Abstract

   Autism spectrum disorder (ASD) is a developmental and neurological
   condition that impacts an individual’s behavior, communication, social
   interaction, and learning abilities. This disease is complex and
   involves different mechanisms, and therefore, modeling is a major
   challenge. Some features can be reproduced in different animal models
   to investigate therapeutic approaches. Here, we proposed a new simple
   model to induce development delay by using the nematode and
   dithiothreitol (DTT) as a chemical agent. In order to investigate a
   complementary treatment, a commercial supplement and its isolated
   components (vit. B12, B1, B6, and B9), curcumin, and
   palmitoylethanolamide (PEA) were used to revert the oxidative stress,
   development impairments, and metabolomic changes caused by DTT.
   Furthermore, computational tools predicted pharmacokinetic properties
   (SwissADME) and possible pathways (enrichment analysis) linked to ASD,
   an important neurodevelopmental disorder. The supplement and its
   components (curcumin, PEA, vit. B12, B6, and B9) partially alleviated
   the delay in larval progression and completely recovered the GABAergic
   neurodevelopmental impairments (supplement, curcumin, vit. B6, B9, and
   B12) caused by DDT. Vitamin B9, B12, and supplement partially protected
   mortality against the oxidative stressor Paraquat. Curcumin, vit. B1,
   B6, and supplement showed potential protection against coexposure to
   DTT by reducing DAF-16 migration to the nucleus compared to DTT.
   Vitamins B1, B9, and B12 coexposure to DTT positively modulated SOD-3
   expression. Amino acids, carnitines, and lipids revealed by LC–MS
   analysis enabled group differentiation and pathway analysis, indicating
   potential signaling molecules. In silico analysis predicted that these
   components may interact with pathways linked to ASD pathogenesis such
   as immunomodulation, synaptic pruning, and complex behavior regulation.
   Our data indicate that DTT is a good chemical model to induce
   developmental disorders and that the supplement, with all its
   components associated, is a promising therapy to be investigated in
   ASD.
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1. Introduction

   Autism spectrum disorder (ASD) encompasses a group of
   neurodevelopmental conditions characterized by deficits in social
   communication alongside restricted and repetitive behaviors or
   interests. Neurodevelopmental disabilities, including autism,
   attention-deficit hyperactivity disorder, dyslexia, and other cognitive
   impairments, affect millions of children worldwide, and some diagnoses
   seem to be increasing in frequency. Some results provide scientific
   evidence of the association that exists between the environmental
   neurotoxins and various neurodevelopmental disorders. Industrial
   chemicals that injure the developing brain are among the known causes
   for this rise in prevalence. Recent data from the Centers for Disease
   Control and Prevention’s Autism and Developmental Disabilities
   Monitoring Network indicate that approximately 1 in 36 children are
   diagnosed with ASD, emphasizing the urgent need for scientific effort.

   The term “spectrum” refers not only to the wide range of symptoms
   observed in ASD, varying in severity and progression, but also to the
   underlying physiological diversity. The significant heterogeneity in
   both clinical presentation and biological underpinnings makes treatment
   decisions particularly challenging, as a one-size-fits-all approach
   fails to address the complexity of the condition. The physiological
   complexity of ASD is attributed to an interplay of environmental,
   genetic, and epigenetic factors, including histone modifications and
   DNA methylation. , Disruptions in the homeostasis of neurotransmitter
   systems, such as gamma-aminobutyric acid (GABA), glutamate, serotonin,
   dopamine, and N-acetyl aspartate, have been linked to ASD, reflecting
   its multifaceted pathophysiology.

   Disrupted folate and transmethylation pathways and other causes of
   methylation impairment have been linked to oxidative stress on an ASD
   group and has specific metabolomic markers, − which suggest that
   oxidative stress and methylation defects may not only contribute to ASD
   development but also serve as biomarkers for defining a metabolic
   endophenotype within the spectrum. This endophenotype, marked by
   disrupted redox homeostasis and impaired metabolic and epigenetic
   resilience, represents a potential target for personalized therapeutic
   interventions, emphasizing the need to address the unique biological
   mechanisms underlying each ASD subtype.

   Despite the advances in understanding the heterogeneity of ASD, there
   is a significant clinical gap for patients’ classification into
   specific endophenotypes. As a result, most interventions remain
   generalized and insufficient to address symptoms in individuals,
   underscoring the urgent need for robust research to stratify ASD based
   on unique neurobiological and metabolic profiles.

   Current drugs used for ASD address behavioral symptoms; however, they
   are ineffective for the core symptoms (social communication
   difficulties, restricted interests, and repetitive behaviors). They
   also fail to address the individuality of endophenotypes, such as
   metabolic subtypes characterized by oxidative stress and impaired
   methylation. Dietary supplements have been proposed as a promising
   therapeutic avenue for addressing symptoms of ASD, particularly for
   subgroups with distinct metabolic vulnerabilities. Among these
   supplements, B-complex vitamins, such as vit. B1, B6, B9, and B12, are
   of particular interest due to their role in correcting methylation
   deficits, a hallmark of the metabolic endophenotype associated with
   methylation defects and disturbances on folate pathways.

   Studies have shown that antioxidants may play a beneficial role in
   addressing oxidative stress and related metabolic dysfunctions in ASD.
   Among these, Modafferi et al. reviewed the potential of
   antioxidant-rich mushrooms as treatments for ASD, proposing as a
   practical model to investigate the molecular mechanisms involved. Other
   natural compounds, such as curcumin, resveratrol, naringenin,
   sulforaphane, and palmitoylethanolamide (PEA), have also demonstrated
   their potential in reducing oxidative stress and targeting oxidative
   stress. Additionally, clinical trials have identified a subgroup of ASD
   patients who respond exceptionally well to supplements, − suggesting
   that some individuals may represent a differential responder group
   linked to specific endophenotypes.

   In order to evaluate these promising molecules, animal models are
   needed. Multiple animal models of ASD, including transgenic mice
   models, have been developed to clarify the etiology and prospect new
   therapeutic strategies. However, the variability on the spectrum poses
   challenges for animal modeling: whereas chemical models such as
   valproic acid (VPA) and polyinosinic/polycytidylic acid (poly I/C) have
   provided valuable insights in animal models, they fail to capture the
   full ASD complexity, particularly endophenotypes associated with other
   metabolic dysregulations.

   Simple experimental models can be used to replicate endophenotype
   mechanisms and explore new therapies. In this perspective, can be used
   due to their conserved neuronal pathways and simplicity in studying
   some of the neurodevelopmental disorder features that are also observed
   in ASD patients. Furthermore, worms are becoming a preferred organism
   model for toxicity testing to comply with the 3R (replace, reduce, and
   refine) concept, reducing the use of vertebrate animal testing. Using
   worms to model neurodevelopmental disorders like ASD is an emerging
   topic that harbors great, untapped potential.

   Here, we propose testing a new chemical model for understanding some
   aspects of development and ASD in . We developed a chemical model in
   using dithiothreitol (DTT), which can modulate methylations to induce
   developmental delay as a phenotype. In , DTT increases the expression
   of R08E5.3, an S-adenosyl-methionine (SAM)-dependent methyltransferase,
   and modulates methionine–homocysteine cycle activity. DTT can also
   cause proteotoxic stress and reductive stress, which are attributed to
   ASD etiology. , We also tested a new supplement based on B-vitamins,
   curcumin extract, and PEA on our model.

