Infant microbes and metabolites point to childhood neurodevelopmental disorders.

This study has followed a birth cohort for over 20 years to find factors associated with neurodevelopmental disorder (ND) diagnosis. Detailed, early-life longitudinal questionnaires captured infection and antibiotic events, stress, prenatal factors, family history, and more. Biomarkers including cord serum metabolome and lipidome, human leukocyte antigen (HLA) genotype, infant microbiota, and stool metabolome were assessed. Among the 16,440 Swedish children followed across time, 1,197 developed an ND. Significant associations emerged for future ND diagnosis in general and for specific ND subtypes, spanning intellectual disability, speech disorder, attention-deficit/hyperactivity disorder, and autism. This investigation revealed microbiome connections to future diagnosis as well as early emerging mood and gastrointestinal problems. The findings suggest links to immunodysregulation and metabolism, compounded by stress, early-life infection, and antibiotics. The convergence of infant biomarkers and risk factors in this prospective, longitudinal study on a large-scale population establishes a foundation for early-life prediction and intervention in neurodevelopment.

Exploring functional genes' correlation with (S)-equol concentration and new daidzein racemase identification.

With its estrogenic activity, (S)-equol plays an important role in maintaining host health and preventing estrogen-related diseases. Exclusive production occurs through the transformation of soy isoflavones by intestinal bacteria, but the reasons for variations in (S)-equol production among different individuals and species remain unclear. Here, fecal samples from humans, pigs, chickens, mice, and rats were used as research objects. The concentrations of (S)-equol, along with the genetic homology and evolutionary relationships of (S)-equol production-related genes [daidzein reductase (DZNR), daidzein racemase (DDRC), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR)], were analyzed. Additionally, in vitro functional verification of the newly identified DDRC gene was conducted. It was found that approximately 40% of human samples contained (S)-equol, whereas 100% of samples from other species contained (S)-equol. However, there were significant variations in (S)-equol content among the different species: rats > pigs > chickens > mice > humans. The distributions of the four genes displayed species-specific patterns. High detection rates across various species were exhibited by DHDR, THDR, and DDRC. In contrast, substantial variations in detection rates among different species and individuals were observed with respect to DZNR. It appears that various types of DZNR may be associated with different concentrations of (S)-equol, which potentially correspond to the regulatory role during (S)-equol synthesis. This enhances our understanding of individual variations in (S)-equol production and their connection with functional genes in vitro. Moreover, the newly identified DDRC exhibits higher potential for (S)-equol synthesis compared to the known DDRC, providing valuable resources for advancing in vitro (S)-equol production. IMPORTANCE: (S)-equol ((S)-EQ) plays a crucial role in maintaining human health, along with its known capacity to prevent and treat various diseases, including cardiovascular diseases, metabolic syndromes, osteoporosis, diabetes, brain-related diseases, high blood pressure, hyperlipidemia, obesity, and inflammation. However, factors affecting individual variations in (S)-EQ production and the underlying regulatory mechanisms remain elusive. This study examines the association between functional genes and (S)-EQ production, highlighting a potential correlation between the DZNR gene and (S)-EQ content. Various types of DZNR may be linked to the regulation of (S)-EQ synthesis. Furthermore, the identification of a new DDRC gene offers promising prospects for enhancing in vitro (S)-EQ production.

The effect of soy isoflavones in brain development: the emerging role of multiple signaling pathways and future perspectives.

