Breast cancer detection using targeted plasma metabolomics Jasbi, Paniz; Wang, Dongfang; Cheng, Sunny Lihua ...
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences,
01/2019, Letnik:
1105
Journal Article
Recenzirano
Breast cancer (BC) is a major cause of human morbidity and mortality, especially among women. Despite the important role of metabolism in the molecular pathogenesis of cancer, robust metabolic ...markers to enable enhanced screening and disease monitoring of BC are still critically needed. In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolic profiling approach is presented for the identification of metabolic marker candidates that could enable highly sensitive and specific detection of all-stage as well as early-stage BC. In this targeted approach, 105 metabolites from >35 metabolic pathways of potential biological relevance were reliably detected in 201 plasma samples taken from two groups of subjects (102 BC patients and 99 healthy controls). The results of our general linear model and partial least squares-discriminant analysis (PLS-DA) informed the construction of a novel 6-metabolite panel of potential biomarkers. A receiver operating characteristic (ROC) curve generated based on an improved PLS-DA model showed relatively high sensitivity (0.80), specificity (0.75), and area under the receiver-operating characteristic curve (AUROC = 0.89). Similar classification performance of the model was observed for detection of early-stage BC (AUROC = 0.87, sensitivity: 0.86, specificity: 0.75). Bioinformatics analyses revealed significant disturbances in arginine/proline metabolism, tryptophan metabolism, and fatty acid biosynthesis. Our univariate and multivariate results indicate the effectiveness of this metabolomics approach for all-stage as well as early-stage BC diagnosis; our bioinformatics results indicate affected pathways related to tumor growth, metastasis, and immune escape mechanisms. Future studies should validate these results using more samples from different locations.
Very little is known about the effect of gut microbiota on the ontogeny of drug-processing genes (DPGs) in liver. In this study, livers were harvested from conventional (CV) and germ-free (GF) male ...and female mice from 1 to 90 days of age. RNA-Seq in livers of 90-day-old male mice showed that xenobiotic metabolism was the most downregulated pathway within the mRNA transcriptome in absence of intestinal bacteria. In male livers, the mRNAs of 67 critical DPGs partitioned into 4 developmental patterns (real-time-quantitative polymerase chain reaction): Pattern-1 gradually increased to adult levels in livers of CV mice and were downregulated in livers of GF mice, as exemplified by the major drug-metabolizing enzymes cytochrome 3a (Cyp3a) family, which are prototypical pregnane X receptor (PXR)-target genes. Genes in Pattern-2 include Cyp1a2 (aryl hydrocarbon receptor-target gene), Cyp2c family, and Cyp2e1, which were all upregulated mainly at 90 days of age; as well as the peroxisome proliferator-activated receptor α (PPARα)-target genes Cyp4a family and Aldh3a2, which were upregulated not only in 90-days adult age, but also between neonatal and adolescent ages (from 1 to 30 days of age). Genes in Pattern-3 were enriched predominantly in livers of 15-day-old mice, among which the sterol-efflux transporter dimers Abcg5/Abcg8 were downregulated in GF mice. Genes in Pattern-4 were neonatal-enriched, among which the transporter Octn1 mRNA tended to be lower in GF mice at younger ages but higher in adult GF mice as compared with age-matched CV mice. Protein assays confirmed the downregulation of the PXR-target gene Cyp3a protein (Western-blot and liquid chromatography tandem mass spectroscopy), and decreased Cyp3a enzyme activities in male GF livers. Increased microsomal-Cyp4a proteins and nuclear-PPARα were also observed in male GF livers. Interestingly, in contrast to male livers, the mRNAs of Cyp2c or Cyp4a were not readily upregulated in female GF livers approaching adult age, suggesting the maturation of female-specific hormones interferes with the interactions between intestinal microbiota and DPG ontogeny. In conclusion, intestinal microbiota markedly impacts the ontogeny of many hepatic DPGs in a gender-specific manner.
