RESUMEN
Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants with various adverse health effects in humans including disruption of lipid metabolism. Aim of the present study was to elucidate the molecular mechanisms of PFAS-mediated effects on lipid metabolism in human cells. Here, we examined the impact of a number of PFAS (PFOS, PFOA, PFNA, PFDA, PFHxA, PFBA, PFHxS, PFBS, HFPO-DA, and PMPP) and of some exposure-relevant PFAS mixtures being composed of PFOS, PFOA, PFNA and PFHxS on lipid metabolism in human HepaRG cells, an in vitro model for human hepatocytes. At near cytotoxic concentrations, the selected PFAS and PFAS mixtures induced triglyceride accumulation in HepaRG cells and consistently affected the expression of marker genes for steatosis, as well as PPARα target genes and genes related to lipid and cholesterol metabolism, pointing to common molecular mechanisms of PFAS in disrupting cellular lipid and cholesterol homeostasis. PPARα activation was examined by a transactivation assay in HEK293T cells, and synergistic effects were observed for the selected PFAS mixtures at sum concentrations higher than 25 µM, whereas additivity was observed at sum concentrations lower than 25 µM. Of note, any effect observed in the in vitro assays occurred at PFAS concentrations that were at least four to five magnitudes above real-life internal exposure levels of the general population.
Asunto(s)
Ácidos Alcanesulfónicos , Contaminantes Ambientales , Fluorocarburos , Humanos , Metabolismo de los Lípidos , PPAR alfa/genética , Células HEK293 , Hepatocitos , Lípidos , Fluorocarburos/toxicidad , Colesterol , Ácidos Alcanesulfónicos/toxicidad , Contaminantes Ambientales/toxicidadRESUMEN
Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals used in many industrial applications. Exposure to PFAS is associated with several health risks, including a decrease in infant birth weight, hepatoxicity, disruption of lipid metabolism, and decreased immune response. We used the in vitro cell models to screen six less studied PFAS [perfluorooctane sulfonamide (PFOSA), perfluoropentanoic acid (PFPeA), perfluoropropionic acid (PFPrA), 6:2 fluorotelomer alcohol (6:2 FTOH), 6:2 fluorotelomer sulfonic acid (6:2 FTSA), and 8:2 fluorotelomer sulfonic acid (8:2 FTSA)] for their capacity to activate nuclear receptors and to cause differential expression of genes involved in lipid metabolism. Cytotoxicity assays were run in parallel to exclude that observed differential gene expression was due to cytotoxicity. Based on the cytotoxicity assays and gene expression studies, PFOSA was shown to be more potent than other tested PFAS. PFOSA decreased the gene expression of crucial genes involved in bile acid synthesis and detoxification, cholesterol synthesis, bile acid and cholesterol transport, and lipid metabolism regulation. Except for 6:2 FTOH and 8:2 FTSA, all tested PFAS downregulated PPARA gene expression. The reporter gene assay also showed that 8:2 FTSA transactivated the farnesoid X receptor (FXR). Based on this study, PFOSA, 6:2 FTSA, and 8:2 FTSA were prioritized for further studies to confirm and understand their possible effects on hepatic lipid metabolism.
RESUMEN
Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are man-made chemicals that are used for the fabrication of many products with water- and dirt-repellent properties. The toxicological potential of both substances is currently under debate. In a recent Scientific Opinion, the European Food Safety Authority (EFSA) has identified increased serum total cholesterol levels in humans as one major critical effect being associated with exposure to PFOA or PFOS. In animal studies, both substances induced a decrease of serum cholesterol levels, and the underlying molecular mechanism(s) for these opposed effects are unclear so far. In the present study, we examined the impact of PFOA and PFOS on cholesterol homoeostasis in the human HepaRG cell line as a model for human hepatocytes. Cholesterol levels in HepaRG cells were not affected by PFOA or PFOS, but both substances strongly decreased synthesis of a number of bile acids. The expression of numerous genes whose products are involved in synthesis, metabolism and transport of cholesterol and bile acids was strongly affected by PFOA and PFOS at concentrations above 10 µM. Notably, both substances led to a strong decrease of CYP7A1, the key enzyme catalyzing the rate-limiting step in the synthesis of bile acids from cholesterol, both at the protein level and at the level of gene expression. Moreover, both substances led to a dilatation of bile canaliculi that are formed by differentiated HepaRG cells in vitro. Similar morphological changes are known to be induced by cholestatic agents in vivo. Thus, the strong impact of PFOA and PFOS on bile acid synthesis and bile canalicular morphology in our in vitro experiments may allow the notion that both substances have a cholestatic potential that is connected to the observed increased serum cholesterol levels in humans in epidemiological studies.
