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Recreational use of nitrous oxide (N2O) has become a major health issue worldwide, with a high number of clinical events, especially in neurology and cardiology. It is essential to be able to detect and monitor N2O abuse to provide effective care and follow-up to these patients. Current recommendations for detecting N2O in cases of recreational misuse and consumption markers are lacking. We aimed to update current knowledge through a review of the literature on N2O measurement and kinetics. We reviewed the outcomes of experiments, whether in preclinical models (in vitro or in vivo), or in humans, with the aim to identify biomarkers of intoxication as well as biomarkers of clinical severity, for laboratory use. Because N2O is eliminated 5â¯min after inhalation, measuring it in exhaled air is of no value. Many studies have found that urine and blood matrices concentrations are connected to ambient concentrations, but there is no similar data for direct exposure. There have been no studies on N2O measurement in direct consumers. Currently, patients actively abusing N2O are monitored using effect biomarkers (biomarkers related to the effects of N2O on metabolism), such as vitamin B12, homocysteine and methylmalonic acid.
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Assessing the potential of a new drug to cause drug-induced liver injury (DILI) is a challenge for the pharmaceutical industry. We therefore determined whether cell models currently used in safety assessment (HepG2, HepaRG, Upcyte and primary human hepatocytes in conjunction with basic but commonly used endpoints) are actually able to distinguish between novel chemical entities (NCEs) with respect to their potential to cause DILI. A panel of thirteen compounds (nine DILI implicated and four non-DILI implicated in man) were selected for our study, which was conducted, for the first time, across multiple laboratories. None of the cell models could distinguish faithfully between DILI and non-DILI compounds. Only when nominal in vitro concentrations were adjusted for in vivo exposure levels were primary human hepatocytes (PHH) found to be the most accurate cell model, closely followed by HepG2. From a practical perspective, this study revealed significant inter-laboratory variation in the response of PHH, HepG2 and Upcyte cells, but not HepaRG cells. This variation was also observed to be compound dependent. Interestingly, differences between donors (hepatocytes), clones (HepG2) and the effect of cryopreservation (HepaRG and hepatocytes) were less important than differences between the cell models per se. In summary, these results demonstrate that basic cell health endpoints will not predict hepatotoxic risk in simple hepatic cells in the absence of pharmacokinetic data and that a multicenter assessment of more sophisticated signals of molecular initiating events is required to determine whether these cells can be incorporated in early safety assessment.
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Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Pruebas de Toxicidad Aguda/métodos , Células Cultivadas , Criopreservación , Células Hep G2/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Humanos , Reproducibilidad de los Resultados , Pruebas de Toxicidad Aguda/normasRESUMEN
UNLABELLED: Drugs induce cholestasis by diverse and still poorly understood mechanisms in humans. Early hepatic effects of chlorpromazine (CPZ), a neuroleptic drug known for years to induce intrahepatic cholestasis, were investigated using the differentiated human hepatoma HepaRG cells. Generation of reactive oxygen species (ROS) was detected as early as 15 minutes after CPZ treatment and was associated with an altered mitochondrial membrane potential and disruption of the pericanalicular distribution of F-actin. Inhibition of [3H]-taurocholic acid efflux was observed after 30 minutes and was mostly prevented by N-acetyl cysteine (NAC) cotreatment, indicating a major role of oxidative stress in CPZ-induced bile acid (BA) accumulation. Moreover, 24-hour treatment with CPZ decreased messenger RNA (mRNA) expression of the two main canalicular bile transporters, bile salt export pump (BSEP) and multidrug resistance protein 3 (MDR3). Additional CPZ effects included inhibition of Na+ -dependent taurocholic cotransporting polypeptide (NTCP) expression and activity, multidrug resistance-associated protein 4 (MRP4) overexpression and CYP8B1 inhibition that are involved in BA uptake, basolateral transport, and BA synthesis, respectively. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Compared to CPZ effects, overloading of HepaRG cells with high concentrations of cholic and chenodeoxycholic acids induced a delayed oxidative stress and, similarly, after 24 hours it down-regulated BSEP and MDR3 in parallel to a decrease of NTCP and CYP8B1 and an increase of MRP4. By contrast, low BA concentrations up-regulated BSEP and MDR3 in the absence of oxidative stress. CONCLUSION: These data provide evidence that, among other mechanisms, oxidative stress plays a major role as both a primary causal and an aggravating factor in the early CPZ-induced intrahepatic cholestasis in human hepatocytes.
