Specific residues and conformational plasticity define the substrate specificity of short-chain dehydrogenases/reductases.

Short-chain dehydrogenases/reductases (SDRs) are one of the most prevalent enzyme families distributed among the sequenced microorganisms. Despite the presence of a conserved catalytic tetrad and high structural similarity, these enzymes exhibit different substrate specificities. The insufficient knowledge regarding the amino acids underlying substrate specificity hinders the understanding of the SDRs' roles in diverse and significant biological processes. Here, we performed bioinformatic analysis, molecular modeling, and mutagenesis studies to identify the key residues that regulate the substrate specificities of two homologous microbial SDRs (i.e., DesE and KduD). Further, we investigated the impact of altering the physicochemical properties of these amino acids on enzyme activity. Interestingly, molecular dynamics simulations also suggest a critical role of enzyme conformational flexibility in substrate recognition and catalysis. Overall, our findings improve the understanding of microbial SDR substrate specificity and shed light on future rational design of more efficient and effective biocatalysts.

Loss of aryl hydrocarbon receptor potentiates FoxM1 signaling to enhance self-renewal of colonic stem and progenitor cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that senses xenobiotics, diet, and gut microbial-derived metabolites, is increasingly recognized as a key regulator of intestinal biology. However, its effects on the function of colonic stem and progenitor cells remain largely unexplored. Here, we observed that inducible deletion of AhR in Lgr5+ stem cells increases the percentage of colonic stem cells and enhances organoid initiating capacity and growth of sorted stem and progenitor cells, while AhR activation has the opposite effect. Moreover, intestinal-specific AhR knockout increases basal stem cell and crypt injury-induced cell proliferation and promotes colon tumorigenesis in a preclinical colitis-associated tumor model by upregulating FoxM1 signaling. Mechanistically, AhR transcriptionally suppresses FoxM1 expression. Activation of AhR in human organoids recapitulates phenotypes observed in mice, such as reduction in the percentage of colonic stem cells, promotion of stem cell differentiation, and attenuation of FoxM1 signaling. These findings indicate that the AhR-FoxM1 axis, at least in part, mediates colonic stem/progenitor cell behavior.

Uniform sized cancer spheroids production using hydrogel-based droplet microfluidics: a review.

Three-dimensional (3D) cell culture models have been extensively utilized in various mechanistic studies as well as for drug development studies as superior in vitro platforms than conventional two-dimensional (2D) cell culture models. This is especially the case in cancer biology, where 3D cancer models, such as spheroids or organoids, have been utilized extensively to understand the mechanisms of cancer development. Recently, many sophisticated 3D models such as organ-on-a-chip models are emerging as advanced in vitro models that can more accurately mimic the in vivo tissue functions. Despite such advancements, spheroids are still considered as a powerful 3D cancer model due to the relatively simple structure and compatibility with existing laboratory instruments, and also can provide orders of magnitude higher throughput than complex in vitro models, an extremely important aspects for drug development. However, creating well-defined spheroids remain challenging, both in terms of throughputs in generation as well as reproducibility in size and shape that can make it challenging for drug testing applications. In the past decades, droplet microfluidics utilizing hydrogels have been highlighted due to their potentials. Importantly, core-shell structured gel droplets can avoid spheroid-to-spheroid adhesion that can cause large variations in assays while also enabling long-term cultivation of spheroids with higher uniformity by protecting the core organoid area from external environment while the outer porous gel layer still allows nutrient exchange. Hence, core-shell gel droplet-based spheroid formation can improve the predictivity and reproducibility of drug screening assays. This review paper will focus on droplet microfluidics-based technologies for cancer spheroid production using various gel materials and structures. In addition, we will discuss emerging technologies that have the potential to advance the production of spheroids, prospects of such technologies, and remaining challenges.

Biphasic chemotaxis of Escherichia coli to the microbiota metabolite indole.

Bacterial chemotaxis to prominent microbiota metabolites such as indole is important in the formation of microbial communities in the gastrointestinal (GI) tract. However, the basis of chemotaxis to indole is poorly understood. Here, we exposed Escherichia coli to a range of indole concentrations and measured the dynamic responses of individual flagellar motors to determine the chemotaxis response. Below 1 mM indole, a repellent-only response was observed. At 1 mM indole and higher, a time-dependent inversion from a repellent to an attractant response was observed. The repellent and attractant responses were mediated by the Tsr and Tar chemoreceptors, respectively. Also, the flagellar motor itself mediated a repellent response independent of the receptors. Chemotaxis assays revealed that receptor-mediated adaptation to indole caused a bipartite response-wild-type cells were attracted to regions of high indole concentration if they had previously adapted to indole but were otherwise repelled. We propose that indole spatially segregates cells based on their state of adaptation to repel invaders while recruiting beneficial resident bacteria to growing microbial communities within the GI tract.

