Single-cell and metabolomic profiling reveals multi-lineage hepatic perturbations and metabolic reprogramming in pregnant mice exposed to dimethomorph

BackgroundPulmonary fibrosis is thought to be driven by recurrent alveolar epithelial injury which leads to the differentiation of fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts and subsequent deposition of extracellular matrix (ECM). Transforming growth factor beta-1 (TGF-β1) plays a key role in fibroblast differentiation, which we have recently shown involves human antigen R (HuR). HuR is an RNA binding protein that also increases the translation of hypoxia inducible factor (HIF-1α) mRNA, a transcription factor critical for inducing a metabolic shift from oxidative phosphorylation towards glycolysis. This metabolic shift may cause fibroblast differentiation. We hypothesized that under hypoxic conditions, HuR controls myofibroblast differentiation and glycolytic reprogramming in human lung fibroblasts (HLFs).MethodsPrimary HLFs were cultured in the presence (or absence) of TGF-β1 (5 ng/ml) under hypoxic (1% O2) or normoxic (21% O2) conditions. Evaluation included mRNA and protein expression of glycolytic and myofibroblast/ECM markers by qRT-PCR and western blot. Metabolic profiling was done by proton nuclear magnetic resonance (1H- NMR). Separate experiments were conducted to evaluate the effect of HuR on metabolic reprogramming using siRNA-mediated knock-down.ResultsHypoxia alone had no significant effect on fibroblast differentiation or metabolic reprogramming. While hypoxia- together with TGFβ1- increased mRNA levels of differentiation and glycolysis genes, such as ACTA2, LDHA, and HK2, protein levels of α-SMA and collagen 1 were significantly reduced. Hypoxia induced cytoplasmic translocation of HuR. Knockdown of HuR reduced features of fibroblast differentiation in response to TGF-β1 with and without hypoxia, including α-SMA and the ECM marker collagen I, but had no effect on lactate secretion.ConclusionsHypoxia reduced myofibroblasts differentiation and lactate secretion in conjunction with TGF-β. HuR is an important protein in the regulation of myofibroblast differentiation but does not control glycolysis in HLFs in response to hypoxia. More research is needed to understand the functional implications of HuR in IPF pathogenesis.

The neurotoxic effects of pesticide residues on nontarget organisms, particularly pregnant women and fetuses, represent a critical concern in environmental and health research. In this study, dimethomorph (DMM) was detected in human cord blood in normal pregnancies at term (without maternal-fetal complications). And neurotoxicity of the fungicide DMM through integrated single-cell RNA sequencing (scRNA-seq) and metabolomic profiling in pregnant mice brain tissue. Our results demonstrate that DMM exposure induces significant alterations in both the proportions and functions of multiple neural cell populations, including microglia, oligodendrocytes, astrocytes, and endothelial cells, accompanied by metabolic reprogramming and blood-brain barrier (BBB) dysfunction. Single-cell analysis revealed cell subtype-specific transcriptional changes and aberrant activation of metabolic pathways (e.g., PI3K-AKT-mTOR signaling), while metabolomic profiling further identified substantial disturbances in amino acid, lipid, and energy metabolism. Furthermore, cell-cell communication analysis indicated enhanced pathological signaling network interactions under DMM exposure. These findings not only elucidate the mechanisms underlying DMM-induced neurotoxicity but also highlight the potential risks of pesticide exposure during pregnancy to maternal and fetal health, providing critical insights for pesticide safety assessment and the development of neuroprotective strategies.