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Carcinoma Hepatocelular/patología , Clorpromazina/efectos adversos , Colestasis/inducido químicamente , Colestasis/fisiopatología , Neoplasias Hepáticas/patología , Estrés Oxidativo/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Actinas/metabolismo , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Clorpromazina/farmacología , Colestasis/metabolismo , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Ácido Taurocólico/metabolismoRESUMEN
Several factors are thought to be implicated in the occurrence of idiosyncratic adverse drug reactions. The present work aimed to question as to whether inflammation is a determinant factor in hepatic lesions induced by chlorpromazine (CPZ) using the human HepaRG cell line. An inflammation state was induced by a 24-hour exposure to proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß; then the cells were simultaneously treated with CPZ and/or cytokine for 24 hours or daily for 5 days. The inflammatory response was assessed by induction of C-reactive protein and IL-8 transcripts and proteins as well as inhibition of CPZ metabolism and down-regulation of cytochrome 3A4 (CYP3A4) and CYP1A2 transcripts, two major cytochrome P450 (P450) enzymes involved in its metabolism. Most effects of cotreatments with cytokines and CPZ were amplified or only observed after five daily treatments; they mainly included increased cytotoxicity and overexpression of oxidative stress-related genes, decreased Na(+)-taurocholate cotransporting polypeptide mRNA levels and activity, a key transporter involved in bile acids uptake, and deregulation of several other transporters. However, CPZ-induced inhibition of taurocholic acid efflux and pericanalicular F-actin distribution were not affected. In addition, a time-dependent induction of phospholipidosis was noticed in CPZ-treated cells, without obvious influence of the inflammatory stress. In summary, our results show that an inflammatory state induced by proinflammatory cytokines increased cytotoxicity and enhanced some cholestatic features induced by the idiosyncratic drug CPZ in HepaRG cells. These changes, together with inhibition of P450 activities, could have important consequences if extrapolated to the in vivo situation.
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Clorpromazina/efectos adversos , Colestasis/metabolismo , Inflamación/metabolismo , Actinas/genética , Actinas/metabolismo , Ácidos y Sales Biliares/genética , Ácidos y Sales Biliares/metabolismo , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Línea Celular , Colestasis/inducido químicamente , Colestasis/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulación hacia Abajo/genética , Humanos , Inflamación/genética , Interleucinas/genética , Interleucinas/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Estrés Oxidativo/genética , ARN Mensajero/genética , Simportadores/genética , Simportadores/metabolismo , Ácido Taurocólico/genética , Ácido Taurocólico/metabolismoRESUMEN
UNLABELLED: Although non-alcoholic fatty liver disease (NAFLD) is currently the most common form of chronic liver disease there is no pharmacological agent approved for its treatment. Since peroxisome proliferator-activated receptors (PPARs) are closely associated with hepatic lipid metabolism, they seem to play important roles in NAFLD. However, the effects of PPAR agonists on steatosis that is a common pathology associated with NAFLD, remain largely controversial. In this study, the effects of various PPAR agonists, i.e. fenofibrate, bezafibrate, troglitazone, rosiglitazone, muraglitazar and tesaglitazar on oleic acid-induced steatotic HepaRG cells were investigated after a single 24-hour or 2-week repeat treatment. Lipid vesicles stained by Oil-Red O and triglycerides accumulation caused by oleic acid overload, were decreased, by up to 50%, while fatty acid oxidation was induced after 2-week co-treatment with PPAR agonists. The greatest effects on reduction of steatosis were obtained with the dual PPARα/γ agonist muraglitazar. Such improvement of steatosis was associated with up-regulation of genes related to fatty acid oxidation activity and down-regulation of many genes involved in lipogenesis. Moreover, modulation of expression of some nuclear receptor genes, such as FXR, LXRα and CAR, which are potent actors in the control of lipogenesis, was observed and might explain repression of de novo lipogenesis. CONCLUSION: Altogether, our in vitro data on steatotic HepaRG cells treated with PPAR agonists correlated well with clinical investigations, bringing a proof of concept that drug-induced reversal of steatosis in human can be evaluated in in vitro before conducting long-term and costly in vivo studies in animals and patients.
