Aryl Hydrocarbon Receptor Activation Research Articles

The role of aryl hydrocarbon receptor in the occurrence and development of periodontitis

Periodontitis is a condition characterized by dysbiosis of microbiota and compromised host immunological responses, resulting in the degradation of periodontal tissues. The aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, plays a crucial role in the pathogenesis of periodontitis. AHR serves as a pivotal mediator for the adverse impacts of exogenous pollutants on oral health. Research indicates elevated expression of AHR in individuals with periodontitis compared to those without the condition. However, subsequent to the identification of endogenous AHR ligands, researches have elucidated numerous significant advantageous roles associated with AHR activation in bone, immune, and epithelial cells. This review concentrates on the modulation of the AHR pathway and the intricate functions that AHR plays in periodontitis. It discusses the characteristics of AHR ligands, detailing the established physiological functions in maintaining alveolar bone equilibrium, regulating immunity, facilitating interactions between the oral microbiome and host, and providing protection to epithelial tissues, while also exploring its potential roles in systemic disorders related to periodontitis.

Telmisartan potentiates the ITE-induced aryl hydrocarbon receptor activity in human liver cell line.

Telmisartan is an angiotensin receptor blocker (ARB) approved by the Food and Drug Administration of the US for the treatment of hypertension. It possesses unique pharmacologic properties, including the longest half-life among all ARBs; this leads to a 24-h sustained reduction of blood pressure. Besides well-known antihypertensive and cardioprotective effects, there is also strong clinical evidence that telmisartan confers renoprotection. Aryl hydrocarbon receptor (AhR) belongs to the steroid receptor family. 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an endogenous ligand of AhR. Cytochrome P450 (CYP) 1A1 is an AhR-target gene. In this article, we demonstrated that telmisartan (2.5-60μM) enhanced CYP1A1 promoter activity and expressions of mRNA and protein. Telmisartan-induced CYP1A1 expression was blocked by the AhR antagonist CH-223191 in liver cell lines and was negligible in the AhR signaling-deficient mutant cells. In addition, telmisartan induced transcriptional activity mediated by aryl hydrocarbon response element in both human and mouse cells, and was able to induce AhR translocation into the nucleus. Accordingly, telmisartan is an AhR agonist. It also acted synergistically with ITE to further enhance the expression of CYP1A1 mRNA and protein. This synergistic effect was more pronounced in cells with AhR overexpression compared to those without. AhR activity has strong association with the progression of chronic renal disease. Our study demonstrated that telmisartan is an AhR agonist and has synergistic effect with ITE, an indole derivative, to potentiate the effect on AhR. This finding may provide additional clues about the mechanism of the protective effect of telmisartan on the kidney.

Effect of Probiotics on Gastrointestinal Health Through the Aryl Hydrocarbon Receptor Pathway: A Systematic Review

Probiotics are living microorganisms recognized for conferring health benefits on the host by modulating the gut microbiota. They interact with various signaling pathways, including the aryl hydrocarbon receptor (AhR), which plays a crucial role in maintaining intestinal homeostasis and immune function. The activation of AhR by probiotics has been associated with benefits such as improved intestinal barrier function, reduced inflammation, and modulation of immune responses. This systematic review aims to summarize current knowledge on the signaling of AhR, mediated by probiotics in physiological conditions and gastrointestinal pathologies. We conducted a comprehensive search across databases, including PubMed and Embase, up until July 2024. Out of 163 studies screened, 18 met the inclusion criteria. Our findings revealed in healthy populations that probiotic consumption increases the production of AhR ligands promoting intestinal immune tolerance. Furthermore, in populations with gastrointestinal pathologies, probiotics ameliorated symptoms through AhR activation by Trp metabolites, leading to the upregulation of the anti-inflammatory response.

