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From 2012 to 2013, approximately 16 New York residents reported vague, nonspecific adverse health effects which included fatigue, loss of scalp hair, and muscle aches. One patient was hospitalized for liver damage. An epidemiological investigation identified a common factor among these patients; the consumption of B-50 vitamin and multimineral supplements from the same supplier. To investigate whether these nutritional supplements might have been responsible for the adverse health effects observed, comprehensive chemical analyses of marketed lots of the supplements were performed. To determine presence of organic components and contaminants, organic extracts of samples were prepared and analyzed using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography high-resolution mass spectrometry (LC-HRMS), and nuclear magnetic resonance (NMR). These analyses revealed the presence of significant levels of methasterone (17ß-hydroxy-2α,17α-dimethyl-5α-androstane-3-one), an androgenic steroid and schedule III-controlled substance; dimethazine, an azine-linked dimer of methasterone; and methylstenbolone (2,17α-dimethyl-17ß-hydroxy-5α-androst-1-en-3-one), a related androgenic steroid. Methasterone and extracts of certain supplement capsules were identified as highly androgenic in luciferase assays by using an androgen receptor promoter construct. This androgenicity persisted for several days after cell exposure to the compounds. The presence of these components in implicated lots were associated with adverse health effects and the hospitalization of one patient and the presentation of symptoms of severe virilization in a child. These findings underscore the need for more rigorous oversight of the nutritional supplement industry.
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Anabolizantes , Doping en los Deportes , Niño , Humanos , Cromatografía de Gases y Espectrometría de Masas/métodos , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Andrógenos/efectos adversos , Suplementos Dietéticos/efectos adversos , Suplementos Dietéticos/análisisRESUMEN
Per- and polyfluoroalkyl substances (PFAS) are contaminants of global concern due to their persistence and associated negative health effects. Considerable attention has been given to monitoring PFAS in the aquatic environment, however, few investigations have done so using freshwater benthic macroinvertebrates (BMIs). As these bottom-dwelling animals are known to bioconcentrate exogenous pollutants to a high degree, studying their PFAS levels may provide a more integrated view of PFAS contamination in the aquatic environment. In this study, BMIs, sediment, and surface water were collected from two streams in the Hudson River Watershed (one historically-impacted by PFAS) and analyzed for 44 PFAS using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Orbitrap high-resolution mass spectrometry (HRMS) was used to confirm the identities of quantitated analytes. Across all matrices, 17 analytes were detected with PFOA dominating in surface water and PFOS in sediment/BMIs. PFOS bioaccumulation factors (BAFs) were approximately one order of magnitude higher than those of PFOA and ranged from 857 to 5151 L kg-1 across different BMI taxa. While PFAS concentrations in surface water and sediment were not excessively high, elevated levels were still measured in most BMI taxa. This observation suggests that the extent of PFAS contamination in a local system may be severely underestimated if only surface water and sediment are used for monitoring. Moreover, these findings have relevance for human exposure assessment considering BMIs are the primary food source of many fish.
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Ácidos Alcanesulfónicos , Fluorocarburos , Contaminantes Químicos del Agua , Ácidos Alcanesulfónicos/análisis , Animales , Cromatografía Liquida , Monitoreo del Ambiente , Fluorocarburos/análisis , Agua Dulce , Humanos , Espectrometría de Masas en Tándem , Contaminantes Químicos del Agua/análisisRESUMEN
The investigation of the 2019-2020 E-cigarette or Vaping Product Use-Associated Lung Injury (EVALI) outbreak in New York State provided a unique opportunity to examine the formulations and chemical components found in clandestine cannabis-containing e-liquids. In this EVALI investigation, it was determined that an unusually high proportion (16%) of the cannabis e-liquids analyzed contained significant levels of ∆8-tetrahydrocannabinol (∆8-THC). Although not thought to be the causative agent in the outbreak, the manufacturing origin of vaping e-liquids containing large concentrations of ∆8-THC was of great interest, since high ∆8-THC concentrations are not observed in the extracts of common cannabis strains. A principal component analysis of multiple cannabinoid concentrations revealed clusters of similar or identical ∆8-THC-containing products. This technique may be useful in identifying common manufacturing sources in this and future investigations. Several possible manufacturing methods to enrich ∆8-THC appear in literature and are discussed based on their likelihood as sources of this cannabinoid in these samples from the EVALI investigation. The presence of high levels of ∆8-THC in numerous illicit vaping products may implicate cannabidiol, which is readily available at low cost, as its synthetic precursor.