Flavone and Hydroxyflavones Are Ligands That Bind the Orphan Nuclear Receptor 4A1 (NR4A1).

It was recently reported that the hydroxyflavones quercetin and kaempferol bind the orphan nuclear receptor 4A1 (NR4A1, Nur77) and act as antagonists in cancer cells and tumors, and they inhibit pro-oncogenic NR4A1-regulated genes and pathways. In this study, we investigated the interactions of flavone, six hydroxyflavones, seven dihydroxyflavones, three trihydroxyflavones, two tetrahydroxyflavones, and one pentahydroxyflavone with the ligand-binding domain (LBD) of NR4A1 using direct-binding fluorescence and an isothermal titration calorimetry (ITC) assays. Flavone and the hydroxyflavones bound NR4A1, and their KD values ranged from 0.36 µM for 3,5,7-trihydroxyflavone (galangin) to 45.8 µM for 3'-hydroxyflavone. KD values determined using ITC and KD values for most (15/20) of the hydroxyflavones were decreased compared to those obtained using the fluorescence assay. The results of binding, transactivation and receptor-ligand modeling assays showed that KD values, transactivation data and docking scores for these compounds are highly variable with respect to the number and position of the hydroxyl groups on the flavone backbone structure, suggesting that hydroxyflavones are selective NR4A1 modulators. Nevertheless, the data show that hydroxyflavone-based neutraceuticals are NR4A1 ligands and that some of these compounds can now be repurposed and used to target sub-populations of patients that overexpress NR4A1.

Loss of aryl hydrocarbon receptor suppresses the response of colonic epithelial cells to IL22 signaling by upregulating SOCS3.

IL22 signaling plays an important role in maintaining gastrointestinal epithelial barrier function, cell proliferation, and protection of intestinal stem cells from genotoxicants. Emerging studies indicate that the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, promotes production of IL22 in gut immune cells. However, it remains to be determined if AhR signaling can also affect the responsiveness of colonic epithelial cells to IL22. Here, we show that IL22 treatment induces the phosphorylation of STAT3, inhibits colonic organoid growth, and promotes colonic cell proliferation in vivo. Notably, intestinal cell-specific AhR knockout (KO) reduces responsiveness to IL22 and compromises DNA damage response after exposure to carcinogen, in part due to the enhancement of suppressor of cytokine signaling 3 (SOCS3) expression. Deletion of SOCS3 increases levels of pSTAT3 in AhR KO organoids, and phenocopies the effects of IL22 treatment on wild-type (WT) organoid growth. In addition, pSTAT3 levels are inversely associated with increased azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon tumorigenesis in AhR KO mice. These findings indicate that AhR function is required for optimal IL22 signaling in colonic epithelial cells and provide rationale for targeting AhR as a means of reducing colon cancer risk.NEW & NOTEWORTHY AhR is a key transcription factor controlling expression of IL22 in gut immune cells. In this study, we show for the first time that AhR signaling also regulates IL22 response in colonic epithelial cells by modulating SOCS3 expression.

Microfluidic Dielectrophoretic Method Enables On-Demand Spatial Arrangement of Bacteria-Encapsulated Agarose Gel Microparticles.

Microbial interactions within a natural or engineered consortium of microbes play an important role in the functions of the consortium. Better understanding these interactions is also important for engineering microbial consortia for specific applications. As such, tools that can enable investigating microbial interactions are highly valuable. One aspect of microbial interactions that impacts community formation is how the spatial organization of individual microbes impacts interactions leading to community formation. Here, we report the development of a tool that can manipulate the spatial organization of microorganisms to investigate the role of these interactions in community formation. Our developed microfluidic platform utilizes dielectrophoretic (DEP) force to perform on-demand spatial arrangement of microorganism-encapsulated agarose gel microparticles. To demonstrate this concept, three gel microparticle manipulators composed of three independently controllable DEP electrodes were utilized for the on-demand spatial arrangement of a specific combination of microparticles, each containing Escherichia coli cells expressing red fluorescence protein, green fluorescent protein, or blank content. The spatially arranged microparticles suspended in carrier oil were first trapped in a downstream particle trapping structure to form a defined microparticle array, followed by the application of an electric field to disrupt the carrier oil barrier so that all gel microparticles were within the same aqueous solution while the individual gel microparticles remain intact, thereby maintaining their spatial arrangements. We demonstrated that this method can be utilized to generate various arrays with differing number of "spacer microparticles", which were blank microparticles, between the two different E. coli-containing microparticles, enabling precise control over spatial distances between the two different cell populations. This method paves the way for more easily investigating bacterial interactions, especially those that depend on their spatial arrangement such as where cell-cell communication plays a major role.

