Different effects of pesticides on transcripts of the endocrine regulation and energy metabolism in honeybee foragers from different colonies.

Honeybees are important pollinators of many crops and contribute to biological biodiversity. For years, a decline in bee populations has been observed in certain areas. This decline in honeybees is accompanied by a decrease in pollinator services. One factor contributing to the decline of bee colonies is the exposure to pesticides. Pesticide exposure of bees, among other effects, can negatively affect orientation, memory, immune system function and gene expression. Among the altered expressed genes are transcripts of endocrine regulation and oxidative phosphorylation. Endocrine regulation plays an important role in the development of nurse bees into foragers and oxidative phosphorylation is involved in energy metabolism. Most of these transcriptional changes were investigated using mixed aged honeybees derived from the same colony. Experiments using nurse bees or foragers of the same age but from different colonies are rare. In the present study, effects of the two pesticides chlorpyrifos and pyraclostrobin on the expression of transcripts linked to endocrine regulation and oxidative phosphorylation in foragers of the same age from three different colonies are investigated to fill this gap. These two pesticides were selected because negative effects at sublethal concentrations on bees are known and because they are found in pollen and nectar of crops and wild plants. For this purpose, 20-22 days old foragers of three different colonies were exposed to different sublethal concentrations of the selected fungicides for 24 h, followed by analysis of the expression of buffy, vitellogenin, hbg-3, ilp-1, mrjp1, 2 and 3, cox5a, cox5b and cox17. Some significant changes in gene expression of both endocrine regulation transcripts and oxidative phosphorylation were shown. Furthermore, it became clear that forager bees from different colonies react differently. This is especially important in relation to the risk analysis of pesticides. In addition, it could be shown that the expression of hbg-3 in the brain of bees is a robust marker to distinguish nurse bees from foragers at the molecular biological level. In summary, this study clearly shows that pesticides, which are often detected in pollen and nectar, display negative effects at sublethal concentrations on bees and that it is important to use bees from different colonies for risk assessment of pesticides.

Developmental neurotoxicity of different pesticides in PC-12 cells in vitro.

The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2ß, gap-43, neurofilament-h, tubulin-α and tubulin-ß. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals.

Cytotoxicity and molecular effects of biocidal disinfectants (quaternary ammonia, glutaraldehyde, poly(hexamethylene biguanide) hydrochloride PHMB) and their mixtures in vitro and in zebrafish eleuthero-embryos.

Frequently used biocidal disinfectants, including quaternary ammonium compounds (QAC), glutaraldehyde and poly(hexamethylene biguanide) hydrochloride (PHMB), occur in the aquatic environment but their potential effects in fish are poorly known, in particular when occurring as mixtures. To investigate their joint activity, we assessed the cytotoxicity of three QACs (BAC, barquat and benzalkonium chloride), glutaraldehyde andPHMB by the MTT assay individually, followed by assessing binary and ternary mixtures in zebrafish liver cells (ZFL) and human liver cells (Huh7). We also analysed molecular effects by quantitative PCR in vitro and in zebrafish eleuthero-embryos employing a targeted gene expression approach. QACs displayed strong cytotoxicity in both cell lines with EC50 values in the low µg/ml range, while glutaraldehyde and PHMB were less cytotoxic. Most of the binary and both ternary mixtures showed synergistic activity at all equi-effective concentrations. A mixture containing all five compounds mixed at their no observed effect concentrations showed strong cytotoxicity, suggesting a synergistic interaction. Additionally, we determined transcriptional alterations of target genes related to endoplasmatic reticulum (ER) stress, general stress, inflammatory action and apoptosis. Induction of ER stress genes occurred at non-cytotoxic concentrations of barquat, glutaraldehyde and BAC in ZFL cells. Barquat and BAC induced tumor necrosis factor alpha (tnf-α). Similar transcriptional alterations were found in vivo upon exposure of zebrafish eleuthero-embryos for 120h. Glutaraldehyde led to induction of ER stress genes and tnf-α, while BAC additionally induced genes indicative of apoptosis, which was also the case with benzalkonium chloride at the highest concentration. We demonstrated strong cytotoxicity of QACs, and synergistic activity of binary, ternary and quintuple mixtures. Barquat and BAC let to induction of ER stress and inflammation in vitro, and BAC and glutaraldehyde at non-toxic concentrations in vivo, while benzalkonium chloride induced expression of tnf-α only.