   Considering the promising findings, further research is required to
   confirm the efficacy and safety of supplements and refine
   stratification methods for targeted interventions. In this context, our
   study aimed to evaluate the effects of one supplement based on
   B-vitamins, curcumin extract, and PEA and these components individually
   on key factors such as methylation, oxidative stress, regulation of
   antioxidant response machinery, developmental delays, behavioral
   impairments, and metabolomic changes induced by DTT as a chemical model
   for ASD-related features in . Additionally, in silico analyses were
   conducted to predict the chemical properties, biological metabolism,
   and activity of these molecules, with the goal of optimizing the
   supplement’s pharmacokinetics.

2. Materials and Methods

2.1. Chemicals

   Sodium chloride P.A (Qumica moderna, SP, Brazil), hydrochloric acid P.A
   A.C.S. (Anidrol, SP, Brazil), sodium biphosphate P.A.C.S (Synth, SP,
   Brazil), potassium phosphate monobasic anhydrous P.A (Synth, SP,
   Brazil), sodium hydroxide (Synth, SP, Brazil), and methanol (xodo
   cientfica, SP, Brazil). NGM medium: agar (EMPROVE, Darmstadt, Germany),
   peptone (Sigma aldrich, St. Louis, EUA), and sodium chloride P.A.
   (Quimica moderna, SP, Brazil). M9 buffer: sodium phosphate dibasic P.A
   A.C.S (Sigma-Aldrich, St. Louis, EUA), potassium phosphate bibasic P.A
   (Synth, SP, Brazil), and sodium chloride P.A (Quimica moderna, SP,
   Brazil). Uranyl acetate (Fisher scientific, EUA) and OP50 (Fisher
   scientific, EUA). Unlabeled amino acid and acylcarnitine standards were
   purchased from Merck (Merck KGaA, Darmstadt, Germany). Acetonitrile and
   methanol HPLC–MS-grade solvents were obtained from J.T. Baker. All
   other reagents were of analytical grade and were obtained from local
   suppliers.

2.2. Strains and Maintenance

   Worms were cultivated in Petri dishes containing a nematode growth
   medium (NGM, 3 g NaCl, 2.5 g peptone, 17 g agar, and 975 mL of
   autoclaved H[2]O; further added 1 mL of 1 mol L^–1 CaCl[2], 1 mL of 5
   mg mL^–1 cholesterol in ethanol, 1 mL of 1 mol L^–1 MgSO[4], and 25 mL
   1 mol L^–1 KPO[4] buffer) and fed with OP50 under controlled
   temperature (20 °C) and humidity (>95%) (Panasonic Healthcare Company
   of North America, MIR-254-PA). The population was synchronized to
   obtain all worms at the same larval stage. For this, a lysis solution
   was prepared (NaOH 1 M, NaClO 1%, and distilled H[2]O) to break the
   cuticle of pregnant hermaphrodites and release the eggs. Eggs were kept
   in the M9 buffer (3 g L^–1 KH[2]PO[4], 6 g L^–1 Na[2]HPO[4], 5 g L^–1
   NaCl, and 1 mM MgSO[4]) until hatching and reaching the first larval
   stage (L1). All the strains employed were submitted to the same
   process. The strains used consisted of N2 (wild-type, Bristol), CF1553
   muIs84 [(pAD76)sod-3p::GFP + rol-6(su1006)], TJ356 zIs356
   [daf-16p::daf-16a/b::GFP + rol-6(su1006)] IV, and EG1285 oxIs12
   [unc-47p::GFP + lin-15(+)]. The strains of and were obtained from the
   Caenorhabditis Genetics Center (CGC, Minnesota, USA).

2.3. Exposure Protocol

   The supplement (303 mg L^–1, Endocan) and the isolated components (PEA,
   Levagen plus, 200 mg L^–1), curcumin (Hydrocure, 80 mg L^–1), and B
   complex vitamins: B1 (1 mg L^–1), B6 (49 mg L^–1), B9 (0.3 mg L^–1),
   and B12 (0.0048 mg L^–1), and DTT (3 mM; Sigma-Aldrich; CAS: 3483-12-3)
   were dissolved in ultrapure water prior to the experiments. PEA and
   curcumin are difficult to be dissolved in water. However, the chosen
   supplement contains a formulation that rapidly disperses in water,
   eliminating the need for toxic solvents for . Both the supplement and
   all of the isolated components were dissolved in ultrapure water to
   prepare a stock solution. The solutions (supplement or its components
   or DTT) were added to the surface of the NGM plate seeded with OP50 and
   incubated in a refrigerator for 24 h prior to starting the experiment.
   Worms at the L1 larval stage were exposed on NGM plates to the
   supplement or its components, with or without DTT, for 44 h or until 72
   h for developmental evaluations. The chosen concentrations were based
   on previous work and standardized to represent the same proportion of
   the supplement ([57]Figure ).

1.

   [58]1
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   Experimental design. Step 1: NGM plates were seeded with OP50 and
   incubated overnight; step 2: the solutions (supplement 303 mg mL^–1 or
   its components (PEA 200 mg mL^–1, curcumin 80 mg mL^–1, B1 1 mg mL^–1,
   B6 49 mg mL^–1, B9 0.3 mg mL^–1, and B12 0.0048 mg mL^–1) or DTT 3 mM)
   were added to the surface of the NGM plate seeded with OP50 and
   incubated in a refrigerator for 24 h; step 3: the synchronized worms at
   the L1 larval stage were transferred to the previously prepared NGM
   plates to the supplement or its components, with or without DTT; step
   4: after 44 h of exposure, developmental analyses, resistance to
   paraquat stress, DAF-16 localization, SOD-3 expression, GABAergic
   neuron development, and metabolomics were performed; step 5: 72 h after
   exposure, the nematode development was analyzed.

2.4. Development and Body Length Measurements

   Worms were exposed to the supplement with isolated compounds with or
   without DTT on plates. They were observed as the larval stage at 44 and
   72 h at 20 °C. The worms were classified as per their stages: L1,
   L2/L3, L4, or adult. The worm size was assessed after image acquisition
   of 10 nematodes per group and quantification using ImageJ software
   (NIH, Bethesda, MD, USA). The experiments were carried out in
   duplicates on three different batches/independent experiments.

2.5. Neurodevelopmental Assessment

   GABAergic neuronal development in exposed to the supplement or
   components was assessed using the GABAergic fluorescent strain EG1285.
   After treatment, nematodes were observed on the slides under a
   fluorescence microscope. The cell bodies of D-type GABAergic neurons
   were counted on the ventral cord to assess neurodevelopment after 44 h
   of exposure. DTT (3 mM) was used to induce GABAergic neurodevelopmental
   delay. The experiments were carried out in triplicate and were repeated
   on three separate days.

2.6. Stress Resistance under Paraquat

   The stress resistance assay was performed using the stressor paraquat
   (1-1′-dimethyl-4-4′-bipyridinium dichloride). The worms (L1) were
   pre-exposed to the treatment and after 44 h (L4) were submitted to
   postexposure to 5 mM paraquat (Gramoxone 200). The protective effect of
   the treatments was assessed by the analysis of survival after exposure
   to paraquat, counted hourly for 6 h following paraquat exposure.
   Survival of a control group without postexposure to the stressor agent
   was also analyzed.

