Diana Teixeira

Flavonoid transport across RBE4 cells: A blood-brain barrier model

There is a growing interest in dietary therapeutic strategies to combat oxidative stress-induced damage to the Central Nervous System (CNS), which is associated with a number of pathophysiological processes, including Alzheimer’s and Parkinson’s diseases and cerebrovascular diseases. Identifying the mechanisms associated with phenolic neuroprotection has been delayed by the lack of information concerning the ability of these compounds to enter the CNS. The aim of this study was to evaluate the transmembrane transport of flavonoids across RBE-4 cells (an immortalized cell line of rat cerebral capillary endothelial cells) and the effect of ethanol on this transport. The detection and quantification of all of the phenolic compounds in the studied samples (basolateral media) was performed using a HPLC-DAD (Diode Array Detector). All of the tested flavonoids (catechin, quercetin and cyanidin-3-glucoside) passed across the RBE-4 cells in a time-dependent manner. This transport was not influenced by the presence of 0.1% ethanol. In conclusion, the tested flavonoids were capable of crossing this blood-brain barrier model.

Blueberry anthocyanins and pyruvic acid adducts: anticancer properties in breast cancer cell lines

The purpose of this study was to investigate the anticancer properties of an anthocyanin‐pyruvic acid adduct extract, which is being developed aiming to be further applied in the food industry. An anthocyanin extract from blueberry (extract I) and an anthocyanin‐pyruvic acid adduct extract (extract II) were tested on two breast cancer cell lines (MDA‐MB‐231 and MCF7). Proliferation was assessed by SRB assay and 3H‐thymidine incorporation. Caspase‐3 activity was determined in the presence of both extracts. Their capacity as chemoattractants and their invasive potential were also assayed.

Estrogen Signaling in Metabolic Inflammation

There is extensive evidence supporting the interference of inflammatory activation with metabolism. Obesity, mainly visceral obesity, is associated with a low-grade inflammatory state, triggered by metabolic surplus where specialized metabolic cells such as adipocytes activate cellular stress initiating and sustaining the inflammatory program. The increasing prevalence of obesity, resulting in increased cardiometabolic risk and precipitating illness such as cardiovascular disease, type 2 diabetes, fatty liver, cirrhosis, and certain types of cancer, constitutes a good example of this association. The metabolic actions of estrogens have been studied extensively and there is also accumulating evidence that estrogens influence immune processes. However, the connection between these two fields of estrogen actions has been underacknowledged since little attention has been drawn towards the possible action of estrogens on the modulation of metabolism through their anti-inflammatory properties. In the present paper, we summarize knowledge on the modification inflammatory processes by estrogens with impact on metabolism and highlight major research questions on the field. Understanding the regulation of metabolic inflammation by estrogens may provide the basis for the development of therapeutic strategies to the management of metabolic dysfunctions.

Persistent organic pollutant levels in human visceral and subcutaneous adipose tissue in obese individuals-Depot differences and dysmetabolism implications

BACKGROUND The role of persistent organic pollutants (POPs) with endocrine disrupting activity in the aetiology of obesity and other metabolic dysfunctions has been recently highlighted. Adipose tissue (AT) is a common site of POPs accumulation where they can induce adverse effects on human health. OBJECTIVES To evaluate the presence of POPs in human visceral (vAT) and subcutaneous (scAT) adipose tissue in a sample of Portuguese obese patients that underwent bariatric surgery, and assess their putative association with metabolic disruption preoperatively, as well as with subsequent body mass index (BMI) reduction. METHODS AT samples (n=189) from obese patients (BMI ≥ 35) were collected and the levels of 13 POPs were determined by gas chromatography with electron-capture detection (GC-ECD). Anthropometric and biochemical data were collected at the time of surgery. BMI variation was evaluated after 12 months and adipocyte size was measured in AT samples. RESULTS Our data confirm that POPs are pervasive in this obese population (96.3% of detection on both tissues), their abundance increasing with age (RS=0.310, p<0.01) and duration of obesity (RS=0.170, p<0.05). We observed a difference in AT depot POPs storage capability, with higher levels of ΣPOPs in vAT (213.9 ± 204.2 compared to 155.1 ± 147.4 ng/g of fat, p<0.001), extremely relevant when evaluating their metabolic impact. Furthermore, there was a positive correlation between POP levels and the presence of metabolic syndrome components, namely dysglycaemia and hypertension, and more importantly with cardiovascular risk (RS=0.277, p<0.01), with relevance for vAT (RS=0.315, p<0.01). Finally, we observed an interesting relation of higher POP levels with lower weight loss in older patients. CONCLUSION Our sample of obese subjects allowed us to highlight the importance of POPs stored in AT on the development of metabolic dysfunction in a context of obesity, shifting the focus to their metabolic effects and not only for their recognition as environmental obesogens.

