Toxicological assessment of photoactivated tetra-cationic porphyrin molecules under white light exposure in a Caenorhabditis elegans model.

Photodynamic therapy (PDT) utilizes the potential of photosensitizing substances to absorb light energy and produce reactive oxygen species. Tetra-cationic porphyrins, which have organic or coordination compounds attached to their periphery, are heterocyclic derivatives with well-described antimicrobial and antitumoral properties. This is due to their ability to produce reactive oxygen species and their photobiological properties in solution. Consequently, these molecules are promising candidates as new and more effective photosensitizers with biomedical, environmental, and other biomedical applications. Prior to human exposure, it is essential to establish the toxicological profile of these molecules using in vivo models. In this study, we used Caenorhabditis elegans, a small free-living nematode, as a model for assessing toxic effects and predicting toxicity in preclinical research. We evaluated the toxic effects of porphyrins (neutral and tetra-cationic) on nematodes under dark/light conditions. Our findings demonstrate that tetra-methylated porphyrins (3TMeP and 4TMeP) at a concentration of 3.3 µg/mL (1.36 and 0.93 µM) exhibit high toxicity (as evidenced by reduced survival, development, and locomotion) under dark conditions. Moreover, photoactivated tetra-methylated porphyrins induce higher ROS levels compared to neutral (3TPyP and 4TPyP), tetra-palladated (3PdTPyP and 4PdTPyP), and tetra-platinated (3PtTPyP and 4PtTPyP) porphyrins, which may be responsible for the observed toxic effects.

Toxicity of copper and zinc alone and in combination in Caenorhabditis elegans model of Huntington's disease and protective effects of rutin.

Copper (Cu) and Zinc (Zn) are required in small concentrations for metabolic functions, but are also toxic. There is a great concern about soil pollution by heavy metals, which may exposure the population to these toxicants, either by inhalation of dust or exposure to toxicants through ingestion of food derived from contaminated soils. In addition, the toxicity of metals in combination is questionable, as soil quality guidelines only assess them separately. It is well known that metal accumulation is often found in the pathologically affected regions of many neurodegenerative diseases, including Huntington's disease (HD). HD is caused by an autosomal dominantly inherited CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. This results in the formation of a mutant huntingtin (mHTT) protein with an abnormally long polyglutamine (polyQ) repeat. The pathology of HD results in loss of neuronal cells, motor changes, and dementia. Rutin is a flavonoid found in various food sources, and previous studies indicate it has protective effects in HD models and acts as a metal chelator. However, further studies are needed to unravel its effects on metal dyshomeostasis and to discern the underlying mechanisms. In the present study, we investigated the toxic effects of long-term exposure to copper, zinc, and their mixture, and the relationship with the progression of neurotoxicity and neurodegeneration in a C. elegans-based HD model. Furthermore, we investigated the effects of rutin post metal exposure. Overall, we demonstrate that chronic exposure to the metals and their mixture altered body parameters, locomotion, and developmental delay, in addition to increasing polyQ protein aggregates in muscles and neurons causing neurodegeneration. We also propose that rutin has protective effects acting through mechanisms involving antioxidant and chelating properties. Altogether, our data provides new indications about the higher toxicity of metals in combination, the chelating potential of rutin in the C. elegans model of HD and possible strategies for future treatments of neurodegenerative diseases caused by the aggregation of proteins related to metals.

Exogenous Adenosine Modulates Behaviors and Stress Response in Caenorhabditis elegans.

Adenosine, a purine nucleoside with neuromodulatory actions, is part of the purinergic signaling system (PSS). Caenorhabditis elegans is a free-living nematode found in soil, used in biological research for its advantages as an alternative experimental model. Since there is a lack of evidence of adenosine's direct actions and the PSS's participation in this animal, such an investigation is necessary. In this research, we aimed to test the effects of acute and chronic adenosine at 1, 5, and 10 mM on nematode's behaviors, morphology, survival after stress conditions, and on pathways related to the response to oxidative stress (DAF-16/FOXO and SKN-1) and genes products downstream these pathways (SOD-3, HSP-16.2, and GCS-1). Acute or chronic adenosine did not alter the worms' morphology analyzed by the worms' length, width, and area, nor interfered with reproductive behavior. On the other hand, acute and chronic adenosine modulated the defecation rate, pharyngeal pumping rate, and locomotion, in addition, to interacting with stress response pathways in C. elegans. Adenosine interfered in the speed and mobility of the worms analyzed. In addition, both acute and chronic adenosine presented modulatory effects on oxidative stress response signaling. Acute adenosine prevented the heat-induced-increase of DAF-16 activation and SOD-3 levels, while chronic adenosine per se induced DAF-16 activation and prevented heat-induced-increase of HSP-16.2 and SKN-1 levels. Together, these results indicate that exogenous adenosine has physiological and biochemical effects on C. elegans and describes possible purinergic signaling in worms.

