Assessing the contribution of the chemical exposome to neurodegenerative disease.

Over the past few decades, numerous environmental chemicals from solvents to pesticides have been suggested to be involved in the development and progression of neurodegenerative diseases. Most of the evidence has accumulated from occupational or cohort studies in humans or laboratory research in animal models, with a range of chemicals being implicated. What has been missing is a systematic approach analogous to genome-wide association studies, which have identified dozens of genes involved in Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases. Fortunately, it is now possible to study hundreds to thousands of chemical features under the exposome framework. This Perspective explores how advances in mass spectrometry make it possible to generate exposomic data to complement genomic data and thereby better understand neurodegenerative diseases.

Determination of bisphenol A and bisphenol S concentrations and assessment of estrogen- and anti-androgen-like activities in thermal paper receipts from Brazil, France, and Spain.

Bisphenol A (BPA) is a high-production-volume chemical with endocrine disrupting properties commonly used as color developer in thermal paper. Concerns about the potential hazards of human BPA exposure have led to the increasing utilization of alternatives such as bisphenol S (BPS) and bisphenol F (BPF). This study was designed to assess: (i) BPA, BPS, and BPF concentrations in 112 thermal paper receipts from Brazil, France, and Spain by liquid chromatography coupled to mass spectrometry (LC-MS); and (ii) hormone-like activities of these receipts using two receptor-specific bioassays, the E-Screen for (anti-)estrogenicity and PALM luciferase assay for (anti-)androgenicity. BPA was present in 95.3% of receipts from Spain, 90.9% of those from Brazil, and 51.1% of those from France at concentrations up to 20.27 mg/g of paper. Only two samples from Brazil, two from Spain, and ten from France had a BPS concentration ranging from 6.46 to 13.29 mg/g; no BPA or BPS was detected in 27.7% of French samples. No BPF was detected in any receipt. Estrogenic activity was observed in all samples from Brazil and Spain and in 74.5% of those from France. Anti-androgenic activity was observed in > 90% of samples from Brazil and Spain and in 53.2% of those from France. Only 25.5% of French samples were negative for both estrogenic and anti-androgenic activity. Estrogenic and anti-androgenic activities per gram of paper were up to 1.411 µM estradiol (E2) equivalent units (E2eq) and up to 359.5 mM procymidone equivalent units (Proceq), respectively. BPA but not BPS concentrations were positively correlated with both estrogenic and anti-androgenic activities. BPA still dominates the thermal paper market in Brazil and Spain, and BPS appears to be one of the main alternatives in France. There is an urgent need to evaluate the safety of alternatives proposed to replace BPA as developer in thermal printing. The large proportion of samples with hormonal activity calls for the adoption of preventive measures.

Epigenetics as a mechanism linking developmental exposures to long-term toxicity.

A variety of experimental and epidemiological studies lend support to the Developmental Origin of Health and Disease (DOHaD) concept. Yet, the actual mechanisms accounting for mid- and long-term effects of early-life exposures remain unclear. Epigenetic alterations such as changes in DNA methylation, histone modifications and the expression of certain RNAs have been suggested as possible mediators of long-term health effects of environmental stressors. This report captures discussions and conclusions debated during the last Prenatal Programming and Toxicity meeting held in Japan. Its first aim is to propose a number of criteria that are critical to support the primary contribution of epigenetics in DOHaD and intergenerational transmission of environmental stressors effects. The main criteria are the full characterization of the stressors, the actual window of exposure, the target tissue and function, the specificity of the epigenetic changes and the biological plausibility of the linkage between those changes and health outcomes. The second aim is to discuss long-term effects of a number of stressors such as smoking, air pollution and endocrine disruptors in order to identify the arguments supporting the involvement of an epigenetic mechanism. Based on the developed criteria, missing evidence and suggestions for future research will be identified. The third aim is to critically analyze the evidence supporting the involvement of epigenetic mechanisms in intergenerational and transgenerational effects of environmental exposure and to particularly discuss the role of placenta and sperm. While the article is not a systematic review and is not meant to be exhaustive, it critically assesses the contribution of epigenetics in the long-term effects of environmental exposures as well as provides insight for future research.

