[Show abstract][Hide abstract] ABSTRACT: Background: Increasingly, there is a move toward using in vitro toxicity testing for assessment of human health risk by chemicals. As with in vivo toxicity testing, an important question for in vitro results is whether there are thresholds for adverse cellular responses. Empirical evaluations may show consistency with thresholds, but the main evidence has to come from mechanistic considerations. Objectives: Cellular response behaviors depend on the molecular pathway and circuitry in the cell and the manner in which chemicals perturb these circuits. Understanding circuit structures that are inherently capable of resisting small perturbations and producing threshold responses is an important step towards mechanistically interpreting in vitro testing data. Methods: Here we have examined dose-response characteristics for several biochemical network motifs. These network motifs are basic building blocks of molecular circuits underpinning a variety of cellular functions, including adaptation, homeostasis, proliferation, differentiation, and apoptosis. For each motif, we present biological examples and models to illustrate how thresholds arise from specific network structures. Discussion and Conclusion: Integral feedback, feedforward and transcritical bifurcation motifs can generate thresholds. Other motifs, e.g., proportional feedback and ultrasensitivity, produce responses where the slope in the low-dose region is small and stays close to the baseline. Feedforward control may lead to nonmonotonic or hormetic responses. We conclude that network motifs provide a basis for understanding thresholds for cellular responses. Computational pathway modeling of these motifs and their combinations occurring in molecular signaling networks will be a key element in new risk assessment approaches based on in vitro cellular assays.
Environmental Health Perspectives 08/2014; · 7.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A lower arsenic methylation capacity is believed to be associated with various arsenic-related diseases. However, the synergistic effect of the arsenic methylation capacity and potential modifiers on arsenicosis risk is unclear. The current study evaluated the joint effect of the arsenic methylation capacity with several risk factors on the risk of arsenicosis characterized by skin lesions. In total, 302 adults (79 arsenicosis and 223 non-arsenicosis) residing in an endemic arsenism area in Huhhot Basin were included. Urinary levels of inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were determined, and the percentages of arsenic species (iAs%, MMA%, and DMA%), as well as two methylation indices (primary methylation index, PMI, and secondary methylation index, SMI), were calculated to assess the arsenic methylation capacity of individuals. The results showed that a lower methylation capacity, which is indicated by higher MMA% values and lower DMA% and SMI values, was significantly associated with arsenicosis after the adjustment for multiple confounders. The relative excess risk for interactions between higher MMA% values and older age was 2.35 (95% CI: -0.56, 5.27), and the relative excess risk for interactions between higher MMA% values and lower BMI was 1.08 (95% CI: -1.20, 3.36). The data also indicated a suggestive synergistic effect of a lower arsenic methylation capacity (lower DMA% and SMI) with older age, lower BMI, and male gender. The findings of the present study suggest that a lower arsenic methylation capacity was associated with arsenicosis and that certain risk factors may enhance the risk of arsenic-induced skin lesions.
