Xenobiotic Metabolism Pathways Research Articles

Metabolomics Simultaneously Derives Benchmark Dose Estimates and Discovers Metabolic Biotransformations in a Rat Bioassay.

Benchmark dose (BMD) modeling estimates the dose of a chemical that causes a perturbation from baseline. Transcriptional BMDs have been shown to be relatively consistent with apical end point BMDs, opening the door to using molecular BMDs to derive human health-based guidance values for chemical exposure. Metabolomics measures the responses of small-molecule endogenous metabolites to chemical exposure, complementing transcriptomics by characterizing downstream molecular phenotypes that are more closely associated with apical end points. The aim of this study was to apply BMD modeling to in vivo metabolomics data, to compare metabolic BMDs to both transcriptional and apical end point BMDs. This builds upon our previous application of transcriptomics and BMD modeling to a 5-day rat study of triphenyl phosphate (TPhP), applying metabolomics to the same archived tissues. Specifically, liver from rats exposed to five doses of TPhP was investigated using liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance spectroscopy-based metabolomics. Following the application of BMDExpress2 software, 2903 endogenous metabolic features yielded viable dose-response models, confirming a perturbation to the liver metabolome. Metabolic BMD estimates were similarly sensitive to transcriptional BMDs, and more sensitive than both clinical chemistry and apical end point BMDs. Pathway analysis of the multiomics data sets revealed a major effect of TPhP exposure on cholesterol (and downstream) pathways, consistent with clinical chemistry measurements. Additionally, the transcriptomics data indicated that TPhP activated xenobiotic metabolism pathways, which was confirmed by using the underexploited capability of metabolomics to detect xenobiotic-related compounds. Eleven biotransformation products of TPhP were discovered, and their levels were highly correlated with multiple xenobiotic metabolism genes. This work provides a case study showing how metabolomics and transcriptomics can estimate mechanistically anchored points-of-departure. Furthermore, the study demonstrates how metabolomics can also discover biotransformation products, which could be of value within a regulatory setting, for example, as an enhancement of OECD Test Guideline 417 (toxicokinetics).

Analysis of the biocorrosion community from ancient wooden constructions at Tianluoshan (7000–6300 cal BP), Zhejiang Province, China

Ancient wooden constructions, also known as wooden cultural relics, refers to ancient wood that has been modified or crafted by human activities. To ensure its preservation, it is crucial to gain further understanding of the decomposition mechanisms affecting archaeological wood. In this study, we investigated the microbiome diversity and cellulose decomposition processes in a 6300-year-old ancient wooden construction at the Tianluoshan site (7000–6300 cal BP) in Zhejiang Province, China. High-throughput sequencing (HTS) was employed to analyze the metagenomic functions, specifically focusing on the microbial communities' cellulose-degrading pathways using bioinformatic approaches. The findings revealed that the excavation of archaeological wood significantly altered the environment, leading to an accelerated deterioration process. This degradation was primarily influenced by carbohydrate metabolism and xenobiotic biodegradation and metabolism pathways within the complex ecosystem consisting of bacteria, archaea, fungi, microfauna, plants, and algae. Proteobacteria, actinobacteria, ascomycota, and basidiomycota were identified as the main sources of bacterial cellulose-degrading enzymes. The results obtained from this evaluation will provide valuable insights for the development of targeted conservation strategies and prioritization of preservation efforts for the ancient wooden constructions found in different regions of the Tianluoshan site.Graphical

A multi-omics approach to elucidate okadaic acid-induced changes in human HepaRG hepatocarcinoma cells.

Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.

Steatotic liver disease induced by TCPOBOP-activated hepatic constitutive androstane receptor: primary and secondary gene responses with links to disease progression.