•51 phospholipids are significantly different between SCZ patients and controls.•These significant phospholipids include PCs, LPCs, PEs, LPEs and SMs.•Extensive disturbances of phospholipids may be ...involved in the development of SCZ.•A panel of 6 metabolites could discriminate SCZ patients from healthy controls.
Schizophrenia (SCZ) is a multifactorial psychiatric disorder. However, the molecular pathogenesis of SCZ remains largely unknown, and no reliable diagnostic test is currently available. Phospholipid metabolism is known to be disturbed during disease processes of SCZ. In this study, we used an untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolic profiling approach to measure lipid metabolites in serum samples from 119 SCZ patients and 109 healthy controls, to identify potential lipid biomarkers for the discrimination between SCZ patients and healthy controls. 51 lipid metabolites were identified to be significant for discriminating SCZ patients from healthy controls, including phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs), phosphatidylethanolamines (PEs), lysophosphatidylethanolamines (LPEs) and sphingomyelins (SMs). Compared to healthy controls, most PCs and LPCs, as well as all PEs in patients were decreased, while most LPEs and all SMs were increased. A panel of six lipid metabolites could effectively discriminate SCZ patients from healthy controls with an area under the receiver-operating characteristic curve of 0.991 in the training samples and 0.980 in the test samples. These findings suggest that extensive disturbances of phospholipids may be involved in the development of SCZ. This LC-MS-based metabolic profiling approach shows potential for the identification of putative serum biomarkers for the diagnosis of SCZ.
Intestine is responsible for the biotransformation of many orally-exposed chemicals.The constitutive androstane receptor(CAR/Nr1i3) is known to up-regulate many genes encoding drugmetabolizing ...enzymes and transporters(drug-processing genes/DPGs) in liver,but less is known regarding its effect in intestine.Sixty-day-old wild-type and Car / mice were administered the CARligand TCPOBOP or vehicle once daily for 4 days.In wild-type mice,Car m RNA was down-regulated by TCPOBOP in liver and duodenum.Car / mice had altered basal intestinal expression of many DPGs in a section-specific manner.Consistent with the liver data(Aleksunes and Klaassen,2012),TCPOBOP upregulated many DPGs(Cyp2b10,Cyp3a11,Aldh1a1,Aldh1a7,Gsta1,Gsta4,Gstm1-m4,Gstt1,Ugt1a1,Ugt2b34,Ugt2b36,and Mrp2–4) in specific sections of small intestine in a CAR-dependent manner.However,the m RNAs of Nqo1 and Papss2 were previously known to be up-regulated by TCPOBOP in liver but were not altered in intestine.Interestingly,many known CAR-target genes were highest expressed in colon where CAR is minimally expressed,suggesting that additional regulators are involved in regulating their expression.In conclusion,CAR regulates the basal expression of many DPGs in intestine,and although many hepatic CAR-targeted DPGs were bona fide CAR-targets in intestine,pharmacological activation of CAR in liver and intestine are not identical.
The gut microbiome is increasingly recognized as a second genome that contributes to the health and diseases of the host. A major function of the gut microbiota is to convert primary bile acids (BAs) ...produced from cholesterol in the liver into secondary BAs that activate distinct host receptors to modulate xenobiotic metabolism and energy homeostasis. The goal of this study was to investigate to what extent oral exposure to an environmentally relevant polychlorinated biphenyl (PCBs mixture), namely the Fox River mixture, impacts gut microbiome and BA homeostasis. Ninety-day-old adult female conventional (CV) and germ-free (GF) C57BL/6 mice were orally exposed to corn oil (vehicle), or the Fox River mixture at 6 or 30 mg/kg once daily for 3 consecutive days. The PCB low dose profoundly increased BA metabolism related bacteria Akkermansia (A.) muciniphila, Clostridium (C.) scindens, and Enterococcus in the large intestinal pellet (LIP) of CV mice (16S rRNA sequencing/qPCR). This correlated with a PCB low dose-mediated increase in multiple BAs in serum and small intestinal content (SIP) in a gut microbiota-dependent manner (UPLC-MS/MS). Conversely, at PCB high dose, BA levels remained stable in CV mice correlated with an increase in hepatic efflux transporters and ileal Fgf15. Interestingly, lack of gut microbiota potentiated the PCB-mediated increase in taurine conjugated α and β muricholic acids in liver, SIP, and LIP. Pearson's correlation identified positive correlations between 5 taxa and most secondary BAs. In conclusion, PCBs dose-dependently altered BA homeostasis through a joint effort between host gut-liver axis and intestinal bacteria.
