Cross-talk between aryl hydrocarbon receptor and the inflammatory response: a role for nuclear factor-κB.

The aryl hydrocarbon receptor (AhR) is involved in the regulation of immune responses, T-cell differentiation, and immunity. Here, we show that inflammatory stimuli such as LPS induce the expression of AhR in human dendritic cells (DC) associated with an AhR-dependent increase of CYP1A1 (cytochrome P4501A1). In vivo data confirmed the elevated expression of AhR by LPS and the LPS-enhanced 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of CYP1A1 in thymus of B6 mice. Inhibition of nuclear factor-κB (NF-κB) repressed both normal and LPS-enhanced, TCDD-inducible, AhR-dependent gene expression and canonical pathway control of RelA-regulated AhR-responsive gene expression. LPS-mediated induction of AhR was NF-κB-dependent, as shown in mouse embryonic fibroblasts (MEFs) derived from Rel null mice. AhR expression and TCDD-mediated induction of CYP1A1 was significantly reduced in RelA-deficient MEF compared with wild type MEF cells and ectopic expression of RelA restored the expression of AhR and induction of CYP1A1 in MEF RelA null cells. Promoter analysis of the human AhR gene identified three putative NF-κB-binding elements upstream of the transcription start site. Mutation analysis of the AhR promoter identified one NF-κB site as responsible for mediating the induction of AhR expression by LPS and electrophoretic shift assays demonstrated that this NF-κB motif is recognized by the RelA/p50 heterodimer. Our results show for the first time that NF-κB RelA is a critical component regulating the expression of AhR and the induction of AhR-dependent gene expression in immune cells illustrating the interaction of AhR and NF-κB signaling.

Depression in Rheumatoid Arthritis and its relation to disease activity.

OBJECTIVES: To determine the level of depression in Rheumatoid Arthritis and its relationship with severity of Rheumatoid Arthritis. METHODS: This cross sectional analytical study was conducted from March 2014 to May 2014. Total 102 cases of Rheumatoid Arthritis (RA) diagnosed as per ACR (American College of Rheumatology) 1987 criteria were enrolled from Rheumatology Department, Fatima Memorial Hospital. Severity of Rheumatoid Arthritis was assessed by Health Assessment Questionnaire (HAQ) and disease activity score (DAS - 28) while severity of depression was measured by Beck Depression Inventory (BDI) scale II questionnaire. The relationship between depression and disease activity (severity) of Rheumatoid Arthritis was assessed by calculating correlation coefficient between depression, disease activity score (DAS 28) of Rheumatoid Arthritis and health assessment questionnaire (HAQ). RESULTS: Out of 102 patients, 77(75.5%) were females and 25(24.5%) males. The mean age of patients was 43.5± 11.9 years and the mean duration of disease was 7.8 ± 5.5 years.71.5% of Rheumatoid Arthritis patients were found to have some degree of depression and this was directly related to the severity of disease. Moderate and severe depression were present in 23 (22.5%) and 19(18.6%) patients respectively. CONCLUSION: In this study, almost three-fourths of Rheumatoid Arthritis patients were found to have depression. There was a strong association between Rheumatoid Arthritis disease activity and the level of depression. So it is imperative for clinicians treating Rheumatoid Arthritis patients to screen them for co morbid depression and manage it accordingly.

Modeling the transplacental transfer of small molecules using machine learning: a case study on per- and polyfluorinated substances (PFAS).