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Cannabinoides , Sistemas Electrónicos de Liberación de Nicotina , Alucinógenos , Vapeo , Dronabinol , New YorkRESUMEN
E-cigarette or vaping product use-associated lung injury (EVALI) is a serious pulmonary condition that is associated with the extended use of certain vaping products. EVALI was first characterized in the summer of 2019 and has since been reported in all 50 U.S. states. From August 2019 through June 2021, the New York State Department of Health has reported more than 197 confirmed cases emanating from all regions of the state. The Wadsworth Center at the New York State Department of Heath received vaping cartridges recovered from EVALI patients for chemical analysis of their contents. Untargeted analytical methods using gas chromatography-mass spectrometry and liquid chromatography-high-resolution mass spectrometry as well as targeted analyses for a variety of analytes including cannabinoids, pesticides, vitamin E acetate (VEA) and mycotoxins were used to characterize the composition of the vaping fluids and several commercial vaping fluid additives. From the analyses of the 284 e-cigarette devices recovered from patients, 82 were found to be nicotine-containing pods, and 202 devices containing cannabis oil, apparently from unauthorized or black-market dealers. The fluids from the cannabis-oil cartridges tended to have lower levels of THCs (Δ9-tetrahydrocannabinol + Δ8-tetrahydrocannabinol) and total cannabinoids compared with those of commercially produced formulations and contained significant levels of diluents including VEA, medium-chain triglycerides, polyethylene glycol, and castor oil. VEA was the diluent most frequently detected, which was present in 132 (65.3%) of the vaping fluids that contained cannabis oil. When present, VEA ranged from 2.0 to 67.8% of the total mass of the oil with a mean content of 37.0%. In some cases, two or three diluents were detected in the same sample. The ratio of VEA to THCs varied widely, from 0.07 to 5.34. VEA and specifically the high ratios of VEA to THCs in black-market vaping fluids may be causative in EVALI. The safety of additional components and additives that are present in vaping fluids are likewise of concern.
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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly used to measure steroids in human saliva. We studied the performance of a conventional LC-MS/MS for measuring dehydroepiandrosterone (DHEA), testosterone and progesterone in human saliva. These three steroids were co-extracted by liquid-liquid extraction and derivatized. Derivatives were resolved on a C18 column and quantified using an LC-MS/MS (AB Sciex API 2000) instrument. The assay's limits of quantification were 0.03 ng/mL for all three steroids. Inter-assay coefficients of variation were 16.6-18.8% (DHEA), 12.0-15.8% (testosterone), and 12.7-19.3% (progesterone). Assay linearity analysis showed R2 of 0.9926, 0.9750 and 0.9949 for DHEA, testosterone and progesterone, respectively. No carry-over between samplings was observed. An ion-enhancement effect of 11.6% for DHEA determination and ion-suppression effects of 13.9% and 20.7% for analysis of progesterone and testosterone, respectively, were determined. No interferences by 9 steroid analogs were detected. Spiked recoveries were 85.5% (DHEA), 86.5% (testosterone), and 92.6% (progesterone). Comparison with laboratory developed test (LDT)-LC-MS/MS methods by other New York State Department of Health certified laboratories revealed R2 = 0.9425 (DHEA, LC-MS/MS = 1.0267 LDT + 21.989), R2 = 0.9849 (testosterone, LC-MS/MS = 0.9447 LDT + 9.8037), and R2 = 0.9736 (progesterone, LC-MS/MS = 1.1196 LDT + 0.0985). Reference intervals for the 3 steroids in saliva for young males and females were estimated. Results of intra-individual salivary progesterone analysis indicated that caution should be exercised when using progesterone concentrations in predicting ovulation for females who are under treatment with birth control pills/devices or has body a weight of > 90 kg.
