Vascular endothelial effects of dibutyl phthalate: In vitro and in vivo evidence.

Dibutyl phthalate (DBP) is widely used in many consumer and personal care products. Here, we report vascular endothelial response to DBP in three different exposure scenarios: after short-term exposure (24 h) of human endothelial cells (ECs) EA.hy926 to 10-6, 10-5, and 10-4 M DBP, long-term exposure (12 weeks) of EA.hy926 cells to 10-9, 10-8, and 10-7 M DBP, and exposure of rats (28 and 90 days) to 100, 500, and 5000 mg DBP/kg food. We examined different vascular functions such as migration of ECs, adhesion of ECs to the extracellular matrix, tube formation, the morphology of rat aorta, as well as several signaling pathways involved in controlling endothelial function. Short-term in vitro exposure to DBP increased migration of ECs through G protein-coupled estrogen receptor, extracellular signal-regulated kinase 1/2, and nitric oxide (NO) signaling and decreased adhesion to gelatin. Long-term in vitro exposure to DBP transiently increased EC migration and had a bidirectional effect on EC adhesion to gelatin and tube formation. These effects were accompanied by a sustained increase in NO production and endothelial NO synthase (eNOS) and Akt activity. In vivo, exposure to DBP for 90 days decreased the aortic wall-to-lumen ratio and increased eNOS and Akt phosphorylation in ECs of rat aorta. This comparative investigation has shown that exposure to DBP may affect vascular function by altering EC migration, adhesion to gelatin, and tube formation after short- and long-term in vitro exposure and by decreasing the aortic wall-to-lumen ratio in vivo. The eNOS-NO and Akt signaling could be important in mediating the effects of DBP in long-term exposure scenarios.

In vitro and in vivo exposure of endothelial cells to dibutyl phthalate promotes monocyte adhesion.

The effect of endothelial cells' exposure to dibutyl phthalate (DBP) on monocyte adhesion is largely unknown. We evaluated monocyte adhesion to DBP-exposed endothelial cells by combining three approaches: short-term exposure (24 h) of EA.hy926 cells to 10-6, 10-5, and 10-4 M DBP, long-term exposure (12 weeks) of EA.hy926 cells to 10-9, 10-8, and 10-7 M DBP, and exposure of rats (28 and 90 days) to 100, 500, and 5000 mg DBP/kg food. Monocyte adhesion to human EA.hy926 and rat aortic endothelial cells, expression of selected cellular adhesion molecules and chemokines, and the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) were analyzed. We observed increased monocyte adhesion to DBP-exposed EA.hy926 cells in vitro and to rat aortic endothelium ex vivo. ERK1/2 inhibitor prevented monocyte adhesion to DBP-exposed EA.hy926 cells in short-term exposure experiments. Increased ERK1/2 phosphorylation in rat aortic endothelium and transient decrease in ERK1/2 activation following long-term exposure of EA.hy926 cells to DBP were also observed. In summary, exposure of endothelial cells to DBP promotes monocyte adhesion, thus suggesting a possible role for this phthalate in the development of atherosclerosis. ERK1/2 signaling could be the mediator of monocyte adhesion to DBP-exposed endothelial cells, but only after short-term high-level exposure.

Differential eigengene network analysis reveals benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin consensus regulatory network in human liver cell line HepG2.

Aryl hydrocarbon receptor (AHR) is one of the main mediators of the toxic effects of benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, a vast number of BaP- and TCDD-affected genes may suggest a more complex transcriptional regulatory network driving common adverse effects of these two chemicals. Unlike TCDD, BaP is rapidly metabolized in the liver, yielding products with a questionable ability to bind and activate AHR. In this study, we used transcriptomics data from the BaP- and TCCD-exposed human liver cell line HepG2, and performed differential eigengene network analysis to understand the correlation among genes and to untangle the common regulatory mechanism in the action of BaP and TCDD. The genes were grouped into 11 meta-modules with an overall preservation of 0.72 and were also segregated into three consensus time clusters: 12, 24, and 48 h. The analysis showed that the consensus genes in each time cluster were either directly regulated by the AHR or the AHR-TF interactions. Some TFs form a direct physical interaction with AHR such as ESR1, FOXA1, and E2F1, whereas others, including CTCF, RXRA, FOXO1, CEBPA, CEBPB, and TP53 show an indirect interaction with AHR. The analysis of biological processes (BPs) identified unique and common BPs in BaP and TCDD samples, with DNA damage response detected in all three time points. In summary, we identified a consensus transcriptional regulatory network common for BaP and TCDD consisting of direct AHR targets and AHR-TF targets. This analysis sheds new light on the common mechanism of action of a genotoxic (BaP) and non-genotoxic (TCDD) chemical in liver cells.

Global gene expression analysis reveals novel transcription factors associated with long-term low-level exposure of EA.hy926 human endothelial cells to bisphenol A.

