Alpha-glycosyl isoquercitrin alleviates subchronic social defeat stress-induced depression symptoms by modulating the microbiota-gut-brain axis in mice.

AIMS: Increasing evidence suggests a link between gut microbial dysbiosis and the pathogenesis of depression. Alpha-glycosyl isoquercitrin (AGIQ), consisting of isoquercitrin and its glycosylated quercetin, has beneficial effects on the gut microbiome and brain function. Here, we detected the potential antidepressant impact of a four-week administration of AGIQ and its underlying mechanisms using a mouse model of depression. MAIN METHODS: Male C57BL/6 mice were orally administered AGIQ (0.05 % or 0.5 % in drinking water) for 28 days; subchronic social defeat stress was performed in the last 10 days. Behavior tests were conducted to assess anxiety and depressive-like behaviors. Additionally, evaluations encompassed 5-hydroxytryptamine (5-HT) levels, the gut microbiota composition, lipopolysaccharide (LPS) concentrations, short-chain fatty acids levels, and intestinal barrier integrity changes. KEY FINDINGS: AGIQ significantly alleviated depression-like behaviors and increased hippocampal 5-HT levels. Further, AGIQ mitigated stress-induced gut microbial abnormalities and reduced the levels of LPS in the serum, which affected the relative gene expression levels of 5-HT biosynthesis enzymes in vitro. Furthermore, AGIQ reversed the reduced butyrate levels in cecal contents and improved the impaired intestinal barrier by increasing the expression of colonic zonula occluden-1 (ZO-1) and occludin, thereby decreasing LPS leakage. SIGNIFICANCE: Our results suggest that AGIQ could improve stress-induced depression by regulating the gut microbiome, which inhibits LPS production and maintains the gut barrier. This is the first report on the potential effect of AGIQ on depression via the gut microbiota-brain axis, shedding new light on treatment options.

Comparison of the fecal bacterial microbiota in mice, rats, and pigs after oral administration of alpha-glycosyl isoquercitrin.

Alpha-glycosyl isoquercitrin (AGIQ) is composed of isoquercitrin and its glucosylated derivatives and has many biological activities, including anti-inflammatory, antioxidant, and anti-cancer properties. However, the effect of AGIQ administered orally on gut microbiota composition remains unclear. The objective of this study was to evaluate the effect of AGIQ on the gut microbiota of animals in different dose groups. Male rats and mice received different doses of AGIQ (1.5%, 3%, or 5% w/v) in diet for carcinogenic or chronic toxicity studies (rasH2 mice: 6 months; Sprague-Dawley rats: 12 months). Male minipigs received 100, 300, or 1000 mg/kg/day for 28 days. Fecal samples were collected from the different animal species and analyzed using 16S-rRNA gene sequencing. No significant changes were observed in alpha and beta diversity of the gut microbiota. Characteristic bacteria that responded to AGIQ were identified in each animal species, and, interestingly, Kineothrix alysoides, a butyrate-producing bacterium, was commonly detected in all three species, suggesting that it may be related to the biological activities of AGIQ. AGIQ selectively modulated the number of beneficial butyrate-producing commensal bacterium beneficial bacteria without changing the diversity of gut microbiota, which further supports the safe use of AGIQ in food products.

DNA methylation-altered genes in the rat hippocampal neurogenic niche after continuous exposure to amorphous curcumin.

Rat offspring who are exposed to an amorphous formula of curcumin (CUR) from the embryonic stage have anti-anxiety-like behaviors, enhanced fear extinction learning, and increased synaptic plasticity in the hippocampal dentate gyrus (DG). In the present study, we investigated the links between genes with altered methylation status in the neurogenic niche and enhanced neural functions after CUR exposure. We conducted methylation and RNA sequencing analyses of the DG of CUR-exposed rat offspring on day 77 after delivery. Methylation status and transcript levels of candidate genes were validated using methylation-sensitive high-resolution melting and real-time reverse-transcription PCR, respectively. In the CUR group, we confirmed the hypermethylation and downregulation of Gpr150, Mmp23, Rprml, and Pcdh8 as well as the hypomethylation and upregulation of Ppm1j, Fam222a, and Opn3. Immunohistochemically, reprimo-like+ hilar cells and protocadherin-8+ granule cells were decreased and opsin-3+ hilar cells were increased by CUR exposure. Both reprimo-like and opsin-3 were partially expressed on subpopulations of glutamic acid decarboxylase 67+ γ-aminobutyric acid-ergic interneurons. Furthermore, the transcript levels of genes involved in protocadherin-8-mediated N-cadherin endocytosis were altered with CUR exposure; this was accompanied by Ctnnb1 and Syp upregulation and Mapk14, Map2k3, and Grip1 downregulation, suggesting that CUR-induced enhanced synaptic plasticity is associated with cell adhesion. Together, our results indicate that functionally different genes have altered methylation and expression in different neuronal populations of the hippocampal neurogenic niche, thus enhancing synaptic plasticity after CUR exposure.

