Toxicological impact of JWH-018 and its phase I metabolite N-(3-hydroxypentyl) on human cell lines

"The emergence and abuse of synthetic cannabinoids has been increasing as an alternative to cannabis, mainly among youth. As their appearance on the drug market has been recent, the pharmacological and toxicological profiles of these psychoactive substances are poorly understood. Current studies suggest that they have stronger effects compared to their natural alternatives and their metabolites retain affinity towards CB1 receptors in CNS. Since studies on its toxicological properties are scarce, the effects of the drug in human derived cell lines were investigated. The present study was designed to explore the toxicological impact of parent drug versus phase I metabolites of synthetic cannabinoids on human cells with and without CB1 receptor. The human cell line of neuroblastoma SH-SY5Y and human kidney cell line HEK-293T were exposed to JWH-018 and to its N-(3-hydroxypentyl) metabolite. Cell toxicity was evaluated using the MTT and LDH assay. Additionally, a dual staining methodology with fluorescent Annexin V-FITC and propidium iodide was performed to address the question of whether JWH-018 N-(3-hydroxypentyl) metabolite is inducing cell death through apoptosis or necrosis, in HEK293T and SH-SY5Y cell lines. The obtained results show that JWH-018 does not cause a statistically significant decrease in cell viability, in contrast to its N-(3-hydroxypentyl) metabolite, which at ≥25μM causes a significant decrease in cell viability. Both cell lines are affected by JWH-018 metabolite. Our results point to higher toxicity of JWH-018 metabolite when compared to its parent drug, suggesting a non-CB1 receptor mediated toxicological mechanism. Comparing the results from Annexin V/PI with MTT and LDH assays of SH-SY5Y and HEK293T in the presence of the synthetic cannabinoid metabolite, emerges the picture that cellular viability decreases and associated death is occurring through necrosis."

Sodium chloride added to transport water and physiological responses of Matrinxã Brycon amazonicus (Teleost: Characidae)

A adição de sal à água tem sido utilizada para a mitigação de estresse e aumento da taxa de sobrevivência em peixes. O presente estudo avaliou o efeito do cloreto de sódio (0,0; 1,0; 3,0 e 6.0 g/l) nas concentrações de cortisol plasmático, glicemia, triglicerídios, proteínas total plasmática, hematócrito, hemoglobina, número de eritrócitos, glicogênio e lipídio hepáticos, e lipídio muscular em matrinxã Brycon amazonicum adultos após quatro horas de transporte e durante período de recuperação de 96 h. Amostras foram coletadas antes e depois do transporte, bem como 24 e 96 h após a chegada. O nível de cortisol plasmático estava mais elevado logo após o transporte quando comparado à condição inicial (pré-transporte), exceto para os peixes transportados com sal nas concentrações 3,0 e 6,0 g/l. Comportamento semelhante foi observado para a glicemia, porém os peixes dos tratamentos 0,0, 1,0 e 3,0 g/l necessitaram de período superior a 24 h para recuperar a condição inicial. Foram registrados níveis mais baixos de glicogênio hepático em peixes do tratamento controle (0,0 g/l). Os parâmetros hemoglobina, número de eritrócitos, proteína plasmática total e lipídio hepático não apresentaram alterações durante o período experimental. Os valores de hematócrito diminuíram logo após o transporte em todos os tratamentos, retornando aos níveis iniciais após 24 h. Todos os tratamentos apresentaram redução nos níveis de lipídio muscular e triglicerídios durante o período de recuperação. Os resultados sugerem que a adição de 6,0 g/l de sal na água de transporte reduz as alterações fisiológicas de estresse e que é necessário período de 96 h após o transporte para a recuperação da condição inicial de matrinxãs transportados sem a adição de sal.

