MeHg exposure impairs both the catecholaminergic and cholinergic systems resulting in motor and non-motor behavioral changes in Drosophila melanogaster.

Human exposure to the natural environmental contaminant methylmercury (MeHg) has been associated to adverse health effects. Importantly, the mechanisms by which this organomercurial exerts its neurotoxicity have yet to be fully clarified. Therefore, the aim of this study was to evaluate whether exposure to MeHg alters dopamine (DA) and octopamine (OA) levels, acetylcholinesterase (AChE) activity and impacts both motor and non-motor behaviours. We studied the effect of MeHg by feeding 1-2 d old flies (male and females) with 25 and 50 µM MeHg for 4 d and determined effects on survival, motor and non-motor behaviours, oxidative stress, AChE and tyrosine hydroxylase (TH) activities, as well as DA and OA levels. We found that Drosophila melanogaster (D. melanogaster) exposed to MeHg showed a reduction in survival rate, associated with the inhibition of AChE and TH activities in head of flies and decreased DA and OA levels. These changes were accompanied by behavioural alterations, such as locomotor deficit and increased grooming behaviour, in addition to an increase in oxidative stress markers both in head and in body of flies, and an increase in glutathione-S-transferase (GST) activity in head of flies. Collectively, our data support the hypothesis that MeHg neurotoxicity is associated with altered OA and DA levels, AChE inhibition, which may serve, at least in part, as the underpinnings of both motor and non-motor behavioural changes.

Neem oil based nanopesticide as an environmentally-friendly formulation for applications in sustainable agriculture: An ecotoxicological perspective.

Sustainable agriculture encourages practices that present low risks to the environment and human health. To this end, zein (corn protein) can be used to develop nanocarrier systems capable of improving the physicochemical properties of biopesticides, reducing their possible toxicity. Neem oil extracted from the Azadirachta indica tree contains many active ingredients including azadirachtin, which is the active ingredient in multiple commercially available biopesticides. In this study, we describe the preparation and characterization of neem oil-loaded zein nanoparticles, together with evaluation of their toxicity towards nontarget organisms, using Allium cepa, soil nitrogen cycle microbiota, and Caenorhabditis elegans aiming to achieve the safer by design strategy. The spherical nanoparticles showed an average diameter of 278 ±â€¯61.5 nm and a good stability during the experiments. In the toxicity assays with A. cepa, the neem oil-loaded zein nanoparticles mitigated the increase in the DNA relative damage index caused by the neem oil. Molecular genetic analysis of the soil nitrogen cycle microbiota revealed that neem oil-loaded zein nanoparticles did not change the number of genes which encode nitrogen-fixing enzymes and denitrifying enzymes. In C. elegans, the neem oil-loaded zein nanoparticles had no toxic effect, while neem oil interfered with pharyngeal pumping and GST-4 protein expression. These neem oil-loaded zein nanoparticles showed promising results in the toxicity studies, opening perspectives for its use in crop protection in organic agriculture.

Organoselenotriazoles attenuate oxidative damage induced by mitochondrial dysfunction in mev-1 Caenorhabditis elegans mutants.

Organic selenium compounds have several pharmacological activities already described, as anti-inflammatory and antitumor activities, which have been attributed to their antioxidant effects. Because they are promising in pharmacology, the synthesis of these compounds has increased significantly. As many new molecules are synthesized the use of a simple model like Caenorhabditis elegans is highly advantageous for initial evaluation of the toxicity and therapeutic potential of these molecules. The objective of this study was to evaluate the toxicity and antioxidant capacity of a series of selenotriazoles compounds in C. elegans. The animals were exposed to the compounds in liquid medium for only 30 min at the first larval stage (L1). The compounds had no toxic effects at the concentrations tested. Treatment with selenotriazoles (10 µM) partially reversed the stress induced by the pesticide paraquat (1 mM). Se-Tz Ia compound partially increased the survival of worms treated with H2O2 (0.5 mM). The compounds also increased the longevity of mev-1 mutants, which have a reduced life span by the production of excessive reactive oxygen species (ROS) in the mitochondria caused by a mutation in complex II of the electron transport chain. In addition, the compounds reduced the levels of ROS determined by the fluorescent probe DCF-DA as well as also reduced catalase enzyme activity in these animals. Based on the results found, it is possible to conclude that the compounds have antioxidant activity mainly in oxidative stress condition generated by a mitochondrial dysfunction in C. elegans.