Soybean is a source of protein, fibers, and phytochemical isoflavones which are considered to have numerous health benefits for children and adulthood. On the other hand, isoflavones are widely known as phytoestrogens that exert their action via the estrogen signaling pathway. With this regard, isoflavones are also considered as endocrine-disrupting chemicals. Endogenous estrogen plays a crucial role in brain development through binding to estrogen receptors (ERs) or G protein-coupled estrogen receptors 1 (GPER1) and regulates morphogenesis, migration, functional maturation, and intracellular metabolism of neurons and glial cells. Soy isoflavones can also bind to ERs, GPER1, and, furthermore, other receptors to modulate their action. Therefore, soy isoflavone consumption may affect brain development during the pre-and post-natal periods. This review summarizes the current knowledge on the mechanisms of isoflavone action, particularly in the early stages of brain development by introducing representative human, and animal models, and in vitro studies, and discusses their beneficial and adverse impact on neurobehavior. As a conclusion, the soy product consumption during the pre-and post-natal periods under proper range of dose showed beneficial effects in neurobehavior development, including improvement of anxiety, aggression, hyperactive behavior, and cognition, whereas their adverse effect by taking higher doses cannot be excluded. We also present novel research lines to further assess the effect of soy isoflavone administration during brain development.

Equol production capability and family history as risk factors for hand osteoarthritis in menopausal and postmenopausal women. Cross-sectional study.

BACKGROUND: Hand disorders are common in menopausal women. Equol is a soy metabolite produced in humans and its production level differs among individuals. The purpose of this research is to investigate the correlative relationship between variables indicating equol production levels and the occurrence of hand disorders in menopausal and postmenopausal women. METHODS: Female subjects were divided into two groups: women 45-70 years of age with hand osteoarthritis (patient group) and women in the same age range without hand osteoarthritis (control group). The equol production level of each subject was estimated by measuring her urine equol concentration. We also surveyed the subjects' family histories of osteoarthritis. RESULTS: Equol levels in the patient group were significantly lower than those in the control group (p < 0.05). This difference was most apparent for women in their 50s. Individuals with family histories of Heberden's and Bouchard's nodes were found to be 48.1 times more likely to develop these conditions than individuals in the control group. CONCLUSIONS: Women with early menopausal hand symptoms (i.e., stiff and arthritic hands) often develop hand osteoarthritis during their late menopausal and postmenopausal periods, which may cause their QoL to significantly deteriorate. Although a link between the probability of women developing hand osteoarthritis and their clinical backgrounds (i.e., family history) had been suspected, it had not been thoroughly investigated. Our survey of women with and without Heberden's nodes and Bouchard's nodes found a significant correlation between the likelihood of women developing osteoarthritis and a family history of this disease. We also found a significant correlation between the likelihood of women developing hand osteoarthritis and their equol production levels. These results indicate that women with family histories of finger osteoarthritis and low equol production have higher risks of developing finger osteoarthritis.

The relationship between equol production status and normal tension glaucoma.

PURPOSE: Equol is metabolized by intestinal bacteria from soy isoflavones and is chemically similar to estrogen. Dietary habits, such as consumption of soy products, influence equol production. A relationship between glaucoma and estrogen has been identified; here, we investigated the relationship between equol production status and glaucoma in Japan. METHODS: We recruited 68 normal-tension glaucoma (NTG) patients (male to female ratio 26:42, average age 63.0 ± 7.6 years) and 31 controls (male to female ratio 13:18, average age 66.0 ± 6.3 years) from our hospital. All women included were postmenopausal. Urinary equol concentration was quantified with the ELISA method. MD was calculated based on the Humphrey visual field. The association between MD and equol was analyzed with Spearman's rank correlation coefficient. The Mann-Whitney U test was used to compare the equol-producing (> 1 µM) and non-producing (< 1 µM) subjects. We also investigated the association between equol and glaucoma with a logistic regression analysis. RESULTS: There was a significant association between equol and MD (r = 0.36, P < 0.01) in the NTG patients. Glaucoma, represented by MD, was significantly milder in the equol-producing subjects than the non-equol producing subjects (P = 0.03). A multivariate analysis revealed the independent contributions of equol, cpRNFLT, and IOP to MD (P = 0.03, P = 0.04, and P < 0.01, respectively). CONCLUSION: Our results suggest that equol, acting through estrogen receptor-mediated neuroprotective effects, might be involved in suppressing the progression of NTG. This result also adds to evidence that glaucoma may be influenced by lifestyle.

