Systems biology approach highlights mechanistic differences between Crohn's disease and ulcerative colitis.

The molecular mechanisms of IBD have been the subject of intensive exploration. We, therefore, assembled the available information into a suite of causal biological network models, which offer comprehensive visualization of the processes underlying IBD. Scientific text was curated by using Biological Expression Language (BEL) and compiled with OpenBEL 3.0.0. Network properties were analysed by Cytoscape. Network perturbation amplitudes were computed to score the network models with transcriptomic data from public data repositories. The IBD network model suite consists of three independent models that represent signalling pathways that contribute to IBD. In the "intestinal permeability" model, programmed cell death factors were downregulated in CD and upregulated in UC. In the "inflammation" model, PPARG, IL6, and IFN-associated pathways were prominent regulatory factors in both diseases. In the "wound healing" model, factors promoting wound healing were upregulated in CD and downregulated in UC. Scoring of publicly available transcriptomic datasets onto these network models demonstrated that the IBD models capture the perturbation in each dataset accurately. The IBD network model suite can provide better mechanistic insights of the transcriptional changes in IBD and constitutes a valuable tool in personalized medicine to further understand individual drug responses in IBD.

Impact of whole-body versus nose-only inhalation exposure systems on systemic, respiratory, and cardiovascular endpoints in a 2-month cigarette smoke exposure study in the ApoE-/- mouse model.

Cigarette smoking is one major modifiable risk factor in the development and progression of chronic obstructive pulmonary disease and cardiovascular disease. To characterize and compare cigarette smoke (CS)-induced disease endpoints after exposure in either whole-body (WB) or nose-only (NO) exposure systems, we exposed apolipoprotein E-deficient mice to filtered air (Sham) or to the same total particulate matter (TPM) concentration of mainstream smoke from 3R4F reference cigarettes in NO or WB exposure chambers (EC) for 2 months. At matching TPM concentrations, we observed similar concentrations of carbon monoxide, acetaldehyde, and acrolein, but higher concentrations of nicotine and formaldehyde in NOEC than in WBEC. In both exposure systems, CS exposure led to the expected adaptive changes in nasal epithelia, altered lung function, lung inflammation, and pronounced changes in the nasal epithelial transcriptome and lung proteome. Exposure in the NOEC caused generally more severe histopathological changes in the nasal epithelia and a higher stress response as indicated by body weight decrease and lower blood lymphocyte counts compared with WB exposed mice. Erythropoiesis, and increases in total plasma triglyceride levels and atherosclerotic plaque area were observed only in CS-exposed mice in the WBEC group but not in the NOEC group. Although the composition of CS in the breathing zone is not completely comparable in the two exposure systems, the CS-induced respiratory disease endpoints were largely confirmed in both systems, with a higher magnitude of severity after NO exposure. CS-accelerated atherosclerosis and other pro-atherosclerotic factors were only significant in WBEC.

Anatabine ameliorates intestinal inflammation and reduces the production of pro-inflammatory factors in a dextran sulfate sodium mouse model of colitis.

BACKGROUND: Inflammatory bowel disease (IBD) is the collective term for chronic immune-mediated diseases of unknown, multifactorial etiology, arising from the interplay between genetic and environmental factors and including two main disease manifestations: ulcerative colitis (UC) and Crohn's disease. In the last few decades, naturally occurring alkaloids have gained interest because of their substantial anti-inflammatory effects in several animal models of disease. Studies on mouse models of IBD have demonstrated the anti-inflammatory action of the main tobacco alkaloid, nicotine. In addition, anatabine, a minor tobacco alkaloid also present in peppers, tomato, and eggplant presents anti-inflammatory properties in vivo and in vitro. In this study, we aimed to evaluate the anti-inflammatory properties of nicotine and anatabine in a dextran sulfate sodium (DSS) mouse model of UC. RESULTS: Oral administration of anatabine, but not nicotine, reduced the clinical symptoms of DSS-induced colitis. The result of gene expression analysis suggested that anatabine had a restorative effect on global DSS-induced gene expression profiles, while nicotine only had limited effects. Accordingly, MAP findings revealed that anatabine reduced the colonic abundance of DSS-associated cytokines and increased IL-10 abundance. CONCLUSIONS: Our results support the amelioration of inflammatory effects by anatabine in the DSS mouse model of UC, and suggest that anatabine constitutes a promising therapeutic agent for IBD treatment.

