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.

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.

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.

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.

Adrenergic regulation of glycogenolysis in rat liver after cholestasis. Modulation of the balance between alpha 1 and beta 2 receptors.

The effects of extrahepatic cholestasis upon adrenergic regulation of glycogenolysis and upon the numbers of adrenoceptors in rat liver were studied using isolated hepatocytes and plasma membranes, respectively. A 60% decrease in the number of alpha 1 adrenoceptors (285 vs. 680 fmol/mg protein) and a simultaneous 2.7-fold increase in the number of beta adrenergic sites (67 vs. 25 fmol/mg protein) were observed beginning 36 h after bile flow obstruction and persisted for at least 68 h. The reciprocal modification of the numbers of alpha 1 and beta adrenoceptors was accompanied by a change in the manner of stimulation of glycogen phosphorylase by catecholamines in hepatocytes; originally alpha 1 adrenergic in normal rats (phenylephrine Ka = 0.9 microM, isoproterenol Ka = 7.1 microM), the stimulation became predominantly beta adrenergic in cholestatic animals (phenylephrine Ka = 3.7 microM, isoproterenol Ka = 0.06 microM). In normal rats, activation of the enzyme by epinephrine was inhibited by the alpha blocker phentolamine, without inhibition by the beta blocker propranolol. In contrast, propranolol was more effective than phentolamine in cholestatic rat hepatocytes. Modification of the regulation of glycogenolysis after cholestasis did not seem to be secondary to an alteration in the metabolism of thyroid hormones or in the action of glucocorticoids. However, cholestasis provoked a 10-fold increase in the number of hepatic mitoses and in the incorporation of thymidine into liver DNA of cholestatic animals. Similar changes were observed in regenerating livers, following two-thirds hepatectomy. We propose that the changes following extrahepatic cholestasis might, as well, be explained by a regenerative process.

An autoregulatory loop controlling CYP1A1 gene expression: role of H(2)O(2) and NFI.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H(2)O(2) catabolism), thus implying that H(2)O(2) is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H(2)O(2), a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H(2)O(2) production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.

A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.

An unusual glucocorticoid-responsive element (called GRE A) was found to mediate the induction of the cytosolic aspartate aminotransferase gene by glucocorticoids and was bound by the glucocorticoid receptor in a DNase I footprinting assay. GRE A consists of two overlapping GREs, each comprising a conserved half-site and an imperfect half-site. The complete unit was able to confer glucocorticoid inducibility to a heterologous promoter (delta MTV-CAT). Mutation of any of the half-sites, including the imperfect ones, abolished inducibility by the hormone, demonstrating that each of the isolated GREs was inactive. In electrophoretic mobility shift assays, purified rat liver glucocorticoid receptor (GR) formed a low-mobility complex with GRE A, presumably containing a GR tetramer. When purified bacterially expressed DBD was used, low-mobility complexes as well as dimer and monomer complexes were formed. In inactive mutated oligonucleotides, no GR tetramer formation was detected. Modification of the imperfect half-sites in order to increase their affinity for GR gave a DNA sequence that bound a GR tetramer in a highly cooperative manner. This activated unit consisting of two overlapping consensus GREs mediated glucocorticoid induction with a higher efficiency than consensus GRE.

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.

Glucocorticoid hormones increase the activity of gamma-glutamyltransferase in a highly differentiated hepatoma cell line.

Gamma-Glutamyltransferase activity was detected in the plasma membrane of the highly differentiated hepatoma cell line Fao, (0.93 mU/mg cell protein). Dexamethasone (1 microM) provoked a 2-3-fold increase in the activity of the enzyme in the presence of fetal calf serum. Maximal induction occurred 48-72 h after addition of the glucocorticoid to the cell culture medium. The hormonal specificity was demonstrated by the relative potencies of several glucocorticoids and sex steroids: hydrocortisone and corticosterone increased gamma-glutamyltransferase activity while tetrahydrocorticosterone and all sex steroids tested were ineffective. The effect of dexamethasone on gamma-glutamyltransferase activity wa specific since the activities of several other plasma membrane enzymes were not modified. The mechanism of the dexamethasone-induced increase in gamma-glutamyltransferase activity was neither by modification of the affinity of the enzyme for its substrates nor by alteration of the subcellular distribution of the enzyme. This increase was prevented by cycloheximide and actinomycin D. The data presented are consistent with a specific glucocorticoid receptor-mediated induction of gamma-glutamyltransferase activity in Fao cells. The kinetic parameters of the induction process by glucocorticoids are very similar to those found in adult rat liver. These results suggest that the Fao cell line is a very convenient system for the study of the molecular mechanisms of glucocorticoid effects on differentiated cells.

Modulation of human mineralocorticoid receptor function by protein kinase A.