Abnormally raised circulating bile acids (BA) during pregnancy threat fetal and offspring health. Our previous study has identified sulfated progesterone metabolites (PMSs) in part account for dysregulation of maternal BA homeostasis during pregnancy, however, limited intervention strategies to remedy increased serum BA through PMSs during pregnancy are available. The purpose of this study is to test the feasibility of manipulating BA homeostasis and progesterone metabolism through steering gut microbiota. A total of 19 pregnant sows were randomly treated with standard diet or vancomycin-supplemented diet, to investigate the intercorrelation of PMSs, intestinal microbiota, and maternal BA metabolism from day 60 of gestation (G60) until farrowing (L0). Pregnant mice orally gavaged with epiallopregnanolone sulfate (PM5S) or vehicle and nonpregnant mice were sampled and further analyzed to verify the effect of PM5S on maternal BA metabolism. The present study revealed that oral vancomycin reduced maternal fasting serum total BA (TBA) levels and postprandial serum TBA levels at day 90 of gestation (G90). BA profile analysis showed the decreased TBA after vancomycin treatment was attributed to the decrease of primary BA and secondary BA, especially hyodeoxycholic acid (HDCA). By using newly developed UPLC-MS/MS methods, we found vancomycin increased fecal excretion of allopregnanolone sulfate (PM4S) and PM5S during late gestation and thus maintaining the relative stability of serum PM4S and PM5S, which play an important role in BA metabolism. Further study in mice showed that pregnant mice have higher serum and liver TBA levels compared with nonpregnant mice, and PM5S administration induced higher gallbladder TBA levels and TBA pool in pregnant mice. In addition, after oral vancomycin, the continuously decreased Parabacteroides genus, potentially enriched with genes encoding steroids sulfatase, may explain the increased fecal PMSs excretion in pregnant sows. Taken together, our study provides the evidence that pregnancy-induced elevation of BA levels in sow is likely regulated by manipulation of gut microbiota, which offer new insights into the prevention and treatment of disrupted BA homeostasis during pregnancy by targeting specific microbiota.

Post-hatch development in pigeons involves dramatic shifts in nutrition and metabolism. However, the underlying systemic metabolic reprogramming remains poorly characterized. Thus, longitudinal analyses of body weight (BW), serum immunoglobulins, biochemical parameters, and untargeted metabolomics at three key developmental stages: postnatal day 5 (PND 5, crop milk dependence; N = 10), PND 15 (transition to grains; N = 10), and PND 25 (independent grain intake; N = 10) were conducted. The BW increased significantly between PND 5 and PND 15 (P < 0.05), concomitant with elevated serum glucose, albumin, total cholesterol, and high-density lipoprotein. Immunoglobulin (Ig) dynamics revealed a significant decline in IgA at PND 15 and PND 25 relative to PND 5 (P < 0.05). The IgG showed a temporary significant decrease at PND 15 compared to PND 5 (P < 0.05), returning to PND 5 levels by PND 25. Metabolomics demonstrated dynamic pathway alterations. Comparing PND 15 to PND 5, differential metabolites were significantly enriched in five pathways (P < 0.05), most prominently alanine, aspartate and glutamate metabolism (P < 0.001) and purine metabolism (P = 0.003). The PND 15 to PND 25 transition featured prominent shifts, notably in glycerophospholipid metabolism (P < 0.001) and tricarboxylic acid (TCA) cycle continuation (P = 0.026). Crucially, PND 25 vs PND 5 analysis identified seven remodeled pathways, with core reprogramming involving alanine, aspartate and glutamate metabolism (P < 0.001), arginine biosynthesis (P < 0.001), and the TCA cycle (P = 0.002), which emerged as a central metabolic hub. The K-means clustering of 25 hub metabolites and physiological parameters delineated seven co-regulation patterns. Notably, BW and nutritional markers (albumin, cholesterol) correlated positively with TCA intermediates (citrate, α-ketoglutarate, malate; P < 0.05), while showing inverse associations with purine catabolites (e.g., guanine, xanthosine) and bile acids. Conversely, immunoglobulins correlated positively with purine metabolites and bile acids. This study identifies TCA cycle intermediates and purine metabolites as dual biomarkers regulating growth and immune function during pigeon development. These findings provide a foundation for targeted nutritional strategies that require adjustments as a function of aging, such as key metabolite supplementation and phospholipid modulation, to optimize pigeon management practices.

