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.

4-(Phenylselanyl)-2H-chromen-2-one-Loaded Nanocapsule Suspension-A Promising Breakthrough in Pain Management: Comprehensive Molecular Docking, Formulation Design, and Toxicological and Pharmacological Assessments in Mice.

Therapies for the treatment of pain and inflammation continue to pose a global challenge, emphasizing the significant impact of pain on patients' quality of life. Therefore, this study aimed to investigate the effects of 4-(Phenylselanyl)-2H-chromen-2-one (4-PSCO) on pain-associated proteins through computational molecular docking tests. A new pharmaceutical formulation based on polymeric nanocapsules was developed and characterized. The potential toxicity of 4-PSCO was assessed using Caenorhabditis elegans and Swiss mice, and its pharmacological actions through acute nociception and inflammation tests were also assessed. Our results demonstrated that 4-PSCO, in its free form, exhibited high affinity for the selected receptors, including p38 MAP kinase, peptidyl arginine deiminase type 4, phosphoinositide 3-kinase, Janus kinase 2, toll-like receptor 4, and nuclear factor-kappa ß. Both free and nanoencapsulated 4-PSCO showed no toxicity in nematodes and mice. Parameters related to oxidative stress and plasma markers showed no significant change. Both treatments demonstrated antinociceptive and anti-edematogenic effects in the glutamate and hot plate tests. The nanoencapsulated form exhibited a more prolonged effect, reducing mechanical hypersensitivity in an inflammatory pain model. These findings underscore the promising potential of 4-PSCO as an alternative for the development of more effective and safer drugs for the treatment of pain and inflammation.

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.

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.

Curcumin and Quercetin-Loaded Lipid Nanocarriers: Development of Omega-3 Mucoadhesive Nanoemulsions for Intranasal Administration.

Curcumin (CUR) and quercetin (QU) are potential compounds for treatment of brain diseases such as neurodegenerative diseases (ND) because of their anti-inflammatory and antioxidant properties. However, low water solubility and poor bioavailability hinder their clinical use. In this context, nanotechnology arises as a strategy to overcome biopharmaceutical issues. In this work, we develop, characterize, compare, and optimize three different omega-3 (ω-3) fatty acids nanoemulsions (NEs) loaded with CUR and QU (negative, cationic, gelling) prepared by two different methods for administration by intranasal route (IN). The results showed that formulations prepared with the two proposed methods exhibited good stability and were able to incorporate a similar amount of CUR and QU. On the other side, differences in size, zeta potential, in vitro release kinetics, and permeation/retention test were observed. Considering the two preparation methods tested, high-pressure homogenization (HPH) shows advantages, and the CQ NE- obtained demonstrated potential for sustained release. Toxicity studies demonstrated that the formulations were not toxic for Caenorhabditis elegans. The developed ω-3 fatty acid NEs have shown a range of interesting properties for the treatment of brain diseases, since they have the potential to increase the nose-to-brain permeation of CUR and QU, enabling enhanced treatments efficiency.

Neurotoxicity induced by toluene: In silico and in vivo evidences of mitochondrial dysfunction and dopaminergic neurodegeneration.

Toluene is an air pollutant widely used as an organic solvent in industrial production and emitted by fossil fuel combustion, in addition to being used as a drug of abuse. Its toxic effects in the central nervous system have not been well established, and how and which neurons are affected remains unknown. Hence, this study aimed to fill this gap by investigating three central questions: 1) How does toluene induce neurotoxicity? 2) Which neurons are affected? And 3) What are the long-term effects induced by airborne exposure to toluene? To this end, a Caenorhabditis elegans model was employed, in which worms at the fourth larval stage were exposed to toluene in the air for 24 h in a vapor chamber to simulate four exposure scenarios. After the concentration-response curve analysis, we chose scenarios 3 (E3: 792 ppm) and 4 (E4: 1094 ppm) for the following experiments. The assays were performed 1, 48, or 96 h after removal from the exposure environments, and an irreversible reduction in neuron fluorescence and morphologic alterations were observed in different neurons of exposed worms, particularly in the dopaminergic neurons. Moreover, a significant impairment in a dopaminergic-dependent behavior was also associated with negative effects in healthspan endpoints, and we also noted that mitochondria may be involved in toluene-induced neurotoxicity since lower adenosine 5'-triphosphate (ATP) levels and mitochondrial viability were observed. In addition, a reduction of electron transport chain activity was evidenced using ex vivo protocols, which were reinforced by in silico and in vitro analysis, demonstrating toluene action in the mitochondrial complexes. Based on these findings model, it is plausible that toluene neurotoxicity can be initiated by complex I inhibition, triggering a mitochondrial dysfunction that may lead to irreversible dopaminergic neuronal death, thus impairing neurobehavioral signaling.

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.

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.

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.

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.

Physicochemical characterization and evaluation of in vitro and in vivo toxicity of goldenberry extract nanoemulsion / Caracterização físico-química e avaliação da toxicidade in vitro e in vivo de nanoemulsão contendo extrato de goldenberry

ABSTRACT: Oil-in-water (O/W) nanoemulsion containing goldenberry extract was elaborated using a high-energy ultrasonic bath method. Physicochemical characterization of the formulation was carried out by determining pH, mean droplet diameter, polydispersity index (PDI) and zeta potential. Nanoemulsion toxicity was assessed using in vitro assays with tumor and non-tumor cell lines, and in vivo using Caenorhabditis elegans. The pH of the nanoemulsion was 3.84, the mean droplet diameter was 268 ± 7 nm, PDI 0.113 and zeta potential -13.94 mV. Results of the cytotoxicity assays employing non-tumor cells indicated that the extract associated or not with nanoemulsion maintained cell viability at different concentrations tested. In the assays using tumor lineage, it is observed that the nanoemulsion containing the extract had higher antitumor activity than the free extract. As for the in vivo tests, there was no change in the survival rate of the worms.

RESUMO: Nanoemulsão óleo/água (O/A) contendo extrato de goldenberry foi elaborada utilizando método de banho ultrassônico de alta energia. A caracterização físico-química da formulação foi realizada pela determinação do pH, diâmetro médio de gotas, índice de polidispersão (PDI) e potencial zeta. A toxicidade das nanoemulsões foi avaliada utilizando ensaios in vitro com linhas celulares tumorais e não tumorais e in vivo utilizando Caenorhabditis elegans. O pH da nanoemulsão foi de 3,84, o diâmetro médio das gotículas foi de 268 ± 7 nm, PDI 0,113 e o potencial zeta -13,94 mV. Os resultados dos ensaios de citotoxicidade empregando células não tumorais indicaram que o extrato associado ou não à nanoemulsão manteve a viabilidade celular em diferentes concentrações testadas. Nos ensaios, utilizando linhagem tumoral, observou-se que a nanoemulsão contendo o extrato apresentou maior atividade antitumoral do que o extrato livre. Quanto aos testes in vivo, não houve mudança na taxa de sobrevivência dos vermes.