Evaluation of toxicity of aerosols from flavored e-liquids in Sprague-Dawley rats in a 90-day OECD inhalation study, complemented by transcriptomics analysis.

The use of flavoring substances is an important element in the development of reduced-risk products for adult smokers to increase product acceptance and encourage switching from cigarettes. In a first step towards characterizing the sub-chronic inhalation toxicity of neat flavoring substances, a study was conducted using a mixture of the substances in a base solution of e-liquid, where the standard toxicological endpoints of the nebulized aerosols were supplemented with transcriptomics analysis. The flavor mixture was produced by grouping 178 flavors into 26 distinct chemical groups based on structural similarities and potential metabolic and biological effects. Flavoring substances predicted to show the highest toxicological effect from each group were selected as the flavor group representatives (FGR). Following Organization for Economic Cooperation and Development Testing Guideline 413, rats were exposed to three concentrations of the FGR mixture in an e-liquid composed of nicotine (23 µg/L), propylene glycol (1520 µg/L), and vegetable glycerin (1890 µg/L), while non-flavored and no-nicotine mixtures were included as references to identify potential additive or synergistic effects between nicotine and the flavoring substances. The results indicated that the inhalation of an e-liquid containing the mixture of FGRs caused very minimal local and systemic toxic effects. In particular, there were no remarkable clinical (in-life) observations in flavored e-liquid-exposed rats. The biological effects related to exposure to the mixture of neat FGRs were limited and mainly nicotine-mediated, including changes in hematological and blood chemistry parameters and organ weight. These results indicate no significant additive biological changes following inhalation exposure to the nebulized FGR mixture above the nicotine effects measured in this sub-chronic inhalation study. In a subsequent study, e-liquids with FGR mixtures will be aerosolized by thermal treatment and assessed for toxicity.

A 90-day OECD TG 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of the aerosol from the carbon heated tobacco product version 1.2 (CHTP1.2) compared with cigarette smoke. I. Inhalation exposure, clinical pathology and histopathology.

Within the framework of a systems toxicology approach, the inhalation toxicity of aerosol from a novel tobacco-heating potentially modified risk tobacco product (MRTP), the carbon-heated tobacco product (CHTP) 1.2, was characterized and compared with that of mainstream smoke (CS) from the 3R4F reference cigarette in a 90-day nose-only rat inhalation study in general accordance with OECD TG 413. CHTP1.2 is a heat-not-burn product using a carbon heat source to produce an aerosol that contains nicotine and tobacco flavor. At equal or twice the nicotine concentration in the test atmospheres, inhalation of CHTP1.2 aerosol led to a significantly lower exposure to harmful constituents and induced less respiratory tract irritation, systemic, and pathological effects compared with CS. Nasal epithelial changes were less pronounced in the CHTP1.2- than in the CS-exposed groups and reverted in the nicotine concentration-matched group after a recovery period. Lung inflammation was minimal in the CHTP1.2-treated groups compared with the moderate extent seen in the 3R4F groups. Many other toxicological endpoints evaluated did not show CHTP1.2 aerosol exposure-related effects, and no effects not seen for 3R4F were observed. These observations were consistent with findings from previous studies in which rats were exposed to MRTP aerosols containing similar nicotine concentrations.

A 90-day OECD TG 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of the aerosol from the carbon heated tobacco product version 1.2 (CHTP1.2) compared with cigarette smoke. II. Systems toxicology assessment.