Isoflavones derived from plant raw materials: bioavailability, anti-cancer, anti-aging potentials, and microbiome modulation.

Isoflavones are secondary metabolites that represent the most abundant category of plant polyphenols. Dietary soy, kudzu, and red clover contain primarily genistein, daidzein, glycitein, puerarin, formononetin, and biochanin A. The structural similarity of these compounds to ß-estradiol has demonstrated protection against age-related and hormone-dependent diseases in both genders. Demonstrative shreds of evidence confirmed the fundamental health benefits of the consumption of these isoflavones. These relevant activities are complex and largely driven by the source, active ingredients, dose, and administration period of the bioactive compounds. However, the preclinical and clinical studies of these compounds are greatly variable, controversial, and still with no consensus due to the non-standardized research protocols. In addition, absorption, distribution, metabolism, and excretion studies, and the safety profile of isoflavones have been far limited. This highlights a major gap in understanding the potentially critical role of these isoflavones as prospective replacement therapy. Our general review exclusively focuses attention on the crucial role of isoflavones derived from these plant materials and critically highlights their bioavailability, possible anticancer, antiaging potentials, and microbiome modulation. Despite their fundamental health benefits, plant isoflavones reveal prospective therapeutic effects that worth further standardized analysis.

Association between equol and non-alcoholic fatty liver disease in Japanese women in their 50s and 60s.

BACKGROUND AND AIM: Equol is a metabolite of soy isoflavone and has estrogenic activity. The incidence of non-alcoholic fatty liver disease (NAFLD) increases after menopause in women, which is thought to result in a decrease in estrogen. This study aimed to evaluate the association between equol and NAFLD. METHODS: We evaluated 1185 women aged 50-69 years who underwent health check-ups at four health centers in Fukushima, Japan. Equol producers were defined by a urinary equol concentration of 1.0 µM or more. In addition to comparison between equol producers and non-producers, the association between equol and NAFLD was estimated using logistic regression analysis adjusting for fast walking and eating habits. RESULTS: Of the 1185 participants, 345 (29.1%) women were equol producers. The proportions of women who had NAFLD (34.8% vs 45.2%) were significantly lower in the equol-producing group than in the non-producing group. Multivariable logistic regression analysis showed that equol production was significantly associated with NAFLD (odds ratio = 0.66, 95% confidence interval: 0.51-0.86). CONCLUSIONS: Equol production was significantly associated with NAFLD in women in their 50s and 60s.

Equol and Resveratrol Improve Bone Turnover Biomarkers in Postmenopausal Women: A Clinical Trial.

Estrogen deficiency is a major cause of loss of postmenopausal bone mineral density (BMD). This study aimed to evaluate the effects of equol and resveratrol on bone turnover biomarkers in postmenopausal women. Sixty healthy postmenopausal women were randomly assigned to receive 200 mg fermented soy containing 10 mg equol and 25 mg resveratrol or a placebo for 12 months. Whole-body BMD and bone turnover biomarkers, such as deoxypyridinoline (DPD), tartrate-resistant acid phosphatase 5b (TRACP-5b), osteocalcin, and bone-specific alkaline phosphatase (BAP), were measured at baseline and after 12 months of treatment. At the end of treatment, DPD, osteocalcin, and BAP significantly improved in the active group (p < 0.0001 for all) compared to the placebo group. Conversely, TRACP-5b levels were unaffected by supplementation (p = 0.051). Statistically significant changes in the concentrations of DPD (p < 0.0001), osteocalcin (p = 0.0001), and BAP (p < 0.0001) compared to baseline were also identified. Overall, the intervention significantly increased BMD measured in the whole body (p = 0.0220) compared with the placebo. These data indicate that the combination of equol and resveratrol may positively modulate bone turnover biomarkers and BMD, representing a potential approach to prevent age-related bone loss in postmenopausal women.