Anti-Inflammatory Activity of Cyanobacterial Serine Protease Inhibitors Aeruginosin 828A and Cyanopeptolin 1020 in Human Hepatoma Cell Line Huh7 and Effects in Zebrafish (Danio rerio).

Intensive growth of cyanobacteria in freshwater promoted by eutrophication can lead to release of toxic secondary metabolites that may harm aquatic organisms and humans. The serine protease inhibitor aeruginosin 828A was isolated from a microcystin-deficient Planktothrix strain. We assessed potential molecular effects of aeruginosin 828A in comparison to another cyanobacterial serine protease inhibitor, cyanopeptolin 1020, in human hepatoma cell line Huh7, in zebrafish embryos and liver organ cultures. Aeruginosin 828A and cyanopeptolin 1020 promoted anti-inflammatory activity, as indicated by transcriptional down-regulation of interleukin 8 and tumor necrosis factor α in stimulated cells at concentrations of 50 and 100 µmol·L(-1) aeruginosin 828A, and 100 µmol·L(-1) cyanopeptolin 1020. Aeruginosin 828A induced the expression of CYP1A in Huh7 cells but did not affect enzyme activity. Furthermore, hatched zebrafish embryos and zebrafish liver organ cultures were exposed to aeruginosin 828A. The transcriptional responses were compared to those of cyanopeptolin 1020 and microcystin-LR. Aeruginosin 828A had only minimal effects on endoplasmic reticulum stress. In comparison to cyanopeptolin 1020 our data indicate that transcriptional effects of aeruginosin 828A in zebrafish are very minor. The data further demonstrate that pathways that are influenced by microcystin-LR are not affected by aeruginosin 828A.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress.

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1µM) and toxic concentrations (5µM) for 24, 48, and 72h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5µM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5nM, while HepG2 cells were insensitive up to 10µM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin.

Molecular Effects of Neonicotinoids in Honey Bees (Apis mellifera).

Neonicotinoids are implicated in the decline of bee populations. As agonists of nicotinic acetylcholine receptors, they disturb acetylcholine receptor signaling leading to neurotoxicity. Several behavioral studies showed the link between neonicotinoid exposure and adverse effects on foraging activity and reproduction. However, molecular effects underlying these effects are poorly understood. Here we elucidated molecular effects at environmental realistic levels of three neonicotinoids and nicotine, and compared laboratory studies to field exposures with acetamiprid. We assessed transcriptional alterations of eight selected genes in caged honey bees exposed to different concentrations of the neonicotinoids acetamiprid, clothianidin, imidacloporid, and thiamethoxam, as well as nicotine. We determined transcripts of several targets, including nicotinic acetylcholine receptor α 1 and α 2 subunit, the multifunctional gene vitellogenin, immune system genes apidaecin and defensin-1, stress-related gene catalase and two genes linked to memory formation, pka and creb. Vitellogenin showed a strong increase upon neonicotinoid exposures in the laboratory and field, while creb and pka transcripts were down-regulated. The induction of vitellogenin suggests adverse effects on foraging activity, whereas creb and pka down-regulation may be implicated in decreased long-term memory formation. Transcriptional alterations occurred at environmental concentrations and provide an explanation for the molecular basis of observed adverse effects of neonicotinoids to bees.

Silica nanoparticles induce endoplasmic reticulum stress response and activate mitogen activated kinase (MAPK) signalling.

Humans may be exposed to engineered silica nanoparticles (SiO2-NPs) but potential adverse effects are poorly understood, in particular in relation to cellular effects and modes of action. Here we studied effects of SiO2-NPs on cellular function in human hepatoma cells (Huh7). Exposure for 24 h to 10 and 50 µg/ml SiO2-NPs led to induction of endoplasmic reticulum (ER) stress as demonstrated by transcriptional induction of DNAJB9, GADD34, CHOP, as well as CHOP target genes BIM, CHAC-1, NOXA and PUMA. In addition, CHOP protein was induced. In addition, SiO2-NPs induced an inflammatory response as demonstrated by induction of TNF-α and IL-8. Activation of MAPK signalling was investigated employing a PCR array upon exposure of Huh7 cells to SiO2-NPs. Five of 84 analysed genes, including P21, P19, CFOS, CJUN and KSR1 exhibited significant transcriptional up-regulation, and 18 genes a significant down-regulation. Strongest down-regulation occurred for the proto-oncogene BRAF, MAPK11, one of the four p38 MAPK genes, and for NFATC4. Strong induction of CFOS, CJUN, FRA1 and CMYC was found after exposure to 50 µg/ml SiO2-NPs for 24 h. To analyse for effects derived from up-regulation of TNF-α, Huh7 cells were exposed to SiO2-NPs in the presence of the TNF-α inhibitor sauchinone, which reduced the induction of the TNF-α transcript by about 50%. These data demonstrate that SiO2-NPs induce ER stress, MAPK pathway and lead to inflammatory reaction in human hepatoma cells. Health implications of SiO2-NPs exposure should further be investigated for a risk assessment of these frequently used nanoparticles.