Modified risk tobacco products (MRTPs) have the potential to reduce smoking-related health risks. The Carbon Heated Tobacco Product 1.2 (CHTP1.2) is a potential MRTP that uses a pressed carbon heat source to generate an aerosol by heating tobacco. Here, we report the results from the systems toxicology arm of a 90-day rat inhalation study (OECD test guideline 413) to assess the effects of CHTP1.2 aerosol compared with cigarette smoke (CS). Transcriptomics, proteomics, and lipidomics analyses complemented the standard endpoints. In the respiratory nasal epithelium, CS induced an adaptive tissue and inflammatory response, which was much weaker after CHTP1.2 aerosol exposure, mostly limited to the highest CHTP1.2 concentration (at twice the 3R4F CS concentration: 50 vs. 23 µg nicotine/L), in female rats. In the lungs, the effects of CS exposure included inflammatory and cellular stress responses, which were absent or much lower after CHTP1.2 aerosol exposure. Outside of the respiratory tract, CS and CHTP1.2 aerosol induced effects that were previously associated with exposure to any nicotine-containing aerosol, e.g., lower lipid concentrations in serum. Overall, this systems toxicology analysis complements and confirms the results from classical toxicological endpoints and further suggests potentially reduced respiratory health risks of CHTP1.2.

Biological changes in C57BL/6 mice following 3 weeks of inhalation exposure to cigarette smoke or e-vapor aerosols.

We compared early biological changes in mice after inhalation exposures to cigarette smoke or e-vapor aerosols (MarkTen® cartridge with Carrier, Test-1, or Test-2 formulations; 4% nicotine). Female C57BL/6 mice were exposed to 3R4F cigarette smoke or e-vapor aerosols by nose-only inhalation for up to 4 hours/day, 5 days/week, for 3 weeks. The 3R4F and e-vapor exposures were set to match the target nose port aerosol nicotine concentration (∼41 µg/L). Only the 3R4F group showed postexposure clinical signs such as tremors and lethargy. At necropsy, the 3R4F group had significant increases in lung weight and changes in bronchoalveolar lavage parameters, as well as microscopic findings in the respiratory tract. The e-vapor groups had minimal microscopic changes, including squamous metaplasia in laryngeal epiglottis, and histiocytic infiltrates in the lung (Test-2 group only). The 3R4F group had a higher incidence and severity of microscopic findings compared to any e-vapor group. Transcriptomic analysis also showed that the 3R4F group had the highest number of differentially expressed genes compared to Sham Control. Among e-vapor groups, Test-2 group had more differentially expressed genes but the magnitude of gene expression-based network perturbations in all e-vapor groups was ∼94% less than the 3R4F group. On proteome analysis in the lung, differentially regulated proteins were detected in the 3R4F group only. In conclusion, 3-weeks of 3R4F exposure induced molecular and microscopic changes associated with smoking-related diseases in the respiratory tract, while e-vapor exposures showed substantially reduced biological activities.

Toxicity of the main electronic cigarette components, propylene glycol, glycerin, and nicotine, in Sprague-Dawley rats in a 90-day OECD inhalation study complemented by molecular endpoints.

While the toxicity of the main constituents of electronic cigarette (ECIG) liquids, nicotine, propylene glycol (PG), and vegetable glycerin (VG), has been assessed individually in separate studies, limited data on the inhalation toxicity of them is available when in mixtures. In this 90-day subchronic inhalation study, Sprague-Dawley rats were nose-only exposed to filtered air, nebulized vehicle (saline), or three concentrations of PG/VG mixtures, with and without nicotine. Standard toxicological endpoints were complemented by molecular analyses using transcriptomics, proteomics, and lipidomics. Compared with vehicle exposure, the PG/VG aerosols showed only very limited biological effects with no signs of toxicity. Addition of nicotine to the PG/VG aerosols resulted in effects in line with nicotine effects observed in previous studies, including up-regulation of xenobiotic enzymes (Cyp1a1/Fmo3) in the lung and metabolic effects, such as reduced serum lipid concentrations and expression changes of hepatic metabolic enzymes. No toxicologically relevant effects of PG/VG aerosols (up to 1.520  mg PG/L + 1.890 mg VG/L) were observed, and no adverse effects for PG/VG/nicotine were observed up to 438/544/6.6 mg/kg/day. This study demonstrates how complementary systems toxicology analyses can reveal, even in the absence of observable adverse effects, subtoxic and adaptive responses to pharmacologically active compounds such as nicotine.