Constitutive Androstane Receptor (CAR, Nr1i3), a liver nuclear receptor and xenobiotic sensor, induces drug, steroid and lipid metabolizing enzymes, stimulates liver hypertrophy and hyperplasia, and ultimately, hepatocellular carcinogenesis. The mechanisms linking early CAR responses to later disease development are poorly understood. Here we show that exposure of CD-1 mice to TCPOBOP, a halogenated xenochemical and selective CAR agonist ligand, induces pericentral steatosis marked by hepatic accumulation of cholesterol and neutral lipid, and elevated circulating alanine aminotransferase, indicating hepatocyte damage. TCPOBOP-induced steatosis was weaker in the pericentral region but stronger in the periportal region in females compared to males. Early (1-day) TCPOBOP transcriptional responses were enriched for CAR-bound primary response genes, and for lipogenesis and xenobiotic metabolism and oxidative stress protection pathways; late (2-wk) TCPOBOP responses included many CAR binding-independent secondary response genes, with enrichment for macrophage activation, immune response and cytokine and reactive oxygen species production. Late upstream regulators specific to TCPOBOP-exposed male liver were linked to pro-inflammatory responses and hepatocellular carcinoma progression. TCPOBOP administered weekly to male mice using a high corn oil vehicle activated carbohydrate-responsive transcription factor (MLXIPL)-regulated target genes, dysregulated mitochondrial respiratory and translation regulatory pathways, and induced more advanced liver pathology. Overall, TCPOBOP exposure recapitulates histological and gene expression changes characteristic of emerging steatotic liver disease, including secondary gene responses in liver non-parenchymal cells indicative of transition to a more advanced disease state. Upstream regulators of both the early and late TCPOBOP response genes include novel biomarkers for foreign chemical-induced metabolic dysfunction-associated steatotic liver disease.

Transcriptomic and epigenetic landscape of nimorazole-enhanced radiochemotherapy in head and neck cancer

BackgroundHypoxia remains a challenge for the therapeutic management of head and neck squamous cell carcinoma (HNSCC). The combination of radiotherapy with nimorazole has shown treatment benefit in HNSCC, but the precise underlying molecular mechanisms remain unclear. PurposeTo assess and to characterize the transcriptomic/epigenetic landscape of HNSCC tumor models showing differential therapeutic response to fractionated radiochemotherapy (RCTx) combined with nimorazole. Materials/methodsBulk RNA-sequencing and DNA methylation experiments were conducted using untreated and treated HNSCC xenografts after 10 fractions of RCTx with and without nimorazole. These tumor models (FaDu, SAS, Cal33, SAT and UT-SCC-45) previously showed a heterogeneous response to RCTx with nimorazole. The prognostic impact of candidate genes was assessed using clinical and gene expression data from HNSCC patients treated with primary RCTx within the DKTK-ROG. ResultsNimorazole responder and non-responder tumor models showed no differences in hypoxia gene signatures However, non-responder models showed upregulation of metabolic pathways. From that, a subset of 15 differentially expressed genes stratified HNSCC patients into low and high-risk groups with distinct outcome. ConclusionIn the present study, we found that nimorazole non-responder models were characterized by upregulation of genes involved in Retinol metabolism and xenobiotic metabolic process pathways, which might contribute to identify mechanisms of resistance to nitroimidazole compounds and potentially expand the repertoire of therapeutic options to treat HNSCC.

Spatial transcriptomics unravels palmitoylation and zonation-dependent gene regulation by AEG-1 in mouse liver

Obesity-induced metabolic dysfunction-associated steatohepatitis (MASH) leads to hepatocellular carcinoma (HCC). Astrocyte-elevated gene-1/Metadherin (AEG-1/MTDH) plays a key role in promoting MASH and HCC. AEG-1 is palmitoylated at residue cysteine 75 (Cys75) and a knock-in mouse representing mutated Cys75 to serine (AEG-1-C75S) showed activation of MASH- and HCC-promoting gene signature when compared to wild-type littermates (AEG-1-WT). The liver consists of 3 zones, periportal, midlobular and pericentral, and zone-specific dysregulated gene expression impairs metabolic homeostasis in the liver, contributing to MASH and HCC. Here, to elucidate how palmitoylation influences AEG-1-mediated gene regulation in regards to hepatic zonation, we performed spatial transcriptomics (ST) in the livers of AEG-1-WT and AEG-1-C75S littermates. ST identified six different clusters in livers and using zone- and cell type-specific markers we attributed specific zones and cell types to specific clusters. Ingenuity Pathway Analysis (IPA) of differentially expressed genes in each cluster unraveled activation of pro-inflammatory and MASH- and HCC-promoting pathways, mainly in periportal and pericentral hepatocytes, in AEG-1-C75S liver compared to AEG-1-WT. Interestingly, in AEG-1-C75S liver, the midlobular zone exhibited widespread inhibition of xenobiotic metabolism pathways and inhibition of PXR/RXR and LXR/RXR activation, vs AEG-1-WT. In conclusion, AEG-1-C75S mutant exhibited zone-specific differential gene expression, which might contribute to metabolic dysfunction and dysregulated drug metabolism leading to MASH and HCC.