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Hígado Graso/tratamiento farmacológico , Metabolismo de los Lípidos/efectos de los fármacos , Lipotrópicos/farmacología , Hígado/efectos de los fármacos , Receptores Activados del Proliferador del Peroxisoma/agonistas , Línea Celular , Receptor de Androstano Constitutivo , Evaluación Preclínica de Medicamentos , Ácidos Grasos no Esterificados/efectos adversos , Hígado Graso/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Lipogénesis/efectos de los fármacos , Hígado/metabolismo , Receptores X del Hígado , Enfermedad del Hígado Graso no Alcohólico , Ácido Oléico/efectos adversos , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Oxazoles/farmacología , Oxidación-Reducción , PPAR alfa/agonistas , PPAR alfa/metabolismo , PPAR gamma/agonistas , PPAR gamma/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Triglicéridos/metabolismoRESUMEN
Drinking water can be contaminated with pharmaceuticals. However, it is uncertain whether this contamination can be harmful for the liver, especially during obesity. Hence, the goal of our study was to determine whether chronic exposure to low doses of pharmaceuticals could have deleterious effects on livers of lean and obese mice. To this end, lean and ob/ob male mice were treated for 4 months with a mixture of 11 drugs provided in drinking water at concentrations ranging from 10 to 106 ng/l. At the end of the treatment, some liver and plasma abnormalities were observed in ob/ob mice treated with the cocktail containing 106 ng/l of each drug. For this dosage, a gene expression analysis by microarray showed altered expression of circadian genes (e.g. Bmal1, Dbp, Cry1) in lean and obese mice. RT-qPCR analyses carried out in all groups of animals confirmed that expression of 8 different circadian genes was modified in a dose-dependent manner. For some genes, a significant modification was observed for dosages as low as 10²-10³ ng/l. Drug mixture and obesity presented an additive effect on circadian gene expression. These data were validated in an independent study performed in female mice. Thus, our study showed that chronic exposure to trace pharmaceuticals disturbed hepatic expression of circadian genes, particularly in obese mice. Because some of the 11 drugs can be found in drinking water at such concentrations (e.g. acetaminophen, carbamazepine, ibuprofen) our data could be relevant in environmental toxicology, especially for obese individuals exposed to these contaminants.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Obesidad/metabolismo , Proteínas Circadianas Period/metabolismo , Preparaciones Farmacéuticas/administración & dosificación , Contaminantes Químicos del Agua/administración & dosificación , Factores de Transcripción ARNTL/agonistas , Factores de Transcripción ARNTL/antagonistas & inhibidores , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Animales , Criptocromos/agonistas , Criptocromos/antagonistas & inhibidores , Criptocromos/genética , Criptocromos/metabolismo , Proteínas de Unión al ADN/agonistas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Perfilación de la Expresión Génica , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/inducido químicamente , Obesidad/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Circadianas Period/agonistas , Proteínas Circadianas Period/antagonistas & inhibidores , Proteínas Circadianas Period/genética , Pruebas de Toxicidad Crónica , Factores de Transcripción/agonistas , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Contaminantes Químicos del Agua/toxicidadRESUMEN
Smoking is an established risk factor for various pathologies including lung cancer. Electronic cigarettes (e-cigs) and heated tobacco products (HTPs) have appeared on the market in recent years, but their safety or, conversely, their toxicity has not yet been demonstrated. This study aimed to compare the metabolome of human lung epithelial cells exposed to emissions of e-cigs, HTPs, or 3R4F cigarettes in order to highlight potential early markers of toxicity. BEAS-2B cells were cultured at the air-liquid interface and exposed to short-term emissions from e-cigs set up at low or medium power, HTPs, or 3R4F cigarettes. Untargeted metabolomic analyses were performed using liquid chromatography coupled with mass spectrometry. Compared to unexposed cells, both 3R4F cigarette and HTP emissions affected the profiles of exogenous compounds, one of which is carcinogenic, as well as those of endogenous metabolites from various pathways including oxidative stress, energy metabolism, and lipid metabolism. However, these effects were observed at lower doses for cigarettes (2 and 4 puffs) than for HTPs (60 and 120 puffs). No difference was observed after e-cig exposure, regardless of the power conditions. These results suggest a lower acute toxicity of e-cig emissions compared to cigarettes and HTPs in BEAS-2B cells. The pathways deregulated by HTP emissions are also described to be altered in respiratory diseases, emphasizing that the toxicity of HTPs should not be underestimated.