Xanthohumol Influences Preadipocyte Differentiation: Implication of Antiproliferative and Apoptotic Effects

There is interest in the research of natural compounds that may interfere with the adipocyte life cycle, due to the growing prevalence of obesity and related complications. We aimed at studying the effect of xanthohumol (XN), a Humulus lupulus L. prenylflavonoid, on adipocytes measuring differentiation, proliferation, and apoptosis in 3T3-L1 cells. XN reduced differentiation, as revealed by decreased lipid content and peroxisome proliferator-activated receptor gamma expression, an effect more pronounced when cells were treated before or during differentiation induction. XN also decreased proliferation, as measured by sulforhodamine staining (IC(50) between 26 and 12 microM for 24, 48, and 72 h), and preadipocyte Ki67 expression. Apoptosis was increased in preadipocytes and adipocytes. NF-kappaB activity was stimulated by XN in preadipocytes. Results suggest that XN may reduce adipocyte number, contributing to adipocyte hypertrophy. Taking into consideration the consequences of adipocyte hypertrophy, XN does not seem to improve the metabolic profile associated with obesity.

Effects of environmental organochlorine pesticides on human breast cancer: Putative involvement on invasive cell ability

Human exposure to persistent organic pollutants (POPs) is a certainty, even to long banned pesticides like o,p′‐dichlorodiphenyltrichloroethane (o,p′‐DDT), and its metabolites p,p′‐dichlorodiphenyldichloroethylene (p,p′‐DDE), and p,p′‐dichlorodiphenyldichloroethane (p,p′‐DDD). POPs are known to be particularly toxic and have been associated with endocrine‐disrupting effects in several mammals, including humans even at very low doses. As environmental estrogens, they could play a critical role in carcinogenesis, such as in breast cancer. With the purpose of evaluating their effect on breast cancer biology, o,p′‐DDT, p,p′‐DDE, and p,p′‐DDD (50–1000 nM) were tested on two human breast adenocarcinoma cell lines: MCF‐7 expressing estrogen receptor (ER) α and MDA‐MB‐231 negative for ERα, regarding cell proliferation and viability in addition to their invasive potential. Cell proliferation and viability were not equally affected by these compounds. In MCF‐7 cells, the compounds were able to decrease cell proliferation and viability. On the other hand, no evident response was observed in treated MDA‐MB‐231 cells. Concerning the invasive potential, the less invasive cell line, MCF‐7, had its invasion potential significantly induced, while the more invasive cell line MDA‐MB‐231, had its invasion potential dramatically reduced in the presence of the tested compounds. Altogether, the results showed that these compounds were able to modulate several cancer‐related processes, namely in breast cancer cell lines, and underline the relevance of POP exposure to the risk of cancer development and progression, unraveling distinct pathways of action of these compounds on tumor cell biology.

Modulation of Adipocyte Biology by Δ9‐Tetrahydrocannabinol

It is recognized that the endocannabinoid system (ECS) plays a crucial role in the modulation of food intake and other aspects of energy metabolism. In this study, we aimed to investigate the effects of Δ9‐tetrahydrocannabinol (THC) on adipocyte biology. 3T3–L1 cells were used to evaluate proliferation by sulforhodamine B (SRB) staining and methyl−3H‐thymidine incorporation after 48 or 72 h of treatment with THC (1–500 nmol/l). Cells were differentiated in the presence or absence of the cannabinoid, and adipogenesis was determined by measuring lipid accumulation and peroxisome proliferator‐activated receptor γ (PPARγ) transcription through reverse transcriptase‐PCR (RT‐PCR). Lipolysis was quantified under basal conditions or after isoproterenol (IP, 100 nmol/l) or insulin (INS, 100 nmol/l) treatment. Transforming growth factor β (TGFβ), diacylglycerol lipase α, and N‐acylphosphatidylethanolamine‐specific phospholipase D (NAPE‐PLD) transcriptions were determined by RT‐PCR in preadipocytes and adipocytes and adiponectin only in adipocytes. THC treatment increased culture protein content and reduced methyl−3H‐thymidine incorporation. Cells treated with THC underwent adipogenesis shown by the expression of PPARγ and had increased lipid accumulation. Basal and IP‐stimulated lipolyses were inhibited by THC and there was no effect on lipolysis of INS‐treated adipocytes. The effects on methyl−3H‐thymidine incorporation and lipolysis seem to be mediated through CB1‐ and CB2‐dependent pathways. THC decreased NAPE‐PLD in preadipocytes and increased adiponectin and TGFβ transcription in adipocytes. These results show that the ECS interferes with adipocyte biology and may contribute to adipose tissue (AT) remodeling. Although these observations point toward increased AT deposition, the stimulation of adiponectin production and inhibition of lipolysis may be in favor of improved INS sensitivity under cannabinoid influence.