BACKGROUND: Despite their large numbers and widespread use, very little is known about the extent to which per- and polyfluoroalkyl substances (PFAS) can cross the placenta and expose the developing fetus. OBJECTIVE: The aim of our study is to develop a computational approach that can be used to evaluate the of extend to which small molecules, and in particular PFAS, can cross to cross the placenta and partition to cord blood. METHODS: We collected experimental values of the concentration ratio between cord and maternal blood (RCM) for 260 chemical compounds and calculated their physicochemical descriptors using the cheminformatics package Mordred. We used the compiled database to, train and test an artificial neural network (ANN). And then applied the best performing model to predict RCM for a large dataset of PFAS chemicals (n = 7982). We, finally, examined the calculated physicochemical descriptors of the chemicals to identify which properties correlated significantly with RCM. RESULTS: We determined that 7855 compounds were within the applicability domain and 127 compounds are outside the applicability domain of our model. Our predictions of RCM for PFAS suggested that 3623 compounds had a log RCM > 0 indicating preferable partitioning to cord blood. Some examples of these compounds were bisphenol AF, 2,2-bis(4-aminophenyl)hexafluoropropane, and nonafluoro-tert-butyl 3-methylbutyrate. SIGNIFICANCE: These observations have important public health implications as many PFAS have been shown to interfere with fetal development. In addition, as these compounds are highly persistent and many of them can readily cross the placenta, they are expected to remain in the population for a long time as they are being passed from parent to offspring. IMPACT: Understanding the behavior of chemicals in the human body during pregnancy is critical in preventing harmful exposures during critical periods of development. Many chemicals can cross the placenta and expose the fetus, however, the mechanism by which this transport occurs is not well understood. In our study, we developed a machine learning model that describes the transplacental transfer of chemicals as a function of their physicochemical properties. The model was then used to make predictions for a set of 7982 per- and polyfluorinated alkyl substances that are listed on EPA's CompTox Chemicals Dashboard. The model can be applied to make predictions for other chemical categories of interest, such as plasticizers and pesticides. Accurate predictions of RCM can help scientists and regulators to prioritize chemicals that have the potential to cause harm by exposing the fetus.

Development and application of a health-based framework for informing regulatory action in relation to exposure of microplastic particles in California drinking water.

Microplastics have been documented in drinking water, but their effects on human health from ingestion, or the concentrations at which those effects begin to manifest, are not established. Here, we report on the outcome of a virtual expert workshop conducted between October 2020 and October 2021 in which a comprehensive review of mammalian hazard studies was conducted. A key objective of this assessment was to evaluate the feasibility and confidence in deriving a human health-based threshold value to inform development of the State of California's monitoring and management strategy for microplastics in drinking water. A tiered approach was adopted to evaluate the quality and reliability of studies identified from a review of the peer-reviewed scientific literature. A total of 41 in vitro and 31 in vivo studies using mammals were identified and subjected to a Tier 1 screening and prioritization exercise, which was based on an evaluation of how each of the studies addressed various quality criteria. Prioritized studies were identified largely based on their application and reporting of dose-response relationships. Given that methods for extrapolating between in vitro and in vivo systems are currently lacking, only oral exposure in vivo studies were identified as fit-for-purpose within the context of this workshop. Twelve mammalian toxicity studies were prioritized and subjected to a Tier 2 qualitative evaluation by external experts. Of the 12 studies, 7 report adverse effects on male and female reproductive systems, while 5 reported effects on various other physiological endpoints. It is notable that the majority of studies (83%) subjected to Tier 2 evaluation report results from exposure to a single polymer type (polystyrene spheres), representing a size range of 0.040 to 20 µm. No single study met all desired quality criteria, but collectively toxicological effects with respect to biomarkers of inflammation and oxidative stress represented a consistent trend. While it was possible to derive a conservative screening level to inform monitoring activities, it was not possible to extrapolate a human-health-based threshold value for microplastics, which is largely due to concerns regarding the relative quality and reliability of current data, but also due to the inability to extrapolate data from studies using monodisperse plastic particles, such as polystyrene spheres to an environmentally relevant exposure of microplastics. Nevertheless, a conservative screening level value was used to estimate a volume of drinking water (1000 L) that could be used to support monitoring activities and improve our overall understanding of exposure in California's drinking water. In order to increase confidence in our ability to derive a human-health-based threshold value in the future, several research recommendations are provided, with an emphasis towards strengthening how toxicity studies should be conducted in the future and an improved understanding of human exposure to microplastics, insights critically important to better inform future risk assessments. Supplementary Information: The online version contains supplementary material available at 10.1186/s43591-022-00030-6.

Neutral sphingomyelinase 2: a novel target in cigarette smoke-induced apoptosis and lung injury.