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Ácidos Alcanesulfónicos/toxicidad , Ácidos y Sales Biliares/metabolismo , Caprilatos/toxicidad , Fluorocarburos/toxicidad , Animales , Carcinoma Hepatocelular , Colesterol , Expresión Génica , Hepatocitos , Homeostasis , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Neoplasias HepáticasRESUMEN
Triazoles are commonly used fungicides which show liver toxicity in rodent studies. While hepatocellular hypertrophy is the most prominent finding, some triazoles have also been reported to cause hepatocellular steatosis. The aim of our study was to elucidate molecular mechanisms of triazole-mediated steatosis. Therefore, we used the two triazoles propiconazole (Pi) and tebuconazole (Te) as test compounds in in vitro assays using the human hepatocarcinoma cell lines HepG2 and HepaRG. Triglyceride accumulation was measured using the Adipored assay and by a gas-chromatographic method. Reporter gene analyses were used to assess the ability of Pi and Te to activate nuclear receptors, which are described as the molecular initiators in the adverse outcome pathway (AOP) for liver steatosis. The expression of steatosis-associated genes was investigated by RT-PCR. Mechanistic analyses of triazole-mediated steatosis were performed using HepaRG subclones that are deficient in different nuclear receptors. Pi and Te both interacted with the constitutive androstane receptor (CAR), the peroxisome proliferator-activated receptor alpha (PPARα), and the pregnane X receptor (PXR). Both compounds induced expression of steatosis-related genes and cellular triglyceride accumulation. The knockout of PXR in HepaRG cells, but not the CAR knockout, abolished triazole-induced triglyceride accumulation, thus underlining the crucial role of PXR in hepatic steatosis resulting from exposure to these fungicides. In conclusion, our findings provide new insight into the molecular mechanisms of steatosis induction by triazole fungicides and identify PXR as a critical mediator of this process.
Asunto(s)
Fungicidas Industriales/toxicidad , Receptor X de Pregnano/metabolismo , Triazoles/toxicidad , Carcinoma Hepatocelular/patología , Línea Celular , Hígado Graso/inducido químicamente , Hígado Graso/fisiopatología , Técnicas de Inactivación de Genes , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Receptor X de Pregnano/genéticaRESUMEN
Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.
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Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Administración Oral , Algoritmos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Simulación por Computador , Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Humanos , Técnicas In Vitro , Dosis Máxima Tolerada , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/sangre , Farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Máquina de Vectores de SoporteRESUMEN
3-Chloro-1,2-propanediol (3-MCPD) and 2-chloro-1,3-propanediol (2-MCPD) are heat-induced food contaminants being present either as free substances or as fatty acid esters in numerous foods. 3-MCPD was classified to be possibly carcinogenic to humans (category 2B) with kidney and testis being the primary target organs according to animal studies. A previous 28-day oral feeding study with rats revealed that the endogenous antioxidant protein DJ-1 was strongly deregulated at the protein level in kidney, liver, and testis of the experimental animals that had been treated either with 3-MCPD, 2-MCPD or their dipalmitate esters. Here we show that this deregulation is due to the oxidation of a conserved, redox-active cysteine residue (Cys106) of DJ-1 to a cysteine sulfonic acid which is equivalent to loss of function of DJ-1. Irreversible oxidation of DJ-1 is associated with a number of oxidative stress-related diseases such as Parkinson, cancer, and type II diabetes. It is assumed that 3-MCPD or 2-MCPD do not directly oxidize DJ-1, but that these substances induce the formation of reactive oxygen species (ROS) which in turn trigger DJ-1 oxidation. The implications of 3-MCPD/2-MCPD-mediated ROS formation in vivo for the ongoing risk assessment of these compounds as well as the potential of oxidized DJ-1 to serve as a novel effect biomarker for 3-MCPD/2-MCPD toxicity are being discussed.