Emissions from plastic incineration induce inflammation, oxidative stress, and impaired bioenergetics in primary human respiratory epithelial cells.

Inhalation exposure to plastic incineration emissions (PIEs) is a problem of increasing human relevance, as plastic production and waste creation have drastically increased since mainstream integration during the 20th century. We investigated the effects of PIEs on human nasal epithelial cells (HNECs) to understand if such exposures cause damage and dysfunction to respiratory epithelia. Primary HNECs from male and female donors were cultured at air-liquid interface (ALI), and 16HBE cells were cultured on coverslips. Smoke condensates were generated from incineration of plastic at flaming (640°C) and smoldering (500°C) temperatures, and cells were subsequently exposed to these materials at 5-50 μg/cm2 concentrations. HNECs were assessed for mitochondrial dysfunction and 16HBE cells for glutathione oxidation in real-time analyses. HNEC culture supernatants and total RNA were collected at 4-h postexposure for cytokine and gene expression analysis, and results show that PIEs can acutely induce inflammation, oxidative stress, and mitochondrial dysfunction in HNECs, and that incineration temperature modifies biological responses. Specifically, condensates from flaming and smoldering PIEs significantly increased HNEC secretion of cytokines IL-8, IL-1β, and IL-13, as well as expression of xenobiotic metabolism pathways and genes such as CYP1A1 and CYP1B1 at 5 and 20 μg/cm2 concentrations. Only 50 μg/cm2 flaming PIEs significantly increased glutathione oxidation in 16HBEs, and decreased respiration and ATP production in HNEC mitochondria. Impact Statement: Our data reveal the impact of incineration temperatures on biological outcomes associated with PIE exposures, emphasizing the importance of temperature as a factor when evaluating respiratory disease associated with PIEs exposure.

Network Pharmacology and Molecular Docking Technology-based Strategy to Explore the Potential Mechanism of Diabecon Formulation Botanicals

This study presents network pharmacology and a molecular docking-centered approach to elucidate the possible mechanism of action underlying the therapeutic effects of diabecon, a formulation containing Gymnema sylvestre, Pterocarpus marsupium, and Asphaltum punjabinum botanicals. Through comprehensive bioinformatics analysis, a collection of bioactive compounds present in these botanicals was identified, and their potential targets were forecasted using Swiss Target Prediction and STITCH. Disease-related genes were retrieved from Genecards and OMIM, and common genes between potential targets and disease-related genes were determined. Pathway analysis using the DAVID database revealed significant enrichment in pathways related to metabolic processes, xenobiotic metabolism, and signaling pathways, such as the insulin signaling pathway and pathways in cancer. Network construction using cytoscape highlighted interactions among identified genes, providing insights into the molecular pathways modulated by diabecon. Molecular docking analysis further elucidated the possible connections amongst biologically active compounds and target proteins, with (2S)-7-hydroxyflavanone exhibiting strong binding affinity with DPP-IV enzyme. Overall, this integrated approach offers valuable insights into the potential mechanisms of action of diabecon, contributing to our understanding of its therapeutic effects in the management of diabetes mellitus

Analysis and Validation of Tyrosine Metabolism-related Prognostic Features for Liver Hepatocellular Carcinoma Therapy.

