RESUMEN
Cyanide (CN = HCN + CN(-)) is a renowned poison and neurotoxicant that is prevalent throughout the environment. Despite a plethora of studies conducted over the last half century, relatively little is known of its potential to cause adverse health outcomes at sublethal exposures. CN exposure is normally determined from blood, but because CN is rapidly metabolized and cleared from this compartment (t(1/2) < 1 h), it is common for several half-lives to have passed before blood samples are drawn for analysis. This variable, coupled with a very narrow toxic index and metabolic diversity within the human population, has rendered accurate assessment of CN exposure, and consequently any predictions of possible adverse health outcomes, highly problematic. Prior studies by us showed the potential of Cys-SCN adducts within human serum albumin (HSA) to act as retrospective surrogates of CN exposure. Here, we report the discovery of a stable, SCN adduct at Cys(567) formed by the reaction of CN with the C-terminal Cys(558)Cys(567) disulfide bond of HSA. Treatment of HSA purified from human serum with base in guanidine hydrochloride releases a readily detectable, uniquely modified, C-terminal-19-mer peptide from Cys(567)-SCN moieties in all the samples examined thus far. Inclusion of a HSA-Cys(567)-S(13)C(15)N labeled internal standard permits quantitation of the Cys(567)-SCN adduct by LC-MS/MS in selective reaction monitoring (SRM) of the surrogate peptide with high sensitivity and good precision. Reaction of CN in vitro with the Cys(558)Cys(567) disulfide bond in HSA is specific, rapid, and concentration dependent within a putative, physiologically relevant range. Data from various human sera demonstrate the potential usefulness of this adduct as a biomarker of CN exposure.
Asunto(s)
Cianuros/química , Albúmina Sérica/química , Biomarcadores/química , Cromatografía Líquida de Alta Presión , Cianuros/toxicidad , Cisteína/química , Deuterio/química , Disulfuros/química , Humanos , Péptidos/química , Espectrometría de Masas en TándemRESUMEN
Arsenite, an environmental cocontaminant of polycyclic aromatic hydrocarbons (PAHs), diminishes the PAH-mediated upregulation of human CYP1A1, the enzyme that bioactivates PAHs to carcinogenic metabolites. Mechanistically, while transcriptional downregulation contributes to these effects, a role for posttranslational regulation has been implicated but not proven. We hypothesize that arsenite induces heme oxygenase-1 (HO-1), which catabolizes CYP1A1 heme or cellular heme pools, thereby downregulating CYP1A1. Arsenite (5 microM), in HepG2 cells, induced HO-1 mRNA 7.4-fold over the 48 h observation period, and it upregulated HO-1 protein expression. Arsenite decreased the induction of CYP1A1 by a PAH, benzo[k]fluoranthene (BKF), by 50%; and transfection of HepG2 cells with siRNA targeting the human HO-1 gene, reduced the arsenite downregulation of BKF-induced CYP1A1 from 54% to 27%, relative to untransfected cells. Reconstituted HO-1 did not significantly catabolize CYP1A1 heme in vitro. Together these findings demonstrate that a posttranslational mechanism involving decreases in the cellular heme pool by arsenite-induced HO-1 may contribute to arsenite-mediated downregulation of CYP1A1.
Asunto(s)
Arsenitos/toxicidad , Citocromo P-450 CYP1A2/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Fluorenos/toxicidad , Hemo-Oxigenasa 1/metabolismo , Teratógenos/toxicidad , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Factores de TiempoRESUMEN
Several systemic and cellular markers of 3'-azido-3'-dideoxythymidine (AZT) metabolism and AZT incorporation into nuclear DNA were measured in cord blood from uninfected infants born to HIV-1-infected mothers receiving prepartum therapies based on AZT or AZT in combination with 2',3'-dideoxy-3'-thiacytidine (3TC). In addition, the relationships among these pharmacological end points, levels of AZT-DNA incorporation, and the previously reported mutagenic responses in these infants were evaluated. AZT- and 3TC-specific radioimmunoassays (RIAs), or HPLC coupled with AZT-RIA, were used to measure plasma levels of AZT and the AZT-glucuronide, and cellular levels of AZT, phosphorylated AZT, and DNA incorporation of AZT or 3TC in cord blood mononuclear cells from treated infants compared with unexposed controls born to HIV-uninfected mothers. Fewer infants had detectable AZT-DNA incorporation levels in the group exposed to AZT (71%; n = 7) compared with those receiving AZT-3TC (100%; n = 21), and the mean AZT-DNA incorporation for AZT-exposed infants (14.6 +/- 6.3 AZT/10(6) nucleotides) was significantly lower than that in AZT-3TC exposed infants (51.6 +/- 10.2 AZT/10(6) nucleotides; P = 0.028). Low levels of 3TC-DNA incorporation found in a few AZT-3TC-exposed newborns correlated with AZT-DNA incorporation values in the same samples. Among the metabolites studied, there were positive correlations between levels of AZT-diphosphate and AZT-triphosphate, and AZT-triphosphate and AZT-DNA incorporation, in nucleoside analog-exposed infants. Levels of AZT-DNA incorporation, however, did not correlate well with the reported frequencies of somatic mutations in the same population of nucleoside analog-treated children. While these data support the continued use of AZT-based therapies during pregnancy, infants receiving prepartum AZT should be monitored long-term for adverse health effects.