[Show abstract][Hide abstract] ABSTRACT: Risk assessment methodologies in toxicology have remained largely unchanged for decades. The default approach uses high dose animal studies, together with human exposure estimates, and conservative assessment (uncertainty) factors or linear extrapolations to determine whether a specific chemical exposure is ‘safe’ or ‘unsafe’. Although some incremental changes have appeared over the years, results from all new approaches are still judged against this process of extrapolating high-dose effects in animals to low-dose exposures in humans. The US National Research Council blueprint for change, entitled Toxicity Testing in the 21st Century: A Vision and Strategy called for a transformation of toxicity testing from a system based on high-dose studies in laboratory animals to one founded primarily on in vitro methods that evaluate changes in normal cellular signalling pathways using human-relevant cells or tissues. More recently, this concept of pathways-based approaches to risk assessment has been expanded by the description of ‘Adverse Outcome Pathways’ (AOPs). The question, however, has been how to translate this AOP/TT21 C vision into the practical tools that will be useful to those expected to make safety decisions. We have sought to provide a practical example of how the TT21 C vision can be implemented to facilitate a safety assessment for a commercial chemical without the use of animal testing. To this end, the key elements of the TT21 C vision have been broken down to a set of actions that can be brought together to achieve such a safety assessment. Such components of a pathways-based risk assessment have been widely discussed, however to-date, no worked examples of the entire risk assessment process exist. In order to begin to test the process, we have taken the approach of examining a prototype toxicity pathway (DNA damage responses mediated by the p53 network) and constructing a strategy for the development of a pathway based risk assessment for a specific chemical in a case study mode. This contribution represents a ‘work-in-progress’ and is meant to both highlight concepts that are well-developed and identify aspects of the overall process which require additional development. To guide our understanding of what a pathways-based risk assessment could look like in practice, we chose to work on a case study chemical (quercetin) with a defined human exposure and to bring a multidisciplinary team of chemists, biologists, modellers and risk assessors to work together towards a safety assessment. Our goal was to see if the in vitro dose response for quercetin could be sufficiently understood to construct a TT21 C risk assessment without recourse to rodent carcinogenicity study data. The data presented include high throughput pathway biomarkers (p-H2AX, p-ATM, p-ATR, p-Chk2, p53, p-p53, MDM2, Wip1) and markers of cell-cycle, apoptosis and micronuclei formation, plus gene transcription in HT1080 cells. Eighteen point dose response curves were generated using flow cytometry and imaging to determine the concentrations that resulted in significant perturbation. NOELs and BMDs were compared to the output from biokinetic modelling and the potential for in vitro to in vivo extrapolation explored. A first tier risk assessment was performed comparing the total quercetin concentration in the in vitro systems with the predicted total quercetin concentration in plasma and tissues. The shortcomings of this approach and recommendations for improvement are described. This paper therefore describes the current progress in an ongoing research effort aimed at providing a pathways-based, proof-of-concept in vitro-only safety assessment for a consumer use product.
[Show abstract][Hide abstract] ABSTRACT: The 2007 NRC Report (")Toxicity Testing in the 21(st) Century: A Vision and A Strategy("), recommended an integrated, toxicity pathway-oriented approach for chemical testing. As an integral component of the recommendation, computational dose response modeling of toxicity pathways promises to provide mechanistic interpretation and prediction of adverse cellular outcomes. Among the many toxicity pathways, the DNA damage response is better characterized and thus more suited for computational modeling. In the present study, we formulated a minimal mathematical model of this pathway to examine the dose response for etoposide (ETP), an anti-cancer drug that causes DNA double strand breaks (DSBs). In the model, DSB results from inhibition of topoisomerase by ETP and p53 is activated by a bistable switch composed of a positive feedback loop between ATM and γH2AX. Our stochastic model recapitulated the dose response for several molecular biomarkers measured with flow cytometry in HT1080 cells, including phosphorylated p53, ATM, γH2AX, and micronuclei. Model simulations were consistent with a bimodal pattern of p53 activation and a graded population-averaged response at high ETP concentrations. The graded response was a result of heterogeneous activation of individual cells due to molecular stochasticity. This work shows the value of combining data collection on single cell responses and mechanistic, stochastic modeling to develop and test hypothesis for the circuitry of important toxicity pathways. Future studies will determine how well this initial modeling effort agrees with a broader set of experimental studies on pathway responses by examining a more diverse group of DNA-damaging compounds.