To explore tyrosine metabolism-related characteristics in liver hepatocellular carcinoma (LIHC) and to establish a risk signature for the prognostic prediction of LIHC. Novel prognostic signatures contribute to the mining of novel biomarkers, which are essential for the construction of a precision medicine system for LIHC and the improvement of survival. Tyrosine metabolism plays a critical role in the initiation and development of LIHC. Based on the tyrosine metabolism-related characteristics in LIHC, this study developed a risk signature to improve the prognostic prediction of patients with LIHC. To investigate the correlation between tyrosine metabolism and progression of LIHC and to develop a tyrosine metabolism-related prognostic model. Gene expression and clinicopathological information of LIHC were obtained from The Cancer Genome Atlas (TCGA) database. Distinct subtypes of LIHC were classified by performing consensus cluster analysis on the tyrosine metabolism-related genes. Univariate and Lasso Cox regression were used to develop a RiskScore prognosis model. Kaplan-Meier (KM) survival analysis with log-rank test and area under the curve (AUC) of receiver operating characteristic (ROC) were employed in the prognostic evaluation and prediction validation. Immune infiltration, tyrosine metabolism score, and pathway enrichment were evaluated using single-sample gene set enrichment analysis (ssGSEA). Finally, a nomogram model was developed with the RiskScore and other clinicopathological features. Based on the tyrosine metabolism genes in the TCGA cohort, we identified 3 tyrosine metabolism-related subtypes showing significant prognostic differences. Four candidate genes selected from the common differentially expressed genes (DEGs) between the 3 subtypes were used to develop a RiskScore model, which could effectively divide LIHC patients into high- and lowrisk groups. In both the training and validation sets, high-risk patients tended to have worse overall survival, less active immunotherapy response, higher immune infiltration and clinical grade, and higher oxidative, fatty, and xenobiotic metabolism pathways. Multivariate analysis confirmed that the RiskScore was an independent indicator for the prognosis of LIHC. The results from pan-- cancer analysis also supported that the RiskScore had a strong prognostic performance in other cancers. The nomogram demonstrated that the RiskScore contributed the most to the prediction of LIHC prognosis. Our study developed a tyrosine metabolism-related risk model that performed well in survival prediction, showing the potential to serve as an independent prognostic predictor for LIHC treatment.

Costs of molecular adaptation to the chemical exposome: a focus on xenobiotic metabolism pathways.

Organisms adapt to their environment through different pathways. In vertebrates, xenobiotics are detected, metabolized and eliminated through the inducible xenobiotic-metabolizing pathways (XMP) which can also generate reactive toxic intermediates. In this review, we will discuss the impacts of the chemical exposome complexity on the balance between detoxication and side effects. There is a large discrepancy between the limited number of proteins involved in these pathways (few dozens) and the diversity and complexity of the chemical exposome (tens of thousands of chemicals). Several XMP proteins have a low specificity which allows them to bind and/or metabolize a large number of chemicals. This leads to undesired consequences, such as cross-inhibition, inefficient metabolism, release of toxic intermediates, etc. Furthermore, several XMP proteins have endogenous functions that may be disrupted upon exposure to exogenous chemicals. The gut microbiome produces a very large number of metabolites that enter the body and are part of the chemical exposome. It can metabolize xenobiotics and either eliminate them or lead to toxic derivatives. The complex interactions between chemicals of different origins will be illustrated by the diverse roles of the aryl hydrocarbon receptor which binds and transduces the signals of a large number of xenobiotics, microbiome metabolites, dietary chemicals and endogenous compounds. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

The Role of Aryl Hydrocarbon Receptor in Bone Biology.

Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is crucial in maintaining the skeletal system. Our study focuses on encapsulating the role of AhR in bone biology and identifying novel signaling pathways in musculoskeletal pathologies using the GEO dataset. The GEO2R analysis identified 8 genes (CYP1C1, SULT6B1, CYB5A, EDN1, CXCR4B, CTGFA, TIPARP, and CXXC5A) involved in the AhR pathway, which play a pivotal role in bone remodeling. The AhR knockout in hematopoietic stem cells showed alteration in several novel bone-related transcriptomes (eg, Defb14, ZNF 51, and Chrm5). Gene Ontology Enrichment Analysis demonstrated 54 different biological processes associated with bone homeostasis. Mainly, these processes include bone morphogenesis, bone development, bone trabeculae formation, bone resorption, bone maturation, bone mineralization, and bone marrow development. Employing Functional Annotation and Clustering through DAVID, we further uncovered the involvement of the xenobiotic metabolic process, p450 pathway, oxidation-reduction, and nitric oxide biosynthesis process in the AhR signaling pathway. The conflicting evidence of current research of AhR signaling on bone (positive and negative effects) homeostasis may be due to variations in ligand binding affinity, binding sites, half-life, chemical structure, and other unknown factors. In summary, our study provides a comprehensive understanding of the underlying mechanisms of the AhR pathway in bone biology.