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Anticonceptivos Orales/farmacología , Deshidroepiandrosterona/análisis , Predicción de la Ovulación , Progesterona/análisis , Testosterona/análisis , Adolescente , Adulto , Peso Corporal/fisiología , Cromatografía Liquida/métodos , Femenino , Humanos , Modelos Lineales , Masculino , Ovulación/efectos de los fármacos , Reproducibilidad de los Resultados , Saliva/química , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem/métodos , Adulto JovenRESUMEN
Due to their unique chemical properties, per- and polyfluoroalkyl substances (PFAS) have been used extensively as industrial surfactants and processing aids. While several types of PFAS have been voluntarily phased out by their manufacturers, these chemicals continue to be of ecological and public health concern due to their persistence in the environment and their presence in living organisms. Moreover, while the compounds referred to as "legacy" PFAS remain in the environment, alternative compounds have emerged as replacements for their legacy predecessors and are now detected in numerous matrices. In this review, we discuss the historical uses of PFAS, recent advances in analytical techniques for analysis of these compounds, and the fate of PFAS in the environment. In addition, we evaluate current biomonitoring studies of human exposure to legacy and emerging PFAS and examine the associations of PFAS exposure with human health impacts, including cancer- and non-cancer-related outcomes. Special focus is given to short-chain perfluoroalkyl acids (PFAAs) and ether-substituted, polyfluoroalkyl alternatives including hexafluoropropylene oxide dimer acid (HFPO-DA; tradename GenX), 4,8-dioxa-3H-perfluorononanoic acid (DONA), and 6:2 chlorinated polyfluoroethersulfonic acid (6:2 Cl-PFESA; tradename F-53B).
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Carcinógenos Ambientales/toxicidad , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/toxicidad , Fluorocarburos/toxicidad , Animales , Biodegradación Ambiental , Carcinógenos Ambientales/química , Contaminantes Ambientales/química , Fluorocarburos/química , HumanosRESUMEN
Perfluoroethercarboxylic acids (PFECAs) have recently emerged as replacements for toxic per- and polyfluorinated alkyl substances (PFAS) including perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS). Compared with other PFAS, many PFECAs including hexafluoropropylene oxide dimer acid (HFPO-DA, trade name GenX) exhibit poor sensitivity during analysis using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and are therefore often difficult to quantify. This study examined changes in ESI probe position, mobile phase additive, and capillary voltage with the goal of enhancing PFECA sensitivity. In addition, the relative contributions of existing mechanistic theories for PFAS ionization during ESI are discussed. Results indicated that the LC-ESI-MS/MS sensitivity for 9 PFECAs can be improved significantly by altering the ESI probe position. At the optimal probe position, lowering the capillary voltage from 2.0 to 0.5 kV universally enhanced the LC-ESI-MS/MS sensitivity for PFAS analysis. For most analytes, the use of ammonium bicarbonate rather than ammonium acetate as a mobile phase additive also enhanced the analytical response. These effects have not been previously reported and suggest that many laboratories may be conducting analyses of PFECAs under suboptimal conditions. Using the strategies outlined in this study, PFECAs can be more easily incorporated into comprehensive methods for PFAS analysis. Here, we describe analytical parameters that enhance the sensitivity for some PFECAs by up to 36-fold while maintaining high sensitivity for legacy PFAS. This work not only highlights solutions to mitigate inadequate PFECA sensitivity but also provides insight into the mechanisms underlying PFAS ionization efficiency during LC-ESI-MS/MS.
