Human exposure to hormone-active chemicals present in personal care products, and risk of onset and development of endometriosis in women of childbearing age

Resum

Introduction Endometriosis is a chronic gynecological disease that affects an increasing number of women worldwide. Despite the growing number of articles published in recent years, there are many gaps in knowledge about the etiology and pathophysiology of this disease. In addition, the difficulties in its diagnosis, mainly surgical, and in its treatment, focused on reducing the symptoms, further hinder its approach. Available knowledge suggests that it is a multifactorial disease, in which genetic, immunological, hormonal and environmental factors interact simultaneously. Along with the increase in the incidence of this disease, in recent decades there has been a parallel increase in the environmental presence of synthetic chemical substances with hormonal activity. Many of these chemicals, endocrine-disrupting chemicals (EDCs), are used in cosmetics and personal care products (PCPs), causing the population to be exposed on a daily basis. Since endometriosis is considered a hormone-dependent disease, it is suspected that human exposure to EDCs could be behind the increased risk of this disease. Objective The main objective of this doctoral thesis was to explore the association between exposure to EDCs, specifically to two families of these compounds, parabens-PB and benzophenones-BP, present in cosmetics and PCPs, and risk of onset and development of endometriosis in women of childbearing age. For this goal, the following specific objectives were proposed: Objective 1: To evaluate the concentrations of PBs and BPs in menstrual blood, a matrix in intimate contact with the endometrium, compare the concentrations of these contaminants in menstrual and peripheral blood, and explore related sociodemographic and lifestyle factors. Objective 2: To investigate the associations between exposure to PBs and BPs and risk of endometriosis, as well as with the frequency of use of cosmetics and PCPs, and to evaluate the influence of oxidative stress on the associations found. Objective 3: To describe the expression profile in endometriotic tissue of different genes related to key cell signaling pathways for the development and progression of endometriosis, and to explore their relationship with the concentrations of PBs and BPs in this tissue. Objective 4: To evaluate the biological activity (estrogenic and anti-androgenic) of cosmetic products and formulations of the company Inves Biofarm, and to analyze the presence of different endocrine disruptors. Material and Methods To answer objective 1, PB and BP concentrations were determined in menstrual and peripheral blood samples from healthy Spanish volunteers. Blood concentrations of PBs and BPs were determined by dispersive liquid-liquid microextraction (DLLME) and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLCMS/ MS). Sociodemographic and lifestyle information was obtained by using different questionnaires. To answer objectives 2 and 3, a case-control study was designed, collecting fasting urine samples from women of childbearing age with a confirmed diagnosis of endometriosis by surgery (cases), and from women without a diagnosis of endometriosis, who underwent laparotomy or laparoscopy in the same hospital as the cases, but with a diagnosis of non-malignant disease (controls). Endometriotic tissue samples were also collected from the cases. Urinary concentrations of PBs and BPs were determined by DLLME and UHPLC-MS/MS. Oxidative stress biomarkers were evaluated in all urine samples using different enzyme kits. The expression profiles of 36 genes involved in 9 cell signaling pathways related to endometriosis were determined in endometriotic tissue samples by real-time PCR. Sociodemographic, clinical and surgical information was obtained using different questionnaires. To answer objective 4, we evaluated the concentrations of PBs and BPs and analyzed the estrogenic and anti-androgenic activity of 6 cosmetic products, 1 cosmetic formulation and 2 plastic packages, provided by the private company Inves Biofarm. Results Article 1: All menstrual blood samples had detectable levels of ≥3 of the selected compounds, and 52.6% of the samples contained ≥6 compounds. MeP, PrP, and BP-3 were the most frequently detected compounds (detection frequencies >90.0%). Age, use of PCPs and consumption of some foods (meat, pasta, cheese or dairy products) were related to menstrual blood concentrations of some PBs/BPs. Concentrations of EDCs measured in menstrual and peripheral blood did not correlate, or correlated weakly, with concentrations in peripheral blood being higher. Article 2: The frequency of use of certain cosmetics and PCPs was significantly associated with urinary concentrations of PBs and BPs. After adjusting for possible confounders, the risk of endometriosis was higher when comparing women in the second versus first tertile of MeP (OR=5.63; p<0.001), BP-1 (OR= 5.12; p=0.011), BP-3 (OR=4.98; p=0.008), and ƩBPs (OR=3.34; p=0.032). No significant associations were found when comparing women in the third versus first tertile of exposure. Oxidative stress did not modify the associations found between exposure to PBs/BPs and risk of endometriosis. Article 3: Although inflammation could play a central role in the pathophysiology of endometriosis and is considered as a possible mechanism of action of EDCs, a systematic review of the scientific literature on human exposure to PBs and BPs and inflammatory biomarkers showed the paucity of studies and information in this area of knowledge and the need for further research to get a better understanding on the underlying mechanisms of action of PBs and BPs, and the key role that inflammation could play in endometriosis. Articles 4 and 5: More than half of the selected genes were found to be expressed in >75% of the endometriotic tissue samples analyzed. The concentration of PB and BP congeners analyzed was positively associated with the expression of genes related to key cellular pathways in the development of endometriosis, such as genes for cell adhesion, migration/invasion/metastasis, inflammation, angiogenesis, cell proliferationhormone stimulation, cell cycle, cell differentiation and lipid metabolism. No associations were found between exposure to PBs and BPs and apoptosis genes. Collaboration with the company Inves Biofarm: no detectable concentrations of PBs and BPs were found in any of the plastic packages tested, or in the cosmetic formulation provided. Detectable concentrations of MeP, EtP and PrP were only found in one sample, and BP-3 was detected in four of the received cosmetic products. The plastic packages showed estrogenic and anti-androgenic activity depending on the chosen extraction methodology. Only some cosmetic products and plastic packages extracted with chloroform and acetonitrile showed estrogenic and antiandrogenic activities. Conclusion Human exposure to hormone-active chemicals, present in some cosmetics and PCPs, could increase the risk of endometriosis in women of childbearing age. This exposure could also be related to the expression of genes involved in key cell signaling pathways for the development of this disease. It is therefore necessary to eliminate or restrict the use of PBs or BPs in the composition of cosmetics and PCPs, which could help to decrease human exposure to these EDCs and, therefore, reduce the risk of onset and development of endometriosis.