Bisphenol A (BPA) is an endocrine disruptor that binds to estrogen receptors (ER); however, studies have shown that the ER pathway was not always the primary molecular mechanism of BPA's action in cells and that gene transcription could be altered by different exposure times and doses. Here, we sought to understand the correlation between the BPA-responsive genes that have associated biological functions and the transcription factors (TFs) involved in their regulation by repeatedly exposing human endothelial cells EA.hy926 to three nanomolar concentrations of BPA (10-9 M, 10-8 M, and 10-7 M) for 14 weeks, after which changes in global gene expression were determined by RNA sequencing. Cytoscape plug-in iRegulon was used to infer TFs involved in the control of BPA-deregulated genes. The results show a minimal overlap in deregulated genes between three concentrations of BPA, with 10-9 M BPA having the highest number of deregulated genes. TF analysis suggests that all three concentrations of BPA were active in the absence of an ER-mediated pathway. A unique set of TFs (NES≥4) has been identified for each BPA concentration, including the NFκB family and CEBPB for 10-9 M BPA, MEF family, AHR/ARNT, and ZBTB33 for 10-8 M BPA, and IRF1-7 and OVOL1/OVOL2 for 10-7 M BPA, whereas STAT1/STAT2 were common TFs for 10-9 M and 10-7 M BPA. Overall, our data suggest that long-term low-level exposure of EA.hy926 cells to BPA leads to concentration-specific changes in gene expression that are not controlled by the ER-mediated signaling but rather by other mechanisms.

Cytotoxic activity of the crude polysaccharides/exopolysaccharides of Coprinus comatus and Coprinellus truncorum.

Fungi are an important source of polysaccharides (PSH) and phenolic compounds (PC). Numerous studies have highlighted the beneficial effects of fungal consumption, but the impact of submerged cultivated mycelia (M) and filtrate (F) has not been fully investigated. We aimed to investigate the cytotoxic activity of isolated crude PSH and exopolysaccharides (ePSH) of submerged cultivated M and F of edible Coprinus comatus and Coprinellus truncorum species. Both PSH and ePSH exhibited significant cytotoxic activity towards HepG2 cancer cells of human origin (three-way ANOVA). The C. truncorum PSH/ePSH was more efficient inducing maximal reduction in cell viability (≈50% at 450 µg/mL) after 24 h while C. comatus PSH/ePSH needed 72 h to reach similar effect (≈60% at 450 µg/mL). Partial least square regression (PLSR) analysis indicated that specific phenolic composition of the PSH/ePSH could be responsible for the difference in their activity.

Adult Rat Liver After Subchronic Acrylamide Treatment: Histological, Stereological and Biochemical Study

SUMMARY: Acrylamide (AA) is a widely used chemical and an important monomer in various industrial and laboratory processes. In addition, AA is formed during processing of starchy food at high temperature. The aim of our study was to examine effects of subchronic AA treatment on adult rat liver using histological, stereological and biochemical methods. Adult male Wistar rats were treated with AA at doses of 25 mg/kg b.w. and 50 mg/kg b.w. for three weeks. Stereological analysis showed decrease of volume density of hepatocyte cytoplasm, and increase of volume density of hepatocyte nuclei and nucleocytoplasmic ratio in AA50mg group. Immunohistochemical analysis of the liver sections showed that treatment with AA50mg increase the percentage of PCNA positive cells, while the percentage of caspase 3 positive cells was not affected by AA. PAS-staining showed that glycogen content in hepatocytes was not affected by AA. Serological examination revealed increase of lipid peroxidation in AA50mg group, while total protein concentration, protein thiol group level, as well as, paraoxonase 1 activity were not changed in AA-exposed animals. Stereological and immunohistochemical analyses of adult liver sections suggest increase of proliferation in AA50mg group, while increase of lipid peroxidation in serum of AA50mg group indicates oxidative stress induction.

La acrilamida (AA) es un químico ampliamente utilizado y un monómero importante en varios procesos industriales y de laboratorio. Además, la AA se forma durante el procesamiento de alimentos ricos en almidón a altas temperaturas. El objetivo de nuestro estudio fue examinar los efectos del tratamiento con AA subcrónica en el hígado de rata adulta utilizando métodos histológicos, estereológicos y bioquímicos. Se trataron ratas Wistar macho adultas con AA a dosis de 25 mg/kg p.v. y 50 mg/kg de peso corporal por tres semanas. El análisis estereológico mostró una disminución de la densidad del volumen del citoplasma de los hepatocitos y un aumento de la densidad del volumen de los núcleos de los hepatocitos y la relación nucleocitoplasmática en el grupo de 50 mg de AA. El análisis inmunohistoquímico de las secciones de hígado mostró que el tratamiento con 50 mg de AA aumentó el porcentaje de células positivas para PCNA, mientras que el porcentaje de células positivas para caspasa 3 no se vio afectado por AA. La tinción con PAS mostró que el contenido de glucógeno en los hepatocitos no se vio afectado por AA. El examen serológico reveló un aumento de la peroxidación de lípidos en el grupo de 50 mg de AA, mientras que la concentración de proteína total, el nivel del grupo tiol de proteína y la actividad de paraoxonasa 1 no cambiaron en los animales expuestos a AA. Los análisis estereológicos e inmunohistoquímicos de secciones de hígado adulto sugieren un aumento de la proliferación en el grupo AA50 mg, mientras que el aumento de la peroxidación lipídica en suero del grupo AA50 mg indica inducción de estrés oxidativo.