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INTRODUCTION: Kratom is derived from the plant Mitragyna speciosa which is indigenous to Southeast Asia. Active compounds, mitragynine and 7-hydroxymitragynine, cause mild stimulant and opioid agonist effects. Although reported to have potential benefits in the treatment of opioid use disorder, efficacy remains uncertain while adverse health effects have been reported. A compounding concern is the presence of adulterants given that this is an unregulated product. CASE DETAILS: A 54-year-old fitness instructor who used an online purchased kratom product regularly for one year developed stimulatory effects and suffered a large hemorrhagic stroke with a close temporal relationship to ingestion of a different kratom product from the one he regularly used. A collaborative investigation by medical toxicologists, a regional poison center, the state public health laboratory, and public health officials determined that his new kratom product was adulterated with phenylethylamine (PEA). DISCUSSION: We report a case of PEA adulterated kratom purchased and used with resultant adverse effects. PEA is structurally similar to amphetamine and is known to produce sympathomimetic effects. It is possible the stimulatory effect of PEA resulted in a marked and transient increase in blood pressure resulting in hemorrhagic stroke. CONCLUSION: Medical toxicologists should form working relationships with laboratories and public health officials to aid in early identification of adulterated products that carry risk to the general population.
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Hemorragia Cerebral/inducido químicamente , Contaminación de Medicamentos , Accidente Cerebrovascular Hemorrágico/inducido químicamente , Fenetilaminas/efectos adversos , Alcaloides de Triptamina Secologanina/efectos adversos , Detección de Abuso de Sustancias , Trastornos Relacionados con Sustancias/diagnóstico , Hemorragia Cerebral/diagnóstico , Accidente Cerebrovascular Hemorrágico/diagnóstico , Humanos , Comunicación Interdisciplinaria , Masculino , Persona de Mediana Edad , Fenetilaminas/análisis , Centros de Control de Intoxicaciones , Valor Predictivo de las Pruebas , Salud Pública , Alcaloides de Triptamina Secologanina/análisis , Trastornos Relacionados con Sustancias/complicaciones , ToxicologíaRESUMEN
Introduction: In the United States, medical marijuana programs have been established in 29 states and the District of Columbia. In 2014, New York State (NYS) approved medical marijuana legislation, and its program became fully operational in January of 2016. Products manufactured under the auspices of the program may be used by certified patients in NYS for the treatment of 1 of 12 qualifying medical conditions. The NYS statute requires rigorous testing of each product lot manufactured in the state for its cannabinoid profile, bacterial and fungal contamination, mycotoxins, heavy metals, plant-growth regulators, and pesticides. Here, we report on the analysis of product cannabinoid profiles. Methods: A method employing a simple extraction/dilution technique and reversed-phase high-performance liquid chromatography with photodiode array detection (HPLC-PDA) was developed for the analysis of 10 cannabinoids: cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol (CBD), tetrahydrocannabivarin, cannabinol, Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene, cannabidivarin, and Δ9-tetrahydrocannabinolic acid-A. The method employed internal standard quantitation and incorporated a surrogate to monitor extraction efficiency and analytical recovery. Results: The HPLC-PDA method was validated using sample matrices composed of medium-chain triglycerides, hemp oil, sesame oil, and an ethanol-propylene glycol tincture. Limits of detection, limits of quantitation, accuracy, precision, and inter- and intraday reproducibility were found to be highly satisfactory. The validated method has been used to analyze over 3500 samples from over 700 lots of medical marijuana products manufactured in NYS from January 2016 through April 2018. Quality-control data showed quantitative spike recoveries and, for the analysis of samples from the same lot, the coefficients of variation for the principal analytes, Δ9-THC and CBD, averaged <3%. Using the HPLC-PDA method, the NYS medical marijuana products were analyzed to verify the potencies on the product labels and to determine the stability of the products. Conclusions: An HPLC-PDA-based method was developed, validated, and employed to analyze 10 cannabinoids in a variety of medical marijuana products. The method has proven to be accurate, precise, stable, and very robust. Its use is an integral part of the NYS Medical Marijuana program for validation of the content and consistency of medical marijuana products.
