AhR Activation Research Articles

Different endurance exercises modulate NK cell cytotoxic and inhibiting receptors

PurposeInduction of IDO depends on the activation of AhR forming the AhR/IDO axis. Activated AhR can transcribe various target genes including cytotoxic and inhibiting receptors of NK cells. We investigated whether AhR and IDO levels as well as activating (NKG2D) and inhibiting (KIR2DL1) NK cell receptors are influenced by acute exercise and different chronic endurance exercise programs.Methods21 adult breast and prostate cancer patients of the TOP study (NCT02883699) were randomized to intervention programs of 12 weeks of (1) endurance standard training or (2) endurance polarized training after a cardiopulmonary exercise test (CPET). Serum was collected pre-CPET, immediately post-CPET, 1 h post-CPET and after 12 weeks post-intervention. Flow cytometry analysis was performed on autologous serum incubated NK-92 cells for: AhR, IDO, KIR2DL1 and NKG2D. Differences were investigated using analysis-of-variance for acute and analysis-of-covariance for chronic effects.ResultsAcute exercise: IDO levels changed over time with a significant increase from post-CPET to 1 h post-CPET (p = 0.03). KIR2DL1 levels significantly decreased over time (p < 0.01). NKG2D levels remained constant (p = 0.31). Chronic exercise: for both IDO and NKG2D a significant group × time interaction, a significant time effect and a significant difference after 12 weeks of intervention were observed (IDO: all p < 0.01, NKG2D: all p > 0.05).ConclusionBoth acute and chronic endurance training may regulate NK cell function via the AhR/IDO axis. This is clinically relevant, as exercise emerges to be a key player in immune regulation.

Behavioural adaptations after antibiotic treatment in male mice are reversed by activation of the aryl hydrocarbon receptor

The intestinal microbiota plays an important role in regulating brain functions and behaviour. Microbiota-dependent changes in host physiology have been suggested to be key contributors to psychiatric conditions. However, specific host pathways modulated by the microbiota involved in behavioural control are lacking. Here, we assessed the role of the aryl hydrocarbon receptor (Ahr) in modulating microbiota-related alterations in behaviour in male and female mice after antibiotic (Abx) treatment. Mice of both sexes were treated with Abx to induce bacterial depletion. Mice were then tested in a battery of behavioural tests, including the elevated plus maze and open field tests (anxiety-like behaviour), 3 chamber test (social preference), and the tail suspension and forced swim tests (despair behaviour). Behavioural measurements in the tail suspension test were also performed after microbiota reconstitution and after administration of an Ahr agonist, β-naphthoflavone. Gene expression analyses were performed in the brain, liver, and colon by qPCR. Abx-induced bacterial depletion did not alter anxiety-like behaviour, locomotion, or social preference in either sex. A sex-dependent effect was observed in despair behaviour. Male mice had a reduction in despair behaviour after Abx treatment in both the tail suspension and forced swim tests. A similar alteration in despair behaviour was observed in Ahr knockout mice. Despair behaviour was normalized by either microbiota recolonization or Ahr activation in Abx-treated mice. Ahr activation by β-naphthoflavone was confirmed by increased expression of the Ahr-target genes Cyp1a1, Cyp1b1, and Ahrr. Our results demonstrate a role for Ahr in mediating the behaviours that are regulated by the crosstalk between the intestinal microbiota and the host. Ahr represents a novel potential modulator of behavioural conditions influenced by the intestinal microbiota.

Aryl Hydrocarbon Receptor Deficiency in Intestinal Epithelial Cells Aggravates Alcohol-Related Liver Disease.