Indium and indium tin oxide induce endoplasmic reticulum stress and oxidative stress in zebrafish (Danio rerio).

Indium and indium tin oxide (ITO) are extensively used in electronic technologies. They may be introduced into the environment during production, use, and leaching from electronic devices at the end of their life. At present, surprisingly little is known about potential ecotoxicological implications of indium contamination. Here, molecular effects of indium nitrate (In(NO3)3) and ITO nanoparticles were investigated in vitro in zebrafish liver cells (ZFL) cells and in zebrafish embryos and novel insights into their molecular effects are provided. In(NO3)3 led to induction of endoplasmic reticulum (ER) stress response, induction of reactive oxygen species (ROS) and induction of transcripts of pro-apoptotic genes and TNF-α in vitro at a concentration of 247 µg/L. In(NO3)3 induced the ER stress key gene BiP at mRNA and protein level, as well as atf6, which ultimately led to induction of the important pro-apoptotic marker gene chop. The activity of In(NO3)3 on ER stress induction was much stronger than that of ITO, which is explained by differences in soluble free indium ion concentrations. The effect was also stronger in ZFL cells than in zebrafish embryos. Our study provides first evidence of ER stress and oxidative stress induction by In(NO3)3 and ITO indicating a critical toxicological profile that needs further investigation.

Additive and synergistic antiandrogenic activities of mixtures of azol fungicides and vinclozolin.

OBJECTIVE: Many pesticides including pyrethroids and azole fungicides are suspected to have an endocrine disrupting property. At present, the joint activity of compound mixtures is only marginally known. Here we tested the hypothesis that the antiandrogenic activity of mixtures of azole fungicides can be predicted by the concentration addition (CA) model. METHODS: The antiandrogenic activity was assessed in MDA-kb2 cells. Following assessing single compounds activities mixtures of azole fungicides and vinclozolin were investigated. Interactions were analyzed by direct comparison between experimental and estimated dose-response curves assuming CA, followed by an analysis by the isobole method and the toxic unit approach. RESULTS: The antiandrogenic activity of pyrethroids deltamethrin, cypermethrin, fenvalerate and permethrin was weak, while the azole fungicides tebuconazole, propiconazole, epoxiconazole, econazole and vinclozolin exhibited strong antiandrogenic activity. Ten binary and one ternary mixture combinations of five antiandrogenic fungicides were assessed at equi-effective concentrations of EC25 and EC50. Isoboles indicated that about 50% of the binary mixtures were additive and 50% synergistic. Synergism was even more frequently indicated by the toxic unit approach. CONCLUSION: Our data lead to the conclusion that interactions in mixtures follow the CA model. However, a surprisingly high percentage of synergistic interactions occurred. Therefore, the mixture activity of antiandrogenic azole fungicides is at least additive. PRACTICE: Mixtures should also be considered for additive antiandrogenic activity in hazard and risk assessment. IMPLICATIONS: Our evaluation provides an appropriate "proof of concept", but whether it equally translates to in vivo effects should further be investigated.

Tissue-, sex- and development-specific transcription profiles of eight UDP-glucuronosyltransferase genes in zebrafish (Danio rerio) and their regulation by activator of aryl hydrocarbon receptor.