Potential Effects of Alpha-Glycosyl Isoquercitrin on Memory by Altering the Gut Microbiota-Blood-Brain Axis in Mice.

Alpha-glycosyl isoquercitrin (AGIQ), composed of isoquercitrin and glycosylated quercetin, has multiple biological effects. Here, we further examined the influence of AGIQ on brain function and provided its potential mechanism. Male C57BL/6 mice were treated with 0, 0.005, and 0.05% AGIQ in drinking water for 4 weeks prior to behavioral testing. Behavior tests showed that 0.05% AGIQ treatment significantly improved learning and memory function without affecting emotion. In the hippocampus, the gene expression of antioxidative defense enzymes was upregulated after 0.05% AGIQ treatment. In contrast, AGIQ caused significant alterations in the microbial abundance of genera Akkermansia, Bifidobacterium, and Alistipes associated with memory function. Metabolomics analysis identified that taurine concentration was significantly increased in serum and hippocampus from AGIQ-treated mice. The correlation analysis suggested that elevated serum taurine levels were closely related to the abundance of Akkermansia, indicating the underlying crosstalk of gut microbiota and serum metabolites. In vitro fecal culture further demonstrated that AGIQ could increase the level of Akkermansia. Taurine could exert antioxidant activity in SH-SY5Y neuroblastoma cell lines in vitro. Finally, vancomycin-induced alterations of gut microbiota attenuated the taurine increases in the serum and the antioxidant gene level in the hippocampus by AGIQ. Taken together, it is likely that AGIQ could increase genus Akkermansia abundance and ultimately increase taurine levels in serum and hippocampus to improve learning and memory function, relying on the gut microbiota-blood-brain axis. Our results supply a new view for understanding effects of AGIQ on brain function.

Exploring the effects of embryonic and neonatal exposure to lipopolysaccharides on oligodendrocyte differentiation in the rat hippocampus and the protective effect of alpha-glycosyl isoquercitrin.

This study compared the effects of embryonic and neonatal lipopolysaccharides (LPS) exposure (E-LPS and N-LPS) on oligodendrocyte (OL) differentiation in the hippocampus of male rats and explored the protective effect of the antioxidant alpha-glycosyl isoquercitrin (AGIQ). Using SD rats, LPS exposure occurred either intraperitoneally in dams between gestational days 15 and 16 (50 µg/kg body weight/time) or in male pups on postnatal day (PND) 3 (1 mg/kg body weight). Under both regimens, AGIQ at 0.5% (w/w) was supplemented, to dams from the gestation period (before LPS exposure) until weaning on PND 21 and to male offspring from weaning until PND 77 (adulthood). Compared with a control treatment, E-LPS treatment resulted in fewer NG2+ OL progenitor cells (OPCs) and an upregulation of Tcf4 at PND 6; by PND 21, low NG2+ OPC number persisted, but OLIG2+ OL lineage cells increased, while CNPase+ mature OLs counts were unchanged. By contrast, N-LPS treatment resulted in fewer OLIG2+ cells and an upregulation of Bmp4 at PND 6; by PND 21, NG2+ OPCs decreased, while GFAP+ astrocytes increased at both PND 6 and 21. After N-LPS treatment, Kl and Yy1 were downregulated and there were fewer Klotho+ and CNPase+ cells at PND 21. Results suggest that E-LPS treatment facilitates OPC differentiation into pre- and immature OLs until weaning, while N-LPS treatment suppresses OPC differentiation into mature OLs but facilitates astrocyte generation; however, these changes spontaneously recovered by adulthood under both regimens. AGIQ treatment ameliorated the effects of LPS treatment of both regimens, suggesting that LPS-induced disruption of OPC/OL differentiation occurs via neuroinflammation.