Asunto(s)
Fármacos Anti-VIH/farmacocinética , Daño del ADN , Leucocitos Mononucleares/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacocinética , Zidovudina/farmacocinética , Fármacos Anti-VIH/sangre , Fármacos Anti-VIH/uso terapéutico , Biomarcadores/análisis , ADN/metabolismo , Femenino , Sangre Fetal/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & control , Humanos , Recién Nacido , Lamivudine/farmacocinética , Intercambio Materno-Fetal , Embarazo , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Complicaciones Infecciosas del Embarazo/prevención & control , Inhibidores de la Transcriptasa Inversa/sangre , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Zidovudina/sangre , Zidovudina/uso terapéuticoRESUMEN
PURPOSE: The regulation of carcinogen metabolism machinery may involve proximate tobacco smoke exposure, hormonal and other endogenous coregulatory factors, and an individual's underlying genetic responsiveness. The mRNA and protein expression patterns of known carcinogen metabolism genes encoding the aromatic hydrocarbon receptor Ahr; the cytochromes P450 CYP1A1 and CYP1B1; glutathione S-transferases GSTM1, GSTM3, GSTP1, and GSTT1; and NADPH quinone oxidoreductase NQO1 were examined. EXPERIMENTAL DESIGN: Paired tumor and nontumor lung tissue from 45 subjects was subject to a recently devised RNA-specific qualitative reverse transcription-PCR strategy, as well as Western immunoblotting. Tobacco exposure measured by plasma biomarkers nicotine and cotinine, plasma estradiol levels, alpha and beta estrogen receptor (ER) expression in the lung, gender, age, and histological diagnosis were then analyzed using multivariate regression models. RESULTS: In nontumor lung tissue, multivariate models identified several correlates of mRNA expression: (a) CYP1B1 in females (positively: smoke status, P=0.024; ER-beta expression, P=0.024); (b) GSTT1 in females (positively: cotinine, P=0.007; negatively: age, P=0.001; ER-beta expression, P=0.005) and in males (positively: plasma estradiol, P=0.015; ER-beta expression, P=0.025); and (c) NQO1 in females (positively: smoke status, P=0.002) and in males (positively: ER-beta expression, P=0.001). CYP1A1 (mRNA, 9.1%) and GSTM1 (mRNA, 17.5%) are uncommonly expressed in human lung. Confirmation by Western immunoassayed protein is described. The results in nontumor tissue differed from that in tumor tissue. CONCLUSIONS: Regulation of carcinogen metabolism genes expressed in human lung seems impacted by hormonal and gender factors, as well as ongoing tobacco exposure. Expression differences between tumor and nontumor tissue in this pathway have both susceptibility and therapeutic implications.