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Although the effects of cigarette smoke (CS) on the development of several intestinal diseases is well documented, the impact of e-cigarette aerosol (e-cig) on digestive health is largely unknown. To compare the effects of e-cig and CS on mouse ileum and colon, animals were chronically exposed for 6 months by nose-only inhalation to e-cig at 18 or 30 W power, or to 3R4F CS. Results showed that e-cig exposure decreased colon cell proliferation. Several other proliferative defects were observed in response to both e-cig and CS exposure, including up- and down-regulation of cyclin D1 protein levels in the ileum and colon, respectively. E-cig and CS exposure reduced myeloperoxidase activity in the ileum. In the colon, both exposures disrupted gene expression of cytokines and T cell transcription factors. For tight junction genes, ZO-1- and occludin-protein expression levels were reduced in the ileum and colon, respectively, by e-cig and CS exposure. The 16S sequencing of microbiota showed specific mild dysbiosis, according to the type of exposure. Overall, e-cig exposure led to altered proliferation, inflammation, and barrier function in both the ileum and colon, and therefore may be a gut hazard on par with conventional CS.
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Metabolite identification in untargeted metabolomics is complex, with the risk of false positive annotations. This work aims to use machine learning to successively predict the retention time (Rt) and the collision cross-section (CCS) of an open-access database to accelerate the interpretation of metabolomic results. Standards of metabolites were tested using liquid chromatography coupled with high-resolution mass spectrometry. In CCSBase and QSRR predictor machine learning models, experimental results were used to generate predicted CCS and Rt of the Human Metabolome Database. From 542 standards, 266 and 301 compounds were detected in positive and negative electrospray ionization mode, respectively, corresponding to 380 different metabolites. CCS and Rt were then predicted using machine learning tools for almost 114,000 metabolites. R2 score of the linear regression between predicted and measured data achieved 0.938 and 0.898 for CCS and Rt, respectively, demonstrating the models' reliability. A CCS and Rt index filter of mean error ± 2 standard deviations could remove most misidentifications. Its application to data generated from a toxicology study on tobacco cigarettes reduced hits by 76%. Regarding the volume of data produced by metabolomics, the practical workflow provided allows for the implementation of valuable large-scale databases to improve the biological interpretation of metabolomics data.
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Electronic cigarettes (e-cig) and heated tobacco products (HTP) are often used as smoking cessation aids, while the harm reduction effects of these alternatives to cigarettes are still the subject of controversial debate, in particular regarding their carcinogenic potential. The objective of this study is to compare the effects of e-cig, HTP and conventional cigarette emissions on the generation of oxidative stress and genetic and epigenetic lesions in human bronchial epithelial BEAS-2B cells. Our results show that HTP were less cytotoxic than conventional cigarettes while e-cig were not substantially cytotoxic in BEAS-2B cells. E-cig had no significant effect on the Nrf2 pathway, whereas HTP and cigarettes increased the binding activity of Nrf2 to antioxidant response elements and the expression of its downstream targets HMOX1 and NQO1. Concordantly, only HTP and cigarettes induced oxidative DNA damage and significantly increased DNA strand breaks and chromosomal aberrations. Neither histone modulations nor global DNA methylation changes were found after acute exposure, regardless of the type of emissions. In conclusion, this study reveals that HTP, unlike e-cig, elicit a biological response very similar to that of cigarettes, but only after a more intensive exposure: both tobacco products induce cytotoxicity, Nrf2-dependent oxidative stress and genetic lesions in human epithelial pulmonary cells. Therefore, the health risk of HTP should not be underestimated and animal studies are required in order to determine the tumorigenic potential of these emerging products.