2.7. DAF-16 Localization

   DAF-16 transcription factor was verified using a strain in which the
   promoter gene of this protein is tagged with a green fluorescent
   protein (GFP). This factor, when located in the cell nucleus, leads to
   the expression of detoxifying and antioxidant enzymes. TJ356 worms were
   exposed for 44 h to the components or supplement, with or without DTT
   (3 mM) coexposure, and then evaluated (L4 stage, 44 h). The worms were
   transferred to glass slides and covered with coverslips for imaging
   using a FLoid Cell Imaging Station fluorescent microscope (Thermo
   Fisher Scientific, Catalog number: 4471136, USA). Results were
   expressed as nuclear, intermediate, or cytosolic. All assays were
   repeated three times independently using 30 worms per group in each
   assay.

2.8. SOD-3 Expression

   To verify the expression of target DAF-16 genes following exposure to
   the dietary supplementation, the expression of superoxide dismutase-3
   (SOD-3) was evaluated by using a strain which the promoter gene is
   tagged with GFP. Therefore, CF1553 worms were exposed to the supplement
   or its components, with or without DTT (3 mM) and after 44 h coexposure
   worms were analyzed (L4 stage). Animals were transferred to glass
   slides containing levamisole (10 mM) and covered with coverslips to
   obtain the images using a FLoid Cell Imaging Station fluorescent
   microscope (Thermo Fisher Scientific, Catalog number: 4471136, USA).
   The head fluorescence intensity of each worm was quantified using
   ImageJ (NIH, Bethesda, MD, USA) software for Windows. We performed
   three independent assays with 10 animals in each group per independent
   assay.

2.9. Metabolomic Analysis

   Approximately 10,000 worms were extracted by protein precipitation and
   physical disruption assisted by freeze and thaw cycles. Extracts were
   analyzed by two LC–MS approaches: untargeted and targeted analyses. For
   the untargeted analysis, we used an ACQUITY UPLC system coupled to a
   XEVO-G2XS Quadrupole Time-of-Flight (QToF) mass spectrometer (Waters,
   Manchester, UK) equipped with an ESI (electrospray ionization) system
   in both positive and negative ionization modes. For the targeted
   analysis, we used a Waters Quattro Micro-triple quadrupole mass
   spectrometer equipped with a Shimadzu SIL-20A LC system. Flow injection
   analysis (FIA) was employed, in a 4 min run, using multiple reaction
   monitoring (MRM) transitions for 20 amino acids and 14 acylcarnitines.
   More information can be accessed in the [60]Supporting Information.

   Untargeted data were processed using the Progenesis QI v2.4 software
   (Nonlinear Dynamics, Newcastle, UK), and the identification of the
   metabolites used fragmentation score, mass accuracy, mass error
   (precursor ions ≤ 5 ppm and fragments ≤ 10 ppm), and isotope
   similarity. External spectra libraries were used, such as LipidMaps
   ([61]http://www.lipidmaps.org/), Human Metabolome Database
   ([62]http://www.hmdb.ca/metabolites), and the MoNAMassBank of North
   America ([63]https://mona.fiehnlab.ucdavis.edu/). To increase the
   number of fragment matches and allow compatibility of PQI to external
   libraries, an in-house-developed free and open-source tool named
   “SDF2PQI” was used and recently detailed. For targeted analysis,
   TargetLynx (Waters) was used for peak integration.

   The Metaboanalyst 5.0 web platform was used for statistical analysis.
   Data were uploaded using the area of the extracted ion chromatograms.
   The selection of the metabolites was based on one-way ANOVA (p < 0.05),
   followed by Tukey’s posthoc analysis (p < 0.05). Midlevel data fusion
   was used for untargeted positive and negative ionization modes.
   Principal component analysis (PCA), heatmaps, and pathway enrichment
   analysis were used as detailed in the [64]Supporting Information.

2.10. Pharmacokinetic Prediction

   Using SwissADME ([65]http://www.swissadme.ch), it is possible to
   predict the human pharmacokinetic profile of the supplement components,
   including absorption, distribution, metabolism, and excretion (ADME) in
   silico. Since curcuminoids extracted from turmeric contain 75–80%
   curcumin, 15–20% demethoxycurcumin, and 4–8% bisdemethoxycurcumin,
   these molecules were also included in the analyses. Analysis of
   physicochemical properties included molecular weight, number of
   H-donors and acceptors, topological polar surface area, lipophilicity
   coefficient, and hydrophilicity coefficient. − The SwissADME tool was
   used to obtain information on the likelihood of gastrointestinal
   absorption, the permeability of the brain–blood barrier, P-glycoprotein
   (P-gp) substrate, and inhibition of CYP enzymes.

2.11. Prediction of Molecule Target Interaction and Enrichment Analysis

   Predictions of the supplement components and their targets were based
   on SwissTargetPrediction ([66]http://www.swisstargetprediction.ch/) for
   human genes. We compiled the predicted targets and submitted them to
   Gene Ontology and UniProt enrichment analysis using FunRich functional
   annotation software. − All molecules were also individually processed
   through PASS Online ([67]www.way2drug.com/passonline/) to predict their
   biological activities ([68]Tables S4–S7).

2.12. Statistical Analysis

   All experiments were performed in duplicate or in triplicate and
   repeated at least three times in independent experiments. Data were
   expressed as mean ± standard error of mean (SEM), with p < 0.05
   considered statistically significant. The normality of data
   distribution was confirmed by the Shapiro–Wilk test (all p’s > 0.05).
   Statistical significance by one-way ANOVA, followed by Tukey’s multiple
   comparison test, was performed using GraphPad Prism Version 8.0.1
   software. The detailed statistical data are exhibited in the Supporting
   Information ([69]Tables S8–S12).

3. Results

3.1. Developmental Parameters (Larval Stage, Body Size, and GABAergic
Neurodevelopment)

   DTT caused a significant delay in the development of nematodes compared
   with the M9 control group. After 44 h (20 °C), the nematodes were
   expected to be in the L4 larval stage. However, animals treated with
   DTT were mostly in the L2 larval stage compared with the M9 control
   worms ([70]Figure A, p < 0.0001). The isolated components and the
   supplement did not generate a delay in worms’ development compared with
   the M9 control group ([71]Figure A). Within 72 h at 20 °C, animals are
   expected to reach the larval stage of gravid adult. The DTT group still
   showed animals in the L2 larval stage, presenting different stages
   compared with the M9 control group (p < 0.0001; [72]Figure B).

2.

   [73]2
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   The supplement and components partially reduced the delay in worm
   development caused by DDT. Worms were exposed to the supplement (S, 303
   mg L^–1), PEA (200 mg L^–1), curcumin (Cur, 80 mg L^–1), vit. B9 (0.3
   mg L^–1), vit. B6 (49 mg L^–1), vit. B12 (0.0048 mg L^–1), and vit. B1
   (1.0 mg L^–1), combined or not to DTT (3 mM). The M9 buffer was used in
   the control groups. The percentage of worms in different larval stages
   (L1 stage to adult) was analyzed at 44 h (worms should achieve the L4
   larval stage) and 72 h (worms should achieve adulthood). Development at
   isolated compounds and supplement without DTT at 44 h (A) and 72 h (B).
   Supplement and compounds with DTT at 44 h (C) and 72 h (D). The body
   length (μm) was evaluated in 44 h without (E) and with DTT (F) (n =
   30). Representative images of treatment plates (44 h, arrow indicates
   worms in L2) (G). Data were expressed as the mean percentage ± standard
   error of the mean (SEM). Three independent assays were performed in
   duplicates. (*) Indicates statistically significant difference compared
   to the control group with *p < 0.05, **p < 0.01, ***p < 0.001, and
   ****p < 0.0001. (#) Denotes statistically significant difference
   compared to the DTT group with #p < 0.05, ##p < 0.01, ###p < 0.001, and
   ####p < 0.0001. The statistical analysis consisted of two-way ANOVA,
   followed by Tukey’s multiple comparison test.