Pyruvate carboxylase deficiency: An underestimated cause of lactic acidosis.

Pyruvate carboxylase (PC) is a biotin-containing mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, thereby being involved in gluconeogenesis and in energy production through replenishment of the tricarboxylic acid (TCA) cycle with oxaloacetate. PC deficiency is a very rare metabolic disorder. We report on a new patient affected by the moderate form (the American type A). Diagnosis was nearly fortuitous, resulting from the revision of an initial diagnosis of mitochondrial complex IV (C IV) defect. The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Intellectual disability was noted at this time. Amino acids in plasma and organic acids in urine did not show patterns of interest for the diagnostic work-up. In skin fibroblasts PC showed no detectable activity whereas biotinidase activity was normal. We had previously reported another patient with the severe form of PC deficiency and we show that she also had secondary C IV deficiency in fibroblasts. Different anaplerotic treatments in vivo and in vitro were tested using fibroblasts of both patients with 2 different types of PC deficiency, type A (patient 1) and type B (patient 2). Neither clinical nor biological effects in vivo and in vitro were observed using citrate, aspartate, oxoglutarate and bezafibrate. In conclusion, this case report suggests that the moderate form of PC deficiency may be underdiagnosed and illustrates the challenges raised by energetic disorders in terms of diagnostic work-up and therapeutical strategy even in a moderate form.

The aryl hydrocarbon receptor regulates focal adhesion sites through a non-genomic FAK/Src pathway.

The aryl hydrocarbon receptor (AhR) is commonly described as a transcription factor, which regulates xenobiotic-metabolizing enzymes. Recent studies have suggested that the binding of ligands to the AhR also activates the Src kinase. In this manuscript, we show that the AhR, through the activation of Src, activates focal adhesion kinase (FAK) and promotes integrin clustering. These effects contribute to cell migration. Further, we show that the activation of the AhR increases the interaction of FAK with the metastatic marker, HEF1/NEDD9/CAS-L, and the expression of several integrins. Xenobiotic exposure, thus, may contribute to novel cell-migratory programs.

Effect of quercetin on paraoxonase 1 activity--studies in cultured cells, mice and humans.

There is increasing evidence that the HDL-associated enzyme paraoxonase 1 (PON1) may have a protective function in the atherosclerotic process. An enhancement of PON1 activity by dietary factors including flavonoids is therefore of interest. Quercetin, a flavonol frequently present in fruits and vegetables has been shown to induce PON1 in cultured liver cells, but the in vivo efficacy of a dietary quercetin supplementation has yet not been evaluated. To this end, we fed laboratory mice quercetin-enriched diets with quercetin concentrations ranging from 0.05 to 2 mg/g diet for 6 weeks and determined the expression of the hepatic PON1 gene and its protein levels. Since we could establish a moderate but significant induction of PON1 mRNA levels by dietary quercetin in mice, we aimed to proof whether healthy human volunteers, given graded supplementary quercetin (50, 100 or 150 mg/day) for two weeks, would respond with likewise enhanced plasma paraoxonase activities. However, PON1 activity towards phenylacetate and paraoxon was not changed following quercetin supplementation in humans. Differences between mice and humans regarding the PON1 inducing activity of quercetin may be related to differences in quercetin metabolism. In mice, unlike in humans, a large proportion of quercetin is methylated to isorhamnetin which exhibits, according to our reporter gene data in cultured liver cells, a potent PON1 inducing activity.

Nedd9/Hef1/Cas-L mediates the effects of environmental pollutants on cell migration and plasticity.

Aryl hydrocarbon receptor (AhR), or dioxin receptor, is a transcription factor that induces adaptive metabolic pathways in response to environmental pollutants. Recently, other pathways were found to be altered by AhR and its ligands. Indeed, developmental defects elicited by AhR ligands suggest that additional cellular functions may be targeted by this receptor, including cell migration and plasticity. Here, we show that dioxin-mediated activation of Ahr induces Nedd9/Hef1/Cas-L, a member of the Cas protein family recently identified as a metastasis marker. The Hef1 gene induction is mediated by two xenobiotic responsive elements present in this gene promoter. Moreover, using RNA interference, we show that Nedd9/Hef1/Cas-L mediates the dioxin-elicited changes related to cell plasticity, including alterations of cellular adhesion and shape, cytoskeleton reorganization, and increased cell migration. Furthermore, we show that both E-cadherin repression and Jun N-terminal kinases activation by dioxin and AhR also depend on the expression of Nedd9/Hef1/Cas-L. Our study unveils, for the first time, a link between pollutants exposure and the induced expression of a metastasis marker and shows that cellular migration and plasticity markers are regulated by AhR and its toxic ligands.