Benzo[a]Pyrene Induces Pyroptotic Colon Damage and Gut Dysbacteriosis by Activating Aryl Hydrocarbon Receptor

Benzo[a]Pyrene Induces Pyroptotic Colon Damage and Gut Dysbacteriosis by Activating Aryl Hydrocarbon Receptor

The uremic toxin indoxyl sulfate decreases osteocyte RANKL/OPG and increases wnt inhibitor RNA expression that is reversed by PTH

Abstract Renal osteodystrophy (ROD) leads to increased fractures, potentially due to underlying low bone turnover in chronic kidney disease (CKD). We hypothesized that indoxyl sulfate (IS), a circulating toxin elevated in CKD and ligand for the aryl hydrocarbon receptor (AhR), may target the osteocytes leading to bone cell uncoupling in ROD. The IDG-SW3 osteocytes were cultured for 14 days (early) and 35 days (mature osteocytes) and incubated with 500 μM of indoxyl sulfate after dose finding studies to confirm AhR activation. Long-term incubation of IS for 14 days led to decreased expression of Tnfsf11/Tnfrsf11b ratio (RANKL/OPG), which would increase osteoclast activity, and increased expression of Wnt inhibitors Sost and Dkk1, which would decrease bone formation in addition to decreased mineralization and alkaline phosphatase (ALP) activity. When osteocytes were incubated with IS and the AhR translocation inhibitor CH223191, mineralization and ALP activity were restored. However, the Tnfsf11/Tnfrsf11b ratio and Sost, Dkk1 expression were not altered compared to IS alone, suggesting more complex signaling. In both early and mature osteocytes, co-culture with PTH and IS reversed the IS-induced upregulation of Sost and Dkk1, and IS enhanced the PTH-induced increase of the Tnfsf11/Tnfrsf11b ratio. Co-culture of IS with PTH additively enhanced the AhR activity assessed by Cyp1a1 and Cyp1b1 expression. In summary, IS in the absence of PTH, increased osteocyte mRNA Wnt inhibitor expression in both early and mature osteocytes and decreased mRNA expression ofTnfsf11/Tnfrsf11b ratio and mineralization in early osteocytes. These changes would lead to decreased resorption and formation resulting in low bone remodeling. These data suggest IS may be important in the underlying low turnover bone disease observed in CKD when PTH is not elevated. In addition, when elevated PTH, IS interacts to further increase Tnfsf11/Tnfrsf11b ratio for osteoclast activity in both early and mature osteocytes which would worsen bone resorption.

2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) elicited dose-dependent shifts in the murine urinary metabolome associated with hepatic AHR-mediated differential gene expression.

Epidemiological evidence suggests an association between dioxin and dioxin-like compound (DLC) exposure and human liver disease. The prototypical DLC, 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), has been shown to induce the progression of reversible hepatic steatosis to steatohepatitis with periportal fibrosis and biliary hyperplasia in mice. Although the effects of TCDD toxicity are mediated by aryl hydrocarbon receptor (AHR) activation, the underlying mechanisms of TCDD-induced hepatotoxicity are unresolved. In the present study, male C57BL/6NCrl mice were gavaged every 4 days for 28 days with 0.03 - 30 μg/kg TCDD and evaluated for liver histopathology and gene expression as well as complementary 1-dimensional proton magnetic resonance (1D- 1 H NMR) urinary metabolic profiling. Urinary trimethylamine (TMA), trimethylamine N -oxide (TMAO), and 1-methylnicotinamide (1MN) levels were altered by TCDD at doses ≤ 3 μg/kg; other urinary metabolites, like glycolate, urocanate, and 3-hydroxyisovalerate, were only altered at doses that induced moderate to severe steatohepatitis. Bulk liver RNA-seq data suggested altered urinary metabolites correlated with hepatic differential gene expression corresponding to specific metabolic pathways. In addition to evaluating whether altered urinary metabolites were liver-dependent, published single-nuclear RNA-seq (snRNA-seq), AHR ChIP-seq, and AHR knockout gene expression datasets provide further support for hepatic cell-type and AHR-regulated dependency, respectively. Overall, TCDD-induced liver effects were preceded by and occurred with changes in urinary metabolite levels due to AHR-mediated changes in hepatic gene expression.

Activation of the aryl hydrocarbon receptor via indole derivatives is a common feature in skin bacterial isolates.

The aryl hydrocarbon receptor (AhR) is a ligand-activated receptor implicated in many inflammatory disorders. The skin microbiota plays a crucial role in maintaining epidermal barrier integrity and is thought to modulate skin homeostasis partly through the production of AhR ligands including metabolites of microbial tryptophan metabolism such as indole derivatives. Here, we report the skin microbiota that activate AhR and their unique tryptophan metabolite profiles. Of the bacteria isolated from healthy human skin and screened for the ability to metabolise tryptophan (18 species, five genera), 14 were positive. The tryptophan metabolites of 10 positive and two negative bacteria were then characterised using liquid chromatography-mass spectrometry. Whole genome sequencing confirmed the presence of key genes involved in the indole-3-pyruvic acid pathway within the genomes of indole-3-acetaldehyde, indole-3-acetic acid, and indole-3-aldehyde-producing organisms. A cell-based luciferase reporter gene assay identified functional agonist activity against human AhR in the culture supernatants of 12 of the 18 species tested. High indole derivative-producing organisms induced potent AhR activation. These data demonstrate the relationship between skin microbiota, tryptophan metabolites, and AhR activation.