Background & AimsThe ligand-activated transcription factor, aryl hydrocarbon receptor (AHR) can sense xenobiotics, dietary, microbial, and metabolic cues. Roles of Ahr in intestinal epithelial cells (IECs) have been much less elucidated compared with those in intestinal innate immune cells. Here, we explored whether the IEC intrinsic Ahr could modulate the development of alcohol-related liver disease (ALD) via the gut–liver axis.MethodsMice with IEC specific Ahr deficiency (AhrΔIEC) were generated and fed with a control or ethanol diet. Alterations of intestinal microbiota and metabolites were investigated by 16S ribosomal RNA sequencing, metagenomics, and untargeted metabolomics. AHR agonists were used to evaluate the therapeutic potentials of intestinal Ahr activation for ALD treatment.ResultsAhrΔIEC mice showed more severe liver injury after ethanol feeding than control mice. Ahr deficiency in IECs altered the intestinal metabolite composition, creating an environment that promoted the overgrowth of Helicobacter hepaticus and Helicobacter ganmani in the gut, enhancing their translocation to mesenteric lymph nodes and liver. Among the altered metabolites, isobutyric acid was increased in the cecum of ethanol-fed AhrΔIEC mice relative to control mice. Furthermore, both H.hepaticus and isobutyric acid administration aggravated ethanol-induced liver injury in vivo and in vitro. Supplementation with AHR agonists, 6-formylindolo[3,2-b]carbazole and indole-3-carbinol, protected mice from ALD development by specifically activating intestinal Ahr without affecting liver Ahr function. Alcoholic patients showed lower intestinal AHR expression and higher H.hepaticus levels compared with healthy individuals.ConclusionsOur results indicate that targeted restoration of IEC intrinsic Ahr function may present as a novel approach for ALD treatment.

Aryl Hydrocarbon Receptor Activation Produces Heat Shock Protein 90 and 70 Overexpression, Prostaglandin E2/Wnt/β-Catenin Signaling Disruption, and Cell Proliferation in MCF-7 and MDA-MB-231 Cells after 24 h and 14 Days of Chlorpyrifos Treatment.

The biocide chlorpyrifos (CPF) was described to increase breast cancer risk in humans, to produce breast cancer in animals, and to induce cell proliferation in MCF-7 and MDA-MB-231 cells after 1 and 14 days of treatment. The entire mechanisms related to these CPF actions remain unknown. CPF induced cell proliferation in MCF-7 and MDA-MB-231 cells after 1 and 14 days of treatment by AhR activation through the PGE2/Wnt/β-catenin pathway and HSP90 and HSP70 overexpression. Our results reveal new information on CPF toxic mechanisms induced in human breast cancer cell lines, which could assist in elucidating its involvement in breast cancer.

Abstract 1785: Inhibition of the IDO-Kyn-AhR cascade optimizes virotherapy in glioblastoma and depends on CD4 T-cell activation

Abstract Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases viral replication and diminishes cell proliferation and induces apoptosis of surrounding effector T-cells. Furthermore, kynurenine (kyn), a metabolite of tryptophan, induces T-cell differentiation into Tregs. Excess kynurenine also elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, D24-RGDOX, clinically known as DNX-2440, triggers IFNγ production contributing to a positive IDO-Kyn-AhR feedback loop. Thus, we hypothesized that combining D24-RGDOX with IDO inhibitors will synergize to treat glioblastoma. We studied the IDO-Kyn-AhR cascade in response to D24-RGDOX infection in human and murine glioblastoma cells. Orthotopic syngeneic glioblastoma mouse models were used to determine efficacy and tumor immune effects of D24-RGDOX and IDO inhibitors as single agents or as a combination treatment. We characterized IDO and AhR in D24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo compared to mock infected controls. D24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. Furthermore, we showed that viral protein expression and viral activity was enhanced in D24-RGDOX infected cultures under IDO inhibition. To test the therapeutic efficacy of combining D24-RGDOX with IDO inhibitors, we used a murine immunocompetent glioblastoma model; the combination produced 30% more long-term survivors compared D24-RGDOX alone (P=0.03, log-rank test). Next, lymphocytic depletion studies illustrated that this anticancer effect was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to D24-RGDOX-treated mice (P=0.009, student t-test). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and MDSCs compared to D24-RGDOX alone (P≤0.05 in all populations, student t-test). This microenvironment remodeling correlated with complete tumor elimination. Altogether, D24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Hong Jiang, Derek A. Wainwright, Sagar Sohoni, Sumit Gupta, Ashley Ossimetha, Frederick Lang, Marta Alonso, Candelaria Gomez-Manzano, Juan Fueyo. Inhibition of the IDO-Kyn-AhR cascade optimizes virotherapy in glioblastoma and depends on CD4 T-cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1785.