Supplement of 1% lithocholic acid (LCA) in the diet for 5-9 days resulted in elevated levels of the marker for liver damage aspartate aminotransferase and alkaline phosphatase activities in both farnesoid X receptor (FXR)-null and wild-type female mice. The levels were clearly higher in wild-type mice than in FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and taurolithocholic acid, were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement. Marked increases in hepatic sulfating activity for LCA (5.5-fold) and hydroxysteroid sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null mice. A 7.4-fold higher 3alpha-sulfated bile acid concentration was observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels of alkaline phosphatase. In contrast, microsomal LCA 6beta-hydroxylation was not increased and was in fact lower in FXR-null mice compared in wild-type mice. Clear decreases in mRNA encoding sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide 1, and liver-specific organic anion transporter-1 function in bile acid import were detected in LCA-fed mice. These transporter levels are higher in FXR-null mice than wild-type mice after 1% LCA supplement. No obvious changes were detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate hydroxysteroid sulfotransferase-mediated LCA sulfation as a major pathway for protection against LCA-induced liver damage. Furthermore, Northern blot analysis using FXR-null, pregnane X receptor-null, and FXR-pregnane X receptor double-null mice suggests a repressive role of these nuclear receptors on basal St2a expression.

Increasing evidence has demonstrated that exposure to endocrine-disrupting chemicals impacts maternal and fetal health, but the underlying mechanisms are still unclear. We previously showed that dietary exposure to 10 µg/kg body weight (bw)/d and 10 mg/kg bw/d of bisphenol A (BPA) during pregnancy induced metabolic abnormalities in F1 male offspring and gestational glucose intolerance in F0 pregnant mice. The aim of this study was to elucidate the underlying etiologies of BPA exposure-induced metabolic disease by analyzing the male fetal liver metabolome. Using the Metabolon Discover HD4 Platform, our laboratory identified metabolic pathways that were altered by BPA exposure, including biochemicals in lipid and amino acid metabolism. Specifically, primary and secondary bile acids were increased in liver from BPA-exposed embryonic day 18.5 male fetuses. We subsequently showed that increased bile acid was associated with a defective farnesoid X receptor-dependent negative feedback mechanism in BPA-exposed fetuses. In addition, through metabolomics, we observed that BPA-exposed fetuses had elevated tryptophan levels. Independent liquid chromatography and mass spectrometry measurement revealed that BPA-exposed dams also had increased tryptophan levels relative to those of controls. Because several key enzymes in tryptophan catabolism are vitamin B6 dependent and vitamin B6 deficiencies have been linked to gestational diabetes, we tested the impact of vitamin B6 supplementation and showed that it rescued gestational glucose intolerance in BPA-exposed pregnant mice. Our study has therefore identified two pathways (bile acid and tryptophan metabolism) that potentially underlie BPA-induced maternal and fetal metabolic disease.