Evaluation of the Tobacco Heating System 2.2. Part 4: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects compared with cigarette smoke.

The objective of the study was to characterize the toxicity from sub-chronic inhalation of test atmospheres from the candidate modified risk tobacco product (MRTP), Tobacco Heating System version 2.2 (THS2.2), and to compare it with that of the 3R4F reference cigarette. A 90-day nose-only inhalation study on Sprague-Dawley rats was performed, combining classical and systems toxicology approaches. Reduction in respiratory minute volume, degree of lung inflammation, and histopathological findings in the respiratory tract organs were significantly less pronounced in THS2.2-exposed groups compared with 3R4F-exposed groups. Transcriptomics data obtained from nasal epithelium and lung parenchyma showed concentration-dependent differential gene expression following 3R4F exposure that was less pronounced in the THS2.2-exposed groups. Molecular network analysis showed that inflammatory processes were the most affected by 3R4F, while the extent of THS2.2 impact was much lower. Most other toxicological endpoints evaluated did not show exposure-related effects. Where findings were observed, the effects were similar in 3R4F- and THS2.2-exposed animals. In summary, toxicological changes observed in the respiratory tract organs of THS2.2 aerosol-exposed rats were much less pronounced than in 3R4F-exposed rats while other toxicological endpoints either showed no exposure-related effects or were comparable to what was observed in the 3R4F-exposed rats.

Evaluation of the Tobacco Heating System 2.2 (THS2.2). Part 5: microRNA expression from a 90-day rat inhalation study indicates that exposure to THS2.2 aerosol causes reduced effects on lung tissue compared with cigarette smoke.

Modified-risk tobacco products (MRTP) are designed to reduce the individual risk of tobacco-related disease as well as population harm compared to smoking cigarettes. Experimental proof of their benefit needs to be provided at multiple levels in research fields. Here, we examined microRNA (miRNA) levels in the lungs of rats exposed to a candidate modified-risk tobacco product, the Tobacco Heating System 2.2 (THS2.2) in a 90-day OECD TG-413 inhalation study. Our aim was to assess the miRNA response to THS2.2 aerosol compared with the response to combustible cigarettes (CC) smoke from the reference cigarette 3R4F. CC smoke exposure, but not THS2.2 aerosol exposure, caused global miRNA downregulation, which may be explained by the interference of CC smoke constituents with the miRNA processing machinery. Upregulation of specific miRNA species, such as miR-146a/b and miR-182, indicated that they are causal elements in the inflammatory response in CC-exposed lungs, but they were reduced after THS2.2 aerosol exposure. Transforming transcriptomic data into protein activity based on corresponding downstream gene expression, we identified potential mechanisms for miR-146a/b and miR-182 that were activated by CC smoke but not by THS2.2 aerosol and possibly involved in the regulation of those miRNAs. The inclusion of miRNA profiling in systems toxicology approaches increases the mechanistic understanding of the complex exposure responses.

Evaluation of the Tobacco Heating System 2.2. Part 7: Systems toxicological assessment of a mentholated version revealed reduced cellular and molecular exposure effects compared with mentholated and non-mentholated cigarette smoke.

Modified risk tobacco products (MRTPs) are being developed with the aim of reducing smoking-related health risks. The Tobacco Heating System 2.2 (THS2.2) is a candidate MRTP that uses the heat-not-burn principle. Here, systems toxicology approaches were engaged to assess the respiratory effects of mentholated THS2.2 (THS2.2M) in a 90-day rat inhalation study (OECD test guideline 413). The standard endpoints were complemented by transcriptomics and quantitative proteomics analyses of respiratory nasal epithelium and lung tissue and by lipidomics analysis of lung tissue. The adaptive response of the respiratory nasal epithelium to conventional cigarette smoke (CS) included squamous cell metaplasia and an inflammatory response, with high correspondence between the molecular and histopathological results. In contrast to CS exposure, the adaptive tissue and molecular changes to THS2.2M aerosol exposure were much weaker and were limited mostly to the highest THS2.2M concentration in female rats. In the lung, CS exposure induced an inflammatory response, triggered cellular stress responses, and affected sphingolipid metabolism. These responses were not observed or were much lower after THS2.2M aerosol exposure. Overall, this system toxicology analysis complements and reconfirms the results from classical toxicological endpoints and further suggests potentially reduced health risks of THS2.2M.