Engineering Selectively Targeting Antimicrobial Peptides.

The rise of antibiotic-resistant strains of bacterial pathogens has necessitated the development of new therapeutics. Antimicrobial peptides (AMPs) are a class of compounds with potentially attractive therapeutic properties, including the ability to target specific groups of bacteria. In nature, AMPs exhibit remarkable structural and functional diversity, which may be further enhanced through genetic engineering, high-throughput screening, and chemical modification strategies. In this review, we discuss the molecular mechanisms underlying AMP selectivity and highlight recent computational and experimental efforts to design selectively targeting AMPs. While there has been an extensive effort to find broadly active and highly potent AMPs, it remains challenging to design targeting peptides to discriminate between different bacteria on the basis of physicochemical properties. We also review approaches for measuring AMP activity, point out the challenges faced in assaying for selectivity, and discuss the potential for increasing AMP diversity through chemical modifications.

Diet-Host-Microbiota Interactions Shape Aryl Hydrocarbon Receptor Ligand Production to Modulate Intestinal Homeostasis.

The aryl hydrocarbon receptor (AhR) is a ligand-activated basic-helix-loop-helix transcription factor that binds structurally diverse ligands and senses cues from environmental toxicants and physiologically relevant dietary/microbiota-derived ligands. The AhR is an ancient conserved protein and is widely expressed across different tissues in vertebrates and invertebrates. AhR signaling mediates a wide range of cellular functions in a ligand-, cell type-, species-, and context-specific manner. Dysregulation of AhR signaling is linked to many developmental defects and chronic diseases. In this review, we discuss the emerging role of AhR signaling in mediating bidirectional host-microbiome interactions. We also consider evidence showing the potential for the dietary/microbial enhancement ofhealth-promoting AhR ligands to improve clinical pathway management in the context of inflammatory bowel diseases and colon tumorigenesis.

Novel Role of Ghrelin Receptor in Gut Dysbiosis and Experimental Colitis in Aging.

Chronic low-grade inflammation is a hallmark of aging, which is now coined as inflamm-aging. Inflamm-aging contributes to many age-associated diseases such as obesity, type 2 diabetes, cardiovascular disease, and inflammatory bowel disease (IBD). We have shown that gut hormone ghrelin, via its receptor growth hormone secretagogue receptor (GHS-R), regulates energy metabolism and inflammation in aging. Emerging evidence suggests that gut microbiome has a critical role in intestinal immunity of the host. To determine whether microbiome is an integral driving force of GHS-R mediated immune-metabolic homeostasis in aging, we assessed the gut microbiome profiles of young and old GHS-R global knockout (KO) mice. While young GHS-R KO mice showed marginal changes in Bacteroidetes and Firmicutes, aged GHS-R KO mice exhibited reduced Bacteroidetes and increased Firmicutes, featuring a disease-susceptible microbiome profile. To further study the role of GHS-R in intestinal inflammation in aging, we induced acute colitis in young and aged GHS-R KO mice using dextran sulfate sodium (DSS). The GHS-R KO mice showed more severe disease activity scores, higher proinflammatory cytokine expression, and decreased expression of tight junction markers. These results suggest that GHS-R plays an important role in microbiome homeostasis and gut inflammation during aging; GHS-R suppression exacerbates intestinal inflammation in aging and increases vulnerability to colitis. Collectively, our finding reveals for the first time that GHS-R is an important regulator of intestinal health in aging; targeting GHS-R may present a novel therapeutic strategy for prevention/treatment of aging leaky gut and inflammatory bowel disease.

Development of a hybrid model for a partially known intracellular signaling pathway through correction term estimation and neural network modeling.