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More than 7 million early deaths/year are attributable to air pollution. Current health concerns are especially focused on air pollution-derived particulate matter (PM). Although oxidative stress-induced airway inflammation is one of the main adverse outcome pathways triggered by air pollution-derived PM, the persistence of both these underlying mechanisms, even after exposure cessation, remained poorly studied. In this study, A/JOlaHsd mice were also exposed acutely (24 h) or sub-chronically (4 weeks), with or without a recovery period (12 weeks), to two urban PM2.5 samples collected during contrasting seasons (i.e., autumn/winter, AW or spring/summer, SS). The distinct intrinsic oxidative potentials (OPs) of AW and SS PM2.5, as evaluated in acellular conditions, were closely related to their respective physicochemical characteristics and their respective ability to really generate ROS over-production in the mouse lungs. Despite the early activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) cell signaling pathway by AW and, in a lesser degree, SS PM2.5, in the murine lungs after acute and sub-chronic exposures, the critical redox homeostasis was not restored, even after the exposure cessation. Accordingly, an inflammatory response was reported through the activation of the nuclear factor-kappa B (NF-κB) cell signaling pathway activation, the secretion of cytokines, and the recruitment of inflammatory cells, in the murine lungs after the acute and sub-chronic exposures to AW and, in a lesser extent, to SS PM2.5, which persisted after the recovery period. Taken together, these original results provided, for the first time, new relevant insights that air pollution-derived PM2.5, with relatively high intrinsic OPs, induced oxidative stress and inflammation, which persisted admittedly at a lower level in the lungs after the exposure cessation, thereby contributing to the occurrence of molecular and cellular adverse events leading to the development and/or exacerbation of future chronic inflammatory lung diseases and even cancers.
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Contaminantes Atmosféricos , Contaminación del Aire , Ratones , Animales , Material Particulado/análisis , Contaminantes Atmosféricos/análisis , Contaminación del Aire/efectos adversos , Contaminación del Aire/análisis , Pulmón , Inflamación/inducido químicamente , Estrés OxidativoRESUMEN
UNLABELLED: Drug-induced liver injury occurs in general after several weeks and is often unpredictable. It is characterized by a large spectrum of lesions that includes steatosis and phospholipidosis. Many drugs such as amiodarone and tetracycline have been reported to cause phospholipidosis and/or steatosis. In this study, acute and chronic hepatic effects of these two drugs were investigated using well-differentiated human hepatoma HepaRG cells. Accumulation of typical lipid droplets, labeled with Oil Red O, was observed in hepatocyte-like HepaRG cells after repeat exposure to either drug. Amiodarone caused the formation of additional intracytoplasmic vesicles that did not stain in all HepaRG cells. At the electron microscopic level, these vesicles appeared as typical lamellar bodies and were associated with an increase of phosphatidylethanolamine and phosphatidylcholine. A dose-dependent induction of triglycerides (TG) was observed after repeat exposure to either amiodarone or tetracycline. Several genes known to be related to lipogenesis were induced after treatment by these two drugs. By contrast, opposite deregulation of some of these genes (FASN, SCD1, and THSRP) was observed in fat HepaRG cells induced by oleic acid overload, supporting the conclusion that different mechanisms were involved in the induction of steatosis by drugs and oleic acid. Moreover, several genes related to lipid droplet formation (ADFP, PLIN4) were up-regulated after exposure to both drugs and oleic acid. CONCLUSION: Our results show that amiodarone causes phospholipidosis after short-term treatment and, like tetracycline, induces vesicular steatosis after repeat exposure in HepaRG cells. These data represent the first demonstration that drugs can induce vesicular steatosis in vitro and show a direct relationship between TG accumulation and enhanced expression of lipogenic genes.