Asunto(s)
Carcinógenos/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmón/metabolismo , Oxidorreductasas/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Femenino , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Pulmón/patología , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , ARN Mensajero/metabolismo , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Estrogen receptor (ER) expression in human lung has been understudied, particularly in light of its potential biological importance in the female lung cancer epidemic. Reverse transcription-polymerase chain reaction was used to probe mRNA expression of wild-type ERalpha and ERbeta and their splice variants in human bronchogenic tumor and adjacent nontumor specimens. In tumor tissue from 13 women and 13 men, ERalpha was expressed in 85% of women versus 15% in men [P=0.001]. ERbeta was expressed equally in tumors from women versus men [92% vs. 69%, P=ns]. Both ERalpha and beta forms were expressed simultaneously in the lung tumors of 77% of women versus 15% of men [P=0.005]. Among adjacent nontumor lung specimens, 31% of the women expressed ERalpha mRNA versus 0% of men [P=0.101], and 39% of women expressed ERbeta mRNA versus 31% of men [P=ns]; only one woman and no men expressed both ERalpha and beta in nontumor tissue. Females expressed ERalpha [P=0.017], ERbeta [P=0.013], and ERalpha+beta [P=0.002] more frequently in tumor versus nontumor tissue, whereas in males expression of ERalpha, beta and both alpha+beta was not clearly different for tumor versus nontumor tissue. In specimens expressing ERalpha mRNA, the transcript lacking exon 7 (delta7) was the major splice variant with varying contributions from the transcripts delta4, delta3+4, delta5 and others unidentified. Alternative splicing of ERbeta mRNA was observed, but not to as great an extent as for ERalpha mRNA. ERalpha promoter usage in tumors varied among individuals. When the ER receptors were co-expressed in tumors, ERalpha was quantitatively more abundant in the majority of cases than ERbeta. Within this small group of 26 patients, no correlation was found between age, smoking history, plasma nicotine, cotinine, estradiol concentrations or histopathologic type with tumor or nontumor estrogen receptor status of any type. However, several positive correlations imply that: (1) ERalpha expression occurs more often in the lungs of women than men; (2) ERbeta is expressed with approximately equal frequency in the lungs of both genders; and (3) tumors display a higher frequency of both receptor types than nontumors in women. We hypothesize that these putative gender-dependent differences in ERalpha and ERbeta expression could contribute unique phenotypic characteristics to lung cancer development or progression in women.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Adenoescamoso/genética , Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Receptores de Estrógenos/genética , Adenocarcinoma/sangre , Adenocarcinoma/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Carcinoma Adenoescamoso/sangre , Carcinoma Adenoescamoso/metabolismo , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/metabolismo , Cartilla de ADN/química , Estradiol/sangre , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Exones , Femenino , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Nicotina/sangre , ARN Mensajero/biosíntesis , Receptores de Estrógenos/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores SexualesRESUMEN
MCF-7 breast tumor cells form multicellular nodules (foci) over a confluent monolayer in an estradiol (E2)-dependent, antiestrogen-sensitive reaction. A cell line cloned from MCF-7 that displays these phenotypes was probed to determine the effects of long term exposure to tamoxifen on the growth of foci, estrogen receptor alpha (ERalpha) status, and gene responsiveness to E2. In one of two experiments, a heterogeneous cell population emerged (TMX2) that over-expressed estrogen receptor alpha wild type mRNA (ERalpha mRNA) (approximately 20-fold) missing exon 3 (ERDelta3 mRNA) and its corresponding protein (ERDelta3P). On a per mRNA to protein basis, ERDelta3P and wild-type ERalpha were equivalently expressed. Return of the TMX2 population to medium without tamoxifen eventually selected for a population that expressed predominately wild-type ERalpha, whereas TMX2 clones over expressing ERDelta3 mRNA and ERDelta3P retained this phenotype in tamoxifen-free media. In both experiments, expression of all ERalpha mRNAs and proteins declined to barely detectable levels during 6-12 months exposure, concomitant with a progressive increase in the ability of the cells to form foci independently of E2 or tamoxifen. Selection for these various populations suggests that tamoxifen can induce and/or support certain cellular changes that lead to altered ERalpha expression, E2-independent cell growth and resistance to antiestrogens.
Asunto(s)
Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores de Estrógenos/genética , Tamoxifeno/farmacología , Empalme Alternativo/efectos de los fármacos , Neoplasias de la Mama/genética , Técnicas de Cultivo de Célula , División Celular , Línea Celular Tumoral , Estradiol/farmacología , Receptor alfa de Estrógeno , Femenino , Humanos , Fenotipo , ARN Mensajero/efectos de los fármacos , Receptores de Estrógenos/análisis , Factores de Tiempo , Transcripción GenéticaRESUMEN