Modulation of aryl hydrocarbon receptor activity by halogenated indoles

The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor integral to various physiological and pathological processes. Among its diverse ligands, indole-based compounds have garnered attention due to their significant biological activity and potential therapeutic applications. This study explores the activation of AhR by structurally diverse halogenated indoles. We evaluated the transcriptional activity of AhR and cell viability in the human LS174T-AhR-luc reporter cell line. Among the tested compounds, 4-FI, 7-FI, 6-BrI, 7-BrI, 6-Cl-2-ox, 5-Br-2-ox, and 6-Br-2-ox activated AhR in a concentration-dependent manner, displaying high efficacy and potency. Molecular docking analysis revealed moderate binding affinities of these compounds to the PAS-B domain of AhR, corroborated by competitive radioligand binding assays. Functional assays showed that halogenated indoles induce the formation of AhR-ARNT heterodimer and enhance the binding of the AhR to the CYP1A1 promoter. Additionally, 4-FI and 7-FI exhibited anti-inflammatory properties in Caco-2 cell models, highlighting their potential for therapeutic applications. This study underscores the significance of the type and position of halogen moiety in indole scaffold, suggesting their potential as candidates for developing therapeutics drugs to treat conditions such as inflammatory bowel disease via AhR activation.

The aryl hydrocarbon receptor shapes monocyte transcriptional responses to interleukin-4 by prolonging STAT6 binding to promoters.

Cytokines induce functional and metabolic adaptations in immune cells, typically through transcriptional responses that can be influenced by other extracellular signals and by intracellular factors. The binding of the cytokine interleukin-4 (IL-4) to its receptor induces the phosphorylation and activation of the transcription factor STAT6. The aryl hydrocarbon receptor (AhR), a transcription factor activated by various endogenous and microbe-derived metabolites, modulates the responses of immune cells to danger signals or inflammatory mediators such as cytokines. Here, we investigated cross-talk between the AhR and signaling stimulated by IL-4 in human and mouse monocytes. AhR activation was required for a subset of IL-4-induced transcriptional responses and inhibited the IL-4-induced metabolic switch to fatty acid β-oxidation. The promoters of the genes that were induced by IL-4 in an AhR-dependent manner lacked canonical AhR binding sites, implying a nongenomic mechanism of AhR action. Mechanistically, AhR activation reduced the activity of SHP-1, a phosphatase that targets and inhibits STAT6, and prolonged STAT6 phosphorylation and binding to specific target loci, thus extending the duration of STAT6 activity. Our results identify AhR as a key player in the molecular control of responses to IL-4 in monocytes and suggest a nongenomic mechanism through which AhR ligands may influence the functional responses of cells to IL-4.

Indole-3-Acetic Acid Esterified with Waxy, Normal, and High-Amylose Maize Starches: Comparative Study on Colon-Targeted Delivery and Intestinal Health Impact.

Background: Accumulating research suggests that metabolites produced by gut microbiota are essential for maintaining a balanced gut and immune system. Indole-3-acetic acid (IAA), one of tryptophan metabolites from gut microbiota, is critical for gut health through mechanisms such as activating aryl hydrocarbon receptor. Delivery of IAA to colon is beneficial for treatment of gastrointestinal diseases, and one promising strategy is IAA esterified starch, which is digested by gut microbes in colon and releases loaded IAA. Amylose content is a key structural characteristic that controls the physicochemical properties and digestibility of starch. In the current study, IAA was esterified with three typical starches with distinct amylose content to obtain indolyl acetylated waxy maize starch (WMSIAA), indolyl acetylated normal maize starch (NMSIAA), and indolyl acetylated high-amylose maize starch (HAMSIAA). The study comparatively analyzed their respective physicochemical properties, how they behave under in vitro digestion conditions, their ability to deliver IAA directly to the colon, and their effects on the properties of the gut microbiota. The new characteristic peak of 1H NMR at 10.83 ppm, as well as the new characteristic peak of FTIR spectra at 1729 cm-1, represented the successful esterification of IAA on starch backbone. The following in vitro digestion study further revealed that treatment with indolyl acetylation significantly elevated the resistant starch content in the starch samples. In vivo experimental results demonstrated that WMSIAA exhibited the most significant increase in IAA levels in the stomach, whereas HAMSIAA and NMSIAA demonstrated the most remarkable increases in IAA levels in the small intestine and colon, respectively. The elevated IAA levels in the colon are conducive to promoting the growth of beneficial intestinal bacteria and significantly alleviating DSS-induced colitis. This research presents innovative insights and options for the advancement of colon-specific drug delivery systems aimed at preventing and curing gastrointestinal disorders.