JM-20 affects GABA neurotransmission in Caenorhabditis elegans.

Along with the discovery of new candidate molecules for pharmaceuticals, several studies have emerged showing different mechanisms of action and toxicological aspects. 3-ethoxycarbonyl-2-methyl-4- (2-nitrophenyl)4,11-dihydro-1 H-pyrido [2,3-b] [1,5] benzodiazepine (JM-20) is a hybrid molecule. It is derived from 1,5-benzodiazepines and structurally differentiated by the addition of 1,4-dihydropyridine bonded to the benzodiazepine ring. This gives this molecule potential neuroprotective, antioxidant, and anxiolytic activity. As this is a promising multi-target molecule, further studies are necessary to improve the knowledge about its mechanism of action. In our study, we used Caenorhabditis elegans (C. elegans) to investigate the effects of chronic treatment with JM-20. Nematodes from the wild-type strain (N2) were treated chronically at different concentrations of JM-20. Our results show that JM-20 does not cause mortality, but higher concentrations can delay the development of worms after 48 h exposure. We assessed basic behaviors in the worm, and our data demonstrate decreased defecation cycle. Our results suggest that JM-20 acts on the C. elegans GABAergic system because GABA neurotransmission is associated with the worm intestine. We also observed increased locomotor activity and decreased egg-laying after JM-20 treatment. When both behaviors were evaluated in mutants with have reduced levels of GABA (unc-25), this effect is no observed, suggesting the GABAergic modulation. Still, the JM-20 exert similar effect of Diazepam in basic behaviors observed. To reinforce neuromodulatory action, computational analysis was performed, and results showed a JM-20 binding on allosteric sites of nematodes GABA receptors. Overall, this work provided a better understanding of the effects of JM-20 in C. elegans as well as showed the effects of this new molecule on the GABAergic system in this animal model.

Neuroprotective effects of rutin on ASH neurons in Caenorhabditis elegans model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant, progressive neurodegenerative disease. It occurs due to a mutated huntingtin gene that contains an abnormal expansion of cytosine-adenine-guanine repeats, leading to a variable-length N-terminal polyglutamine (polyQ) chain. The mutation confers toxic functions to mutant huntingtin protein, causing neurodegeneration. Rutin is a flavonoid found in various plants, such as buckwheat, some teas, and apples. Our previous studies have indicated that rutin has protective effects in HD models, but more studies are needed to unravel its effects on protein homeostasis, and to discern the underlying mechanisms. In the present study, we investigated the effects of rutin in a Caenorhabditis elegans model of HD, focusing on ASH neurons and antioxidant defense. We tested behavioral changes (touch response, movement, and octanol response), measured neuronal polyQ aggregates, and assessed degeneration using a dye-filling assay. In addition, we analyzed expression levels of heat-shock protein-16.2 and superoxide dismutase-3. Overall, our data demonstrate that chronic rutin treatment maintains the function of ASH neurons, and decreases the degeneration of their sensory terminations. We propose that rutin does so in a mechanism that involves antioxidant activity by controlling the expression of antioxidant enzymes and other chaperones regulating proteostasis. Our findings provide new evidence of rutin's potential neuroprotective role in the C. elegans model and should inform treatment strategies for neurodegenerative diseases and other diseases caused by age-related protein aggregation.

Antimycobacterial activity of Achyrocline flaccida (Asteraceae) aqueous extract from Southern Brazil.

