Cannabidiol Modulation of Nicotine-Induced Toxicity: Assessing Effects on Behavior, Brain-Derived Neurotrophic Factor, and Oxidative Stress in C57BL/6 Male Mice.

High doses of nicotine administered to rodents serve as a model for studying anxiety and test compounds' potential anxiolytic effects. At these doses, anxiety in rodents is accompanied by disruption of brain-derived neurotrophic factor (BDNF). The endocannabinoids and nicotine modulate several central nervous system processes via their specific receptors, impacting locomotion, anxiety, memory, nociception, and reward. Cannabidiol (CBD), an active ingredient of Cannabis sativa L., is devoid of psychoactive actions and has gained attention for its anxiolytic, antioxidant, and anti-inflammatory properties, among others. This work aims to examine the potential anxiety-reducing properties of CBD in a well-established experimental mouse model of anxiety-like behavior induced by high doses of nicotine on male C57BL/6 mice. In this context, the open-field behavioral test was specially conducted to assess CBD's effects on anxiety-like behavior and locomotion. Brain neuronal plasticity, modulated by BDNF, along with a diverse array of blood's metabolic markers, was examined as a means of evaluating systemic toxicity under various treatments. Finally, oxidative stress was evaluated through the measurement of glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA), while pro-inflammatory cytokine assessments were conducted to evaluate redox status and immune system function. Our research suggests that CBD shows potential in reducing anxiety-like behaviors induced by high doses of nicotine, by mitigating changes in BDNF protein levels in cerebral hemispheres and cerebellum. At the same time, CBD targets specific liver enzymes, maintains tissue's systemic toxicity (i.e., renal, kidney, and pancreatic), balances redox status (SOD, GSH, and MDA), and regulates the secretion of pro-inflammatory cytokines (TNF-alpha and IL-6).

Prox1 inhibits neurite outgrowth during central nervous system development.

During central nervous system (CNS) development, proper and timely induction of neurite elongation is critical for generating functional, mature neurons, and neuronal networks. Despite the wealth of information on the action of extracellular cues, little is known about the intrinsic gene regulatory factors that control this developmental decision. Here, we report the identification of Prox1, a homeobox transcription factor, as a key player in inhibiting neurite elongation. Although Prox1 promotes acquisition of early neuronal identity and is expressed in nascent post-mitotic neurons, it is heavily down-regulated in the majority of terminally differentiated neurons, indicating a regulatory role in delaying neurite outgrowth in newly formed neurons. Consistently, we show that Prox1 is sufficient to inhibit neurite extension in mouse and human neuroblastoma cell lines. More importantly, Prox1 overexpression suppresses neurite elongation in primary neuronal cultures as well as in the developing mouse brain, while Prox1 knock-down promotes neurite outgrowth. Mechanistically, RNA-Seq analysis reveals that Prox1 affects critical pathways for neuronal maturation and neurite extension. Interestingly, Prox1 strongly inhibits many components of Ca2+ signaling pathway, an important mediator of neurite extension and neuronal maturation. In accordance, Prox1 represses Ca2+ entry upon KCl-mediated depolarization and reduces CREB phosphorylation. These observations suggest that Prox1 acts as a potent suppressor of neurite outgrowth by inhibiting Ca2+ signaling pathway. This action may provide the appropriate time window for nascent neurons to find the correct position in the CNS prior to initiation of neurites and axon elongation.

In vivo quantification and pharmacokinetic studies of cotinine in mice after smoke exposure by LC-MS/MS.

A sensitive analytical method was developed and validated for the quantification of cotinine in mouse plasma after exposure to smoke of 0.5, 1.0, and 1.5 commercially available cigarettes, using liquid chromatography tandem mass spectrometry. The method was validated over a linear concentration range of 0.075-20.0 ng/mL with the R2 value being higher than 0.99. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. The lower limit of quantification (LLOQ) for cotinine was 0.075 ng/mL with sufficient specificity, accuracy, and precision. Following exposure to 0.5, 1.0, and 1.5 cigarette smoke, it was observed that the AUC and the Cmax increased linearly as the doses increased. The pharmacokinetics of cotinine was found linear for the range of 0.5-1.5 commercial cigarette smoke. The quantification of the concentration of cotinine in mouse plasma after smoke exposure will facilitate future behavioral and toxicological experiments in animals and may prove useful in predicting cotinine levels in humans during smoking.

