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Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.
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Neoplasias Hepáticas Experimentais/induzido quimicamente , PPAR alfa/fisiologia , Animais , Benchmarking , Peso Corporal/efeitos dos fármacos , Proliferação de Células , Dietilexilftalato/toxicidade , Relação Dose-Resposta a Droga , Modelos Lineares , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Estresse Oxidativo , Ácidos Ftálicos/toxicidade , Reação em Cadeia da PolimeraseRESUMO
The 2 objectives of this subchronic study were to determine the arsenite drinking water exposure dependent increases in female C3H mouse liver and lung tissue arsenicals and to characterize the dose response (to 0, 0.05, 0.25, 1, 10, and 85 ppm arsenite in drinking water for 30 days and a purified AIN-93M diet) for genomic mouse lung expression patterns. Mouse lungs were analyzed for inorganic arsenic, monomethylated, and dimethylated arsenicals by hydride generation atomic absorption spectroscopy. The total lung mean arsenical levels were 1.4, 22.5, 30.1, 50.9, 105.3, and 316.4 ng/g lung tissue after 0, 0.05, 0.25, 1, 10, and 85 ppm, respectively. At 85 ppm, the total mean lung arsenical levels increased 14-fold and 131-fold when compared to either the lowest noncontrol dose (0.05 ppm) or the control dose, respectively. We found that arsenic exposure elicited minimal numbers of differentially expressed genes (DEGs; 77, 38, 90, 87, and 87 DEGs) after 0.05, 0.25, 1, 10, and 85 ppm, respectively, which were associated with cardiovascular disease, development, differentiation, apoptosis, proliferation, and stress response. After 30 days of arsenite exposure, this study showed monotonic increases in mouse lung arsenical (total arsenic and dimethylarsinic acid) concentrations but no clear dose-related increases in DEG numbers.
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The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher's algorithm (p-value ≤ 10(-4))) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of inflammation including acetaminophen, concanavalin A, lipopolysaccharide, and 300 nm silica particles. In conclusion, we have shown that a CAR biomarker signature coupled with a rank-based similarity method accurately predicts CAR activation. This analytical approach, when applied to a gene expression compendium, increased the universe of known chemicals that directly or indirectly activate CAR, highlighting the promiscuous nature of CAR activation and signaling through activation of other xenobiotic-activated receptors.
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Regulação da Expressão Gênica/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Animais , Biomarcadores/metabolismo , Receptor Constitutivo de Androstano , Avaliação Pré-Clínica de Medicamentos , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Environmental exposures occurring early in life may have an important influence on cancer risk later in life. Here, we investigated carryover effects of dichloroacetic acid (DCA), a small molecule analog of pyruvate with metabolic programming properties, on age-related incidence of liver cancer. The study followed a stop-exposure/promotion design in which 4-week-old male and female B6C3F1 mice received the following treatments: deionized water alone (dH2O, control); dH2O with 0.06% phenobarbital (PB), a mouse liver tumor promoter; or DCA (1.0, 2.0 or 3.5g/l) for 10 weeks followed by dH2O or PB (n = 20-30/group/sex). Pathology and molecular assessments were performed at 98 weeks of age. In the absence of PB, early-life exposure to DCA increased the incidence and number of hepatocellular tumors in male and female mice compared with controls. Significant dose trends were observed in both sexes. At the high dose level, 10 weeks of prior DCA treatment induced comparable effects (≥85% tumor incidence and number) to those seen after continuous lifetime exposure. Prior DCA treatment did not enhance or inhibit the carcinogenic effects of PB, induce persistent liver cytotoxicity or preneoplastic changes on histopathology or alter DNA sequence variant profiles within liver tumors compared with controls. Distinct changes in liver messenger RNA and micro RNA profiles associated with prior DCA treatment were not apparent at 98 weeks. Our findings demonstrate that early-life exposure to DCA may be as carcinogenic as life-long exposures, potentially via epigenetic-mediated effects related to cellular metabolism.