Achyrocline flaccida aqueous extract was obtained by macerating wildflowers. The phytochemical profile present in the A. flaccida aqueous extract was elucidated by HPLC-ESI-MS/MS. Toxicity was evaluated in vitro by comet assay in peripheral blood mononuclear cells (PBMCs) and in vivo using Caenorhabditis elegans as a model. The antioxidant activity was also evaluated, and antimycobacterial activity was assessed by the broth microdilution method. The compounds present in the aqueous extract mainly belonged to the flavonoid class (89%). The concentrations that showed protective effects in C. elegans against oxidative stress and antimycobacterial activity had no toxic effects. The antimycobacterial activity test demonstrated that the concentration of 1,560 µg mL-1 inhibited the growth and eradication of the mycobacterial tested strains. Based on our findings, the A. flaccida aqueous extract presents a viable potential in developing new phytotherapeutic drugs against mycobacteria of clinical relevance.

Ilex paraguariensis extract provides increased resistance against oxidative stress and protection against Amyloid beta-induced toxicity compared to caffeine in Caenorhabditis elegans.

Ilex paraguariensis is a plant from South America, used to prepare a tea-like beverage rich in caffeine and polyphenols with antioxidant proprieties. Caffeine consumption is associated with a lower risk of age-associated neuropathologies, besides several extracts that have antioxidant proprieties are known to be neuroprotective, and oxidative stress strongly correlates with Aß-toxicity. This study aims to investigate the neuroprotective effects of the Ilex paraguariensis hydroalcoholic extract (IPHE) and to evaluate if caffeine agent present in IPHE exerts neuroprotective effects in an amyloid beta-peptide (Aß)-induced toxicity in Caenorhabditis elegans. The wild-type and CL2006 worms were treated with IPHE (2 and 4 mg/mL) or caffeine (200 and 400 µM) since larval stage 1 (L1) until they achieved the required age for each assay. IPHE and caffeine increased the lifespan and appeared to act directly by reactive oxygen species (ROS) scavenger in both wild-type and CL2006 worms, also conferred resistance against oxidative stress in wild-type animals. Furthermore, both treatments delayed Aß-induced paralysis and decreased AChE activity in CL2006. The protective effect of IPHE against Aß-induced paralysis was found to be dependent on heat shock factor hsf-1 and FOXO-family transcription factor daf-16, which are respectively involved in aging-related processes and chaperone synthesis, while that of caffeine was dependent only on daf-16. Mechanistically, IPHE and caffeine decreased the levels of Aß mRNA in the CL2006 worms; however, only IPHE induced expression of the heat shock chaperonin hsp-16.2, involved in protein homeostasis. The results were overall better when treated with IPHE than with caffeine.

Diphenyl diselenide protects a Caenorhabditis elegans model for Huntington's disease by activation of the antioxidant pathway and a decrease in protein aggregation.

Huntington's disease (HD) is an autosomal dominant, progressive neurodegenerative disease with a distinct phenotype. It occurs due to a mutation in the huntingtin (or IT19) gene with an abnormal CAG repeat, leading to a variable length N-terminal polyglutamine chain (poly-Q). Like most neurodegenerative diseases, HD is characterized by the abnormal deposition and aggregation of proteins in the cell, which impairs the proteostasis and disrupts cellular homeostasis. In this study, we used Caenorhabditis elegans as an animal model due to its easy genetic manipulation and high homology of genes and signaling pathways with mammals. Worms were exposed to diphenyl diselenide (PhSe)2 at 25, 50 and 100 µM, and then we analyzed the polyQ aggregation, neurodegeneration, touch response, reactive oxygen species (ROS) levels, lifespan and health span. In addition, we analyzed the involvement of the transcription factor DAF-16, a FOXO-ortholog, and the downstream heat-shock protein-16.2 (HSP-16.2) and superoxide dismutase-3 (SOD-3). Our data demonstrate that chronic treatment with (PhSe)2 reduced polyQ aggregation in muscle and polyQ mediated neuronal cell death of sensory neurons ASH, as well as maintaining the neuronal function. In addition, (PhSe)2 decreased ROS levels and extended the lifespan and health span of wild type and PolyQ mutant worms. The mechanism proposed is the activation of DAF-16, HSP-16.2 and SOD-3 in whole body tissues to increase the antioxidant capacity and regulation of proteostasis, decreasing PolyQ aggregation and toxicity and reducing ROS levels, leading to an increase in lifespan, and healthspan. Our findings provide new clues for treatment strategies for neurodegenerative diseases and other diseases caused by age-related protein aggregation.