Metabolic Side Effects from Antipsychotic Treatment with Clozapine Linked to Aryl Hydrocarbon Receptor (AhR) Activation.

Metabolic syndrome (MetS) is the most common adverse drug reaction from psychiatric pharmacotherapy. Neuroreceptor blockade by the antipsychotic drug clozapine induces MetS in about 30% of patients. Similar to insulin resistance, clozapine impedes Akt kinase activation, leading to intracellular glucose and glutathione depletion. Additional cystine shortage triggers tryptophan degradation to kynurenine, which is a well-known AhR ligand. Ligand-bound AhR downregulates the intracellular iron pool, thereby increasing the risk of mitochondrial dysfunction. Scavenging iron stabilizes the transcription factor HIF-1, which shifts the metabolism toward transient glycolysis. Furthermore, the AhR inhibits AMPK activation, leading to obesity and liver steatosis. Increasing glucose uptake by AMPK activation prevents dyslipidemia and liver damage and, therefore, reduces the risk of MetS. In line with the in vitro results, feeding experiments with rats revealed a disturbed glucose-/lipid-/iron-metabolism from clozapine treatment with hyperglycemia and hepatic iron deposits in female rats and steatosis and anemia in male animals. Decreased energy expenditure from clozapine treatment seems to be the cause of the fast weight gain in the first weeks of treatment. In patients, this weight gain due to neuroleptic treatment correlates with an improvement in psychotic syndromes and can even be used to anticipate the therapeutic effect of the treatment.

Individual and combined antagonism of aryl hydrocarbon receptor (AhR) and estrogen receptors (ERs) offers distinct level of protection against Bisphenol A (BPA)-induced pancreatic islet cell toxicity in mice.

Bisphenol A (BPA), a pervasive endocrine-disrupting chemical, is known to convey harmful impact on pancreatic islets through estrogen receptors (ERs). Conversely, BPA can activate aryl hydrocarbon receptor (AhR) in certain contexts and has raised concerns about potential toxicological effects. However, BPA-AhR interaction in the context of pancreatic islet toxicity is yet to be reported. We demonstrated the specific role of AhR and its interaction with ERs to mediate BPA toxicity in pancreatic islets. In vitro, isolated islet cells treated with BPA (1 nM), with or without CH22319 (10 mM) and ICI182780 (1 mM) and insulin release, glucose-stimulated insulin secretion (GSIS), cell viability, and pERK1/2 and pAkt expression were measured. In vivo, mice were treated with BPA (10 and 100 µg/kg body weight/day for 21 days) with or without intraperitonial co-treatment of CH22319 (AhR antagonist, 10mg/kg), and ICI182780 (ER antagonist, 500 µg/kg). Glucose homeostasis, insulin resistance, oxidative stress, and inflammatory markers were measured. In vitro data revealed the involvement of AhR in the BPA-mediated alteration in insulin secretion, GSIS, and pERK1/2 and pAkt expression which were counteracted by CH223191 (AhR antagonist) alone or with ICI182780 (ER antagonist). Further, CH223191 alone or with ICI182780 modulated BPA-induced oxidative stress and pro-inflammatory cytokines and alleviated islet cell dysfunction and impaired insulin secretion. In conclusion, therapeutic targeting of AhR and ER combined might be a promising target against diabetogenic action of BPA.

