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IntroductionPhytoestrogen found in soy, fruits, peanuts, and other legumes, has been identified as a metabolite capable of providing beneficial effect in multiple pathological conditions due to its ability to mimic endogenous estrogen. Interestingly, the health promoting effect of phytoestrogens such as isoflavone is dependent on the presence of gut bacteria with ability to metabolize dietary phytoestrogen into metabolite such as equol which have higher affinity for endogenous estrogen receptors. We have previously shown that patients with multiple sclerosis (MS), a neuroinflammatory disease lack gut bacteria with the ability to metabolize phytoestrogen. We have validated the importance of phytoestrogen and gut bacteria in disease protection utilizing an animal model of MS. Specifically, we have shown that an isoflavone rich diet can protect from neuroinflammatory diseases and that protection was dependent on the ability of gut bacteria to metabolize isoflavone into equol. Additionally, mice on a diet with isoflavone showed an anti-inflammatory response compared to the mice on a phyto-free diet. However, it is unknown how isoflavone and/or equol mediates its protective effect especially the effect of isoflavone on host metabolomics. ObjectivesIn this study we utilized untargeted metabolomics to identify the metabolites modulated by the presence of dietary isoflavone. ResultsWe found that the consumption of isoflavone increases anti-inflammatory monounsaturated fatty acids and beneficial polyunsaturated fatty acids while reducing pro-inflammatory glycerophospholipids, sphingolipids, phenylalanine metabolism, and arachidonic acid derivatives. ConclusionIsoflavone consumption alters the systemic metabolic landscape through resultant increase in monounsaturated fatty acids and beneficial polyunsaturated fatty acids while reducing proinflammatory metabolites and pathways. This highlights a potential mechanism by which an isoflavone diet modulate immune mediated disease.

As geographical location can impact the gut microbiome, it is important to study region-specific microbiome signatures of various diseases. Therefore, we profiled the gut microbiome of breast cancer (BC) patients of the Midwestern region of the United States. The bacterial component of the gut microbiome was profiled utilizing 16S ribosomal RNA sequencing. Additionally, a gene pathway analysis was performed to assess the functional capabilities of the bacterial microbiome. Alpha diversity was not significantly different between BC and healthy controls (HC), however beta diversity revealed distinct clustering between the two groups at the species and genera level. Wilcoxon Rank Sum test revealed modulation of several gut bacteria in BC specifically reduced abundance of those linked with beneficial effect such as Faecalibacterium prausnitzii. Machine learning analysis confirmed the significance of several of the modulated bacteria found by the univariate analysis. The functional analysis showed a decreased abundance of SCFA (propionate) production in BC compared to HC. In conclusion, we observed gut dysbiosis in BC with the depletion of SCFA-producing gut bacteria suggesting their role in the pathobiology of breast cancer. Mechanistic understanding of gut bacterial dysbiosis in breast cancer could lead to refined prevention and treatment.

The role of the human gut microbiome has been increasingly appreciated in respect to its influence on human health and disease. It has been proposed that dysbiosis in the gut microbiome and subsequent induction of chronic inflammatory state might have an influence on breast cancer development and prognosis. Therefore this study was undertaken to determine the role of gut microbiome in breast cancer. The Breast Molecular Epidemiology Resource (BMER) resources of the Holden Comprehensive Cancer Center (HCCC) were used to recruit 23 women with breast cancer. Fecal samples were used to characterize the gut microbiome of our patient population using 16S rRNA amplification and Illumina sequencing. Reads were then mapped back to amplicon sequence variant using DADA2 pipeline and compared to a cohort of race, age, and gender matched cohort of regional healthy controls. In our preliminary analysis, we observed that breast cancer patients have distinct microbiota compared to age and gender matched healthy controls. We identified depletion of the anti-inflammatory short chain fatty acid producing bacteria specifically Faecalibacterium, Anaerostipes, Parabacteroides and Phascolarctobacterium in feces from BC patients. Additionally, breast cancer cohort showed enrichment of the pro-inflammatory bacteria such as Eubacterium (dolichum), Bifidobacterium, and Blautia. These bacteria have been previously been identified to change with clinical disease stage and/or BMI, as has genus Bifidobacterium (increased in BC cohort). Within BC group, patients with high grade showed lower levels of Eggerthella lenta and Desulfovibrio whereas patients receiving chemotherapy showed depletion of Sulphur reducing Desulfovibrio and methane producing archaea Methanobrevibacter. Further analysis may lead to insights into the interplay between gut microbiome and breast cancer, as well as the feasibility of therapeutic strategies in the form of novel and existing probiotics. Citation Format: Praveen Vikas, Nicole Cady, Jessica Knobbe, Jemmie Hoang, Meeta Yadav, Melissa Curry, Cathernine Cherwin, Sonia Sugg, Sneha Phadke, Edward Filardo, Ashuthosh Mangalam. Breast cancer patients have reduced levels of short chain fatty acid producing beneficial gut bacteria [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-57.