Asunto(s)
Glicerol/análogos & derivados , Proteína Desglicasa DJ-1/metabolismo , alfa-Clorhidrina/toxicidad , Animales , Antioxidantes/metabolismo , Línea Celular Tumoral , Cisteína/metabolismo , Contaminación de Alimentos , Glicerol/administración & dosificación , Glicerol/toxicidad , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Oxidación-Reducción , Proteína Desglicasa DJ-1/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Ratas , alfa-Clorhidrina/administración & dosificaciónRESUMEN
Transcriptomics is developing into an invaluable tool in toxicology. The aim of this study was, using a transcriptomics approach, to identify genes that respond similar to many different chemicals (including drugs and industrial compounds) in both rat liver in vivo and in cultivated hepatocytes. For this purpose, we analyzed Affymetrix microarray expression data from 162 compounds that were previously tested in a concentration-dependent manner in rat livers in vivo and in rat hepatocytes cultivated in sandwich culture. These data were obtained from the Japanese Toxicogenomics Project (TGP) and North Rhine-Westphalian (NRW) data sets, which represent 138 and 29 compounds, respectively, and have only 5 compounds in common between them. The in vitro gene expression data from the NRW data set were generated in the present study, while TGP is publicly available. For each of the data sets, the overlap between up- or down-regulated genes in vitro and in vivo was identified, and named in vitro-in vivo consensus genes. Interestingly, the in vivo-in vitro consensus genes overlapped to a remarkable extent between both data sets, and were 21-times (upregulated genes) or 12-times (down-regulated genes) enriched compared to random expectation. Finally, the genes in the TGP and NRW overlap were used to identify the upregulated genes with the highest compound coverage, resulting in a seven-gene set of Cyp1a1, Ugt2b1, Cdkn1a, Mdm2, Aldh1a1, Cyp4a3, and Ehhadh. This seven-gene set was then successfully tested with structural analogues of valproic acid that are not present in the TGP and NRW data sets. In conclusion, the seven-gene set identified in the present study responds similarly in vitro and in vivo to a wide range of different chemicals. Despite these promising results with the seven-gene set, transcriptomics with cultivated rat hepatocytes remains a challenge, because in general many genes are up- or downregulated by in vitro culture per se, respond differently to test compounds in vitro and in vivo, and/or show higher variability in the in vitro system compared to the corresponding in vivo data.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatocitos/efectos de los fármacos , Pruebas de Toxicidad/métodos , Toxicogenética/métodos , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/genética , Expresión Génica , Perfilación de la Expresión Génica/métodos , Hígado/efectos de los fármacos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Ratas , Ratas Wistar , Regulación hacia Arriba/genéticaRESUMEN
Numerous Maillard reaction and lipid oxidation products are present in processed foods such as heated cereals, roasted meat, refined oils, coffee, and juices. Due to the lack of experimental toxicological data, risk assessment is hardly possible for most of these compounds. In the present study, an in silico approach was employed for the prediction of the toxicological endpoints mutagenicity and carcinogenicity on the basis of the structure of the respective compound, to examine (quantitative) structure-activity relationships for more than 800 compounds. Five software tools for mutagenicity prediction (T.E.S.T., SARpy, CAESAR, Benigni-Bossa, and LAZAR) and three carcinogenicity prediction tools (CAESAR, Benigni-Bossa, and LAZAR) were combined to yield so-called mutagenic or carcinogenic scores for every single substance. Alcohols, ketones, acids, lactones, and esters were predicted to be mutagenic and carcinogenic with low probability, whereas the software tools tended to predict a considerable mutagenic and carcinogenic potential for thiazoles. To verify the in silico predictions for the endpoint mutagenicity experimentally, twelve selected compounds were examined for their mutagenic potential using two different validated in vitro test systems, the bacterial reverse mutation assay (Ames test) and the in vitro micronucleus assay. There was a good correlation between the results of the Ames test and the in silico predictions. However, in the case of the micronucleus assay, at least three substances, 2-amino-6-methylpyridine, 6-heptenoic acid, and 2-methylphenol, were clearly positive although they were predicted to be non-mutagenic. Thus, software tools for mutagenicity prediction are suitable for prioritization among large numbers of substances, but these predictions still need experimental verification.
