Methylliberine (1,O2,7,9-tetramethyluric acid; 1,2,7,9-MU) and theacrine (1,3,7,9-tetramethyluric acid; 1,3,7,9-MU) are purine alkaloids found in a variety of botanicals and products marketed as dietary supplements. Despite their widespread use, extensive data on their human health effects are lacking. Therefore, methylliberine and theacrine were profiled using multiple new approach methods (NAMs): in silico metabolite predictions, in silico blood-brain barrier (BBB) permeability predictions, and in vitro assays for adenosine receptor binding (A1, A2A), functional activity (A2A), cytochrome P450 inhibition (CYP1A2), and phosphodiesterase inhibition (PDE3A, PDE4B, PDE4D). Like caffeine, methylliberine and theacrine were predicted to be metabolized via desmethylation by multiple in silico models. In silico predictions and cluster analysis also suggested that methylliberine and theacrine may permeate the BBB like caffeine. In vitro assays revealed that methylliberine and its metabolite 1,2,9-MU were CYP1A2 inhibitors (IC50, 9.6 and 27 μM, respectively) that agreed with clinical observations and could lead to interactions with drugs and xenobiotics. Both methylliberine and theacrine were A2A receptor antagonists, suggesting a potential for stimulant effects, although their potencies (52–62% inhibition at 100 μM) were weaker than theophylline, paraxanthine, and caffeine (IC50, 4.9–23.3 μM). Overall, this study highlights integration of multiple NAMs for profiling two purine alkaloids relevant to human health.
Silicon dioxide nanoparticles (SiO2NPs) are widely used as anti-caking agents (food additive E551). Their impact on the human gut microbiota is poorly understood. We evaluated the short-term effects of amorphous SiO2NPs using a computer-controlled in vitro gastrointestinal digestion and batch culture fermentation model inoculated with fecal microbiota from a single healthy adult donor. Three doses were applied, low 3.15 mg, medium 63 mg and high 1260 mg, corresponding to human-equivalent intakes of 0.09, 1.8 and 36 mg/kg body weight per day, spanning the reported mean dietary intake of E551 and an upper-bound exposure scenario; controls received deionized water. We monitored microbial community composition and metabolic activity by 16S rRNA sequencing, quantification of short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs), ammonium concentration, and antioxidant capacity (FRAP and DPPH) over 12 h. Over this window and at these doses, SiO2NP treatment did not significantly alter α- or β-diversity, total SCFA or BCFA concentrations, ammonium, or FRAP/DPPH activity. However, taxonomic shifts were dose-dependent: low doses enriched Collinsella and depleted Flavonifractor, the medium dose enriched an Escherichia–Shigella amplicon sequence variant, and the high dose enriched Faecalibacterium and unclassified Clostridia while reducing Subdoligranulum, Faecalitalea and Flavonifractor (all false discovery rate < 0.05); an increase in Akkermansia at the high dose did not survive correction for multiple testing. Integrated multi-omics analysis (DIABLO) revealed that high-dose exposure weakened linkages between butyrate-producing Firmicutes and SCFAs and shifted the metabolic profile from butyrate toward propionate fermentation, accompanied by strengthened associations between proteolytic taxa, BCFAs and ammonium. These findings suggest that short-term exposure to SiO2NPs induces subtle, concentration-dependent functional restructuring of the gut microbiota without overall diversity loss, although, as a single-donor in vitro study, the results are hypothesis-generating and require confirmation across multiple donors and longer exposures.
This study investigated the effects of MGO exposure at dietary-relevant concentrations on cognitive function and the underlying mechanisms using zebrafish larvae. Behavioral assessments demonstrated that MGO induced dose-dependent cognitive impairment, with the medium and high dose groups showing a remarkable reduction in spatial memory and locomotor activity. Mechanistic investigations revealed that MGO triggered substantial oxidative stress, evidenced by suppressed antioxidant enzyme activities (SOD and CAT decreased by approximately 30% and 28%, respectively, in the high-dose group) and elevated lipid peroxidation (MDA increased by 3.5-fold). These changes were accompanied by dopaminergic system impairment, including neurotransmitter depletion (dopamine levels reduced to 0.27 ± 0.01 μg/g prot in high dose group) and neuronal degeneration (neuronal density decreased by 39%). Metabolomics analysis identified 37 differential metabolites across all groups, with significant enrichment in glycerophospholipid metabolism pathways. Our findings establish a comprehensive mechanistic pathway linking MGO exposure to cognitive deficits through oxidative stress-mediated dopaminergic damage and metabolic dysregulation, suggesting potential neurotoxic risks of dietary carbonyl compounds and underscoring the need for further mechanistic and translational studies. This study provides a preliminary toxicological basis for food safety evaluation, while acknowledging that validation in higher vertebrate systems is essential for human risk assessment.
Prenatal exposure to organophosphate pesticides has been increasingly associated with long-term reproductive failure; nevertheless, their effects on uterine developmental programming are still little understood. This study examined whether in utero exposure to acephate causes lasting uterine damage in female rat progeny and analyzed the associated molecular processes. Pregnant Wistar rats were administered oral acephate (14 or 28 mg/kg) from gestational days 6 to 21, and uterine tissues from female progeny were analyzed at postnatal day 28. Prenatal exposure to acephate caused significant histopathological changes, including endometrial glandular hyperplasia. Oxidative stress was indicated by increased malondialdehyde and protein carbonyl levels, alongside substantial decreases in the activity of superoxide dismutase, catalase, and glutathione peroxidase. Molecular investigations revealed elevated expression of LC3 and caspase-3 at both the mRNA and protein levels, corroborated by immunofluorescence, signifying the activation of autophagy and apoptosis. Acephate altered PI3K/Akt/mTOR signaling in a dose-dependent manner, exhibiting suppression of PI3K/mTOR at lower doses and compensatory Akt activation despite ongoing mTOR inhibition at higher doses. These findings collectively indicate that prenatal exposure to acephate alters uterine development via oxidative stress-induced, dysregulation of PI3K/Akt/mTOR signaling, leading to autophagy, apoptosis, and abnormal proliferative remodeling that could jeopardize long-term female reproductive health.