[Show abstract][Hide abstract] ABSTRACT: Transcriptional signaling through the antioxidant response element (ARE), orchestrated by the Nuclear factor E2-related factor 2 (Nrf2), is a major cellular defense mechanism against oxidative or electrophilic stress. Here, we reported that isoniazid (INH), a widely used antitubercular drug, displays a substantial inhibitory property against ARE activities in diverse mouse and human cells. In 3T3-L1 preadipocytes, INH concentration-dependently suppressed the ARE-luciferase reporter activity and mRNA expression of various ARE-dependent antioxidant genes under basal and oxidative stressed conditions. In keeping with our previous findings that Nrf2-ARE plays a critical role in adipogenesis by regulating expression of CCAAT/enhancer-binding protein β (C/EBPβ) and peroxisome proliferator-activated receptor γ (PPARγ), suppression of ARE signaling by INH hampered adipogenic differentiation of 3T3-L1 cells and human adipose-derived stem cells (ADSCs). Following adipogenesis induced by hormonal cocktails, INH-treated 3T3-L1 cells and ADSCs displayed significantly reduced levels of lipid accumulation and attenuated expression of C/EBPα and PPARγ. Time-course studies in 3T3-L1 cells revealed that inhibition of adipogenesis by INH occurred in the early stage of terminal adipogenic differentiation, where reduced expression of C/EBPβ and C/EBPδ was observed. To our knowledge, the present study is the first to demonstrate that INH suppresses ARE signaling and interrupts with the transcriptional network of adipogenesis, leading to impaired adipogenic differentiation. The inhibition of ARE signaling may be a potential underlying mechanism by which INH attenuates cellular antioxidant response contributing to various complications.
Toxicology and Applied Pharmacology 10/2013; · 3.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001-0.1μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1-IKKβ-NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. Graohical abstract.
Toxicology and Applied Pharmacology 07/2013; · 3.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: Oxidative stress is implicated in pancreatic β-cell dysfunction, yet clinical outcomes of antioxidant therapies on diabetes are inconclusive. Since reactive oxygen species (ROS) can function as signaling intermediates for glucose-stimulated insulin secretion (GSIS), we hypothesize that exogenously boosting cellular antioxidant capacity dampens signaling ROS and GSIS. METHODS: To test the hypothesis, we formulated a mathematical model of redox homeostatic control circuit comprising known feedback and feedforward loops and validated model predictions with plant-derived antioxidant sulforaphane (SFN). RESULTS: SFN acutely (30-min treatment) stimulated basal insulin secretion in INS-1(832/13) cells and cultured mouse islets, which could be attributed to SFN-elicited ROS as N-acetylcysteine or glutathione ethyl ester suppressed SFN-stimulated insulin secretion. The mathematical model predicted an adapted redox state characteristic of strong induction of endogenous antioxidants but marginally increased ROS under prolonged SFN exposure, a state that attenuates rather than facilitates glucose-stimulated ROS and GSIS. We validated the prediction by demonstrating that although 24-h treatment of INS-1(832/13) cells with low, non-cytotoxic concentrations of SFN (2-10 μM) protected the cells from cytotoxicity by oxidative insult, it markedly suppressed insulin secretion stimulated by 20 mM glucose. CONCLUSIONS: Our study indicates that adaptive induction of endogenous antioxidants by exogenous antioxidants, albeit cytoprotective, inhibits GSIS in β-cells.
Pharmaceutical Research 03/2013; · 4.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many environmental contaminants can disrupt the adaptive immune response. Exposure to the ubiquitous aryl hydrocarbon receptor (AhR) ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other agonists suppresses the antibody response. The underlying pathway mechanism by which TCDD alters B cell function is not well understood. The present study investigated the mechanism of AhR-mediated pathways and mode of suppression by which TCDD perturbs terminal differentiation of B cells to plasma cells and thereby impairs antibody production. An integrated approach combining computational pathway modeling and in vitro assays with primary mouse B cells activated by lipopolysaccharide was employed. We demonstrated that suppression of the IgM response by TCDD occurs in an all-or-none (binary) rather than graded mode: i.e., it reduces the number of IgM-secreting cells in a concentration-dependent manner without affecting the IgM content in individual plasma cells. The mathematical model of the gene regulatory circuit underpinning B cell differentiation revealed that two previously identified AhR-regulated pathways, inhibition of signaling protein AP-1 and activation of transcription factor Bach2, could account for the all-or-none mode of suppression. Both pathways disrupt the operation of a bistable-switch circuit that contains transcription factors Bcl6, Prdm1, Pax5, and Bach2 and regulates B cell fate. The model further predicted that by transcriptionally activating Bach2, TCDD might delay B cell differentiation and increase the likelihood of isotype switching, thereby altering the antibody repertoire. In conclusion, the present study revealed the mode and specific pathway mechanisms by which the environmental immunosuppressant TCDD suppresses B cell differentiation.