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Ácido Dicloroacético/farmacologia , Neoplasias Hepáticas/induzido quimicamente , Animais , Metilação de DNA/efeitos dos fármacos , Ácido Dicloroacético/administração & dosagem , Ácido Dicloroacético/toxicidade , Relação Dose-Resposta a Droga , Ingestão de Alimentos , Poluentes Ambientais/toxicidade , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos , MicroRNAs , Fenobarbital/toxicidade , RNA MensageiroRESUMO
OBJECTIVES: We evaluated a community-based, translational lifestyle program to reduce diabetes risk in lower-socioeconomic status (SES) and ethnic minority adults. METHODS: Through an academic-public health department partnership, community-dwelling adults at risk for diabetes were randomly assigned to individualized lifestyle counseling delivered primarily via telephone by health department counselors or a wait-list control group. Primary outcomes (6 and 12 months) were fasting glucose level, triglycerides, high- and low-density lipoprotein cholesterol, weight, waist circumference, and systolic blood pressure. Secondary outcomes included diet, physical activity, and health-related quality of life. RESULTS: Of the 230 participants, study retention was 92%. The 6-month group differences for weight and triglycerides were significant. The intervention group lost 2 pounds more than did the control group (P=.03) and had decreased triglyceride levels (difference in change, 23 mg/dL; P=.02). At 6 months, the intervention group consumed 7.7 fewer grams per day of fat (P=.05) and more fruits and vegetables (P=.02) than did control participants. CONCLUSIONS: Despite challenges designing effective translational interventions for lower-SES and minority communities, this program modestly improved some diabetes risk factors. Thus, individualized, telephone-based models may be a promising alternative to group-based interventions.
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Diabetes Mellitus/prevenção & controle , Etnicidade , Promoção da Saúde/métodos , Grupos Minoritários , Classe Social , Adulto , Idoso , California , Aconselhamento , Feminino , Seguimentos , Humanos , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Características de Residência , Fatores de Risco , Inquéritos e Questionários , TelefoneRESUMO
Conazoles are fungicides used as agricultural pesticides and pharmaceutical products. We investigated whether a common core of toxicological and transcriptional responses underlies the observed carcinogenic effects of three conazoles: cyproconazole, epoxiconazole, and propiconazole. In studies where mice were fed diets of these conazoles for 30 days, we found a common set of toxicological effects altered by these conazoles: hepatomegaly, hepatocellular hypertrophy, decreased serum cholesterol, decreased hepatic levels of all-trans-retinoic acid, and increased hepatic cell proliferation. Microarray-based transcriptional analysis revealed 330 significantly altered probe sets common to these conazoles, many of which showed strong dose responses for cytochrome P450, glutathione S-transferase, and oxidative stress genes. More detailed analyses identified a subset of 80 altered genes common to the three conazoles that were associated with cancer. Pathways associated with these genes included xenobiotic metabolism, oxidative stress, cell signaling, and cell proliferation. A common TGFα-centric pathway was identified within the 80-gene set, which, in combination with the toxicological and other transcriptomic findings, provides a more refined toxicity profile for these carcinogenic conazoles.
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Carcinógenos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fungicidas Industriais/toxicidade , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Triazóis/toxicidade , Administração Oral , Animais , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Relação Dose-Resposta a Droga , Compostos de Epóxi/toxicidade , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular fractions from control, MVAL or propiconazole-treated cells revealed increased Ras protein in the cytoplasmic fraction of L-744,832-treated cells, while propiconazole or MVAL reversed these effects. Western blot analysis indicated that phosphorylation of Erk1/2, a protein downstream of Ras, was increased by propiconazole. These data indicate that propiconazole increases cell proliferation by increasing the levels of cholesterol biosynthesis intermediates presumably through a negative feedback mechanism within the pathway, a result of CYP51 inhibition. This feedback mechanism increases Erk1/2 signaling through mevalonate-mediated Ras activation. These results provide an explanation for the observed effects of propiconazole on hepatic cholesterol pathways and on the increased hepatic cell proliferation induced by propiconazole in mice.