The toxicity of many “inert” ingredients of pesticide formulations, such as safeners, is poorly characterized, despite evidence that humans may be exposed to these chemicals. Analysis of ToxCast data for dichloroacetamide safeners with the ToxPi tool identified benoxacor as the safener with the highest potential for toxicity, especially liver toxicity. Benoxacor was subsequently administered to mice via oral gavage for 3 days at concentrations of 0, 0.5, 5, and 50 mg/kg bodyweight (b.w.). Bodyweight-adjusted liver and testes weights were significantly increased in the 50 mg/kg b.w. group. There were no overt pathologies in either the liver or the intestine. 16S rRNA analysis of the cecal microbiome revealed no effects of benoxacor on α- or β-diversity; however, changes were observed in the abundance of certain bacteria. RNAseq analysis identified 163 hepatic genes affected by benoxacor exposure. Benoxacor exposure expressed a gene regulation profile similar to dichloroacetic acid and the fungicide sedaxane. Metabolomic analysis identified 9 serum and 15 liver metabolites that were affected by benoxacor exposure, changes that were not significant after correcting for multiple comparisons. The activity of antioxidant enzymes was not altered by benoxacor exposure. In vitro metabolism studies with liver microsomes and cytosol from male mice demonstrated that benoxacor is enantioselectively metabolized by cytochrome P450 enzymes, carboxylesterases, and glutathione S-transferases. These findings suggest that the minor toxic effects of benoxacor may be due to its rapid metabolism to toxic metabolites, such as dichloroacetic acid. This result challenges the assumption that inert ingredients of pesticide formulations are safe.

Isoflavone-metabolizing gut bacteria ameliorate CNS autoimmunity in mice., The gut microbiota is a potential environmental factor that influences the development of multiple sclerosis (MS). We and others have demonstrated that patients with MS and healthy individuals have distinct gut microbiomes. However, the pathogenic relevance of these differences remains unclear. Previously, we showed that bacteria that metabolize isoflavones are less abundant in patients with MS, suggesting that isoflavone-metabolizing bacteria might provide protection against MS. Here, using a mouse model of MS, we report that an isoflavone diet provides protection against disease, which is dependent on the presence of isoflavone-metabolizing bacteria and their metabolite equol. Notably, the composition of the gut microbiome in mice fed an isoflavone diet exhibited parallels to healthy human donors, whereas the composition in those fed an isoflavone-free diet exhibited parallels to patients with MS. Collectively, our study provides evidence that dietary-induced gut microbial changes alleviate disease severity and may contribute to MS pathogenesis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Despite IL-17A being linked to the immunopathogenesis of MS, the mechanism of IL-17A in disease development and maintenance is unclear. In the present study, we investigated the association of IL-17A and gut microbiota in disease development and severity, utilizing HLA-DR3 transgenic mice lacking IL-17A (IL-17A−/−). We found that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ Treg cells and develop milder EAE compared to IL-17A suffcient HLA-DR3 mice. Utilizing 16S metagenocmic sequencing and analysis of gut microbiota we observed that HLA-DR3.IL-17A−/− mice had an increased abundance of regulatory T cells promoting bacteria in the gut such as Lactobacillus and Clostridia. Fecal transplantation followed by co-housing of HLA.DR3 mice with HLA-DR3.IL17A−/− resulted in development of milder EAE and an increase in CD4+CD25+FoxP3+ Treg cells in HLA-DR3 mice. Further whole-genome shotgun metagenomic analysis showed modulation of pathogen-associated molecular patterns (PAMPs) and O-antigen pathways linked with the host innate immune responses in HLA-DR3.IL-17A−/− mice. Thus our study suggests that gut microbiota changes due to the absence of IL17A induce regulatory T cells and suppresses disease in an animal model of MS by modulating the host-bacterial dynamics via PRR-PAMPS interactions.