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Amiodarona/farmacología , Carcinoma Hepatocelular/metabolismo , Hígado Graso/metabolismo , Lipogénesis/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , Tetraciclina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Amiodarona/efectos adversos , Carcinoma Hepatocelular/patología , Proteínas Portadoras , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Hígado Graso/inducido químicamente , Hígado Graso/patología , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Lipogénesis/genética , Lipogénesis/fisiología , Neoplasias Hepáticas/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ácido Oléico/farmacología , Perilipina-2 , Perilipina-4 , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Tetraciclina/efectos adversos , Triglicéridos/metabolismo , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Tobacco smoking is classified as a human carcinogen. A wide variety of new products, in particular electronic cigarettes (e-cigs), have recently appeared on the market as an alternative to smoking. Although the in vitro toxicity of e-cigs is relatively well known, there is currently a lack of data on their long-term health effects. In this context, the aim of our study was to compare, on a mouse model and using a nose-only exposure system, the in vivo genotoxic and mutagenic potential of e-cig aerosols tested at two power settings (18 W and 30 W) and conventional cigarette (3R4F) smoke. The standard comet assay, micronucleus test and Pig-a gene mutation assay were performed after subacute (4 days), subchronic (3 months) and chronic (6 months) exposure. The generation of oxidative stress was also assessed by measuring the 8-hydroxy-2'-deoxyguanosine and by using the hOGG1-modified comet assay. Our results show that only the high-power e-cig and the 3R4F cigarette induced oxidative DNA damage in the lung and the liver of exposed mice. In return, no significant increase in chromosomal aberrations or gene mutations were noted whatever the type of product. This study demonstrates that e-cigs, at high-power setting, should be considered, contrary to popular belief, as hazardous products in terms of genotoxicity in mouse model.
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Sistemas Electrónicos de Liberación de Nicotina , Productos de Tabaco , Aerosoles/toxicidad , Animales , Daño del ADN , Electrónica , RatonesRESUMEN
A preliminary ethnopharmacological survey, achieved in French Polynesia, led to the collection of the most cited plants among 63 species used to treat "infectious" diseases, with a description of their medicinal uses. Bibliographical investigations and antimicrobial screening permitted the selection of the botanical species Syzygium malaccense (Myrtaceae) for phytochemical analysis. Leaves of Syzygium malaccense were usually used in mixture with rhizomes of Curcuma longa to treat infectious diseases such as cystitis. The methanolic plant extracts were tested in vitro with an agar microdilution method on 33 bacteria strains and 1 yeast to obtain their Minimal Inhibitory Concentration (MIC), and cytotoxicity against HepG2 cells were evaluated. Antimicrobial synergistic effects of methanolic plant extracts from leaves of Syzygium malaccense and rhizomes from Curcuma longa were also evaluated. The bio-guided isolation of leaf extract from Syzygium malaccense led to the identification of seven alkyl-salicylic acids (anacardic acids or ginkgolic acids C15:0, C15:1, C17:0, C17:1, C17:2, C17:3 and C19:1) described for the first time in this species. All compounds were tested against Staphylococcus aureus (18.75 < MIC < 75.0 µg/mL), Streptococcus pyogenes (2.34 < MIC < 18.75 µg/mL) and Pseudomonas aeruginosa (MIC = 150 µg/mL), and their structure−activity relationships were discussed. The methanolic extract and salicylic derivatives from S. malaccense showed an interesting antimicrobial activity against Gram+ bacteria, without toxicity on hepG2 cells at 400 µg/mL. Moreover, these antibacterial compounds have already been studied for their anti-inflammatory activity, which supports the therapeutic interest of S. malaccense against infectious diseases.
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The electronic cigarettes (e-cigs) and more recently the heated tobacco products (HTP) provide alternatives for smokers as they are generally perceived to be less harmful than conventional cigarettes. However, it is crucial to compare the health risks of these different emergent devices, in order to determine which product should be preferred to substitute cigarette. The present study aimed to compare the composition of emissions from HTP, e-cigs and conventional cigarettes, regarding selected harmful or potentially harmful compounds, and their toxic impacts on the human bronchial epithelial BEAS-2B cells. The HTP emitted less polycyclic aromatic hydrocarbons and carbonyls than the conventional cigarette. However, amounts of these compounds in HTP aerosols were still higher than in e-cig vapours. Concordantly, HTP aerosol showed reduced cytotoxicity compared to cigarette smoke but higher than e-cig vapours. HTP and e-cig had the potential to increase oxidative stress and inflammatory response, in a manner similar to that of cigarette smoke, but after more intensive exposures. In addition, increasing e-cig power impacted levels of certain toxic compounds and related oxidative stress. This study provides important data necessary for risk assessment by demonstrating that HTP might be less harmful than tobacco cigarette but considerably more harmful than e-cig.
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Sistemas Electrónicos de Liberación de Nicotina , Productos de Tabaco , Aerosoles/toxicidad , Humanos , Humo/efectos adversos , Nicotiana , Productos de Tabaco/toxicidadRESUMEN
HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.