Chronic obstructive pulmonary disease (COPD) is caused by exposure to cigarette smoke (CS). One mechanism of CS-induced lung injury is aberrant generation of ceramide, which leads to elevated apoptosis of epithelial and endothelial cells in the alveolar spaces. Recently, we discovered that CS-induced ceramide generation and apoptosis in pulmonary cells is governed by neutral sphingomyelinase (nSMase) 2. In the current experiments, we expanded our studies to investigate whether nSMase2 governs ceramide generation and apoptosis in vivo using rodent and human models of CS-induced lung injury. We found that exposure of mice or rats to CS leads to colocalizing elevations of ceramide levels and terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling-positive cells in lung tissues. These increases are nSMase2 dependent, and are abrogated by treatment with N-acetyl cysteine or anti-nSMase2 small interfering RNA (siRNA). We further showed that mice that are heterozygous for nSMase2 demonstrate significant decrease in ceramide generation after CS exposure, whereas acidic sphingomyelinase (aSMase) knockout mice maintain wild-type ceramide levels, confirming our previous findings (in human airway epithelial cells) that only nSMase2, and not aSMase, is activated by CS exposure. Lastly, we found that lung tissues from patients with emphysema (smokers) display significantly higher levels of nSMase2 expression compared with lung tissues from healthy control subjects. Taken together, these data establish the central in vivo role of nSMase2 in ceramide generation, aberrant apoptosis, and lung injury under CS exposure, underscoring its promise as a novel target for the prevention of CS-induced airspace destruction.

Neutral sphingomyelinase 2 is activated by cigarette smoke to augment ceramide-induced apoptosis in lung cell death.

Cigarette smoke (CS) induces a rapid, sustained upregulation of ceramide production in human bronchial epithelial cells, leading to increased apoptosis. Using loss-of-function and overexpression analyses, we show that neutral sphingomyelinase 2 (nSMase2) is required for CS-mediated ceramide generation and apoptosis. Glutathione (GSH), a crucial antioxidant in lung defense, blocks nSMase2 activity and thus inhibits apoptosis triggered by CS. We found that the exposure to CS, as with exposure to H(2)O(2), results in increased nSMase2 activation leading to ceramide generation and therefore increased apoptosis. Interestingly, exposure of cells to GSH abolishes nSMase2 activation caused by CS and leads to a decrease in CS-induced apoptosis. This suggests that the effects of CS oxidants on nSMase2 are counteracted by GSH. Our data support a model where CS induces nSMase2 activation thereby increasing membrane-sphingomyelin hydrolysis to ceramide. In turn, elevated ceramide enhances airway epithelial cell death, which causes bronchial and alveolar destruction and lung injury in pulmonary diseases.

Epidermal growth factor receptor exposed to cigarette smoke is aberrantly activated and undergoes perinuclear trafficking.

Exposure to hydrogen peroxide (H2O2), one of the reactive oxidants in the gas phase of cigarette smoke (CS), induces aberrant phosphorylation of the epidermal growth factor receptor (EGFR), resulting in the lack of ubiquitination by c-Cbl, and impaired degradation. EGFR activation without the feedback regulation of normal degradation leads to uncontrolled cell growth and tumor promotion. Using immunoprecipitation, immunoblotting, and confocal microscopy, we now demonstrate that the pattern of EGFR activation by CS is similar to H2O2. We found that exposure of human airway epithelial cells to CS, as with exposure to H2O2, not only results in an increase in EGFR activation over time, but the EGFR activated by H2O2 or CS is neither ubiquitinated nor subsequently degraded due to its inability to bind the E3 ubiquitin ligase, c-Cbl, either directly or indirectly via the Grb2 adapter protein. Moreover, the stabilized H2O2- and CS-activated EGFR remains plasma membrane-bound, while a population of the receptor is trafficked to a perinuclear region. Concomitantly, CS exposure results in the activation of downstream Akt and ERK1/2 survival and proliferation pathways. Therefore, exposure to CS, like exposure to H2O2, results in prolonged signaling by the EGFR and may contribute to uncontrolled lung cell growth.

Monte Carlo simulation of cell death signaling predicts large cell-to-cell stochastic fluctuations through the type 2 pathway of apoptosis.