   To evaluate the effectiveness of the components or supplement in
   protecting against the developmental damage caused by DTT, the animals
   were exposed simultaneously to both DTT and the compound. After 44 h of
   treatment, none of the compounds tested were able to completely reverse
   the damage caused by DTT ([75]Figure C). However, the supplement,
   curcumin (p < 0.0001), vit. B6 (p < 0.05), and B1 (p < 0.01)
   significantly reduced the number of animals in the L2 larval stage when
   compared to the DTT group ([76]Figure C). Conversely, the number of
   animals in the L3 larval stage significantly increased in the curcumin
   and supplement groups (p < 0.0001) compared with that in the DTT group.
   Therefore, within 44 h, a partial reversal of the developmental delay
   caused by DTT was observed, particularly with curcumin and the
   supplement component.

   After 72 h, the groups treated with DTT in combination with the
   supplement, PEA, curcumin, and vitamin B9 had fewer animals in the L2
   larval stage compared to the DTT group (p < 0.0001; [77]Figure D).
   Vitamins B6, B12 (p < 0.01), and B1 (p < 0.05) also caused a reduction
   in the percentage of animals in the L2 larval stage when coexposed with
   DTT ([78]Figure D). In terms of the L4 larval stage, the groups treated
   with DTT together with supplements such as vit. B9 and other
   supplements (p < 0.0001), PEA (p < 0.001), curcumin (p < 0.05), vit. B6
   (p < 0.01), and vit. B12 (p < 0.05), exhibited an increased percentage
   of animals in the L4 larval stage, thereby reducing the damage caused
   by DTT ([79]Figure D).

   The body length was reduced by DTT when compared to the control group
   (p < 0.0001, [80]Figure E). However, this reduction was partially
   reversed by the addition of vitamins (p < 0.0001 compared to the DTT
   group, [81]Figure F). Representative images illustrating the effects of
   DTT and DTT + S are shown in [82]Figure G. Both the supplement and
   isolated compounds did not cause any per se effect on development
   ([83]Figure A,B,E). Therefore, while the isolated components and the
   supplement were partially effective in mitigating the damage caused by
   DTT on the developmental end points of , they did not completely
   restore normal development.

   The cell bodies of GABAergic D-type neurons were counted after 44 h of
   exposure ([84]Figure A,B, p < 0.0001 compared to control). Notably,
   this neurodevelopmental impairment was partially restored by PEA (p <
   0.0001), vit. B1 (p < 0.0001), and vit. B12 (p < 0.05) ([85]Figure C,
   compared to the DTT group and M9 control group). In addition, complete
   protection from DTT damage was achieved with curcumin, vit. B6, vit.
   B9, and the supplement ([86]Figure C, p < 0.0001 compared to the DTT
   group). As expected, the supplement and compounds showed no per se
   effects on the GABAergic D-type neuron cell bodies ([87]Figure B).

3.

   [88]3
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   DTT impaired the GABAergic development and while the dietary supply
   protected the delay in . Worms were exposed to the supplement (S, 303
   mg L^–1), PEA (200 mg L^–1), curcumin (Cur, 80 mg L^–1), vit. B9 (0.3
   mg L^–1), vit. B6 (49 mg L^–1), vit. B12 (0.0048 mg L^–1), and vit. B1
   (1.0 mg L^–1), combined or not to DTT (3 mM). The M9 buffer was used in
   the control groups. GABA D-type neuron cell bodies were quantified at
   44 h of exposure at isolated compounds and the supplement without DTT
   (B) and supplement and compounds with DTT (C) (n = 40). Representative
   images of treatments (A). Data were expressed as mean ± standard error
   of the mean (SEM). Three independent assays were performed in
   duplicates. (*) Indicates statistically significant difference compared
   to the control group M9 with ****p < 0.0001. (#) Denotes statistically
   significant difference compared to the DTT group with #p < 0.05, ####p
   < 0.0001. The statistical analysis consisted of two-way ANOVA, followed
   by Tukey’s multiple comparison test.

3.2. Stress Responses

3.2.1. Stress Resistance with Paraquat

   Exposure to the stressor paraquat (5 mM) significantly decreased the
   survival rate of the worms compared to the M9 control group from the
   first hour onward in all situations ([90]Figure A–G, p < 0.001).
   Pretreatments with PEA, curcumin, vit. B1, and vit. B6 ([91]Figure A–C)
   did not lead a significant improvement in survival rates at any of the
   4 h analyzed, when compared to the group exposed to paraquat. However,
   the survival rate from treatment with vit. B9 ([92]Figure E) induced a
   significant difference in survival rates at 2 (p < 0.05), 3 (p <
   0.001), and 4 (p < 0.05) h in comparison to paraquat. Moreover, vit.
   B12 treatment also protected against paraquat at 1 (p < 0.05), 2, 3,
   and 4 h (p < 0.001) ([93]Figure F). The treatment with the supplement
   showed a significant reduction in mortality only at the fourth hour (p
   < 0.05, [94]Figure G). Despite these protective effects, the compounds
   only partially mitigated the mortality caused by paraquat as they did
   not completely prevent it. The supplement and compounds did not affect
   the survival rate per se.

4.

   [95]4
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   Stress resistance with Paraquat. Quantification of alive worms (L4
   stage) (n = 8–10) after exposure to (A) PEA (200 mg L^–1), (B) curcumin
   (Cur, 80 mg L^–1), (C) vit. B1 (1.0 mg L^–1), (D) vit. B6 (49 mg L^–1),
   (E) vit. B9 (0.3 mg L^–1), (F) vit. B12 (0.0048 mg L^–1) and (G)
   supplement (S, 303 mg L^–1) with or without paraquat (5 mM)
   postexposure. Data were expressed as mean ± standard error of mean
   (SEM). The results were analyzed by Two-way ANOVA followed by Tukey’s
   multiple comparison test. (*) Indicates a statistically significant
   difference compared to the control group M9 with ****p < 0.001. (#)
   Denotes a statistically significant difference compared to the Paraquat
   with #p < 0.05, ##p < 0.01, ####p < 0.001.

3.2.2. DAF-16 Subcellular Localization

   The migration of DAF-16 transcription factor from the cytosol to the
   cell nucleus is associated with stress response in . Initially, we
   found that the components had no per se effect, as there was no
   significant difference when compared to the M9 control ([97]Figure
   A–G). Subsequently, we observed that DTT significantly induced the
   migration of DAF-16 from the cytosol to the nucleus compared to the M9
   control ([98]Figure B p < 0.05, 5C p < 0.05, 5D p < 0.01, 5G p < 0.01).
   This suggests that the presence of this agent triggers a stress
   response. Furthermore, curcumin (p < 0.05), vit. B1 (p < 0.05), B6 (p <
   0.05), and the supplement (p < 0.05) induced a possible protective
   effect against coexposure to DTT, as they reduced the migration of the
   transcription factor to the nucleus when compared to the positive
   control, maintaining levels similar to those of the M9 control
   ([99]Figure B,C,D,G). However, no significant differences were observed
   between PEA, vit. B9, and B12 components when cotreated with DTT
   ([100]Figure A,E,F).

5.