Twelve-month in utero safety assessment of gardenia blue, a natural food colorant.

Toxicity assessment of the food colorant Gardenia jasminoides Ellis at dietary exposures of 0.0%, 0.1%, 0.5%, 1.5%, 3.0% and 5.0% included measures of T-cell- dependent antibody response, neurotoxicity, and clinical and anatomic pathology in Sprague Dawley rats during mating, gestation, lactation, postnatal development, and following weaning for up to 12 months including 3- and 6-month interim evaluations. Blue coloration of the gastrointestinal tract, mesenteric lymph nodes and kidneys was present in treated rats only at necropsy with minimal blue coloration at the lowest dose and without histopathological correlates in any of the tissues. There was good survival with no consistent treatment-related changes in hematology, clinical chemistry, enhanced evaluation of lymphoid tissues, or tissue histopathology at interim and final time points. T-cell dependent antibody response and neurotoxicity screening were negative in treated rats. The no-observed-adverse-effect level (NOAEL) was determined to be 5.0% gardenia blue (2,854.5 and 3,465.4 mg/kg/day in parental males and females, respectively, prior to mating; 3,113.5 and 4,049.6 mg/kg/day in male and female offspring, respectively, following up to 12 months of exposure.

Extended one-generation reproductive toxicity study evaluating gardenia blue in Sprague Dawley rats.

Gardenia blue powder was administered at 0.5%, 2.5%, or 5.0% in feed to male and female Sprague Dawley rats in an Extended One-Generation Reproductive Toxicity Study (OECD Test Guideline 443). The dosed diet began 14 days before mating and was continued at the same concentration level for the entire study for all parental animals (P0) and offspring (F1). At weaning, offspring were allocated into one of 5 cohorts for different endpoints. P0 and F1 animals had blue urine, blue or black feces, and blue discolorations in gastrointestinal organs, mesenteric lymph nodes, and kidneys. This treatment-related finding was not considered adverse as there were no histopathologic correlates. There was a dose-related increase in sperm concentration in P0 and F1 males. There were dose-related increases in heart weights of F1 postnatal day (PND) 21 males, male and female thyroid weights, and female TSH levels of PND 91 F1 offspring, with no histopathological correlate. There were no consistent treatment-related adverse effects on any other parameters evaluated for general toxicity, reproductive toxicity, developmental neurotoxicity, or developmental immunotoxicity. The highest dietary concentration (5.0%) of gardenia blue powder was the no observed adverse effect level (NOAEL) for male and female rats at all life stages evaluated.

Amelioration of lipopolysaccharides-induced impairment of fear memory acquisition by alpha-glycosyl isoquercitrin through suppression of neuroinflammation in rats.

This study investigated the role of neuroinflammation in a lipopolysaccharides (LPS)-induced cognitive dysfunction model in rats using an antioxidant, α-glycosyl isoquercitrin (AGIQ). Six-week-old rats were dietary treated with 0.5% (w/w) AGIQ for 38 days, and LPS at 1 mg/kg body weight was administered intraperitoneally once daily on Days 8 and 10. On Day 11, LPS alone increased or tended to increase interleukin-1ß and tumor necrosis factor-α in the hippocampus and cerebral cortex. Immunohistochemically, LPS alone increased the number of Iba1+ and CD68+ microglia, and GFAP+ astrocytes in the hilus of the hippocampal dentate gyrus (DG). AGIQ treatment decreased or tended to decrease brain proinflammatory cytokine levels and the number of CD68+ microglia in the DG hilus. In the contextual fear conditioning test during Day 34 and Day 38, LPS alone impaired fear memory acquisition, and AGIQ tended to recover this impairment. On Day 38, LPS alone decreased the number of DCX+ cells in the neurogenic niche, and AGIQ increased the numbers of PCNA+ cells in the subgranular zone and CALB2+ hilar interneurons. Additionally, LPS alone decreased or tended to decrease the number of synaptic plasticity-related FOS+ and COX2+ granule cells and AGIQ recovered them. The results suggest that LPS administration induced acute neuroinflammation and subsequent impairment of fear memory acquisition caused by suppressed synaptic plasticity of newborn granule cells following disruptive neurogenesis. In contrast, AGIQ exhibited anti-inflammatory effects and ameliorated LPS-induced adverse effects. These results suggest that neuroinflammation is a key factor in the development of LPS-induced impairment of fear memory acquisition.