UDP-Glucuronosyltransferases (Ugts) are phase II biotransformation enzymes that glucuronidate numerous endogenous and xenobiotic substrates. Based on the reported zebrafish Ugt gene repertoire, primers for the Ugt1a and Ugt1b family and for individual Ugt5a1, Ugt5a3, Ugt5a4, Ugt5a5, Ugt5c2 and Ugt5c3 were designed and applied in RT-qPCR analyses. Transcriptional expression profiles of these Ugts were analyzed in intestine, liver, gonad and brain of female and male adult zebrafish and at different embryonic developmental stages. We found tissue-, sex- and developmental-specific expression patterns for all isoforms. Throughout all tissues, the most abundant Ugts were Ugt1a, Ugt1b, Ugt5a1 and Ugt5a3. Expression during embryonic development was assessed between 24 and 120 hpf. Ugts showed a development-dependent expression. The pattern of Ugt1a, Ugt1b, Ugt5a1, Ugt5a3 and Ugt5a4 were similar with highest expression at 24 hpf followed by a decrease and rebound increase up to 120 hpf. To analyze for transcriptional regulation of Ugts by the arylhydrocarbon receptor (ahr2), zebrafish eleuthero-embryos were exposed to 5, 25 and 50µg/L benzo(a)pyrene (BaP), a model ahr2 regulator for cyp1a. Besides transcriptional induction of ahr2 and cyp1a, BaP produced a significant induction of Ugt1a, Ugt5a1, Ugt5a3 and Ugt5a5 as well as a down-regulation of Ugt1b. These data demonstrate the link between ahr2 signalling and transcriptional expression of Ugt genes. This is the first study showing transcriptional expression of eight different Ugts in tissues and during embryonic development and offers new perspectives on the involvement of Ugts in fish xenobiotic metabolism.

Silica nanoparticles induce endoplasmic reticulum stress response, oxidative stress and activate the mitogen-activated protein kinase (MAPK) signaling pathway.

Application of silica nanoparticles (SiO2-NPs) may result in human exposure. Here we investigate unexplored modes of action by which SiO2-NPs with average size of 225 nm act on human hepatoma cells (Huh7). We focused on the endoplasmic (ER) stress response and on mitogen-activated protein kinase (MAPK) signaling pathways. Both pathways were induced. ER stress and the associated three unfolded protein response (UPR) pathways were activated as demonstrated by significant inductions of BiP and XBP-1s and a moderate but significant induction of ATF-4 at 0.05 and 0.5 mg/ml. In addition to activation of NFкB interferon stimulated genes IP-10, IRF-9, and ISG-15 were up-regulated. As a consequence of ER stress, the pro-inflammatory cytokine TNFα and PP2Ac were induced following exposure to 0.05 mg/ml SiO2-NPs. Additionally, this occurred at 0.005 mg/ml SiO2-NPs for TNFα at 24 h. This in turn led to a strong transcriptional induction of MAP-kinases and its target genes cJun, cMyc and CREB. A strong transcriptional down-regulation of the proapoptotic gene p53 occurred at 0.05 and 0.5 mg/ml SiO2-NP. Exposure of Huh7 cells to the anti-oxidant N-acetyl cysteine reduced transcriptional induction of ER stress markers demonstrating a link between the induction of oxidative stress and ER stress. Our study demonstrates that SiO2-NPs lead to strong ER stress and UPR induction, oxidative stress, activation of MAPK signaling and down-regulation of p53. All of these activated pathways, which are analyzed here for the first time in detail, inhibit apoptosis and induce cell proliferation, which may contribute to a hepatotoxic, inflammatory and tumorigenic action of SiO2-NPs.

Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish.

Silver nanoparticles (AgNPs) find increasing applications, and therefore humans and the environment are increasingly exposed to them. However, potential toxicological implications are not sufficiently known. Here we investigate effects of AgNPs (average size 120 nm) on zebrafish in vitro and in vivo, and compare them to human hepatoma cells (Huh7). AgNPs are incorporated in zebrafish liver cells (ZFL) and Huh7, and in zebrafish embryos. In ZFL cells AgNPs lead to induction of reactive oxygen species (ROS), endoplasmatic reticulum (ER) stress response, and TNF-α. Transcriptional alterations also occur in pro-apoptotic genes p53 and Bax. The transcriptional profile differed in ZFL and Huh7 cells. In ZFL cells, the ER stress marker BiP is induced, concomitant with the ER stress marker ATF-6 and spliced XBP-1 after 6h and 24h exposure to 0.5 g/L and 0.05 g/L AgNPs, respectively. This indicates the induction of different pathways of the ER stress response. Moreover, AgNPs induce TNF-α. In zebrafish embryos exposed to 0.01, 0.1, 1 and 5mg/L AgNPs hatching was affected and morphological defects occurred at high concentrations. ER stress related gene transcripts BiP and Synv are significantly up-regulated after 24h at 0.1 and 5mg/L AgNPs. Furthermore, transcriptional alterations occurred in the pro-apoptotic genes Noxa and p21. The ER stress response was strong in ZFL cells and occurred in zebrafish embryos as well. Our data demonstrate for the first time that AgNPs lead to induction of ER stress in zebrafish. The induction of ER stress can have several consequences including the activation of apoptotic and inflammatory pathways.