Evaluation of the Tobacco Heating System 2.2. Part 6: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of a mentholated version compared with mentholated and non-mentholated cigarette smoke.

The toxicity of a mentholated version of the Tobacco Heating System (THS2.2M), a candidate modified risk tobacco product (MRTP), was characterized in a 90-day OECD inhalation study. Differential gene and protein expression analysis of nasal epithelium and lung tissue was also performed to record exposure effects at the molecular level. Rats were exposed to filtered air (sham), to THS2.2M (at 15, 23 and 50 µg nicotine/l), to two mentholated reference cigarettes (MRC) (at 23 µg nicotine/l), or to the 3R4F reference cigarette (at 23 µg nicotine/l). MRCs were designed to meet 3R4F specifications. Test atmosphere analyses demonstrated that aldehydes were reduced by 75%-90% and carbon monoxide by 98% in THS2.2M aerosol compared with MRC smoke; aerosol uptake was confirmed by carboxyhemoglobin and menthol concentrations in blood, and by the quantities of urinary nicotine metabolites. Systemic toxicity and alterations in the respiratory tract were significantly lower in THS2.2M-exposed rats compared with MRC and 3R4F. Pulmonary inflammation and the magnitude of the changes in gene and protein expression were also dramatically lower after THS2.2M exposure compared with MRCs and 3R4F. No menthol-related effects were observed after MRC mainstream smoke-exposure compared with 3R4F.

Toxicity of aerosols of nicotine and pyruvic acid (separate and combined) in Sprague-Dawley rats in a 28-day OECD 412 inhalation study and assessment of systems toxicology.

Toxicity of nebulized nicotine (Nic) and nicotine/pyruvic acid mixtures (Nic/Pyr) was characterized in a 28-day Organization for Economic Co-operation and Development 412 inhalation study with additional transcriptomic and lipidomic analyses. Sprague-Dawley rats were nose-only exposed, 6 h/day, 5 days/week to filtered air, saline, nicotine (50 µg/l), sodium pyruvate (NaPyr, 33.9 µg/l) or equimolar Nic/Pyr mixtures (18, 25 and 50 µg nicotine/l). Saline and NaPyr caused no health effects, but rats exposed to nicotine-containing aerosols had decreased body weight gains and concentration-dependent increases in liver weight. Blood neutrophil counts were increased and lymphocyte counts decreased in rats exposed to nicotine; activities of alkaline phosphatase and alanine aminotransferase were increased, and levels of cholesterol and glucose decreased. The only histopathologic finding in non-respiratory tract organs was increased liver vacuolation and glycogen content. Respiratory tract findings upon nicotine exposure (but also some phosphate-buffered saline aerosol effects) were observed only in the larynx and were limited to adaptive changes. Gene expression changes in the lung and liver were very weak. Nic and Nic/Pyr caused few significant changes (including Cyp1a1 gene upregulation). Changes were predominantly related to energy metabolism and fatty acid metabolism but did not indicate an obvious toxicity-related response. Nicotine exposure lowered plasma lipids, including cholesteryl ester (CE) and free cholesterol and, in the liver, phospholipids and sphingolipids. Nic, NaPyr and Nic/Pyr decreased hepatic triacylglycerol and CE. In the lung, Nic and Nic/Pyr increased CE levels. These data suggest that only minor biologic effects related to inhalation of Nic or Nic/Pyr aerosols were observed in this 28-day study.

Transcriptional profiling and targeted proteomics reveals common molecular changes associated with cigarette smoke-induced lung emphysema development in five susceptible mouse strains.

