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Reports on the enzyme cytosolic aspartate aminotransferase which displays liver-specific hormonal regulation. Increase of the activity and mRNA level of cAspAT by glucocorticoids; Insertion of deletion fragments; Stable transfection experiments; Physical separation of the positive and negative control elements.

BACKGROUND: Exposure to persistent organic pollutants (POPs) has been associated with the progression of chronic liver diseases, yet the contribution of POPs to the development of fibrosis in non-alcoholic fatty liver disease (NAFLD), a condition closely linked to obesity, remains poorly documented. [ABSTRACT FROM AUTHOR]

Cytochrome P450 2E1 (CYP2E1) gene expression is known to be induced by interleukin-4 (IL4) and repressed by inflammatory cytokines, such as interleukin-1β3 (IL1β3) in human hepatocytes. The mechanisms involved in these transcriptional regulations remain elusive. In order to study these mechanisms, various constructs of the human CYP2E1 promoter were prepared and transfected into the human HepG2 hepatoma cell line. Our findings revealed that an IL4-responsive region of 128 bp (-671/-544) was required to mediate induction by IL4. IL1β caused moderate but significant decrease of the promoter activity, which was abolished when the two cytokines were combined. The IL1β inhibitory effect is mediated through a regulatory sequence independent of that of IL4. Furthermore, by using specific signaling pathway inhibitors, we demonstrated that IL4 activation required protein kinase C (PKC) activation. In addition, our results suggest that induction by IL4 was not dependent on a single binding site but rather on a complex region which includes putative binding sites for signal transducer and activator of transcription (STAT)6, activator protein (AP)-1, nuclear factor kappa-B (NFκB), nuclear factor of activated T cells (NFAT) and CCAAT enhancer binding protein (C/EBP). Electrophoretic mobility shift assays suggest that AP1 and NFAT transcription factors are able to bind to three sites in the IL4-responsive region. [Copyright &y& Elsevier]

Chlordane compounds (CHLs) are components of technical chlordane listed in the Stockholm convention on persistent organic pollutants identified as endocrine disrupting chemicals (EDCs) and may interfere with hormone biosynthesis, metabolism or action resulting in an unbalanced hormonal function. There is increasing scientific evidence showing EDCs as risk factors in the pathogenesis and development of obesity and obesity-related metabolic syndromes such as type 2 diabetes, but there is no systematized information on the effect of CHLs in humans. Our aim is to identify the epidemiological data on the association between CHLs with adiposity and diabetes using a systematic approach to identify the available data and summarizing the results through meta-analysis. We searched PubMed and Web of Science from inception up to 15 February 2021, to retrieve original data on the association between chlordanes, and adiposity or diabetes. For adiposity, regression coefficients and Pearson or Spearman correlation coefficients were extracted and converted into standardized regression coefficients. Data were combined using fixed effects meta-analyses to compute summary regression coefficients and corresponding 95% confidence intervals (95% CI). For the association between chlordanes and diabetes, Odds ratios (ORs) were extracted and the DerSimonian and Laird method was used to compute summary estimates and respective 95% CI. For both, adjusted estimates were preferred, whenever available. Among 31 eligible studies, mostly using a cross-sectional approach, the meta-analysis for adiposity was possible only for oxychlordane and transchlordane, none of them were significantly associated with adiposity [(β = 0.04, 95% CI 0.00; 0.07, I2 = 89.7%)] and (β = 0.02, 95% CI − 0.01; 0.06), respectively. For diabetes, the estimates were positive for all compounds but statistically significant for oxychlordane [OR = 1.96 (95% CI 1.19; 3.23)]; for trans-nonachlor [OR = 2.43 (95% CI 1.64; 3.62)] and for heptachlor epoxide [OR = 1.88 (95% CI 1.42; 2.49)]. Our results support that among adults, the odds of having diabetes significantly increase with increasing levels of chlordanes. The data did not allow to reach a clear conclusion regarding the association with adiposity. [ABSTRACT FROM AUTHOR]