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Antifúngicos/farmacologia , Colesterol/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fígado/efeitos dos fármacos , Triazóis/farmacologia , Proteínas ras/metabolismo , Animais , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Metionina/análogos & derivados , Metionina/farmacologia , Camundongos , Prenilação/efeitos dos fármacos , Triazóis/antagonistas & inibidoresRESUMO
Perinatal loss can be emotionally devastating for couples who experience miscarriage, fetal or neonatal death. Nurses in a variety of settings can assist couples through their grief by providing emotional support, giving information about the grief process, and in planning for a future pregnancy or deciding to forego future childbearing. This article explicates the relationship between grief and perinatal loss and its effects on couples, specifically in the interconception period, when the initial grief and distress have begun to subside. Interconception care focuses on bridging the couple from the end of the postpartum period to the subsequent pregnancy or decision not to conceive again. Nurses assist couples in distinguishing between uncomplicated grief, complicated grief, and depression, and make appropriate referrals. Openness to expressions of grief, helping couples mobilize support, considering readiness for another pregnancy, and directing couples to useful Internet sites are essential nursing interventions.
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Morte Fetal , Pesar , Papel do Profissional de Enfermagem , Relações Pais-Filho , Pais/psicologia , Cuidado Pré-Concepcional/métodos , Adulto , Ansiedade/prevenção & controle , Atitude Frente a Morte , Depressão/prevenção & controle , Feminino , Humanos , Masculino , Memória , Gravidez , Apoio Social , Transtornos de Estresse Pós-Traumáticos/prevenção & controle , Adulto JovemRESUMO
Propiconazole is a mouse hepatotumorigenic fungicide and has been the subject of recent investigations into its carcinogenic mechanism of action. The goals of this study were (1) to identify metabolomic changes induced in the liver by increasing doses of propiconazole in mice, (2) to interpret these results with key previously reported biochemical, transcriptomic, and proteomic findings obtained from mouse liver under the same treatment conditions, and (3) to relate these alterations to those associated with the carcinogenesis process. Propiconazole was administered to male CD-1 mice in the feed for 4 days with six mice per feed level (500, 1250, and 2500 ppm). The 2500 ppm dose level had previously been shown to induce both adenocarcinomas and adenomas in mouse liver after a 2-year continuous feed regimen. Endogenous biochemicals were profiled using liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry methods and 261 were detected. The most populous biochemical class detected was lipids, followed by amino acids and then carbohydrates. Nucleotides, cofactors and vitamins, energy, peptides, and xenobiotics were also represented. Of the biochemicals detected, 159 were significantly altered by at least one dose of propiconazole and many showed strong dose responses. Many alterations in the levels of biochemicals were found in the glycogen metabolism, glycolysis, lipolysis, carnitine, and the tricarboxylic acid cycle pathways Several groups of metabolomic responses were ascribed to the metabolism and clearance of propiconazole: glucuronate, glutathione, and cysteine pathways. Groups of metabolic responses supported previous hypotheses on key events that can lead to propiconazole-induced tumorigenesis: oxidative stress and increases in the cholesterol biosynthesis pathway. Groups of metabolomic responses identified biomarkers associated with neoplasia: increases in glycolysis and increases in the levels of spermidine, sarcosine, and pseudouridine. These results extended the companion transcriptomic and proteomic studies and provided a more complete understanding of propiconazole's effects in mouse liver.
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Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fungicidas Industriais/toxicidade , Fígado/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Triazóis/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fungicidas Industriais/química , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Estrutura Molecular , Tamanho do Órgão/efeitos dos fármacos , Triazóis/químicaRESUMO
Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0mg/kg. Lungs and livers were harvested after 24h, the DNA extracted and subjected to (32)P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.