Isoflavones Mediate Dendritogenesis Mainly through Estrogen Receptor α.

The nuclear estrogen receptor (ER) and G-protein-coupled ER (GPER1) play a crucial role during brain development and are involved in dendrite and spine growth as well as synapse formation. Soybean isoflavones, such as genistein, daidzein, and S-equol, a daidzein metabolite, exert their action through ER and GPER1. However, the mechanisms of action of isoflavones on brain development, particularly during dendritogenesis and neuritogenesis, have not yet been extensively studied. We evaluated the effects of isoflavones using mouse primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture with neurons and astrocytes. Soybean isoflavone-augmented estradiol mediated dendrite arborization in Purkinje cells. Such augmentation was suppressed by co-exposure with ICI 182,780, an antagonist for ERs, or G15, a selective GPER1 antagonist. The knockdown of nuclear ERs or GPER1 also significantly reduced the arborization of dendrites. Particularly, the knockdown of ERα showed the greatest effect. To further examine the specific molecular mechanism, we used Neuro-2A clonal cells. Isoflavones also induced neurite outgrowth of Neuro-2A cells. The knockdown of ERα most strongly reduced isoflavone-induced neurite outgrowth compared with ERß or GPER1 knockdown. The knockdown of ERα also reduced the mRNA levels of ER-responsive genes (i.e., Bdnf, Camk2b, Rbfox3, Tubb3, Syn1, Dlg4, and Syp). Furthermore, isoflavones increased ERα levels, but not ERß or GPER1 levels, in Neuro-2A cells. The co-culture study of Neuro-2A cells and astrocytes also showed an increase in isoflavone-induced neurite growth, and co-exposure with ICI 182,780 or G15 significantly reduced the effects. In addition, isoflavones increased astrocyte proliferation via ER and GPER1. These results indicate that ERα plays an essential role in isoflavone-induced neuritogenesis. However, GPER1 signaling is also necessary for astrocyte proliferation and astrocyte-neuron communication, which may lead to isoflavone-induced neuritogenesis.

Taxonomic distribution and evolutionary analysis of the equol biosynthesis gene cluster.

BACKGROUND: Equol, an isoflavonoid metabolite with possible health benefits in humans, is known to be produced by some human gut bacteria. While the genes encoding the equol production pathway have been characterized in a few bacterial strains, a systematic analysis of the equol production pathway is currently lacking. RESULTS: This study presents an analysis of the taxonomic distribution and evolutionary history of the gene cluster encoding the equol production pathway. A survey for equol gene clusters within the Genome Taxonomy Database bacterial genomes and human gut metagenomes resulted in the identification of a highly conserved gene cluster found in nine bacterial species from the Eggerthellaceae family. The identified gene clusters from human gut metagenomes revealed potential variations in the equol gene cluster organization and gene content within the equol-producing Eggerthellaceae clades. Subsequent analysis showed that in addition to the four genes directly involved in equol production, multiple other genes were consistently found in the equol gene clusters. These genes were predicted to encode a putative electron transport complex and hydrogenase maturase system, suggesting potential roles for them in the equol production pathway. Analysis of the gene clusters and a phylogenetic reconstruction of a putative NAD kinase gene provided evidence of the recent transfer of the equol gene cluster from a basal Eggerthellaceae species to Slackia_A equolifaciens, Enteroscipio sp000270285, and Lactococcus garvieae 20-92. CONCLUSIONS: This analysis demonstrates that the highly conserved equol gene cluster is taxonomically restricted to the Eggerthellaceae family of bacteria and provides evidence of the role of horizontal gene transfer in the evolutionary history of these genes. These results provide a foundation for future studies of equol production in the human gut and future efforts related to bioengineering and the use of equol-producing bacteria as probiotics.

Prospective association of dietary soy and fibre intake with puberty timing: a cohort study among Chinese children.