The mechanisms by which pollutants participate in the development of diverse pathologies are not completely understood. The pollutant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) activates the AhR (aryl hydrocarbon receptor) signaling pathway. We previously showed that TCDD (25 nM, 30 h) decreased the expression of several alcohol metabolism enzymes (cytochrome P450 2E1, alcohol dehydrogenases ADH1, 4 and 6) in differentiated human hepatic cells (HepaRG). Here, we show that, as rapidly as 8 h after treatment (25 nM TCDD) ADH expression decreased 40 % ( p < 0.05). ADH1 and 4 protein levels decreased 40 and 27 %, respectively ( p < 0.05), after 72 h (25 nM TCDD). The protein half-lives were not modified by TCDD which suggests transcriptional regulation of expression. The AhR antagonist CH-223191 or AhR siRNA reduced the inhibitory effect of 25 nM TCDD on ADH1A, 4 and 6 expression 50-100 % ( p < 0.05). The genomic pathway (via the AhR/ARNT complex) and not the non-genomic pathway involving c-SRC mediated these effects. Other AhR ligands (3-methylcholanthrene and PCB 126) decreased ADH1B, 4 and 6 mRNAs by more than 78 and 55 %, respectively ( p < 0.01). TCDD also regulated the expression of ADH4 in the HepG2 human hepatic cell line, in primary human hepatocytes and in C57BL/6J mouse liver. In conclusion, activation of the AhR/ARNT signaling pathway by AhR ligands represents a novel mechanism for regulating the expression of ADHs. These effects may be implicated in the toxicity of AhR ligands as well as in the alteration of ethanol or retinol metabolism and may be associated further with higher risk of liver diseases or/and alcohol abuse disorders. [ABSTRACT FROM AUTHOR]

It is well known that several metals, such as lead, mercury, cadmium, and vanadium, can mimic the effects of estrogens (metallo-estrogens). Nevertheless, there are only a few studies that have assessed the effects of toxic metals on the female genital tract and, in particular, endometrial tissue. In this context, we measured the concentrations of several trace elements in human endometrial tissue samples from individuals with hyperplasia or adenocarcinoma and in normal tissues. Hyperplasic endometrial tissue has a 4-fold higher concentration of mercury than normal tissue. Mercury can affect both the AhR and ROS signaling pathways. Thus, we investigated the possible toxic effects of mercury by in vitro studies. We found that mercury increases oxidative stress (increased HO1 and NQO1 mRNA levels) and alters the cytoskeleton in the human endometrial Ishikawa cell line and to a lesser extent, in the “less-differentiated” human endometrial Hec-1b cells. The results might help to explain a potential link between this metal and the occurrence of endometrial hyperplasia. [ABSTRACT FROM AUTHOR]

Individuals, typically, are exposed to mixtures of environmental xenobiotics affecting multiple organs and acting through different xenosensors and pathways in species and cell-type specific manners. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and α-endosulfan are Persistent Organic Pollutants (POPs) and endocrine disruptors which act through different xenosensors and accumulate in the liver. Our objective in this HEALS study was to investigate the effects of the mixture of these POPs on gene expression in a human-derived hepatocyte cell line, HepaRG. We found that, in spite of having largely uncorrelated effects, TCDD and α-endosulfan, when mixed, alter the expression of genes. The combined effects of the mixture of the POPs significantly altered the expression of 100 genes (42 up- and 58 down-regulated) whereas the same concentration of either POP alone did not alter significantly the expression of these genes. For 32 other genes, selective inhibitory crosstalk between TCDD and α-endosulfan was observed. One of the POPs inhibited the effect, on gene expression, of the other in the mixture although, when used alone, that POP did not affect expression. The expression of another 82 genes was significantly altered (up- or down-regulated) by a single POP. The addition of the second POP either increased, in the same direction, the effect on gene expression or had no further effect. At low concentrations (0.2 nM TCDD and 1 μM α-endosulfan), the POPs still had significant effects and the levels of expression of the corresponding proteins were found to be affected for some genes. Particularly striking was the 80–90% inhibition, by the mixture, of the expression of a number of genes of several hepatic intermediary metabolic pathways (glycerolipid metabolism, FXR/RXR activation, glycolysis/gluconeogenesis, retinoid and bile acid biosynthesis), whereas each pollutant alone had only a moderate effect. [ABSTRACT FROM AUTHOR]

The contribution of environmental pollutants to liver fibrosis is an important and poorly explored issue. In vitro studies suggest that the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands induce several genes that are known to be upregulated during liver fibrosis. Our aim was to determine whether exposure to such pollutants can lead to liver fibrosis and to characterize the mechanisms of action. Mice were treated for 2, 14, or 42 days, once a week with 25 µg/kg of TCDD. Gene and protein expression, in vitro and in vivo, as well as liver histology were investigated for each treatment. Treatment of mice with TCDD for 2 weeks modified the hepatic expression of markers of fibrosis such as collagen 1A1 and α-smooth muscle actin. This is not observed in AhR knockout mice. Following 6 weeks of treatment, histological features of murine hepatic fibrosis became apparent. In parallel, the levels of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor α) and of markers of activated fibroblasts(fibroblast-specific protein 1) were found to be upregulated. Interestingly, we also found increased expression of genes of the TGF-β pathway and a concomitant decrease of miR-200a levels. Because the transcription factors of the Snail family were shown to be involved in liver fibrosis, we studied their regulation by TCDD. Two members of the Snail family were increased, whereas their negative targets, the epithelial marker E-cadherin and Claudin 1, were decreased. Further, the expression of mesenchymal markers was increased. Finally, we confirmed that Snai2 is a direct transcriptional target of TCDD in the human hepatocarcinoma cell line, HepG2. The AhR ligand, TCDD, induces hepatic fibrosis by directly regulating profibrotic pathways. [ABSTRACT FROM AUTHOR]