Environmental factors correlated with the metabolite profile of Vitis vinifera cv. Pinot Noir berry skins along a European latitudinal gradient

Mature berries of Pinot Noir grapevines were sampled across a latitudinal gradient in Europe, from southern Spain to central Germany. Our aim was to study the influence of latitude-dependent environmental factors on the metabolite composition (mainly phenolic compounds) of berry skins. Solar radiation variables were positively correlated with flavonols and flavanonols and, to a lesser extent, with stilbenes and cinnamic acids. The daily means of global and erythematic UV solar radiation over long periods (bud break-veraison, bud break-harvest, and veraison-harvest), and the doses and daily means in shorter development periods (5–10 days before veraison and harvest) were the variables best correlated with the phenolic profile. The ratio between trihydroxylated and monohydroxylated flavonols, which was positively correlated with antioxidant capacity, was the berry skin variable best correlated with those radiation variables. Total flavanols and total anthocyanins did not show any correlation with radiation variables. Air temperature, degree days, rainfall, and aridity indices showed fewer correlations with metabolite contents than radiation. Moreover, the latter correlations were restricted to the period veraison-harvest, where radiation, temperature, and water availability variables were correlated, making it difficult to separate the possible individual effects of each type of variable. The data show that managing environmental factors, in particular global and UV radiation, through cultural practices during specific development periods, can be useful to promote the synthesis of valuable nutraceuticals and metabolites that influence wine quality.

Exploiting molecular simulations as an atomic-level microscope to elucidate clinically relevant cation chloride cotransporters

The cation chloride cotransporters (CCCs) represent a vital family of ion transporters, with several members implicated in significant neurological disorders. Specifically, conditions such as cerebrospinal fluid accumulation, epilepsy, Down’s syndrome, Asperger’s syndrome, and certain cancers have been attributed to various CCCs. This thesis delves into these pharmacological targets using advanced computational methodologies. I primarily employed GPU-accelerated all-atom molecular dynamics simulations, deep learning-based collective variables, enhanced sampling methods, and custom Python scripts for comprehensive simulation analyses. Our research predominantly centered on KCC1 and NKCC1 transporters. For KCC1, I examined its equilibrium dynamics in the presence/absence of an inhibitor and assessed the functional implications of different ion loading states. In contrast, our work on NKCC1 revealed its unique alternating access mechanism, termed the rocking-bundle mechanism. I identified a previously unobserved occluded state and demonstrated the transporter's potential for water permeability under specific conditions. Furthermore, I confirmed the actual water flow through its permeable states. In essence, this thesis leverages cutting-edge computational techniques to deepen our understanding of the CCCs, a family of ion transporters with profound clinical significance.

Autologous Platelet Gel: Five Cases Illustrating Use On Sickle Cell Disease Ulcers.

Leg ulcers represent a particularly disabling complication in patients with sickle cell disease (SCD). Platelet gel (PG) is a novel therapeutic strategy used for accelerating wound healing of a wide range of tissues through the continuous release of platelet growth factors. Here, we describe the use of PG preparation according to Anitua's PRGF (preparations rich in growth factors) protocol for treating chronic nonhealing ulcers in patients with SCD. A positive response occurred in 3 patients with an area reduction of 85.7% to 100%, which occurred within 7 to 10 weeks, and a 35.2% and 20.5% of area reduction in 2 other patients, who however, had large ulcers. After calcium chloride addition, the platelet-rich plasmas demonstrated enhanced platelet-derived growth factors-BB (P < .001), transforming growth factor-β1 (P = .015), vascular endothelial growth factors (P = .03), and hepatocyte growth factors (nonsignificant) secretion. Furthermore, calcium chloride addition induced a significant decrease in platelet number (P = .0134) and there was no leukocyte detection in the PG product. These results demonstrate that PG treatment might impact the healing of leg ulcers in sickle cell disease, especially in patients with small ulcers.

Corrosion Kinetics And Topography Analysis Of Ti-6al-4v Alloy Subjected To Different Mouthwash Solutions.