Monocytic differentiation and AHR signaling as Primary Nodes of BET Inhibitor Response in Acute Myeloid Leukemia.

To understand mechanisms of response to BET inhibitors (BETi), we mined the Beat AML functional genomic dataset and performed genome-wide CRISPR screens on BETi- sensitive and BETi- resistant AML cells. Both strategies revealed regulators of monocytic differentiation, SPI1, JUNB, FOS, and aryl-hydrocarbon receptor signaling (AHR/ARNT), as determinants of BETi response. AHR activation synergized with BETi while inhibition antagonized BETi-mediated cytotoxicity. Consistent with BETi sensitivity dependence on monocytic differentiation, ex vivo sensitivity to BETi in primary AML patient samples correlated with higher expression of monocytic markers CSF1R, LILRs, and VCAN. In addition, HL-60 cell line differentiation enhanced its sensitivity to BETi. Further, screens to rescue BETi sensitivity identified BCL2 and CDK6 as druggable vulnerabilities. Finally, monocytic AML patient samples refractory to venetoclax ex vivo were significantly more sensitive to combined BETi + venetoclax. Together, our work highlights mechanisms that could predict BETi response and identifies combination strategies to overcome resistance.

P0225 - AhR activation attenuates calcium oxalate nephrocalcinosis-mediated kidney injury and crystals deposition by promoting M2 macrophage polarization

P0225 - AhR activation attenuates calcium oxalate nephrocalcinosis-mediated kidney injury and crystals deposition by promoting M2 macrophage polarization

Cross-Model Comparison of Transcriptomic Dose-Response of Short-Chain Chlorinated Paraffins.

Short-chain chlorinated paraffins (SCCPs) have attracted attention because of their toxicological potential in humans and wildlife at environmentally relevant doses. However, limited information is available regarding mechanistic differences across species in terms of the biological pathways that are impacted by SCCP exposure. Here, a concentration-dependent reduced human transcriptome (RHT) approach was conducted to evaluate 15 SCCPs in HepG2 cells and compared with our previous results using a reduced zebrafish transcriptome (RZT) approach in zebrafish embryos (ZFEs). Generally, SCCPs induced a broader suite of biological pathways in ZFEs than HepG2 cells, and all of the 15 SCCPs were more potent in HepG2 cells compared to ZFEs. Despite these general differences, the transcriptional potency of SCCPs in both model systems showed a significant linear relationship (p = 0.0017, r2 = 0.57), and the average ratios of transcriptional potency for each SCCP in RZT to that in RHT were ∼100,000. C10H14Cl8 was the most potent SCCP, while C10H17Cl5 was the least potent in both ZFEs and HepG2 cells. An adverse outcome pathway network-based analysis demonstrated model-specific responses, such as xenobiotic metabolism that may be mediated by different nuclear receptor-mediated pathways between HepG2 cells (e.g., CAR and AhR activation) and ZFEs (e.g., PXR activation). Moreover, induced transcriptional changes in ZFEs associated with pathways and molecular initiating events (e.g., activation of nicotinic acetylcholine receptor) suggest that SCCPs may disrupt neural development processes. The cross-model comparison of concentration-dependent transcriptomics represents a promising approach to assess and prioritize SCCPs.

The Enigma of AHR Activation in the Skin: Interplay among Ligands, Metabolism, and Bioavailability

AHR is expressed in a variety of skin cell types and contributes to skin homeostasis. Therapeutic targeting of AHR in dermatology was first described for the treatment of inflammatory skin diseases using coal tar ointment. In addition to therapies involving active ligands such as coal tar and tapinarof, the inhibition of AHR-dependent enzymatic activities in the skin may be an alternative approach to resolving skin inflammation.