BACKGROUND: Mouse models are useful for studying cigarette smoke (CS)-induced chronic pulmonary pathologies such as lung emphysema. To enhance translation of large-scale omics data from mechanistic studies into pathophysiological changes, we have developed computational tools based on reverse causal reasoning (RCR). OBJECTIVE: In the present study we applied a systems biology approach leveraging RCR to identify molecular mechanistic explanations of pathophysiological changes associated with CS-induced lung emphysema in susceptible mice. METHODS: The lung transcriptomes of five mouse models (C57BL/6, ApoE (-/-) , A/J, CD1, and Nrf2 (-/-) ) were analyzed following 5-7 months of CS exposure. RESULTS: We predicted 39 molecular changes mostly related to inflammatory processes including known key emphysema drivers such as NF-κB and TLR4 signaling, and increased levels of TNF-α, CSF2, and several interleukins. More importantly, RCR predicted potential molecular mechanisms that are less well-established, including increased transcriptional activity of PU.1, STAT1, C/EBP, FOXM1, YY1, and N-COR, and reduced protein abundance of ITGB6 and CFTR. We corroborated several predictions using targeted proteomic approaches, demonstrating increased abundance of CSF2, C/EBPα, C/EBPß, PU.1, BRCA1, and STAT1. CONCLUSION: These systems biology-derived candidate mechanisms common to susceptible mouse models may enhance understanding of CS-induced molecular processes underlying emphysema development in mice and their relevancy for human chronic obstructive pulmonary disease.

Assessment of a novel multi-array normalization method based on spike-in control probes suitable for microRNA datasets with global decreases in expression.

BACKGROUND: High-quality expression data are required to investigate the biological effects of microRNAs (miRNAs). The goal of this study was, first, to assess the quality of miRNA expression data based on microarray technologies and, second, to consolidate it by applying a novel normalization method. Indeed, because of significant differences in platform designs, miRNA raw data cannot be normalized blindly with standard methods developed for gene expression. This fundamental observation motivated the development of a novel multi-array normalization method based on controllable assumptions, which uses the spike-in control probes to adjust the measured intensities across arrays. RESULTS: Raw expression data were obtained with the Exiqon dual-channel miRCURY LNA™ platform in the "common reference design" and processed as "pseudo-single-channel". They were used to apply several quality metrics based on the coefficient of variation and to test the novel spike-in controls based normalization method. Most of the considerations presented here could be applied to raw data obtained with other platforms. To assess the normalization method, it was compared with 13 other available approaches from both data quality and biological outcome perspectives. The results showed that the novel multi-array normalization method reduced the data variability in the most consistent way. Further, the reliability of the obtained differential expression values was confirmed based on a quantitative reverse transcription-polymerase chain reaction experiment performed for a subset of miRNAs. The results reported here support the applicability of the novel normalization method, in particular to datasets that display global decreases in miRNA expression similarly to the cigarette smoke-exposed mouse lung dataset considered in this study. CONCLUSIONS: Quality metrics to assess between-array variability were used to confirm that the novel spike-in controls based normalization method provided high-quality miRNA expression data suitable for reliable downstream analysis. The multi-array miRNA raw data normalization method was implemented in an R software package called ExiMiR and deposited in the Bioconductor repository.

Discovery of Emphysema Relevant Molecular Networks from an A/J Mouse Inhalation Study Using Reverse Engineering and Forward Simulation (REFS™).

Chronic obstructive pulmonary disease (COPD) is a respiratory disorder caused by extended exposure of the airways to noxious stimuli, principally cigarette smoke (CS). The mechanisms through which COPD develops are not fully understood, though it is believed that the disease process includes a genetic component, as not all smokers develop COPD. To investigate the mechanisms that lead to the development of COPD/emphysema, we measured whole genome gene expression and several COPD-relevant biological endpoints in mouse lung tissue after exposure to two CS doses for various lengths of time. A novel and powerful method, Reverse Engineering and Forward Simulation (REFS™), was employed to identify key molecular drivers by integrating the gene expression data and four measured COPD-relevant endpoints (matrix metalloproteinase (MMP) activity, MMP-9 levels, tissue inhibitor of metalloproteinase-1 levels and lung weight). An ensemble of molecular networks was generated using REFS™, and simulations showed that it could successfully recover the measured experimental data for gene expression and COPD-relevant endpoints. The ensemble of networks was then employed to simulate thousands of in silico gene knockdown experiments. Thirty-three molecular key drivers for the above four COPD-relevant endpoints were therefore identified, with the majority shown to be enriched in inflammation and COPD.