Background: It is a major clinical challenge to predict which patients, with advanced stage head and neck squamous cell carcinoma, will not exhibit a reduction in tumor size following induction chemotherapy in order to avoid toxic effects of ineffective chemotherapy and delays for instituting other therapeutic options. Further, it is of interest to know to what extent a gene signature, which identifies patients with tumors that will not respond to a particular induction chemotherapy, is applicable when additional chemotherapeutic agents are added to the regimen. Methodology/Principal Findings: To identify genes that predict tumor resistance to induction with cisplatin/5-fluorouracil (PF) or PF and a taxane, we analyzed patient tumor biopsies with whole genome microarrays and quantitative reverse transcriptase-PCR (TLDA) cards. A leave one out cross-validation procedure allowed evaluation of the prediction tool. A ten- gene microarray signature correctly classified 12/13 responders and 7/10 non-responders to PF (92% specificity, 82.6% accuracy). TLDA analysis (using the same classifier) of the patients correctly classified 12/12 responders and 8/10 non-responders (100% specificity, 90.9% accuracy). Further, TLDA analysis correctly predicted the response of 5 new patients and, overall, 12/12 responders and 13/15 non-responders (100% specificity, 92.6% accuracy). The protein products of the genes constituting the signature physically associate with 27 other proteins, involved in regulating gene expression, constituting an interaction network. In contrast, TLDA-based prediction (with the same gene signature) of responses to induction with PF and either of two taxanes was poor (0% specificity, 25% accuracy and 33.3% specificity, 25% accuracy). Conclusions/Significance: Successful transfer of the microarray-based gene signature to an independent, PCR-based technology suggests that TLDA-based signatures could be a useful hospital-based technology for determining therapeutic options. Although highly specific for tumor responses to PF induction, the gene signature is unsuccessful when taxanes are added. The results illustrate the subtlety in developing "personalized medicine". [ABSTRACT FROM AUTHOR]

Abstract: TCDD (2,3,7,8-tetrachlorodibenzodioxin), a highly persistent environmental pollutant and a human carcinogen, is the ligand with the highest affinity for the Aryl Hydrocarbon Receptor (AhR) that induces via the AhR, xenobiotic metabolizing enzyme genes as well as several other genes. This pollutant elicits a variety of systemic toxic effects, which include cancer promotion and diverse cellular alterations that modify cell cycle progression and cell proliferation. Large-scale studies have shown that the expression of Son of Sevenless 1 (SOS1), the main mediator of Ras activation, is one of the targets of dioxin in human cultured cells. In this study, we investigated the regulation of the previously uncharacterized SOS1 gene promoter by the AhR and its ligands in the human hepatocarcinoma cell line, HepG2. We found that several environmental pollutants (AhR ligands) induce SOS1 gene expression by increasing its transcription. Chromatin immunoprecipitation experiments demonstrated that the AhR binds directly and activates the SOS1 gene promoter. We also showed that dioxin treatment leads to an activated Ras-GTP state, to ERK activation and to accelerated cellular proliferation. All these effects were mediated by SOS1 induction as shown by knock down experiments. Our data indicate that dioxin-induced cellular proliferation is mediated, at least partially, by SOS1 induction. Remarkably, our studies also suggest that SOS1 induction leads to functional effects similar to those elicited by the well-characterized oncogenic Ras mutations. [ABSTRACT FROM AUTHOR]