Asunto(s)
Pruebas de Carcinogenicidad/métodos , Contaminación de Alimentos , Modelos Biológicos , Pruebas de Mutagenicidad/métodos , Alcoholes/toxicidad , Aminopiridinas/toxicidad , Animales , Simulación por Computador , Cresoles/toxicidad , Glicerol/análogos & derivados , Glicerol/toxicidad , Humanos , Cetonas/toxicidad , Lactonas/toxicidad , Reacción de Maillard , Pruebas de Micronúcleos , Programas InformáticosRESUMEN
The chlorinated propanols 2- and 3-monochloropropanediol (MCPD), and their fatty acid esters have gained public attention due to their frequent occurrence as heat-induced food contaminants. Toxic properties of 3-MCPD in kidney and testis have extensively been characterized. Other 3-MCPD target organs include heart and liver, while 2-MCPD toxicity has been observed in striated muscle, heart, kidney, and liver. Inhibition of glycolysis appears to be important in 3-MCPD toxicity, whereas mechanisms of 2-MCPD toxicity are still unknown. It is thus not clear whether toxicity by the two isomeric compounds is dependent on similar or dissimilar modes of action. A 28-day oral feeding study in rats was conducted using daily non-toxic doses of 2-MCPD or 3-MCPD [10 mg/kg body weight], or an equimolar (53 mg/kg body weight) or a lower (13.3 mg/kg body weight) dose of 2-MCPD dipalmitate. Comprehensive comparative proteomic analyses of substance-induced alterations in the common target organ heart revealed striking similarities between effects induced by 2-MCPD and its dipalmitate ester, whereas the degree of effect overlap between 2-MCPD and 3-MCPD was much less. The present data demonstrate that even if exerting effects in the same organ and targeting similar metabolic networks, profound differences between molecular effects of 2-MCPD and 3-MCPD exist thus warranting the necessity of separate risk assessment for the two substances. This study for the first time provides molecular insight into molecular details of 2-MCPD toxicity. Furthermore, for the first time, molecular data on 3-MCPD toxicity in the heart are presented.
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Cardiotoxicidad/metabolismo , Glicerol/análogos & derivados , Proteínas/metabolismo , alfa-Clorhidrina/toxicidad , Administración Oral , Animales , Cardiotoxicidad/etiología , Glicerol/administración & dosificación , Glicerol/toxicidad , Masculino , Proteómica , Ratas Wistar , alfa-Clorhidrina/administración & dosificaciónRESUMEN
Environmental emissions of organic contaminants are caused by man-made and natural combustion processes, industrial production facilities, and the release from products. Food represents the main source of human exposure for some of these compounds. This is the case for three groups of persistent organic contaminants: (1) per- and polyfluoroalkyl substances (PFAS), (2) polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and for (3) polycyclic aromatic hydrocarbons (PAHs). The issues regarding PCDD/F emissions were already recognized as a problem in the 1970s and have since then been effectively regulated; the impact of PFAS as global anthropogenic environmental contaminants was identified much later.A system of toxicity equivalency factors (TEF system) was established for the assessment of the toxic potency of a mixed exposure to PCDD/F and certain PCBs. For the health assessment and regulation of PAHs and PFAS, no such system has been implemented so far. For PFAS, a re-evaluation of the present tolerable daily intake values (TDI values) is currently being discussed, as new insights into toxicology and epidemiology have been gained.The persistence in the environment of the compound groups discussed here leads to entry into the food chain over long periods of time, even if access into the environment is minimized. This requires a long-term continuation of the monitoring of food stuffs and forward-looking risk assessment approaches and regulatory measures.