Saffron (Crocus sativus L.) Tea Intake Prevents Learning/Memory Defects and Neurobiochemical Alterations Induced by Aflatoxin B1 Exposure in Adult Mice.

This study aimed to investigate the potential neurotoxic effects of aflatoxin B1 (AFB1) and the preventive effects of saffron. Male Balb-c mice received AFB1 (0.6 mg/kg/day intraperitoneally for 4 days), saffron infusion (90 mg styles/200 mL, ad libitum access for 2 weeks) or saffron infusion plus AFB1 (saffron treatment as previously plus 0.6 mg AFB1/kg/day intraperitoneally for the last 4 days). Control mice were intraperitoneally injected with DMSO:saline (1:1, v/v) during AFB1 treatment. Learning/memory was assessed by passive avoidance task. The activity of acetylcholinesterase [AChE, salt-(SS)/detergent-soluble(DS) isoforms], butyrylcholinesterase (BuChE, SS/DS isoforms), monoamine oxidase (MAO-A, MAO-B), the levels of lipid peroxidation (malondialdehyde, MDA) and reduced glutathione (GSH), were determined in whole brain (minus cerebellum) and cerebellum. We demonstrate for the first time that AFB1 administration impaired the memory of adult mice and decreased significantly whole brain AChE and BuChE activity, cerebellar AChE activity and cerebral GSH content. Moreover, MAO isoforms activity in whole brain, MAO-B activity in cerebellum and MDA levels of both tissues were significantly higher after AFB1 treatment. Pre-treatment with saffron prevented memory decline, activation of MAO-A and MAO-B in whole brain and cerebellum, respectively, and lipid peroxidation triggered by AFB1. Interestingly, the activity of AChE isoforms in whole brain, DS-AChE in cerebellum and GSH levels of both tissues were further significantly decreased in saffron +AFB1-treated mice compared with AFB1 group. Our findings support the neuroprotective efficacy of saffron against AFB1 in adult mice.

An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding.

Protein misfolding and aggregation are common pathological features of several human diseases, including Alzheimer's disease and type 2 diabetes. Here, we report an integrated and generalizable bacterial system for the facile discovery of chemical rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocyclic molecules are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding and aggregation using a flow cytometric assay. We demonstrate the effectiveness of this approach by identifying drug-like, head-to-tail cyclic peptides that modulate the aggregation of the Alzheimer's disease-associated amyloid ß peptide. Biochemical, biophysical and biological assays using isolated amyloid ß peptide, primary neurons and various established Alzheimer's disease nematode models showed that the selected macrocycles potently inhibit the formation of neurotoxic amyloid ß peptide aggregates. We also applied the system to the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase-an enzyme linked with inherited forms of amyotrophic lateral sclerosis. Overall, the system enables the identification of molecules with therapeutic potential for rescuing the misfolding of disease-associated polypeptides.

Cerebral Area Differential Redox Response of Neonatal Rats to Selenite-Induced Oxidative Stress and to Concurrent Administration of Highbush Blueberry Leaf Polyphenols.

Our goal was to delineate the mechanisms of selenite-induced oxidative stress in neonatal rats and investigate the potential of blueberry leaf polyphenols to counteract the induced stress. Vaccinium corymbosum leaf decoction (BLD) was analyzed by UPLC-MS and LC-DAD, along with its in vitro antioxidant activity (DPPH radical scavenging, FRAP, ferrous chelation). Newborn suckling Wistar rats were randomly divided into three groups: 'Se' and 'SeBLD' received 20 µmol Na2SeO3/kg BW subcutaneously (PN day 10); 'SeBLD' received 100 mg dry BLD/kg BW intraperitoneally (PN11 and 12) and Group 'C' received normal saline. Βiochemical analysis revealed tissue-specific effects of selenite. Brain as a whole was more resistant to selenite toxicity in comparison to liver; midbrain and cerebellum were in general not affected, but cortex was moderately disturbed. Liver lipid peroxidation, GSH, SOD, CAT, GPx were significantly affected, whereas proteolytic activity was not. BLD, which is rich in chlorogenic acid and flavonols (especially quercetin derivatives), exerted significant antioxidant protective effects in all regions. In conclusion, we provide for the first time an insight to the neonatal rat cerebral and liver redox response against a toxic selenite dose and blueberry leaf polyphenols.