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7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/química , Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/biossíntese , Adutos de DNA/química , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Acetilcisteína/farmacologia , Animais , Benzo(a)pireno/química , Benzo(a)pireno/metabolismo , Carcinógenos/química , Carcinógenos/metabolismo , Sequestradores de Radicais Livres/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos A , Modelos Biológicos , Radioisótopos de Fósforo , Hidrocarbonetos Policíclicos Aromáticos/química , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidadeRESUMO
BACKGROUND: Simian Virus 40 (SV40) immortalization followed by treatment of cells with 3-methylcholanthrene (3-MC) has been used to elicit tumors in athymic mice. 3-MC carcinogenesis has been thoroughly studied, however gene-level interactions between 3-MC and SV40 that could have produced the observed tumors have not been explored. The commercially-available human uroepithelial cell lines were either SV40-immortalized (HUC) or SV40-immortalized and then 3-MC-transformed (HUC-TC). RESULTS: To characterize the SV40 - 3MC interaction, we compared human gene expression in these cell lines using a human cancer array and confirmed selected changes by RT-PCR. Many viral Large T Antigen (Tag) expression-related changes occurred in HUC-TC, and it is concluded that SV40 and 3-MC may act synergistically to transform cells. Changes noted in IFP 9-27, 2'-5' OAS, IF 56, MxA and MxAB were typical of those that occur in response to viral exposure and are part of the innate immune response. Because interferon is crucial to innate immune host defenses and many gene changes were interferon-related, we explored cellular growth responses to exogenous IFN-gamma and found that treatment impeded growth in tumor, but not immortalized HUC on days 4 - 7. Cellular metabolism however, was inhibited in both cell types. We conclude that IFN-gamma metabolic responses were functional in both cell lines, but IFN-gamma anti-proliferative responses functioned only in tumor cells. CONCLUSIONS: Synergism of SV40 with 3-MC or other environmental carcinogens may be of concern as SV40 is now endemic in 2-5.9% of the U.S. population. In addition, SV40-immortalization is a generally-accepted method used in many research materials, but the possibility of off-target effects in studies carried out using these cells has not been considered. We hope that our work will stimulate further study of this important phenomenon.
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We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this fungicide. Utilizing two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we identified 62 proteins that were altered. Several of these protein changes detected by 2-DE/MS were verified by Western blot analyses. These differentially expressed proteins were mapped using Ingenuity Pathway Analyses (IPA) canonical pathways and IPA tox lists. Forty-four pathways/lists were identified. IPA was also used to create networks of interacting protein clusters. The protein-generated IPA canonical pathways and IPA tox lists were compared to those pathways and lists previously generated from genomic analyses from livers of mice treated with propiconazole under the same experimental conditions. There was a significant overlap in the specific pathways and lists generated from the proteomic and the genomic data with 27 pathways common to both proteomic and genomic analyses. However, there were also 17 pathways/lists identified only by proteomics analysis and 21 pathways/lists only identified by genomic analysis. The protein network analysis produced interacting subnetworks centered around hepatocyte nuclear factor 4 alpha (HNF4 alpha), MYC, proteasome subunit type 4 alpha, and glutathione S-transferase (GST). The HNF4 alpha network hub was also identified by genomic analysis. Five GST isoforms were identified by proteomic analysis and GSTs were present in 10 of the 44 protein-based pathways/lists. Hepatic GST activities were compared between mice treated with propiconazole and 2 additional conazoles and higher GST activities were found to be associated with the tumorigenic conazoles. Overall, this comparative proteomic and genomic study has revealed a series of alterations in livers induced by propiconazole: nuclear receptor activation, metabolism of xenobiotics, metabolism of biochemical intermediates, biosynthesis of biochemical intermediates, and oxidative stress in mouse liver. The present study provides novel insights into toxic mechanisms and/or modes of action of propiconazole which are required for human health risk assessment of this environmental chemical.