   [101]5
   [102]Open in a new tab

   Localization of DAF-16 after exposure to the components or supplement.
   Cellular localization of the transcription factor daf-16::GFP in strain
   TJ356 (L4 stage) (n = 3–4) after exposure to (A) PEA (200 mg L^–1), (B)
   curcumin (Cur, 80 mg L^–1), (C) vit. B1 (1.0 mg L^–1), (D) vit. B6 (49
   mg L^–1), (E) vit. B9 (0.3 mg L^–1), (F) vit. B12 (0.0048 mg L^–1), and
   (G) supplement (S, 303 mg L^–1), with or without DTT (3 mM) coexposure.
   (H) Representative images of the TJ356 strain exposed to components or
   the supplement. Data were expressed as mean ± standard error of mean
   (SEM). (*) Indicates statistically significant difference compared with
   the control M9; *p < 0.05, **p < 0.01. (#) Denotes statistically
   significant difference compared with the positive control DTT; #p <
   0.05, ##p < 0.01. The statistical analysis consisted of one-way ANOVA,
   followed by Tukey’s multiple comparison test.

3.2.3. SOD-3 Expression

   To evaluate the antioxidant system in , SOD-3 protein expression was
   assessed through GFP quantification. The isolated components maintained
   similar expression levels to the M9 control ([103]Figure A,C–F). Of
   note, the supplement showed a per se decrease in SOD-3 expression when
   compared to the M9 control, showing similar expression levels to the
   group exposed to DTT ([104]Figure G, p < 0.05).

6.

   [105]6
   [106]Open in a new tab

   Relative fluorescence of SOD-3 after exposure to the components or
   supplement. (A) Quantification of sod-3::GFP in strain CF1553 (L4
   stage) (n = 30) after exposure to (A) PEA (200 mg L^–1), (B) curcumin
   (Cur, 80 mg L^–1), (C) vit. B1 (1.0 mg L^–1), (D) vit. B6 (49 mg L^–1),
   (E) vit. B9 (0.3 mg L^–1), (F) vit. B12 (0.0048 mg L^–1), and (G)
   supplement (S, 303 mg L^–1), with or without DTT (3 mM) coexposure. (H)
   Representative images of the CF1553 strain exposed to the components or
   supplement. Data were expressed as mean ± standard error of mean (SEM).
   (*) Indicates statistically significant difference compared with the
   control M9; *p < 0.05, **p < 0.01, ***p < 0.001. (#) Denotes
   statistically significant difference compared with the positive control
   DTT; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001. The
   statistical analysis consisted of one-way ANOVA, followed by Tukey’s
   multiple comparison test.

   We observed that exposure to DTT decreased SOD-3 expression in
   ([107]Figure A–G, p < 0.001). We found that vit. B1 (p < 0.05), B9 (p <
   0.001), and B12 (p < 0.001) coexposed to DTT reached the levels of the
   M9 control, suggesting the positive modulatory effect of the vitamins
   on protein expression ([108]Figure C,E,F). Worms exposed to the other
   components (PEA, curcumin, and vit. B6) and supplement coexposed to DTT
   did not show a significant difference in SOD-3 expression when compared
   to DTT ([109]Figure A,B,D,G).

3.3. Metabolomic Analysis

   Targeted analysis included 15 amino acids and 12 acylcarnitines after
   the removal of 7 molecules, with relative standard deviation (RSD) >
   15% ([110]Tables S1 and S2). These analytes were able to cluster the
   groups when a PCA score plot was created, explaining 57% of the
   variance of the samples ([111]Figure S1A). In the heatmap, when
   comparing DTT and control groups, proline, threonine, and serine were
   less abundant in the control group, while arginine, C4, and C14 were
   more abundant ([112]Figure , p < 0.05). When comparing DTT and
   supplement + DTT groups, serine, leucine, threonine, C16, methionine,
   and alanine were found to be more abundant in the DTT group, while
   arginine, histidine, proline, and lysine were found to be abundant in
   the supplement + DTT group.

7.

   7
   [113]Open in a new tab

   Heatmap visualization of significantly altered amino acids and
   acylcarnitines between the studied groups in . Red color indicates high
   analytical signal intensity and blue indicates low intensity.
   Acylcarnitines are represented by their carbon number (A). Pathway
   analysis (p ≤ 0.05) based on the altered amino acids and acylcarnitines
   comparing the supplemented DTT group (supplement + DTT) with the DTT
   group (B) and the control group with the DTT group (C). ^1Valine,
   leucine, and isoleucine biosynthesis; ^2cysteine and methionine
   metabolism; ^3glycine, serine, and threonine metabolism; ^4arginine and
   proline metabolism; ^5arginine biosynthesis. DTT: dithiothreitol; S:
   supplement.

   The supplement led to an increase in arginine, histidine, lysine, and
   proline compared to the control group and an increase in arginine,
   histidine, lysine, and alanine when compared to the DTT group.
   Interestingly, the arginine level was reduced in the DTT group (DTT vs
   control) and recovered in the supplement + DTT group (supplement + DTT
   vs DTT). Indeed, the arginine biosynthesis pathway was found altered
   when comparing the control versus DTT groups (p = 0.035, [114]Figure C)
   and not impacted in the comparison of supplement + DTT versus DTT
   ([115]Figure B). A decrease in leucine, C4, C14, and asparagine levels
   was observed when comparing the supplemented group with the control one
   (supplement vs control), while a decrease in leucine, alanine,
   threonine, methionine, serine, and C16 was observed when comparing
   supplement + DTT versus DTT. Notably, threonine and serine levels were
   restored to lower levels in the presence of the supplement, as they had
   decreased when comparing the control and DTT groups.

   For the untargeted analysis, a total of 1298 features were identified
   in negative ionization mode and 3028 in positive ionization mode after
   processing the raw data. The features with RSD >30% were removed,
   leaving 2226 for statistical analysis and midlevel data fusion. The
   metabolic variation between the groups was evidenced by the clustering
   of the groups in the PCA score plot, explaining 45.5% of the variance
   of the samples ([116]Figure S1B). One-way ANOVA (p < 0.05), followed by
   Tukey’s posthoc analysis (p < 0.05), resulted in the detection of 839
   statistically significant compounds. Among these, focus was given to
   compounds identified as phosphatidylcholines (PC) and lyso-PC (LPC),
   phosphatidylethanolamine (PE), and lyso-PE, as these compounds are
   known to be altered in ASD children. ,

   Lyso-PC and PC species (LPC 18:0, LPC 18:2, LPC 14:1, LPC 16:1, LPC
   18:0, LPC 18:1, LPC 18:4, PC 12:0, and PC 22:1) were, in general, more
   abundant in the DTT group compared to the control group, while only
   three PC presented the opposite trend (LPC 16:0, LPC 20:5, and PC 14:0)
   ([117]Figure ). For the PE class, 15 species were detected, of which 9
   were lower in the DTT group than in the control
   (lysophosphatidylethanolamine (LPE) 14:0, LPE 16:1, LPE 18:0, LPE 18:1,
   LPE 20:0, LPE 20:1, LPE 20:5, PE 16:0, and PE 30:5), and 3 showed the
   opposite trend LPE 18:3, PE 16:1, and PE 22:3 ([118]Table S3).

8.

   8
   [119]Open in a new tab

   Heat map visualization of significantly altered LPC, PC, LPE, and PE
   between the studied groups. Red color indicates high analytical signal
   intensity, and blue indicates low intensity. LPC:
   lysophosphatidylcholine; PC: phosphatidylcholine; LPE:
   lysophosphatidylethanolamine; PE: phosphatidylethanolamine; S:
   supplement; DTT: dithiothreitol.