Toxicology and Applied Pharmacology 01/2013; · 3.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multi-component signal transduction pathways and gene regulatory circuits underpin integrated cellular responses to perturbations. A recurring set of network motifs serve as the basic building blocks of these molecular signalling networks. This review focuses on ultrasensitive response motifs (URMs) that amplify small percentage changes in the input signal into larger percentage changes in the output response. URMs generally possess a sigmoid input-output relationship that is steeper than the Michaelis-Menten type of response and is often approximated by the Hill function. Six types of URMs can be commonly found in intracellular molecular networks and each has a distinct kinetic mechanism for signal amplification. These URMs are: (i) positive cooperative binding, (ii) homo-multimerization, (iii) multistep signalling, (iv) molecular titration, (v) zero-order covalent modification cycle and (vi) positive feedback. Multiple URMs can be combined to generate highly switch-like responses. Serving as basic signal amplifiers, these URMs are essential for molecular circuits to produce complex nonlinear dynamics, including multistability, robust adaptation and oscillation. These dynamic properties are in turn responsible for higher-level cellular behaviours, such as cell fate determination, homeostasis and biological rhythm.
Open Biology 01/2013; 3(4):130031. · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that functions as a master regulator of the cellular adaptive response to oxidative stress. Our previous studies showed that Nrf2 plays a critical role in adipogenesis by regulating expression of CCAAT/enhancer-binding protein β and peroxisome proliferator-activated receptor γ. To determine the role of Nrf2 in the development of obesity and associated metabolic disorders, the incidence of metabolic syndrome was assessed in whole-body or adipocyte-specific Nrf2-knockout mice on a leptin-deficient ob/ob background, a model with an extremely positive energy balance. On the ob/ob background, ablation of Nrf2, globally or specifically in adipocytes, led to reduced white adipose tissue (WAT) mass, but resulted in an even more severe metabolic syndrome with aggravated insulin resistance, hyperglycemia, and hypertriglyceridemia. Compared with wild-type mice, WAT of ob/ob mice expressed substantially higher levels of many genes related to antioxidant response, inflammation, adipogenesis, lipogenesis, glucose uptake, and lipid transport. Absence of Nrf2 in WAT resulted in reduced expression of most of these factors at mRNA or protein levels. Our findings support a novel role for Nrf2 in regulating adipose development and function, by which Nrf2 controls the capacity of WAT expansion and insulin sensitivity and maintains glucose and lipid homeostasis.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: There is growing evidence that chronic exposure to inorganic arsenic (iAs) is associated with increased prevalence of Type 2 diabetes (T2D). However, the mechanisms for the diabetogenic effect of iAs are still largely unknown. White adipose tissue (WAT) actively stores and releases energy and maintains lipid and glucose homeostasis. METHODS: The effects and associated mechanisms of iAs and its major metabolites on adipogenesis were determined in 3T3-L1 preadipocytes, mouse adipose-derived stromal-vascular fraction cells (ADSVFCs) and human adipose tissue-derived stem cells (ADSCs). RESULTS: Exposure of 3T3-L1 preadipocytes to noncytotoxic levels of arsenic, including inorganic arsenite (iAs3+, up to 5 μM), inorganic arsenate (up to 20 μM), trivalent monomethylated arsenic (MMA3+, up to 1 μM) and trivalent dimethylated arsenic (DMA3+, up to 2 μM) decreased adipogenic hormone-induced adipogenesis in a concentration-dependent manner. In addition, iAs3+, MMA3+ and DMA3+ exhibited a strong inhibitory effect on adipogenesis in primary cultured mouse ADSVFCs and human ADSCs. Time-course studies in 3T3-L1 cells revealed that inhibition of adipogenesis by arsenic occurred in the early stage of terminal adipogenic differentiation and was highly correlated with induction of C/EBP homologous protein (CHOP10), an endoplasmic reticulum (ER) stress response protein. Induction of CHOP10 by arsenic is associated with reduced DNA-binding activity of CCAAT/enhancer-binding protein β (C/EBPβ), which regulates the transcription of peroxisome proliferator-activated receptor γ and C/EBPα. CONCLUSIONS: Low-level iAs and MMA3+ trigger the ER stress response and upregulate CHOP10, which inhibits C/EBPβ transcriptional activity, and thus suppresses adipogenesis. Arsenic-induced dysfunctional adipogenesis may be associated with reduced capacity of WAT to store lipids and insulin resistance.