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Genômica/métodos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteoma/efeitos dos fármacos , Triazóis/farmacologia , Animais , Western Blotting , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Fígado/química , Masculino , Espectrometria de Massas , Camundongos , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteômica/métodos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacosRESUMO
Propiconazole, a widely used fungicide, is hepatotoxic and hepatotumorigenic in mice. Previous genomic analysis of liver tissues from propiconazole-treated mice identified genes and pathways involved in oxidative stress, suggesting that oxidative stress may play a role in propiconazole-induced toxicity. To understand the contribution of oxidative stress on toxicity at the protein level, we developed an integrated approach for the systematic measurement of protein oxidation in the livers from propiconazole-treated mice. Liver protein carbonylation increased significantly after treatment with propiconazole, demonstrating propiconazole-associated induction of oxidative stress. Utilizing two-dimensional gel electrophoresis (2-DE), immunoblotting, and mass spectrometry, we identified 17 carbonylated proteins that were altered with varying intensities by propiconazole treatment. The potential effects of protein carbonylation on protein functions and cellular activities in the liver of propiconazole-treated mice were further investigated. A significant negative correlation between protein carbonylation and cytochrome c reductase activity was found. We conclude that glycolysis, mitochondrial respiratory chain, ATP production, amino acid metabolism, CO2 hydration, cellular antioxidant defense and detoxification system, and tetrahydrobiopterin pathways are affected by oxygen radicals in the livers of propiconazole-treated mice. This study suggests a mode of propiconazole-induced toxicity in mouse liver which primarily involves oxidative damage to cellular proteins.
Assuntos
Fungicidas Industriais/farmacologia , Fígado/metabolismo , Estresse Oxidativo/fisiologia , Carbonilação Proteica/fisiologia , Triazóis/farmacologia , Animais , Eletroforese em Gel Bidimensional , Fungicidas Industriais/toxicidade , Fígado/efeitos dos fármacos , Camundongos , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triazóis/toxicidadeRESUMO
Conazoles are fungicides used to control fungal growth in environmental settings and to treat humans with fungal infections. Mouse hepatotumorigenic conazoles display many of the same hepatic toxicologic responses as the mouse liver carcinogen phenobarbital (PB): constitutive androstane receptor (CAR) activation, hypertrophy, Cyp2b induction, and increased cell proliferation. The goal of this study was to apply transcriptional analyses to hepatic tissues from mice exposed to PB, propiconazole (Pro) or triadimefon (Tri) at tumorigenic exposure levels to reveal similarities and differences in response among these treatments. Mice were administered diets containing PB (850 ppm), Pro (2500 ppm), or Tri (1800 ppm) for 4 and 30 days. Targeted transcriptomic analyses were conducted at the gene level examining differentially expressed genes (DEGs), and subsets of DEGs: cell cycle genes, and transcription factors. Analyses were also conducted on function, pathway and network levels examining Ingenuity Pathway Analysis Tox Lists and Canonical Pathways, and Gene-Go MetaCore dynamic networks and their central hubs. Genes expressed by PB or the two conazoles were also compared with those genes associated with human hepatocellular cancer. The results from these analyses indicated greater differences between PB and the two conazoles than similarities. Significant commonalities between the two conazole treatments were also noted. We posit that the transcriptional profiles of tissues exposed to toxic chemicals inherently contain their mechanisms of toxicity. We conclude that although PB and these 2 conazoles induce mouse liver tumors and exhibit similar toxicological responses, their transcriptional profiles are significantly different and thus their mechanisms of tumorigenic action are likely to differ.