Chronic toxicity and carcinogenicity study of dietary gardenia blue in Sprague Dawley rats.

In this combined chronic toxicity/carcinogenicity study of gardenia blue as a natural food color additive, Sprague Dawley rats were administered 0.5%, 2.5%, or 5.0% gardenia blue via the feed or carrier diet (0.0% gardenia blue) for 12 (chronic toxicity cohort) or 24 (carcinogenicity cohort) months. No abnormal clinical, ophthalmological, neurotoxicity or clinical pathology changes were attributed to treatment, and there was no increase in mortality due to gardenia blue exposure. The only treatment-related change was grossly observed blue discoloration of the stomach, intestines, and mesenteric lymph nodes as well as reversible dark discoloration of the kidneys all without associated histopathology. The no-observed-adverse-effect level (NOAEL) for gardenia blue exposure via the diet for one or two years was determined to be 5.0% (2175.3 mg/kg body weight/day in male rats and 3075.4 mg/kg body weight/day in female rats).

Continuous exposure to alpha-glycosyl isoquercitrin from mid-gestation ameliorates polyinosinic-polycytidylic acid-disrupted hippocampal neurogenesis in rats.

Polyinosinic-polycytidylic acid (PIC) provides a model of developmental neuropathy by inducing maternal immune activation. We investigated the effects of an antioxidant, alpha-glycosyl isoquercitrin (AGIQ), on PIC-induced developmental neuropathy in rats, focusing on postnatal hippocampal neurogenesis. On gestational day 15, PIC at 4 mg/kg body weight was administered to dams intravenously. AGIQ either at 0.25% or 0.5% was administered through the diet to dams from gestational day 10 until weaning on day 21 post-delivery and, thereafter, to offspring until postnatal day 77 (adult stage). At weaning, the numbers of TBR2+ cells and PCNA+ cells in the subgranular zone and reelin+ cells in the dentate gyrus hilus in offspring of dams treated with PIC only were decreased compared with untreated controls. In contrast, 0.5% AGIQ ameliorated these changes and increased the transcript levels of genes related to signaling of reelin (Reln and Vldlr), growth factors (Bdnf, Cntf, Igf1, and Igf1r), and Wnt/ß-catenin (Wnt5a, Lrp6, Fzd1, and Fzd3). In adults, AGIQ increased the number of FOS+ granule cells at 0.25% and the transcript levels of NMDA-type glutamate receptor genes, Grin2a and Grin2b, at 0.25% and 0.5%, respectively. These results suggest that mid-gestation PIC treatment decreased the abundance of type-2b neural progenitor cells (NPCs) by reducing NPC proliferation in relation with suppression of reelin signaling at weaning. We suggest that AGIQ ameliorated the PIC-induced suppressed neurogenesis by enhancing reelin, growth factor, and Wnt/ß-catenin signaling at weaning to rescue NPC proliferation and increased synaptic plasticity by enhancing glutamatergic signaling via NMDA-type receptors after maturation.

Progressive disruption of neurodevelopment by mid-gestation exposure to lipopolysaccharides and the ameliorating effect of continuous alpha-glycosyl isoquercitrin treatment.

We investigated the effect of lipopolysaccharide (LPS)-induced maternal immune activation used as a model for producing neurodevelopmental disorders on hippocampal neurogenesis and behaviors in rat offspring by exploring the antioxidant effects of alpha-glycosyl isoquercitrin (AGIQ). Pregnant Sprague-Dawley rats were intraperitoneally injected with LPS (50 µg/kg body weight) at gestational days 15 and 16. AGIQ was administered in the diet to dams at 0.5% (w/w) from gestational day 10 until weaning at postnatal day 21 and then to offspring until adulthood at postnatal day 77. During postnatal life, offspring of LPS-injected animals did not show neuroinflammation or oxidative stress in the brain. At weaning, LPS decreased the numbers of type-2b neural progenitor cells (NPCs) and PCNA+ proliferating cells in the subgranular zone, FOS-expressing granule cells, and GAD67+ hilar interneurons in the dentate gyrus. In adulthood, LPS decreased type-1 neural stem cells, type-2a NPCs, and GAD67+ hilar interneurons, and downregulated Dpysl3, Sst, Fos, Mapk1, Mapk3, Grin2a, Grin2b, Bdnf, and Ntrk2. In adults, LPS suppressed locomotor activity in the open field test and suppressed fear memory acquisition and fear extinction learning in the contextual fear conditioning test. These results indicate that mid-gestation LPS injections disrupt programming of normal neurodevelopment resulting in progressive suppression of hippocampal neurogenesis and synaptic plasticity of newborn granule cells by suppressing GABAergic and glutamatergic neurotransmitter signals and BDNF/TrkB signaling to result in adult-stage behavioral deficits. AGIQ ameliorated most aberrations in hippocampal neurogenesis and synaptic plasticity, as well as behavioral deficits. Effective amelioration by continuous AGIQ treatment starting before LPS injections may reflect both anti-inflammatory and anti-oxidative stress effects during gestation and neuroprotective effects of continuous exposure through adulthood.

