Daiana Silva de Ávila

Extracellular dopamine potentiates mn-induced oxidative stress, lifespan reduction, and dopaminergic neurodegeneration in a BLI-3-dependent manner in Caenorhabditis elegans

Parkinsons disease (PD)-mimicking drugs and pesticides, and more recently PD-associated gene mutations, have been studied in cell cultures and mammalian models to decipher the molecular basis of PD. Thus far, a dozen of genes have been identified that are responsible for inherited PD. However they only account for about 8% of PD cases, most of the cases likely involving environmental contributions. Environmental manganese (Mn) exposure represents an established risk factor for PD occurrence, and both PD and Mn-intoxicated patients display a characteristic extrapyramidal syndrome primarily involving dopaminergic (DAergic) neurodegeneration with shared common molecular mechanisms. To better understand the specificity of DAergic neurodegeneration, we studied Mn toxicity in vivo in Caenorhabditis elegans. Combining genetics and biochemical assays, we established that extracellular, and not intracellular, dopamine (DA) is responsible for Mn-induced DAergic neurodegeneration and that this process (1) requires functional DA-reuptake transporter (DAT-1) and (2) is associated with oxidative stress and lifespan reduction. Overexpression of the anti-oxidant transcription factor, SKN-1, affords protection against Mn toxicity, while the DA-dependency of Mn toxicity requires the NADPH dual-oxidase BLI-3. These results suggest that in vivo BLI-3 activity promotes the conversion of extracellular DA into toxic reactive species, which, in turn, can be taken up by DAT-1 in DAergic neurons, thus leading to oxidative stress and cell degeneration.

Swimming training prevents pentylenetetrazol-induced inhibition of Na+, K+-ATPase activity, seizures, and oxidative stress

Purpose:  In the present study we decided to investigate whether physical exercise protects against the electrographic, oxidative, and neurochemical alterations induced by subthreshold to severe convulsive doses of pentyltetrazole (PTZ).

Baker yeast-induced fever in young rats: characterization and validation of an animal model for antipyretics screening.

In this study we describe a low-cost and reliable method for inducing fever in young male rats (28-30 days of age, 75-90 g), which seems suitable for the screening of new antipyretics. The effects of temperature measuring procedure-induced stress on the basal rectal temperature and on Baker yeast-induced hyperthermia was assessed. Rectal temperature (T) was recorded every hour for 12 h (07:00-19:00 h) with a lubricated thermistor probe. The animals were injected intraperitoneally with baker yeast (0.25, 0.135, 0.05 g/kg) or the equivalent volume of saline at 7:00 h. The administration of 0.135 g/kg baker yeast induced a sustained increase in rectal temperature for 4 h. Classical (dipyrone and acetaminophen) and novel (MPCA and FPCA) antipyretics, at doses that had no effect per se, reverted baker yeast-induced fever. The method presented induces a clear-cut fever, which is reverted by antipyretics commonly used in human beings and selected novel antipyretics in small animals. The method also allows antipyretic evaluation with low amount of drugs, due to the use of small animals and to the small variability of the pyretic response, which ultimately causes a significant reduction in the number of animals necessary for antipyretic evaluation. Therefore, this study describes an animal model of fever that is not only advantageous from the economical and technical point of view, but that also bears ethical concerns.

Valeriana officinalis does not alter the orofacial dyskinesia induced by haloperidol in rats: role of dopamine transporter.

Chronic treatment with classical neuroleptics in humans can produce a serious side effect, known as tardive dyskinesia (TD). Here, we examined the effects of V. officinalis, a medicinal herb widely used as calming and sleep-promoting, in an animal model of orofacial dyskinesia (OD) induced by long-term treatment with haloperidol. Adult male rats were treated during 12 weeks with haloperidol decanoate (38 mg/kg, i.m., each 28 days) and with V. officinalis (in the drinking water). Vacuous chewing movements (VCMs), locomotor activity and plus maze performance were evaluated. Haloperidol treatment produced VCM in 40% of the treated rats and the concomitant treatment with V. officinalis did not alter either prevalence or intensity of VCMs. The treatment with V. officinalis increased the percentage of the time spent on open arm and the number of entries into open arm in the plus maze test. Furthermore, the treatment with haloperidol and/or V. officinalis decreased the locomotor activity in the open field test. We did not find any difference among the groups when oxidative stress parameters were evaluated. Haloperidol treatment significantly decreased [(3)H]-dopamine uptake in striatal slices and V. officinalis was not able to prevent this effect. Taken together, our data suggest a mechanism involving the reduction of dopamine transport in the maintenance of chronic VCMs in rats. Furthermore, chronic treatment with V. officinalis seems not produce any oxidative damage to central nervous system (CNS), but it also seems to be devoid of action to prevent VCM, at least in the dose used in this study.