Disruption of Estrogenic and Androgenic Bioactivities in Human Fetuses Exposed to Maternal Smoking

Endocrine disruptors (EDs) interfere with hormonal signalling and, given that multiple developmental processes are hormone-driven, the prenatal period is a window of increased sensitivity. Maternal smoking is a real-life model of in utero exposure to a complex mixture of EDs. Cigarette smoke contains of >7,000 pollutants, including polycyclic aromatic hydrocarbons (PAHs), which are AhR ligands and cross-talk with the estrogen receptor (ER) system. Prenatal exposure to cigarette smoke is associated with adverse outcomes, including intrauterine growth restriction and increased risk of metabolic syndrome later in life. We aimed to evaluate ED effects associated with smoke exposure in human fetuses. Fetal tissues (plasma, n=48; placenta, n=30; liver, n=29) from elective terminations of normally progressing pregnancies, ranging from 10 to 20 gestation weeks, were collected (SAFeR and FEGO studies: REC 15/NS/0123, REC 04/S0802/21). PAHs and PAH-like compounds were extracted from placenta and fetal liver. Bioactivity levels in plasma, placenta and liver extracts were determined using ER and androgen receptor (AR) transactivation reporter gene assays. PAH burden was evaluated using the AhR-responsive DRhp-CALUX assay. Smoke exposure was associated with a 1.3-fold increase in plasma estrogenic activity. The developmental trajectory of androgenic activity was altered in plasma of smoke-exposed fetuses, with significant anti-androgenic activity in older fetuses (>16 weeks of gestation). In males, plasma androgenic activity was positively associated with testes weight and anogenital distance. In contrast, placentas from smoking mothers had significantly increased androgenic potential. Furthermore, AhR-like activity was 2.9-fold higher in smoke-exposed placentas compared to controls, and 2.3-fold higher in female compared to male fetal livers. Overall, all bioactivity levels were higher in placentas compared to fetal liver. Prenatal exposure to cigarette smoke is associated with higher placental AhR activation, indicative of increased xenotoxicants burden. We also report that smoke-exposed fetuses showed increased circulating estrogenic activity and disrupted androgenic potential, across 10-20 weeks of gestation, in both fetal plasma and placenta. This demonstrates that EDs present in cigarette smoke are able to interfere with hormonal signalling and alter dynamic endocrine activity profiles, which are critical to ensure appropriate, sex-specific, development. These ED effects are likely to disturb placental function and reprogramme fetal development and thus impacting on life-long health.

Lipopolysaccharide-Enhanced Responses against Aryl Hydrocarbon Receptor in FcgRIIb-Deficient Macrophages, a Profound Impact of an Environmental Toxin on a Lupus-Like Mouse Model.

Fc gamma receptor IIb (FcgRIIb) is the only inhibitory-FcgR in the FcgR family, and FcgRIIb-deficient (FcgRIIb−/−) mice develop a lupus-like condition with hyper-responsiveness against several stimulations. The activation of aryl hydrocarbon receptor (Ahr), a cellular environmental sensor, might aggravate activity of the lupus-like condition. As such, 1,4-chrysenequinone (1,4-CQ), an Ahr-activator, alone did not induce supernatant cytokines from macrophages, while the 24 h pre-treatment by lipopolysaccharide (LPS), a representative inflammatory activator, prior to 1,4-CQ activation (LPS/1,4-CQ) predominantly induced macrophage pro-inflammatory responses. Additionally, the responses from FcgRIIb−/− macrophages were more prominent than wild-type (WT) cells as determined by (i) supernatant cytokines (TNF-α, IL-6, and IL-10), (ii) expression of the inflammation associated genes (NF-κB, aryl hydrocarbon receptor, iNOS, IL-1β and activating-FcgRIV) and cell-surface CD-86 (a biomarker of M1 macrophage polarization), and (iii) cell apoptosis (Annexin V), with the lower inhibitory-FcgRIIb expression. Moreover, 8-week-administration of 1,4-CQ in 8 week old FcgRIIb−/− mice, a genetic-prone lupus-like model, enhanced lupus characteristics as indicated by anti-dsDNA, serum creatinine, proteinuria, endotoxemia, gut-leakage (FITC-dextran), and glomerular immunoglobulin deposition. In conclusion, an Ahr activation worsened the disease severity in FcgRIIb−/− mice possibly through the enhanced inflammatory responses. The deficiency of inhibitory-FcgRIIb in these mice, at least in part, prominently enhanced the pro-inflammatory responses. Our data suggest that patients with lupus might be more vulnerable to environmental pollutants.