Assuntos
Carcinógenos/toxicidade , Fungicidas Industriais/toxicidade , Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Fenobarbital/farmacologia , Transcrição Gênica/efeitos dos fármacos , Triazóis/toxicidade , Animais , Carcinoma Hepatocelular/genética , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Hibridização Genética , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Triadimefon, propiconazole and myclobutanil are conazoles, an important class of agricultural and therapeutic fungicides. Triadimefon and propiconazole are mouse liver tumorigens, while myclobutanil is not. All three conazoles are generally inactive in short-term genotoxicity tests. We studied the in vivo mutagenicity of these three conazoles using the Big Blue mouse assay system. Groups of mice were fed either control diet or diet containing 1800 p.p.m. triadimefon, 2500 p.p.m. propiconazole or 2000 p.p.m. myclobutanil. After 4 days of feeding, mice were immediately euthanized, livers were removed, DNA isolated and lacI genes recovered into infectious bacteriophage lambda particles by in vitro packaging. Bacteriophage with mutations in the lacI gene was detected by infecting into Escherichia coli, and mutant frequencies were determined using a colorimetric plaque assay. Propiconazole induced a 1.97-fold increase in mutant frequency compared to concurrent controls (P = 0.018) and triadimefon induced a 1.94-fold increase compared to concurrent controls (P = 0.009). Myclobutanil did not induce any change in mutant frequency (P = 0.548). These results provide the first evidence that the hepatotumorigenic conazoles are capable of inducing mutations in liver in vivo while the non-tumorigen myclobutanil is not, suggesting that mutagenicity may represent a key event in conazoles tumorigenic mode of action.
Assuntos
Antifúngicos/toxicidade , Fungicidas Industriais/toxicidade , Neoplasias/patologia , Animais , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Mutagenicidade , Mutação/genéticaRESUMO
Conazoles are fungicides used in agriculture and as pharmaceuticals. In a previous toxicogenomic study of triazole-containing conazoles we found gene expression changes consistent with the alteration of the metabolism of all trans-retinoic acid (atRA), a vitamin A metabolite with cancer-preventative properties (Ward et al., Toxicol. Pathol. 2006; 34:863-78). The goals of this study were to examine effects of propiconazole, triadimefon, and myclobutanil, three triazole-containing conazoles, on the microsomal metabolism of atRA, the associated hepatic cytochrome P450 (P450) enzyme(s) involved in atRA metabolism, and their effects on hepatic atRA levels in vivo. The in vitro metabolism of atRA was quantitatively measured in liver microsomes from male CD-1 mice following four daily intraperitoneal injections of propiconazole (210 mg/kg/d), triadimefon (257 mg/kg/d) or myclobutanil (270 mg/kg/d). The formation of both 4-hydroxy-atRA and 4-oxo-atRA were significantly increased by all three conazoles. Propiconazole-induced microsomes possessed slightly greater metabolizing activities compared to myclobutanil-induced microsomes. Both propiconazole and triadimefon treatment induced greater formation of 4-hydroxy-atRA compared to myclobutanil treatment. Chemical and immuno-inhibition metabolism studies suggested that Cyp26a1, Cyp2b, and Cyp3a, but not Cyp1a1 proteins were involved in atRA metabolism. Cyp2b10/20 and Cyp3a11 genes were significantly over-expressed in the livers of both triadimefon- and propiconazole-treated mice while Cyp26a1, Cyp2c65 and Cyp1a2 genes were over-expressed in the livers of either triadimefon- or propiconazole-treated mice, and Cyp2b10/20 and Cyp3a13 genes were over-expressed in the livers of myclobutanil-treated mice. Western blot analyses indicated conazole induced-increases in Cyp2b and Cyp3a proteins. All three conazoles decreased hepatic atRA tissue levels ranging from 45-67%. The possible implications of these changes in hepatic atRA levels on cell proliferation in the mouse tumorigenesis process are discussed.