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant that binds the aryl hydrocarbon receptor (AhR), a transcription factor that triggers various biological responses. In this study, we show that TCDD treatment counteracts the p53 activation (phosphorylation and acetylation) elicited by a genotoxic compound, etoposide, in the human hepatocarcinoma cell line HepG2 and we delineated the mechanisms of this interaction. Using small interfering RNA knockdown experiments, we found that the newly described metastasis marker, anterior gradient-2 (AGR2), is involved in this effect. Both AGR2 messenger RNA (mRNA) and protein levels were increased (sixfold and fourfold, respectively) by TCDD treatment, and this effect was mediated by the AhR receptor. The half-life of AGR2 mRNA was unchanged by TCDD treatment. Analysis of the promoter of the AGR2 gene revealed three putative xenobiotic-responsive elements (XREs) in the proximal 3.5-kb promoter. Transient transfection of HepG2 cells by the Gaussia luciferase reporter gene driven by various deleted and mutated fragments of the promoter indicated that only the most proximal XRE was active. Binding of the AhR to the endogenous AGR2 promoter was also triggered by TCDD treatment. These results suggest that AhR ligands such as TCDD might contribute to tumor progression by inhibiting p53 regulation (phosphorylation and acetylation) triggered by genotoxicants via the increased expression of the metastasis marker AGR2. [ABSTRACT FROM PUBLISHER]

Abstract: In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) α agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPARα agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at −574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum. [Copyright &y& Elsevier]

We show that cytosolic aspartate aminotransferase (cAspAT) is involved in adipocyte glyceroneogenesis, a regulated pathway that controls fatty acid homeostasis by promoting glycerol 3-phosphate formation for fatty acid re-esterification during fasting. cAspAT activity, as well as the incorporation of [14C]aspartate into the neutral lipid fraction of 3T3-F442A adipocytes was stimulated by the thiazolidinedione (TZD) rosiglitazone. Conversely, the ratio of fatty acid to glycerol released into the medium decreased. Regulation of cAspAT gene expression was specific to differentiated adipocytes and did not require any peroxisome proliferator-activated receptor γ (PPARγ)/retinoid X receptor-α direct binding. Nevertheless, PPARγ is indirectly necessary for both cAspAT basal expression and TZD responsiveness because they are, respectively, diminished and abolished by ectopic overexpression of a dominant negative PPARγ. The cAspAT TZD-responsive site was restricted to a single AGGACA hexanucleotide located at -381 to -376 bp whose mutation impaired the specific RORα binding. RORα ectopic expression activated the cAspAT gene transcription in absence of rosiglitazone, and its protein amount in nuclear extracts is 1.8-fold increased by rosiglitazone treatment of adipocytes. Finally, the amounts of RORα and cAspAT mRNAs were similarly increased by TZD treatment of human adipose tissue explants, confirming coordinated regulation. Our data identify cAspAT as a new member of glyceroneogenesis, transcriptionally regulated by TZD via the control of RORα expression by PPARγ in adipocytes. [ABSTRACT FROM AUTHOR]

Abstract: The uptake of oxidized LDL by macrophages is a key event in the development of atherosclerosis. The scavenger receptor CD36 is one major receptor that internalizes oxidized LDL. In differentiated human macrophages, we compared the regulation of CD36 expression by copper-oxidized LDL or their products. Only oxidized derivatives of cholesteryl ester (CEOOH) increased the amount of CD36 mRNA (2.5-fold). Both oxidized LDL and CEOOH treatment increased two to fourfold the transcription of promoters containing peroxisome-proliferator-activated-receptor responsive elements (PPRE) in the presence of PPARα or γ. Electrophoretic-mobility-shift-assays with nuclear extracts prepared from macrophages treated by either oxidized LDL or CEOOH showed increased binding of PPARα to the CD36 gene promoter PPRE. In conclusion, CEOOH present in oxidized LDL increase CD36 gene expression in a pathway involving PPARα. [Copyright &y& Elsevier]

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) activator, increases the expression of the cytosolic aspartate aminotransferase (cAspAT) gene in human liver cells, which may partially explain the increase of this enzyme in the serum of individuals undergoing fenofibrate treatment. Conversely, in rodents, fenofibrate represses the expression of the cAspAT gene. We compared the mechanisms of fenofibrate action in human and rat hepatoma cells. Transfection assays of the wild-type and mutated rat promoters in Fao and H4IIEC3 cells established a critical role for sequences similar to nuclear receptor responsive elements in the −404/−366 bp region. Nuclear proteins bound to these sequences and the amounts of protein bound were decreased by fenofibrate treatment, probably accounting for the decreased gene expression. Pharmacological studies confirmed the involvement of PPARα. However, this receptor did not bind directly to these sequences. The human promoter was cloned and the regulatory region localized between −2663/−706 bp. Co-transfection assays suggested that, in humans, the PPARα was also involved in the increase in expression of the cAspAT gene due to fibrates, without the presence of a canonical PPAR responsive element. [Copyright &y& Elsevier]