   When examining the effect of supplementation on the overall PC and PE
   species profile, some specific trends were noted for PC species (LPC
   14:0; LPC 18:2; LPC 18:0; LPC 18:1; PC 22:1; and PC 12:0) that had
   their levels reduced in the DTT group (DTT vs control) and increased
   after supplementation (DTT vs supplement + DTT). Increased levels of
   LPE 18:0 and PE 16:0 were observed after supplementation (DTT vs
   supplement + DTT).

3.4. Computational Analysis

3.4.1. ADME Properties

   The prediction of the pharmacokinetics for the compounds is shown in
   [120]Table . However, due to the size of the molecule, SwissADME was
   unable to process results for vit. B12. As expected, the analyzed
   vitamins have log S compatibility with hydrosoluble molecules higher
   than −4. , These results present data compatible with the high
   hydrophobic profile of PEA and curcuminoids (log Ps greater than −4),
   which can negatively impact their bioavailability.

1. SwissADME Numerical Results^ .

   molecule MW (g mol^–1) H-bond donors H-bond acceptors MlogP (o/w) log S
   (Ali) WlogP (o/w) TPSA (Å^2)
   PEA 299.49 2 2 3.39 –7.00 4.58 49.33
   curcumin 368.38 2 6 1.47 –4.83 3.15 93.06
   demethoxycurcumin 338.35 2 5 1.80 –4.76 3.14 83.83
   bisdemethoxycurcumin 308.33 2 4 2.13 –4.50 3.13 74.60
   vitamin B1 (thiamine) 265.35 2 3 0.05 –2.80 0.62 104.15
   vitamin B6 (pyridoxine) 169.18 3 4 –0.91 –0.30 –0.22 73.58
   vitamin B9 (methylfolate) 441.40 6 9 –0,62 –2.91 –0.38 213.23
   [121]Open in a new tab
   ^a

   MW: molecular weight; TPSA: topological polar surface area.

   [122]Table indicates that PEA and curcuminoids have the potential to
   inhibit CYP enzymes, specifically, CYP1A2, CYP2C9, CYP2D6, and CYP2A4.
   Among all the tested components, vit. B9 is expected to have low
   gastrointestinal absorption. Curcumin, demethoxycurcumin, and vit. B1,
   and vit. B9 are not predicted to be permeable through the brain blood
   barrier (BBB). Vitamin B1 is also predicted to be a P-gp substrate. The
   prediction for PEA and curcuminoids shows inhibition of CYP enzymes.

2. SwissADME Nominal Results^ .

   molecule GI absorption BBB permeant P-gp substrate CYP1A2 inhibitor
   CYP2C19 inhibitor CYP2C9 inhibitor CYP2D6 inhibitor CYP3A4 inhibitor
   PEA high yes no yes no no yes no
   curcumin high no no no no yes no yes
   demethoxycurcumin high no no yes no yes no yes
   bisdemethoxycurcumin high yes no yes no yes no yes
   vitamin B1 (thiamine) high no yes no no no no no
   vitamin B6 (pyridoxine) high no no no no no no no
   vitamin B9 (methylfolate) low no no no no no no no
   [123]Open in a new tab
   ^a

   GI: gastrointestinal; BBB: brain blood barrier; P-gp: P-glycoprotein.

3.4.2. Enrichment Analysis

   The Reactome pathways are shown in [124]Figure S2 based on UniProt
   enrichment analysis conducted through FunRich. Reversible hydration of
   carbon dioxide, prostanoid ligand receptors, G-alpha (q) signaling,
   interleukin 4 and 13 signaling, G-alpha (i) signaling, nuclear receptor
   transcription pathway, EPH-Ephrin signaling, PIP3-activated AKT
   signaling, VEGFR2-mediated cell proliferation, and muscarinic
   acetylcholine receptors are presented to be involved in the Reactome
   pathway related to these molecules’ target predictions.

4. Discussion

   According to the Center for Disease Control and Prevention (CDC), the
   prevalence of ASD in the population has reached 1%. The mechanisms that
   trigger ASD are complex, and no specific pharmacological treatment for
   core symptoms is available. This has led to the emergence of new
   possibilities for management. Currently, the most common methods of
   support for ASD include occupational, behavioral, speech, and play
   therapies. Although drugs such as antidepressants, anticonvulsants,
   antipsychotics, and stimulants have been used to treat ASD symptoms,
   none of them have been able to fully reverse the main symptoms.
   Additionally, these drugs can cause important side effects. On the
   other hand, natural products and dietary supplements, such as vitamins
   and minerals, have been suggested as potential treatments for various
   conditions, including ASD and other neurodevelopmental disorders.

   For ASD research, another crucial limitation is the reduced
   availability of animal models to investigate new treatments. Here, a
   new model is proposed for fast testing of a new approach to induce some
   features and metabolic markers of an ASD endophenotype: the nematode
   exposed to DTT. DTT is a demethylating and stress-inducing agent, a
   chemical model produced in specific biochemical and molecular changes
   present in some ASD patients, especially changes in enzymes that
   catalyze methylation, such as methionine synthase, resulting in
   developmental delays in the nematode. This model may help us understand
   the potential association between altered methylation and ASD. DTT
   exposure triggers specific responses, activating the hypoxia response
   pathway and modulating the methionine–homocysteine cycle. The induction
   of the hypoxia response pathway, mediated by the hypoxia-inducible
   factor (HIF-1), indicates DTT as a stressor by mimicking low-load
   conditions. DTT influences the metabolic cycle by altering the SAM
   levels and upregulating the SAM-dependent methyltransferase gene,
   rips-1. These changes impact growth, development, and life cycle
   transitions, revealing the crucial role of DTT as a reducing agent in
   model organisms. Our results corroborate the literature once DTT also
   caused a delay in larval progression and body size. In agreement, DTT
   delayed and impaired the GABAergic neuron development. In , there are
   19 ventral cord D-type GABA motor neurons whose function is to inhibit
   contraction of the body wall muscles during movement, being crucial for
   the animals’ survival (food foraging and escaping from adverse
   environments).

   Alterations in the GABAergic system have been linked to the development
   of both genetic and nongenetic endophenotypes of ASD in humans,
   although the precise molecular mechanisms remain unclear. In children
   with ASD, GABAergic dysfunction has been associated with impairments in
   cognitive processes such as somatosensory integration, attention, and
   social behavior. This notion is further supported by studies in other
   animal models of ASD, which demonstrate compromised GABAergic and
   glutamatergic neurotransmission. Additionally, lower GABA levels and
   functional changes in GABAergic receptors have been implicated in ASD
   pathophysiology.

   Conversely, increased glutamate levels have been observed in postmortem
   and body fluid samples from both pediatric and adult patients with ASD.
   Dysregulation of glutamatergic signaling, similarly documented in ASD
   patients and animal models, is thought to contribute to the higher
   prevalence of epilepsy in this population, given the critical role of
   glutamatergic pathways in seizure activity.

   The disruption of GABAergic and glutamatergic signaling creates a
   significant E/I imbalance, which underlies the core symptoms of ASD,
   including social difficulties, repetitive behaviors, and heightened
   susceptibility to epilepsy.