The mineralocorticoid receptor (MR) acts as a ligand-dependent transcription factor modulating specific gene expression in sodium-transporting epithelia. Physiological evidence suggest a cross-talk between the cAMP- and aldosterone-signaling pathways. We provide evidence that protein kinase A (PKA), a major mediator of signal transduction pathways, modulates transcriptional activity of the human MR (hMR). Using transient transfection assays in HepG2 cells, we show that 8-bromo-cAMP, a protein kinase A activator, stimulates glucocorticoid response element (GRE)-containing promoters in a ligand-independent manner. This effect was strictly MR dependent since no activation of the reporter gene was observed in the absence of cotransfected hMR expression plasmid. Furthermore, a synergistic activation was achieved when cells were treated with both aldosterone and cAMP. This synergistic effect was also observed in the CV1 and the stable hMR-expressing M cells but was dependent on the promoter used. In particular, synergism was less pronounced in promoters containing several GREs. We show that (protein kinase-inhibiting peptide (PKI), the peptide inhibitor of PKA, prevented both cAMP and aldosterone induction, which indicates that a functional cAMP pathway is required for stimulation of transcription by aldosterone. Using MR-enriched baculovirus extracts in gel shift assays, we have shown that the binding of the MR to a GRE-containing oligonucleotide was enhanced by PKA. Increased DNA binding of hMR is likely to reflect an increase in the number of active receptors, as measured by Scatchard analysis. Using a truncated MR, we show that the N-terminal domain is required for the effect. Finally, the N-terminal truncated MR was not directly phosphorylated by PKA in vitro. We conclude that PKA acts indirectly, probably by relieving the effect of an MR repressor.

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.

Identification of an adipocyte-specific negative glucose response region in the cytosolic aspartate aminotransferase gene.

Cytosolic aspartate aminotransferase (cAspAT) participates in gluconeogenesis in the liver and is expected to exert a glyceroneogenic function in the adipose tissue when the supply of glucose is limited. Here we demonstrate that adipose cAspAT messenger RNA (mRNA) is increased when rats are fed a low carbohydrate diet. In the 3T3-F442A, BFC-1 adipocyte cell lines and differentiated adipocytes in primary culture, a 24 h glucose deprivation induces approximately a 4-fold increase in cytosolic AspAT (cAspAT) mRNA, whereas mitochondrial AspAT mRNA remains unchanged. cAspAT activity is also increased in a weaker but reproducible manner. Addition of glucose within a physiological range of concentrations reverses the increase of cAspAT mRNA in 8 h (EC50 = 1.25 g/liter). Such a regulation requires protein synthesis and is specific for adipocytes differentiated in culture. It does not occur in Fao or H4IIE hepatoma cells, in C2 muscle cells, or in 293 kidney cells. 2-deoxyglucose mimicks glucose, while 3-orthomethyl-glucose has no effect, suggesting that glucose-6-phosphate is the effector. cAspAT mRNA stability is not affected by glucose deprivation. To ascertain the transcriptional nature of the glucose effect, we have stably transfected 3T3-F442A adipoblasts with constructs containing the chloramphenicol acetyltransferase reporter gene under the control of either 5'-deletions of the cAspAT gene promoter or internal fragments in an heterologous context. We demonstrate that a glucose response element(s) is present in the region between -1838 and -1702 bp relative to the translation start site. In this region, three DNA sequences bind nuclear proteins from adipocytes as shown by footprinting experiments. Our results indicate that cAspAT gene transcription is repressed by glucose selectively in adipocytes.

Effects of ethanol, dexamethasone and RU 486 on expression of cytochromes P450 2B, 2E, 3A and glutathione transferase pi in a rat hepatoma cell line (Fao).

The aim of our study was to investigate the suitability of Fao cells, derived from the Reuber H35 rat hepatoma as a tool for studying regulation of drug-metabolizing enzymes and drug metabolism. Fao cells express P450 2B, 2E, 3A and GST pi and were used to study the effects different inducers on these enzymes. Ethanol considerably increased the amounts of P450 2E and, to a lesser extent, P450 2B and GST pi mRNA and protein. Dexamethasone decreased the amounts of P450 2B, 3A and GST pi mRNAs, but had no appreciable effect per se upon the protein concentration of these enzymes. However, it antagonized the induction of P450 2E, 2B and GST pi by ethanol, even at the protein level. RU 486 decreased P450 2B protein and P450 2E mRNA and protein levels without effecting P450 3A and GST pi expression. RU 486 did not antagonize the dexamethasone effects, suggesting that at least some of these effects are not mediated by the glucocorticoid receptor. These data indicate that these cells constitute a suitable tool for studying the regulation of drug-metabolizing enzyme expression and drug metabolism.

A single d(GpG) cisplatin adduct on the estrogen response element decreases the binding of the estrogen receptor.

