Browsing by Author "Olivares, Belén"
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Item A Novel Morphine Drinking Model of Opioid Dependence in Rats(2022) Berríos, Pablo; Quezada, Mauricio; Santapau, Daniela; Morales, Paola; Olivares, Belén; Ponce, Carolina; Ávila, Alba; De Gregorio, Cristian; Ezquer, Marcelo; Quintanilla, María; Herrera, Mario; Israel, Yedy; Ezquer, FernandoAbstract: An animal model of voluntary oral morphine consumption would allow for a pre-clinical evaluation of new treatments aimed at reducing opioid intake in humans. However, the main limitation of oral morphine consumption in rodents is its bitter taste, which is strongly aversive. Taste aversion is often overcome by the use of adulterants, such as sweeteners, to conceal morphine taste or bitterants in the alternative bottle to equalize aversion. However, the adulterants’ presence is the cause for consumption choice and, upon removal, the preference for morphine is not preserved. Thus, current animal models are not suitable to study treatments aimed at reducing consumption elicited by morphine itself. Since taste preference is a learned behavior, just-weaned rats were trained to accept a bitter taste, adding the bitterant quinine to their drinking water for one week. The latter was followed by allowing the choice of quinine or morphine (0.15 mg/mL) solutions for two weeks. Then, quinine was removed, and the preference for morphine against water was evaluated. Using this paradigm, we show that rats highly preferred the consumption of morphine over water, reaching a voluntary morphine intake of 15 mg/kg/day. Morphine consumption led to significant analgesia and hyperlocomotion, and to a marked deprivation syndrome following the administration of the opioid antagonist naloxone. Voluntary morphine consumption was also shown to generate brain oxidative stress and neuroinflammation, signs associated with opioid dependence development. We present a robust two-bottle choice animal model of oral morphine self-administration for the evaluation of therapeutic interventions for the treatment of morphine dependence.Item Betaine-urea deep eutectic solvent improves imipenem antibiotic activity(2022) Olivares, Belén; Martínez, Fabián; Ezquer, Marcelo; Morales, Bernardo; Fuentes, Ignacia; Calvo, Margarita; Campodónico, PaolaBeta-lactam antibiotics are highly unstable in aqueous media, which may lead to subclinical concentrations, antimicrobial resistance and therapeutic failure. In previous work we demonstrated that a natural deep eutectic solvent consisting of betaine and urea (BU) is capable of improving the stability of some beta-lactams, including imipenem (IMP), the most unstable antibiotic of the family. Here, IMP-BU was studied by selective protonic Nuclear Overhauser Effect Spectroscopy Magnetic Resonance (H1 NOESY NMR) to gain insight into the mechanism by which BU protects IMP. The kinetics of IMP release and its antibacterial activity were evaluated in diffusional, time-kill and antibiofilm assays. It was found that BU is a protective matrix which allows a fast release of IMP, resulting in superior antibacterial activity when compared to IMP in aqueous solution, both against bacteria growing in planktonic form and in biofilms. Furthermore, it was shown that BU is nontoxic when evaluated in fibroblast primary cell cultures and in organotypic skin cultures, and is not immunogenic when tested in vitro in macrophage cultures, suggesting that BU has potential application as a biomaterial or excipientPublication Chronic Voluntary Morphine Intake Is Associated with Changes in Brain Structures Involved in Drug Dependence in a Rat Model of Polydrug Use(2023) Ezquer, Fernando; Ezquer, Marcelo; Gallardo, Javiera; Quintanilla, María; Morales, Paola; Santapau, Daniela; Ávila, Alba; Ponce, Carolina; Berrios, Pablo; Olivares, Belén; Herrera, Mario; Israel, YedyChronic opioid intake leads to several brain changes involved in the development of dependence, whereby an early hedonistic effect (liking) extends to the need to self-administer the drug (wanting), the latter being mostly a prefrontal-striatal function. The development of animal models for voluntary oral opioid intake represents an important tool for identifying the cellular and molecular alterations induced by chronic opioid use. Studies mainly in humans have shown that polydrug use and drug dependence are shared across various substances. We hypothesize that an animal bred for its alcohol preference would develop opioid dependence and further that this would be associated with the overt cortical abnormalities clinically described for opioid addicts. We show that Wistar-derived outbred UChB rats selected for their high alcohol preference additionally develop: (i) a preference for oral ingestion of morphine over water, resulting in morphine intake of 15 mg/kg/day; (ii) marked opioid dependence, as evidenced by the generation of strong withdrawal signs upon naloxone administration; (iii) prefrontal cortex alterations known to be associated with the loss of control over drug intake, namely, demyelination, axonal degeneration, and a reduction in glutamate transporter GLT-1 levels; and (iv) glial striatal neuroinflammation and brain oxidative stress, as previously reported for chronic alcohol and chronic nicotine use. These findings underline the relevance of polydrug animal models and their potential in the study of the wide spectrum of brain alterations induced by chronic morphine intake. This study should be valuable for future evaluations of therapeutic approaches for this devastating condition.Item Experimental Analyses Emphasize the Stability of the Meisenheimer Complex in a S N Ar Reaction Toward Trends in Reaction Pathways(2020-07) Campodónico, Paola; Olivares, Belén; Tapia, Ricardo A.The mechanism of SNAr reactions between 2-chloro-5-nitropyrimidine with primary and secondary alicyclic amines, respectively, have been studied by kinetic measurements. The kinetic data