   Another etiopathological mechanism of ASD that has been evidenced in
   our DTT model is the impairment of the antioxidant response. Numerous
   studies have indicated the presence of oxidative stress in individuals
   with ASD, which implicates a possible pathogenesis mechanism. Thus, an
   imbalance in oxidative metabolism may contribute to ASD by causing
   oxidative damage during embryonic and fetal development as well as in
   early childhood, ultimately leading to neurodegeneration. Children with
   ASD exhibit impaired endogenous antioxidant defenses, including reduced
   total GSH levels and altered activities of key enzymes such as GPx,
   SOD, and CAT. From this perspective, the use of supplements that
   enhance the activity of these enzymes could serve as a potential
   strategy to reduce the incidence of new ASD cases or as a therapeutic
   option for affected individuals. Our findings demonstrated that the
   supplement, along with some of its isolated components, significantly
   decreased the mortality rate induced by the pro-oxidant paraquat in
   nematodes. Furthermore, it modulated the nuclear migration of
   transcription factor DAF-16, contributing to the prevention of stress
   responses triggered by DTT.

   PEA is known for its benefits against neuroinflammation and
   neurodegeneration caused by glutamate. , It is already used in some
   European countries as a supplement due to its antioxidant and
   neuroprotective properties. PEA is now clearly related to the
   endocannabinoid system, sharing metabolic pathways and targets that
   help maintain body homeostasis. In the context of ASD, PEA has improved
   irritability and hyperactivity in children. In the model, PEA partially
   recovered the development and GABAergic neurodevelopment delay caused
   by DTT, likely via mechanisms other than affecting antioxidant
   responses. Our study also demonstrates that PEA treatment increases the
   expression of the antioxidant enzyme SOD-3, indicating its ability to
   stimulate this important protective enzyme.

   Curcumin, an active compound in turmeric (), shows promise as a
   treatment for ASD in animal studies. Curcumin’s antioxidant and
   anti-inflammatory properties may benefit individuals with ASD. When DTT
   toxicity was induced, larval development experienced partial
   protection, while curcumin completely restored GABAergic
   neurodevelopment. Moreover, it re-established the DAF-16 transcription
   factor localization to the control levels, possibly through this
   pathway, indicating that it can modulate antioxidant responses and
   development. A study conducted on examined the effects of curcumin
   supplementation, revealing that it enhances the longevity of these
   nematodes by upregulating antioxidant genes such as sod-1, sod-2, and
   sod-3. Moreover, another study highlighted curcumin’s role in aging due
   to the activation of antioxidant enzymes such as SOD in rats. These
   evidence support our results, which show that curcumin not only
   increased the nuclear migration of DAF-16 but also elevated the
   expression of SOD-3 when coexposed with DTT.

   Vitamin B1 is a trace element that is important to carbohydrate
   metabolism and energy production. Its role in transporting and
   eliminating heavy metals in the body prevents damage from these
   toxicants. Additionally, its neuroprotective properties warrant further
   exploration. Some studies suggest that a deficiency in vit. B1 may lead
   to neurological disturbances, including ASD. , In , vit. B1
   supplementation can improve mitochondrial stress and enhance survival
   rates. Under DTT exposure, vit. B1 partially mitigated development
   delays and increased body length, restored DAF-16 translocation, and
   increased the expression of SOD-3 in the nematodes. The antioxidant
   activity of vit. B1 by the increase of SOD and CAT activities has
   already been reported. Therefore, the deficiency of this vitamin
   contributes to oxidative stress and decreases the activity of these
   enzymes. The relation of vit. B1 with the GABAergic system is vital in
   neuronal development and modulation of excitatory neurotransmission
   impacting GABA and glutamate activities, and the perinatal deficiency
   of this vitamin leads to disruption of the neuromotor behavior and
   decrease in the concentration of GABA and glutamate. Conversely, in our
   study, vit. B1 could not rescue the GABAergic damage in treated with
   DTT, probably due to the low concentration used.

   Vitamin B6 can positively affect ASD individuals without causing
   notable side effects. Studies have shown that vit. B6 treatment can
   lead to a decrease in behavioral problems by improving appropriate
   behavior and brain wave activity. It is worth noting that vit. B6 is
   crucial for the synthesis of several neurotransmitters such as GABA,
   noradrenalin, serotonin, histamine, dopamine, glycine, and d-serine.
   This implies that vit. B6 supplements could potentially enhance
   multiple neurotransmitter systems. In experiments with ,
   supplementation with vit. B6 was linked to an extended lifespan. Here,
   vit. B6 ameliorated the development and GABAergic delay, rescued the
   DAF-16 transition to control levels, and increased the expression of
   SOD-3 in relation to the positive control DTT, returning its levels the
   same as the control. Our findings are consistent with the literature,
   as many studies have reported that vit. B6 fulfills antioxidant goals
   by quenching ROS.

   Vitamin B9 (folate) is an essential micronutrient that helps prevent
   congenital disabilities, particularly neural tube defects. It is
   necessary for forming several coenzymes in many metabolic systems,
   including purine, pyrimidine, and nucleoprotein synthesis, as well as
   maintaining erythropoiesis. Therefore, its deficiency may lead to
   neurological symptoms in ASD. , Vitamin B9 treatment protected the mice
   from paraquat stress and improved DTT-induced development delay,
   GABAergic neurodevelopment, and SOD expression. In , vit. B9 has been
   reported for increasing oxidative stress resistance and longevity by
   elevating the expression levels of relevant genes in stress response
   and lifespan, including daf-16, skn-1, and sir 2.1, as well as
   glutathione S-transferase 4 (GST-4) and SOD-3 enzymes.

   Vitamin B12 is a water-soluble essential micronutrient implicated in
   two main metabolic pathways: the canonical propionate breakdown pathway
   and the methionine/S-adenosylmethionine (Met/SAM) cycle. In , low
   levels of vit. B12 or genetic disruption of the canonical propionate
   breakdown pathway lead to an accumulation of propionate and the
   transcriptional activation of a propionate shunt pathway. Vitamin B12
   can be transported by the ABC transporter mrp-5 from the intestine to
   support embryonic development. In addition, vit. B12 could alleviate
   the development delay induced by DTT in . Our results also demonstrated
   that supplementation with vit. B12 protects the nematodes from stress
   induced by paraquat; however, it does not show antioxidant activity in
   the studied pathways. The deficiency of vit. B12 can cause cellular
   accumulation of ROS and nitric oxide and decrease in antioxidants and
   CAT/SOD activity, resulting in oxidative damage and memory impairment
   in worms. Even though our results did not indicate a significant
   increase in the expression of SOD or modulation of the nuclear
   translocation of DAF-16, it is essential to note that vit. B12 may
   still play a crucial role as a neuroprotective agent against ASD.
   Finally, vit. B12 provided partial protection against GABAergic
   impairment caused by DTT, underscoring its potential as a beneficial
   treatment option.

   Notably, the supplement showed the best performance in improving end
   points compared to all the isolated compounds tested. When exposed to
   the supplement, showed significant improvement in the antioxidant
   responses, development, and GABAergic neurodevelopment after DTT
   damage, which can be attributed to the combination of benefits of each
   component in the supplement. The findings support the benefits of
   combined supplements in humans and lead to the necessity of further
   studies targeting ASD children.

   To enhance the agility and accuracy of biological information
   processing prior to in vivo testing, this study applied bioinformatics.
   Therefore, as there is an intention to apply this supplement to humans,
   it is essential to consider these molecules’ pharmacokinetics and
   biological properties that can affect their physiological functions.
   Vitamin B1, as predicted through SwissADME, is a P-gp substrate, which
   correlates with scientific literature that shows the cells prevent its
   accumulation by pumping it out. Vitamin B9 is predicted to have low GI
   absorption; however, the human GI tract has a carrier-mediated
   transport mechanism to absorb it.