AHR activation relieves deoxynivalenol-induced disruption of porcine intestinal epithelial barrier functions

Mycotoxins are ubiquitous natural pollutants that pose a serious threat to public health. Deoxynivalenol (DON) as one of the most prominent mycotoxins has a noticeable adverse effect on intestinal barrier function, which depends on the intestinal barrier integrity. However, the potential mechanisms and effective therapeutic strategies remain unclear. Aryl hydrocarbon receptor (AHR) has been implicated in the modulation of intestinal barrier function and inflammation. The study aims to investigate the unique role of AHR in mediating DON-induced intestinal epithelial barrier function. In the current study, we revealed that DON triggered mitochondrial structural damage and functional impairment, leading to oxidative stress and apoptosis in porcine jejunal epithelial cells (IPEC-J2). DON altered the integrity of IPEC-J2 cells by disrupting the distribution and function of tight junction proteins. Additionally, DON activated TNF-α/NF-κB/MLCK signaling pathway, thereby eliciting inflammatory response. Notably, DON inhibited AHR nuclear translocation and attenuated xenobiotic response element promoter activity and its target genes. However, overexpression of AHR mitigated DON-induced disruption of intestinal epithelial barrier functions by suppressing TNF-α/NF-κB/MLCK pathway in IPEC-J2 cells. Our findings indicate that AHR regulates intestinal epithelial barrier function and therefore is a novel therapeutic molecule for intestinal disorders.

8743 Regulation Of Tryptophan 2,3-Dioxygenase 2 And Androgen Receptor In Triple-Negative Breast Cancer

Abstract Disclosure: L. Kuo: None. K. Lacey: None. N. Spoelstra: None. J.K. Richer: None. Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype with a peak recurrence rate within 3-5 years after initial diagnosis. Androgen receptor (AR) and Tryptophan 2,3-dioxygenase 2 (TDO2) are elevated in TNBC cells grown under anchorage-independent conditions and facilitate anoikis resistance. Several studies reported that the luminal AR subset of TNBC showed a positive enrichment of the tryptophan (TRP) catabolism pathway gene signature. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) inhibition decreases AR promoter transcriptional activity in prostate cancer. We have observed that TNBC with high AR have high levels of genes/proteins in the tryptophan catabolism pathway including the rate limiting enzyme TDO2. Hypothesis: TDO2 activity regulates AR expression through the downstream tryptophan catabolite kynurenine (KYN), which binds and activates AhR and AhR and AR may work together to promote TNBC progression. Methods: Human TNBC cell lines: MDA-MB-453, BT549, SUM159PT ​ were used in this study and the expression of TDO2 and AR were examined by immunohistochemistry (IHC) and western blot. TDO2 was stably knocked down (KD) via shRNA or overexpressed (OE) using lentivirus transduction. Mass spectrometry-based metabolomics were used to measure changes in TRP catabolite levels, and a cytokine array (R&D) was utilized to analyze conditioned media. Results: TDO2 and AR protein are positively correlated in human TNBC cell lines. Although overexpression of TDO2 did not alter AR protein expression in SUM159PT, in the human MDA-MB-453, which have high levels of AR, knockdown of TDO2 (shTDO2) reduced AR protein by 40% and production of kynurenine (KYN) was reduced by 50% (p<0.0001). This suggests that TDO2 activity, specifically KYN binding to AhR, may influence AR protein levels. Conditioned media (CM) from TDO2 OE and KD respectively increased and decreased CXCL5 and IGFBP2. Lastly, TCGA database analysis of TNBC tumors with high levels of both AhR and AR showed significantly shorter patient survival (HR=1.47, log rank p=0.046) compared to those with tumors with lower amounts of both genes. Conclusion: TDO2 and AR are positively correlated in human TNBC cell lines. Tumors with high levels of both AR and AhR result in significantly shorter survival time for TNBC patients. Knockdown of TDO2 significantly reduced KYN production and AR expression in luminal AR TNBC cell lines and future experiments will focus on the underlying mechanism behind this and whether it is dependent on KYN-mediated activation of AhR and if AhR stabilizes AR in TNBC. Presentation: 6/3/2024