Apoptosis, or genetically programmed cell death, is a crucial cellular process that maintains the balance between life and death in cells. The precise molecular mechanism of apoptosis signaling and the manner in which type 1 and type 2 pathways of the apoptosis signaling network are differentially activated under distinct apoptotic stimuli is poorly understood. Based on Monte Carlo stochastic simulations, we show that the type 1 pathway becomes activated under strong apoptotic stimuli, whereas the type 2 mitochondrial pathway dominates apoptotic signaling in response to a weak death signal. Our results also show signaling in the type 2 pathway is stochastic; the population average over many cells does not capture the cell-to-cell fluctuations in the time course (approximately 1-10 h) of downstream caspase-3 activation. On the contrary, the probability distribution of caspase-3 activation for the mitochondrial pathway shows a distinct bimodal behavior that can be used to characterize the stochastic signaling in type 2 apoptosis and other similar complex signaling processes. Interestingly, such stochastic fluctuations in apoptosis signaling occur even in the presence of large numbers of signaling molecules.

Life and death decisions: ceramide generation and EGF receptor trafficking are modulated by oxidative stress.

Reactive oxidants are associated with the pathogenesis of pulmonary diseases and affect various cell functions, from proliferation to apoptosis. We have shown that oxidants exert growth control on airway epithelial cells by modulating upstream receptor function. Additionally, hydrogen peroxide-mediated oxidative stress modulates ceramide levels to induce apoptosis in lung epithelium. Depletion of glutathione in lung epithelial cells results in ceramide accumulation, suggesting that ceramide elevation, coupled to oxidative stress, initiates apoptosis. While it is desirable to prevent cell death and tissue injury induced by oxidants in diseases such as asthma or acute respiratory distress syndrome, the opposite is sought in cancer. But oxidants may also activate growth factor receptors, enhancing cell proliferation and facilitating tumor promotion. Under oxidative stress, phosphorylation of the epidermal growth factor receptor (EGFR) is abrogated at tyrosine 1,045, the docking site for the ubiquitin ligase c-Cbl, rendering EGFR unable to recruit c-Cbl and be ubiquitylated and degraded. We thus proposed that this deficiency, which confers prolonged receptor signaling at the plasma membrane, links oxidative stress, EGFR, and tumorigenesis. Decoding the molecular interactions between oxidative stress and ceramide pathways and characterizing ubiquitylation control of receptor desensitization should provide new strategies for intervention in diverse pulmonary diseases and in diagnosing and eradicating cancer.

Activation of the Ah receptor by extracts of dietary herbal supplements, vegetables, and fruits.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by a structurally diverse range of synthetic and natural chemicals, and it mediates the toxic and biological effects of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The spectrum of chemicals that bind to and activate the AhR signal transduction pathway and the identity of materials containing AhR active chemicals is only now being defined. Utilizing AhR-dependent gel retardation and reporter gene bioassays, the screening of extracts of 22 dietary herbal supplements and 21 food products (vegetables and fruits) was performed to identify those containing AhR agonists. Several herbal extracts (ginseng, Fo-Ti, white oak bark, licorice, ginkgo biloba, and black cohosh) stimulated AhR DNA binding and gene expression to levels between 20 and 60% of that produced by TCDD. Although some food extracts (corn, jalapeño pepper, green bell pepper, apple, Brussels sprout, and potato) were relatively potent activators of AhR DNA binding (30-50% of TCDD), only corn and jalapeño pepper extracts induced AhR-dependent luciferase reporter gene expression. However, dilution of corn, jalapeño pepper, bell pepper, and potato extracts dramatically increased their ability to induce luciferase activity, suggesting that these extracts contained AhR antagonists whose effectiveness was overcome by dilution. Overall, these results demonstrate that dietary products can be a major source of naturally occurring AhR ligands to which animals and humans are chronically exposed.

Cultural change has to continue for the sake of patient care and safety.

I read with interest the interview with Colin Ovington, director of nursing and midwifery at Mid Staffordshire NHS Foundation Trust (features June 20).

Cigarette smoke exposure causes changes in Scavenger Receptor B1 level and distribution in lung cells.