Rutin protects Huntington's disease through the insulin/IGF1 (IIS) signaling pathway and autophagy activity: Study in Caenorhabditis elegans model.

Huntington's disease (HD) is inherited neurodegenerative disease, it is characterized by excessive motor movements and cognitive and emotional deficits. HD is caused by an abnormally long polyglutamine (polyQ) expansion in the huntingtin (Htt) protein, which confers toxic functions to mutant Htt leading to neurodegeneration. Rutin is a flavonoid found in plants, buckwheat, some teas and also in apples. Although previous studies have already indicated that rutin has some protective effects in HD's models, the underlying mechanisms are still unknown. In our study, we investigated the effects of rutin in Caenorhabditis elegans model of HD. We assessed polyQ aggregation, oxidative damage, neurodegeneration level and lifespan, and investigated the possible role of autophagy and insulin/IGF1 (IIS) signaling pathways in the beneficial effects induced by rutin. Overall, our data demonstrate that chronic rutin treatment reduced polyglutamine (polyQ) protein aggregation in muscle, reduced polyQ-mediated neuronal death in ASH sensory neurons, and extended lifespan. The possible mechanisms involved are antioxidant activity, activation of protein degradation (autophagy) and insulin/IGF1 (IIS) signaling pathways. These findings indicate that rutin consumption might be helpful in preventing HD and also provide possible pathways to be explored to search for new therapies against proteinopathies related to aging.

Guarana (Paullinia cupana Mart.) protects against amyloid-ß toxicity in Caenorhabditis elegans through heat shock protein response activation.

Alzheimer disease (AD) is a progressive neurodegenerative brain disorder that causes significant disruption in normal brain functioning, representing the most common cause of dementia in the elderly. The main hallmark of AD is the presence of amyloid plaques in the brain formed by the deposition of insoluble amyloid protein (Aß) outside of neurons. Despite intensive investigation of the mechanisms of AD pathogenesis during the past three decades, little has been achieved in terms of effective treatments or ways to prevent the disease. Paullinia cupana, known as guarana, is a plant endemic to the Amazon region in Brazil with several beneficial effects reported, including delayed aging. In this study, we investigated the effects of chronic consumption of guarana ethanolic extract (GEE) on Aß toxicity using a C. elegans model of AD. We analyzed the behavioral phenotype, oxidative damage and Aß protein expression in worms treated with GEE. In addition, we investigated the possible role of the heat shock response on the beneficial effects induced by GEE. Overall, our data demonstrate that chronic GEE treatment decreased the formation of Aß aggregates in C. elegans, preventing the behavioral deficits and the oxidative damage inducible by Aß expression, due to activation of the heat shock protein (HSP) response. This finding provides a new alternative against amyloidogenic neurodegenerative diseases and other diseases caused by protein accumulation during aging.

Guanosine Prevents against Glutamatergic Excitotoxicity in C. elegans.

Glutamatergic neurotransmission is present in most mammalian excitatory synapses and plays a key role in central nervous system homeostasis. When over-activated, it can induce excitotoxicity, which is present in several neuropathologies. The nucleoside guanosine (GUO) is a guanine-based purine known to have neuroprotective effects by modulating glutamatergic system during glutamate excitotoxicity in mammals. However, GUO action in Caenorhabditis elegans, as well as on C. elegans glutamatergic excitotoxicity model, is not known. The GUO effects on behavioral parameters in Wild Type (WT) and knockouts worms for glutamate transporters (GLT-3, GLT-1), glutamate vesicular transporter (EAT-4), and NMDA and non-NMDA receptors were used to evaluate the GUO modulatory effects. The GUO tested concentrations did not alter the animals' development, but GUO reduced pharyngeal pumps in WT animals in a dose-dependent manner. The same effect was observed in pharyngeal pumps, when the animals were treated with 4 mM of GUO in glr-1, nmr-1 and eat-4, but not in glt-3 and glt-3;glt-1 knockouts. The double mutant glt-3; glt-1 for GluTs had decreased body bends and an increased number of reversions. This effect was reverted after treatment with GUO. Furthermore, GUO did not alter the sensory response in worms with altered glutamatergic signaling. Thus, GUO seems to modulate the worm's glutamatergic system in situations of exacerbated glutamatergic signaling, which are represented by knockout strains to glutamate transporters. However, in WT animals, GUO appears to reinforce glutamatergic signaling in specific neurons. Our findings indicate that C. elegans strains are useful models to study new compounds that could be used in glutamate-associated neurodegenerative diseases.