Microcystin-LR induces endoplasmatic reticulum stress and leads to induction of NFκB, interferon-alpha, and tumor necrosis factor-alpha.

Microcystins (MCs) are hepatotoxins produced by cyanobacteria responsible for toxicity in humans and animals. Here, we investigate unexplored molecular pathways by which microcystin-LR (MC-LR) acts on hepatocytes to elucidate unknown modes of action. We focus on the endoplasmatic reticulum (ER) stress response or unfolded protein response (UPR), and on mechanisms that may contribute to the tumor-promoting effect of MCs in animals, including the activation of NFκB, the expression of interferon alpha (IFN-α) and the induction of interferon stimulated genes (ISGs), as well as the expression of tumor necrosis factor alpha (TNF-α). To this end, we exposed human hepatoma cells (Huh7) to 0.5 µM (nontoxic concentration), 5 µM (EC50 concentration), 25 µM and 50 µM (cytotoxic concentrations) MC-LR for 6, 24, 48, and 72 h. The expression of phosphatase 2A (PP2A) mRNA and protein was induced at 5 µM MC-LR. Phosphorylated P-CREB, a transcription factor for PP2A, leads to elevated expression of PP2A. Furthermore, all of the three ER stress pathways, the UPR and the endoplasmic reticulum-associated degradation were activated after exposure to 5, 25, and 50 µM MC-LR. Additionally, the expression of NFκB, IFN-α, and several INF-α-stimulated genes was strongly activated. The proinflammatory cytokine TNF-α was also induced. Our data demonstrate that MC-LR induces all ER stress response pathways. Consequently NFκB is activated, which in turn induces the expression of IFN-α and TNF-α. All of these activated pathways, which are analyzed here for the first time in detail, may contribute to the hepatotoxic, inflammatory, and tumorigenic action of MC-LR.

Silica nanoparticles and silver-doped silica nanoparticles induce endoplasmatic reticulum stress response and alter cytochrome P4501A activity.

Engineered silica nanoparticles (SiO(2)-NPs) find widespread application and may lead to exposure of humans and the environment. Here we compare the effects of SiO(2)-NPs and SiO(2)-NPs doped with silver (SiO(2)-Ag-NPs) on survival and cellular function of human liver cells (Huh7) and Pimephales promelas (fathead minnow) fibroblast cells (FMH). In Huh7 cells we investigate effects on the endoplasmatic reticulum (ER), including ER stress, and interactions of nanoparticles (NPs) with metabolizing enzymes and efflux transporters. The NPs formed agglomerates/aggregates in cell culture media as revealed by SEM and TEM. SiO(2) and SiO(2)-1% Ag-NPs were taken up into cells as demonstrated by agglomerates occurring in vesicular-like structures or freely dispersed in the cytosol. Cytotoxicity was more pronounced in Huh7 than in FMH cells, and increased with silver content in silver-doped NPs. Dissolved silver was the most significant factor for cytotoxicity. At toxic and non-cytotoxic concentrations SiO(2)-NPs and SiO(2)-1% Ag-NPs induced perturbations in the function of ER. In Huh7 cells NPs induced the unfolded protein response (UPR), or ER stress response, as demonstrated in induced expression of BiP and splicing of XBP1 mRNA, two selective markers of ER stress. Additionally, SiO(2)-1% Ag-NPs and AgNO(3) induced reactive oxygen species. Pre-treatment of Huh7 cells with SiO(2)-1% Ag-NPs followed by exposure to the inducer benzo(a)pyrene caused a significant reduced induction of CYP1A activity. NPs did not alter the activity of ABC transporters. These data demonstrate for the first time that SiO(2)-NPs and SiO(2)-1% Ag-NPs result in perturbations of the ER leading to the ER stress response. This represents a novel and significant cellular signalling pathway contributing to the cytotoxicity of NPs.