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Exposición a Riesgos Ambientales/efectos adversos , Exposición a Riesgos Ambientales/análisis , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Compuestos Orgánicos/efectos adversos , Compuestos Orgánicos/análisis , Alemania , Factores de Riesgo , Pruebas de ToxicidadRESUMEN
The production and preparation of foodstuffs may entail at high temperatures the generation of undesirable, potentially harmful compounds. Among the best investigated heat-induced contaminants are acrylamide, furan, and the fatty acid esters of glycidol and the monochloropropanediols. This article presents the main insights into the formation, toxicology, and exposure of these compounds. Acrylamide and glycidol were characterized as carcinogens with a genotoxic mechanism in animal experiments. Their content in foods should be minimized. For 3monochloropropanediol (3-MCPD), a tolerable daily intake can be derived. In contrast, a complete risk assessment is currently not possible for furan and 2MCPD owing to insufficient data.Many other heat-induced substances in foodstuffs were identified in addition to the compounds mentioned above, but for most no data on their toxicological properties and human exposure is available. Therefore, no risk assessment can currently be undertaken for these compounds. To prioritize this large number of compounds according to their possible hazard potential, it is reasonable to utilize computer modeling programs for the prediction of defined toxicological endpoints based on the molecular chemical structures. However, substances classed as a priority must be further investigated with regard to the toxicology and quantification of the food content of these compounds to allow a meaningful risk assessment.
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Carcinógenos/análisis , Carcinógenos/toxicidad , Culinaria , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Calefacción/efectos adversos , Acrilamida/análisis , Acrilamida/toxicidad , Simulación por Computador , Compuestos Epoxi/análisis , Compuestos Epoxi/toxicidad , Furanos/análisis , Furanos/toxicidad , Propanoles/análisis , Propanoles/toxicidad , Medición de Riesgo , alfa-Clorhidrina/análisis , alfa-Clorhidrina/toxicidadRESUMEN
The acute toxicity of cyanide is determined by its peak levels reached in the body. Compared to the ingestion of free cyanide, lower peak levels may be expected after consumption of foods containing cyanogenic glycosides with the same equivalent dose of cyanide. This is due to possible delayed and/or incomplete release of cyanide from the cyanogenic glycosides depending on many factors. Data on bioavailability of cyanide after consumption of foods containing high levels of cyanogenic glycosides as presented herein were necessary to allow a meaningful risk assessment for these foods. A crossover study was carried out in 12 healthy adults who consumed persipan paste (equivalent total cyanide: 68 mg/kg), linseed (220 mg/kg), bitter apricot kernels (about 3250 mg/kg), and fresh cassava roots (76-150 mg/kg), with each "meal" containing equivalents of 6.8 mg cyanide. Cyanide levels were determined in whole blood using a GC-MS method with K(13)C(15)N as internal standard. Mean levels of cyanide at the different time points were highest after consumption of cassava (15.4 µM, after 37.5 min) and bitter apricot kernels (14.3 µM, after 20 min), followed by linseed (5.7 µM, after 40 min) and 100 g persipan (1.3 µM, after 105 min). The double dose of 13.6 mg cyanide eaten with 200 g persipan paste resulted in a mean peak level of 2.9 µM (after 150 min). An acute reference dose of 0.075 mg/kg body weight was derived being valid for a single application/meal of cyanides or hydrocyanic acid as well as of unprocessed foods with cyanogenic glycosides also containing the accompanying intact ß-glucosidase. For some of these foods, this approach may be overly conservative due to delayed release of cyanide, as demonstrated for linseed. In case of missing or inactivated ß-glucosidase, the hazard potential is much lower.