Ilex paraguariensis modulates fat metabolism in Caenorhabditis elegans through purinergic system (ADOR-1) and nuclear hormone receptor (NHR-49) pathways.

Ilex paraguariensis is a well-known plant that is widely consumed in South America, primarily as a drink called mate. Mate is described to have stimulant and medicinal properties. Considering the potential anti-lipid effects of I. paraguariensis infusion, we used an extract of this plant as a possible modulator of fat storage to control lipid metabolism in worms. Herein, the I. paraguariensis-dependent modulation of fat metabolism in Caenorhabditis elegans was investigated. C. elegans were treated with I. paraguariensis aqueous extract (1 mg/ml) from L1 larvae stage until adulthood, to simulate the primary form of consumption. Expression of adipocyte triglyceride lipase 1 (ATGL-1) and heat shock protein 16.2, lipid accumulation through C1-BODIPY-C12 (BODIPY) lipid staining, behavioral parameters, body length, total body energy expenditure and overall survival were analyzed. Total body energy expenditure was determined by the oxygen consumption rate in N2, nuclear hormone receptor knockout, nhr-49(nr2041), and adenosine receptor knockout, ador-1(ox489) strains. Ilex paraguariensis extract increased ATGL-1 expression 20.06% and decreased intestinal BODIPY fat staining 63.36%, compared with the respective control group, without affecting bacterial growth and energetic balance, while nhr-49(nr2041) and ador-1(ox489) strains blocked the worm fat loss. In addition, I. paraguariensis increased the oxygen consumption in N2 worms, but not in mutant strains, increased N2 worm survival following juglone exposure, and did not alter hsp-16.2 expression. We demonstrate for the first time that I. paraguariensis can decrease fat storage and increase body energy expenditure in worms. These effects depend on the purinergic system (ADOR-1) and NHR-49 pathways. Ilex paraguariensis upregulated the expression of ATGL-1 to modulate fat metabolism. Furthermore, our data corroborates with other studies that demonstrate that C. elegans is a useful tool for studies of fat metabolism and energy consumption.

Quinolinic acid and glutamatergic neurodegeneration in Caenorhabditis elegans.

Quinolinic acid (QUIN) is an endogenous neurotoxin that acts as an N-methyl-D-aspartate receptor (NMDAR) agonist generating a toxic cascade, which can lead to neurodegeneration. The action of QUIN in Caenorhabditis elegans and the neurotoxins that allow the study of glutamatergic system disorders have not been carefully addressed. The effects of QUIN on toxicological and behavioral parameters in VM487 and VC2623 transgenic, as well as wild-type (WT) animals were performed to evaluate whether QUIN could be used as a neurotoxin in C. elegans. QUIN reduced survival of WT worms in a dose-dependent manner. A sublethal dose of QUIN (20 mM) increased reactive oxygen species (ROS) levels in an nmr-1/NMDAR-dependent manner, activated the DAF-16/FOXO transcription factor, and increased expression of the antioxidant enzymes, superoxide dismutase-3, glutathione S-transferase-4, and heat shock protein-16.2. QUIN did not change motor behavioral parameters, but altered the sensory behavior in N2 and VM487 worms. Notably, the effect of QUIN on the sensory behavioral parameters might occur, at least in part, secondary to increased ROS. However, the touch response behavior indicates a mechanism of action that is independent of ROS generation. In addition, non-lethal doses of QUIN triggered neurodegeneration in glutamatergic neurons. Our findings indicate that C. elegans might be useful as a model for studies of QUIN as a glutamatergic neurotoxin in rodent models.