Antiandrogenic activity of phthalate mixtures: validity of concentration addition.

Phthalates and bisphenol A have very widespread use leading to significant exposure of humans. They are suspected to interfere with the endocrine system, including the androgen, estrogen and the thyroid hormone system. Here we analyzed the antiandrogenic activity of six binary, and one ternary mixture of phthalates exhibiting complete antiandrogenic dose-response curves, and binary mixtures of phthalates and bisphenol A at equi-effective concentrations of EC(10), EC(25) and EC(50) in MDA-kb2 cells. Mixture activity followed the concentration addition (CA) model with a tendency to synergism at high and antagonism at low concentrations. Isoboles and the toxic unit approach (TUA) confirmed the additive to synergistic activity of the binary mixtures BBP+DBP, DBP+DEP and DEP+BPA at high concentrations. Both methods indicate a tendency to antagonism for the EC(10) mixtures BBP+DBP, BBP+DEP and DBP+DEP, and the EC(25) mixture of DBP+BPA. A ternary mixture revealed synergism at the EC(50), and weak antagonistic activity at the EC(25) level by the TUA. A mixture of five phthalates representing a human urine composition and reflecting exposure to corresponding parent compounds showed no antiandrogenic activity. Our study demonstrates that CA is an appropriate concept to account for mixture effects of antiandrogenic phthalates and bisphenol A. The interaction indicates a departure from additivity to antagonism at low concentrations, probably due to interaction with the androgen receptor and/or cofactors. This study emphasizes that a risk assessment of phthalates should account for mixture effects by applying the CA concept.

Some flame retardants and the antimicrobials triclosan and triclocarban enhance the androgenic activity in vitro.

Contaminants including flame retardants, antimicrobial agents and phthalates, occurring as residues in human tissues were associated with altered endocrine function. In our study we analysed the flame retardants tetrabromobisphenol A (TBBPA), hexabromocyclodecane (HBCD), penta-bromodiphenylether (BDE-100) and hexa-BDE (BDE-155), the antimicrobial compounds triclosan (TCS) and triclocarban (TCC) and eight phthalates for their androgenic and antiandrogenic activity in vitro in the MDA-kb2 cell line. No or only weak androgenic activity was observed for all the tested compounds. TBBPA showed weak antiandrogenic activity, which was demonstrated for the first time. The flame retardants HBCD, BDE-100 and BDE-155 enhanced the dihydrotestosterone-dependent activation of androgen receptor-responsive gene expression but exhibited little or no agonistic activity. The enhancement reached 150%, which was similar to the antimicrobials (TCS up to 180%, and TCC up to 130%). This enhancement of androgenic activity represents a novel mode of action of the endocrine activity of flame retardants. In contrast, most phthalates showed antiandrogenic activity. Butylbenzyl phthalate (BBP), dibutyl phthalate (DBP) and diethyl phthalate (DEP) showed strong antiandrogenicity, whereas the action of diethylhexyl phthalate (DEHP), dipentyl phthalate (DPP), dimethyl phthalate (DMP), and the DEHP metabolite monoethylhexyl phthalate (MEHP) was lower. Our in vitro study demonstrates for the first time a weak antiandrogenic activity of TBBPA, and a significant enhancement of the androgenic activity of HBCD, BDE-100 and BDE-155, which represents a novel mechanism of hormonal activity of flame retardants.

Hepatitis C virus-induced up-regulation of protein phosphatase 2A inhibits histone modification and DNA damage repair.