Lead-induced effects on learning/memory and fear/anxiety are correlated with disturbances in specific cholinesterase isoform activity and redox imbalance in adult brain.

The aim of the present study was to investigate whether the underlying mechanism of lead (Pb)-induced effects on learning/memory and fear/anxiety behavior involves changes either on AChE G4 (most abundant in brain) or on G1 isoform activity, and/or to a putative local disruption of oxidant/antioxidant balance. Adult male mice were randomly divided into two groups (18 animals/group): a vehicle group [500ppm (mg/L) CH3COONa/day for 4weeks in their drinking water] and a Pb-treated group [500ppm Pb(CH3COO)2/day for 4weeks in their drinking water]. At the end of the treatment period, mice were subjected to the behavioral tasks. Learning/memory was tested by step-through passive avoidance test, whereas fear/anxiety was studied using the elevated plus-maze and thigmotaxis tests. Pb levels in mice brain were determined using atomic absorption spectrometry. AChE activity was determined colorimetrically, and GSH and MDA levels fluorometrically in whole brain minus cerebellum, cerebral cortex, midbrain, hippocampus, striatum and cerebellum. The possible correlations between learning/memory or fear/anxiety behavior with the AChE activity and/or the lipid peroxidation levels and GSH content were also examined. Pb consumption caused significant deficits on mice learning/memory ability and increased anxiety. The consumption of the Pb solution inhibited the activity of the two AChE isoforms in all brain regions tested. Moreover, Pb exposure increased lipid peroxidation and decreased GSH levels in all brain regions examined. Spearman correlation analysis revealed that the coefficients between the particular behaviors, AChE activity and redox balance were brain region- and AChE isoform-specific.

Phytochemical composition of "mountain tea" from Sideritis clandestina subsp. clandestina and evaluation of its behavioral and oxidant/antioxidant effects on adult mice.

PURPOSE: The goals of this study were to monitor the effect of drinking of herbal tea from Sideritis clandestina subsp. clandestina for 6 weeks on behavioral and oxidant/antioxidant parameters of adult male mice and also to evaluate its phytochemical composition. METHODS: The phytochemical profile of the Sideritis tea was determined by liquid chromatography-UV diode array coupled to ion-trap mass spectrometry with electrospray ionization interface. The effects of two doses of the herbal infusion (2 and 4% w/v, daily) intake on anxiety-like state in mice were studied by the assessment of their thigmotactic behavior. The oxidant/antioxidant status of brain (-Ce), liver and heart of adult male Balb-c mice following the consumption of Sideritis tea was also evaluated via the measurement of malondialdehyde (MDA) and reduced glutathione (GSH) levels using fluorometric assays. Our study was further extended to determine the antioxidant effects of the herbal tea on specific brain regions (cerebral cortex, cerebellum and midbrain). RESULTS: The identified compounds were classified into several natural product classes: quinic acid derivatives, iridoids, phenylethanol glycosides and flavonoids. Our results showed that only the 4% Sideritis tea exhibited anxiolytic-like properties as evidenced by statistically significant (p < 0.05) decrease in the thigmotaxis time and increase in the number of entries to the central zone in comparison with the control group. Consumption of both tea doses (2 and 4% w/v) elevated GSH (12 and 28%, respectively, p < 0.05) and decreased MDA (16 and 29%, p < 0.05) levels in brain (-Ce), while liver and heart remained unaffected. In regard to the effect of herbal tea drinking (2 and 4% w/v) on specific brain regions, it caused a significant increase in GSH of cerebellum (13 and 36%, respectively, p < 0.05) and midbrain (17 and 36%, p < 0.05). Similarly, MDA levels were decreased in cerebellum (45 and 79%, respectively, p < 0.05) and midbrain (50 and 63%, respectively, p < 0.05), whereas cerebral cortex remained unaffected. CONCLUSIONS: Mountain tea drinking prevents anxiety-related behaviors and confers antioxidant protection to rodent's tissues in a region-specific, dose-dependent manner, and its phytochemical constituents are shown for the first time.

Investigation of the neuroprotective action of saffron (Crocus sativus L.) in aluminum-exposed adult mice through behavioral and neurobiochemical assessment.