Little is known regarding the effect of intestinal microbiota modifiers, such as probiotics and conventionalization with exogenous bacteria, on host hepatic drug metabolism. Therefore, the goal of ...this study was to determine the effect of these modifiers on the expression of various drug-metabolizing enzymes of the host liver. VSL3 is a probiotic that contains eight live strains of bacteria. Five groups of mice were used: 1) conventional mice (CV), 2) conventional mice treated with VSL3 in drinking water, 3) germ-free (GF) mice, 4) GF mice treated with VSL3, and 5) GF mice exposed to the conventional environment for 2 months. All mice were 3 months old at tissue collection. GF conditions markedly downregulated the cytochrome P450 (P450) 3a gene cluster, but upregulated the Cyp4a cluster, whereas conventionalization normalized their expression to conventional levels reverse-transcription quantitative polymerase chain reaction (qPCR) and western blot. Changes in the Cyp3a and 4a gene expression correlated with alterations in the pregnane X receptor and peroxisome proliferator-activated receptor α-DNA binding, respectively (chromatin immunoprecipitation-qPCR). VSL3 increased each bacterial component in the large intestinal content of the CV mice, and increased these bacteria even more in GF mice, likely due to less competition for growth in the GF environment. VSL3 given to conventional mice increased the mRNAs of Cyp4v3, alcohol dehydrogenase 1, and carboxyesterase 2a, but decreased the mRNAs of multiple phase II glutathione-S-transferases. VSL3 given to germ-free mice decreased the mRNAs of UDP-glucuronosyltransferases 1a9 and 2a3. In conclusion, conventionalization and VSL3 alter the expression of many drug-metabolizing enzyme s in the liver, suggesting the importance of considering "bacteria-drug" interactions for various adverse drug reactions in patients.
The constitutive androstane receptor (CAR/Nr1i3) is an important xenobiotic-sensing nuclear receptor that is highly expressed in the liver and is well known to have species differences. During ...development, age-specific activation of CAR may lead to modified pharmacokinetics and toxicokinetics of drugs and environmental chemicals, leading to higher risks for adverse drug reactions in newborns and children. The goal of this study was to systematically investigate the age- and species-specific regulation of various drug-processing genes (DPGs) after neonatal or adult CAR activation in the livers of wild-type, CAR-null, and humanized CAR transgenic mice. At either 5 or 60 days of age, the three genotypes of mice were administered a species-appropriate CAR ligand or vehicle once daily for 4 days (i.p.). The majority of DPGs were differentially regulated by age and/or CAR activation. Thirty-six DPGs were commonly upregulated by CAR activation regardless of age or species of CAR. Although the cumulative mRNAs of uptake transporters were not readily altered by CAR, the cumulative phase I and phase II enzymes as well as efflux transporters were all increased after CAR activation in both species. In general, mouse CAR activation produced comparable or even greater fold increases of many DPGs in newborns than in adults; conversely, humanized CAR activation produced weaker induction in newborns than in adults. Western blotting and enzyme activity assays confirmed the age and species specificities of selected CAR-targeted DPGs. In conclusion, this study systematically compared the effect of age and species of CAR proteins on the regulation of DPGs in the liver and demonstrated that the regulation of xenobiotic biotransformation by CAR is profoundly modified by age and species.