Protective effects of resveratrol against the cardiac developmental toxicity of trichloroethylene in zebrafish embryos

Trichloroethylene (TCE), a prevalent environmental contaminant, has been shown to induce cardiac malformations. Resveratrol (RSV) is a natural polyphenolic compound exhibiting protective effects on heart development. To investigate if RSV could protect against TCE-induced heart defects, we exposed zebrafish embryos to TCE (10 ppb) in the presence or absence of RSV (1 μg/mL). Our results showed that RSV significantly attenuated TCE-induced heart defects in zebrafish embryos. The TCE-induced ROS (reactive oxygen species) generation, 8-OHdG (8-hydroxy-2`-deoxyguanosine) formation and cell proliferation were significantly counteracted by RSV. Moreover, RSV attenuated the TCE-induced changes in mRNA expression or activity of genes involved in AHR and Nrf2 signal pathways. We further showed that RSV might inhibit TCE-enhanced cell proliferation by rescuing the downregulation of the p53/p21 axis. In conclusion, our data demonstrates that RSV protects against the cardiac developmental toxicity of TCE by inhibiting AHR activity, oxidative stress and cell proliferation.

Curcumin exerts hepatoprotection via overexpression of Paraoxonase-1 and its regulatory genes in rats undergone bile duct ligation.

Curcumin is described as an antioxidant, hepato-protective and antifibrotic in liver fibrosis, although its mechanism is still not known. One of the models of the chronic liver disease stemming from oxidative stress and the generation of free radical has been considered to be bile duct ligation (BDL). Paraoxonase 1 (PON1) is a prominent antioxidant enzyme. Therefore, the objective of the present research is to assess the effects of curcumin on upregulation of PON1 in BDL rats. As predicted, the rats have been divided into the four groups of Sham, Sham+Cur (curcumin), BDL and BDL+Cur. We evaluated the efficacy of curcumin (100mg/kg/day) on protein and gene expression of PON1 and regulatory genes contributed to the gene expression PON1 such as Sp1, PKCα, SREBP-2, AhR, JNK and regulation PON1 activity gene expression of Apo A1. Curcumin attenuated alterations in liver histology, hepatic enzymes and the mRNA expression of fibrotic markers (p<0.05). In addition, curcumin increased significantly mRNA, protein expression of PON1 and mRNA of the genes that are contributed to the expression of PON1 such as Sp1, PKCα, SREBP-2, AhR, JNK and increased PON1 activity through upregulation of Apo A1 (p<0.05). Cirrhosis progression may be inhibited by treatment with curcumin through the increased influence the expression and activity of PON1.

Berberine improves colitis by triggering AhR activation by microbial tryptophan catabolites

Inflammatory bowel diseases (IBD) are kind of recurrent inflammatory issues that occur in the gastrointestinal tract, and currently clinical treatment is still unideal due to the complex pathogenesis of IBD. Basically, gut barrier dysfunction is triggered by gut microbiota dysbiosis that is closely associated with the development of IBD, we thus investigated the therapeutic capacity of berberine (BBR) to improve the dysregulated gut microbiota, against IBD in rats, using a combinational strategy of targeted metabolomics and 16 s rDNA amplicon sequencing technology. Expectedly, our data revealed that BBR administration could greatly improve the pathological phenotype, gut barrier disruption, and the colon inflammation in rats with dextran sulfate sodium (DSS)-induced colitis. In addition, 16S rDNA-based microbiota analysis demonstrated that BBR could alleviate gut dysbiosis in rats. Furthermore, our targeted metabolomics analysis illustrated that the levels of microbial tryptophan catabolites in the gastrointestinal tract were significantly changed during the development of the colitis in rats, and BBR treatment can significantly restore such changes of the tryptophan catabolites accordingly. At last, our in vitro mechanism exploration was implemented with a Caco-2 cell monolayer model, which verified that the modulation of the dysregulated gut microbiota to change microbial metabolites coordinated the improvement effect of BBR on gut barrier disruption in the colitis, and we also confirmed that the activation of AhR induced by microbial metabolites is indispensable to the improvement of gut barrier disruption by BBR. Collectively, BBR has the capacity to treat DSS-induced colitis in rats through the regulation of gut microbiota associated tryptophan metabolite to activate AhR, which can greatly improve the disrupted gut barrier function. Importantly, our finding elucidated a novel mechanism of BBR to improve gut barrier function, which holds the expected capacity to promote the BBR derived drug discovery and development against the colitis in clinic setting.