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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurements of steroids in human saliva has garnered increased interest in the area of clinical psychoneuroendocrinological research. However, performance characteristics of LC-MS/MS methods for the analysis of steroids in saliva are limited. Human saliva samples were collected via passive drool. Cortisol and dehydroepiandrosterone sulfate (DHEA-S) in the samples were extracted together, resolved on a C18-A column, and analyzed using tandem mass spectrometry. The LC-MS/MS method had limits of quantitation of 0.03 and 0.06 ng/mL for DHEA-S and cortisol, respectively. Method evaluations showed coefficient variation (%CV) of inter-assay ranging 4.6-17.9% for DHEA-S and cortisol, recoveries of 102.4-109.5% for DHEA-S and 94.6-98.3% for cortisol, and assay linearity with R2 = 0.9964 for DHEA-S (1.0-25.0 ng/mL) and R2 = 0.997 (1.0-25.0 ng/mL) for cortisol. No cross contamination among samples was observed. Human saliva showed 20% and 18% ion enhancement effect for DHEA-S and cortisol assay, respectively. No interference by ten common steroids was detected. Regression analysis of method comparisons with laboratory-developed test (LDT) method revealed R2 = 0.9688 (LC-MS/MS = 0.9665 LDT-LC-MS/MS - 0.7355) for cortisol, and R2 = 0.9039 (LC-MS/MS = 1.0173 LDT-LC-MS/MS + 3.6797) for DHEA-S. Reference ranges for young adults were determined to be 0.3-5.9 ng/mL for females and 0.1-5.6 ng/mL for males for salivary cortisol, and 0.6-7.4 ng/mL for females and 0.6-10.1 ng/mL for males for salivary DHEA-S. An LC-MS/MS method for quantifying cortisol and DHEA-S in human saliva was developed and validated for clinical and psychoneuroendocrinological research that require noninvasive means of measuring these hormones.
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Sulfato de Deshidroepiandrosterona/análisis , Hidrocortisona/análisis , Saliva/química , Espectrometría de Masas en Tándem/métodos , Adolescente , Adulto , Cromatografía Liquida/métodos , Femenino , Humanos , Límite de Detección , Masculino , Valores de Referencia , Adulto JovenRESUMEN
Immunoassays for measuring 17-hydroxyprogesterone (17-OHP) produce high rates of false positives that impact the identification of congenital adrenal hyperplasia (CAH) in neonates. A confirmatory test with high analytical specificity employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology is needed in newborn screening for CAH. 17-OHP and cortisol were extracted from dried blood spot (DBS) samples, resolved on a C18 column, and measured using tandem mass spectrometry. The results were compared with those determined using the AutoDELFIA immunoassay. The LC-MS/MS method had a limit of quantitation of 10.0 and 5.0 ng/mL for 17-OHP and cortisol, respectively. The method characteristics showed coefficient variation (%CV) ≤ 11.9% for both 17-OHP and cortisol, recoveries ranging from 83.1 to 101.5% for 17-OHP and from 95.1 to 102.8% for cortisol, and linearity with R2 = 0.9994 for 17-OHP and R2 = 0.9996 for cortisol, clinical sensitivity of 100.0% and a specificity of 96.4% as obtained by receiver operating characteristic analysis on 45 patient samples when 17-OHP > 39.1 ng/mL was selected as the cutoff value. Comparison between the LC-MS/MS and the AutoDELFIA immunoassay methods revealed a poor correlation for patient DBS samples (R2 = 0.6784); however, an excellent correlation was obtained for QC and proficiency test (PT) DBS samples (R2 = 0.9797). The LC-MS/MS method produced reliable results for 17-OHP and cortisol for the diagnosis of CAH. The AutoDELFIA immunoassay appears to be subject to matrix effects in the analysis for 17-OHP in DBS patient samples. The DBS samples of non-patient origin may not be suitable for assessing analytical accuracy of immunoassays.