8743 Regulation of Tryptophan 2,3-Dioxygenase 2 and Androgen Receptor in Triple-Negative Breast Cancer

Abstract Disclosure: L. Kuo: None. K. Lacey: None. N. Spoelstra: None. J.K. Richer: None. Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype with a peak recurrence rate within 3-5 years after initial diagnosis. Androgen receptor (AR) and Tryptophan 2,3-dioxygenase 2 (TDO2) are elevated in TNBC cells grown under anchorage-independent conditions and facilitate anoikis resistance. Several studies reported that the luminal AR subset of TNBC showed a positive enrichment of the tryptophan (TRP) catabolism pathway gene signature. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) inhibition decreases AR promoter transcriptional activity in prostate cancer. We have observed that TNBC with high AR have high levels of genes/proteins in the tryptophan catabolism pathway including the rate limiting enzyme TDO2. Hypothesis: TDO2 activity regulates AR expression through the downstream tryptophan catabolite kynurenine (KYN), which binds and activates AhR and AhR and AR may work together to promote TNBC progression. Methods: Human TNBC cell lines: MDA-MB-453, BT549, SUM159PT ​ were used in this study and the expression of TDO2 and AR were examined by immunohistochemistry (IHC) and western blot. TDO2 was stably knocked down (KD) via shRNA or overexpressed (OE) using lentivirus transduction. Mass spectrometry-based metabolomics were used to measure changes in TRP catabolite levels, and a cytokine array (R&D) was utilized to analyze conditioned media. Results: TDO2 and AR protein are positively correlated in human TNBC cell lines. Although overexpression of TDO2 did not alter AR protein expression in SUM159PT, in the human MDA-MB-453, which have high levels of AR, knockdown of TDO2 (shTDO2) reduced AR protein by 40% and production of kynurenine (KYN) was reduced by 50% (p<0.0001). This suggests that TDO2 activity, specifically KYN binding to AhR, may influence AR protein levels. Conditioned media (CM) from TDO2 OE and KD respectively increased and decreased CXCL5 and IGFBP2. Lastly, TCGA database analysis of TNBC tumors with high levels of both AhR and AR showed significantly shorter patient survival (HR=1.47, log rank p=0.046) compared to those with tumors with lower amounts of both genes. Conclusion: TDO2 and AR are positively correlated in human TNBC cell lines. Tumors with high levels of both AR and AhR result in significantly shorter survival time for TNBC patients. Knockdown of TDO2 significantly reduced KYN production and AR expression in luminal AR TNBC cell lines and future experiments will focus on the underlying mechanism behind this and whether it is dependent on KYN-mediated activation of AhR and if AhR stabilizes AR in TNBC. Presentation: 6/3/2024

Serotonin Availability Shapes the Effects of Phenelzine on Inflammatory Response and Gene Expression in Macrophages.

Background Serotonin (5-HT) is a neurotransmitter with extensive physiological influence in the central nervous system (CNS) and various behavioral and biological functions, including immune regulation through 5-HT receptors (5-HTRs) expressed by immune cells. A variety of serotonin-modulating drugs have been developed to treat neurological disorders. Phenelzine, a drug indicated for the management of treatment-resistant depression, is a potent, non-selectiveinhibitor of monoamine oxidase (MAO), the enzyme that metabolizes serotonin to 5-hydroxyindole acetic acid (5-HIAA). Given the emerging evidence of a bidirectional link between depression and inflammation, as well as the potential therapeutic applications of serotonin-modulating drugs in autoimmune diseases and cancer, our study investigated the pro-inflammatory and anti-inflammatory factors influenced by serotonin and phenelzine. Methodology We conducted experiments on RAW264.7 macrophages, exposing them to various combinations and concentrations of serotonin, 5-HIAA, and phenelzine. We assessed the relative gene expression of monoamine oxidase-A (MAO-A), CYP1B1, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) using real-time PCR and measured the production of IL-6, TNF-α, and IL-10 cytokines using enzyme-linked immunosorbent assays (ELISA). Results Our findings revealed that phenelzine can downregulate genes associated with the production of reactive oxygen and nitrogen species, reduce aryl hydrocarbon receptor (AHR)-related gene expression induced by serotonin, and enhance the production of inflammatory cytokines. These effects were significantly influenced by the concentration of available serotonin. Conclusions Our study demonstrates that various mechanisms, including AHR activation, modulation of reactive oxygen and nitrogen speciesproduction, and others, in addition to the increased availability of serotonin due to phenelzine treatment, can significantly influence the inflammatory state.