Comparison of quantitation methods in proteomics to define relevant toxicological information on AhR activation of HepG2 cells by BaP

The application of quantitative proteomics provides a new and promising tool for standardized toxicological research. However, choosing a suitable quantitative method still puzzles many researchers because the optimal method needs to be determined.In this study, we investigated the advantages and limitations of two of the most commonly used global quantitative proteomics methods, namely label-free quantitation (LFQ) and tandem mass tags (TMT). As a case study, we exposed hepatocytes (HepG2) to the environmental contaminant benzo[a]pyrene (BaP) using a concentration of 2 μM. Our results revealed that both methods yield a similar proteome coverage, in which for LFQ a wider range of fold changes was observed but with less significant p-values compared to TMT. We detected 37 and 47 significantly enriched pathways by LFQ and TMT, respectively, with 17 overlapping pathways.To define the minimally required effort in proteomics as a benchmark, we artificially reduced the LFQ, and TMT data sets stepwise and compared the pathway enrichment. Thereby, we found that fewer proteins are necessary for detecting significant enrichment of pathways in TMT compared to LFQ, which might be explained by the higher reproducibility of the TMT data that was observed.In summary, we showed that the TMT approach is the preferable one when investigating toxicological questions because it offers a high reproducibility and sufficient proteome coverage in a comparably short time.

EXTH-45. DELTA-24-RGDOX ACTIVATION OF THE IDO-KYN-AHR CASCADE IN GLIOBLASTOMA: OLD TARGETS FOR A NEW THERAPY

Abstract Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases proliferation and induces apoptosis of surrounding effector T-cells. Kynurenine, a metabolite of tryptophan, induces T-cell differentiation into immunosuppressive Tregs. Excess kynurenine elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, Delta-24-RGDOX, triggers IFNγ production contributing to a positive IDO-Kynurenine-AhR feedback loop. We hypothesized that combining Delta-24-RGDOX with IDO inhibitors will synergize to effectively treat glioblastoma. We characterized IDO and AhR in Delta-24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo. We also observed induction of AhR in CD4+ and CD8+ T-cells by Delta-24-RGDOX in vivo. Delta-24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. We used a murine glioblastoma model to test the efficacy of combining Delta-24-RGDOX with IDO inhibitor, 1MT/indoximod; the combination produced 30% more long-term survivors compared Delta-24-RGDOX alone (P=0.03), which we showed, through lymphocytic depletion studies, was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and myeloid derived suppressor cells compared to Delta-24-RGDOX single treatment (P≤0.05). This microenvironment remodeling correlated with complete tumor elimination. Altogether, Delta-24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression.

Resilience of Micropollutant and Biological Effect Removal in an Aerated Horizontal Flow Treatment Wetland

The performance of an aerated horizontal subsurface flow treatment wetland was investigated before, during and after a simulated aeration failure. Conventional wastewater parameters (e.g., carbonaceous biological oxygen demand, total nitrogen, and Escherichia coli) as well as selected micropollutants (caffeine, ibuprofen, naproxen, benzotriazole, diclofenac, acesulfame, and carbamazepine) were investigated. Furthermore, the removal of biological effects was investigated using in vitro bioassays. The six bioassays selected covered environmentally relevant endpoints (indicative of activation of aryl hydrocarbon receptor, AhR; binding to the peroxisome proliferator-activated receptor gamma, PPARγ; activation of estrogen receptor alpha, ERα; activation of glucocorticoid receptor, GR; oxidative stress response, AREc32; combined algae test, CAT). During the aeration interruption phase, the water quality deteriorated to a degree comparable to that of a conventional (non-aerated) horizontal subsurface flow wetland. After the end of the aeration interruption, the analytical and biological parameters investigated recovered at different time periods until their initial treatment performance. Treatment efficacy for conventional parameters was recovered within a few days, but no complete recovery of treatment efficacy could be observed for bioassays AhR, AREc32 and CAT in the 21 days following re-start of the aeration system. Furthermore, the removal efficacy along the flow path for most of the chemicals and bioassays recovered as it was observed in the baseline phase. Only for the activation of AhR and AREc32 there was a shift of the internal treatment profile from 12.5% to 25% (AhR) and 50% (AREc32) of the fractional length.