The aim of this study is to investigate the metabolic alterations associated with pheochromocytomas and paragangliomas (PPGLs) and the impact of surgical resection on the serum metabolome using untargeted nuclear magnetic resonance (NMR) metabolomics. For this, the study included 34 patients diagnosed with PPGLs. Pre-operative and postoperative serum samples were analyzed using 1D-proton NMR spectroscopy, and NMR spectral data were processed using Bruker software Topspin. The quantitative metabolic profiles were estimated using CHENOMX NMR-Suite, and multivariate data were analyzed using partial least squares discriminant analysis (PLS-DA) and orthogonal PLS-DA followed by random forest (RF) classification method (a machine learning approach). The multivariate analysis revealed distinct metabolic differences between pre-operative and postoperative samples with respect to normal control (NC) samples, indicating a metabolic shift following tumor resection. RF classification, with an out-of-bag error rate of 0.186, effectively distinguished between NC, presurgery, and postsurgery groups, underscoring the distinct metabolic states in PPGL and the restorative effect of surgical intervention. Pre-operative serum profiles of PPGL patients were characterized by decreased levels of key metabolites, including glucose, citrate, amino acids (glutamine, glycine, leucine, valine, tyrosine, and alanine), histidine, myo-inositol, and creatinine, suggesting altered energy metabolism, and amino acid catabolism induced by catecholamine excess. Postsurgical profiles showed partial metabolic restoration, with significant increases in proline, glutamate, and 3-hydroxybutyrate (3-HB) (p < 0.01), indicating normalization involving lipid oxidation and amino acid metabolism. Although plasma metanephrines normalized postsurgery, full biochemical recovery lagged, as metabolic profiles of postoperative patients remained distinct from healthy controls. In conclusion, the present untargeted NMR metabolomics revealed significant metabolic reprogramming in PPGL patients and captured the partial normalization of metabolic pathways following tumor resection. Metabolites such as proline, glutamate, and 3-HB emerged as potential biomarkers of treatment response. These findings underscore the utility of metabolomics to identify biomarkers for monitoring disease progression, assessing postsurgical recovery, and improving our understanding of PPGL pathophysiology.

Di-(2-ethylhexyl)-phthalate (DEHP) is a ubiquitous environmental pollutant and is widely used in industrial plastics. Intrahepatic cholestasis of pregnancy (ICP), distinguished by maternal pruritus and elevated serum bile acid levels, is linked to unfavorable pregnancy consequences. Few studies have investigated the potential effect of gestational DEHP exposure on the cholestasis in pregnant female mice, and the underlying mechanisms remain unclear. In the present study, a mouse model of cholestasis during pregnancy was established by DEHP exposure. We found that DEHP induces elevated bile acid levels by affecting bile acid synthesis and transporter receptor expression in the maternal liver and placenta of pregnant female mice, ultimately leading to intrauterine growth restriction (IUGR). In addition, DEHP changed the bile acid composition of maternal serum and liver as well as placenta and amniotic fluid in pregnant female mice; Importantly, we found that DEHP down-regulates the expression of farnesoid X receptor (FXR), which is considered to be a bile acid receptor. FXR agonist obeticholic acid (OCA) effectively alleviated the adverse effects of DEHP on pregnant female mice. While, OCA itself had no adverse effects on normal pregnant female mice. In summary, DEHP could induces bile acid disorder and IUGR in pregnant female mice by affect FXR, which was reversed by OCA.

During pregnancy, proper hepatobiliary transport and bile acid synthesis protect the liver from cholestatic injury and regulate the maternal and fetal exposure to bile acids, drugs, and environmental chemicals. The objective of this study was to determine the temporal messenger RNA (mRNA) and protein profiles of uptake and efflux transporters as well as bile acid synthetic and conjugating enzymes in livers from virgin and pregnant mice on gestational days (GD) 7, 11, 14, and 17 and postnatal days (PND) 1, 15, and 30. Compared with virgins, the mRNAs of most transporters were reduced approximately 50% in pregnant dams between GD11 and 17. Western blot and immunofluorescence staining confirmed the downregulation of Mrp3, 6, Bsep, and Ntcp proteins. One day after parturition, the mRNAs of many uptake and efflux hepatobiliary transporters remained low in pregnant mice. By PND30, the mRNAs of all transporters returned to virgin levels. mRNAs of the bile acid synthetic enzymes in the classic pathway, Cyp7a1 and 8b1, increased in pregnant mice, whereas mRNA and protein expression of enzymes in the alternative pathway of bile acid synthesis (Cyp27a1 and 39a1) and conjugating enzymes (Bal and Baat) decreased. Profiles of transporter and bile acid metabolism genes likely result from coordinated downregulation of transcription factor mRNA (CAR, LXR, PXR, PPARα, FXR) in pregnant mice on GD14 and 17. In conclusion, pregnancy caused a global downregulation of most hepatic transporters, which began as early as GD7 for some genes and was maximal by GD14 and 17, and was inversely related to increasing concentrations of circulating 17β-estradiol and progesterone as pregnancy progressed.