BACKGROUND: Dietary phytoestrogens have been suggested to influence puberty timing, a critical stage for well-being in adulthood. We hypothesized that childhood soy intake might prospectively influence puberty timing and that dietary fibre and the key isoflavone metabolite equol might play roles. METHODS: Cox proportional hazard regression models were performed in 4781 children (2152 girls and 2629 boys) aged 6-8 years old from the Chinese Adolescent Cohort Study for whom a food frequency questionnaire at baseline and information about potential confounders were available. Anthropometry and pubertal status including age at Tanner stage 2 for breast development (B2) or age at the initiation of gonadal growth (G2), and age at menarche (M) or voice break (VB) were assessed annually. Equol excretion was determined by urine samples from 1311 participants. RESULTS: Among girls and boys, higher soy intake was associated with later puberty timing (hazard ratio (HR)-B2: 0.88 (95% CI, 0.80-0.96), p=0.02; HR-M, 0.87 (0.77-0.94), p=0.01; HR-G2, 0.91 (0.82-0.98), p=0.013; HR-VB, 0.90 (0.82-0.9), p=0.02), independent of prepubertal body fatness and fibre intake. These associations were more pronounced among children with a high urinary equol level (pfor-interaction ≤ 0.04) or with a high cereal fibre intake (pfor-interaction ≤ 0.06). Intake of dietary fibre or its subtype was not prospectively associated with puberty onset after adjusting for dietary soy intake (p≥0.06). CONCLUSION: Higher childhood soy intake is prospectively associated with later puberty timing in both Chinese girls and boys, independent of prepubertal body fatness, and the association is particularly pronounced among individuals with a higher urinary equol level.

(S)-Equol Is More Effective than (R)-Equol in Inhibiting Osteoclast Formation and Enhancing Osteoclast Apoptosis, and Reduces Estrogen Deficiency-Induced Bone Loss in Mice.

BACKGROUND: Equol, a metabolite of daidzein, binds to the estrogen receptor with greater affinity than daidzein and exhibits various biological properties. It exists as an enantiomer, either (S)-equol or (R)-equol. OBJECTIVES: We have previously shown that the inhibitory effect of (S)-equol on bone fragility is stronger than that of racemic equol in ovariectomized (OVX) mice; however, the effect of (R)-equol has not been elucidated. The aim of this study was to compare the activities of equol enantiomers on bone metabolism in vitro and in vivo. METHODS: Bone marrow cells (BMCs) and RAW 264.7 cells were treated with equol enantiomers. The number of osteoclasts and caspase-3/7 activity were measured. We examined the effect of equol enantiomers on osteoblast differentiation in MC3T3-E1 cells. In vivo, 8-wk-old female ddY mice were assigned to 4 groups: sham-operated (sham), OVX, OVX + 0.5 mg/d of (S)-equol (S-eq), and OVX + 0.5 mg/d of (R)-equol (R-eq). Four weeks after the intervention, femoral bone mineral density (BMD) and osteoclastic gene expression were analyzed, along with concentrations of equol enantiomers in the serum and tissues. RESULTS: (S)-equol and (R)-equol inhibited osteoclast differentiation in BMCs (97% and 60%, P < 0.05) and RAW 264.7 cells (83% and 68%, P < 0.05). (S)-equol promoted apoptosis of mature osteoclasts by inducing caspase-3/7 activity (29%, P < 0.05) and enhanced osteoblast differentiation (29%, P < 0.05). In OVX mice, BMD was ameliorated in (S)-equol-treated mice (11%, P < 0.05), but not in (R)-equol-treated mice. The concentrations of (S)-equol were greater than those of (R)-equol in the serum, tibia, liver, and kidney (by 148%, 80%, 22%, and 139%, respectively). CONCLUSIONS: These results suggest that (S)-equol is more effective than (R)-equol in inhibiting osteoclast formation and enhancing osteoclast apoptosis in vitro, supporting the beneficial effect of (S)-equol to reduce estrogen deficiency-induced bone loss in OVX mice.