PCB126 Exposure Revealed Alterations in m6A RNA Modifications in Transcripts Associated With AHR Activation.

Chemical modifications of proteins, DNA, and RNA moieties play critical roles in regulating gene expression. Emerging evidence suggests the RNA modifications (epitranscriptomics) have substantive roles in basic biological processes. One of the most common modifications in mRNA and noncoding RNAs is N6-methyladenosine (m6A). In a subset of mRNAs, m6A sites are preferentially enriched near stop codons, in 3' UTRs, and within exons, suggesting an important role in the regulation of mRNA processing and function including alternative splicing and gene expression. Very little is known about the effect of environmental chemical exposure on m6A modifications. As many of the commonly occurring environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Hence, the objective of this study was to characterize the acute effects of developmental exposure to PCB126, an environmentally relevant dioxin-like PCB, on m6A methylation patterns. We exposed zebrafish embryos to PCB126 for 6 h starting from 72 h post fertilization and profiled m6A RNA using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq). Our analysis revealed 117 and 217 m6A peaks in the DMSO and PCB126 samples (false discovery rate5%), respectively. The majority of the peaks were preferentially located around the 3' UTR and stop codons. Statistical analysis revealed 15 m6A marked transcripts to be differentially methylated by PCB126 exposure. These include transcripts that are known to be activated by AHR agonists (eg, ahrra, tiparp, nfe2l2b) as well as others that are important for normal development (vgf, cebpd, sned1). These results suggest that environmental chemicals such as dioxin-like PCBs could affect developmental gene expression patterns by altering m6A levels. Further studies are necessary to understand the functional consequences of exposure-associated alterations in m6A levels.

Indoxylsulfate, a Metabolite of the Microbiome, Has Cytostatic Effects in Breast Cancer via Activation of AHR and PXR Receptors and Induction of Oxidative Stress.

Simple SummaryThe aim of our study was to identify metabolites of bacterial origin that play role in the pathogenesis of breast cancer. We identified indoxylsulfate, a metabolite of the amino acid tryptophan, as a metabolite with cytostatic properties on breast cancer cells. Cytostatic properties were dependent on increasing oxidative stress blocking the capacity of cancer cells to migrate, enter blood vessels and to form metastases. Furthermore, indoxylsulfate reduced the proportions of cancer stem cells that are highly resistant to chemotherapy and have vital role in initiating recurrence. We identified that indoxylsulfate exert its effects through the pregnane-X receptor and the aryl-hydrocarbon receptor. The expression of these receptors decrease with the progression of the disease, furthermore, the expression of these receptors is low in cases with poor prognosis.Changes to bacterial metabolite-elicited signaling, in oncobiosis associated with breast cancer, plays a role in facilitating the progression of the disease. We show that indoxyl-sulfate (IS), a tryptophan metabolite, has cytostatic properties in models of breast cancer. IS supplementation, in concentrations corresponding to the human serum reference range, suppressed tumor infiltration to the surrounding tissues and metastasis formation in a murine model of breast cancer. In cellular models, IS suppressed NRF2 and induced iNOS, leading to induction of oxidative and nitrosative stress, and, consequently, reduction of cell proliferation; enhanced oxidative and nitrosative stress are crucial in the subsequent cytostasis. IS also suppressed epithelial-to-mesenchymal transition vital for suppressing cellular movement and diapedesis. Furthermore, IS rendered cells hypometabolic, leading to a reduction in aldehyde-dehydrogenase positive cells. Pharmacological inhibition of the pregnane-X receptor using CH223191 and the aryl-hydrocarbon receptor using ketoconazole diminished the IS-elicited effects, suggesting that these receptors were the major receptors of IS in these models. Finally, we showed that increased expression of the human enzymes that form IS (Cyp2E1, Sult1A1, and Sult1A2) is associated with better survival in breast cancer, an effect that is lost in triple negative cases. Taken together, IS, similar to indolepropionic acid (another tryptophan metabolite), has cytostatic properties and higher expression of the metabolic machinery responsible for the formation of IS supports survival in breast cancer.