Differential transcriptomic alterations in nasal versus lung tissue of acrolein-exposed rats

Introduction: Acrolein is a significant component of anthropogenic and wildfire emissions, as well as cigarette smoke. Although acrolein primarily deposits in the upper respiratory tract upon inhalation, patterns of site-specific injury in nasal versus pulmonary tissues are not well characterized. This assessment is critical in the design of in vitro and in vivo studies performed for assessing health risk of irritant air pollutants.Methods: In this study, male and female Wistar-Kyoto rats were exposed nose-only to air or acrolein. Rats in the acrolein exposure group were exposed to incremental concentrations of acrolein (0, 0.1, 0.316, 1 ppm) for the first 30 min, followed by a 3.5 h exposure at 3.16 ppm. In the first cohort of male and female rats, nasal and bronchoalveolar lavage fluids were analyzed for markers of inflammation, and in a second cohort of males, nasal airway and left lung tissues were used for mRNA sequencing.Results: Protein leakage in nasal airways of acrolein-exposed rats was similar in both sexes; however, inflammatory cells and cytokine increases were more pronounced in males when compared to females. No consistent changes were noted in bronchoalveolar lavage fluid of males or females except for increases in total cells and IL-6. Acrolein-exposed male rats had 452 differentially expressed genes (DEGs) in nasal tissue versus only 95 in the lung. Pathway analysis of DEGs in the nose indicated acute phase response signaling, Nrf2-mediated oxidative stress, unfolded protein response, and other inflammatory pathways, whereas in the lung, xenobiotic metabolism pathways were changed. Genes associated with glucocorticoid and GPCR signaling were also changed in the nose but not in the lung.Discussion: These data provide insights into inhaled acrolein-mediated sex-specific injury/inflammation in the nasal and pulmonary airways. The transcriptional response in the nose reflects acrolein-induced acute oxidative and cytokine signaling changes, which might have implications for upper airway inflammatory disease susceptibility.

Transcriptome and gut microbiota analyses reveal a possible mechanism underlying rifampin-mediated interruption of the larval development of chironomid Propsilocerus akamusi (Diptera: Chironomidae)

Chironomids, the most abundant insect group found in freshwater habitats, are known to be pollution tolerate and serve as important bioindicators of contaminant stress. Gut microbiota has recently been shown to potentially provide a number of beneficial services to insect hosts. However, the antibiotic-mediated interruption of chironomid gut microbial community and its subsequent influence on host body are still unclear. In the present study, the effects of rifampin on chironomid larvae were investigated at both transcriptome and microbiome level to assess the relationship between gut bacteria and associated genes. Our data indicated that the rifampin-induced imbalance of gut ecosystem could inhibit the development of chironomid larvae via decreasing the body weight, body length and larval eclosion rate during 96-h treatment. Both the community structure and taxonomic composition were significantly altered due to the invasion of rifampin in digestive tracts. The relative abundance of phylum Deferribacterota and Bacteroidota were dramatically increased with rifampin exposure. A set of genes involved in amino acid synthesis as well as xenobiotic metabolism pathways were greatly changed and proved to have tight correlation with certain genus. Bacterial genus Tyzzerella was positively correlated with detoxifying PaCYP6GF1 and PaCYP9HL1 genes. This study provides a reference for understanding the environmental risks of antibiotic and aims to accelerate new biological insights into the effects of antibiotic on the fitness of chironomids and into the microbe mediated-regulatory mechanism of aquatic insects.