Soy isoflavone-specific biotransformation product S-equol in the colon: physiological functions, transformation mechanisms, and metabolic regulatory pathways.

Epidemiological data suggest that regular intake of soy isoflavones may reduce the incidence of estrogen-dependent and aging-associated disorders. Equol is a metabolite of soy isoflavone (SI) produced by specific gut microbiota and has many beneficial effects on human health due to its higher biological activity compared to SI. However, only 1/3 to 1/2 of humans are able to produce equol in the body, which means that not many people can fully benefit from SI. This review summarizes the recent advances in equol research, focusing on the chemical properties, physiological functions, conversion mechanisms in vitro and vivo, and metabolic regulatory pathways affecting S-equol production. Advanced experimental designs and possible techniques in future research plan are also fully discussed. Furthermore, this review provides a fundamental basis for researchers in the field to understand individual differences in S-equol production, the efficiency of metabolic conversion of S-equol, and fermentation production of S-equol in vitro.

Metabolomics profiles of premenopausal women are different based on O-desmethylangolensin metabotype.

Urinary O-desmethylangolensin (ODMA) concentrations provide a functional gut microbiome marker of dietary isoflavone daidzein metabolism to ODMA. Individuals who do not have gut microbial environments that produce ODMA have less favourable cardiometabolic and cancer risk profiles. Urinary metabolomics profiles were evaluated in relation to ODMA metabotypes within and between individuals over time. Secondary analysis of data was conducted from the BEAN2 trial, which was a cross-over study of premenopausal women consuming 6 months on a high and a low soya diet, each separated by a 1-month washout period. In all of the 672 samples in the study, sixty-six of the eighty-four women had the same ODMA metabotype at seven or all eight time points. Two or four urine samples per woman were selected based on temporal metabotypes in order to compare within and across individuals. Metabolomics assays for primary metabolism and biogenic amines were conducted in sixty urine samples from twenty women. Partial least-squares discriminant analysis was used to compare metabolomics profiles. For the same ODMA metabotype across different time points, no profile differences were detected. For changes in metabotype within individuals and across individuals with different metabotypes, distinct metabolomes emerged. Influential metabolites (variables importance in projection score > 2) included several phenolic compounds, carnitine and derivatives, fatty acid and amino acid metabolites and some medications. Based on the distinct metabolomes of producers v. non-producers, the ODMA metabotype may be a marker of gut microbiome functionality broadly involved in nutrient and bioactive metabolism and should be evaluated for relevance to precision nutrition initiatives.

Biocatalytic synthesis and evaluation of antioxidant and antibacterial activities of hydroxyequols.

Hydroxyequols are promising analogues of the biologically active flavonoid, equol. We recently found that the flavin-dependent monooxygenase HpaBro-3 of Rhodococcus opacus regioselectively synthesizes 3'-hydroxyequol from equol, whereas HpaBpl-1 of Photorhabdus luminescens synthesizes 6-hydroxyequol. In this study, we investigated the cascade synthesis of a dihydroxyequol compound from equol using these two enzymes. When Escherichia coli cells expressing HpaBro-3 and cells expressing HpaBpl-1 were simultaneously incubated with equol, the cells efficiently synthesized 6,3'-dihydroxyequol (8.7 mM, 2.4 g/L) via 3'- and 6-hydroxyequols in one pot. The antioxidant activity of the equol derivatives increased with an increase in the number of hydroxyl groups on the equol scaffold. 6,3'-Dihydroxyequol exhibited potent antioxidant activity. In addition, 6-hydroxyequol significantly inhibited the growth of E. coli. Cell survival studies suggested that 6-hydroxyequol is a bactericidal rather than bacteriostatic compound. To our knowledge, this is the first report describing the antibacterial activity of hydroxyequols.

Potential Protective Mechanisms of S-equol, a Metabolite of Soy Isoflavone by the Gut Microbiome, on Cognitive Decline and Dementia.