Thimerosal inhibits Drosophila melanogaster tyrosine hydroxylase (DmTyrH) leading to changes in dopamine levels and impaired motor behavior: implications for neurotoxicity.

Thimerosal (THIM) is a well-established antifungal and antiseptic agent widely used as a preservative in vaccines. Recent studies identified the neurotoxic effects of THIM, including malfunction of the monoaminergic system. However, the underlying cytotoxic mechanisms are not well understood. Here we used the fruit fly Drosophila melanogaster to investigate the mechanisms of THIM-induced neurotoxicity. We focused on the dopaminergic system, and the rate-limiting enzyme tyrosine hydroxylase (DmTyrH), to test the hypothesis that THIM can impair dopamine (DA) homeostasis and subsequently cause dysfunction. We studied the effect of THIM by feeding 1-2 day old flies (both sexes) food supplemented with 25 µM THIM for 4 days and determined THIM-induced effects on survival, oxidative stress, and metabolic activity based on MTT assay and acetylcholinesterase (AChE) activity. Our results demonstrate that D. melanogaster exposed to THIM present changes in DmTyrH expression and activity, together with altered DA levels that led to impaired motor behavior. These phenotypes were accompanied by an increase in oxidative stress, with a decrease in MTT reduction, in AChE activity, and also in survival rate. These findings suggest an initiating and primary role for THIM-mediated DmTyrH dysfunction that leads to impaired DA function and behavioral abnormalities, ultimately causing oxidative stress-related neurotoxicity.

Cellular Responses in Drosophila melanogaster Following Teratogen Exposure.

Studies focusing on the teratogenicity of a series of new chemicals that are produced in a daily basis represent an important focus in toxicological/pharmaceutical research, particularly due to the risks arising from occupational exposure of the subjects. However, the complex mating procedures, scheduling of treatments, requirements for trained personnel, and elevated costs of traditional teratological assays with mammals hamper this type of assessments. Accordingly, the use of Drosophila melanogaster as a model for teratological studies has received considerable attention. Here some general protocols about Drosophila exposure-at different stages of their life cycle-to any chemical with putative teratological activity are presented. Importantly, some details about D. melanogaster embryonic, larval, pupal, or adult endpoints, that can be used to assess teratogenicity using flies as a model organism, are presented.

Metabolic effects of manganese in the nematode Caenorhabditis elegans through DAergic pathway and transcription factors activation.

Manganese (Mn) is an essential trace element for physiological functions since it acts as an enzymatic co-factor. Nevertheless, overexposure to Mn has been associated with a pathologic condition called manganism. Furthermore, Mn has been reported to affect lipid metabolism by mechanisms which have yet to be established. Herein, we used the nematode Caenorhabditis elegans to examine Mn's effects on the dopaminergic (DAergic) system and determine which transcription factors that regulate with lipid metabolism are affected by it. Worms were exposed to Mn for four hours in the presence of bacteria and in a liquid medium (85 mM NaCl). Mn increased fat storage as evidenced both by Oil Red O accumulation and triglyceride levels. In addition, metabolic activity was reduced as a reflection of decreased oxygen consumption caused by Mn. Mn also affected feeding behavior as evidenced by decreased pharyngeal pumping rate. DAergic neurons viability were not altered by Mn, however the dopamine levels were significantly reduced following Mn exposure. Furthermore, the expression of sbp-1 transcription factor and let-363 protein kinase responsible for lipid accumulation control was increased and decreased, respectively, by Mn. Altogether, our data suggest that Mn increases the fat storage in C. elegans, secondary to DAergic system alterations, under the control of SBP-1 and LET-363 proteins.

Insights into the differential toxicological and antioxidant effects of 4-phenylchalcogenil-7-chloroquinolines in Caenorhabditis elegans.

