG2-specific inhibitors for clinical use are yet available. Here, we report the evaluation of sixteen tetrahydroquinoline/4,5-dihydroisoxazole derivatives as a new class of ABCG2 inhibitors. The affinity of the five best inhibitors was further investigated by the vanadate-sensitive ATPase assay. Molecular modelling data, proposing a potential binding mode, suggest that they can inhibit the ABCG2 activity by binding on site S1, previously reported as inhibitors binding region, as well targeting site S2, a selective region for substrates, and by specifically interacting with residues Asn436, Gln398, and Leu555. Altogether, this study provided new insights into THQ/4,5-dihydroisoxazole molecular hybrids, generating great potential for the development of novel most potent ABCG2 inhibitors.

Design of a Repellent Against Aedes aegypti (Diptera: Culicidae) Using in silico Simulations With AaegOBP1 Protein.

Skin irritation has been reported to be the main adverse effect of excessive use of N,N-diethyl-m-toluamide (DEET) and ethyl 3-acetyl(butyl)amino (IR3535) commercial repellents. Therefore, there is an interest in alternatives of natural origin such as essential oils (EOs) and major compounds, which have repellent effects but have no contraindications. The main purpose of the present study was to identify the repellent effect of selected terpenes on Aedes aegypti Linnaeus, 1762 (Diptera: Culicidae) by in silico analysis based on their affinity with the odorant protein AaegOBP1. The protein-metabolite interactions in 20 terpenes were analyzed using the SwissDock tool. Terpenes presenting the highest affinity compared with commercial repellents were selected to evaluate repellent activity at concentrations 0.1, 10, and 25% against Ae. aegypti. Different periods (0-2, 2-15, 15-60 min) were evaluated with DEET as a positive control. The toxicity of terpenes was verified through Osiris and Molinspiration Cheminformatics Software, and cytotoxicity assays in Vero and HepaRG cells were performed using the MTT method. Two formulations were prepared with polyethylene glycol to evaluate skin long-lasting in vivo assay. The results showed four terpenes: geranyl acetate, nerolidol, α-bisabolol, and nerol, with affinity to AaegOBP1 comparable with DEET and IR3535. Geranyl acetate, nerolidol, and their mixtures showed no cytotoxicity and protection percentages close to 100% during the test at concentrations 10 and 25%. Long-lasting assays with geranyl acetate and nerolidol formulate showed 3 h as maximum protection time with 100% protection percentage. These metabolites and their mixtures are candidates to repellent formulations with times and protection percentages similar to DEET.

Mitochondrial affectation, DNA damage and AChE inhibition induced by Salvia officinalis essential oil on Aedes aegypti larvae.

The aim of this research study was to understand the mechanism of action of Salvia officinalis (Lamiaceae) essential oil (EO) on Aedes aegypti larvae. We evaluated the effect on DNA damage, acetylcholinesterase (AChE) inhibition and mitochondrial enzymatic alterations. The major components were analyzed in silico using OSIRIS and Molispiration free software. Aedes aegypti DNA was extracted from mosquito larvae between third (L3) and fourth (L4) instars to determine the DNA fragmentation or degradation at S. officinalis EO lethal concentrations (LC10, LC20, LC50, and LC90). DNA integrity was assessed in both LCs in larvae treated for 24 h and in larvae homogenized with EO; we also assessed purified DNA larvae by a densitometric analysis. The AChE inhibition was quantified in protein larvae L3-L4 following Ellman's method and the enzymatic activities related to the mitochondrial respiratory chain of mitochondrial proteins was estimated by spectrophotometry. In silico analysis of 1,8-cineol and of α-thujone, major EO components, showed that they were highly permeable in biological membranes without mutagenic risks. Alterations in the integrity of DNA were observed in larvae exposed and homogenized with S. officinalis EO. The EO induced an AChE inhibition of 37 ±â€¯2.6% to IC50. On the other hand, mitochondrial bioenergetics suggest that EO inhibits electrons entry to the respiratory chain, via Complex II. AChE activity alteration causes mortality of individuals, by blocking the insect cholinergic functions. These results indicate that EO affects the integrity of DNA, the mitochondrial respiration chain and the AChE activity.

Effects of new tetrahydroquinoline-isoxazole hybrids on bioenergetics of hepatocarcinoma Hep-G2 cells and rat liver mitochondria.