Introduction. DNA damage response (DDR) inhibitors are effective across multiple cancers; however, the long-term consequences of sustained DDR checkpoint blockade, especially in terms of genomic stability and resistance mechanisms, remain poorly understood. Here, we model chronic dual CHK1/CHK2 inhibition in B-acute lymphoblastic leukemia (B-ALL) to define evolutionary trajectories of resistance and uncover actionable vulnerabilities.Methods. NALM-6 cells were exposed long-term to the CHK1/CHK2 inhibitor PF-0477736, generating stepwise resistant subclones (N6R-PF4, N6R-PF8). We profiled viability, cell cycle, apoptosis, DDR signaling, cytogenetics/FISH, genome-wide copy number (CytoScan HD), whole-exome sequencing, RNA-seq, and DIA proteomics. Seahorse flux, comet assays, and γH2AX foci quantified mitochondrial and genotoxic responses. Combination studies tested ATM inhibition (KU-60019) and metabolic co-targeting with atorvastatin.Results. Prolonged CHK1/CHK2 inhibition selected for resistant clones restoring copy-number neutrality at the 11q22 (ATM) locus, in contrast to parental cells with heterozygous ATM loss. Resistant cells exhibited impaired G1 checkpoint activation, reduced DDR signaling and apoptosis in response to PF-0477736, and partial cross-resistance to a second CHK1/CHK2 inhibitor. Exome profiling revealed increased mutation burden with enrichment of DDR gene lesions (e.g., TP53BP1, NBN, FANCD2, PMS2) and mismatch-repair-like mutational signatures. Multi-omics and metabolic analysis converged on metabolic rewiring: resistant cells upregulated oxidative phosphorylation and sterol/fatty-acid biosynthesis, with suppression of global protein translation. This metabolic shift could be targeted using atorvastatin (sterol biosynthesis inhibitor) but not with metformin (oxidative phosphorylation inhibitor), indicating selective dependence on mevalonate metabolism. Proteomics showed increased expression of several repair pathways (NER, MMR, NHEJ) alongside reduced BER/HR proteins. These DDR-alterations modified significantly the tolerance to DNA damages in the resistant clone. Indeed, N6R-PF8 cells accumulated significantly less DNA damage and underwent reduced apoptosis following oxidative (H₂O₂) or chemotherapeutic stress (anthracyclines, antimetabolites, vinca alkaloids). Finally, combination treatment demonstrated strong synergy between PF-0477736 and ATM inhibition in N6R-PF8 cells compared to NALM-6 confirming the importance of ATM kinase in the sensitivity to PF-0477736. Conclusions. Chronic CHK1/CHK2 inhibition promotes genomic instability, highlighting the need for clinical strategies that prevent adaptive escape and avoid aggravating the intrinsic instability of leukemic cells. Our data reveal a tight functional link between metabolic rewiring and DNA repair pathways, suggesting that dual targeting of DDR and metabolic vulnerabilities may be essential to optimize the safe and effective use of DDR inhibitors in leukemia.