Environmental Health Perspectives 12/2012; · 7.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The nuclear factor-E2-related factor 2 (NRF2) serves as a master regulator in cellular defense against oxidative stress and chemical detoxification. However, persistent activation of NRF2 resulting from mutations in NRF2 and/or downregulation of or mutations in its suppressor, Kelch-like ECH-associated protein 1 (KEAP1), is associated with tumorigenicity and chemoresistance of non-small-cell lung carcinomas (NSCLCs). Thus, inhibiting the NRF2-mediated adaptive antioxidant response is widely considered a promising strategy to prevent tumor growth and reverse chemoresistance in NSCLCs. Unexpectedly, stable knockdown of KEAP1 by lentiviral shRNA sensitized three independent NSCLC cell lines (A549, HTB-178, and HTB-182) to multiple chemotherapeutic agents, including arsenic trioxide (As(2)O(3)), etoposide, and doxorubicin, despite moderately increased NRF2 levels. In lung adenocarcinoma epithelial A549 cells, silencing of KEAP1 augmented the expression of peroxisome proliferator-activated receptor γ (PPARγ) and genes associated with cell differentiation, including E-cadherin and gelsolin. In addition, KEAP1-knockdown A549 cells displayed attenuated expression of the proto-oncogene cyclin D1 and markers for cancer stem cells (CSCs) and reduced nonadherent sphere formation. Moreover, deficiency of KEAP1 led to elevated induction of PPARγ in response to As(2)O(3). Pretreatment of A549 cells with PPARγ agonists activated PPARγ and augmented the cytotoxicity of As(2)O(3). A mathematical model was formulated to advance a hypothesis that differential regulation of PPARγ and detoxification enzymes by KEAP1 and NRF2 may underpin the observed landscape changes in chemosensitivity. Collectively, suppression of KEAP1 expression in human NSCLC cells resulted in sensitization to chemotherapeutic agents, which may be attributed to activation of PPARγ and subsequent alterations in cell differentiation and CSC abundance.
Free Radical Biology and Medicine 06/2012; 53(4):758-68. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nuclear factor E2-related factors (NRFs), including NRF2 and NRF1, play critical roles in mediating the cellular adaptive response to oxidative stress. Human exposure to inorganic arsenic, a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer.
We investigated the cross-regulations among NRF2, NRF1, and KEAP1, a cullin-3-adapter protein that allows NRF2 to be ubiquinated and degraded by the proteasome complex, in arsenic-induced antioxidant responses.
In human keratinocyte HaCaT cells, selective knockdown (KD) of NRF2 by lentiviral short hairpin RNAs (shRNAs) significantly reduced the expression of many antioxidant enzymes and sensitized the cells to acute cytotoxicity of inorganic arsenite (iAs(3+)). In contrast, silencing KEAP1 led to a dramatic resistance to iAs(3+)-induced apoptosis. Pretreatment of HaCaT cells with NRF2 activators, such as tert-butylhydroquinone, protects the cells against acute iAs(3+) toxicity in an NRF2-dependent fashion. Consistent with the negative regulatory role of KEAP1 in NRF2 activation, KEAP1-KD cells exhibited enhanced transcriptional activity of NRF2 under nonstressed conditions. However, deficiency in KEAP1 did not facilitate induction of NRF2-target genes by iAs(3+). In addition, NRF2 silencing reduced the expression of KEAP1 at transcription and protein levels but increased the protein expression of NRF1 under the iAs(3+)-exposed condition. In contrast, silencing KEAP1 augmented protein accumulation of NRF2 under basal and iAs3+-exposed conditions, whereas the iAs(3+)-induced protein accumulation of NRF1 was attenuated in KEAP1-KD cells.