New molecular hybrids were synthesized by combining tetrahydroquinoline (THQ) and isoxazole (ISX) scaffolds, in search for chemical structures with improved pharmacological properties. Our tetrahydroquinoline (THQ) and isoxazole (ISX) hybrids differ in the X and Y substituents: FM53 (X = H; Y= H), FM49 (X = CH3; Y= OCH3), FM50 (X = Cl; Y= H) and FM48 (X = Cl; Y= OCH3). Aiming at exploring their bioactivity in liver cancer cells, in this paper we report the effect of four THQ-ISX hybrids on viability, respiration and oxidative stress in Hep-G2 human hepatoma cells. In addition, we measured the alterations induced by these compounds on oxygen uptake and respiratory chain enzymes in isolated mitochondria. Cell viability assay indicated that these THQ-ISX hybrids displayed antiproliferative activity on Hep-G2 cells. Among these, FM50 (IC50 = 5.2 ±â€¯1.9 µM) and FM53 (IC50 = 6.8 ±â€¯0.7 µM) had the highest cytotoxicity. These four hybrids also inhibited the Hep-G2 cells respiration in the uncoupled state, with FM50 decreasing all respiratory states (basal, leak, uncoupled). While only FM49 and FM53 altered the Hep-G2 cells redox function. In terms of mitochondrial bioenergetics, THQ-ISX hybrids decreased the oxygen consumption in state 3 (via complex I and II), and also inhibited NADH oxidase and NADH cytochrome c reductase enzyme activities. In these experiments, the structural homologues FM50 and FM53 had a remarkable inhibitory effect (~50%) with respect to FM49 and FM48. These results show that THQ-ISX hybrids are promising compounds for hepatoma cancer treatment and that the phenyl substituent (Y= H) in the ISX scaffold intensifies both, the cytotoxicity in Hep-G2 cells and, inhibition of electron transport through complex I of the mitochondrial respiratory chain.

Efecto del linalool sobre la bioenergética de mitocondrias de hígado de rata / Linalool effect on mitochondrial bioenergetics rat liver

Antecedentes: El linalool (2,6-dimetil-2,7-octadien-6-ol) es un monoterpeno presente en más de 200 tipos de plantas, y constituye el componente mayoritario de varios aceites esenciales. El linalool posee una amplia variedad de potenciales propiedades farmacológicas; que van desde propiedades antisépticas, ansiolíticas, anti-inflamatorias, antifúngicas, antimicrobianas y proapotóticas en diferentes líneas de células tumorales. Sin embargo, su principal uso está relacionado como una molécula odorante o fragancia en productos de uso tópico en productos cosméticos y en la industria del perfume. Objetivo: En este estudio se evaluó el efecto del linalool sobre la bioenergética de mitocondrias aisladas de hígado de rata. Métodos: El linalool en concentraciones entre (5-100µg/mL) fue evaluado en mitocondrias aisladas de ratas machos albinas raza Wistar, de acuerdo al método descrito por Voss et al (1961). Inicialmente, se determinó el consumo de oxígeno en mitocondrias intactas mediante un método polarográfico por medio de un oxígrafo. Posteriormente, se determinó el efecto del compuesto sobre las enzimas de la cadena respiratoria mitocondrial usando métodos polarográficos y espectrofotométricos. Resultados: Los resultados obtenidos indican que el linalool en las concentraciones de 5, 10, 50 y 100µg/mL, afecta la velocidad de consumo de oxígeno en el estado 3 y 4 de mitocondrias energizadas con glutamato-malato y succinato. Sin embargo, el efecto inhibitorio es mayor al usar glutamato-malato como sustrato. Por su parte, el análisis de la actividad de las enzimas de la cadena respiratoria mitocondrial mostró que el linalool (50µg/mL) inhibe los complejos I, III y la actividad ATPasa. Conclusiones: El linalool inhibe el complejo I, complejo III y la ATPasa, esta inhibición podría aumentar la generación de especies reactivas de oxígeno.

Background: The linalool (2,6-dimethyl-2 ,7-octadiene-6-ol) is a monoterpene present in more than 200 types of plants, and it is the major component of several essential oils. The linalool has a wide range of potential pharmacological properties, such as antiseptic, anxiolytic, anti-inflammatory, antifungal, antimicrobial and proapoptotic in different tumor cell lines. However, its primary use is associated as an odorant molecule or fragrance in topical products, in cosmetics products and fragrance industry. Objective: In this study the effect of linalool on the bioenergetics of isolated rat liver mitochondria was evaluated. Methods: Linalool in concentrations between (5-100µg/mL) was evaluated in isolated mitochondria of male albino Wistar rats, according to the method described by Voss et al (1961). Initially, the oxygen consumption by intact mitochondria was determined by polarographic method using an oxygraph. Subsequently, the effect of the compound on mitochondrial respiratory chain enzymes was evaluated, by using polarographic and spectrophotometric methods. Results: The results obtained indicated that linalool, at concentrations of 5, 10, 50 and 100µg/mL, affects the rate of oxygen consumption in state 3 and 4 of mitochondria energized with glutamate-malate and succinate. However, the inhibitory effect was greater when using glutamate-malate as substrate. For its part, the analysis of the enzyme activities of the mitochondrial respiratory chain showed that linalool (50µg/mL) inhibits complex I, III, and ATPase activity. Conclusions: linalool inhibits complex I, complex III and ATPase, this inhibition could increase the generation of reactive oxygen species.

Essential oils with insecticidal activity against larvae of Aedes aegypti (Diptera: Culicidae).

