Desarrollo de biomarcadores del grado de exposición y activación para la evaluación de la neurotoxicidad de la MDMA en humanos

La 3,4-Metilendioximetanfetamina (MDMA, éxtasis) es un derivado anfetamínico sintético ampliamente usado como droga recreativa, que produce neurotoxicidad serotonérgica en animales y posiblemente también en humanos. El mecanismo subyacente de neurotoxicidad, incluye la formación de especies reactivas de oxigeno (ROS), pero la fuente de generación de estos es un punto de controversia. Se postula que la neurotoxicidad inducida por la MDMA es mediada por la formación de metabolitos bioreactivos. Específicamente, los metabolitos primarios de tipo catecol, la 3,4- dihidroximetanfetamina (HHMA) y la 3,4-dihidroxianfetamina (HHA), que luego dan lugar a la formación de conjugados con el glutatión y la N-acetilcisteína, y que conservan la capacidad de entrar en el ciclo redox y presentan neurotoxicidad serotonérgica en ratas. Aunque la presencia de dichos metabolitos se demostró recientemente en microdialisados de cerebros de ratas, su formación en humanos no se ha reportado aun. Este trabajo describe la detección de N-acetil-cisteína-HHMA (NAC-HHMA) y N-acetil-cisteína-HHA (NAC-HHA) en orina humana de 15 consumidores recreacionales de MDMA (1.5 mg/kg) en un entorno controlado. Los resultados revelan que en las primeras 4 horas después del consumo de MDMA aproximadamente el 0.002% de la dosis administrada es recuperada como aductos tioéter. Los polimorfismos genéticos en la expresión de las enzimas CYP2D6 y COMT, que en conjunto son las principales determinantes de los niveles estables de HHMA y HHA, posiblemente expliquen la variabilidad interindividual observada en la recuperación de la NAC-HHMA y la NAC-HHA en orina. Resumiendo, por primera vez se demuestra la formación de aductos tioéteres neurotóxicos de la MDMA en humanos. Estos resultados apoyan la hipótesis de que la bioactivación de la MDMA a metabolitos neurotóxicos es el mecanismo relevante para la generación de la neurotoxicidad en humanos.

Synthesis, binding affinity, and molecular docking analysis of new benzofuranone derivative as pontential antipsychotics

The complex etiology of schizophrenia has prompted researchers to develop clozapine-related multitargetstrategies to combat its symptoms. Here we describe a series of new 6-aminomethylbenzofuranones in aneffort to find new chemical structures with balanced affinities for 5-HT2 and dopamine receptors. Throughbiological and computational studies of 5-HT2A and D2 receptors, we identified the receptor serine residuesS3.36 and S5.46 as the molecular keys to explaining the differences in affinity and selectivity betweenthese new compounds for this group of receptors. Specifically, the ability of these compounds to establishone or two H-bonds with these key residues appears to explain their difference in affinity. In addition, wedescribe compound 2 (QF1004B) as a tool to elucidate the role of 5-HT2C receptors in mediating antipsychoticeffects and metabolic adverse events. The compound 16a (QF1018B) showed moderate to high affinitiesfor D2 and 5-HT2A receptors, and a 5-HT2A/D2 ratio was predictive of an atypical antipsychotic profile.

A general decomposition formula for derivative prices in stochastic volatility models

We see that the price of an european call option in a stochastic volatilityframework can be decomposed in the sum of four terms, which identifythe main features of the market that affect to option prices: the expectedfuture volatility, the correlation between the volatility and the noisedriving the stock prices, the market price of volatility risk and thedifference of the expected future volatility at different times. We alsostudy some applications of this decomposition.