This study evaluated the corrosion kinetics and surface topography of Ti-6Al-4V alloy exposed to mouthwash solutions (0.12% chlorhexidine digluconate, 0.053% cetylpyridinium chloride and 3% hydrogen peroxide) compared to artificial saliva (pH6.5) (control). Twenty Ti-6Al-4V alloy disks were used and divided into 4 groups (n=5). For the electrochemical assay, standard tests as open circuit potential and electrochemical impedance spectroscopy (EIS) were applied at baseline, 7 and 14days after immersion in the solutions. Scanning electron microscopy, atomic force microscopy and profilometry (average roughness - Ra) were used for surface characterization. Total weight loss of disks was calculated. Data were analyzed by ANOVA and Bonferroni's test (α=0.05). Hydrogen peroxide generated the lowest polarization resistance (Rp) values for all periods (P<0.05). For the capacitance (Cdl), similar results were observed among groups at baseline (P=0.098). For the 7 and 14-day periods, hydrogen peroxide promoted the highest Cdl values (P<0.0001). Hydrogen peroxide promoted expressive superficial changes and greater Ra values than the others (P<0.0001). It could be concluded that solutions containing cetylpyridinium chloride and chlorhexidine digluconate might be the mouthwashes of choice during the post-operatory period of dental implants. However, hydrogen peroxide is counter-indicated in these situations. Further studies evaluating the dynamics of these solutions (tribocorrosion) and immersing the disks in daily cycles (two or three times a day) to mimic a clinical situation closest to the application of mouthwashes in the oral cavity are warranted to prove our results.

Bay 41-2272, A Soluble Guanylate Cyclase Stimulator, Relaxes Isolated Human Ureter In A Standardized In Vitro Model.

To characterize the relaxation induced by BAY 41-2272 in human ureteral segments. Ureter specimens (n = 17) from multiple organ human deceased donors (mean age 40 ± 3.2 years, male/female ratio 2:1) were used to characterize the relaxing response of BAY 41-2272. Immunohistochemical analysis for endothelial and neuronal nitric oxide synthase, guanylate cyclase stimulator (sGC) and type 5 phosphodiesterase was also performed. The potency values were determined as the negative log of the molar to produce 50% of the maximal relaxation in potassium chloride-precontracted specimens. The unpaired Student t test was used for the comparisons. Immunohistochemistry revealed the presence of endothelial nitric oxide synthase in vessel endothelia and neuronal nitric oxide synthase in urothelium and nerve structures. sGC was expressed in the smooth muscle and urothelium layer, and type 5 phosphodiesterase was present in the smooth muscle only. BAY 41-2272 (0.001-100 μM) relaxed the isolated ureter in a concentration dependent manner, with a potency and maximal relaxation value of 5.82 ± 0.14 and 84% ± 5%, respectively. The addition of nitric oxide synthase and sGC inhibitors reduced the maximal relaxation values by 21% and 45%, respectively. However, the presence of sildenafil (100 nM) significantly potentiated (6.47 ± 0.10, P <.05) this response. Neither glibenclamide or tetraethylammonium nor ureteral urothelium removal influenced the relaxation response by BAY 41-2272. BAY 41-2272 relaxes the human isolated ureter in a concentration-dependent manner, mainly by activating the sGC enzyme in smooth muscle cells rather than in the urothelium, although a cyclic guanosine monophosphate-independent mechanism might have a role. The potassium channels do not seem to be involved.

Membrane Lipid Profile Monitored By Mass Spectrometry Detected Differences Between Fresh And Vitrified In Vitro-produced Bovine Embryos.

Summary This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2-20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase 'e' and 'p', respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

Brij Detergents Reveal New Aspects Of Membrane Microdomain In Erythrocytes.

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4 °C and 37 °C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs - a detergent that preferentially solubilizes the membrane inner leaflet - while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.

[should Pediatric Parenteral Nutrition Be Individualized?].