Continuous Exposure to Alpha-Glycosyl Isoquercitrin from Gestation Ameliorates Disrupted Hippocampal Neurogenesis in Rats Induced by Gestational Injection of Valproic Acid.

This study examined the ameliorating effect of alpha-glycosyl isoquercitrin (AGIQ), an antioxidant, on disrupted hippocampal neurogenesis in the dentate gyrus (DG) in a rat model of autism spectrum disorder induced by prenatal valproic acid (VPA) exposure. Dams were intraperitoneally injected with 500 mg/kg VPA on gestational day 12. AGIQ was administered in the diet at 0.25 or 0.5% to dams from gestational day 13 until weaning at postnatal day (PND) 21 and then to pups until PND 63. At PND 21, VPA-exposed offspring showed decreased numbers of type-2a and type-3 neural progenitor cells (NPCs) among granule cell lineage subpopulations. AGIQ treatment at both doses rescued the reduction in type-3 NPCs. AGIQ upregulated Reln and Vldlr transcript levels in the DG at 0.5% and ≥ 0.25%, respectively, and increased the number of reelin+ interneurons in the DG hilus at 0.5%. AGIQ at 0.25% and/or 0.5% also upregulated Ntrk2, Cntf, Igf1, and Chrnb2. At PND 63, there were no changes in the granule cell lineage subpopulations in response to VPA or AGIQ. AGIQ at 0.25% increased the number of FOS+ granule cells, accompanied by Gria2 and Gria3 upregulation and increasing trend in the number of FOS+ granule cells at 0.5%. There was no definitive evidence of VPA-induced oxidative stress in the hippocampus throughout postnatal life. These results indicate that AGIQ ameliorates the VPA-induced disruption of hippocampal neurogenesis at weaning involving reelin, BDNF-TrkB, CNTF, and IGF1 signaling, and enhances FOS-mediated synaptic plasticity in adulthood, potentially through AMPA-receptor upregulation. The ameliorating effects of AGIQ may involve direct interactions with neural signaling cascades rather than antioxidant capacity.

Ameliorating effect of continuous alpha-glycosyl isoquercitrin treatment starting from late gestation in a rat autism model induced by postnatal injection of lipopolysaccharides.

The present study investigated the role of neuroinflammation and brain oxidative stress induced by neonatal treatment with lipopolysaccharides (LPS) on the development of autism spectrum disorder (ASD)-like behaviors and disruptive hippocampal neurogenesis in rats by exploring the chemopreventive effects of alpha-glycosyl isoquercitrin (AGIQ) as an antioxidant. AGIQ was dietary administered to dams at 0.25% or 0.5% (w/w) from gestational day 18 until postnatal day (PND) 21 on weaning and then to pups until the adult stage on PND 77. The pups were intraperitoneally injected with LPS (1 mg/kg body weight) on PND 3. At PND 6, LPS alone increased Iba1+ and CD68+ cell numbers without changing the CD163+ cell number and strongly upregulated pro-inflammatory cytokine gene expression (Il1a, Il1b, Il6, Nfkb1, and Tnf) in the hippocampus, and increased brain malondialdehyde levels. At PND 10, pups decreased ultrasonic vocalization (USV), suggesting the induction of pro-inflammatory responses and oxidative stress to trigger communicative deficits. By contrast, LPS alone upregulated Nfe2l2 expression at PND 6, increased Iba1+, CD68+, and CD163+ cell numbers, and upregulated Tgfb1 at PND 21, suggesting anti-inflammatory responses until the weaning period. However, LPS alone disrupted hippocampal neurogenesis at weaning and suppressed social interaction parameters and rate of freezing time at fear acquisition and extinction during the adolescent stage. On PND 77, neuroinflammatory responses had mostly disappeared; however, disruptive neurogenesis and fear memory deficits were sustained. AGIQ ameliorated most changes on acute pro-inflammatory responses and oxidative stress at PND 6, and the effects on USVs at PND 10 and neurogenesis and behavioral parameters throughout the adult stage. These results suggested that neonatal LPS treatment induced acute but transient neuroinflammation, triggering the progressive disruption of hippocampal neurogenesis leading to abnormal behaviors in later life. AGIQ treatment was effective for ameliorating LPS-induced progressive changes by critically suppressing initial pro-inflammatory responses and oxidative stress.