Our studies suggest that NRF2, KEAP1, and NRF1 are coordinately involved in the regulation of the cellular adaptive response to iAs(3+)-induced oxidative stress.
Environmental Health Perspectives 04/2012; 120(4):583-9. · 7.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We provide an overview of computational systems biology approaches as applied to the study of chemical- and drug-induced toxicity. The concept of "toxicity pathways" is described in the context of the 2007 US National Academies of Science report, "Toxicity testing in the 21st Century: A Vision and A Strategy." Pathway mapping and modeling based on network biology concepts are a key component of the vision laid out in this report for a more biologically based analysis of dose-response behavior and the safety of chemicals and drugs. We focus on toxicity of the liver (hepatotoxicity) - a complex phenotypic response with contributions from a number of different cell types and biological processes. We describe three case studies of complementary multi-scale computational modeling approaches to understand perturbation of toxicity pathways in the human liver as a result of exposure to environmental contaminants and specific drugs. One approach involves development of a spatial, multicellular "virtual tissue" model of the liver lobule that combines molecular circuits in individual hepatocytes with cell-cell interactions and blood-mediated transport of toxicants through hepatic sinusoids, to enable quantitative, mechanistic prediction of hepatic dose-response for activation of the aryl hydrocarbon receptor toxicity pathway. Simultaneously, methods are being developing to extract quantitative maps of intracellular signaling and transcriptional regulatory networks perturbed by environmental contaminants, using a combination of gene expression and genome-wide protein-DNA interaction data. A predictive physiological model (DILIsym™) to understand drug-induced liver injury (DILI), the most common adverse event leading to termination of clinical development programs and regulatory actions on drugs, is also described. The model initially focuses on reactive metabolite-induced DILI in response to administration of acetaminophen, and spans multiple biological scales.
[Show abstract][Hide abstract] ABSTRACT: Nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper transcription factor that is involved in the cellular adaptive response to oxidative stress. Our previous study reported that targeted disruption of the Nrf2 gene in mice decreases adipose tissue mass and protects against obesity induced by a high-fat diet. Deficiency of Nrf2 in preadipocytes and mouse embryonic fibroblasts led to impaired adipogenesis. Consistent with these findings, the current study found that lack of Nrf2 in primary cultured mouse preadipocytes and 3T3-L1 cells hampered adipogenic differentiation induced by hormonal cocktails. Stable knockdown of Nrf2 in 3T3-L1 cells blocked the enhanced adipogenesis caused by deficiency of kelch-like ECH-associated protein 1 (Keap1), a Cul3-adapter protein that allows for Nrf2 to be ubiquinated and degraded by the 26S protesome complex. In addition, increased production of reactive oxygen species (ROS) and activation of Nrf2 occurred at the very early stage upon adipogenic hormonal challenge in 3T3-L1 cells, followed by an immediate induction of CCAAT/enhancer-binding protein β (C/EBPβ). Knockdown of Nrf2 led to reduced expression of C/EBPβ induced by adipogenic hormonal cocktails, chemical Nrf2 activators or Keap1 silencing. Cebpβ promoter-driven reporter assays and chromatin immunoprecipitation suggested that Nrf2 associates with a consensus antioxidant response element (ARE) binding site in the promoter of the Cebpβ gene during adipogenesis and upregulates its expression. These findings demonstrate a novel role of Nrf2 beyond xenobiotic detoxification and antioxidant response, and suggest that Nrf2 is one of the transcription factors that control the early events of adipogenesis by regulating expression of Cebpβ.
Free Radical Biology and Medicine 10/2011; 52(2):462-72. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 μM) inorganic arsenite (iAs³(+)) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs³(+) exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs³(+) exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4 expression may also be involved in arsenic-induced insulin resistance in adipocytes. Taken together our studies suggest that prolonged low-level iAs³(+) exposure activates the cellular adaptive oxidative stress response, which impairs insulin-stimulated ROS signaling that is involved in ISGU, and thus causes insulin resistance in adipocytes.