Pressure regulated basis for gene transcription by delta-cell micro-compliance modeled in silico: Biphenyl, bisphenol and small molecule ligand models of cell contraction-expansion.

Molecular diameter, lipophilicity and hydrophilicity exclusion affinity limits exist for small molecule carrier-mediated diffusion or transport through channel pores or interaction with the cell surface glycocalyx. The molecular structure lipophilicity limit for non-specific carrier-mediated transmembrane diffusion through polarity-selective transport channels of the cell membrane is Lexternal structure ∙ Hpolar group-1 of ≥ 1.07. The cell membrane channel pore size is > 0.752 and < 0.758 nm based on a 3-D ellipsoid model (biphenyl), and within the molecular diameter size range 0.744 and 0.762 nm based on a 2-D elliptical model (alkanol). The adjusted van der Waals diameter (vdWD, adj; nm) for the subset of halogenated vapors is predictive of the required MAC for anesthetic potency at an initial (-) Δ Cmicro effect. The molecular structure L ∙ Hpolar group-1 for Neu5Ac is 0.080, and the L ∙ Hpolar group-1 interval range for the cell surface glycocalyx hydrophilicity barrier interaction is 0.101 (Saxitoxin, Stx; Linternal structure ∙ Hpolar group-1) - 0.092 (m-xylenediamine, Lexternal structure · Hpolar group). Differential predictive effective pressure mapping of gene activation or repression reveals that p-dioxin exposure results in activation of AhR-Erβ (Arnt)/Nrf-2, Pparδ, Errγ (LxRα), Dio3 (Dio2) and Trα limbs, and due to high affinity Dio2 and Dio3 (OH-TriCDD, Lext · H-1: 1.91–4.31) exothermy-antagonism (Δ contraction) with high affinity T4/rT3-TRα-mediated agonism (Δ expansion). co-planar PCB metabolite exposure (Lext · H-1: 1.95–3.91) results in activation of AhR (Erα/β)/Nrf2, Rev-Erbβ, Errα, Dio3 (Dio2) and Trα limbs with a Δ Cmicro contraction of 0.89 and Δ Cmicro expansion of 1.05 as compared to p-dioxin. co-, ortho-planar PCB metabolite exposure results in activation of Car/PxR, Pparα (Srebf1,—Lxrβ), Arnt (AhR-Erβ), AR, Dio1 (Dio2) and Trβ limbs with a Δ Cmicro contraction of 0.73 and Δ Cmicro expansion of 1.18 (as compared to p-dioxin). Bisphenol A exposure (Lext struct ∙ H-1: 1.08–1.12, BPA–BPE, Errγ; BPAF, Lext struct ∙ H-1: 1.23, CM Erα, β) results in increased duration at Peff for Timm8b (Peff 0.247) transcription and in indirect activation of the AhR/Nrf-2 hybrid pathway with decreased duration at Peff 0.200 (Nrf1) and increased duration at Peff 0.257 (Dffa). The Bpa/Bpaf convergent pathway Cmicro contraction-expansion response increase in the lower Peff interval is 0.040; in comparison, small molecule hormone Δ Cmicro contraction-expansion response increases in the lower Peff intervals for gene expression ≤ 0.168 (Dex· GR) ≥ 0.156 (Dht · AR), with grade of duration at Peff (min·count) of 1.33x105 (Dex/Cort) and 1.8–2.53x105 (Dht/R1881) as compared to the (-) coupled (+) Δ Cmicro Peff to 0.136 (Wnt5a, Esr2) with applied DES (1.86x106). The subtype of trans-differentiated cell as a result of an applied toxin or toxicant is predictable by delta-Cmicro determined by Peff mapping. Study findings offer additional perspective on the basis for pressure regulated gene transcription by alterations in cell micro-compliance (Δ contraction-expansion, Cmicro), and are applicable for the further predictive modeling of gene to gene transcription interactions, and small molecule modulation of cell effective pressure (Peff) and its potential.