Developing an accurate first-principle model is an important step in employing systems biology approaches to analyze an intracellular signaling pathway. However, an accurate first-principle model is difficult to be developed since it requires in-depth mechanistic understandings of the signaling pathway. Since underlying mechanisms such as the reaction network structure are not fully understood, significant discrepancy exists between predicted and actual signaling dynamics. Motivated by these considerations, this work proposes a hybrid modeling approach that combines a first-principle model and an artificial neural network (ANN) model so that predictions of the hybrid model surpass those of the original model. First, the proposed approach determines an optimal subset of model states whose dynamics should be corrected by the ANN by examining the correlation between each state and outputs through relative order. Second, an L2-regularized least-squares problem is solved to infer values of the correction terms that are necessary to minimize the discrepancy between the model predictions and available measurements. Third, an ANN is developed to generalize relationships between the values of the correction terms and the system dynamics. Lastly, the original first-principle model is coupled with the developed ANN to finalize the hybrid model development so that the model will possess generalized prediction capabilities while retaining the model interpretability. We have successfully validated the proposed methodology with two case studies, simplified apoptosis and lipopolysaccharide-induced NFκB signaling pathways, to develop hybrid models with in silico and in vitro measurements, respectively.

Dopamine is an aryl hydrocarbon receptor agonist.

Tryptophan metabolites exhibit aryl hydrocarbon receptor (AhR) agonist activity and recent studies show that the phenylalanine metabolites serotonin and carbidopa, a drug used in treating Parkinson's disease, activated the AhR. In this study, we identified the neuroactive hormone dopamine as an inducer of drug-metabolizing enzymes CYP1A1, CYP1B1, and UGT1A1 in colon and glioblastoma cells and similar results were observed for carbidopa. In contrast, carbidopa but not dopamine exhibited AhR activity in BxPC3 pancreatic cancer cells whereas minimal activity was observed for both compounds in Panc1 pancreatic cancer cells. In contrast with a previous report, the induction responses and cytotoxicity of carbidopa was observed only at high concentrations (100 µM) in BxPC3 cells. Our results show that similar to serotonin and several tryptophan metabolites, dopamine is also an AhR-active compound.

Microbiota-Mediated Immune Regulation in Atherosclerosis.

There is a high level of interest in identifying metabolites of endogenously produced or dietary compounds generated by the gastrointestinal (GI) tract microbiota, and determining the functions of these metabolites in health and disease. There is a wealth of compelling evidence that the microbiota is linked with many complex chronic inflammatory diseases, including atherosclerosis. Macrophages are key target immune cells in atherosclerosis. A hallmark of atherosclerosis is the accumulation of pro-inflammatory macrophages in coronary arteries that respond to pro-atherogenic stimuli and failure of digesting lipids that contribute to foam cell formation in atherosclerotic plaques. This review illustrates the role of tryptophan-derived microbiota metabolites as an aryl hydrocarbon receptor (AhR) ligand that has immunomodulatory properties. Also, microbiota-dependent trimethylamine-N-oxide (TMAO) metabolite production is associated with a deleterious effect that promotes atherosclerosis, and metabolite indoxyl sulfate has been shown to exacerbate atherosclerosis. Our objective in this review is to discuss the role of microbiota-derived metabolites in atherosclerosis, specifically the consequences of microbiota-induced effects of innate immunity in response to atherogenic stimuli, and how specific beneficial/detrimental metabolites impact the development of atherosclerosis by regulating chronic endotoxemic and lipotoxic inflammation.

Molecular Mechanism for Attractant Signaling to DHMA by E. coli Tsr.

The attractant chemotaxis response of Escherichia coli to norepinephrine requires that it be converted to 3,4-dihydroxymandelic acid (DHMA) by the monoamine oxidase TynA and the aromatic aldehyde dehydrogenase FeaB. DHMA is sensed by the serine chemoreceptor Tsr, and the attractant response requires that at least one subunit of the periplasmic domain of the Tsr homodimer (pTsr) has an intact serine-binding site. DHMA that is generated in vivo by E. coli is expected to be a racemic mixture of the (R) and (S) enantiomers, so it has been unclear whether one or both chiral forms are active. Here, we used a combination of state-of-the-art tools in molecular docking and simulations, including an in-house simulation-based docking protocol, to investigate the binding properties of (R)-DHMA and (S)-DHMA to E. coli pTsr. Our studies computationally predicted that (R)-DHMA should promote a stronger attractant response than (S)-DHMA because of a consistently greater-magnitude piston-like pushdown of the pTsr α-helix 4 toward the membrane upon binding of (R)-DHMA than upon binding of (S)-DHMA. This displacement is caused primarily by interaction of DHMA with Tsr residue Thr156, which has been shown by genetic studies to be critical for the attractant response to L-serine and DHMA. These findings led us to separate the two chiral species and test their effectiveness as chemoattractants. Both the tethered cell and motility migration coefficient assays validated the prediction that (R)-DHMA is a stronger attractant than (S)-DHMA. Our study demonstrates that refined computational docking and simulation studies combined with experiments can be used to investigate situations in which subtle differences between ligands may lead to diverse chemotactic responses.