Scavenger Receptor B1 has been shown to play a prominent role in the uptake and delivery of vitamin E from HDL and is likely involved in regulating vitamin E in the lung. We have previously demonstrated that lung Scavenger Receptor B1 levels (protein and mRNA) are modulated by cigarette smoke in mice and this was accompanied by changes in lung vitamin E. To further characterize the molecular mechanism(s) involved in this process, human alveolar epithelial cells were exposed to cigarette smoke and Scavenger Receptor B1 cellular levels and distribution were assessed. Results demonstrated that Scavenger Receptor B1 localizes in patches on the cellular membrane and in the per nuclear area of control cells. Upon cigarette smoke exposure, Scavenger Receptor B1 first translocated to the cell surface (within the first 12h of exposure) and then cell levels (protein and mRNA levels) decreased significantly at 24h. This decline was accompanied by increased Scavenger Receptor B1 ubiquitination which may explain the decrease in the protein levels. Cigarette smoke induced changes in both sub-cellular redistribution and ubiquitination of Scavenger Receptor B1 together with our previous in vivo data provides evidence that cigarette smoke exposure may alter lung's ability to control its tocopherol levels.

EGF receptor exposed to oxidative stress acquires abnormal phosphorylation and aberrant activated conformation that impairs canonical dimerization.

Crystallographic studies have offered understanding of how receptor tyrosine kinases from the ErbB family are regulated by their growth factor ligands. A conformational change of the EGFR (ErbB1) was shown to occur upon ligand binding, where a solely ligand-mediated mode of dimerization/activation was documented. However, this dogma of dimerization/activation was revolutionized by the discovery of constitutively active ligand-independent EGFR mutants. In addition, other ligand-independent activation mechanisms may occur. We have shown that oxidative stress (ox-stress), induced by hydrogen peroxide or cigarette smoke, activates EGFR differently than its ligand, EGF, thereby inducing aberrant phosphorylation and impaired trafficking and degradation of EGFR. Here we demonstrate that ox-stress activation of EGFR is ligand-independent, does not induce "classical" receptor dimerization and is not inhibited by the tyrosine kinase inhibitor AG1478. Thus, an unprecedented, apparently activated, state is found for EGFR under ox-stress. Furthermore, this activation mechanism is temperature-dependent, suggesting the simultaneous involvement of membrane structure. We propose that ceramide increase under ox-stress disrupts cholesterol-enriched rafts leading to EGFR re-localization into the rigid, ceramide-enriched rafts. This increase in ceramide also supports EGFR aberrant trafficking to a peri-nuclear region. Therefore, the EGFR unprecedented and activated conformation could be sustained by simultaneous alterations in membrane structure under ox-stress.

Enhancing the response of CALUX and CAFLUX cell bioassays for quantitative detection of dioxin-like compounds.

Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications. Over the past 10 years, reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices, such as biological, environmental, food and feed samples, given that high-resolution instrumental analysis techniques are impractical for large-scale screening analysis. The most sensitive cell-based reporter gene bioassay systems developed are the mechanism-based CALUX (Chemically Activated Luciferase Expression) and CAFLUX (Chemically Activated Fluorescent Expression) bioassays, which utilize recombinant cell lines containing stably transfected dioxin (AhR)-responsive firefly luciferase or enhanced green fluorescent protein (EGFP) reporter genes, respectively. While the current CALUX and CAFLUX bioassays are very sensitive, increasing their lower limit of sensitivity, magnitude of response and dynamic range for chemical detection would significantly increase their utility, particularly for those samples that contain low levels of dioxin-like HAHs (i.e., serum). In this study, we report that the addition of modulators of cell signaling pathways or modification of cell culture conditions results in significant improvement in the magnitude and overall responsiveness of the existing CALUX and CAFLUX cell bioassays.

Nitric oxide-enhanced caspase-3 and acidic sphingomyelinase interaction: a novel mechanism by which airway epithelial cells escape ceramide-induced apoptosis.

Reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease. The molecular mechanisms and signaling events involved in lung cell injury by RNS are still poorly understood. In the current study, we observe a novel anti-apoptotic response to nitric oxide (NO) exposure (via the NO donors 3-morpholine-syndnonimine (SIN1) or papa-NONOate) of human airway epithelial (HAE) cells. NO exposure via the NO donors increased cellular ceramide levels via ceramide synthase but did not trigger an apoptotic response. Rather, exposure to the NO donors promoted an increase in the protein-protein interaction between acidic sphingomyelinase (aSMase) and caspase-3, with aSMase sequestering caspase-3 and preventing its cleavage. In contrast, when aSMase was silenced in HAE cells or was knocked out in mice, an increase in cleaved caspase-3 was observed. This elevated caspase-3 cleavage was further augmented upon NO exposure (via SIN1 or papa-NONOate) of HAE cells and could be prevented by an inhibitor to ceramide synthase. These results demonstrate a novel mechanism of NO modulation of apoptosis, in which HAE cells exposed to NO via an NO donor induces ceramide generation via ceramide synthase. However, this ceramide induction does not lead to apoptosis unless aSMase is knocked down, allowing the release of caspase-3, its activation and execution of apoptosis.

Epidermal growth factor receptor exposed to oxidative stress undergoes Src- and caveolin-1-dependent perinuclear trafficking.

The epidermal growth factor (EGF) receptor (EGFR) has been found to be overexpressed in several types of cancer cells, and the regulation of its oncogenic potential has been widely studied. The paradigm for EGFR down-regulation involves the trafficking of activated receptor molecules from the plasma membrane, through clathrin-coated pits, and into the cell for lysosomal degradation. We have previously shown that oxidative stress generated by H2O2 results in aberrant phosphorylation of the EGFR. This leads to the loss of c-Cbl-mediated ubiquitination of the EGFR and, consequently, prevents its degradation. However, we have found that c-Cbl-mediated ubiquitination is required solely for degradation but not for internalization of the EGFR under oxidative stress. To further examine the fate of the EGFR under oxidative stress, we used confocal analysis to show that the receptor not only remains co-localized with caveolin-1 at the plasma membrane, but at longer time points, is also sorted to a perinuclear compartment via a clathrin-independent, caveolae-mediated pathway. Our findings indicate that although the EGFR associates with caveolin-1 constitutively, caveolin-1 is hyperphosphorylated only under oxidative stress, which is essential in transporting the EGFR to a perinuclear location, where it is not degraded and remains active. Thus, oxidative stress may have a role in tumorigenesis by not only activating the EGFR but also by promoting prolonged activation of the receptor both at the plasma membrane and within the cell.

c-Cbl-mediated ubiquitinylation is required for epidermal growth factor receptor exit from the early endosomes.

Epidermal growth factor receptor (EGFR) controls cell growth and has a key role in tumorigenic processes. The extent of EGFR signaling is tightly regulated by post-transcriptional modifications leading to down-regulation of the levels of the receptor. Previous studies from our laboratory demonstrated that the reactive oxidant hydrogen peroxide activates the EGFR, yet, without down-regulation of the receptor levels, which results in prolonged receptor signaling. In the present study we examined the role of the E3 ligase c-Cbl, as a possible link between oxidative stress, EGFR signaling, and tumorigenic responses. First, we ectopically expressed a mutant EGFR (Tyr-1045 --> Phe) in cells lacking endogenous receptor, to determine whether the lack of phosphorylation at this site is the cause for EGFR retention at the membrane under oxidative stress, as we have previously suggested. Our findings suggest that abrogation of tyrosine 1045 phosphorylation alone is not enough to retain the EGFR at the plasma membrane under oxidative stress. Second, through the use of the Src inhibitor PP1, our findings establish EGFR movement out of the early endosomes as the exact location where c-Cbl-mediated ubiquitinylation is essential for EGFR trafficking. Finally, our studies substantiate the findings that c-Cbl-mediated ubiquitinylation is needed for degradation, but not for internalization of the EGFR in both transfection-dependent Chinese hamster ovary cells and transfection-independent A549 lung epithelial cells. These findings only begin to explain the features seen under oxidative stress, but they yield a greater understanding of the role of c-Cbl in EGFR trafficking.