Expression of inosine monophosphate dehydrogenase type I and type II after mycophenolate mofetil treatment: a 2-year follow-up in kidney transplantation.

The objective of the study was to evaluate the effect of mycophenolate mofetil (MMF) on the regulation of inosine monophosphate dehydrogenase (IMPDH) during the first 2 years after renal transplantation. Twelve patients were enrolled, and 10-h time-course evaluations of the effects of MMF were regularly performed during the study. IMPDH activity and gene expression were measured in whole blood and in mononuclear cells, respectively. Type I IMPDH (IMPDH-I) mRNA was increased during the first 3 months following transplantation and reached its maximal level during acute rejection episodes, whereas type II IMPDH mRNA was stable. Furthermore, although no alteration in the predose samples was observed, patients with prolonged MMF treatment exhibited an increase in the induction potency of both IMPDH activity and gene expression. In vitro experiments confirmed that IMPDH-I is inducible, but preferentially in monocytes than in lymphocytes. This finding suggests that the measurement of IMPDH mRNAs may provide reliable information to predict acute rejection.

Activation of the dioxin/aryl hydrocarbon receptor (AhR) modulates cell plasticity through a JNK-dependent mechanism.

Environmental chemicals such as dioxin adversely affect immune, neurological and reproductive functions and have been implicated in cancer development. However, the mechanisms responsible for dioxin toxicity are still poorly understood. Here, we show that dioxin and related pollutants trigger a marked morphological change in epithelial cells that remodel their cytoskeleton to increase interaction with extra cellular matrix while loosening cell-cell contacts. Furthermore, dioxin-treated cells show increased motility. These dioxin-mediated effects are mimicked by constitutive expression and activation of the intracellular dioxin receptor (aryl hydrocarbon receptor (AhR)). They correlate with activation of the Jun NH2-terminal kinase (JNK) and are reverted by treatment with a JNK inhibitor. Dioxin-induced effects occur 48 h post-treatment initiation, a time scale, which argues for a genomic effect of the AhR, linked to induction of target genes. This novel Ahr action on cell plasticity points to a role in cancer progression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces insulin-like growth factor binding protein-1 gene expression and counteracts the negative effect of insulin.

Recent epidemiological studies have revealed a possible correlation between exposure to high levels of dioxins or dioxin-like compounds and diabetes. Yet the interaction between insulin and dioxin actions remains elusive. We studied the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), a protein involved in glucose homeostasis and whose expression is down-regulated by insulin. We showed that 2,3,7,8-tetrachorodibenzo-p-dioxin (TCDD) specifically induced IGFBP-1 mRNA in human hepatocytes and HepG2 human hepatoma cells (2.5- and 8-fold, respectively). Cellular and secreted IGFBP-1 protein levels were also up-regulated. Transfection and reporter assays showed that the IGFBP-1 promoter was activated by TCDD and that this activation was dependent on the integrity of a proximal xenobiotic-responsive element (XRE). This XRE, located near the insulin-glucocorticoid regulatory region, binds the aryl-hydrocarbon receptor. In agreement with previous studies, the IGFBP-1 promoter was down-regulated by insulin (50%); we show here that although TCDD activated the IGFBP-1 promoter 5- to 6-fold, the combination of TCDD and insulin led to an expression level of IGFBP-1 that was higher than basal level (2- to 3-fold activation). Similar regulations were observed for the endogenous IGFBP-1 mRNA. These data suggest that the xenobiotic-hormonal regulatory region of the IGFBP-1 promoter mediates an up-regulation of IGFBP-1 expression by TCDD even in the presence of insulin. Because IGFBP-1 modulates blood glucose levels, the up-regulation of IGFBP-1 by dioxins might account for the disruptive effects of these pollutants on glucose metabolism.