Adipose tissue dysfunction as a central mechanism leading to dysmetabolic obesity triggered by chronic exposure to p , p ’-DDE

Endocrine-disrupting chemicals such as p,p’-dichlorodiphenyldichloroethylene (p,p’-DDE), are bioaccumulated in the adipose tissue (AT) and have been implicated in the obesity and diabetes epidemic. Thus, it is hypothesized that p,p’-DDE exposure could aggravate the harm of an obesogenic context. We explored the effects of 12 weeks exposure in male Wistar rats’ metabolism and AT biology, assessing a range of metabolic, biochemical and histological parameters. p,p’-DDE -treatment exacerbated several of the metabolic syndrome-accompanying features induced by high-fat diet (HF), such as dyslipidaemia, glucose intolerance and hypertension. A transcriptome analysis comparing mesenteric visceral AT (vAT) of HF and HF/DDE groups revealed a decrease in expression of nervous system and tissue development-related genes, with special relevance for the neuropeptide galanin that also revealed DNA methylation changes at its promoter region. Additionally, we observed an increase in transcription of dipeptidylpeptidase 4, as well as a plasmatic increase of the pro-inflammatory cytokine IL-1β. Our results suggest that p,p’-DDE impairs vAT normal function and effectively decreases the dynamic response to energy surplus. We conclude that p,p’-DDE does not merely accumulate in fat, but may contribute significantly to the development of metabolic dysfunction and inflammation. Our findings reinforce their recognition as metabolism disrupting chemicals, even in non-obesogenic contexts.

Effects of xenoestrogens in human M1 and M2 macrophage migration, cytokine release, and estrogen-related signaling pathways

Bisphenol A (BPA), bis(2‐ethylhexyl)phthalate (DEHP) and di(n‐butyl)phthalate (DBP) are environmental estrogens that have been associated with a wide range of adverse health outcomes for which inflammation has also been hypothesized as a potentially involved mechanism and where macrophages play a central role. This study was carried out to evaluate if xenoestrogen (XE) treatment of classically (M1) or alternatively (M2) activated macrophages could affect their behavior. For this purpose, human peripheral blood monocyte‐derived macrophages either unstimulated or activated with lipopolysaccharide (100 ng/mL, M1) or with interleukin (IL) 4 (15 ng/mL, M2) were treated with 17β‐estradiol (E2), BPA, DEHP and DBP alone or in combination with selective ERα or ERβ antagonists. Migratory capability, cytokine release, and estrogen‐associated signaling pathways were evaluated to assess macrophage function. All tested XEs had a tendency to stimulate M2 migration, an effect that followed the same direction than E2. Moreover, all XEs significantly induced IL10 in M1 and decreased IL6 and globally decreased IL10, IL6, TNFα, and IL1β release by M2 macrophages. However, DEHP and DBP significantly increased IL1β release in M1 and M2 macrophages, respectively. Some of the effects described above were shown to be mediated by either ERα or ERβ and were simultaneous to modulation of NF‐κB, AP1, JNK, or ERK signaling pathways. We provide new evidence of the effect of XE on macrophage behavior and their mechanisms with relevance to the understanding of the action of environmental chemicals on the immune system and inflammation‐associated diseases.

Inflammatory and Cardiometabolic Risk on Obesity: Role of Environmental Xenoestrogens

CONTEXT Some chemicals used in consumer products or manufacturing (eg, plastics, pesticides) have estrogenic activities; these xenoestrogens (XEs) may affect immune responses and have recently emerged as a new risk factors for obesity and cardiovascular disease. However, the extent and impact on health of chronic exposure of the general population to XEs are still unknown. OBJECTIVE The objective of the study was to investigate the levels of XEs in plasma and adipose tissue (AT) depots in a sample of pre- and postmenopausal obese women undergoing bariatric surgery and their cardiometabolic impact in an obese state. DESIGN AND PARTICIPANTS We evaluated XE levels in plasma and visceral and subcutaneous AT samples of Portuguese obese (body mass index ≥ 35 kg/m(2)) women undergoing bariatric surgery. Association with metabolic parameters and 10-year cardiovascular disease risk was assessed, according to menopausal status (73 pre- and 48 postmenopausal). Levels of XEs were determined by gas chromatography with electron-capture detection. Anthropometric and biochemical data were collected prior to surgery. Adipocyte size was determined on tissue sections obtained during surgery. RESULTS Our data show that XEs are pervasive in this obese population. Distribution of individual and concentration of total XEs differed between plasma, visceral AT, and subcutaneous AT, and the pattern of accumulation was different between pre- and postmenopausal women. Significant associations between XE levels and metabolic and inflammatory parameters were found. In premenopausal women, XEs in plasma seem to be a predictor of 10-year cardiovascular disease risk. CONCLUSIONS Our findings point toward a different distribution of XE between plasma and AT in pre- and postmenopausal women, and reveal the association between XEs on the development of metabolic abnormalities in obese premenopausal women.