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17-alfa-Hidroxiprogesterona/sangre , Cromatografía Liquida , Inmunoensayo/métodos , Espectrometría de Masas en Tándem , 17-alfa-Hidroxiprogesterona/química , Humanos , Recién Nacido , Estructura Molecular , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC)n, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC)2 alleles were observed; however, in western gorilla, (GGGGC)n alleles with n=2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC)n was n=4>5â«2, 6. When frequencies of the (GGGGC)n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC)2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC)n short tandem repeats are inherited, and that the (GGGGC)2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Neoplasias de la Mama/genética , Neoplasias del Colon/genética , Neoplasias Pulmonares/genética , Repeticiones de Microsatélite , Polimorfismo Genético , Regiones Promotoras Genéticas , Receptores de Hidrocarburo de Aril/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/biosíntesis , Citocromo P-450 CYP1B1/genética , Inducción Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica , Frecuencia de los Genes , Genes Reporteros , Predisposición Genética a la Enfermedad , Herencia , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Receptores de Hidrocarburo de Aril/metabolismo , Factores de Riesgo , TransfecciónRESUMEN
BACKGROUND: Unconjugated estriol (uE3) is routinely analyzed in clinical laboratories as risk assessment for Down syndrome. Immunoassays of various types are the most commonly used methods. The accuracies of RIAs and ELISAs for uE3 have been questioned, and to date there have been no independent studies investigating the accuracy of the relatively new chemiluminescent immunoassays. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for uE3 measurements in serum. METHODS: Serum samples from patients in the second trimester of pregnancy were used, and uE3 concentrations were measured by LC-MS/MS and the Beckman Coulter Access® 2 and Siemens IMMULITE 2000 automatic chemiluminescent immunoassay analyzers. RESULTS: The LC-MS/MS method was validated and showed limit of detection 0.05 ng/mL; limit of quantification 0.2 ng/mL; linearity of response to 32 ng/mL; total imprecision of 16.2%, 10.4%, and 8.2% for uE3 at 1.10, 4.18, and 8.32 ng/mL, respectively; and analytical recoveries of 95.9%-104.2%. ANOVA of the correlation for LC-MS/MS results vs chemiluminescent immunoassays results showed R(2) = 0.9678 (Access 2 = 0.9305 LC-MS/MS + 0.2177, Sy|x = 0.1786, P < 0.0001), and R(2) = 0.9663 (IMMULITE 2000 = 0.8849 LC-MS/MS - 0.0403, Sy|x = 0.1738, P < 0.0001). Bland-Altman plots of uE3 results revealed concentration-dependent immunoassay biases. Mock risk analysis for Down syndrome showed no apparent difference in the risk assessment outcomes if the adjusted method-specific multiples of the median were used, and the assay imprecision was <10% CV. CONCLUSIONS: Standardization of immunoassay methods for uE3 analysis is needed to improve the accuracy of the measurements.