Organic selenium and tellurium compounds are known for their broad-spectrum effects in a variety of experimental disease models. However, these compounds commonly display high toxicity and the molecular mechanisms underlying these deleterious effects have yet to be elucidated. Thus, the need for an animal model that is inexpensive, amenable to high-throughput analyses, and feasible for molecular studies is highly desirable to improve organochalcogen pharmacological and toxicological characterization. Herein, we use Caenorhabdtis elegans (C. elegans) as a model for the assessment of pharmacological and toxicological parameters following exposure to two 4-phenylchalcogenil-7-chloroquinolines derivatives (PSQ for selenium and PTQ for tellurium-containing compounds). While non-lethal concentrations (NLC) of PTQ and PSQ attenuated paraquat-induced effects on survival, lifespan and oxidative stress parameters, lethal concentrations (LC) of PTQ and PSQ alone are able to impair these parameters in C. elegans. We also demonstrate that DAF-16/FOXO and SKN-1/Nrf2 transcription factors underlie the mechanism of action of these compounds, as their targets sod-3, gst-4 and gcs-1 were modulated following exposures in a daf-16- and skn-1-dependent manner. Finally, in accordance with a disturbed thiol metabolism in both LC and NLC, we found higher sensitivity of trxr-1 worm mutants (lacking the selenoprotein thioredoxin reductase 1) when exposed to PSQ. Finally, our study suggests new targets for the investigation of organochalcogen pharmacological effects, reinforcing the use of C. elegans as a powerful platform for preclinical approaches.

"Manganese-induced neurotoxicity: a review of its behavioral consequences and neuroprotective strategies".

Manganese (Mn) is an essential heavy metal. However, Mn's nutritional aspects are paralleled by its role as a neurotoxicant upon excessive exposure. In this review, we covered recent advances in identifying mechanisms of Mn uptake and its molecular actions in the brain as well as promising neuroprotective strategies. The authors focused on reporting findings regarding Mn transport mechanisms, Mn effects on cholinergic system, behavioral alterations induced by Mn exposure and studies of neuroprotective strategies against Mn intoxication. We report that exposure to Mn may arise from environmental sources, occupational settings, food, total parenteral nutrition (TPN), methcathinone drug abuse or even genetic factors, such as mutation in the transporter SLC30A10. Accumulation of Mn occurs mainly in the basal ganglia and leads to a syndrome called manganism, whose symptoms of cognitive dysfunction and motor impairment resemble Parkinson's disease (PD). Various neurotransmitter systems may be impaired due to Mn, especially dopaminergic, but also cholinergic and GABAergic. Several proteins have been identified to transport Mn, including divalent metal tranporter-1 (DMT-1), SLC30A10, transferrin and ferroportin and allow its accumulation in the central nervous system. Parallel to identification of Mn neurotoxic properties, neuroprotective strategies have been reported, and these include endogenous antioxidants (for instance, vitamin E), plant extracts (complex mixtures containing polyphenols and non-characterized components), iron chelating agents, precursors of glutathione (GSH), and synthetic compounds that can experimentally afford protection against Mn-induced neurotoxicity.

Reversible reprotoxic effects of manganese through DAF-16 transcription factor activation and vitellogenin downregulation in Caenorhabditis elegans.

AIMS: Vitellogenesis is the yolk production process which provides the essential nutrients for the developing embryos. Yolk is a lipoprotein particle that presents lipids and lipid-binding proteins, referred to as vitellogenins (VIT). The Caenorhabditis elegans nematode has six genes encoding VIT lipoproteins. Several pathways are known to regulate vitellogenesis, including the DAF-16 transcription factor. Some reports have shown that heavy metals, such as manganese (Mn), impair brood size in C. elegans; however the mechanisms associated with this effect have yet to be identified. Our aim was to evaluate Mn's effects on C. elegans reproduction and better understand the pathways related to these effects. MAIN METHODS: Young adult larval stage worms were treated for 4h with Mn in 85mM NaCl and Escherichia coli OP50 medium. KEY FINDINGS: Mn reduced egg-production and egg-laying during the first 24h after the treatment, although the total number of progenies were indistinguishable from the control group levels. This delay may have occurred due to DAF-16 activation, which was noted only after the treatment and was not apparent 24h later. Moreover, the expression, protein levels and green fluorescent protein (GFP) fluorescence associated with VIT were decreased soon after Mn treatment and recovered after 24h. SIGNIFICANCE: Combined, these data suggest that the delay in egg-production is likely regulated by DAF-16 and followed by the inhibition of VIT transport activity. Further studies are needed to clarify the mechanisms associated with Mn-induced DAF-16 activation.

Are delta-aminolevulinate dehydratase inhibition and metal concentrations additional factors for the age-related cognitive decline?