alpha-Glycosyl Isoquercitrin (AGIQ) and its lack of carcinogenicity in rasH2 mice.

alpha-Glycosyl Isoquercitrin (AGIQ), is used in Japan as a food additive and was granted generally recognized as safe (GRAS) status in 2005 (FEMA) and 2007 (FDA). The safety and toxicity information for AGIQ is sparse and therefore, the carcinogenicity potential of AGIQ was examined in the CByB6F1-Tg(HRAS)2Jic (rasH2) model. One hundred female and male rasH2 mice, each, were allocated to one of four designated dose groups; 0 (control)%, 1.5%, 3.0% or 5.0% AGIQ. Animals were administered the diets for six months and an additional 10 females and 10 males, each, were administered a positive control, N-methyl-N-nitrosourea (MNU). Body weights and clinical observations were collected. A full screen necropsy, organ weights, clinical chemistry, urinalysis and histopathology were performed. The positive control animals elicited appropriate responses specific to this strain (rasH2) of mice. There were statistically significant sporadic non-dose-dependent changes in clinical chemistries without corresponding pathological correlation. No microscopic AGIQ-related findings were noted; the range of pathology observations were all considered background findings, either specific to rasH2 mice or common to inbred strains of mice. Therefore, under the study conditions, the no-observed-adverse-effect level (NOAEL) was determined to be more than 5.0% (7215.4 mg/kg BW/day in male mice and 14685.5 mg/kg/day in female mice).

Continuous exposure to amorphous formula of curcumin from the developmental stage facilitates anti-anxiety-like behavior and fear-extinction learning in rats.

An amorphous formula of curcumin (CUR) has shown to enable an improved bioavailability after ingestion. The aim of this study was to investigate the hypothesis that exogenously administered CUR has an advantage in ameliorating post-traumatic stress disorder at low doses. To this end, Long-Evans rats were dietary exposed to CUR at 0.1% or 0.5% from gestational day 6 to postnatal day (PND) 74 or 77. Offspring exposed to 0.1% CUR revealed facilitation of anti-anxiety-like behavior in the open field test and fear-extinction learning tested during PND 62 to 74, increases in hippocampal granule cells expressing immediate-early gene proteins and a decrease in prelimbic cortical neurons expressing phosphorylated extracellular signal-regulated kinase 1/2 after the last trial of the fear-extinction learning test on PND 74. The constitutive gene expression levels of Gria1, Gria2, Grin2d, Slc17a6, and Slc17a7 were altered in the hippocampal dentate gyrus and amygdala on PND 77. These results suggest alterations in synaptic plasticity to strengthen neural circuits in promoting the behavioral effects by 0.1%-CUR. In contrast, 0.5% CUR revealed a lack of any of the changes in behavioral tests that were observed at 0.1%; however, this dose upregulated oxidative stress and neuroinflammation-related genes in the hippocampal dentate gyrus, and increased neural stem cells and proliferation activity of the subgranular zone in the dentate gyrus. These results suggest a possible preventive use of CUR at low doses in mitigating some stress disorders; however, excessively absorbed doses may prevent behavioral changes by inducing neuroinflammation that affects hippocampal neurogenesis involving neural stem cells.

A chemical structure-based approach for estimating the added levels of flavourings to foods for the purpose of assessing consumer intake.