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A major function of the gut microbiota is to convert primary bile acids (BAs), which are produced from cholesterol in liver, into secondary BAs generally thought to be more lipophilic ...and toxic. Key microbial reactions for BA metabolism include dehydroxylation and deconjugation. Accumulating evidence suggests that environmental exposure to xenobiotics may adversely impact various intermediary metabolism pathways. The goal of the present study was to investigate how oral exposure to polychlorinated biphenyls (PCBs), a class of persistent organic pollutants, impacts the BA homeostasis, and to what extent such interactions depend on gut microbiota. Ninety‐day‐old adult female conventional (CV) and germ‐free (GF) mice on a C57BL/6 background were orally exposed to corn oil (vehicle), or PCB mixture (Fox River Mix) at 6 or 30 mg/kg, once daily for 3‐consecutive days. The GF status was confirmed using 16S rRNA qPCR of universal bacteria in intestinal content of GF mice. Targeted BA metabolomics of 19 major BA metabolites was performed in liver, serum, small intestinal content (SIC), and large intestinal content (LIC) of these mice (n=5~6 per group). GF conditions markedly altered the BA homeostasis in various compartments. In liver and serum of GF mice, there was an increase in total BAs, conjugated BAs, as well as non‐12α‐OH BAs (which were produced through a Cyp8b1‐dependent pathway) compared to CV mice; conversely, total secondary BAs were diminished in GF mice. Intestinal compartment was influenced by GF conditions, with a marked increase in almost all host‐derived BAs but a decrease in microbial BAs (namely the total unconjugated and secondary BAs) in SIC and LIC (GF vehicle vs CV vehicle). PCBs did not affect the hepatic BA profiles or composition in CV or GF mice. However, PCBs at low dose increased serum total BAs, primary BAs, conjugated BAs, 12α‐OH BAs and non‐12α‐OH BAs in both CV and GF mice. PCBs at low dose also increased serum unconjugated BAs in CV mice but not in GF mice. PCBs at high dose did not affect serum BAs in either CV or GF mice. In SIC, PCBs at high dose also did not have any major effect on BA profiles in either CV or GF mice. However, PCBs at low dose increased total unconjugated and secondary BAs in a gut microbiota‐dependent manner. Although PCBs at low dose did not alter total BAs, primary BAs, conjugated BAs, and non‐12α‐OH BAs in SIC of CV mice, these BAs were increased in SIC of GF mice. In LIC, total conjugated BAs were increased by PCBs at both doses and in both strains of mice. In contrast, other types of BAs were not altered by PCBs in CV mice, but they were all markedly increased by PCBs at both doses in GF mice, suggesting that the lack of gut microbiota unmasked the PCB‐mediated effects on BA profiles. In summary, the present study has demonstrated that there is a novel interaction between PCB exposure and BA homeostasis that partially depends on the presence of gut microbiota. Whereas lack of gut microbiota alters the basal levels of BAs in multiple compartments of mice, both gut microbiota‐dependent and GF‐potentiation effects were observed on PCB‐mediated changes in BA homeostasis.