obtained in aqueous media opens a controversial discussion based on Brönsted-type plots analysis. The first approach based on the kinetic data reveals a non-catalyzed pathway. Then, the subtlety of the mathematical treatment of the kinetic data is discussed over a concerted or stepwise mechanism, respectively.Publication Exploring the behavior of Candida antarctica lipase B in aqueous mixtures of an imidazolium ionic liquid and its surfactant analogue(2024) Campodónico, Paola; Calderón, Cristian; Alcázar, Jackson; Olivares, Belén; Jaldin, Limberg; Suárez, CristianThe performance of Candida antarctica lipase B (CALB) has been evaluated in 1- butyl-3-methylimidazolium tetrafluoroborate (BMIMBF4)/water mixtures in a wide range of molar fractions (χBMIMBF4) with and without 1-dodecyl-3- methylimidazolium tetrafluoroborate (C12-MIMBF4), a surfactant derived from BMIMBF4. The main aim of this work is to evaluate the influence of χBMIMBF4 over micellar aggregates to assess the activity of enzymatic reactions. The investigated reaction corresponds to the hydrolysis of the substrate p-nitrophenyl laureate in each χBMIMBF4. The kinetic study for χBMIMBF4 at around 0.2 proved to be a border point in enzymatic activity. At χBMIMBF4 = 0.1, the lipase activity increases in thepresence of C12-MIMBF4. However, at higher concentrations, BMIMBF4 has a negligible effect over the lipase activity. These results suggest specific interactions between water and BMIMBF4 molecules in relation to CALB. This research highlights the superactivity phenomenon driven by the reaction media and the micelle interface. In this interfacial interaction, BMIMBF4 acts directly on the changes induced on the enzyme upon its interaction with the micellar interface. This study opens a green perspective toward the biocatalysis field.Item Innate gut microbiota predisposes to high alcohol consumption(2021) Ezquer, Fernando; Quintanilla, Maria Elena; Moya-Flores, Francisco; Morales, Paola; Munita, José; Olivares, Belén; Landskron, Glauben; Hermoso, Marcela A.; Ezquer, Marcelo; Israel, Yedy; Herrera-Marschitz, MarioGut microbiota is known to be transferred from the mother to their offspring. This study determines whether the innate microbiota of rats selectively bred for generations as high alcohol drinkers play a role in their alcohol intake. Wistar-derived high-drinker UChB rats (intake 10-g ethanol/kg/day) administered nonabsorbable oral antibiotics before allowing access to alcohol, reducing their voluntary ethanol intake by 70%, an inhibition that remained after the antibiotic administration was discontinued. Oral administration of Lactobacillus rhamnosus Gorbach–Goldin (GG) induced the synthesis of FGF21, a vagal β-Klotho receptor agonist, and partially re-invoked a mechanism that reduces alcohol intake. The vagus nerve constitutes the main axis transferring gut microbiota information to the brain (“microbiota-gut-brain” axis). Bilateral vagotomy inhibited rat alcohol intake by 75%. Neither antibiotic treatment nor vagotomy affected total fluid intake. A microbiota-mediated marked inflammatory environment was observed in the gut of ethanol-naïve high-drinker rats, as gene expression of proinflammatory cytokines (TNF-α; IL-6; IL-1β) was significantly reduced by nonabsorbable antibiotic administration. Gut cytokines are known to activate the vagus nerve, while vagal activation induces pro-rewarding effects in nucleus accumbens. Both alcoholics and alcohol-preferring rats share a marked preference for sweet tastes—likely an evolutionary trait to seek sweet fermented fruits. Saccharin intake by UChB rats was inhibited by 75%–85% by vagotomy or oral antibiotic administration, despite saccharin-induced polydipsia. Overall, data indicate that the mechanisms that normally curtail heavy drinking are inhibited in alcohol-preferring animals and inform a gut microbiota origin. Whether it applies to other mammals and humans merits further investigation.Publication Kinetics and Reaction Mechanism of Biothiols Involved in SNAr Reactions: An Experimental Study(2022) Campodónico, Paola; Alarcón-Espósito, Jazmín; Olivares, BelénFew kinetic parameters, or reaction rates, are known up to date in detail about 1-chloro and 1-fluoro-2,4-dinitrobenzene (ClDNB and FDNB, respectively) with a series of biothiols in aqueous media. These biological nucleophiles with thiol groups have been widely used as a reference in nucleophile reactivity assays due to their prevalence and cellular abundance. The main aim of this study was to elucidate the reaction mechanism based on Brönsted-type plots and reactivity patterns of the electrophile/nucleophile pairs. A complete kinetic study was performed in terms of the comparison of Brönsted-type slope parameters (βnuc) for the reactions and was used for assigning the mechanism and the rate-determining step associated with the reaction route. A mass spectrometry analysis demonstrated that the nucleophilic center of the biothiols is the -SH group and there is only one kinetic product. The kinetic study suggests that the reaction mechanism might be the borderline between concerted and stepwise pathways. An amine–enol equilibrium for the most reactive nucleophiles appears to be the main determining factor controlling the nucleophilic attack in the nucleophilic aromatic substitution reactions investigated, highlighting the anionic form for these nucleophiles. This amine–enol equilibrium involves a hydrogen bond which stabilizes the intermediate species in the reaction pathway. Thus, intramolecular bonds are formed and enhance the nucleophilic strength through the contribution of the solvent surrounding the electrophile/ nucleophile pairs. Finally, we highlight the importance of the formation of electrophile/ nucleophile adducts that could modify structures and/or functions of biological systems with potential toxic effects. Therefore, it is essential to know all these kinetic and reactivity patterns and their incidence on other studies.