Intake assessment and hazard profile of chemical substances are the two critical inputs in a safety assessment. Human intake assessment presents challenges that stem either from the absence of data or from numerous sources of variability and uncertainty, which have led regulators to adopt conservative approaches that inevitably overestimate intake. Refinements of intake assessments produce more realistic estimates and help prioritise areas of concern and better direct investment of resources. However, use levels (ULs), which represent the usual added amount of flavourings to food products, are the starting point for refined intake assessments, are data-intensive, and data availability is often a limitation. The work presented here was undertaken to investigate the use level patterns of substances used as flavourings in foods and to develop a systematic tool for data extrapolation based on chemical structure. The available dataset consists of use levels reported through eight industry surveys and hence are representative of industry uses rather than regulatory limits, which are higher by design and not realistic. A systematic statistical analysis was undertaken to determine whether the industry-reported UL data can be used to estimate use levels of flavouring substances belonging to the same chemical group for which such data are not available. Predictive modelling approaches were explored to evaluate relationships in the data and utilised additional variables relevant to technological considerations, such as volatility losses upon heat treatment, and Tanimoto index-based pair-wise structural similarity scores to determine whether more granular similarity information can reduce the within-group variability. The analyses indicated that the use levels of flavouring substances can reasonably be estimated based on the available data using chemical group classifications stratified by food category. Source of uncertainty and limitations are discussed.

Continuous exposure to α-glycosyl isoquercitrin from developmental stages to adulthood is necessary for facilitating fear extinction learning in rats.

We previously reported that exposure to α-glycosyl isoquercitrin (AGIQ) from the fetal stage to adulthood facilitated fear extinction learning in rats. The present study investigated the specific AGIQ exposure period sufficient for inducing this behavioral effect. Rats were dietarily exposed to 0.5% AGIQ from the postweaning stage to adulthood (PW-AGIQ), the fetal stage to postweaning stage (DEV-AGIQ), or the fetal stage to adulthood (WP-AGIQ). Fear memory, anxiety-like behavior, and object recognition memory were assessed during adulthood. Fear extinction learning was exclusively facilitated in the WP-AGIQ rats. Synaptic plasticity-related genes showed a similar pattern of constitutive expression changes in the hippocampal dentate gyrus and prelimbic medial prefrontal cortex (mPFC) between the DEV-AGIQ and WP-AGIQ rats. However, WP-AGIQ rats revealed more genes constitutively upregulated in the infralimbic mPFC and amygdala than DEV-AGIQ rats, as well as FOS-immunoreactive(+) neurons constitutively increased in the infralimbic cortex. Ninety minutes after the last fear extinction trial, many synaptic plasticity-related genes (encoding Ephs/Ephrins, glutamate receptors/transporters, and immediate-early gene proteins and their regulator, extracellular signal-regulated kinase 2 [ERK2]) were upregulated in the dentate gyrus and amygdala in WP-AGIQ rats. Additionally, WP-AGIQ rats exhibited increased phosphorylated ERK1/2+ neurons in both the prelimbic and infralimbic cortices. These results suggest that AGIQ exposure from the fetal stage to adulthood is necessary for facilitating fear extinction learning. Furthermore, constitutive and learning-dependent upregulation of synaptic plasticity-related genes/molecules may be differentially involved in brain regions that regulate fear memory. Thus, new learning-related neural circuits for facilitating fear extinction can be established in the mPFC.

Identification of compound causing yellow bone discoloration following alpha-glycosyl isoquercitrin exposure in Sprague-Dawley rats.

Previous rat toxicity studies of alpha-glycosyl isoquercitrin (AGIQ), a water-soluble flavonol glycoside derived from rutin, revealed systemic yellow bone discoloration. This investigative study was conducted to determine the AGIQ metabolite(s) responsible for the discoloration. Female Sprague-Dawley rats were administered dietary AGIQ at doses of 0%, 1.5%, 3.0%, or 5.0% (0, 1735.0, 3480.8, and 5873.7 mg/kg/day, respectively) for 14 days, followed by a 14- or 28-day recovery period. Measurements of quercetin in urine and quercetin, quercetin 3-O-glucuronide, kaempferol, and 3-o-methylquercetin metabolites of AGIQ in bone (femur), white and brown fat, and cerebrum samples were conducted following the exposure period and each recovery period. Gross examination of the femur revealed yellow discoloration that increased in intensity with dose and was still present in a dose-related manner following both recovery periods. Quercetin, at levels correlating with AGIQ dose, was measured in the urine following the 14-day exposure period and, at lower concentrations, 14 or 28 days following cessation of AGIQ exposure. All four metabolites were present in a dose-dependent manner in the femur following 14 days of dietary exposure; only quercetin, quercetin 3-O-glucuronide, and 3-o-methylquercetin were present during the recovery periods. Quercetin, quercetin 3-O-glucuronide, and 3-o-methylquercetin were detected in white fat (along with kaempferol), brown fat (excluding quercetin due to analytical interference), and cerebrum samples, indicating systemic availability of the metabolites. Collectively, these data implicate quercetin, quercetin 3-O-glucuronide, or 3-o-methylquercetin (or a combination thereof) as the most likely metabolite of AGIQ causing the yellow discoloration of bone in rats administered dietary AGIQ.