   The current research predicted that curcuminoids can inhibit CYPs.
   Curcumin can inhibit CYP enzymes, specifically CYP2C9 and CYP3A4
   enzymes, through noncompetitive and competitive inhibition,
   respectively. In vivo studies have further confirmed that curcuminoids
   can inhibit CYP enzymes. − This raises concerns regarding the
   concurrent use of this supplement with other drugs that are metabolized
   by the same enzymes, such as aspirin, ibuprofen, and other nonsteroidal
   anti-inflammatory drugs. ,

   PEA- and curcumin-poor water solubility are known to disfavor their
   bioavailability in the bloodstream. , Therefore, to enhance their
   unfavorable pharmacokinetic properties, the supplement contains PEA and
   curcumin with LipiSperse technology (commercially referred to as
   Levagen plus and Hydrocure, respectively). These new formulations
   promote improved absorption of PEA and curcumin in the gastrointestinal
   tract. ,

   Enrichment analysis predicted that this supplement targets pathways
   associated with neurodevelopmental impairment that are observed in ASD.
   The prediction suggests that some pathways are related to
   immunomodulation, such as prostanoid ligand receptors and interleukin 4
   and 13 signalings. Prostanoids and interleukins play pivotal roles in
   immune modulation, which is a critical factor in ASD development,
   especially triggered by neuroinflammation dysfunction. Additionally,
   prostanoids, especially prostaglandin E2, are also crucial regulators
   of synaptic plasticity in the central nervous system and might be
   significant factors in ASD pathogenesis.

   EPH-Ephrin signaling and muscarinic acetylcholine receptors
   demonstrated in our results to be pathways related to the supplement’s
   activity; although not clearly associated with immunomodulation, these
   pathways also show an association with ASD in in vivo animal models
   through different mechanisms. In mutant mice, depletion of EPH
   receptors results in autistic-like phenotypes, which can be reduced by
   artificially activating the remaining EPH receptors and rescuing
   synaptic pruning. The muscarinic acetylcholine receptor regulates
   complex behaviors. Consequently, their agonists have been shown to
   reduce the restrictive repetitive behavior in the animal models of ASD.
   − In relation to VEGFR2, the connection to ASD remains unclear.
   However, recent studies indicate a potential relationship due to the
   role of VEGFR2 in cerebrovascular modulation. − In addition, enrichment
   analysis revealed additional pathways that have not been described yet
   for ASD or neurodevelopment.

   In silico analysis pointed out that PEA and the curcuminoids present
   some pharmacokinetic properties that are unfavorable to their use as
   oral drugs. The tested supplement effectively addresses the problem of
   hydrophobicity by utilizing the LipiSperse technology. In addition, the
   activity of the supplement components has been predicted to positively
   affect some pathways involved in ASD pathogenesis, such as
   immunomodulation, synaptic pruning, and regulation of complex
   behaviors. The relation between these pathways suggests that the
   supplement may be a potential complementary strategy for managing ASD.

   Metabolomic data in ASD patients are rare and conflicting due to the
   heterogeneity of the spectrum. In , experiments can be controlled, and
   uniformity can be guaranteed. Some changes in the amino acid levels in
   patients with ASD have been reported. However, the profile identity is
   variable between different studies. Proline has been found to increase
   in ASD patients. This increase is hypothesized to be related to the
   low-activity alleles of catechol O-methyltransferase (COMT) and proline
   dehydrogenase (oxidase) 1 (PRODH), enzymes involved in the degradation
   of proline. Serine also has been reported as increased in ASD patients.
   Despite DTT-increased threonine levels, ASD patients present lower
   levels of this amino acid. In addition, lower levels of threonine and
   serine were re-established by the supplement. Although arginine was
   found to be reduced in the DTT group, studies on ASD children have
   shown that this amino acid is increased.

   Regarding lipid metabolism, DTT reduced the acylcarnitines (C14) in ,
   which are crucial for mitochondria for β-oxidation and healthy brain
   development. Carnitine biosynthesis defects have been linked to an
   increased risk for ASD among males. The supplement was effective in
   increasing C14 levels in the worms. Studies have found that children
   with ASD have decreased levels of PE in their erythrocyte membranes.
   The present study also found some of those species following the same
   profile of ASD children. Reduced levels of PC and lyso-PC, which are
   involved in glycerophospholipid metabolism, could impact language
   ability in children with ASD. Metabolomic analysis showed that at least
   three species of PC were reduced. DTT can cause the increase in
   SAM-dependent methyltransferase, while induces SAM depletion. This
   could impact the metabolism of choline and methionine, verified in the
   metabolomic analysis by DTT modulation on their probably related
   products (PC and PE). Additionally, phosphatidylethanolamine
   methyltransferase also utilizes SAM to convert PE to PC through
   methylation activity in the brain.

   Our study brings relevant findings on a new model for exploring
   therapeutic strategies for an ASD subgroup and proposes a safety
   management approach for the condition. However, it is essential to
   carefully analyze these results and recognize the limitations inherent
   in any experimental model. In this case, the DTT model addresses some
   of the known and still undisclosed mechanisms of ASD. Behavioral
   observations are still challenging due to the heterogeneity of larval
   stages, which makes the individual picking of worms required for some
   tests unfeasible. Conversely, it provides a rapid method for screening
   new drugs and advancing precision medicine approaches, which are
   essential given the heterogeneity of ASD pathophysiology and the
   increasing prevalence of diagnoses within the spectrum.

   Regarding the supplement, we focused on achieving the highest possible
   concentration of dispersion while maintaining the same concentration
   ratios as those found in the supplement. Therefore, some vitamins were
   tested in concentrations lower than those supplied to worms by other
   authors (i.e., vit. B12 at 0.2 mg L^–1, while in our study, 0.0048 mg
   L^–1). This difference might explain why we did not observe some of the
   effects of the isolated components.

   Moreover, the multiple mechanisms involved in the development of ASD
   can contribute to heterogeneous responses to alternative therapies,
   which may be implicated in the inefficiency of vitamins and natural
   products for some individuals. However, this variability also occurs
   with established drug therapies, which can often be more toxic.
   Therefore, it is essential to consider vitamins and natural products as
   potential initial alternatives for treatment.

5. Conclusion

   Here, we present a novel chemical model using the nematode ,
   considering some characteristics of ASD, to seek both mechanisms and
   management strategies. DTT delayed worm development, impaired GABAergic
   morphology, and altered the biochemical profile related to amino acids,
   carnitines, and lipids. All of these specific metabolic vulnerabilities
   within the methylation endophenotype can be observed in a group of ASD
   patients. The newly tested supplement has proven to be profitable in
   addressing the development and stress associated with the metabolic
   vulnerabilities of some ASD endophenotypes. Bioinformatic analysis
   predicted PEA and the curcuminoid’s hydrophobic properties, which can
   be enhanced using LipiSperse technology in the supplement. In addition,
   the components present in the supplement can interact with certain
   human pathways associated with ASD, such as prostanoid and EPH-Ephrin
   signaling. Considering all of the outcomes presented in this study, the
   tested supplement may be a potential complementary strategy in managing
   ASD. Furthermore, additional research could be useful in consolidating
   the DTT model in studying ASD and other environmental endophenotypes of
   neurodevelopmental delays. Since the metabolomic analysis has uncovered
   several avenues to be pursued regarding important pathway changes
   affected by this chemical, the DTT model offers a promising model for
   further investigation.

Supplementary Material

   [125]ao4c10748_si_001.pdf^ (308.6KB, pdf)

Acknowledgments