In the present study, the possible reversal effects of saffron against established aluminum (Al)-toxicity in adult mice, were investigated. Control, Al-treated (50 mg AlCl(3)/kg/day diluted in the drinking water for 5 weeks) and Al+saffron (Al-treatment as previously plus 60 mg saffron extract/kg/day intraperitoneally for the last 6 days), groups of male Balb-c mice were used. We assessed learning/memory, the activity of acetylcholinesterase [AChE, salt-(SS)/detergent-soluble(DS) isoforms], butyrylcholinesterase (BuChE, SS/DS isoforms), monoamine oxidase (MAO-A, MAO-B), the levels of lipid peroxidation (MDA) and reduced glutathione (GSH), in whole brain and cerebellum. Brain Al was determined by atomic absorption spectrometry, while, for the first time, crocetin, the main active metabolite of saffron, was determined in brain after intraperitoneal saffron administration by HPLC. Al intake caused memory impairment, significant decrease of AChE and BuChE activity, activation of brain MAO isoforms but inhibition of cerebellar MAO-B, significant elevation of brain MDA and significant reduction of GSH content. Although saffron extract co-administration had no effect on cognitive performance of mice, it reversed significantly the Al-induced changes in MAO activity and the levels of MDA and GSH. AChE activity was further significantly decreased in cerebral tissues of Al+saffron group. The biochemical changes support the neuroprotective potential of saffron under toxicity.

Cell-line specific protection by berry polyphenols against hydrogen peroxide challenge and lack of effect on metabolism of amyloid precursor protein.

Amyloid precursor protein (APP) altered metabolism, Aß-overproduction/aggregation and oxidative stress are implicated in the development of Alzheimer's disease pathology. Based on our previous data indicating that administration of a polyphenol-rich (PrB) blueberry extract (from wild Vaccinium angustifolium) is memory enhancing in healthy mice and in order to delineate the neuroprotective mechanisms, this study investigated the antioxidant effects of PrB in H2O2-induced oxidative damage, Aß peptide fibrillogenesis and APP metabolism. PrB suppressed H2O2-initiated oxidation (DCF assay) and cell death (MTT assay) in SH-SY5Y cells. Protective effects were observed on Chinese hamster ovary (CHO) cells overexpressing APP770 carrying the mutation Val717Phe only at high concentrations, while further damage on HEK293 cells was induced after co-treatment with 250 µM H2O2 and PrB in comparison with H2O2 alone. Using the thioflavine T assay, blueberry polyphenols inhibited Aß-aggregation (~70%, 15 µg/mL) in a time-dependent manner, while in the CHO(APP770) cells it had no effect on APP metabolism as assessed by western blot. The results suggest that blueberry polyphenols exhibit antioxidant and/or pro-oxidant properties according to the cellular environment and have no effect on APP metabolism.

Differential antioxidant effects of consuming tea from Sideritis clandestina subsp. peloponnesiaca on cerebral regions of adult mice.

Oxidative stress is involved in the pathophysiology of neurodegenerative diseases and aging. Many species of the genus Sideritis (mountain tea) are widely consumed in the Mediterranean region as herbal tea. This study evaluated the effect of supplementation of mice with herbal tea from Sideritis clandestina subsp. peloponnesiaca on the antioxidant status of different brain regions. To select the most bioactive herbal tea, the polyphenolic content (Folin-Ciocalteu method) and the antioxidant properties (ferric reducing antioxidant power [FRAP] and 2,2-diphenyl-1-picrylhydrazyl assays) of several taxa and different populations of the S. clandestina infusions were measured in vitro. Male adult mice had ad libitum access to water (control) or the herbal tea (4% w/v) for 6 weeks. At the end of the treatment period we assessed the total antioxidant power (FRAP assay) and the levels of malondialdehyde (indicator of lipid peroxidation) and reduced glutathione in the cerebral cortex, cerebellum, and midbrain. These biochemical measures have also been determined in liver samples used as a comparative reference peripheral tissue. Consumption of 4% herbal tea increased the total antioxidant power of the midbrain by 72% (P<.05); a significant (P<.05) decrease in malondialdehyde levels and increase in reduced glutathione content of the cerebellum (78% and 27%, respectively) and midbrain (59% and 32%, respectively) were also observed. These findings indicate that mountain tea consumption enhances the antioxidant defense of the adult rodent brain in a region-specific manner.

Memory enhancing effects of saffron in aged mice are correlated with antioxidant protection.