A Possible Neuroprotective Action of a Vinylic Telluride against Mn-Induced Neurotoxicity

Manganese (Mn) is a metal required by biological systems. However, environmental or occupational exposure to high levels of Mn can produce a neurological disorder called manganism, which has similarities to Parkinsons disease. Diethyl-2-phenyl-2-tellurophenyl vinylphosphonate (DPTVP) is an organotellurium compound with a high antioxidant activity, especially in the brain. The present study was designed to investigate the effects of long-term low-dose exposure to Mn in drinking water on behavioral and biochemical parameters in rats and to determine the effectiveness of vinylic telluride in attenuating the effects of Mn. After 4 months of treatment with MnCl(2) (13.7 mg/kg), rats exhibited clear signs of neurobehavioral toxicity, including a decrease in the number of rearings in the open field and altered motor performance in rotarod. The administration of DPTVP (0.150 micromol/kg, ip, 2 weeks) improved the motor performance of Mn-treated rats, indicating that the compound could be reverting Mn neurotoxicity. Ex vivo, we observed that Mn concentrations in the Mn-treated group were highest in the striatum, consistent with a statistically significant decrease in mitochondrial viability and [(3)H]glutamate uptake, and increased lipid peroxidation. Mn levels in the hippocampus and cortex were indistinguishable from controls, and no significant differences were noted in the ex vivo assays in these areas. Treatment with DPTVP fully reversed the biochemical parameters altered by Mn. Furthermore, DPTVP treatment was also associated with a reduction in striatal Mn levels. Our results demonstrate that DPTVP has neuroprotective activity against Mn-induced neurotoxicity, which may be attributed to its antioxidant activity and/or its effect on striatal Mn transport.

Organotellurium and organoselenium compounds attenuate Mn-induced toxicity in Caenorhabditis elegans by preventing oxidative stress.

Organochalcogens have been widely studied given their antioxidant activity, which confers neuroprotection, antiulcer, and antidiabetic properties. Given the complexity of mammalian models, understanding the cellular and molecular effects of organochalcogens has been hampered. The nematode worm Caenorhabditis elegans is an alternative experimental model that affords easy genetic manipulations, green fluorescent protein tagging, and in vivo live analysis of toxicity. We previously showed that manganese (Mn)-exposed worms exhibit oxidative-stress-induced neurodegeneration and life-span reduction. Here we use Mn-exposed worms as a model for an oxidatively challenged organism to investigate the underlying mechanisms of organochalcogen antioxidant properties. First, we recapitulate in C. elegans the effects of organochalcogens formerly observed in mice, including their antioxidant activity. This is followed by studies on the ability of these compounds to afford protection against Mn-induced toxicity. Diethyl-2-phenyl-2-tellurophenyl vinyl phosphonate (DPTVP) was the most efficacious compound, fully reversing the Mn-induced reduction in survival and life span. Ebselen was also effective, reversing the Mn-induced reduction in survival and life span, but to a lesser extent compared with DPTVP. DPTVP also lowered Mn-induced increases in oxidant levels, indicating that the increased survival associated with exposure to this compound is secondary to a decrease in oxidative stress. Furthermore, DPTVP induced nuclear translocation of the transcriptional factor DAF-16/FOXO, which regulates stress responsiveness and aging in worms. Our findings establish that the organochalcogens DPTVP and ebselen act as antiaging agents in a model of Mn-induced toxicity and aging by regulating DAF-16/FOXO signaling and attenuating oxidative stress.

α-Tocopherol protects against pentylenetetrazol- and methylmalonate-induced convulsions