A 28-day rat inhalation study with an integrated molecular toxicology endpoint demonstrates reduced exposure effects for a prototypic modified risk tobacco product compared with conventional cigarettes.

Towards a systems toxicology-based risk assessment, we investigated molecular perturbations accompanying histopathological changes in a 28-day rat inhalation study combining transcriptomics with classical histopathology. We demonstrated reduced biological activity of a prototypic modified risk tobacco product (pMRTP) compared with the reference research cigarette 3R4F. Rats were exposed to filtered air or to three concentrations of mainstream smoke (MS) from 3R4F, or to a high concentration of MS from a pMRTP. Histopathology revealed concentration-dependent changes in response to 3R4F that were irritative stress-related in nasal and bronchial epithelium, and inflammation-related in the lung parenchyma. For pMRTP, significant changes were seen in the nasal epithelium only. Transcriptomics data were obtained from nasal and bronchial epithelium and lung parenchyma. Concentration-dependent gene expression changes were observed following 3R4F exposure, with much smaller changes for pMRTP. A computational-modeling approach based on causal models of tissue-specific biological networks identified cell stress, inflammation, proliferation, and senescence as the most perturbed molecular mechanisms. These perturbations correlated with histopathological observations. Only weak perturbations were observed for pMRTP. In conclusion, a correlative evaluation of classical histopathology together with gene expression-based computational network models may facilitate a systems toxicology-based risk assessment, as shown for a pMRTP.

Physical and functional interaction of the HECT ubiquitin-protein ligases E6AP and HERC2.

Deregulation of the ubiquitin-protein ligase E6AP contributes to the development of the Angelman syndrome and to cervical carcinogenesis suggesting that the activity of E6AP needs to be under tight control. However, how E6AP activity is regulated at the post-translational level under non-pathologic conditions is poorly understood. In this study, we report that the giant protein HERC2, which is like E6AP a member of the HECT family of ubiquitin-protein ligases, binds to E6AP. The interaction is mediated by the RCC1-like domain 2 of HERC2 and a region spanning amino acid residues 150-200 of E6AP. Furthermore, we provide evidence that HERC2 stimulates the ubiquitin-protein ligase activity of E6AP in vitro and within cells and that this stimulatory effect does not depend on the ubiquitin-protein ligase activity of HERC2. Thus, the data obtained indicate that HERC2 acts as a regulator of E6AP.

Regulation of p27 degradation and S-phase progression by Ro52 RING finger protein.

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27 provides a powerful route for enforcing normal progression through the mammalian cell cycle. According to a current model, the ubiquitination of p27 during S-phase progression is mediated by SCF(Skp2) E3 ligase that captures Thr187-phosphorylated p27 by means of the F-box protein Skp2, which in turn couples the bound substrate via Skp1 to a catalytic core complex composed of Cul1 and the Rbx/Roc RING finger protein. Here we identify Skp2 as a component of an Skp1-cullin-F-box complex that is based on a Cul1-Ro52 RING finger B-box coiled-coil motif family protein catalytic core. Ro52-containing complexes display E3 ligase activity and promote the ubiquitination of Thr187-phosphorylated p27 in a RING-dependent manner in vitro. The knockdown of Ro52 expression in human cells with small interfering RNAs causes the accumulation of p27 and the failure of cells to enter S phase. Importantly, these effects are abrogated by the simultaneous removal of p27. Taken together, these data suggest a key role for Ro52 RING finger protein in the regulation of p27 degradation and S-phase progression in mammalian cells and provide evidence for the existence of a Cul1-based catalytic core that utilizes Ro52 RING protein to promote ubiquitination.

The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation.

The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.