1. Introduction
Plastic pollution is one of the leading environmental problems facing our society, which is currently living at a new turning point in the history of humanity, called the “Anthropocene” or the “Plastic Age” [
1]. Plastic polymers have become the basis of numerous industries thanks to their versatility and affordability; therefore, their demand and productivity have maintained constant growth in recent decades. In 2019, a production volume of 368 million tons [
2] was recorded, which is expected to double in just 20 years [
3]. Consequently, the storage of urban waste and the continuous production and recycling of plastic polymers pose a potentially severe impact at numerous trophic levels [
4], ultimately leading to chronic human exposure. Recent studies have confirmed plastic monomers’ presence in water, soil, and air worldwide [
5,
6,
7].
One of the primary monomers studied by the scientific community, due to its environmental ubiquity and high level of detection in human biological fluids, is bisphenol A (BPA). Due to its high production volume and multiplicity of uses, BPA is considered one of the most widely used monomers in the world [
8]. Thanks to its chemical properties [
9], it can move throughout the human body and cross biological barriers, so it is commonly found in urine, blood, breast milk, and even amniotic fluid [
7,
10,
11]. Furthermore, it is a known endocrine disruptor with the potential ability to affect numerous organs and systems, including the cardiovascular system [
12,
13,
14]. The growing amount of evidence provided by the scientific community on the multiple and heterogeneous potential effects of BPA on health has made it essential to introduce new rules and regulations restricting the use of BPA.
Faced with the imperative need to replace BPA, industries have adopted structurally similar monomers, which still need to be studied in detail. The two most used molecules in the replacement process are bisphenol S (BPS) and bisphenol F (BPF) [
15,
16], whose presence has already been detected in water, air, and food in several parts of the world [
5,
17,
18]. Both monomers have—like BPA—two phenols, and they differ only in their interphenolic linkers (C(CH3)2 for BPA; CH2 for BPF; SO2 for BPS) [
19]. Due to their respective high degrees of structural homology, similarities have been observed in their potential endocrine disruptor abilities, which have even been described as hormonally active as BPA itself [
15]. Furthermore, evidence in the academic literature suggests that BPS and BPF could exert a similar effect to BPA on adipocytes [
20] and the GC-2 spermatocyte cell line [
21]. In GC-2 cells, Sidorkiewicz et al. [
21] manifested that the mixture of bisphenols causes effects comparable to the individual ones.
At the cardiovascular level, few works have explored the possible implications of the new BPA substitute molecules on human health. A limited amount of evidence has been developed in some cellular lines [
22,
23], animal models [
24,
25,
26,
27,
28], and human cohorts [
29,
30,
31]; in all of them, the perspective of the study contemplates only the individual effect of each one of the phenolic monomers. Nevertheless, it is evident that in the real world, outside the regulated laboratory setting, co-exposure to bisphenols does occur, as demonstrated in human cohorts that quantify the presence of a mixture of bisphenols in urine [
32].
Consequently, the present manuscript pretends, for the first time, to study the possible cardiovascular implications of the bisphenols used mainly by the industry, considering their possible additive or synergistic effect through a retrospective cohort study of the mixture of urinary bisphenols in the NHANES cohort. After performing statistical analyses to identify links between joint exposure to these monomers and cardiovascular diseases, the effect of the mixture of bisphenols on endothelial cells will be studied using the realistic proportion of bisphenols found in the urine of subjects in the NHANES cohort.
The present work proposes two central hypotheses: first, actual exposure to the bisphenol mixture (the concentration present in the urine of the general population) is statistically related to heart disease; second, the bisphenol mixture may exert a deleterious effect on the cardiovascular endothelium, which would help to explain the cardiovascular pathological relationship with this class of endocrine disruptors. Our study will help redefine the way of studying BPA and its emerging substitute molecules and propose an update of the work methodology in investigating endocrine disruptors.
3. Discussion
For the first time, the present manuscript has demonstrated significant associations between the bisphenol mixture and heart disease. Our initial hypothesis, based on [
15,
20,
21], suggests that BPS and BPF could exert an additive (or synergistic) effect on BPA in the context of heart disease. Since the three phenolic molecules have a high degree of structural homology and hormonal activity (BPF and BPS have been described as hormonally active as BPA itself [
15]), it is coherent to consider BP
mix as a single quantitative variable. Thus, the statistical analysis performed on the NHANES cohort (a
retrospective cohort study) has yielded sufficient evidence to justify the subsequent in vitro experimental model.