Calcineurin activity as a functional index of immunosuppression after allogeneic stem-cell transplantation.

BACKGROUND: The authors have previously shown that mononuclear cells derived from patients with resistant chronic graft-versus-host-disease (GVHD) express high calcineurin (CN) activity, suggesting that in vitro assessment of CN activity may be a useful index to estimate the degree of immunosuppression afforded by cyclosporine A (CsA). The goal of this study was to assess CN activity during the first 2 months after allogeneic stem-cell transplantation (SCT) and to correlate its evolution with the occurrence of acute GVHD. METHODS: Thirty-one allogeneic SCT recipients were enrolled during a 21-month period. All received GVHD prophylaxis with CsA (2 mg/kg/day) and methotrexate (on days 1, 3, and 6). CN activity was measured before transplant, and then once weekly, for at least 2 months. RESULTS: Eighteen patients developed acute grade II or higher GVHD at a median time of 22.5 days and were treated with steroids. CN activity was significantly increased in these 18 patients when compared with 13 patients who did not develop GVHD. Analysis involving the receiver operating characteristic curve demonstrated that acute grade II or higher GVHD can be predicted with a sensitivity of 89% and a specificity of 54% with the use of a cutoff value of 28 pmol RII/mg proteins/min of CN activity. CONCLUSIONS: CN activity appears to be a promising therapeutic test to predict acute GVHD after allogeneic SCT. This functional assessment of the in vivo efficacy of CsA opens new insights for CsA dose adjustment-in particular, the administration of its most efficient dose instead of its maximal tolerated dose, as is currently performed.

Regulation of NAD(P)H:quinone oxidoreductase 1 gene expression by CYP1A1 activity.

The dioxin 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) induces phase I and II xenobiotic metabolizing enzymes (XME) which act sequentially to eliminate different classes of xenobiotics. The transcriptional effects of TCDD are generally mediated by the arylhydrocarbon receptor (AhR). We hypothesized that TCDD could also act indirectly, by increasing the activity of cytochrome P450 1A1 (CYP1A1), a phase I gene, which could then mediate the induction of other XME genes, such as the NAD(P)H:quinone oxidoreductase 1 (NQO1). To test this hypothesis, NQO1 gene expression was monitored after either overexpression of CYP1A1 or siRNA-mediated knock-down of CYP1A1 activity in the hepatoma cell line HepG2. Overexpression of CYP1A1 in the absence of TCDD was carried out using either adenoviral infection or the "Tet-off" system. Recombinant adenoviruses were produced encoding no protein, CYP1A1 (Ad1A1), or a mutated inactive CYP1A1 (Ad1A1mut). In the HepG2 Tet-off cell line, CYP1A1 expression was induced by the removal of doxycycline (dox) from the cell medium. Ad1A1 infection or dox removal induced CYP1A1 activity and H(2)O(2) production similarly to TCDD treatment. Moreover, in both systems, the amount of NQO1 mRNA increased to the same level as after TCDD treatment (approximately 2-fold). The UDP-glucuronosyl transferase 1A6 (UGT1A6) gene is also similarly regulated. NQO1 gene expression was not induced when mutant, inactive CYP1A1 was overexpressed or when the antioxidant N-acetyl cysteine (NAC) was added to Ad1A1. Finally, either NAC or siRNA directed against CYP1A1 mRNA decreased the induction of NQO1 gene expression by TCDD. We conclude that, after exposure to TCDD, the NQO1 gene expression can be controlled by CYP1A1 activity through an oxidative stress mediated pathway.

Organic compounds from diesel exhaust particles elicit a proinflammatory response in human airway epithelial cells and induce cytochrome p450 1A1 expression.