Increased excitatory amino acid transmission and decreased GABAergic inhibitory responses seem to be important mechanisms in the genesis of convulsions, where reactive oxygen species (ROS) have recently been suggested to play a critical role. Therefore, administration of antioxidants may be potentially beneficial for the treatment of convulsive states. In the current study we investigated the effect of the systemic Vitamin E administration, an antioxidant, on the convulsions and oxidative damage induced by two convulsant agents with different mechanisms of action: methylmalonic acid (MMA), which induces convulsions through energy depletion and secondary activation of glutamatergic mechanisms and ROS production and pentylenetetrazol (PTZ), which is a chemical convulsant that causes convulsions by blocking the GABAA receptor-coupled chloride ionophore. Adult male Wistar rats (270-300 g) were injected with vehicle (5% Tween 80 in 0.9% NaCl; 1 ml/kg, i.p.) or alpha-tocopherol (25, 75 or 225 mg/kg, i.p.), once a day for 7 days. On the seventh day of antioxidant treatment, the animals were injected with the antioxidant (or vehicle) and, 30 min later, they were intrastriatally injected with NaCl (9 micromol/2 microl) or with MMA (6 micromol/2 microl) or PTZ (3.26 mmicromol/2 microl). The animals were observed for the appearance of convulsive behavior and the striatal content of thiobarbituric acid-reactive substances (TBARS) and total protein carbonylation were determined. Intrastriatal injection of increasing amounts of PTZ and of MMA caused the appearance of convulsive behavior. PTZ- and MMA-induced convulsions, TBARS production and total protein carbonylation were attenuated by alpha-tocopherol in a dose-dependent manner.

Additive anticonvulsant effects of creatine supplementation and physical exercise against pentylenetetrazol-induced seizures.

Although physical activity and creatine supplementation have been a documented beneficial effect on neurological disorders, its implications for epilepsy are still controversial. Thus, we decided to investigate the effects of 6 weeks swimming training, creatine supplementation (300 mg/kg; p.o.) or its combination seizures and neurochemical alterations induced by pentylenetetrazol (PTZ). We found that 6 weeks of physical training or creatine supplementation decreased the duration of PTZ-induced seizures in adult male Wistar rats, as measured by cortical and hippocampal electroencephalography and behavioral analysis. Importantly, the combination between physical training and creatine supplementation had additive anticonvulsant effects, since it increased the onset latency for PTZ-induced seizures and was more effective in decrease seizure duration than physical training and creatine supplementation individually. Analysis of selected parameters of oxidative stress and antioxidant defenses in the hippocampus revealed that physical training, creatine supplementation or its combination abrogated the PTZ-elicited increase in levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonylation, as well as decrease in non-protein-thiols content, catalase (CAT) and SOD activities. In addition, this protocol of physical training and creatine supplementation prevented the PTZ-induced decrease in hippocampal Na+,K+-ATPase activity. Altogether, these results suggest that protection elicited physical training and creatine supplementation of selected targets for reactive species-mediated damage decrease of neuronal excitability and consequent oxidative damage elicited by PTZ. In conclusion, the present study shows that physical training, creatine supplementation or its combination attenuated PTZ-induced seizures and oxidative damage in vivo, and provide evidence that combination between creatine supplementation and physical exercise may be a useful strategy in the treatment of convulsive disorders.

The Caenorhabiditis elegans model as a reliable tool in neurotoxicology

Caenorhabiditis elegans (C. elegans) offers an attractive experimental platform as it has a short life cycle, is inexpensive to maintain and most importantly has high degree of evolutionary conservation with higher eukaryotes. Understanding the contribution of inherent genes that regulate neurotoxicity and antioxidant stress responses in the worm provides critical insight into mechanisms of mammalian neurotoxicity. The C. elegans model readily enables multi-gene approach, allowing for combinatorial genetic variation to be studied within the context of the influence of multigenic polymorphisms in environmental risk and vulnerability. This review provides a synopsis of recent studies on metal and pesticides toxicity in C. elegans, highlighting the utility of the model system in understanding molecular mechanisms that underlie developmental, reproductive and neuronal damage. The continuation of these investigations combining basic toxicological experimentation with novel genetic and high throughput methods will continue to make C. elegans an invaluable tool for future research, providing insight into molecular and cellular mechanisms of toxicity.

Utility of Caenorhabditis elegans in high throughput neurotoxicological research.

Caenorhabditis elegans is a nematode that has been used as a valuable research tool in many facets of biological research. Researchers have used the many tools available to investigate this well-studied nematode, including a cell lineage map, sequenced genome, and complete wiring diagram of the nervous system, making in-depth investigation of the nervous system practical. These tools, along with other advantages, such as its small size, short life cycle, transparency, and ability to generate many progeny, have made C. elegans an attractive model for many studies, including those investigating toxicological paradigms and those using high throughput techniques. Researchers have investigated a number of endpoints, such as behavior and protein expression using a green fluorescent protein (GFP) marker following toxicant exposure and have explored the mechanisms of toxicity using techniques such as microarray, RNA interference (RNAi), and mutagenesis. This review discusses the benefits of using C. elegans as a model system and gives examples of the uses of C. elegans in toxicological research. High throughput techniques are discussed highlighting the advantages of using an in vivo system that has many advantageous characteristics of an in vitro system while emphasizing endpoints relating to developmental and adult neurotoxicity.