Insecticidal activity of the essential oils (EOs) isolated from Tagetes lucida, Lippia alba, Lippia origanoides, Eucalyptus citriodora, Cymbopogon citratus, Cymbopogon flexuosus, Citrus sinensis, Swinglea glutinosa, and Cananga odorata aromatic plants, grown in Colombia (Bucaramanga, Santander), and of a mixture of L. alba and L. origanoides EOs were evaluated on Aedes (Stegomyia) aegypti Rockefeller larvae. The EOs were extracted by microwave-assisted hydrodistillation and characterized by gas chromatography-mass spectrometry (GC-MS). The main components of the EOs were identified using their linear retention indices and mass spectra. The lethal concentrations (LCs) of the EOs were determined between the third and fourth instar of A. aegypti. LC50 was determined by probit analysis using mortality rates of bioassays. All essential oils tested showed insecticidal activity. The following values were obtained for C. flexuosus (LC50 = 17.1 ppm); C. sinensis (LC50 = 20.6 ppm); the mixture of L. alba and L. origanoides (LC50 = 40.1 ppm); L. alba (LC50 = 42.2 ppm); C. odorata (LC50 = 52.9 ppm); L. origanoides (LC50 = 53.3 ppm); S. glutinosa (LC50 = 65.7 ppm); T. lucida (LC50 = 66.2 ppm); E. citriodora (LC50 = 71.2 ppm); and C. citratus (LC50 = 123.3 ppm). The EO from C. flexuosus, with citral (geranial + neral) as main component, showed the highest larvicidal activity.

Two galactomannan preparations from seeds from Mimosa scabrella (bracatinga): Complexation with oxovanadium(IV/V) and cytotoxicity on HeLa cells.

Two galactomannans, GALMAN-A and GALMAN-B, were isolated from seeds of Mimosa scabrella (bracatinga), with deactivation and exposure to native enzymes, respectively. They were treated with oxovanadium(IV) and oxovanadium(V), designated (VO(2+)/VO(3+)) to form GALMAN-A:VO(2+)/VO(3+) and GALMAN-B:VO(2+)/VO(3+) complexes, respectively. The potentiometric studies provided the binding constants for the complexes and the resulting complexed species were a function of pH. (51)V NMR spectra of GALMAN-A:VO(2+)/VO(3+) and GALMAN-B:VO(2+)/VO(3+) at pH 7.8 and at 30 degrees C indicated the occurrence of two types of complexes formed by oxovanadium ions and galactomannans. GALMAN-A:VO(2+)/VO(3+) and GALMAN-B:VO(2+)/VO(3+) caused loss of HeLa cells viability at concentrations of 50-200microg/mL. GALMAN-A:VO(2+)/VO(3+) exhibited low toxicity for 24h, although GALMAN-B:VO(2+)/VO(3+) was extremely toxic, since 50microg/mL was sufficient to decrease HeLa cell viability after 48h by 60%. GALMAN-A gave rise to a slight increase in cell proliferation after 48h at 100microg/mL, whereas GALMAN-B promoted a slight decrease at concentrations of 50-100microg/mL. GALMAN-A:VO(2+)/VO(3+) and GALMAN-B:VO(2+)/VO(3+) exhibited a significant decrease in cell proliferation after 48h, each reaching 60% inhibition at 5-10microg/mL. The complexes which caused this effect were at concentrations 10 times lower than the uncomplexed polymers.

The inhibition of lipoperoxidation by mesoionic compound MI-D: a relationship with its uncoupling effect and scavenging activity.

Important biological activities have been described for mesoionic compounds. We previously reported that MI-D (4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride) inhibited the respiratory chain, collapsed the transmembrane potential, and stimulated ATPase activity in intact rat liver mitochondria. It is known that drugs that affect mitochondrial membrane potential may facilitate the induction of cell death by apoptosis. Mitochondria have also a central role in the generation of reactive oxygen species, therefore it would be important to investigate how MI-D could affect processes related to oxidative stress. In this work, we evaluated the effects of MI-D on the lipoperoxidation and its ability to scavenge free radicals. Interestingly, it was observed that MI-D promoted a strong inhibition of the lipoperoxidation induced by Fe(3+)-ADP/2-oxoglutarate in isolated mitochondria (95%+/-0.27 at the highest concentration of 80 nmol mg(-1) protein) in a dose-dependent manner. However, at the same concentration its effect was less intense (22%+/-3.46) when the lipoperoxidation was initiated by peroxyl radicals generated from the azocompound AAPH. Lipid peroxidation in both coupled and uncoupled submitochondrial particles initiated with Fe(2+)/NADH was also inhibited by MI-D. The inhibition was about four times greater in coupled particles (approximately 34% at 80 nmol mg(-1) protein) in relation to uncoupled. MI-D inhibited the soybean phosphatidylcholine liposomes lipoperoxidation in a dose-dependent manner (5-80 microM) regardless of the radical being generated in lipid or aqueous phase. The mesoionic compound showed ability of scavenging superoxide radical (7, 11 and 31% for 25, 38 and 80 microM, respectively). Our results strongly suggest that the inhibition of lipoperoxidation promoted by MI-D is due to its scavenger action and to its previously described uncoupling effect.