Assuntos
Fungicidas Industriais/toxicidade , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Tretinoína/metabolismo , Animais , Western Blotting , Peso Corporal/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Nitrilas/toxicidade , Tamanho do Órgão/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização por Electrospray , Tretinoína/análogos & derivados , Triazóis/toxicidadeRESUMO
Large-scale differential gene expression analysis was used to examine the biological effects of disinfected surface waters on cultured rat hepatocytes. Source water from East Fork Lake (Harsha Lake), a reservoir on the Little Miami River in Ohio, was spiked with iodide and bromide and disinfected by chlorination or ozonation/postchlorination. The chlorinated and ozonated/postchlorinated waters were concentrated, respectively, 136- and 124-fold (full strength) by reverse-osmosis membrane techniques. Volatile disinfection by-products (DBP) lost during concentration were restored to the extent possible. Primary rat hepatocytes were exposed to either full-strength or 1:10 or 1:20 dilutions of the concentrates for 24 h and assayed for cytotoxicity and gene expression alterations. The full-strength concentrates were cytotoxic, whereas the diluted samples exhibited no detectable cytotoxicity. Differential gene expression analysis provided evidence for the underlying causes of the severe cytotoxicity observed in rat hepatocytes treated with the full-strength ozonation/postchlorination concentrate (e.g., cell cycle arrest, metabolic stasis, oxidative stress). Many gene expression responses were shared among the hepatocyte cultures treated with dilutions of the ozonation/ postchlorination and chlorination concentrates. The shift in the character of the response between the full-strength concentrates and the diluted samples indicated a threshold for toxicity. A small subset of gene expression changes was identified that was observed in the response of hepatocytes to peroxisome proliferators, phthalate esters, and haloacetic acids, suggesting a peroxisome proliferative response.
Assuntos
Compostos Clorados/farmacologia , Desinfetantes/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Halogenação , Hepatócitos/efeitos dos fármacos , Ozônio/toxicidade , Poluentes Químicos da Água/farmacologia , Animais , Células Cultivadas , Compostos Clorados/toxicidade , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
Propiconazole is a triazole-containing fungicide that is used agriculturally on grasses, fruits, grains, seeds, hardwoods, and conifers. Propiconazole is a mouse liver hepatotoxicant and a hepatocarcinogen that has adverse reproductive and developmental toxicities in experimental animals. The goal of this study was to investigate the cytotoxic responses of propiconazole and its metabolites to determine if metabolism of this agent differentially affected its cytotoxic activities in hepatic tumor cell lines and in primary hepatocytes. To this end the cytotoxic effects of propiconazole and five of its metabolites were examined in three hepatic cell types: The mouse hepatoma Hepa1c1c7 cell line, the human hepatoma HepG2 cell line, and primary cultures of mouse hepatocytes. We initially compared the responses of propiconazole exposure in both Hepa1c1c7 and HepG2 cell lines over a concentration range of 0-200 microM using two assay systems: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the neutral red assay. Concentration-related cytotoxic responses were evident in both cell lines using both endpoints with the MTT assay providing enhanced sensitivity. The relative cytotoxic effects of propiconazole and five propiconazole metabolites were further assessed by the MTT assay using Hepa1c1c7 and HepG2 tumor cell lines. The cell cultures were exposed to various concentrations of propiconazole and five of its metabolites over a range of 0-400 microM. Propiconazole was cytotoxic in both cell lines in a dose-dependent manner. All five metabolites were less cytotoxic in both cell lines compared to the parent compound. The most cytotoxic metabolites in Hepa1c1c7 and HepG2 cells among the five were 3-(2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)propan-1-ol and 1-(2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)propan-2-ol. Propiconazole was cytotoxic in primary mouse hepatocytes; however none of the five propiconazole metabolites exerted cytotoxic activities. There was a linear relationship between the cLogP and the cytotoxic effects of propiconazole and its five metabolites in Hepa1c1c7 cells. We conclude that these propiconazole metabolites would not contribute to the propiconazole-induced cytotoxicity process in primary mouse hepatocytes. Furthermore, since in tumor cell lines the metabolites were less cytotoxic than the parent propiconazole, our results suggest that in the tumorigenesis process as tumor cells are formed they would be more susceptible to the cytotoxic effects of propiconazole compared to the metabolites.