Cyanide (CN) is a ubiquitous environmental toxicant. The measurement of CN in whole blood is a common exposure assay, but values are error prone because of CN's rapid metabolism and clearance (t1/2 < 1 h) from this compartment. This study was undertaken to determine whether CN forms covalent adduct(s) with plasma proteins that could serve as stable biomarker(s) and potential surrogate(s) of exposure. When added to human blood, plasma, or serum, CN formed covalent adducts with immunoglobulin G (IgG) and serum albumin (HSA) in the plasma fraction. Covalent adducts were not detected in the cellular, primarily erythrocyte, fraction. With human, mouse, and rabbit IgGs, the reaction with CN occurred at intra- and/or interchain disulfide linkages in the heavy and light chains. Digestion of CN-treated HSA with trypsin or the endoproteinase Lys-C at basic pH produced tautomeric 2-iminothiazoline-4-carboxylyl/2-aminothiazolidine-4-carboxylyl (itcCys) N-terminal peptides exclusively, consistent with prior model peptide/protein studies showing that under basic conditions internal S-cyanylated-Cys residues cyclize with concomitant release of the upstream peptide. The most readily detectable reaction of CN with purified HSA was at Cys34, the only Cys of the 35 present not connected as internal cystines. Because CN does not react with free sulfhydryl groups, it is probable that S-cyanylation at Cys34 occurs at those residues that carry GSH, Cys, or other small molecules as mixed disulfides. Relatively less detectable, modified Cys residues were also identified at positions 53, 124, 392, 477, and 487. When 14CN was added to human serum or whole blood at concentrations spanning a putative nontoxic to lethal range, stable adduct formation with HSA occurred in a linear, concentration-dependent reaction that was complete within 2 h. These attributes of the reaction, coupled with a plasma compartment location, suggest that quantitation of CN bound to HSA would provide a much more reliable assessment of exposure than does measurement of CN in blood.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Cianuros/química , Secuencia de Aminoácidos , HumanosRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) and heavy metals are often environmental cocontaminants that could interact to alter PAH carcinogenicity. The heavy metal, arsenite, and the PAH, benzo[k]fluoranthene, were used as prototypes to investigate, in human HepG2 cells, mechanisms whereby the bioactivation of benzo[k]fluoranthene by human CYP1A1 could be diminished by arsenite-mediated decreases in CYP1A1 induction by benzo[k]fluoranthene. To determine whether arsenite down-regulates CYP1A1 transcription, quantitative real-time reverse transcriptase-polymerase chain reaction assays and luciferase reporter gene expression assays were used with HepG2 cells treated with benzo[k]fluoranthene and arsenite, separately and as a mixture. Benzo[k]fluoranthene (0.5 microM) and arsenite (5 microM) markedly decreased benzo[k]fluoranthene-mediated induction of CYP1A1 mRNA by 45%. Plasmids containing the CYP1A1 promoter region (pHu-1A1-FL) were induced 7.4-fold over vehicle by benzo[k]fluoranthene (0.5 microM), whereas arsenite (1, 2.5, or 5 microM) decreased reporter gene expression by 46%, 45%, and 61%, respectively. The plasmid, pHu-1A1-Delta100-FL, lacked xenobiotic response element (XRE) sites at -1061 and -981 and showed greater responsiveness relative to pHu-1A1-FL, by 1.7-fold. Benzo[k]fluoranthene (0.5 microM) and arsenite (1, 2.5, or 5 microM) decreased reporter gene expression by 0%, 27%, and 39%, respectively, relative to expression levels produced by benzo[k]fluoranthene alone. Arsenite is stable for at least 48 h in the HepG2 cell medium with respect to its ability to diminish CYP1A1 benzo[k]fluoranthene induction. Arsenite did not affect benzo[k]fluoranthene induction directly through XRE sites, nor did it affect the stability of CYP1A1 mRNA. Thus, arsenite affects the transcriptional regulation of the benzo[k]fluoranthene-mediated induction of CYP1A1 and could diminish PAH carcinogenicity by decreasing bioactivation by CYP1A1.
Asunto(s)
Arsenitos/farmacología , Citocromo P-450 CYP1A1/biosíntesis , Contaminantes Ambientales/antagonistas & inhibidores , Fluorenos/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Carcinógenos Ambientales/toxicidad , Línea Celular Tumoral , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/genética , Contaminantes Ambientales/toxicidad , Inducción Enzimática , Genes Reporteros , Humanos , Luciferasas/genética , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that have been extensively studied for multiple toxicological endpoints in both laboratory animals and humans. The purpose of this study was to investigate the estrogenicity of PAHs in the human breast cancer cell line MCF-7. We investigated 14 PAHs for their ability to bind either the estrogen receptor (ER) or the aryl hydrocarbon receptor (AhR) and to activate target gene expression. PAHs were tested in a human recombinant estrogen receptor (hrER) competitive binding assay, and in both an estrogen response element (ERE)- and xenobiotic response element (XRE)-mediated reporter gene assay. We used quantitative RT-PCR to examine selected PAHs that showed activity in the ERE reporter gene assay for their ability to upregulate estrogen-responsive genes HEM45, progesterone receptor, and pS2, and the aryl hydrocarbon-responsive CYP1A1 gene. None of the 14 PAHs bound the hrER, but five of the PAHs (anthracene, B[a]A, chrysene, B[b]F, and B[a]P) induced ER-reporter activity. This activity was dependent on the metabolism of PAHs in MCF-7 cells via the AhR pathway, which resulted in the formation of metabolites that bound the ER. None of the five PAHs that induced the ER-reporter were found to upregulate estrogen-responsive genes, yet four of the five PAHs induced AhR-dependent CYP1A1 gene expression. In contrast, a metabolite of B[a]P, 3'OH-B[a]P, and a PCB metabolite, 4'OH-2,4,6-BP, did weakly upregulate all three estrogen-responsive genes. Data from these studies indicate that induction of ER-reporter activity alone does not necessarily parallel endogenous gene transcription, and that the reporter gene assay may detect interactions that are not functional in vivo.