Support or Funding Information
Grace Liejun Guo
glg48@eohsi.rutgers.edu
Abstract only
Pharmacological activation of the constitutive androstane receptor (CAR) has been shown to protect against bile acid (BA)‐induced liver injury in adults (Beilke et al., 2009). During ...development, targeting CAR by agonists has been suggested to be one of the ultimate goals in the management and treatment of total parenteral nutrition related cholestasis in newborns (Hendaus, 2013). However, very little is known regarding the age‐ and species‐specific effects of CAR activation on BA homeostasis during development. The goal of this study was to use RNA sequencing and BA metabolomics to investigate the effects of CAR activation on the expression of genes involved in BA homeostasis and the BA profiles in 5‐ and 60‐day‐old wild‐type (WT) and humanized CAR transgenic (hCAR‐TG) mice. Male mice were i.p. administered corn oil or a species‐appropriate CAR ligand (TCPOBOP at 3mg/kg for mCAR, and CITCO at 30mg/kg for hCAR) once daily for 4 days. In control mice of both genotypes, from newborns to adulthood, there was a marked decrease in total serum BAs likely due to the maturation of the enterohepatic system. Specifically, serum total primary BAs and conjugated BAs were down‐regulated with age in both WT and hCAR‐TG mice. Conversely, there was a marked increase in serum total secondary BAs and unconjugated BAs, likely due to an ontogenic increase in intestinal bacteria for BA‐dehydroxylation and deconjugation. Following mCAR‐activation, there was an increase in serum total BAs, primary BAs, secondary BAs, and conjugated BAs at both Day 5 and 60. However, the increase in serum BAs was not observed following hCAR activation. In fact, serum cholic acid was decreased by CITCO in 60‐day‐old hCAR‐TG mice. Regarding hepatic BA synthesis, Cyp7a1 mRNA was minimally influenced by CAR activation regardless of the age or species. Conversely, the regulation of Cyp8b1 mRNA seems age‐specific, because it was up‐regulated by CAR of both species at Day 5, but was down‐regulated at Day 60. The regulation of many other BA‐synthetic enzymes at mRNA level was mCAR‐specific, evidenced by an induction of Akr1d1, but a reduction of Amacr, Hsd17b4, Hsd3b7, and Scp2 at both ages only by activation of mCAR but not hCAR. Age‐ and/or species‐specific regulatory effects of CAR activation were also observed for genes involved in BA conjugation and transport. However, several BA‐processing genes appeared to be “universal targets” of CAR of both species and at both ages, including a consistent up‐regulation of Cyp39a1, Akr1c14, Mrp3, and Mrp4. However, in general, mCAR activation produced greater degree of induction as compared to hCAR activation at the same age. In conclusion, the present study suggests that hCAR activation has less effects on serum BA metabolites as compared to mCAR, whereas mCAR and hCAR activation produce both age‐ and species‐specific effects on the hepatic expression of genes involved in BA homeostasis. Understanding the age‐ and species‐differences in the pharmacodynamics of CAR activation is essential in precision medicine for pediatric patients.
Support or Funding Information
Grace Liejun Guo
glg48@eohsi.rutgers.edu
Safety concerns have emerged regarding the potential long-lasting effects due to developmental exposure to xenobiotics. The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are ...critical xenobiotic-sensing nuclear receptors that are highly expressed in liver. The goal of this study was to test our hypothesis that neonatal exposure to PXR- or CAR-activators not only acutely but also persistently regulates the expression of drug-processing genes (DPGs). A single dose of the PXR-ligand PCN (75 mg/kg), CAR-ligand TCPOBOP (3 mg/kg), or vehicle (corn oil) was administered intraperitoneally to 3-day-old neonatal wild-type mice. Livers were collected 24 h post-dose or from adult mice at 60 days of age, and global gene expression of these mice was determined using Affymetrix Mouse Transcriptome Assay 1.0. In neonatal liver, PCN up-regulated 464 and down-regulated 449 genes, whereas TCPOBOP up-regulated 308 and down-regulated 112 genes. In adult liver, there were 15 persistently up-regulated and 22 persistently down-regulated genes following neonatal exposure to PCN, as well as 130 persistently up-regulated and 18 persistently down-regulated genes following neonatal exposure to TCPOBOP. Neonatal exposure to both PCN and TCPOBOP persistently down-regulated multiple Cyp4a members, which are prototypical-target genes of the lipid-sensor PPARα, and this correlated with decreased PPARα-binding to the Cyp4a gene loci. RT-qPCR, western blotting, and enzyme activity assays in livers of wild-type, PXR-null, and CAR-null mice confirmed that the persistent down-regulation of Cyp4a was PXR and CAR dependent. In conclusion, neonatal exposure to PXR- and CAR-activators both acutely and persistently regulates critical genes involved in xenobiotic and lipid metabolism in liver.
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