Aging is often accompanied by cognitive impairments and influenced by oxidative status and chemical imbalances. Thus, this study was conducted to examine whether age-related cognitive deficit is associated with oxidative damage, especially with inhibition of the enzyme delta-aminolevulinate dehydratase (ALA-D), as well as to verify the influence of some metals in the enzyme activity and cognitive performance. Blood ALA-D activity, essential (Fe, Zn, Cu, Se) and non-essential metals (Pb, Cd, Hg, As, Cr, Ni, V) were measured in 50 elderly and 20 healthy young subjects. Cognitive function was assessed by tests from Consortium to Establish a Registry for Alzheimer's Disease (CERAD) battery and other. The elderly group presented decreased ALA-D activity compared to the young group. The index of ALA-D reactivation was similar to both study groups, but negatively associated with metals. The mean levels of essential metals were within the reference values, while the most toxic metals were above them in both groups. Cognitive function impairments were observed in elderly group and were associated with decreased ALA-D activity, with lower levels of Se and higher levels of toxic metals (Hg and V). Results suggest that the reduced ALA-D activity in elderly can be an additional factor involved in cognitive decline, since its inhibition throughout life could lead to accumulation of the neurotoxic compound ALA. Toxic metals were found to contribute to cognitive decline and also to influence ALA-D reactivation.

Ilex paraguariensis Extract Increases Lifespan and Protects Against the Toxic Effects Caused by Paraquat in Caenorhabditis elegans.

Recent studies have shown that phenolic compounds present in yerba mate have antioxidant defense properties. To verify whether Ilex paraguariensis extracts are capable of increasing the lifespan of an organism, we have used the free-living nematode Caenorhabditis elegans. Notably, this is the first study that analyzes the effects of the extracts of yerba mate obtained from an extraction method that mimics the manner that the plant is consumed by the population by using a live organism. Yerba mate was purchased from commercial markets from Argentina, Brazil, and Uruguay. Ilex paraguariensis extracts significantly increased the life span of C. elegans. Moreover, the extracts reduced the ROS levels per se, and protected from the reduced survival and reproduction rate induced by paraquat exposure. Considering molecular aspects, we observed that the worms pretreated with the extracts depicted higher translocation of the transcription factor DAF-16::GFP to the nucleus. However, there was no increase in the levels of the DAF-16 target genes, SOD-3 and catalase. Our results suggest that the increase of lifespan caused by the different extracts is associated to the antioxidant potential of yerba mate, however this effect is not completely mediated by daf-16.

Seleno- and telluro-xylofuranosides attenuate Mn-induced toxicity in C. elegans via the DAF-16/FOXO pathway.

Organochalcogens are promising pharmacological agents that possess significant biological activities. Nevertheless, because of the complexity of mammalian models, it has been difficult to determine the molecular pathways and specific proteins that are modulated in response to treatments with these compounds. 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. Abundant evidence points to oxidative stress in mediating manganese (Mn)-induced toxicity. In this study we challenged worms with Mn, and investigated the efficacy of inedited selenium- and tellurium-xylofuranosides in reversing and/or protecting the worms from Mn-induced toxicity. In addition, we investigated their putative mechanism of action. First, we determined the lethal dose 50% (LD50) and the effects of the xylofuranosides on various toxic parameters. This was followed by studies on the ability of xylofuranosides to afford protection against Mn-induced toxicity. Both Se- and Te-xylofuranosides increased the expression of superoxide dismutase (SOD-3). Furthermore, we observed that the xylofuranosides induced nuclear translocation of the transcription factor DAF-16/FOXO, which in the worm is known to regulate stress responsiveness, aging and metabolism. These findings suggest that xylofuranosides attenuate toxicity Mn-induced, by regulating the DAF-16/FOXO signaling pathway.

The influence of Bauhinia forficata Link subsp. pruinosa tea on lipid peroxidation and non-protein SH groups in human erythrocytes exposed to high glucose concentrations.