Effects of high-fat diet and intestinal aryl hydrocarbon receptor deletion on colon carcinogenesis.

Consumption of a high-fat diet has been associated with an increased risk of developing colorectal cancer (CRC). However, the effects of the interaction between dietary fat content and the aryl hydrocarbon receptor (AhR) on colorectal carcinogenesis remain unclear. Mainly known for its role in xenobiotic metabolism, AhR has been identified as an important regulator for maintaining intestinal epithelial homeostasis. Although previous research using whole body AhR knockout mice has revealed an increased incidence of colon and cecal tumors, the unique role of AhR activity in intestinal epithelial cells (IECs) and modifying effects of fat content in the diet at different stages of sporadic CRC development are yet to be elucidated. In the present study, we have examined the effects of a high-fat diet on IEC-specific AhR knockout mice in a model of sporadic CRC. Although loss of AhR activity in IECs significantly induced the development of premalignant lesions, in a separate experiment, no significant changes in colon mass incidence were observed. Moreover, consumption of a high-fat diet promoted cell proliferation in crypts at the premalignant colon cancer lesion stage and colon mass multiplicity as well as ß-catenin expression and nuclear localization in actively proliferating cells in colon masses. Our data demonstrate the modifying effects of high-fat diet and AhR deletion in IECs on tumor initiation and progression.NEW & NOTEWORTHY Through the use of an intestinal-specific aryl hydrocarbon receptor (AhR) knockout mouse model, this study demonstrates that the expression of AhR in intestinal epithelial cells is required to reduce the formation of premalignant colon cancer lesions. Furthermore, consumption of a high-fat diet and the loss of AhR in intestinal epithelial cells influences the development of colorectal cancer at various stages.

Ah receptor ligands and their impacts on gut resilience: structure-activity effects.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and structurally related halogenated aromatics modulate gene expression and induce biochemical and toxic responses that are mediated by initial binding to the aryl hydrocarbon receptor (AhR). The AhR also binds structurally diverse compound including pharmaceuticals, endogenous biochemicals, health-promoting phytochemicals, and microbial metabolites. Many of these AhR ligands do not induce TCDD-like toxic responses and some AhR ligands such as microbial metabolites of tryptophan play a role in maintaining gut health and protecting against intestinal inflammation and cancer. Many AhR ligands exhibit tissue- and response-specific AhR agonist or antagonist activities, and act as selective AhR modulators (SAhRMs) and this SAhRM-like activity has also been observed in AhR-ligand-mediated effects in the intestine. This review summarizes studies showing that several AhR ligands including phytochemicals and TCDD protect against dextran sodium sulfate-induced intestinal inflammation. In contrast, AhR ligands such as oxazole compounds enhance intestinal inflammation suggesting that AhR-mediated gut health can be enhanced or decreased by selective AhR modulators and this needs to be considered in development of AhR ligands for therapeutic applications in treating intestinal inflammation.

Effect of diet and intestinal AhR expression on fecal microbiome and metabolomic profiles.

