Katiane Roversi

Polyphenolic Composition and Evaluation of Antioxidant Activity, Osmotic Fragility and Cytotoxic Effects of Raphiodon echinus (Nees & Mart.) Schauer

Raphiodon echinus (R. echinus) is used in Brazilian folk medicine for the treatment of inflammation, coughs, and infectious diseases. However, no information is available on the potential antioxidant, cytotoxicity and genotoxicity of this plant. In this study, the polyphenolic constituents, antioxidant capacity and potential toxic effects of aqueous and ethanolic extracts of R. echinus on human erythrocytes and leukocytes were investigated for the first time. R. echinus extracts showed the presence of Gallic, chlorogenic, caffeic and ellagic acids, rutin, quercitrin and quercetin. Aqueous and ethanolic extracts of R. echinus exhibited antioxidant activity in DPPH radical scavenging with IC50 = 111.9 μg/mL (EtOH extract) and IC50 = 227.9 μg/mL (aqueous extract). The extracts inhibited Fe2+ (10 μM) induced thiobarbituric acid reactive substances (TBARS) formation in rat brain and liver homogenates. The extracts (30–480 μg/mL) did not induce genotoxicity, cytotoxicity or osmotic fragility in human blood cells. The findings of this present study therefore suggest that the therapeutic effect of R. echinus may be, in part, related to its antioxidant potential. Nevertheless, further in vitro and in vivo studies are required to ascertain the safety margin of its use in folk medicine.

Neonatal handling prevents anxiety-like symptoms in rats exposed to chronic mild stress: Behavioral and oxidative parameters

This study investigated the influence of neonatal handling on behavioral and biochemical consequences of chronic mild stress (CMS) in adulthood. Male rat pups were submitted to daily tactile stimulation (TS) or maternal separation (MS), from postnatal day 1 (PND1) to postnatal day 21 (PND21), for 10 min/day. In adulthood, half the number of animals were exposed to CMS for 3 weeks and submitted to behavioral testing, including sucrose preference (SP), elevated plus maze (EPM), and defensive burying tasks (DBTs), followed by biochemical assessments. CMS reduced SP, increased anxiety in EPM and DBT, and increased adrenal weight. In addition, CMS decreased plasma vitamin C (VIT C) levels and increased protein carbonyl (PC) levels, catalase (CAT) activity in hippocampus and cortex, and superoxide dismutase (SOD) levels in cortex. In contrast, both forms of neonatal handling were able to prevent reduction in SP, anxiety behavior in DBT, and CMS-induced adrenal weight increase. Furthermore, they were also able to prevent plasma VIT C reduction, hippocampal PC levels increase, CAT activity increase in hippocampus and cortex, and SOD levels increase in cortex following CMS. Only TS was able to prevent CMS-induced anxiety symptoms in EPM and PC levels in cortex. Taken together, these findings show the protective role of neonatal handling, especially TS, which may enhance ability to cope with stressful situations in adulthood.

Influence of trans fat and omega-3 on the preference of psychostimulant drugs in the first generation of young rats

The current Western diet often provides considerable amounts of saturated and trans fatty acids (TFA), whose incorporation into neuronal membranes has been implicated in changes of brain neurochemical functions. Such influence has caused concerns due to precipitation of neuropsychiatric disorders, whose data are still unclear. Here we evaluated the influence of different fats on preference parameters for amphetamine (AMPH): adolescent rats were orally supplemented with soybean oil (SO, rich in n-6 FA, which was considered an isocaloric control group), fish oil (FO, rich in n-3 FA) and hydrogenated vegetable fat (HVF, rich in saturated and trans FA) from weaning, which were born of dams supplemented with the same fat from pregnancy and lactation. AMPH preference, anxiety-like symptoms and locomotor index were evaluated in conditioned place preference (CPP), elevated plus maze (EPM) and open-field (OF), respectively, while brain oxidative status was determined in cortex, striatum and hippocampus. HVF increased AMPH-CPP and was associated with withdrawal signs, as observed by increased anxiety-like symptoms. Moreover, SO and FO were not associated with AMPH preference, but only FO-supplemented rats did not show any anxiety-like symptoms or increased locomotion. FO supplementation was related to lower oxidative damages to proteins and increased CAT activity in striatum and hippocampus, as well as increased GSH levels in blood, while HVF was related to increased oxidative status. In conclusion, our study showed the harmful influence of TFA on AMPH-CPP and drug craving symptoms, which can be related to dopaminergic neurotransmission.

Cross-generational trans fat intake modifies BDNF mRNA in the hippocampus: Impact on memory loss in a mania animal model.