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Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Xenobióticos/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular Transformada , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Glucuronosiltransferasa/biosíntesis , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Omeprazol/farmacología , Fenobarbital/farmacología , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Reproducibilidad de los Resultados , Rifampin/farmacología , Factores de TiempoRESUMEN
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are two of the most common heterocyclic aromatic amines (HAA) produced during cooking of meat, fish and poultry. Both HAA produce different tumor profiles in rodents and are suspected to be carcinogenic in humans. In order to better understand the molecular basis of HAA toxicity, we have analyzed gene expression profiles in the metabolically competent human HepaRG cells using pangenomic oligonucleotide microarrays, after either a single (24-h) or a repeated (28-day) exposure to 10 µM PhIP or MeIQx. The most responsive genes to both HAA were downstream targets of the arylhydrocarbon receptor (AhR): CYP1A1 and CYP1A2 after both time points and CYP1B1 and ALDH3A1 after 28 days. Accordingly, CYP1A1/1A2 induction in HAA-treated HepaRG cells was prevented by chemical inhibition or small interference RNA-mediated down-regulation of the AhR. Consistently, HAA induced activity of the CYP1A1 promoter, which contains a consensus AhR-related xenobiotic-responsive element (XRE). In addition, several other genes exhibited both time-dependent and compound-specific expression changes with, however, a smaller magnitude than previously reported for the prototypical AhR target genes. These changes concerned genes mainly related to cell growth and proliferation, apoptosis, and cancer. In conclusion, these results identify the AhR gene battery as the preferential target of PhIP and MeIQx in HepaRG cells and further support the hypothesis that intake of HAA in diet might increase human cancer risk.
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Imidazoles/administración & dosificación , Quinoxalinas/administración & dosificación , Receptores de Hidrocarburo de Aril/biosíntesis , Receptores de Hidrocarburo de Aril/genética , Aminas/administración & dosificación , Aminas/toxicidad , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/toxicidad , Quinoxalinas/toxicidadRESUMEN
Human exposure to heterocyclic aromatic amines (HAA) usually occurs through mixtures rather than individual compounds. However, the toxic effects and related mechanisms of co-exposure to HAA in humans remain unknown. We compared the effects of two of the most common HAA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), individually or in combination, in the metabolically competent human hepatoma HepaRG cells. Various endpoints were measured including cytotoxicity, apoptosis, oxidative stress and DNA damage by the comet assay. Moreover, the effects of PhIP and/or MeIQx on mRNA expression and activities of enzymes involved in their activation and detoxification pathways were evaluated. After a 24h treatment, PhIP and MeIQx, individually and in combination, exerted differential effects on apoptosis, oxidative stress, DNA damage and cytochrome P450 (CYP) activities. Only PhIP induced DNA damage. It was also a stronger inducer of CYP1A1 and CYP1B1 expression and activity than MeIQx. In contrast, only MeIQx exposure resulted in a significant induction of CYP1A2 activity. The combination of PhIP with MeIQx induced an oxidative stress and showed synergistic effects on apoptosis. However, PhIP-induced genotoxicity was abolished by a co-exposure with MeIQx. Such an inhibitory effect could be explained by a significant decrease in CYP1A2 activity which is responsible for PhIP genotoxicity. Our findings highlight the need to investigate interactions between HAA when assessing risks for human health and provide new insights in the mechanisms of interaction between PhIP and MeIQx.