S-equol, a metabolite of soy isoflavone daidzein transformed by the gut microbiome, is the most biologically potent among all soy isoflavones and their metabolites. Soy isoflavones are phytoestrogens and exert their actions through estrogen receptor-ß. Epidemiological studies in East Asia, where soy isoflavones are regularly consumed, show that dietary isoflavone intake is inversely associated with cognitive decline and dementia; however, randomized controlled trials of soy isoflavones in Western countries did not generally show their cognitive benefit. The discrepant results may be attributed to S-equol production capability; after consuming soy isoflavones, 40-70% of East Asians produce S-equol, whereas 20-30% of Westerners do. Recent observational and clinical studies in Japan show that S-equol but not soy isoflavones is inversely associated with multiple vascular pathologies, contributing to cognitive impairment and dementia, including arterial stiffness and white matter lesion volume. S-equol has better permeability to the blood-brain barrier than soy isoflavones, although their affinity to estrogen receptor-ß is similar. S-equol is also the most potent antioxidant among all known soy isoflavones. Although S-equol is available as a dietary supplement, no long-term trials in humans have examined the effect of S-equol supplementation on arterial stiffness, cerebrovascular disease, cognitive decline, or dementia.

Soy-Derived Equol Induces Antioxidant Activity in Zebrafish in an Nrf2-Independent Manner.

Antioxidant effects of soy-derived isoflavones are predicted to be mediated by the Keap1-Nrf2 pathway. Recently, we constructed an assay system to evaluate the antioxidant effects of dietary phytochemicals in zebrafish and revealed a relationship between these effects and the Keap1-Nrf2 pathway. In this study, we used this system to examine the antioxidant effects of seven isoflavones. Among those seven, equol showed strong antioxidant effects when arsenite was used as an oxidative stressor. The antioxidant effect of equol was also shown in Nrf2-mutant zebrafish nfe2l2afh318, suggesting that this effect was not mediated by the Keap1-Nrf2 pathway. To elucidate this unidentified mechanism, the gene expression profiles of equol-treated larvae were analyzed using RNA-seq and qRT-PCR, while no noticeable changes were detected in the expression of genes related to antioxidant effects, except weak induction of Nrf2 target genes. Because nfe2l2afh318 is an amino acid-substitution mutant (Arg485Lue), we considered that the antioxidant effect of equol in this mutant might be due to residual Nrf2 activity. To examine this possibility, we generated an Nrf2-knockout zebrafish nfe2l2ait321 using CRISPR-Cas9 and analyzed the antioxidant effect of equol. As a result, equol showed strong antioxidant effects even in Nrf2-knockout larvae, suggesting that equol indeed upregulates antioxidant activity in zebrafish in an Nrf2-independent manner.

[Equol and its enantiomers inhibited urethane-induced lung cancer in mice].

OBJECTIVE: To investigate the effects and mechanisms of equol and its enantiomers on urethane-induced lung cancer in mice. METHODS: A total of 120 5-week-old male C57BL/6 mice were randomly divided into 8 groups: lung cancer tumor control group (CG), genistein control group (GCG), low dose racemic equol group (LEG), high dose racemic equol group (HEG), low dose R-equol group (LRE), high dose R-equol group (HRE), low dose S-equol group (LSE) and high dose S-equol group (HSE). Urethane was injected subcutaneously twice a week for 4 weeks to induce lung cancer and then the mice were fed for 4 months. The body weight and food intake of each group were measured and recorded weekly. After the mice were sacrificed, the blood, livers and lungs of the mice were collected. The incidence of lung cancer in each group was recorded. The concentration of serum superoxide dismutase (SOD), malondialdehyde (MDA) and 8-hydroxydeoxygunosine (8-OHdG) were detected by the corresponding kits. Western blotting was used to detect the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the livers. Between-group differences in body weight and food intake of the mice were compared using repeated measures ANOVA, and ANOVA for the differences between non-repeated measurements, with post hoc analysis using Tukey's method if there were between-group differences. Comparisons of categorical data were performed by chi-square test, and if there were differences between the groups, the Bonferroni method was used for pairwise comparison. RESULTS: A total of 49 in the 120 mice developed lung cancer. The overall incidence of lung cancer was 40.8%. Compared with the control group, the incidence of lung cancers in each experimental group was lower, and the difference was statistically significant. The incidence of lung cancer in the high-dose experimental group was significantly lower than that in the low-dose experimental group. However, the incidence of lung cancer was similar in the three equol groups and the genistein group at the same dose. Compared with the control group, the high-dose experimental group had higher serum SOD concentration, lower MDA and 8-OHdG concentrations, and the differences were statistically significant. Western blotting analysis showed that the expression levels of Nrf2 protein in the experimental groups were higher than those in the control group except the low-dose racemic equol group, and the Nrf2 protein expression level in the high-dose equol groups was higher than that in the low-dose equol groups. CONCLUSION: Racemic equol and its enantiomers mayinhibit lung carcinogenesis through antioxidant effects.