ETHNOPHARMACOLOGICAL RELEVANCE: Bauhinia forficata (BF) has been traditionally used as tea in folk medicine of Brazil for treatment of Diabetes mellitus (DM). AIM OF THE STUDY: To evaluate the effects of BF leaf tea on markers of oxidative damage and antioxidant levels in an experimental model of hyperglycemia in human erythrocytes in vitro. MATERIALS AND METHODS: Human erythrocytes were incubated with high glucose concentrations or glucose and BF tea for 24h and 48h. After incubation lipid peroxidation and non-protein SH levels were analyzed. Moreover, quantification of polyphenols and flavonoids, iron chelating property, scavenging of DPPH, and prevention of lipid peroxidation in isolated lipids were also assessed. RESULTS: A significant amount of polyphenols and flavonoids was observed. The main components found by LC-MS analysis were quercetin-3-O-(2-rhamnosyl) rutinoside, kaempferol-3-O-(2-rhamnosyl) rutinoside, quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside. BF tea presents important antioxidant and chelating properties. Moreover, BF tea was effective to increase non-protein SH levels and reduce lipid peroxidation induced by high glucose concentrations in human erythrocytes. CONCLUSION: The antioxidant effects of BF tea could be related to the presence of different phenolic and flavonoids components. We believe that these components can be responsible to protect human erythrocytes exposed to high glucose concentrations against oxidative damage.

Gene-environment interactions: neurodegeneration in non-mammals and mammals.

The understanding of how environmental exposures interact with genetics in central nervous system dysfunction has gained great momentum in the last decade. Seminal findings have been uncovered in both mammalian and non-mammalian model in large result of the extraordinary conservation of both genetic elements and differentiation processes between mammals and non-mammalians. Emerging model organisms, such as the nematode and zebrafish have made it possible to assess the effects of small molecules rapidly, inexpensively, and on a miniaturized scale. By combining the scale and throughput of in vitro screens with the physiological complexity and traditional animal studies, these models are providing relevant information on molecular events in the etiology of neurodegenerative disorders. The utility of these models is largely driven by the functional conservation seen between them and higher organisms, including humans so that knowledge obtained using non-mammalian model systems can often provide a better understanding of equivalent processes, pathways, and mechanisms in man. Understanding the molecular events that trigger neurodegeneration has also greatly relied upon the use of tissue culture models. The purpose of this summary is to provide-state-of-the-art review of recent developments of non-mammalian experimental models and their utility in addressing issues pertinent to neurotoxicity (Caenorhabditis elegans and Danio rerio). The synopses by Aschner and Levin summarize how genetic mutants of these species can be used to complement the understanding of molecular and cellular mechanisms associated with neurobehavioral toxicity and neurodegeneration. Next, studies by Suñol and Olopade detail the predictive value of cultures in assessing neurotoxicity. Suñol and colleagues summarize present novel information strategies based on in vitro toxicity assays that are predictive of cellular effects that can be extrapolated to effects on individuals. Olopade and colleagues describe cellular changes caused by sodium metavanadate (SMV) and demonstrate how rat primary astrocyte cultures can be used as predicitive tools to assess the neuroprotective effects of antidotes on vanadium-induced astrogliosis and demyelination.

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 Parkinson's 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.

Comparative studies on dicholesteroyl diselenide and diphenyl diselenide as antioxidant agents and their effect on the activities of Na+/K+ ATPase and delta-aminolevulinic acid dehydratase in the rat brain.

The present study sought to evaluate the effect of a newly synthesized selenium compound, dicholesteroyl diselenide (DCDS) and diphenyl diselenide (DPDS) on the activities of delta-aminolevulinate dehydratase and Na+/K+-ATPase in the rat brain. The glutathione peroxidase mimetic activity of the two compounds as well as their ability to oxidize mono- and di- thiols were also evaluated. The antioxidant effects were tested by measuring the ability of the compounds to inhibit the formation of thiobarbituric acid reactive species and also their ability to inhibit the formation of protein carbonyls. The results show that DPDS exhibited a higher glutathione peroxidase mimetic activity as well as increased ability to oxidize di-thiols than DCDS. In addition, while DPDS inhibited the formation of thiobarbituric acid reactive species and protein carbonyls, DCDS exhibited a prooxidant effect in all the concentration range (20-167 microM) tested. Also the activities of cerebral delta-aminolevulinate dehydratase and Na+/K+ ATPase were significantly inhibited by DPDS but not by DCDS. In addition, the present results suggested that the inhibition of Na+/K+ ATPase by organodiselenides, possibly involves the modification of the thiol group at the ATP binding site of the enzyme. In conclusion, the results of the present investigation indicated that the non-selenium moiety of the organochalcogens can have a profound effect on their antioxidant activity and also in their reactivity towards SH groups from low-molecular weight molecules and from brain proteins.