Brain aging is characterized by cognitive decline and memory deficits that could be the result of oxidative stress and impaired cholinergic function. In this study, the effects of a daily, 7-day, intraperitoneal administration of saffron on cognitive functions were examined in both healthy adult (4 months old) and aged (20 months old), male Balb-c mice (n=8/group), by passive avoidance test. Whole brain homogenates (minus cerebellum) were collected for examination of brain oxidative markers, caspase-3 and acetylcholinesterase (AChE) activity. Results showed that saffron-treated mice exhibited significant improvement in learning and memory, accompanied by reduced lipid peroxidation products, higher total brain antioxidant activity and reduced caspase-3 activity in both age groups of mice. Furthermore, salt- and detergent-soluble AChE activity was significantly decreased only in adult mice. Thus, we showed, for the first time, that the significant cognitive enhancement conferred by saffron administration in mice, is more closely related to the antioxidant reinforcement. Next, we compared the effect of saffron (1-250 µg/mL), crocetin and safranal (1-125 µM) on H(2)O(2)-induced toxicity in human neuroblastoma SH-SY5Y cells. Both saffron and crocetin provided strong protection in rescuing cell viability (MTT assay), repressing ROS production (DCF assay) and decreasing caspase-3 activation. These data, together with earlier studies suggest that crocetin is a unique and potent antioxidant, capable of mediating the in vivo effects of saffron.

Effect of a polyphenol-rich wild blueberry extract on cognitive performance of mice, brain antioxidant markers and acetylcholinesterase activity.

The aim of this study was to examine the effect of a polyphenol-rich extract (PrB) of Vaccinium angustifolium (wild blueberries) introduced intraperitoneally (i.p.) at 30 (PrB30) and 60 (PrB60) mg/kg body weight for 7 days, on cognitive performance, brain oxidative status and acetylcholinesterase activity in adult, male, 3-4-month-old Balb-c mice. Evaluation of rodent learning and memory was assessed by a step-through test on day 6 after a double training and an initial acquisition trial on day 5. Antioxidant status was determined by ferric reducing antioxidant power (FRAP), ascorbic acid concentration (FRASC), malondialdehyde and reduced glutathione levels in whole brain homogenates. Acetylcholinesterase (AChE) activity was determined by Ellman's colorimetric method. Results showed that the PrB60-treated mice exhibited a significant improvement in learning and memory (step-through latency time of 228+/-38 s compared to 101+/-32 s of the control group). PrB extract administration also resulted in reduced lipid peroxidation products (38 and 79%) and higher brain ascorbic acid levels (21 and 64%) in both PrB30 and PrB60-treated groups, respectively, and higher glutathione levels (28%) in the PrB60-treated group. Furthermore, salt- and detergent soluble AChE activity significantly decreased in both PrB-treated groups. Thus, the significant cognitive enhancement observed in adult mice after short-term i.p. supplementation with the blueberry extract concentrated in polyphenols, is closely related to higher brain antioxidant properties and inhibition of AChE activity. These findings stress the critical impact of wild blueberry bioactive components on brain function.

Inhibitory activity on amyloid-beta aggregation and antioxidant properties of Crocus sativus stigmas extract and its crocin constituents.

Crocus sativus stigmas are one of the widely known spices (saffron) and consist of unusually polar carotenoids. Alzheimer's disease is characterized pathologically by deposition of amyloid beta-peptide (Abeta) fibrils. Oxidation is thought to promote Abeta fibril formation and deposition. To identify agents inhibiting the pathogenesis of Alzheimer's disease, we examined in vitro the antioxidant properties of extract of C. sativus stigmas and its effect on Abeta(1-40) fibrillogenesis. The antioxidant properties were determined by measuring the ferric-reducing antioxidant power and Trolox-equivalent antioxidant capacity, while its effects on Abeta-aggregation and fibrillogenesis were studied by thioflavine T-based fluorescence assay and by DNA binding shift assay. The water:methanol (50:50, v/v) extract of C. sativus stigmas possesses good antioxidant properties, higher than those of tomatoes and carrots, and inhibited Abeta fibrillogenesis in a concentration and time-dependent manner. The main carotenoid constituent, trans-crocin-4, the digentibiosyl ester of crocetin, inhibited Abeta fibrillogenesis at lower concentrations than dimethylcrocetin, revealing that the action of the carotenoid is enhanced by the presence of the sugars. Our findings suggest the possible use of C. sativus stigma constituents for inhibition of aggregation and deposition of Abeta in the human brain.