Anti-bacterial and Anti-biofilm Effects of Equol on Yersinia enterocolitica.

Yersinia enterocolitica has clinical significance due to its etiological role in yersiniosis and gastroenteritis. This study was designed to assess anti-bacterial and anti-biofilm effects of equol on Y. enterocolitica via phenotypic and genetic analyses. To determine its anti-bacterial activity, minimum inhibitory concentrations (MICs) of equol against clinically isolated Y. enterocolitica strains were analyzed. Subsequently, it was confirmed that the sub-MIC90 value of equol could inhibit biofilm formation and reduce preformed biofilm. Furthermore, it was found that equol could reduce the expression of biofilm-related (hmsT) gene in Y. enterocolitica. This study also demonstrated that equol not only reduced levels of bacterial motility, but also decreased the expression of a motility-related (flhDC) gene in Y. enterocolitica. XTT [2,3-bis (2-metoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction analysis revealed that equol attenuated cellular metabolic activities in Y. enterocolitica biofilm. Additionally, changes in biomass and cell density in equol-treated biofilms were visualized using a confocal laser scanning microscope. In conclusion, this study suggests that equol is a potential anti-bacterial and anti-biofilm agent to treat Y. enterocolitica.

Effect of isoflavones on breast cancer cell development and their impact on breast cancer treatments.

PURPOSE: Epidemiological studies have suggested that intake of soy isoflavones is associated with a reduced risk of development of breast cancer and an improved prognosis in patients with breast cancer. In addition, basic research has demonstrated the antitumor effects of these compounds on breast cancer cell lines. However, the detailed effects of the intake of equol, which is one of the metabolites of the soy isoflavones, are yet to be clarified on the risk of development and recurrence of breast cancer and its interactions with drugs used for treating breast cancer. This study aimed to determine the antitumor effects of equol and investigate the impact of adding equol to therapeutic agents for breast cancer using breast cancer cell lines. METHODS: We examined the antitumor effect of equol on breast cancer cell lines using MTS assay. We also studied the combined effect of equol and the existing hormonal or chemotherapeutic agents using combination index. We evaluated the expressions of the related proteins by Western blot analysis and correlated the findings with the antitumor effect. RESULTS: Equol showed bi-phasic protumor and antitumor effects; at a low concentration, it promoted the tumor growth in hormone receptor-positive cell lines, whereas antitumor effects were generally observed when an excessive amount of dose unexpected in the blood and the tissue was administered. When used with tamoxifen, equol might have some antagonistic effect, although it depends on equol concentration and the type of cancer cells. CONCLUSIONS: We confirmed that equol has dual action, specifically a tumor growth-promoting effect and an antitumor effect. Although the results suggested that equol might exert an antagonistic effect against tamoxifen depending on the concentration, equol did not exert an antagonistic effect on other therapeutic agents.