Diesel exhaust particles (DEP) are known to enhance inflammatory responses in human volunteers. In cultured human bronchial epithelial (16HBE) cells, they induce the release of proinflammatory cytokines after triggering transduction pathways, including nuclear factor (NF)-kappaB activation and mitogen-activated protein kinase (MAPK) phosphorylation. This study compares the effects of native DEP (nDEP), organic extracts of DEP (OE-DEP), and carbonaceous particles, represented by stripped DEP (sDEP) and carbon black particles (CB), in order to clarify their respective roles. OE-DEP and nDEP induce granulocyte macrophage colony-stimulating factor (GM-CSF) release, NF-kappaB activation, and MAPK phosphorylation. The carbonaceous core generally induces less intense effects. Reactive oxygen species are produced in 16HBE cells and are involved in GM-CSF release and in the stimulation of NF-kappaB DNA binding by nDEP and OE-DEP. We demonstrate, for the first time, in airway epithelial cells in vitro that nDEP induce the expression of the CYP1A1, a cytochrome P450 specifically involved in polycyclic aromatic hydrocarbons metabolism, thereby demonstrating the critical role of organic compounds in the DEP-induced proinflammatory response. Understanding the respective contributions of DEP components in these effects is important for vehicle manufacturers in order to improve their exhaust gas post-treatment technologies. In conclusion, the DEP-induced inflammatory response in airway epithelial cells mainly involves organic compounds such as PAH, which induce CYP1A1 gene expression.

Differential regulation of cytochrome P450 1A1 and 1B1 by a combination of dioxin and pesticides in the breast tumor cell line MCF-7.

Dioxin and pesticides with xenoestrogenic activity are environmental contaminants that are suspected of promoting human diseases such as cancers. However, few studies have addressed the molecular consequences of a combination of these contaminants, a situation that is likely to occur in the environment. We investigated the effects of natural and xenoestrogens on basal and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 (CYP) 1A1 and 1B1. The CYP1B1/1A1 ratio is a critical determinant of the metabolism and toxicity of estradiol in mammary cells. Here we show that in MCF-7 cells, 17beta-estradiol and alpha-endosulfan can repress whole cell ethoxyresorufin-O-deethylase activity, lowering CYP1A1 mRNA levels as well as promoter activity as assessed by transient transfection assays. These negative effects are observed at both the basal and tetrachlorodibenzo-p-dioxin-induced levels. Under the same conditions, CYP1B1 mRNA levels and promoter activity are not affected. The effects on mRNA-induced levels are also observed in another mammary cell line, T47D, but not in mammary cell lines that do not express aryl hydrocarbon receptor and estrogen receptor (ER). Moreover, the use of ER antagonists shows that these effects are ER dependent in MCF-7 cells. In human hepatoma HepG2 cells, which lack functional ER, alpha-endosulfan, but not 17beta-estradiol, displays a repressive effect on CYP1A1 through a different mechanism. These results show that xenoestrogens, by altering the ratio of CYP1B1/CYP1A1, could redirect estradiol metabolism in a more toxic pathway in the breast cell line MCF-7.

Repression of cytochrome P450 1A1 gene expression by oxidative stress: mechanisms and biological implications.

Cytochrome P450 1A1 (CYP1A1) is a member of a multigenic family of xenobiotic-metabolizing enzymes. Beyond its usual role in the detoxification of polycyclic aromatic compounds, the activity of this enzyme can be deleterious since it can generate mutagenic metabolites and oxidative stress. The CYP1A1 gene is highly inducible by the environmental contaminants dioxin and benzo[a]pyrene. We discuss here the regulatory mechanisms that limit this induction. Several feedback loops control the activation of this gene and the subsequent potential toxicity. The oxidative repression of the CYP1A1 gene seems to play a central role in these regulations. The transcription factor Nuclear Factor I/CCAAT Transcription Factor (NFI/CTF), which is important for the transactivation of the CYP1A1 gene promoter, is particularly sensitive to oxidative stress. A critical cysteine within the transactivating domain of NFI/CTF appears to be the target of H(2)O(2). The DNA-binding domains of several transcription factors have been described as targets of oxidative stress. However, recent studies described here suggest that more attention should be given to transactivating domains that may represent biologically relevant redox targets of cellular signaling.

Regulation of human beta-glucuronidase by A23187 and thapsigargin in the hepatoma cell line HepG2.