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Cianuros/farmacocinética , Enfermedades Transmitidas por los Alimentos , Glicósidos/farmacocinética , Manihot/química , Adulto , Disponibilidad Biológica , Estudios Cruzados , Cianuros/sangre , Cianuros/envenenamiento , Femenino , Lino/química , Glicósidos/toxicidad , Humanos , Masculino , Persona de Mediana Edad , Prunus armeniaca/químicaRESUMEN
3-Chloropropane-1,2-diol (3-MCPD) and its fatty acid esters are formed during thermal treatment of fat-containing foodstuff in the presence of salt. Toxicological studies indicate a carcinogenic potential of 3-MCPD, pointing to the kidney as the main target organ. It is assumed that the toxicological property of 3-MCPD esters is constituted by the release of 3-MCPD during digestion. In a repeated-dose 28-day oral toxicity study using Wistar rats, animals were treated with equimolar doses of either 3-MCPD (10 mg/kg body weight) or 3-MCPD dipalmitate (53 mg/kg body weight). A lower dose of 3-MCPD dipalmitate (13.3 mg/kg body weight) was also applied. No histopathologically visible toxicity was observed in the study. To address molecular mechanisms leading to toxicity of 3-MCPD and its esters, kidney samples were analyzed by a comparative, two-dimensional gel electrophoresis/mass spectrometry proteomic approach. After either 3-MCPD or 3-MCPD dipalmitate treatment, alterations in proteins related to various metabolic pathways, including carbohydrate, amino acid, and fatty acid metabolism, were detected. These findings confirm and complement previous data on the inhibition of glucose metabolism by 3-MCPD. Altogether, broad overlap of 3-MCPD- and 3-MCPD dipalmitate-induced proteomic changes was observed. Further analyses revealed that the observed induction of glutathione S-transferase pi 1 (Gstp1) occurred at the transcriptional level and was not related to nuclear factor (erythroid-derived 2)-like 2 activation. Overall, the results indicate common mechanisms of toxicity for 3-MCPD and its dipalmitate ester. Furthermore, data suggest Gstp1 as a sensitive marker for early 3-MCPD-induced effects in rat kidney.
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Gutatión-S-Transferasa pi/metabolismo , Riñón/efectos de los fármacos , Palmitatos/toxicidad , Proteoma/metabolismo , alfa-Clorhidrina/toxicidad , Animales , Western Blotting , Culinaria , Electroforesis en Gel Bidimensional , Contaminación de Alimentos , Riñón/enzimología , Riñón/metabolismo , Riñón/patología , Masculino , Metaboloma/efectos de los fármacos , Proteómica , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Pruebas de Toxicidad SubagudaRESUMEN
BACKGROUND: The estrogen receptor-positive M13SV1 breast epithelial cell line was proposed to be a suitable in vitro model for breast cancer research since two derivatives with graduated tumorigenicity-M13SV1-R2-2 and M13SV1-R2-N1-are available for this cell line. In the present study, these three cell lines were comparatively examined for their morphological and their biochemical properties on the molecular level. METHODS: A transcriptomic approach (gene array analysis) was chosen to unravel differences in gene expression among the three cell lines. Network analysis was conducted to identify deregulated signaling pathways. Cellular viability was determined by impedance measurements as well as by neutral red uptake assay. Apoptosis was determined by using a caspase assay. For morphological characterization, cells were grown in three-dimensional cell culture, and cellular differentiation and spheroid formation was followed by immunofluorescence staining by using confocal laser scanning microscopy. RESULTS: The gene array results indicated that there were only marginal differences in gene expression among the three cell lines. Network analysis predicted the R2-N1 derivative (1) to display enhanced apoptosis and (2) to have a higher migration capability compared to its parent cell line M13SV1. Enhanced apoptosis was confirmed by elevated caspase activity, and increased migration was observed in 3D culture when cells migrated out of the globular spheroids. In 3D cell culture, all three cell lines similarly formed spheroids within three days, but there was no acini formation until day 21 which is indicated by a growth arrest around day 15, cellular polarization, and the formation of hollow lumen inside the spheroids. These characteristics, however, are crucial to study, e.g., the differentiation process of breast epithelial cells in vitro. CONCLUSION: Due to the molecular and morphological features, the M13SV1 cell line and its tumorigenic derivatives seem to be less suitable as in vitro models than other cell lines such as the MCF-10A cell line which displays proper acini formation in 3D culture.