Assessment of the potential skin irritation of lysine-derivative anionic surfactants using mouse fibroblast and human keratinocytes as an alternative to animal testing

Purpose. The aim of this study was to identify new surfactants with low skin irritant properties for use in pharmaceutical and cosmetic formulations, employing cell culture as an alternative method to in vivo testing. In addition, we sought to establish whether potential cytotoxic properties were related to the size of the counterions bound to the surfactants. Methods. Cytotoxicity was assessed in the mouse fibroblast cell line 3T6, and the human keratinocyte cell line NCTC 2544, using the MTT assay and uptake of the vital dye neutral red 24 h after dosing (NRU). Results. Lysine-derivative surfactants showed higher IC50s than did commercial anionic irritant compounds such as sodium dodecyl sulphate, proving to be no more harmful than amphoteric betaines. The aggressiveness of the surfactants depended upon the size of their constituent counterions: surfactants associated with lighter counterions showed a proportionally higher aggressivity than those with heavier ones. Conclusions. Synthetic lysine-derivative anionic surfactants are less irritant than commercial surfactants such as sodium dodecyl sulphate and Hexadecyltrimethylammonium bromide and are similar to Betaines. These surfactants may offer promising applications in pharmaceutical and cosmetic preparations, representing a potential alternative to commercial anionic surfactants as a result of their low irritancy potential.

Effect of chronic ethanol feeding on rat hepatocytic glutathione. Compartmentation, efflux, and response to incubation with ethanol.

Hepatocytes from rats that were fed ethanol chronically for 6-8 wk were found to have a modest decrease in cytosolic GSH (24%) and a marked decrease in mitochondrial GSH (65%) as compared with pair-fed controls. Incubation of hepatocytes from ethanol-fed rats for 4 h in modified Fisher's medium revealed a greater absolute and fractional GSH efflux rate than controls with maintenance of constant cellular GSH, indicating increased net GSH synthesis. Inhibition of gamma-glutamyltransferase had no effect on these results, which indicates that no degradation of GSH had occurred during these studies. Enhanced fractional efflux was also noted in the perfused livers from ethanol-fed rats. Incubation of hepatocytes in medium containing up to 50 mM ethanol had no effect on cellular GSH, accumulation of GSH in the medium, or cell viability. Thus, chronic ethanol feeding causes a modest fall in cytosolic and a marked fall in mitochondrial GSH. Fractional GSH efflux and therefore synthesis are increased under basal conditions by chronic ethanol feeding, whereas the cellular concentration of GSH drops to a lower steady state level. Incubation of hepatocytes with ethanol indicates that it has no direct, acute effect on hepatic GSH homeostasis.

Inhibition of glutathione efflux in the perfused rat liver and isolated hepatocytes by organic anions and bilirubin. Kinetics, sidedness, and molecular forms.

Using isolated, in situ, single-pass perfused rat livers, incubations of freshly isolated hepatocytes, and sinusoidal membrane-enriched vesicles, we and others have shown the saturability of transport (efflux) of hepatic glutathione (GSH). These observations have implicated a carrier mechanism. Our present studies were designed to provide further evidence in support of a carrier mechanism for hepatic GSH efflux by demonstrating competition by liver-specific ligands which are taken up by hepatocytes. Perfusing livers with different substances, we found that: (a) sulfobromophthalein-GSH (BSP-GSH) had a dose-dependent and fully reversible inhibitory effect on GSH efflux, while GSH alone did not have any effect; (b) taurocholate had no inhibitory effect; (c) all of the organic anions studied, i.e., BSP, rose bengal, indocyanine green, and unconjugated bilirubin (UCB), manifested potent, dose-dependent inhibitory effects, with absence of toxic effects and complete reversibility of inhibition in the case of UCB. The inhibitory effects of UCB could be overcome partially by raising (CoCl2-induced) hepatic GSH concentration. Because of the physiological importance of UCB, we conducted a detailed study of its inhibitory kinetics in the isolated hepatocyte model in the range of circulating concentrations of UCB. Studies with Cl- -free media, to inhibit the uptake of UCB by hepatocytes, showed that the inhibition of GSH efflux by UCB is apparently from inside the cell. This point was confirmed by showing that the inhibition is overcome only when bilirubin-loaded cells are cleared of bilirubin (incubation with 5% bovine serum albumin). Using Gunn rat hepatocytes and purified bilirubin mono- and diglucuronides, we found that both UCB and glucuronide forms of bilirubin inhibit GSH efflux in a dose-dependent manner. We conclude that the organic anions, although taken up by a mechanism independent of GSH, may competitively inhibit the carrier for GSH efflux from inside the hepatocyte.

Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress.

Isolated hepatocytes incubated with [35S]-methionine were examined for the time-dependent accumulation of [35S]-glutathione (GSH) in cytosol and mitochondria, the latter confirmed by density gradient purification. In GSH-depleted and -repleted hepatocytes, the increase of specific activity of mitochondrial GSH lagged behind cytosol, reaching nearly the same specific activity by 1-2 h. However, in hepatocytes from ethanol-fed rats, the rate of increase of total GSH specific radioactivity in mitochondria was markedly suppressed. In in vivo steady-state experiments, the mass transport of GSH from cytosol to mitochondria and vice versa was 18 nmol/min per g liver, indicating that the half-life of mitochondrial GSH was approximately 18 min in controls. The fractional transport rate of GSH from cytosol to mitochondria, but not mitochondria to cytosol, was significantly reduced in the livers of ethanol-fed rats. Thus, ethanol-fed rats exhibit a decreased mitochondrial GSH pool size due to an impaired entry of cytosol GSH into mitochondria. Hepatocytes from ethanol-fed rats exhibited a greater susceptibility to the oxidant stress-induced cell death from tert-butylhydroperoxide. Incubation with glutathione monoethyl ester normalized the mitochondrial GSH and protected against the increased susceptibility to t-butylhydroperoxide, which was directly related to the lowered mitochondrial GSH pool size in ethanol-fed cells.

Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria in periportal and perivenous rat hepatocytes.

Chronic ethanol feeding selectively impairs the translocation of cytosol GSH into the mitochondrial matrix. Since ethanol-induced liver cell injury is preferentially localized in the centrilobular area, we examined the hepatic acinar distribution of mitochondrial GSH transport in ethanol-fed rats. Enriched periportal (PP) and perivenous (PV) hepatocytes from pair- and ethanol-fed rats were prepared as well as mitochondria from these cells. The mitochondrial pool size of GSH was decreased in both PP and PV cells from ethanol-fed rats either as expressed per 10(6) cells or per microliter of mitochondrial matrix volume. The rate of reaccumulation of mitochondrial GSH and the linear relationship of mitochondrial to cytosol GSH from ethanol-fed mitochondria were lower for both PP and PV cells, effects observed more prominently in the PV cells. Mitochondrial functional integrity was lower in both PP and PV ethanol-fed rats, which was associated with decreased cellular ATP levels and mitochondrial membrane potential, effects which were greater in the PV cells. Mitochondrial GSH depletion by ethanol feeding preceded the onset of functional changes in mitochondria, suggesting that mitochondrial GSH is critical in maintaining a functionally competent organelle and that the greater depletion of mitochondrial GSH by ethanol feeding in PV cells could contribute to the pathogenesis of alcoholic liver disease.

Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics

Using the Xenopus oocyte expression system, we have previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein-glutathione (BSP-GSH)-insensitive reduced glutathione (GSH) transport (Fernandez-Checa, J., J. R. Yi, C. Garcia-Ruiz, Z. Knezic, S. Tahara, and N. Kaplowitz. 1993. J. Biol. Chem. 268:2324-2328). Starting with a cDNA library constructed from this fraction, we have now isolated a single clone that expresses GSH transporter activity. The cDNA for the rat canalicular GSH transporter (RcGshT) is 4.05 kb with an open reading frame of 2,505 nucleotides encoding for a polypeptide of 835 amino acids (95,785 daltons). No identifiable homologies were found in searching various databases. An approximately 96-kD protein is generated in in vitro translation of cRNA for RcGshT. Northern blot analysis reveals a single 4-kb transcript in liver, kidney, intestine, lung, and brain. The abundance of mRNA for RcGshT in rat liver increased 3, 6, and 12 h after a single dose of phenobarbital. Insensitivity to BSP-GSH and induction by phenobarbital, unique characteristics of canalicular GSH secretion, suggest that RcGshT encodes for the canalicular GSH transporter.