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Imidazoles/toxicidad , Quinoxalinas/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Daño del ADN , Relación Dosis-Respuesta a Droga , Humanos , Estrés Oxidativo/efectos de los fármacos , Reacción en Cadena de la PolimerasaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: An extensive ethnopharmacological survey was carried out in the Peruvian Amazonian district of Loreto with informants of various cultural origins from the surroundings of Iquitos (capital city of Loreto) and from 15 isolated riverine Quechua communities of the Pastaza River. A close attention was paid to the medical context and plant therapy, leading to the selection of 35 plant species (45 extracts). The extracts were tested for antiviral activity against HCV with counting of Huh-7 cellular death in case of toxicity, and cytotoxicity was evaluated in HepG2 cells. AIM OF THE STUDY: The aim of the study was to inventory the plants used against hepatitis in Loreto, then to evaluate their antiviral activity and to suggest a way to improve local therapeutic strategy against viral hepatitis, which is a fatal disease that is still increasing in this area. MATERIALS AND METHODS: An ethnographic survey was carried out using "participant-observation" methodology and focusing on plant therapy against hepatitis including associated remedies. 45 parts of plant were extracted with methanol and tested in vitro for anti-HCV activity in 96-well plate, using HCV cell culture system with immunofluorescent detection assisted by automated confocal microscopy. Toxicity of plant extracts was also evaluated in microplates on hepatic cells by immunofluorescent detection, for the Huh-7 nuclei viability, and by UV-absorbance measurement of MTT formazan for cytotoxicity in HepG2 cells. RESULTS: In vitro assay revealed interesting activity of 18 extracts (50% infection inhibition at 25⯵g/mL) with low cytotoxicity for 15 of them. Result analysis showed that at least 30% of HCV virus were inhibited at 25⯵g/mL for 60% of the plant extracts. Moreover, the ethnomedical survey showed that remedies used with low and accurate dosing as targeted therapy against hepatitis are usually more active than species indicated with more flexible dosing to alleviate symptoms of hepatic diseases. CONCLUSION: Together with bibliographic data analysis, this study supported the traditional medicinal uses of many plants and contributed to a better understanding of the local medical system. It also permitted to refine the therapeutic plant indications regarding patients' liver injuries and vulnerability. Only 2 of the 15 most active plant species have already been studied for antiviral activity against hepatitis suggesting new avenues to be followed for the 13 other species.
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Antivirales/farmacología , Etnofarmacología , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/farmacología , Plantas Medicinales/química , Antivirales/aislamiento & purificación , Células Hep G2 , Hepatitis C/virología , Humanos , Perú , Extractos Vegetales/aislamiento & purificación , Bosque LluviosoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The plant species reported here are used in contemporary phytotherapies by native and neo-urban societies from the Iquitenian surroundings (district of Loreto, Peruvian Amazon) for ailments related to microbial infections. Inhabitants of various ethnic origins were interviewed and 81 selected extracts were evaluated for their antimicrobial properties against a panel of 36 sensitive and multi-resistant bacteria or yeast. Medicinal plant researches in the Peruvian Amazon are now significant, but none of them has focused on an exhaustive listing of identified species tested on so many microbes with standardized experiments (to obtain MIC value). AIM OF THE STUDY: The aim of the study was to inventory the plants used against infections in the Loreto, an Amazonian region of Peru. It led to the new identification of secondary metabolites in two plant species. MATERIALS AND METHODS: Ethnographic survey was carried out using "participant-observation" methodology and focus on bioprospecting of antimicrobial remedies. Selected plant extracts and antimicrobial drugs were tested in vitro with agar dilution method on 35 bacteria strains and 1 yeast to evaluate their Minimal Inhibitory Concentration (MIC). Microdilution methods using 96-well microtiter plates were used for the determination of MIC from isolated compounds, and cytotoxicity in HepG2 cells from some selected extracts were also evaluated. Activity-guided isolation and identification of compounds were performed by various chromatographic methods and structural elucidations were established using HRMS and NMR spectroscopy. RESULTS: This study outlined antimicrobial activities of 59 plant species from 33 families (72 single plant extracts and 2 fermented preparations), 7 mixtures, and one insect nest extract against 36 microorganisms. Of the 59 species analysed, 12 plants showed relevant antibacterial activity with MIC ≤0.15 mg/mL for one or several of the 36 micro-organisms (Aspidosperma excelsum, Brosimum acutifolium, Copaifera paupera, Erythrina amazonica, Hura crepitans, Myrciaria dubia, Ocotea aciphylla, Persea americana, Spondias mombin, Swartzia polyphylla, Virola pavonis, Vismia macrophylla). Examination by bioautography of E. amazonica, M. dubia and O. aciphylla extracts allowed the phytochemical characterization of antimicrobial fractions and compounds. CONCLUSION: This study suggested an a posteriori correlation of the plant extract antimicrobial activity with the chemosensory cues of the drugs and attested that those chemosensory cues may be correlated with the presence of antimicrobial compounds (alkaloids, tannins, saponosids, essential oil, oleoresin ). It also led to the first isolation and identification of three secondary metabolites from E. amazonica and M. dubia.