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Fungicidas Industriais/toxicidade , Neoplasias Hepáticas/metabolismo , Triazóis/toxicidade , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fungicidas Industriais/administração & dosagem , Fungicidas Industriais/metabolismo , Humanos , Camundongos , Especificidade da Espécie , Triazóis/administração & dosagem , Triazóis/metabolismoRESUMO
Dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P) are carcinogenic polycyclic aromatic hydrocarbons (PAHs) that are each capable of forming a variety of covalent adducts with DNA. Some of the DNA adducts formed by these PAHs have been demonstrated to spontaneously depurinate, producing apurinic (AP) sites. The significance of the formation of AP sites as a key event in the production of mutations and tumours by PAHs has been a subject of ongoing investigations. Because cells have efficient and accurate mechanisms for repairing background levels of AP sites, the contribution of PAH-induced AP site mutagenesis is expected to be maximal in conditions where those induced AP sites are produced in significant excess of the endogenous AP sites. In this study, we investigated the effect of two dosing regimens on the mutagenicity of DB[a,l]P and B[a]P in vivo using the Big Blue(R) transgenic mouse system. We compared administration of a single highly tumorigenic dose of each PAH with a fractionated delivery of the same total dose administered over 5 days, with the expectation that PAH-induced AP sites would be produced at a greater margin above background levels in animals receiving the high single dose than in the animals receiving the fractionated doses. Treatment with DB[a,l]P yielded a 2.5-fold (single dose) to 3-fold (fractionated dose) increase in mutant frequencies relative to controls. Both single-dose and fractionated dose treatment regimens with B[a]P produced about a 15-fold increase in mutant frequencies compared to controls. The mutations induced by B[a]P and DB[a,l]P correlated with the stable covalent DNA adducts produced by each. These mutation results are consistent with the previously identified stable covalent DNA adducts being the promutagenic lesions produced by these two PAHs and do not support a major role for depurinating adducts, contributing to PAH-induced mutagenesis in mouse lung in vivo.
Assuntos
Proteínas de Bactérias/genética , Benzo(a)pireno/toxicidade , Benzopirenos/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/análise , Pulmão/efeitos dos fármacos , Mutação , Proteínas Repressoras/genética , Animais , Proteínas de Bactérias/metabolismo , Benzo(a)pireno/administração & dosagem , Benzopirenos/administração & dosagem , Carcinógenos/administração & dosagem , Dano ao DNA , Feminino , Repressores Lac , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Repressoras/metabolismoRESUMO
Perfluorooctanoic acid (PFOA) is a chemical used in the production of fluoropolymers. Its persistence in the environment and presence in humans and wildlife has raised health concerns. Liver tumor induction by PFOA is thought to be mediated in rodents by PPAR-alpha. A recent US EPA scientific advisory board questioned the contribution of PPAR-alpha in PFOA-induced liver tumors. Liver response in CD-1, SV/129 wild-type (WT), and PPAR-alpha knockout (KO) SV/129 mice was evaluated after seven daily treatments of PFOA-NH4(+) (1, 3, or 10 mg/kg, p.o.) or the prototype PPARalpha-agonist Wyeth 14,643 (WY, 50 mg/kg). Livers were examined by light and electron microscopy. Proliferation was quantified after PCNA immunostaining. PFOA treatment induced a dose-dependent increase in hepatocyte hypertrophy and labeling index (LI) similar to WY in WT mice. Ultrastructural alterations of peroxisome proliferation were similar between WY-treated and 10 mg/kg PFOA-treated WT mice. KO mice had a dose-dependent increase in hepatocyte vacuolation but increased LI only at 10 mg PFOA/kg. WY-treated KO mice were not different from KO control. These data suggest that PPAR-alpha is required for WY- and PFOA-induced cellular alterations in WT mouse liver. Hepatic enlargement observed in KO mice may be due to an accumulation of cytoplasmic vacuoles that contain PFOA.