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Cromatografía Liquida , Pruebas de Química Clínica/normas , Estriol/sangre , Inmunoensayo/normas , Mediciones Luminiscentes/normas , Espectrometría de Masas en Tándem , Síndrome de Down/diagnóstico , Estriol/química , Femenino , Humanos , Masculino , Embarazo , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
The AhR was initially identified as a ligand-activated transcription factor mediating effects of chlorinated dioxins and polycyclic aromatic hydrocarbons on cytochrome P450 1 (CYP1) expression. Recently, evidence supporting involvement of the AhR in cell-cycle regulation and tumorigenesis has been presented. To further define the roles of the AhR in cancer, we investigated the effects of AhR expression on cell proliferation, migration, invasion, and tumorigenesis of MCF-7 human breast cancer cells. In these studies, the properties of MCF-7 cells were compared with those of two MCF-7-derived sublines: AH(R100) , which express minimal AhR, and AhR(exp) , which overexpress AhR. Quantitative PCR, Western immunoblots, 17ß-estradiol (E2 ) metabolism assays, and ethoxyresorufin O-deethylase assays showed the lack of AhR expression and AhR-regulated CYP1 expression in AH(R100) cells, and enhanced AhR and CYP1 expression in AhR(exp) cells. In the presence of 1 nM E2 , rates of cell proliferation of the three cell lines showed an inverse correlation with the levels of AhR mRNA. In comparison with MCF-7 and AhR(exp) cells, AH(R100) cells produced more colonies in soft agar and showed enhanced migration and invasion in chamber assays with E2 as the chemoattractant. Despite the lack of significant AhR expression, AH(R100) cells retained the ability to form tumors in severe combined immunodeficient mice when supplemented with E2 , producing mean tumor volumes comparable to those observed with MCF-7 cells. These studies indicate that, while CYP1 expression and inducibility are highly dependent on AhR expression, the proliferation, invasion, migration, anchorage-independent growth, and estrogen-stimulated tumor formation of MCF-7 cells do not require the AhR.
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Neoplasias de la Mama/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/patología , Estrógenos/farmacología , Neovascularización Patológica , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Apoptosis/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Femenino , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones SCID , Receptores de Hidrocarburo de Aril/genética , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Células Tumorales CultivadasRESUMEN
The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 â« 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.
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Epigénesis Genética , Secuencia Rica en GC , Regulación Neoplásica de la Expresión Génica , Regiones Promotoras Genéticas , Receptores de Hidrocarburo de Aril/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Cromatografía Liquida , Dicroismo Circular , Citocromo P-450 CYP1A1/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Mutagénesis Sitio-Dirigida , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción Sp/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Genes MHC Clase II/fisiología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Intestino Delgado/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Animales , Secuencia de Bases , Antígenos de Histocompatibilidad Clase II/genética , Lanosterol/genética , Lanosterol/metabolismo , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , NADPH-Ferrihemoproteína Reductasa/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Life-long estrogen exposure is recognized as a major risk factor for the development of breast cancer. While the initial events in the regulation of gene expression by estrogen have been described in detail, far less is known of the role of estrogen in the long-term regulation of gene expression. In this study, we investigated the effects of long-term exposure of MCF-7 breast cancer cells to 1nM 17ß-estradiol on gene expression with the goal of distinguishing between gene expression that is continually reliant on estrogen receptor (ER) function as opposed to secondary and persistent effects that are downstream of ER. To assess the direct involvement of ER in the differential gene expression of long-term estrogen exposed (LTEE) cells in comparison with that of control cells, we exposed cultures to the selective estrogen receptor modulator raloxifene (RAL). cDNA microarray analysis showed that exposure to RAL inhibited expression of numerous characterized estrogen-regulated genes, including PGR, GREB1, and PDZK1. Genes that were increased in expression in LTEE cells yet were unaffected by RAL exposure included the aryl hydrocarbon receptor (AHR) and numerous other genes that were not previously reported to be regulated by estrogen. Epigenetic regulation was evident for the AHR gene; AhR transcript levels remained elevated for several cell passages after the removal of estrogen. Signal transducer and activator of transcription 1 (STAT1); STAT1-regulated genes including ISG15, IFI27, and IFIT1; and MHC class I genes were also up-regulated in LTEE cells and were unaffected by RAL exposure. STAT1 is commonly overexpressed in breast and other cancers, and is associated with increased resistance to radiation and chemotherapy. This is the first study to relate estrogen exposure to increased STAT1 expression in breast cancer cells, an effect that may represent an additional role of estrogen in the pathogenesis of breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/genética , Línea Celular Tumoral , Epigenómica , Femenino , Genes MHC Clase I , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismoRESUMEN
A novel method was established for simultaneous quantitation of testosterone (T) and dihydrotestosterone (DHT) in murine tissue and serum samples. Endogenous T and DHT, together with the internal standards 17alpha-methyl-T and 17alpha-methyl-DHT, were extracted from tissues and then derivatized by reaction with 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP). Analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) resulted in product ion spectra of HTP derivatives of both T and DHT that showed analyte-specific fragmentations; the latter fragmentations were characterized by the use of high-resolution Orbitrap MS/MS. These specific fragmentations enabled quantitation of T and DHT in the multiple-reaction monitoring (MRM) mode. The method was validated with charcoal-stripped serum as the matrix. The lower limit of quantitation (LLOQ) was 0.10ng/ml for T and 0.50ng/ml for DHT. The method was then used for determination of serum and tissue levels of T and DHT in transgenic mice carrying a hypomorphic NADPH-cytochrome P450 reductase gene (Cpr-low mice). Remarkably, ovarian T levels in Cpr-low mice were found to be 25-fold higher than those in wild-type mice, a finding that at least partly explains the female infertility seen in the Cpr-low mice. In conclusion, our method provides excellent sensitivity and selectivity for determination of endogenous levels of T and DHT in mouse tissues.
Asunto(s)
Dihidrotestosterona/sangre , Dihidrotestosterona/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Testosterona/sangre , Testosterona/metabolismo , Animales , Cromatografía Liquida/métodos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NADPH-Ferrihemoproteína Reductasa/genética , Distribución TisularRESUMEN
The cumulative exposure to estrogens is an important determinant in the risk of breast cancer, yet the full range of mechanisms involving estrogens in the genesis and progression of breast cancer remains a subject of debate. Interactions of estrogens and environmental toxicants have received attention as putative factors contributing to carcinogenesis. Mechanistic studies have demonstrated interactions between estrogen receptor alpha (ERalpha) and the aryl hydrocarbon receptor (AhR), with consequences on the genes that they regulate. Many studies of ERalpha and AhR-mediated effects and crosstalk between them have focused on the initial molecular events. In this study, we investigated ERalpha- and AhR-mediated effects in long-term estrogen exposed (LTEE) MCF-7 human breast cancer cells, which were obtained by continuous culturing for at least 12 weeks in medium supplemented with 1 nM of 17beta-estradiol (E(2)). With these LTEE cells and with parallel control cells cultured without E(2) supplementation, we performed an extensive study of cytochrome P450 (CYP) induction, carcinogen bioactivation, global gene expression, and tumorigenicity in immunocompromised mice. We found that LTEE cells, in comparison with control cells, had higher levels of AhR mRNA and protein, greater responsiveness for AhR-regulated CYP1A1 and CYP1B1 induction, a 6-fold higher initial level of benzo(a)pyrene-DNA adducts as determined by liquid chromatography tandem mass spectrometry, marked differences in the expression of numerous genes, and a higher rate of E(2)-dependent tumor growth as xenografts. These studies indicate that LTEE causes adaptive responses in MCF-7 cells, which may reflect processes that contribute to the overall carcinogenic effect of E(2).
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinógenos/farmacocinética , Estrógenos/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas/genética , Secuencia de Bases , Biotransformación , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromatografía Liquida , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1 , Aductos de ADN , Cartilla de ADN , Estradiol/análogos & derivados , Estradiol/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Estrógenos/farmacología , Femenino , Fulvestrant , Humanos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 microM benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17beta-estradiol (E(2)) metabolism, whereas BKF levels greater than 1 muM inhibited E(2) metabolism. Time course studies showed that induction of CYP1-catalyzed E(2) metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.