Both cisplatin and the estrogen receptor (ER) are known to bend DNA. The influence of the bending of sequences by the d(GpG)cisPt adduct binding of ER to estrogen response element (ERE)-like sequences was examined. Three ERE-like oligonucleotides with different affinities for ER and which include a GG in the linker sequence were designed in order to form a single central d(GpG)cisPt adduct. Using electrophoretic mobility shift assay and Scatchard analysis, it was shown that the presence of a single d(GpG)cisPt adduct in the linker sequence decreases the ER affinity for DNA. These results do not support a critical role of a DNA bend in the initial recognition of ERE by ER. Then, the platination of DNA outside of the ERE half-sites decreases the interaction of ER with ERE.

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.

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.

Glucocorticoid hormones prevent the induction of gamma-glutamyl transpeptidase by ethanol in a rat hepatoma cell line.

The increase in serum gamma-glutamyl transpeptidase (GGT) is a well known marker of chronic alcoholism in man. We have previously shown that ethanol (180 mM) induces GGT activity 2-3-fold in the C2 rat hepatoma cell line. In this study, we have analyzed the interaction of ethanol with steroid hormones and drugs in this well defined cell culture system. Dexamethasone (100 nM), a synthetic glucocorticoid agonist, completely prevented the induction of GGT by ethanol, but had no effect when added alone. This inhibitory effect was also observed with other corticosteroids, but not with sex steroids; it was prevented by RU 486, a glucocorticoid antagonist. These observations suggest that dexamethasone acts through a high affinity glucocorticoid receptor. Conversely, ethanol did not interfere with the glucocorticoid induction of alanine aminotransferase in the same cell. We have analyzed the metabolism of ethanol in the C2 cells. These cells lack significant alcohol dehydrogenase activity as well as any cytochrome P-450 Alc immunoreactivity. Dexamethasone did not modify the disappearance of ethanol in the culture medium of those cells. We conclude that glucocorticoid hormones interact with ethanol at the cellular level, and that this interaction does not involve a modification of alcohol metabolism.

An assay for the detection of xenoestrogens based on a promoter containing overlapping EREs.

Xenoestrogens could be implicated in the decrease of male fertility and in the increased incidence of testicular and breast cancers in humans. To predict their deleterious effects, various in vivo or in vitro tests have been proposed to assay the xenoestrogenic activity. We have designed an assay for the detection of xenoestrogens based on a novel estrogen responsive unit formed by two overlapping estrogen response elements (overEREs). This construct is able to mediate a synergistic activation of transcription by 17ss-estradiol. We have used the overERE unit to assay the estrogenic activity of synthetic compounds, mostly organochlorine compounds. By using the overERE construct, we were able to detect the estrogenic activity of compounds at concentrations 10- to 100-fold lower than a single ERE (i.e., we detected the estrogenic effect of endosulfan at a concentration of 10(-5) M with ERE, whereas the overERE unit allowed us to detect a significant estrogenic activity of endosulfan at a lower concentration (10(-6) M). Some compounds did not exhibit any estrogenic activity when tested with a classical ERE, whereas they were potent xenoestrogens when the overERE was used (i.e., Betanal). The assays we have developed are very sensitive and can be performed quickly. Moreover, because the promoter that we used contains only an overlapping ERE as a regulatory unit, the interference of the tested molecules with other regulatory pathways can be avoided.

Fenofibrate modifies transaminase gene expression via a peroxisome proliferator activated receptor alpha-dependent pathway.

Fibrates modify the expression of genes implicated in lipoprotein and fatty acid metabolism via the peroxisome proliferator-activated receptor alpha(PPARalpha), leading to reductions in serum triglycerides and cholesterol. The expression of certain genes regulated by PPARalpha have been shown to be modified in a species dependent manner. Aspartate aminotransferase (AspAT or GOT) and alanine aminotransferase (AlaAT or GPT) are enzymes involved in intermediate metabolism in all cells and in hepatic gluconeogenesis. These enzymes are also widely used as serum markers of possible tissue damage. This study investigated whether fenofibrate could modify the expression of liver AspAT and/or AlaAT and thus possibly alter transaminase levels independently of a cytotoxic effect. In human Hep G2 cells, fenofibrate increased cytosolic AspAT (cAspAT) activity by 40% and AlaAT activity by 100%, as well as both mRNAs. Nuclear run on assays showed that this effect was, at least in part, transcriptional. Increases in mRNA were also observed in human hepatocyte cultures at concentrations of the drug attained in patients. In C57BL/6 mice, fenofibrate decreased cAspAT and cAlaAT mRNA, while these effects were abolished in PPARalpha knock-out mice. In conclusion, fenofibrate has been shown to modify cAspAT and AlaAT gene expression in a species and PPARalpha dependent manner. This is the first demonstration that cAspAT and AlaAT activities may be pharmacologically altered, independently of a toxic phenomenon.