UNLABELLED: The molecular mechanisms underlying hepatocarcinogenesis in chronic viral hepatitis are poorly understood. A potential tumorigenic pathway could involve protein phosphatase 2A (PP2A) and protein arginine methyltransferase 1 (PRMT1), because both enzymes are dysregulated in chronic hepatitis C, and both enzymes have been involved in chromatin remodeling and DNA damage repair. We used cell lines that allow the inducible expression of hepatitis C virus proteins (UHCV57.3) and of the catalytic subunit of PP2A (UPP2A-C8) as well as Huh7.5 cells infected with recombinant cell culture-derived hepatitis C virus (HCVcc) to study epigenetic histone modifications and DNA damage repair. The induction of viral proteins, the overexpression of PP2Ac, or the infection of Huh7.5 cells with HCVcc resulted in an inhibition of histone H4 methylation/acetylation and histone H2AX phosphorylation, in a significantly changed expression of genes important for hepatocarcinogenesis, and inhibited DNA damage repair. Overexpression of PP2Ac in NIH-3T3 cells increased anchorage-independent growth. These changes were partially reversed by the treatment of cells with the methyl-group donor S-adenosyl-L-methionine (SAMe). CONCLUSION: Hepatitis C virus-induced overexpression of PP2Ac contributes to hepatocarcinogenesis through dysregulation of epigenetic histone modifications. The correction of defective histone modifications by S-adenosyl-L-methionine makes this drug a candidate for chemopreventive therapies in patients with chronic hepatitis C who are at risk for developing hepatocellular carcinoma.

Interferon signaling and treatment outcome in chronic hepatitis C.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFNalpha) and ribavirin. It achieves a sustained viral clearance in only 50-60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFNalpha. In patients with a rapid virological response to treatment, pegIFNalpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFNalpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.

Virus-induced over-expression of protein phosphatase 2A inhibits insulin signalling in chronic hepatitis C.

BACKGROUND/AIMS: Hepatitis C virus (HCV) infection disturbs glucose and lipid metabolism contributing to the development of liver steatosis, insulin resistance and type 2 diabetes mellitus. On the other hand, insulin resistance and steatosis have been found to be associated with increased rates of fibrosis progression and lower rates of response to interferon therapy in chronic hepatitis C (CHC). The molecular mechanisms contributing to insulin resistance in CHC are not well understood. We have shown previously that protein phosphatase 2A (PP2A) is over-expressed in biopsies from patients with CHC. In this study, we tested if PP2A over-expression leads to insulin resistance. METHODS: We studied insulin signalling in cell lines that allow the regulated over-expression of HCV proteins and of the PP2A catalytic subunit (PP2Ac). Insulin signalling and PP2Ac expression were also studied in HCV transgenic mice and in liver biopsies from patients with CHC. RESULTS: Over-expression of PP2Ac in cells inhibited insulin signalling by dephosphorylation of PKB/Akt. PP2Ac over-expression and impaired insulin signalling were found in the liver of HCV transgenic mice and in liver biopsies of patients with CHC. CONCLUSIONS: HCV-induced over-expression of PP2A in the liver contributes to the pathogenesis of insulin resistance in patients with CHC.

Inhibition of alpha interferon signaling by hepatitis B virus.

Alpha interferon (IFN-alpha) and pegylated IFN-alpha (pegIFN-alpha) are used for the treatment of chronic hepatitis B (CHB). Unfortunately, only a minority of patients can be cured. The mechanisms responsible for hepatitis B virus (HBV) resistance to pegIFN-alpha treatment are not known. pegIFN-alpha is also used to treat patients with chronic hepatitis C (CHC). As with chronic hepatitis B, many patients with chronic hepatitis C cannot be cured. In CHC, IFN-alpha signaling has been found to be inhibited by an upregulation of protein phosphatase 2A (PP2A). PP2A inhibits protein arginine methyltransferase 1 (PRMT1), the enzyme that catalyzes the methylation of the important IFN-alpha signal transducer STAT1. Hypomethylated STAT1 is less active because it is bound by its inhibitor, PIAS1. In the present work, we investigated whether similar molecular mechanisms are also responsible for the IFN-alpha resistance found in many patients with chronic hepatitis B. We analyzed the expression of PP2A, the enzymatic activity of PRMT1 (methylation assays), the phosphorylation and methylation of STAT1, the association of STAT1 with PIAS1 (via coimmunoprecipitation assays), the binding of activated STAT1 to interferon-stimulated response elements (via electrophoretic mobility shift assays), and the induction of interferon target genes (via real-time RT-PCR) in human hepatoma cells expressing HBV proteins as well as in liver biopsies from patients with chronic hepatitis B and from controls. We found an increased expression of PP2A and an inhibition of IFN-alpha signaling in cells expressing HBV proteins and in liver biopsies of patients with CHB. The molecular mechanisms involved are similar to those found in chronic hepatitis C.