Parenteral nutrition (PN) formulations are commonly individualized, since their standardization seem inadequate for the pediatric population. This study aimed to evaluate the nutritional state and the reasons for PN individualization in pediatric patients using PN hospitalized in a tertiary hospital in Campinas, São Paulo. This longitudinal study comprised patients using PN followed by up to 67 days. Nutritional status was classified according to the criteria established by the World Health Organization (WHO) (2006) and WHO (2007). The levels of the following elements on blood were analyzed: sodium, potassium, ionized calcium, chloride, magnesium, inorganic phosphorus and triglycerides (TGL). Among the criteria for individualization, were considered undeniable: significant reduction in blood levels of potassium (<3 mEq/L), sodium (<125 mEq/)L, magnesium (<1 mEq/L), phosphorus (<1.5 mEq/L), ionic calcium (<1 mmol) and chloride (<90 mEq/L) or any value above the references. Twelve pediatric patients aged 1 month to 15 years were studied (49 individualizations). Most patients were classified as malnourished. It was observed that 74/254 (29.2%) of examinations demanded individualized PN by indubitable reasons. The nutritional state of patients was considered critical in most cases. Thus, the individualization performed in the beginning of PN for energy protein adequacy was indispensable. In addition, the individualized PN was indispensable in at least 29.2% of PN for correction of alterations found in biochemical parameters.

Nasal Potential Difference In Cystic Fibrosis Considering Severe Cftr Mutations.

The gold standard for diagnosing cystic fibrosis (CF) is a sweat chloride value above 60 mEq/L. However, this historical and important tool has limitations; other techniques should be studied, including the nasal potential difference (NPD) test. CFTR gene sequencing can identify CFTR mutations, but this method is time-consuming and too expensive to be used in all CF centers. The present study compared CF patients with two classes I-III CFTR mutations (10 patients) (G1), CF patients with classes IV-VI CFTR mutations (five patients) (G2), and 21 healthy subjects (G3). The CF patients and healthy subjects also underwent the NPD test. A statistical analysis was performed using the Mann-Whitney, Kruskal-Wallis, χ(2), and Fisher's exact tests, α = 0.05. No differences were observed between the CF patients and healthy controls for the PDMax, Δamiloride, and Δchloride + free + amiloride markers from the NPD test. For the finger value, a difference between G2 and G3 was described. The Wilschanski index values were different between G1 and G3. In conclusion, our data showed that NPD is useful for CF diagnosis when classes I-III CFTR mutations are screened. However, if classes IV-VI are considered, the NPD test showed an overlap in values with healthy subjects.

Enzymatic Biodiesel Synthesis From Yeast Oil Using Immobilized Recombinant Rhizopus Oryzae Lipase.

The recombinant Rhizopus oryzae lipase (1-3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6% was obtained with microbial oils lower than olive oil 54.3%. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.

Hypobromous Acid, A Powerful Endogenous Electrophile: Experimental And Theoretical Studies.

Hypobromous acid (HOBr) is an inorganic acid produced by the oxidation of the bromide anion (Br(-)). The blood plasma level of Br(-) is more than 1,000-fold lower than that of chloride anion (Cl(-)). Consequently, the endogenous production of HOBr is also lower compared to hypochlorous acid (HOCl). Nevertheless, there is much evidence of the deleterious effects of HOBr. From these data, we hypothesized that the reactivity of HOBr could be better associated with its electrophilic strength. Our hypothesis was confirmed, since HOBr was significantly more reactive than HOCl when the oxidability of the studied compounds was not relevant. For instance: anisole (HOBr, k2=2.3×10(2)M(-1)s(-1), HOCl non-reactive); dansylglycine (HOBr, k2=7.3×10(6)M(-1)s(-1), HOCl, 5.2×10(2)M(-1)s(-1)); salicylic acid (HOBr, k2=4.0×10(4)M(-1)s(-1), non-reactive); 3-hydroxybenzoic acid (HOBr, k2=5.9×10(4)M(-1)s(-1), HOCl, k2=1.1×10(1)M(-1)s(-1)); uridine (HOBr, k2=1.3×10(3)M(-1)s(-1), HOCl non-reactive). The compounds 4-bromoanisole and 5-bromouridine were identified as the products of the reactions between HOBr and anisole or uridine, respectively, i.e. typical products of electrophilic substitutions. Together, these results show that, rather than an oxidant, HOBr is a powerful electrophilic reactant. This chemical property was theoretically confirmed by measuring the positive Mulliken and ChelpG charges upon bromine and chlorine. In conclusion, the high electrophilicity of HOBr could be behind its well-established deleterious effects. We propose that HOBr is the most powerful endogenous electrophile.