RESUMEN
The food contaminants 3-chloro-1,2-propanediol (3-MCPD) and 3-MCPD fatty acid esters have attracted considerable attention in the past few years due to their toxic properties and their occurrence in numerous foods. Recently, significant amounts of the isomeric compounds 2-chloro-1,3-propanediol (2-MCPD) fatty acid esters have been detected in refined oils. Beside the interrogation which toxic effects might be related to the core compound 2-MCPD, the key question from the risk assessment perspective is again-as it was discussed for 3-MCPD fatty acid esters before-to which degree these esters are hydrolyzed in the gut, thereby releasing free 2-MCPD. Here, we show that free 2-MCPD but not 2-MCPD fatty acid esters were able to cross a monolayer of differentiated Caco-2 cells as an in vitro model for the human intestinal barrier. Instead, the esters were hydrolyzed by the cells, thereby releasing free 2-MCPD which was neither absorbed nor metabolized by the cells. Cytotoxicity assays revealed that free 2-MCPD as well as free 3-MCPD was not toxic to Caco-2 cells up to a level of 1 mM, whereas cellular viability was slightly decreased in the presence of a few 2-MCPD and 3-MCPD fatty acid esters at concentrations above 10 µM. The observed cytotoxic effects correlated well with the induction of caspase activity and might be attributed to the induction of apoptosis by free fatty acids which were released from the esters in the presence of Caco-2 cells.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Ácidos Grasos/toxicidad , Absorción Intestinal , alfa-Clorhidrina/toxicidad , Células CACO-2 , Relación Dosis-Respuesta a Droga , Ésteres , Ácidos Grasos/administración & dosificación , Ácidos Grasos/farmacocinética , Humanos , Hidrólisis , Medición de Riesgo , alfa-Clorhidrina/administración & dosificación , alfa-Clorhidrina/farmacocinéticaRESUMEN
Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals used in various industrial and consumer products. They have gained attention due to their ubiquitous occurrence in the environment and potential for adverse effects on human health, often linked to immune suppression, hepatotoxicity, and altered cholesterol metabolism. This study aimed to explore the impact of ten individual PFAS, 3â¯H-perfluoro-3-[(3-methoxypropoxy) propanoic acid] (PMPP/Adona), ammonium perfluoro-(2-methyl-3-oxahexanoate) (HFPO-DA/GenX), perfluorobutanoic acid (PFBA), perfluorobutanesulfonic acid (PFBS), perfluorodecanoic acid (PFDA), perfluorohexanoic acid (PFHxA), perfluorohexanesulfonate (PFHxS), perfluorononanoic acid (PFNA), perfluorooctanoic acid (PFOA), and perfluorooctanesulfonic acid (PFOS) on the lipid metabolism in human hepatocyte-like cells (HepaRG). These cells were exposed to different concentrations of PFAS ranging from 10⯵M to 5000⯵M. Lipids were extracted and analyzed using liquid chromatography coupled with mass spectrometry (LC- MS-QTOF). PFOS at 10⯵M and PFOA at 25⯵M increased the levels of ceramide (Cer), diacylglycerol (DAG), N-acylethanolamine (NAE), phosphatidylcholine (PC), and triacylglycerol (TAG) lipids, while PMPP/Adona, HFPO-DA/GenX, PFBA, PFBS, PFHxA, and PFHxS decreased the levels of these lipids. Furthermore, PFOA and PFOS markedly reduced the levels of palmitic acid (FA 16.0). The present study shows distinct concentration-dependent effects of PFAS on various lipid species, shedding light on the implications of PFAS for essential cellular functions. Our study revealed that the investigated legacy PFAS (PFOS, PFOA, PFBA, PFDA, PFHxA, PFHxS, and PFNA) and alternative PFAS (PMPP/Adona, HFPO-DA/GenX and PFBS) can potentially disrupt lipid homeostasis and metabolism in hepatic cells. This research offers a comprehensive insight into the impacts of legacy and alternative PFAS on lipid composition in HepaRG cells.