Descriptive statistics and comparative analysis of the 3014 subjects in the cohort have shown the first indications of the relationship between BP
mix and heart disease. In addition, it has confirmed significant differences in the covariates included in the study of heart disease (gender, age, BMI, ethnicity, and poverty/income ratio. The analysis of the BP
mix quartiles also showed interesting statistical relationships with all the study covariates except BMI. The serum cotinine levels suggested that tobacco use is a significant source of environmental exposure to bisphenols. This effect is especially significant since bisphenol enters the bloodstream directly, avoiding the classic hepatic detoxification mechanisms (glucuronidation and sulfation) traditionally related to the oral route [
9].
The subsequent binomial and multinomial logistic regression models confirmed the trends described in the previous analyses, determining that bisphenol mixture is an environmental factor related to the risk of developing heart disease, independent of other factors such as age, gender, BMI, ethnicity, poverty/income ratio, and serum cotinine. Statistical evidence does not shed light on the possible causal relationship between the variables; it only allows for determining a relative risk or odds ratio [
37]. The greatest weakness of retrospective cross-sectional studies is their inefficiency in determining causal relationships. However, the high and heterogeneous number of individuals allows for the development of a robust base for future translational studies. Of the total number of individuals in the 2013–2014 and 2015–2016 NHANES cohorts, 3014 subjects were included in the study because they had all the necessary data for the correct development of the statistical study. Furthermore, WQS, quantile g-computation, and BKMR analysis showed interesting results that reaffirm the positive association between the mixture of bisphenols and heart disease risk.
Consequently, the retrospective cohort study allowed us to delve deeper into the issue by developing an in vitro experimental model on the primary culture of murine aortic endothelial cells. The most exciting element of the present manuscript is the use of realistic proportions of a bisphenol mixture mimicking human exposure. For the first time in the academic literature, an endocrine disruptor study was contextualized in the real world to extrapolate the results with new potential human risks.
The subsequent
in vitro experimental model demonstrated that exposure to a mixture of bisphenols in a proportion analogous to the average of a human cohort induces an effect different from BPA alone. Previous work from our laboratory linked murine endothelial cell exposure to BPA with necroptosis, a mechanism of programmed necrosis cell death [
13]. However, the evidence presented in this article demonstrates that the mixture of bisphenols induces apoptosis. Beginning with the MTT viability cell assay, the individual comparative study showed that BPS and BPF have a lower ability to affect cells than BPA. This phenomenon is consistent with the work of Prudencio et al. [
38], who observed that BPS and BPF are less disruptive to cardiac electrophysiology. In other cellular contexts, Castellini et al. [
39] work demonstrated that BPS and BPF are safe alternatives for sperm biology. Russo et al. [
40] also observed this pattern and partially attributed it to phospholipid affinity, implying that toxicity would increase with increasing membrane affinity. However, there are also certain contradictions in the literature, such as in the work of Ji et al. [
23]. They observed that BPF could induce higher vascular toxicity and oxidative stress than BPA and BPS.
The results of MTT at higher concentrations showed significant differences between BPS and BPF, showing that BPS is the compound with the least capacity to modulate cell viability in murine endothelial cells. In human hepatocarcinoma cells and peripheral blood mononuclear cells, it has been observed that BPS is the phenol that generates less genotoxicity [
41,
42]. However, there are studies with BPS in animal models that have related it to atherosclerotic cardiovascular disease [
43] and cardiotoxicity [
44] in zebrafish, and altered cardiac function in mice [
27] and rats [
26].
When studying the combined effect, the results show a more significant effect than individual BPA. It is essential to highlight that in the comparative study, we used a fixed concentration of BPA and the corresponding proportionality determined in the NHANES cohort. For example, the concentration of 100 µM of BPA was compared with a mixture that includes 100 µM of BPA + 38 µM of BPF + 36 µM of BPS. In this way, it is easy to determine if the bisphenols added to the mixture act as agonists or antagonists. In this case, the results show a potentiating effect of the mixture of urinary bisphenols compared to the individual effect. This fact defined the cellular experimental model, using doses of 1 to 100 µM of BPA and its equivalent proportions of BPS and BPF.