A peroxisomal glutathione transferase of Saccharomyces cerevisiae is functionally related to sulfur amino acid metabolism

Saccharomyces cerevisiae cells contain three omega-class glutathione transferases with glutaredoxin activity (Gto1, Gto2, and Gto3), in addition to two glutathione transferases (Gtt1 and Gtt2) not classifiable into standard classes. Gto1 is located at the peroxisomes, where it is targeted through a PTS1-type sequence, whereas Gto2 and Gto3 are in the cytosol. Among the GTO genes, GTO2 shows the strongest induction of expression by agents such as diamide, 1-chloro-2,4-dinitrobenzene, tert-butyl hydroperoxide or cadmium, in a manner that is dependent on transcriptional factors Yap1 and/or Msn2/4. Diamide and 1-chloro-2,4-dinitrobenzene (causing depletion of reduced glutathione) also induce expression of GTO1 over basal levels. Phenotypic analyses with single and multiple mutants in the S. cerevisiae glutathione transferase genes show that, in the absence of Gto1 and the two Gtt proteins, cells display increased sensitivity to cadmium. A gto1-null mutant also shows growth defects on oleic acid-based medium, which is indicative of abnormal peroxisomal functions, and altered expression of genes related to sulfur amino acid metabolism. As a consequence, growth of the gto1 mutant is delayed in growth medium without lysine, serine, or threonine, and the mutant cells have low levels of reduced glutathione. The role of Gto1 at the S. cerevisiae peroxisomes could be related to the redox regulation of the Str3 cystathionine -lyase protein. This protein is also located at the peroxisomes in S. cerevisiae, where it is involved in transulfuration of cysteine into homocysteine, and requires a conserved cysteine residue for its biological activity.

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

Synthesis of a tetrahydroimidazo-[2',1':2,3]thiazolo[5,4-c]pyridine derivative with Met inhibitory activity

A straightforward synthesis of the Met antagonist JLK1360 involving an alkylationcyclocondensation process using aminothiazole 1 and nitrophenacyl bromide 2, reduction of the nitro group, and coupling of the resulting tetracyclic aniline 5 with an appropriate N-acyl alanine derivative, is reported.

Synthesis of a tetrahydroimidazo-[2',1':2,3]thiazolo[5,4-c]pyridine derivative with Met inhibitory activity

A straightforward synthesis of the Met antagonist JLK1360 involving an alkylationcyclocondensation process using aminothiazole 1 and nitrophenacyl bromide 2, reduction of the nitro group, and coupling of the resulting tetracyclic aniline 5 with an appropriate N-acyl alanine derivative, is reported.

Embedding theorems of funcitional classes, III

In this paper we obtain the necessary and sufficient conditions for embedding results of different function classes. The main result is a criterion for embedding theorems for the so-called generalized Weyl-Nikol'skii class and the generalized Lipschitz class. To define the Weyl-Nikol'skii class, we use the concept of a (λ,β)-derivative, which is a generalization of the derivative in the sense of Weyl. As corollaries, we give estimates of norms and moduli of smoothness of transformed Fourier series.

Cubic spline population density functions and subcentre delimitation. The case of Barcelona

The presence of subcentres cannot be captured by an exponential function. Cubic spline functions seem more appropriate to depict the polycentricity pattern of modern urban systems. Using data from Barcelona Metropolitan Region, two possible population subcentre delimitation procedures are discussed. One, taking an estimated derivative equal to zero, the other, a density gradient equal to zero. It is argued that, in using a cubic spline function, a delimitation strategy based on derivatives is more appropriate than one based on gradients because the estimated density can be negative in sections with very low densities and few observations, leading to sudden changes in estimated gradients. It is also argued that using as a criteria for subcentre delimitation a second derivative with value zero allow us to capture a more restricted subcentre area than using as a criteria a first derivative zero. This methodology can also be used for intermediate ring delimitation.