Effects of Chromium Exposure on the Gene Expression of the Midgut in Silkworms, Bombyx mori.

Chromium is a severe heavy metal pollutant with significant environmental risks. The effects of Chromium on the digestion of Bombyx mori (silkworms) are of particular importance due to their ecological and economic significance. Herein, RNA sequencing was conducted on nine midgut samples from silkworms exposed to control, 12 g/kg and 24 g/kg Chromium chemical diets. Comparative transcriptomics revealed that under moderate Chromium exposure, there was a significant increase in up-regulated genes (1268 up-regulated to 857 down-regulated), indicating a stimulation response. At higher stress levels, a weakened survival response was observed, with a decrease in up-regulated genes and an increase in down-regulated genes (374 up-regulated to 399 down-regulated). A notable shift in cellular responses under medium chromium exposure was exposed, signifying the activation of crucial metabolic and transport systems and an elevation in cellular stress and toxicity mechanisms. The observation of up-regulated gene expression within xenobiotic metabolism pathways suggests a heightened defense against Chromium-induced oxidative stress, which was primarily through the involvement of antioxidant enzymes. Conversely, high-dose Chromium exposure down-regulates the folate biosynthesis pathway, indicating biological toxicity. Two novel genes responsive to pressure were identified, which could facilitate future stress adaptation understanding. The findings provide insights into the molecular mechanisms underlying silkworms' digestion response to Chromium exposure and could inform its biological toxicity.

User-friendly microfluidic system reveals native-like morphological and transcriptomic phenotypes induced by shear stress in proximal tubule epithelium.

Drug-induced nephrotoxicity is a leading cause of drug attrition, partly due to the limited relevance of pre-clinical models of the proximal tubule. Culturing proximal tubule epithelial cells (PTECs) under fluid flow to mimic physiological shear stress has been shown to improve select phenotypes, but existing flow systems are expensive and difficult to implement by non-experts in microfluidics. Here, we designed and fabricated an accessible and modular flow system for culturing PTECs under physiological shear stress, which induced native-like cuboidal morphology, downregulated pathways associated with hypoxia, stress, and injury, and upregulated xenobiotic metabolism pathways. We also compared the expression profiles of shear-dependent genes in our in vitro PTEC tissues to that of ex vivo proximal tubules and observed stronger clustering between ex vivo proximal tubules and PTECs under physiological shear stress relative to PTECs under negligible shear stress. Together, these data illustrate the utility of our user-friendly flow system and highlight the role of shear stress in promoting native-like morphological and transcriptomic phenotypes in PTECs in vitro, which is critical for developing more relevant pre-clinical models of the proximal tubule for drug screening or disease modeling.

GHR disruption in mature adult mice alters xenobiotic metabolism gene expression in the liver.

Lifelong reduction of growth hormone (GH) action extends lifespan and improves healthspan in mice. Moreover, congenital inactivating mutations of GH receptor (GHR) in mice and humans impart resistance to age-associated cancer, diabetes, and cognitive decline. To investigate the consequences of GHR disruption at an adult age, we recently ablated the GHR at 6-months of age in mature adult (6mGHRKO) mice. We found that both, male and female 6mGHRKO mice have reduced oxidative damage, with males 6mGHRKO showing improved insulin sensitivity and cancer resistance. Importantly, 6mGHRKO females have an extended lifespan compared to controls. To investigate the possible mechanisms leading to health improvements, we performed RNA sequencing using livers from male and female 6mGHRKO mice and controls. We found that disrupting GH action at an adult age reduced the gap in liver gene expression between males and females, making gene expression between sexes more similar. However, there was still a 6-fold increase in the number of differentially expressed genes when comparing male 6mGHRKO mice vs controls than in 6mGHRKO female vs controls, suggesting that GHR ablation affects liver gene expression more in males than in females. Finally, we found that lipid metabolism and xenobiotic metabolism pathways are activated in the liver of 6mGHRKO mice. The present study shows for the first time the specific hepatic gene expression profile, cellular pathways, biological processes and molecular mechanisms that are driven by ablating GH action at a mature adult age in males and females. Importantly, these results and future studies on xenobiotic metabolism may help explain the lifespan extension seen in 6mGHRKO mice.