Asunto(s)
Fluorocarburos , Hepatocitos , Metabolismo de los Lípidos , Humanos , Fluorocarburos/toxicidad , Metabolismo de los Lípidos/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Línea Celular , Contaminantes Ambientales/toxicidad , Ácidos Alcanesulfónicos/toxicidadRESUMEN
Trans fatty acids (TFA) are hypothesized to have an impact not only on coronary heart diseases but also on the development of colon cancer. To analyze if TFA exhibit cellular and molecular effects which could be involved in colon tumor progression, cells of the human colorectal adenocarcinoma-derived cell line Caco-2 were treated with various TFA isomers differing in the number and position of trans double bonds. The TFA tested in this study did not increase cellular proliferation but displayed growth-inhibitory effects at concentrations higher than 500 µM. In case of the TFA isomer C18:3 t9, t11, t13, an IC50 value of 23 µM was estimated for cytotoxicity indicating a high cytotoxic potential of this compound. In addition to the cytotoxicity studies, the TFA isomers were tested for their ability to activate peroxisome proliferator-activated receptors (PPAR) by taking advantage of a PPAR-dependent reporter gene assay. In contrast to PPARγ that was not activated by the TFA isomers tested in this study, the substances were shown to moderately activate PPARα, and strong activation was observed for PPARδ. The putative impact of TFA on colon cancer development with respect to PPARδ activation is being discussed.
Asunto(s)
PPAR alfa/metabolismo , PPAR delta/metabolismo , Ácidos Grasos trans/farmacología , Células CACO-2/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Humanos , Isomerismo , L-Lactato Deshidrogenasa/metabolismo , PPAR alfa/genética , PPAR delta/genética , PPAR gamma/genética , PPAR gamma/metabolismo , Ácidos Grasos trans/químicaRESUMEN
3-Chloro-1,2-propanediol (3-MCPD) and its fatty acid esters (FE) are present as contaminants in different processed foods. Based on the available toxicological data the potential risk of 3-MCPD and its FE to human health was assessed by risk assessment authorities, including the European Food Safety Authority (EFSA). Considering the available data, EFSA concluded that 3-MCPD is a non-genotoxic compound exhibiting secondary carcinogenic effects in rodents. A tolerable daily intake of 2 µg/kg body weight and day was derived by EFSA for free and ester-bound 3-MCPD in 2018. However, there are still different pending issues that have remained unclear until now. Here, we summarize the current knowledge regarding 3-MCPD and its FE with a focus on pending issues regarding exposure assessment via biomarkers as well as the identification of (toxic) metabolites formed after exposure to FE of 3-MCPD and their modes of action.
Asunto(s)
alfa-Clorhidrina , Humanos , alfa-Clorhidrina/toxicidad , alfa-Clorhidrina/análisis , Ésteres/análisis , Ácidos Grasos , Medición de Riesgo , Inocuidad de los Alimentos , Contaminación de Alimentos/análisisRESUMEN
The human intestinal cell line Caco-2 is a well-established model system to study cellular differentiation of human enterocytes of intestinal origin, because these cells have the capability to differentiate spontaneously into polarized cells with morphological and biochemical features of small intestinal enterocytes. Therefore, the cells are widely used as an in vitro model for the human intestinal barrier. In this study, a proteomic approach was used to identify the molecular marker of intestinal cellular differentiation. The proteome of proliferating Caco-2 cells was compared with that of fully differentiated cells. Two-dimensional gel analysis yielded 53 proteins that were differently regulated during the differentiation process. Pathway analysis conducted with those 34 proteins that were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed subsets of proteins with common molecular and cellular function. It was shown that proteins involved in xenobiotic and drug metabolism as well as in lipid metabolism were upregulated upon cellular differentiation. In parallel, proteins associated with proliferation, cell growth and cancer were downregulated, reflecting the loss of the tumorigenic phenotype of the cells. Thus, the proteomic approach in combination with a literature-based pathway analysis yielded valuable information about the differentiation process of Caco-2 cells on the molecular level that contributes to the understanding of the development of colon cancer or inflammatory diseases such as ulcerative colitis--diseases associated with an imbalanced differentiation process of intestinal cells.