A novel approach to reducing organ toxicity of anticancer agents is the application of nontoxic glucuronide prodrugs from which the active drug is released by human beta-glucuronidase, an enzyme present at high levels in many tumors. In view of high interindividual variability in beta-glucuronidase expression, regulation of this enzyme is an essential factor modulating bioactivation of glucuronide prodrugs. However, data on regulation of human beta-glucuronidase expression are not available. Preliminary evidence from animal experiments points to a role of intracellular calcium in regulation of beta-glucuronidase activity. Therefore, we investigated regulation of beta-glucuronidase by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. The enzyme was characterized on activity, protein, and mRNA levels by cleavage of 4-methylumbelliferyl-beta-D-glucuronide, Western blotting, Northern blotting, and nuclear run-on transcription. Incubation of HepG2 cells with A23187 and thapsigargin, respectively, revealed a time and concentration dependent down-regulation of beta-glucuronidase activity to about 50% of the control level. This effect could also be demonstrated in several other cell lines (e.g., HL-60, ECV 304, 32M1, Caco-2/TC7). Effects on protein and mRNA levels paralleled those obtained on enzymatic activity. In line with these data, A23187 and thapsigargin decreased beta-glucuronidase transcriptional rate. Our data demonstrate regulation of human beta-glucuronidase by xenobiotics. Down-regulation of beta-glucuronidase by A23187 and thapsigargin is at least partly mediated by a transcriptional mechanism. Based on our findings, we speculate that beta-glucuronidase activity and hence bioactivation of glucuronide prodrugs in humans can be modulated by exogenous factors.

[Oxidative stress and gene expression]. / Stress oxydant et expression des gènes.

Oxidative stress elicits cellular toxicity implicating various targets such as DNA, RNA, proteins, lipids.... Similarly to other stress conditions, it triggers cellular adaptation and the induction of defense mechanisms. Adaptation to this stress is ubiquitous but it displays cell specificity. Adaptation to stress requires the regulation of a large number of genes. Those encoding anti-oxidant and repair genes are induced while others are repressed. The activity of several transcription factors is altered by oxidative stress. Many transcription factors are inhibited because of the oxidation of cysteine residues in their DNA binding domains. In the case of NFI, a cysteine in the transactivating domain is oxidized which leads to inactivation. The mechanisms of transcription factor activation are often complex, as in the case of cJun. This factor is activated by a kinase signalling cascade. Paradoxically, it is also inhibited by stress since one of its cysteines is oxidized; however, this oxidation is repaired in the nucleus by the ref1-thioredoxine system. A similar mechanism is observed for NF kappa B. Alteration of the redox status is observed in several physiological pathological and toxic conditions. It is also provoked by other cellular stress, in particular endoplasmic reticulum stress, hypoxia, shear stress, osmotic and heat shocks. The actual role of oxidative stress during other stress conditions remains to be elucidated. This widespread implication of oxidative stress could account for its role in a variety of disease such as neurodegenerative diseases, cancer, atherosclerosis, and diabetes. The efficacy of anti-oxidant therapy depends on the actual contribution of oxidative stress to these diseases.

Nuclear factor I/CCAAT box transcription factor trans-activating domain is a negative sensor of cellular stress.

The adaptive response to cellular stress requires the reprogramming of gene expression. So far, research has focused on induction mechanisms; several transcription factors activated by cellular stress have been shown to trigger the induction of repair and detoxification enzymes. Using the hepatoma cell line HepG2, we report that the trans-activating function of the nuclear factor I/CCAAT box transcription factor (NFI/CTF-1) is, on the contrary, repressed by various stress conditions, including inflammatory cytokine treatment, glutathione depletion, heat and osmotic shocks, and chemical stress. Under the same conditions, other transcription factors were not affected. We show that when Cys-427 within the trans-activating domain of NFI/CTF-1 is mutated into a serine, the repressive effect triggered by cellular stresses is no longer observed. In addition, this effect is abolished in cells transfected with a thioredoxin expression vector. Using the dichlorofluorescein fluorescent probe, we provide direct evidence that the stress conditions elicit an intracellular reactive oxygen species generation, which can, in turn, negatively regulate NFI/CTF-1. In agreement with these observations, we show that the CYP1A1 mRNA and the CYP1A1 gene promoter, which is a target of NFI/CTF-1, are repressed by stress conditions. Thus, through the redox regulation of its trans-activating function, NFI/CTF-1 constitutes a novel biologically relevant negative sensor of several stress stimuli.