Activation of Aryl Hydrocarbon Receptor by an Indolic Uremic Toxin Increases the Risk of Deep Venous Thrombosis

Activation of Aryl Hydrocarbon Receptor by an Indolic Uremic Toxin Increases the Risk of Deep Venous Thrombosis

The mode of toxic action of ionic liquids: Narrowing down possibilities using high-throughput, in vitro cell-based bioassays

Growing concerns about the environmental impact of ionic liquids (ILs) have spurred research into their (eco)toxic effects, but studies on their mode of toxic action (MOA) still remain limited. However, understanding the MOA and identifying structural features responsible for enhanced toxicity is crucial for characterising the hazard and designing safer alternatives. Therefore, 45 ILs, with systematically varied chemical structures, were tested for cytotoxicity and two specific endpoints in reporter gene assays targeting the Nrf2-ARE mediated oxidative stress response (AREc32) and aryl hydrocarbon receptor activation (AhR-CALUX). While none of the ILs activated the reporter genes, cytotoxicity was high and markedly different between cell lines. Seven and 25 ILs proved more cytotoxic than predicted by baseline toxicity model in the AREc32 and the AhR-CALUX assays, respectively. The length of the side chain and headgroup structures of ILs altered the MOA of ILs. Cellular metabolism of the ILs, investigated by LC-MS/MS, showed side-chain oxidation of the long-chain quaternary ammonium compounds in AhR-CALUX cells and, to a lower extent, in AREc32 cells, however, this transformation could not explain the high cytotoxicity. Effect data for 72 ILs for ten endpoints retrieved from the Tox21 database identified the inhibition of aromatase activity and of mitochondrial membrane potential as potential MOAs. However, in vitro fluorimetric assays for these endpoints demonstrated that effects were activated in a non-specific manner, probably through cytotoxicity. Although many of the ILs tested induced cytotoxicity at concentrations lower than baseline toxicity, the specific MOAs responsible could not be identified. Alternatively, we suggest that the descriptors currently used may fail to define the affinity of ILs for cells. Testing of the affinity of ILs for a diverse range of biomolecules is needed to accurately describe their interactions with cells.

Ineffectiveness of 6,2',4'-trimethoxyflavone in mitigating cerebral ischemia/reperfusion injury after post-reperfusion administration in rats.

Pharmacological inhibition of aryl hydrocarbon receptor (AhR) activation after ischemia alleviates cerebral ischemia/reperfusion (IR) injury. To investigate whether AhR antagonist administration after reperfusion was also effective in attenuating cerebral IR injury. A total of 24 Sprague-Dawley rats were divided into the sham-operated group (no IR), control group (IR), and 6,2',4'-trimethoxyflavone (TMF) group (IR + TMF administration), with 10 rats assigned to each group. Cerebral IR injury was induced by 60 min of middle cerebral artery occlusion followed by reperfusion. TMF (5 mg/kg) was used as the AhR antagonist and was administered intraperitoneally immediately after reperfusion. Cerebral IR injury was observed using magnetic resonance imaging (MRI) and neurobehavioral assessments at baseline, immediately after ischemia, and at 3 days after ischemia. On MRI, the TMF group showed no significant differences in relative apparent diffusion coefficient (ADC), T2, and fractional anisotropy (FA) values; midline shift value; and infarct volume. In terms of neurobehavioral function, factors such as grip strength, contralateral forelimb use, time to touch, and time to remove adhesive tape from the forepaw, were also not significantly different between the control and TMF groups. This study demonstrated that AhR treatment after reperfusion had no noticeable effect on reducing cerebral IR injury in rats.

Molecular Interactions Governing the Rat Aryl Hydrocarbon Receptor Activities of Polycyclic Aromatic Compounds and Predictive Model Development.

Polycyclic aromatic compounds (PACs) exhibit rat aryl hydrocarbon receptor (rAhR) activities, leading to diverse biological or toxic effects. In this study, the key amino residues and molecular interactions that govern the rAhR activity of PACs were investigated using in silico strategies. The homology model of rAhR was first docked with 90 PACs to yield complexes, and the results of the molecular dynamics simulations of 16 typical complexes showed that the binding energies of the complexes range from -7.37 to -26.39 kcal/mol. The major contribution to the molecular interaction comes from van der Waals forces, and Pro295 and Arg316 become the key residues involved in most complexes. Two QSAR models were further developed to predict the rAhR activity of PACs (in terms of log IEQ for PACs without halogen substitutions and log%-TCDD-max for halogenated PACs). Both models have good predictive ability, robustness, and extrapolation ability. Molecular polarizability, electronegativity, size, and nucleophilicity are identified as the important factors affecting the rAhR activity of PACs. The developed models could be employed to predict the rAhR activity of other reactive PACs. This work provides insight into the mechanisms and interactions of the rAhR activity of PACs and assists in the assessment of their fate and risk in organisms.