Carga obesogénica total efectiva: nuevo biomarcador de efecto combinado a obesógenos como factor de riesgo en obesidad

Abstract

The prevalence of overweight and obesity, as well as other metabolic diseases such as metabolic syndrome (MetS), is increasing worldwide. This continuous increase cannot be due to a single cause, but everything suggests that multiple risk factors are involved. The environmental obesogens hypothesis suggests that human exposure to "obesogenic" EDCs may inappropriately interfere with lipid homeostasis and metabolism and promote adipogenesis, modifying both hormonal and neuronal signalling pathways, thereby predisposing exposed individuals to increased fat mass and excess weight. EDCs that not only increase adipose tissue mass but also result in other metabolic dysfunctions which are also referred to as metabolism disrupting chemicals (MDCs). Environmental obesogens are defined as chemicals capable of increasing the quantity of adipocytes (hyperplasia), promoting the fat storage in cells (hypertrophy), affecting preadipocyte differentiation or function, and initiating and/or deregulating basal metabolism and weight gain. Obesogens might also act indirectly by disrupting energy balance (promoting calorie storage), and altering appetite and satiety signals, besides the central mechanisms that keep the body's response to daily nutritional changes. The list of chemicals with obesogenic potential has been growing steadily in recent years, with humans being exposed to this multitude of EDCs from a variety of sources. To date, current developments in environmental epidemiology and toxicological risk assessment are inadequate and unsatisfactory for evaluating the exposure to multiple obesogenic EDCs, and for knowing their impact on human development and health. Although there have been some attempts to consider the combined effect of several chemicals or compounds which belong to the same chemical family, the methods available so far to quantify the combined effect of exposure to multiple residues are still scarce and not fully validated and conclusive. Taking into account all of the above, the starting hypothesis of this International Doctoral Thesis was that exposure to mixtures of EDCs constitutes a risk factor for obesity, and that the development of methodologies based on the measurement of the combined effect of multiple obesogenic EDCs known to occur in human adipose tissue, through in vitro assays, would serve as biomarker of combined effect in metabolic diseases. Therefore, the main objective of this International Doctoral Thesis was to investigate human exposure to specific endocrine disrupting chemicals (EDCs), specifically to those with obesogenic activities, by analysing both the presence of these contaminants in the human population and the individual and combined effect of residues (EDCs) present in human adipose tissue, through the development of in vitro biomarkers, which could be used as risk markers for obesity and other metabolic diseases. The specific objectives defined for this purpose were the following: 1. To develop and validate an in vitro human mesenchymal stem cell model to assess the effect of obesogenic EDCs. This tool could serve as a way to compare the potency of different obesogens, as well as to identify new compounds with obesogenic activity. 2. To study ad hoc mixtures of environmental chemicals on the process of adipogenesis, investigating the possibility of synergistic, additive or even antagonistic effects of the individual components. 3. To deepen into the knowledge on mechanisms and signaling pathways through which obesogen-EDCs contribute to an obesogenic phenotype (analyzing gene and protein expression of specific adipogenic markers - PPARγ, C/EBPα, LPL and FABP4). 4. To evaluate the exposure to obesogens in adipose tissue samples from adult hospitalized patients in Southern Spain, with normal weight and obesity and to investigate the relationship between the accumulated concentrations in adipose tissue and the risk of clinical diagnosis of MetS. 5. To evaluate the developed total load biomarker ("O-Screen") in adipose tissue samples from adult patients with normal weight and obesity, and to investigate its possible association with obesity and other metabolic disorders. To answer the first three objectives, hASCs (human adipose-derived stem cells) in vitro were exposed to different concentrations of bisphenol-F (BPF), bisphenol-S (BPS) and bisphenol-A (BPA) (0.01-25 µM), individually and in combination (equimolar mixture of the three bisphenols), during 7 and 14 days of adipocyte differentiation. The effect of exposure on lipid accumulation and adipogenesis (Oil Red O), in addition to gene (mRNA) and protein (WB, western blot) expression of the lipogenic markers PPARγ, C/EBPα, LPL and FABP4 were investigated. The epidemiological study investigated the real exposure of the population to persistent EDCs using the biological matrix for which they have the greatest appetite, that is, adipose tissue; in particular, visceral adipose tissue was selected, as it is the tissue associated with the greatest risk of metabolic diseases. The possible associations between pollutants and clinical diagnosis of MetS were also evaluated, using both exposure biomarker information and clinical effect biomarkers available for the study population. The main results of this doctoral thesis are as follows: Article #1 (Reina-Pérez et al., 2021): Exposure of hASCs to BPF and BPS promotes adipogenesis by altering adipocyte developmental programming and increasing intracellular lipid accumulation. In particular, cell exposure with BPF, at concentrations of 10 or 25 M for 7 or 14 days, potentiates stem cell differentiation into adipocytes in a dose-dependent manner, which is confirmed by verifying an increase in the expression of selected adipogenic genes (PPARγ, C/EBPα, LPL and FABP4), as well as their corresponding proteins. Our results show for the first time the obesogenic potential of BPF on human adipocyte differentiation, and it corroborates the previous data obtained in murine preadipocytes. Article #2 (Reina-Pérez et al., 2022): The equimolar ternary mixture of the three bisphenols (BPF, BPS and BPA), interferes with the programming of hASCs by increasing their ability to differentiate into adipocytes. However, the obesogenic effect of the mixture differed from that of the individual compounds by showing higher gene expression levels than those obtained for the individual components of the mixture. Exposure of the cells resulted in a dose-dependent effect on lipid accumulation, while gene expression showed a non-monotonic response, adopting a U-shape. This work is one of the few in vitro studies that have investigated the combined obesogenic activity of EDCs-obesogen mixtures on adipogenic differentiation, lipid accumulation, gene expression and protein synthesis, in an attempt to emulate real exposure situations in human population. Article #3 (Reina-Pérez et al., 2023): The Spanish adult population continues to be exposed to persistent environmental pollutants (persistent organic pollutants, POPs), with significantly higher levels of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in the group of patients studied with MetS compared to individuals without this syndrome. Positive associations were found between certain POPs and the clinical diagnosis of MetS or some of its components. Specifically, higher levels of HCB and γ-HCH were associated with higher fasting glucose levels, higher blood pressure, and increased risk of MetS, both in linear regression models and in logistic models adjusted for age and sex. Exposure to the mixture of the 7 POPs found in adipose tissue is also positively and significantly associated with MetS risk. The increasing prevalence of MetS in the world makes necessary the identification of preventable risk factors, besides stablishing intervention measures to stop and reverse the progression of this syndrome. Within this context, once the role of exposure to environmental-obesogenic pollutants in metabolic disease has been identified, more strictly regulations in their uses and applications should be implemented. The participation of clinical professionals in this area of research will be essential to deal with the environmental health challenges that we are facing and will face through precautionary measures.