Abstract A007: Proteogenomic landscape of KRASG12C lung adenocarcinomas reveals new subtypes and potential combination therapies

Abstract Despite the presence of the KRASG12C oncogene, not all patients respond to KRASG12C inhibitors (e.g., sotorasib, adagrasib) and duration of response has been variable. We hypothesized that subtypes of KRASG12C lung cancers could be responsible for such differential effects. To define subtypes, we performed targeted exon sequencing, transcriptomics, proteomics, and phosphoproteomics on 46 frozen surgically resected KRASG12C lung adenocarcinomas. We employed unbiased consensus clustering methods, pathway analysis, and PTM-SEA to identify kinase signatures from phosphoproteomics data. Transcriptomics data revealed 4 clusters. Pathway enrichment showed that gene cluster (GC) 1 was enriched for immune pathways including interferon gamma response. Epithelial-mesenchymal transition (EMT), assessed by a TGF-β gene signature, was also enriched in GC1. 100% of GC1 overlapped the Proximal-Inflammatory subtype (Wilkerson et al.) and 53% with the mesenchymal subtype (Daemen et al.). GC2 was enriched for inflammatory response and TNF-alpha signaling. GC3 represented a metabolic phenotype with enriched xenobiotic metabolism and other metabolism pathways. 83% of GC3 overlapped with the Terminal Respiratory Unit subtype (Wilkerson et al.) and 56% with the proliferative subtype (Daemen et al.). GC4 only had 5 samples and did not have any significant enrichment. We next identified four proteomic clusters with distinct biology. Protein Cluster 1 (PC1) was enriched for immune pathways, PC2 was enriched for EMT, and PC3 was enriched for TNF/NF-κβ signaling. PC4 was enriched for xenobiotic and fatty acid metabolism and had significantly more KEAP1 mutations. We observed concordance of protein based clusters with mRNA based clusters, as PC1 largely overlapped with GC1 (70%) and PC4 largely overlapped with GC3 (86%). Cell type inference using xCell largely recapitulated the immune subtypes identified by transcriptomics and proteomics. GC1/PC1 had significantly more CD8+ effector memory T-cells, CD4+ Th2 T-cells, and M1 macrophages, while GC3/PC4 had significantly less of these same cell types. Collectively, GC1/PC1 represents 43% of the cohort and was defined by enrichment of immune pathways, elevated immune populations, and significantly higher PD-L1. Finally, we leveraged the phosphoproteomics data to determine whether signaling activity differs across subtypes. Using PTM-SEA, we identified phosphoproteomic signatures of activity for 50 kinases and pathways. We identified 26% of samples (N=12) with high EGFR activity, which is known to affect sensitivity of cells to KRASG12C inhibitors. The EGFR-high samples were observed across all genomic, transcriptomic, and proteomic subtypes. Our results suggest concordance of some transcriptomic and proteomic subtypes, including an immune subtype, while a metabolic protein subtype and EGFR high signaling subtype may provide additional subclasses with potential relevance for treatment responses. Additional phosphoproteomics signatures such as PLK1 and AKT1 may identify subtypes amenable to combination therapy approaches. Citation Format: Paul A. Stewart, Hitendra Solanki, Eric Welsh, Yaakov Stern, Denis Imbody, Yonghong Zhang, Bruna Pellini, Bin Fang, Sean Yoder, Steven Eschrich, Jamie Teer, John Koomen, Eric B. Haura. Proteogenomic landscape of KRASG12C lung adenocarcinomas reveals new subtypes and potential combination therapies [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A007.

Microbiome Diversity and Cellulose Decomposition Processes by Microorganisms on the Ancient Wooden Seawall of Qiantang River of Hangzhou, China.