Effect of Acrylamide Treatment on Cyp2e1 Expression and Redox Status in Rat Hepatocytes.

Acrylamide (AA) toxicity is associated with oxidative stress. During detoxification, AA is either coupled to gluthatione or biotransformed to glycidamide by the enzyme cytochrome P450 2E1 (CYP2E1). The aim of our study was to examine the hepatotoxicity of AA in vivo and in vitro. Thirty male Wistar rats were treated with 25 or 50 mg/kg b.w. of AA for 3 weeks. Qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), CYP2E1, catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 expression in liver was carried out. Bearing in mind that the liver is consisted mainly of hepatocytes, in a parallel study, we used the rat hepatoma cell line H4IIE to investigate the effects of AA at IC20 and IC50 concentrations on the redox status and the activity of CAT, SOD, and glutathione-S-transferase (GST), their gene expression, and CYP2E1 and iNOS expression. Immunohistochemically stained liver sections showed that treatment with AA25mg induced a significant decrease of CYP2E1 protein expression (p < 0.05), while treatment with AA50mg led to a significant increase of iNOS protein expression (p < 0.05). AA treatment dose-dependently elevated SOD2 protein expression (p < 0.05), while SOD1 protein expression was significantly increased only at AA50mg (p < 0.05). CAT protein expression was not significantly affected by AA treatments (p > 0.05). In AA-treated H4IIE cells, a concentration-dependent significant increase in lipid peroxidation and nitrite levels was observed (p < 0.05), while GSH content and SOD activity significantly decreased in a concentration-dependent manner (p < 0.05). AA IC50 significantly enhanced GST activity (p < 0.05). The level of mRNA significantly increased in a concentration-dependent manner for iNOS, SOD2, and CAT in AA-treated H4IIE cells (p < 0.05). AA IC50 significantly increased the transcription of SOD1, GSTA2, and GSTP1 genes (p < 0.05), while AA IC20 significantly decreased mRNA for CYP2E1 in H4IIE cells (p < 0.05). Obtained results indicate that AA treatments, both in vivo and in vitro, change hepatocytes; drug-metabolizing potential and disturb its redox status.

Acrylamide and Potential Risk of Diabetes Mellitus: Effects on Human Population, Glucose Metabolism and Beta-Cell Toxicity.

Diabetes mellitus is a frequent endocrine disorder characterized by hyperglycemia. Acrylamide (AA) is food contaminant formed during the high-temperature processing of food rich in carbohydrates and low in proteins. Recent human epidemiological studies have shown a potential association between AA exposure and the prevalence of diabetes in the general population. In male rats, AA treatment promoted pancreatic islet remodeling, which was determined by alpha-cell expansion and beta-cell reduction, while in female rats AA caused hyperglycemia and histopathological changes in pancreatic islets. In vitro and in vivo rodent model systems have revealed that AA induces oxidative stress in beta cells and that AA impairs glucose metabolism and the insulin signaling pathway. Animal studies have shown that diabetic rodents are more sensitive to acrylamide and that AA aggravates the diabetic state. In this review, we provide an overview of human epidemiological studies that examined the relation between AA exposure and glucose disorders. In addition, the effects of AA treatment on pancreatic islet structure, beta-cell function and glucose metabolism in animal models are comprehensively analyzed with an emphasis on sex-related responses. Furthermore, oxidative stress as a putative mechanism of AA-induced toxicity in beta cells is explored. Finally, we discuss the effects of AA on diabetics in a rodent model system.

Thyroid Gland Alterations in Old-Aged Wistar Rats: A Comprehensive Stereological, Ultrastructural, Hormonal, and Gene Expression Study.

The aim of the present study was to determine and elaborate on all changes in old-aged (OA) versus young-aged (YA) rat thyroids by using stereological, ultrastructural, hormonal, and gene expression analyses. We used 4- and 24-month-old male Wistar rats in our evaluation, presenting all changes in comparison with YA rats. Results showed that the thyroid parenchyma was characterized by higher absolute volumes of the gland, colloid, epithelium, and interstitium by 135, 135, 140, and 142% (p < 0.05) respectively, while the relative volumes of colloid and glands were unchanged. Ultrastructural analysis revealed less active glands, with smaller amounts of lysosomes, thyroglobulin (Tg) granules, and microvilli in the luminal colloid. Optical density values for thyroid peroxidase (TPO), Tg, and vascular-endothelial growth factor immunostaining remained unchanged; however, TPO and Tg exhibited visually stronger expression in small active follicles. Thyroxine (T4)-Tg, the relative intensity of fluorescence (RIF), serum T4, and the sodium-iodide symporter immunohistochemical and gene expressions decreased by 20, 40, 29, and 31% (p < 0.05), respectively, in OA thyroids. Pituitary thyroid-stimulating hormone (TSH) RIF increased by 44% (p < 0.05), but the TSH serum concentration remained unchanged. In conclusion, the obtained results indicate depression of the thyroid gland synthetic and secretory capacity with advanced age.