BACKGROUND: Diet, loss of aryl hydrocarbon receptor (AhR) expression and their modification of the gut microbiota community composition and its metabolites affect the development of colorectal cancer (CRC). However, the concordance between fecal microbiota composition and the fecal metabolome is poorly understood. Mice with specific AhR deletion (AhRKO) in intestinal epithelial cell and their wild-type littermates were fed a low-fat diet or a high-fat diet. Shifts in the fecal microbiome and metabolome associated with diet and loss of AhR expression were assessed. Microbiome and metabolome data were integrated to identify specific microbial taxa that contributed to the observed metabolite shifts. RESULTS: Our analysis shows that diet has a more pronounced effect on mouse fecal microbiota composition than the impact of the loss of AhR. In contrast, metabolomic analysis showed that the loss of AhR in intestinal epithelial cells had a more pronounced effect on metabolite profile compared to diet. Integration analysis of microbiome and metabolome identified unclassified Clostridiales, unclassified Desulfovibrionaceae, and Akkermansia as key contributors to the synthesis and/or utilization of tryptophan metabolites. CONCLUSIONS: Akkermansia are likely to contribute to the synthesis and/or degradation of tryptophan metabolites. Our study highlights the use of multi-omic analysis to investigate the relationship between the microbiome and metabolome and identifies possible taxa that can be targeted to manipulate the microbiome for CRC treatment.

MODELING INTER-KINGDOM REGULATION OF INFLAMMATORY SIGNALING IN HUMAN INTESTINAL EPITHELIAL CELLS.

The human gastrointestinal (GI) tract is colonized by a highly diverse and complex microbial community (i.e., microbiota). The microbiota plays an important role in the development of the immune system, specifically mediating inflammatory responses, however the exact mechanisms are poorly understood. We have developed a mathematical model describing the effect of indole on host inflammatory signaling in HCT-8 human intestinal epithelial cells. In this model, indole modulates transcription factor nuclear factor κ B (NF-κB) and produces the chemokine interleukin-8 (IL-8) through the activation of the aryl hydrocarbon receptor (AhR). Phosphorylated NF-κB exhibits dose and time-dependent responses to indole concentrations and IL-8 production shows a significant down-regulation for 0.1 ng/mL TNF-α stimulation. The model shows agreeable simulation results with the experimental data for IL-8 secretion and normalized NF-κB values. Our results suggest that microbial metabolites such as indole can modulate inflammatory signaling in HTC-8 cells through receptor-mediated processes.

Aryl Hydrocarbon Receptor (AHR) Ligands as Selective AHR Modulators (SAhRMs).

The aryl hydrocarbon receptor (AhR) was first identified as the intracellular protein that bound and mediated the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and dioxin-like compounds (DLCs). Subsequent studies show that the AhR plays an important role in maintaining cellular homeostasis and in pathophysiology, and there is increasing evidence that the AhR is an important drug target. The AhR binds structurally diverse compounds, including pharmaceuticals, phytochemicals and endogenous biochemicals, some of which may serve as endogenous ligands. Classification of DLCs and non-DLCs based on their persistence (metabolism), toxicities, binding to wild-type/mutant AhR and structural similarities have been reported. This review provides data suggesting that ligands for the AhR are selective AhR modulators (SAhRMs) that exhibit tissue/cell-specific AhR agonist and antagonist activities, and that their functional diversity is similar to selective receptor modulators that target steroid hormone and other nuclear receptors.

Isoflavones as Ah Receptor Agonists in Colon-Derived Cell Lines: Structure-Activity Relationships.

Many of the protective responses observed for flavonoids in the gastrointestinal track resemble aryl hydrocarbon receptor (AhR)-mediated effects. Therefore, we examined the structure-activity relationships of isoflavones and isomeric flavone and flavanones as AhR ligands on the basis of their induction of CYP1A1, CYP1B1, and UGT1A1 gene expression in colon cancer Caco2 cells and young adult mouse colonocyte (YAMC) cells. Caco2 cells were significantly more Ah-responsive than YAMC cells, and this was due, in part, to flavonoid-induced cytotoxicity in the latter cell lines. The structure-activity relationships for the flavonoids were complex and both response and cell context specific; however, there was significant variability in the AhR activities of the isomeric substituted isoflavones and flavones. For example, 4',5,7-trihydroxyisoflavone (genistein) was AhR-inactive whereas 4',5,7-trihydroxyflavone (apigenin) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells. In contrast, both 5,7-dihydroxy-4-methoxy substituted isoflavone (biochanin A) and flavone (acacetin) induced all three AhR-responsive genes; 4',5,7-trimethoxyisoflavone was a potent AhR agonist, and the isomeric flavone was AhR-inactive. These results coupled with simulation studies modeling flavonoid interaction within the AhR binding pocket demonstrate that the orientation of the substituted phenyl ring at C-2 (flavones) or C-3 (isoflavones) on the common 4-H-chromen-4-one ring strongly influences the activities of isoflavones and flavones as AhR agonists.