Cell adhesion could not explain the alterations in the viability assays since no changes induced by any BP
mix concentrations were observed. However, these preliminary studies focused on short-term exposure to the mixture of bisphenols (24 h) and we cannot exclude the possibility that chronic exposure to BP
mix, the same type of exposure that occurs in the human population, may alter cell adhesion [
45]. Indeed, previous work in our laboratory determined that BPA can substantially affect the cell adhesion capacity (as well as the structural integrity) of the human podocyte [
33].
The flow cytometry assay showed that the mixture of bisphenols induces an increase in cell apoptosis that was not observed in the quadrant associated with necroptosis, showing an essential difference with exposure to BPA alone. In the cardiovascular context, previous work realized by our team determined that BPA exposure could promote cell death by necroptosis through CAMKII [
13]. Nomura et al. [
46] observed that increased concentrations of cytoplasmic calcium promote necroptosis through the activation of CAMKII, which is coherent with evidence that relates BPA exposure with alterations in intracellular calcium [
47], ionic channels [
48,
49], or even intestinal absorption ability [
50]. However, the evidence in the present manuscript strongly suggests a differential effect promoted by the realistic mixture of bisphenols.
It has been described that CASP8 can inactivate the TNFRSF1A complex activity by cleaving necroptotic protein RIP1 and, consequently, favor apoptosis through the activation of CAS3 or CAS7 [
36]. The Western blot and immunofluorescence analysis showed an increased expression of both CAS8 and CAS3, confirming the significant increase in cell apoptosis observed by flow cytometry. Furthermore, the relative expression of crucial proteins implicated in necroptosis, RIP3, and MLKL, was analyzed by Western blot. As expected, no changes were observed in the assays, confirming that BP
mix could promote cell death through different cellular mechanisms better than BPA alone.
In previous studies, we found that inflammation and oxidative stress in BPA induced cardiac effects and as a consequence of endothelial necroptosis [
14]. We have not tested the inflammatory potential of BP
mix. Consequently, further work on future cellular and animal study models will allow us to demonstrate the molecular mechanisms underlying the new pathological evidence related to the BPA mixture.
The results described in the present work are novel due to the focus on combining bisphenols. In the literature, it is usual to find works developed in the context of a single molecule; however, the approach developed in the NHANES cohort, with the combined “real” exposure, coupled with the in vitro model proportional to the translational model, has provided completely novel evidence. Thus, despite the reduced effect on cell viability observed with BPS and BPF, individually, their combination with BPA in the human mean proportionality produces a potentiating effect. Furthermore, the observed change in the type of cell death associated with BPA or BPmix opens a new avenue for studying endocrine disruptors.
The clinical applicability of the findings is fundamentally related to the type of materials commonly used in the clinical context. Due to the physical properties of plastic polymers, they are commonly used in the clinical environment. Our results demonstrate the need to replace polymers derived from bisphenols or to limit their use as enhancers in the plastics industry.
The results of the present work fundamentally represent the breaking of two paradigms: firstly, it proposes the need to modify the methodological approach related to the study of endocrine disruptors individually; and secondly, it implies an impact on the current system of plastics manufacturing. Companies are beginning to replace BPA with monomers such as BPS or BPF; however, the evidence points to the need to apply the precautionary principle. Today, many products are marked as “BPA-free”. It is commonly found in most plastic products related to breastfeeding and newborns. However, the new substitute molecules likely do not provide a health benefit, as they could pose a risk comparable to that of BPA itself. The heterogeneity of uses of bisphenols is a problem for the consumer since they are found in most food packaging in varying proportions, as well as in everyday objects and even sanitary equipment. As a result, it is impossible to avoid contact with them. However, consumers must know they can replace plastic packaging and containers with glass. Consumers must not heat plastic containers due to the increased release of endocrine disruptors by heat. For example, using glass bottles in the baby’s early life stages could significantly reduce exposure to bisphenols.