Exposure to environmental chemicals has become one of the major concerns in the past decades. Phthalates are a family of synthetic organic chemicals used in the manufacture of plastics, solvents, and personal care products. These compounds are considered as endocrine-disrupting compounds (EDCs) since they may interfere with the endocrine system and disrupt its physiologic function. The purpose of this work is to synthesize results from published literature on the association between the exposure to phthalates and adiposity in adults and children. We searched PubMed from inception up to 01 August 2019, to retrieve original papers reporting data on the association between EDCs and adiposity, using the following search expression: (("Endocrine disruptor" OR Endocrine disruptor[mh] OR phthalate) AND (Obesity OR Overweight OR BMI OR "Body fat" OR Adipose tissue[mh] OR Body size[mh] OR "body size" OR "body weight" OR Anthropometry OR "anthropometric measures")) AND (humans[mh]). The study variables and characteristics were collected during data extraction, namely the study design, sample, exposure, outcome, descriptive and association measures. Study quality was assessed using the STROBE template for observational studies. Although studies examined several adiposity measures, Body Mass Index (BMI) and Waist Circumference (WC) were the most commonly used, therefore, we used the beta coefficients regarding BMI and WC, and odds ratios when BMI outcome was categorical to perform the meta-analysis. Data from the studies were combined using fixed effects meta-analyses to compute summary regression coefficients or odds ratios and corresponding 95% confidence intervals (95% CI). Heterogeneity between studies was assessed by the I2 statistic. In the systematic review we found 29 publications addressing the association between phthalate compounds and adiposity. The vast majority of the included studies reported associations that were not statistically significant. For most of the phthalate compounds there were few studies providing compatible measures and therefore it was not possible to combine the results in a meta-analysis. Both for BMI and WC, the meta-analysis for MiBP, MCPP and MbzP showed negative associations and null association for MBP in children, although none of them was significant. For MEP, positive but not significant associations were found both in children and adults. Conversely, for MEHP a negative association was found also in children and adults although it did not reach statistical significance. Only for MECPP a significant association was found for obesity in adults (OR = 1.67 (95% CI 1.30; 2.16). In general, a positive association between phthalates and adiposity measures was found, especially in adults. However, most of the results did not reach statistical significance and the inconsistencies found between studies did not allow to reach a definitive conclusion. Additionally, we cannot exclude a possible effect of publication bias. • The results tend to be consistent whatever the measure of adiposity used. • The associations were strong for adults than for children. • In general, MEP, MiBP, MMP, MEOHP and MEHHP showed a positive association. • Overall inconsistent results were found for MbP, MEHP and MbzP. • The scarcity of longitudinal studies hind the interpretation of causality. [ABSTRACT FROM AUTHOR]

Individuals as well as entire ecosystems are exposed to mixtures of Persistent Organic Pollutants (POPs). Previously, we showed, by a non-targeted approach, that the expression of several genes involved in carbohydrate metabolism was almost completely inhibited in the human hepatic cell line HepaRG following exposure to a mixture of the organochlorine insecticide alpha-endosulfan and 2,3,7,8 tetrachlorodibenzo-p-dioxin. In this European HEALS project, which studies the effects of the exposome on human health, we used a Physiologically Based BioKinetic model to compare the concentrations previously used in vitro with in vivo exposures for humans. We investigated the effects of these POPs on the levels of proteins, on glycogen content, glucose production and the oxidation of glucose into CO 2 and correlated them to the expression of genes involved in carbohydrate metabolism as measured by RT-qPCR. Exposure to individual POPs and the mixture decreased the expression of the proteins investigated as well as glucose output (up to 82%), glucose oxidation (up to 29%) and glycogen content (up to 48%). siRNAs that specifically inhibit the expression of several xenobiotic receptors were used to assess receptor involvement in the effects of the POPs. In the HepaRG model, we demonstrate that the effects are mediated by the aryl hydrocarbon receptor and the estrogen receptor alpha, but not the pregnane X receptor or the constitutive androstane receptor. These results provide evidence that exposure to combinations of POPs, acting through different signaling pathways, may affect, more profoundly than single pollutants alone, metabolic pathways such as carbohydrate/energy metabolism and play a potential role in pollutant associated metabolic disorders. • TCDD and α-endosulfan mixture decreases carbohydrate metabolism in liver cells. • PBBK model of TCDD and α-endosulfan in human liver. • α-endosulfan acts via estrogen α receptor in HepaRG cells. • Glucose oxidation and production and glycogen content are decreased by the mixture. [ABSTRACT FROM AUTHOR]