Recently, we have described the influence of dietary fatty acids (FA) on mania‐like behavior of first generation animals. Here, two sequential generations of female rats were supplemented with soybean oil (SO, rich in n‐6 FA, control group), fish oil (FO, rich in n‐3 FA) and hydrogenated vegetable fat (HVF, rich in trans FA) from pregnancy and during lactation. In adulthood, half of each group was exposed to an amphetamine (AMPH)‐induced mania animal model for behavioral, biochemical and molecular assessments. FO supplementation was associated with lower reactive species (RS) generation and protein carbonyl (PC) levels and increased dopamine transporter (DAT) levels, while HVF increased RS and PC levels, thus decreasing catalase (CAT) activity and DAT levels in hippocampus after AMPH treatment. AMPH impaired short‐ (1 h) and long‐ (24 h) term memory in the HVF group. AMPH exposure was able to reduce hippocampal BDNF‐ mRNA expression, which was increased in FO. While HVF was related to higher trans FA (TFA) incorporation in hippocampus, FO was associated with increased percentage of n‐3 polyunsaturated FA (PUFA) together with lower n‐6/n‐3 PUFA ratio. Interestingly, our data showed a positive correlation between brain‐derived neurotrophic factor (BDNF) mRNA and short‐ and long‐term memory (r2 = 0.53; P = 0.000/r2 = 0.32; P = 0.011, respectively), as well as a negative correlation between PC and DAT levels (r2 = 0.23; P = 0.015). Our findings confirm that provision of n‐3 or TFA during development over two generations is able to change the neuronal membrane lipid composition, protecting or impairing the hippocampus, respectively, thus affecting neurothrophic factor expression such as BDNF mRNA. In this context, chronic consumption of trans fats over two generations can facilitate the development of mania‐like behavior, so leading to memory impairment and emotionality, which are related to neuropsychiatric conditions.

Toxicological aspects of trans fat consumption over two sequential generations of rats: Oxidative damage and preference for amphetamine

Chronic consumption of processed food causes structural changes in membrane phospholipids, affecting brain neurotransmission. Here we evaluated noxious influences of dietary fats over two generations of rats on amphetamine (AMPH)-conditioned place preference (CPP). Female rats received soybean oil (SO, rich in n-6 fatty acids (FA)), fish oil (FO, rich in n-3 FA) and hydrogenated vegetable fat (HVF, rich in trans fatty acids (TFA)) for two successive generations. Male pups from the 2nd generation were maintained on the same supplementation until 41 days of age, when they were conditioned with AMPH in CPP. While the FO group showed higher incorporation of n-3 polyunsaturated-FA (PUFA) in cortex/hippocampus, the HVF group showed TFA incorporation in these same brain areas. The SO and HVF groups showed AMPH-preference and anxiety-like symptoms during abstinence. Higher levels of protein carbonyl (PC) and lower levels of non-protein thiols (NPSH) were observed in cortex/hippocampus of the HVF group, indicating antioxidant defense system impairment. In contrast, the FO group showed no drug-preference and lower PC levels in cortex. Cortical PC was positively correlated with n-6/n-3 PUFA ratio, locomotion and anxiety-like behavior, and hippocampal PC was positively correlated with AMPH-preference, reinforcing connections between oxidative damage and AMPH-induced preference/abstinence behaviors. As brain incorporation of trans and n-6 PUFA modifies its physiological functions, it may facilitate drug addiction.

Chronic consumption of trans fat can facilitate the development of hyperactive behavior in rats

In recent decades, the increased consumption of processed foods, which are rich in hydrogenated vegetable fat (HVF), has led to a decreased consumption of fish and oilseed, rich in omega-3 fatty acids. This eating habit provides an increased intake of trans fatty acids (TFA), which may be related to neuropsychiatric conditions, including inattention and hyperactivity. In this study, we evaluated the potential connection between prolonged trans fat consumption and development of hyperactivity-like symptoms in rats using different behavioral paradigms. Trans fat intake for 10 months (Experiment 1), as well as during pregnancy and lactation across two sequential generations of rats, (Experiment 4) induced active coping in the forced swimming task (FST). In addition, HVF supplementation was associated with increased locomotion before and after amphetamine (AMPH) administration (Experiment 2). Similarly, HVF supplementation during pregnancy and lactation were associated with increased locomotion in both young and adult rats (Experiment 3). Furthermore, trans fat intake across two sequential generations increased locomotor and exploratory activities following stressors (Experiment 4). From these results, we suggest that chronic consumption of trans fat is able to enhance impulsiveness and reactivity to novelty, facilitating hyperactive behaviors.

Cross-Generational trans Fat Consumption Favors Self-Administration of Amphetamine and Changes Molecular Expressions of BDNF, DAT, and D1/D2 Receptors in the Cortex and Hippocampus of Rats.

Amphetamine (AMPH) is an addictive psychostimulant drug whose use has been related to neurotoxicity. Experimentally, AMPH increases anxiety-like symptoms, showing addictive properties. In the last decades, the growing consumption of processed foods has provided an excess of saturated and trans fats in detriment of essential fatty acids, which may modify the lipid profile of brain membranes, thus modifying its permeability and dopaminergic neurotransmission. Here, we assessed the influence of brain incorporation of different fatty acids (FA) on AMPH self-administration. Three groups of young male rats were orally supplemented from weaning with a mixture of soybean oil (SO, rich in n-6 FA) and fish oil (FO, rich in n-3 FA), hydrogenated vegetable fat (HVF, rich in trans fatty acids—TFA), or water (control group). These animals were born from dams that were supplemented with the same fat from pregnancy to lactation. Anxiety-like symptoms and locomotor index were assessed in elevated plus maze and open-field (OF), respectively, while brain molecular expressions of dopaminergic receptors, dopamine transporter (DAT), and BDNF were determined in the cortex and hippocampus. HVF increased the frequency of AMPH self-administration and was associated with reinforcement and withdrawal signs as observed by increased anxiety-like symptoms. Contrarily, SO/FO decreased these parameters. Increased BDNF protein together with decreased DAT expression was observed in the hippocampus of HVF group. Based on these findings, our study points to a harmful influence of trans fats on drug addiction and craving symptoms, whose mechanism may be related to changes in the dopaminergic neurotransmission.