Archaeological wood, also known as wooden cultural relics, refers to ancient wood that has been worked by humans. Further insights into the decomposition mechanism of archaeological wood are needed for its preventive conservation. In this study, we assessed the microbiome diversity and cellulose decomposition processes on a 200-year-old ancient wooden seawall - the Qiantang River of Hangzhou, China. We used high-throughput sequencing (HTS) to deduce the metagenomic functions, particularly the cellulose-decomposing pathway of the microbial communities, through bioinformatical approaches. The predominant cellulose-decomposing microorganisms were then verified with traditional isolation, culture, and identification method. The results showed that the excavation of archaeological wood significantly altered the environment, accelerating the deterioration process of the archaeological wood through the carbohydrate metabolism and the xenobiotic biodegradation and metabolism pathways, under the comprehensive metabolism of complex ecosystem formed by bacteria, archaea, fungi, microfauna, plants, and algae. Bacteroidetes, Proteobacteria, Firmicutes, and Actinobacteria were found to be the predominant source of bacterial cellulose-decomposing enzymes. Accordingly, we suggest relocating the wooden seawall to an indoor environment with controllable conditions to better preserve it. In addition, these results provide further evidence for our viewpoints that HTS techniques, combined with rational bioinformatical data interpretation approaches, can serve as powerful tools for the preventive protection of cultural heritage.

Biomarkers of oxidative stress, diet and exercise distinguish soldiers selected and non-selected for special forces training

IntroductionThe metabolomic profiles of Soldiers entering the U.S. Special Forces Assessment and Selection course (SFAS) have not been evaluated.ObjectivesTo compare pre-SFAS blood metabolomes of Soldiers selected during SFAS versus those not selected, and explore the relationships between the metabolome, physical performance, and diet quality. MethodsFasted blood samples and food frequency questionnaires were collected from 761 Soldiers prior to entering SFAS to assess metabolomic profiles and diet quality, respectively. Physical performance was assessed throughout SFAS.ResultsBetween-group differences (False Discovery Rate < 0.05) in 108 metabolites were detected. Selected candidates had higher levels of compounds within xenobiotic, pentose phosphate, and corticosteroid metabolic pathways, while non-selected candidates had higher levels of compounds potentially indicative of oxidative stress (i.e., sphingomyelins, acylcarnitines, glutathione, amino acids). Multiple compounds higher in non-selected versus selected candidates included: 1-carboxyethylphenylalanine; 4-hydroxy-nonenal-glutathione; α-hydroxyisocaproate; hexanoylcarnitine; sphingomyelin and were associated with lower diet quality and worse physical performance. ConclusionCandidates selected during SFAS had higher pre-SFAS levels of circulating metabolites that were associated with resistance to oxidative stress, higher physical performance and higher diet quality. In contrast, non-selected candidates had higher levels of metabolites potentially indicating elevated oxidative stress. These findings indicate that Soldiers who were selected for continued Special Forces training enter the SFAS course with metabolites associated with healthier diets and better physical performance. Additionally, the non-selected candidates had higher levels of metabolites that may indicate elevated oxidative stress, which could result from poor nutrition, non-functional overreaching/overtraining, or incomplete recovery from previous physical activity.

Kras oncogene ablation prevents resistance in advanced lung adenocarcinomas.

KRASG12C inhibitors have revolutionized the clinical management of patients with KRASG12C-mutant lung adenocarcinoma. However, patient exposure to these inhibitors leads to the rapid onset of resistance. In this study, we have used genetically engineered mice to compare the therapeutic efficacy and the emergence of tumor resistance between genetic ablation of mutant Kras expression and pharmacological inhibition of oncogenic KRAS activity. Whereas Kras ablation induces massive tumor regression and prevents the appearance of resistant cells in vivo, treatment of KrasG12C/Trp53-driven lung adenocarcinomas with sotorasib, a selective KRASG12C inhibitor, caused a limited antitumor response similar to that observed in the clinic, including the rapid onset of resistance. Unlike in human tumors, we did not observe mutations in components of the RAS-signaling pathways. Instead, sotorasib-resistant tumors displayed amplification of the mutant Kras allele and activation of xenobiotic metabolism pathways, suggesting that reduction of the on-target activity of KRASG12C inhibitors is the main mechanism responsible for the onset of resistance. In sum, our results suggest that resistance to KRAS inhibitors could be prevented by achieving a more robust inhibition of KRAS signaling mimicking the results obtained upon Kras ablation.