Metabolism alters markedly with advancing gestation, characterized by progressive insulin resistance, dyslipidemia, and raised serum bile acids. The nuclear receptor farnesoid X receptor (FXR) has an integral role in bile acid homeostasis and modulates glucose and lipid metabolism. FXR is known to be functionally suppressed in pregnancy. The FXR agonist, obeticholic acid (OCA), improves insulin sensitivity in patients with type 2 diabetes with nonalcoholic fatty liver disease. We therefore hypothesized that OCA treatment during pregnancy could improve disease severity in a mouse model of gestational diabetes mellitus (GDM). C57BL/6J mice were fed a high-fat diet (HFD; 60% kcal from fat) for 4 wk before and throughout pregnancy to induce GDM. The impact of the diet supplemented with 0.03% OCA throughout pregnancy was studied. Pregnant HFD-fed mice displayed insulin resistance and dyslipidemia. OCA significantly reduced plasma cholesterol concentrations in nonpregnant and pregnant HFD-fed mice (by 22.4%, P < 0.05 and 36.4%, P < 0.001, respectively) and reduced the impact of pregnancy on insulin resistance but did not change glucose tolerance. In nonpregnant HFD-fed mice, OCA ameliorated weight gain, reduced mRNA expression of inflammatory markers in white adipose tissue, and reduced plasma glucagon-like peptide 1 concentrations (by 62.7%, P < 0.01). However, these effects were not evident in pregnant mice. OCA administration can normalize plasma cholesterol levels in a mouse model of GDM. However, the absence of several of the effects of OCA in pregnant mice indicates that the agonistic action of OCA is not sufficient to overcome many metabolic consequences of the pregnancy-associated reduction in FXR activity.

Multiple hormonal activities of UV filters and comparison of in vivo and in vitro estrogenic activity of ethyl-4-aminobenzoate in fish

Background/Objectives: Metabolic dysregulation underlies a myriad of chronic diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD) and obesity, and bile acids emerge as an important mediator in their etiology. Weight control by improving diet quality is the standard of care in prevention and control of these metabolic diseases. Inclusion of pulses, such as common bean, is an affordable yet neglected approach to improving diet quality and metabolic outcomes. Thus, this study evaluated the possibility that common bean alters bile acid metabolism in a health-beneficial manner. Methods: Using biospecimens from several similarly designed studies, cecal content, feces, liver tissue, and plasma samples from C57BL/6 mice fed an obesogenic diet lacking (control) or containing cooked common bean were subjected to total bile acid analysis and untargeted metabolomics. RNA-seq, qPCR, and Western blot assays of liver tissue complemented the bile acid analyses. Microbial composition and predicted function in the cecal contents were evaluated using 16S rRNA gene amplicon and shotgun metagenomic sequencing. Results: Bean-fed mice had increased cecal bile acid content and excreted more bile acids per gram of feces. Consistent with these effects, increased synthesis of bile acids in the liver was observed. Microbial composition and capacity to metabolize bile acids were markedly altered by bean, with greater prominence of secondary bile acid metabolites in bean-fed mice, i.e., microbial metabolites of chenodeoxycholate/lithocholate increased while metabolites of hyocholate were reduced. Conclusions: In rendering mice resistant to obesogenic diet-induced MASLD and obesity, cooked bean consumption sequesters bile acids, increasing their hepatic synthesis and enhancing their diversity through microbial metabolism. Bean-induced changes in bile acid metabolism have potential to improve dyslipidemia.

Untargeted and spatial-resolved metabolomics characterize serum and tissue-specific metabolic reprogramming in acute kidney injury

Ovarian cancer is the most lethal gynecological malignancy and is often associated with both DNA repair deficiency and extensive metabolic reprogramming. While still emerging, the interplay between these pathways can affect ovarian cancer phenotypes, including therapeutic resistance to the DNA damaging agents that are standard-of-care for this tumor type. In this review, we will discuss what is currently known about cellular metabolic rewiring in ovarian cancer that may impact DNA damage and repair in addition to highlighting how specific DNA repair proteins also promote metabolic changes. We will also discuss relevant data from other cancers that could be used to inform ovarian cancer therapeutic strategies. Changes in the choice of DNA repair mechanism adopted by ovarian cancer are a major factor in promoting therapeutic resistance. Therefore, the impact of metabolic reprogramming on DNA repair mechanisms in ovarian cancer has major clinical implications for targeted combination therapies for the treatment of this devastating disease.