Influence of neonatal tactile stimulation on amphetamine preference in young rats: parameters of addiction and oxidative stress.

This study investigated the influence of neonatal handling on amphetamine-induced conditioned place preference (CPP), as well as the consequent anxiety-like symptoms and oxidative status related to drug abstinence in young rats. Male pups were exposed to tactile stimulation (TS) or neonatal isolation (NI) for 10 min every day from postnatal day one (PND1) to PND21. After being weaned (PND22), pups were separated by handling type until PND40, when treatment with amphetamine (AMPH-4 mg/kg/mL ip, for 8 days) or vehicle (NaCl 0.9% ip, 1 mL/Kg) in CPP started. AMPH-conditioning evoked drug-preference (in 24h and 96 h) and abstinence symptoms in unhandled (UH) animals, followed by oxidative damage in the cortex, hippocampus and striatum. TS showed beneficial influence, as observed by the decreased drug-preference (24 and 96 h) in relation to UH and NI, showing no abstinence symptoms in this last period, as observed by the reduced anxiety-like symptoms. The oxidative status indicated a protective influence of TS on brain tissues: lower lipid peroxidation (LP) and reduced protein carbonylation (PC) in the cortex, hippocampus and striatum. Furthermore, TS also increased antioxidant defenses in brain tissues and blood: i) increased plasma levels of vitamin C; ii) increased activity of catalase (CAT) and iii) higher levels of glutathione (GSH) in red blood cells (RBC). Moreover, there were positive correlations of AMPH-CPP with PC and LP levels in all the brain areas assessed. In summary, TS modifies AMPH-preference in the CPP paradigm, reducing drug abstinence behaviors, and stimulating the antioxidant defense system, thus protecting the brain areas closely related to addiction in young rats. Studies about TS and addiction in animal models should be extended to the molecular level.

Tactile stimulation and neonatal isolation affect behavior and oxidative status linked to cocaine administration in young rats

We investigated the influence of neonatal handling on cocaine-induced conditioned place preference (CPP), anxiety-like symptoms and oxidative status related to drug abstinence in young rats. Pups were submitted to tactile stimulation (TS) or neonatal isolation (NI10 or NI60) after birth, and then were submitted to CPP performed with cocaine. TS group did not show place preference, while unhandled (UH), NI10 and NI60 rats did. Handling was related to anxiety-like symptoms per se in UH and NI60 groups and this behavior was also observed in the cocaine-conditioned rats exposed to the same handlings. Both TS and NI10 pups treated or not with cocaine showed less anxiety-like behavior than animals submitted to other handlings. TS reduced protein carbonyl (PC) in cortex and NI60 increased PC in both striatum and hippocampus of cocaine-treated rats. Among cocaine-treated rats, both times of NI increased plasma lipoperoxidation levels, which was reduced by TS in erythrocytes. TS increased the catalase activity in brain areas, while other handlings did not change this. Both TS and NI10 increased plasma vitamin C levels. These findings indicate that neonatal handling can modify anxiety-like symptoms related to cocaine preference and abstinence, and its protective influence, especially TS, on the antioxidant system.

Neonatal tactile stimulation changes anxiety-like behavior and improves responsiveness of rats to diazepam.

In this study we evaluated the influence of neonatal tactile stimulation (TS) on behavioral and biochemical effects related to a low dose of diazepam (DZP) in adult rats. Male pups of Wistar rats were handled (TS) daily from PND1 to PND21 for 10 min, while unhandled (UH) rats were not touched. In adulthood, half the animals of each group received a single administration of diazepam (0.25mg/kg body weight i.p.) or vehicle and then were submitted to behavioral and biochemical evaluations. In the TS group, DZP administration reduced anxiety-like symptoms in different behavioral paradigms (elevated plus maze, EPM; staircase and open-field and defensive burying) and increased exploratory behavior. These findings show that neonatal TS increased DZP pharmacological responses in adulthood compared to neonatally UH animals, as observed by reduced anxiety-like symptoms and lower levels of plasma cortisol. TS also changed plasma levels of antioxidant defenses such as vitamin C and glutathione peroxidase, whose increase may be involved in lower oxidative damages to proteins in cortex, subthalamic region and hippocampus of these animals. Here we are showing for the first time that neonatal TS is able to change responsiveness to benzodiazepine drugs in adulthood and provides better pharmacological responses in novel situations of stress.