Biochemical and Biophysical Research Communications 03/2011; 407(2):360-5. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The image of the "epigenetic landscape", with a series of branching valleys and ridges depicting stable cellular states and the barriers between those states, has been a popular visual metaphor for cell lineage specification - especially in light of the recent discovery that terminally differentiated adult cells can be reprogrammed into pluripotent stem cells or into alternative cell lineages. However the question of whether the epigenetic landscape can be mapped out quantitatively to provide a predictive model of cellular differentiation remains largely unanswered.
Here we derive a simple deterministic path-integral quasi-potential, based on the kinetic parameters of a gene network regulating cell fate, and show that this quantity is minimized along a temporal trajectory in the state space of the gene network, thus providing a marker of directionality for cell differentiation processes. We then use the derived quasi-potential as a measure of "elevation" to quantitatively map the epigenetic landscape, on which trajectories flow "downhill" from any location. Stochastic simulations confirm that the elevation of this computed landscape correlates to the likelihood of occurrence of particular cell fates, with well-populated low-lying "valleys" representing stable cellular states and higher "ridges" acting as barriers to transitions between the stable states.
This quantitative map of the epigenetic landscape underlying cell fate choice provides mechanistic insights into the "forces" that direct cellular differentiation in the context of physiological development, as well as during artificially induced cell lineage reprogramming. Our generalized approach to mapping the landscape is applicable to non-gradient gene regulatory systems for which an analytical potential function cannot be derived, and also to high-dimensional gene networks. Rigorous quantification of the gene regulatory circuits that govern cell lineage choice and subsequent mapping of the epigenetic landscape can potentially help identify optimal routes of cell fate reprogramming.
BMC Systems Biology 01/2011; 5:85. · 2.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gene networks that regulate the choice of cell fate in living organisms contain a set of ubiquitous design motifs. We discuss the role of one such motif, the double-negative feedback loop, in producing discrete and stable steady states during the development of mammalian cell lineages. Several examples of this motif are described from the literature. Thereafter, we use an ordinary differential equation-based computational model of the double-negative feedback loop motif to illustrate the mechanism by which it generates stable cellular states. Stochastic simulations show how this motif ensures robustness of cell states to stochastic fluctuations in gene expression.
Int. J. of Design Engineering. 01/2011; 4(1):41 - 57.
[Show abstract][Hide abstract] ABSTRACT: The approaches to quantitatively assessing the health risks of chemical exposure have not changed appreciably in the past 50 to 80 years, the focus remaining on high-dose studies that measure adverse outcomes in homogeneous animal populations. This expensive, low-throughput approach relies on conservative extrapolations to relate animal studies to much lower-dose human exposures and is of questionable relevance to predicting risks to humans at their typical low exposures. It makes little use of a mechanistic understanding of the mode of action by which chemicals perturb biological processes in human cells and tissues. An alternative vision, proposed by the U.S. National Research Council (NRC) report Toxicity Testing in the 21(st) Century: A Vision and a Strategy, called for moving away from traditional high-dose animal studies to an approach based on perturbation of cellular responses using well-designed in vitro assays. Central to this vision are (a) "toxicity pathways" (the innate cellular pathways that may be perturbed by chemicals) and (b) the determination of chemical concentration ranges where those perturbations are likely to be excessive, thereby leading to adverse health effects if present for a prolonged duration in an intact organism. In this paper we briefly review the original NRC report and responses to that report over the past 3 years, and discuss how the change in testing might be achieved in the U.S. and in the European Union (EU). EU initiatives in developing alternatives to animal testing of cosmetic ingredients have run very much in parallel with the NRC report. Moving from current practice to the NRC vision would require using prototype toxicity pathways to develop case studies showing the new vision in action. In this vein, we also discuss how the proposed strategy for toxicity testing might be applied to the toxicity pathways associated with DNA damage and repair.
PLoS ONE 01/2011; 6(6):e20887. · 3.53 Impact Factor