Lack of preventive effect of maternal exposure to α-glycosyl isoquercitrin and α-lipoic acid on developmental hypothyroidism-induced aberrations of hippocampal neurogenesis in rat offspring.

Hypothyroidism during the developmental stage induces disruption of hippocampal neurogenesis in later life, as well as inducing oxidative stress in the brain. The present study investigated the preventive effect of co-exposure to an antioxidant on disruptive neurogenesis induced by developmental exposure to anti-thyroid agent in rats. For this purpose, we used two antioxidants, α-glycosyl isoquercitrin (AGIQ) and α-lipoic acid (ALA). Mated female Sprague Dawley rats were either untreated (control) or treated with 12 ppm 6-propyl-2-thiouracil (PTU), an anti-thyroid agent, in drinking water from gestational day 6 to postnatal day (PND) 21, the latter group being subjected to feeding basal diet alone or diet containing AGIQ at 5,000 ppm or ALA at 2,000 ppm during PTU exposure. On PND 21, PTU-exposed offspring showed reductions in a broad range of granule cell lineage subpopulations and a change in the number of GABAergic interneuron subpopulations. Co-exposure of AGIQ or ALA with PTU altered the transcript levels of many genes across multiple functions, suggestive of enhancement of synaptic plasticity and neurogenesis. Nevertheless, immunohistochemical results did not support these changes. PTU exposure and co-exposure of AGIQ or ALA with PTU did not alter the hippocampal lipid peroxidation level. The obtained results suggest a possibility that thyroid hormone depletion itself primarily disrupts neurogenesis and that oxidative stress may not be involved in the disruption during development. Transcript expression changes of many genes caused by antioxidants may be the result of neuroprotective actions of antioxidants rather than their antioxidant activity. However, no preventive effect on neurogenesis suggested impairment of protein synthesis via an effect on mRNA translation due to hypothyroidism.

Ameliorating effect of postweaning exposure to antioxidant on disruption of hippocampal neurogenesis induced by developmental hypothyroidism in rats.

Developmental hypothyroidism as a model of autism spectrum disorders disrupts hippocampal neurogenesis through the adult stage. The present study investigated the ameliorating effect of postweaning exposure to antioxidant on the hypothyroidism-induced disruptive neurogenesis. Mated female Sprague-Dawley rats were treated with 0 or 10 ppm 6-propyl-2-thiouracil (PTU) as an anti-thyroid agent in drinking water from gestational day 6 to postnatal day (PND) 21 on weaning. PTU-exposed male offspring were fed either basal diet, diet containing α-glycosyl isoquercitrin (AGIQ) at 5,000 ppm or α-lipoic acid (ALA) at 1,000 ppm as an antioxidant from PND 21 to PND 77. PTU-exposure decreased DCX+ and NeuN+ granule cell lineage subpopulations, synaptic plasticity-related FOS+ granule cells, and hilar PVALB+ and GAD67+ GABAergic interneurons, increased hilar SST+ and CALB2+ interneurons, and upregulated Gria3, Otx2, and antioxidant enzyme genes in the dentate gyrus on PND 77. These results suggest disruption of neurogenesis remained in relation with increase of oxidative stress and compensatory responses to the disruption at the adult stage. AGIQ recovered expression of some antioxidant enzyme genes and was effective for restoration of NeuN+ postmitotic granule cells and PVALB+ and SST+ interneurons. In contrast, ALA was effective for restoration of all interneuron subpopulations, as well as postmitotic granule cells, and upregulated Grin2a that may play a role for the restoration. Both antioxidants recovered expression of Otx2 and AGIQ-alone recovered Gria3, suggesting a reversal of disruptive neurogenesis by compensatory responses. Thus, postweaning antioxidant exposure may be effective for ameliorating developmental hypothyroidism-induced disruptive neurogenesis by restoring the function of regulatory system.