In the present study, we found an independent and positive association between urinary MBzP, the major metabolite of BBP, and cognitive impairment in elderly NHANES subjects, while the association was nonsignificant or negative-significant for other phthalate metabolites. More importantly, the results of two-stage PCA-WQSR combined association analysis revealed that LWM phthalates were linked to cognitive dysfunctions and that MBzP contributed most to this potential cognitive detrimental effect. Considering the relatively low levels of urinary MBzP among all phthalates, these findings collectively suggested the critical role of MBzP/BBP in the process of cognitive impairment. In addition, BBP decreased learning and memory functions by impairing hippocampal neurons and especially disrupting the neuroactive ligand − receptor interaction pathway in mice. To the best of our knowledge, we are the first to provide population and confirmatory experimental evidence that cognitive function exhibits susceptibility to BBP toxicity and that the disruption of hippocampal neurons and the inhibition of synaptic transmission may be the underlying mechanism for this.
Elderly individuals are a particularly vulnerable population among whom neurodegenerative diseases, including cognitive impairment largely develop (Hou et al., 2019). As a nonnegligible and modifiable risk factor for cognitive impairment, environmental pollution has raised dramatic public awareness and its implication for the prevention of neurodegenerative disorders has been extensively explored (Marras et al., 2019; Periñán et al., 2022). Nevertheless, evidence regarding the association between phthalate and cognitive impairment in elders is rather insufficient. In a study utilizing the same database as the present study – data from NHANES 2011–2014 cycles (Weng et al., 2022) – the authors found inconsistent and confusing results for different tests and methods: MECPP, MnBP, MCOP and MCPP were negatively linked to IR z scores, while MCPP and MBzP were negatively linked to DR z scores. Additionally, the main effect of phthalate mixture was remarkably altered between the WQSR and Bayesian kernel machine regression (BKMR) models. In contrast, our findings of single phthalate based logistic regression analysis and mixed phthalate based two-stage PCA-WQSR analysis collectively revealed a consistent and positive association of the same phthalate, MBzP, from LMW phthalates (PC3), with cognitive impairment in elderly individuals. The discrepancy between Weng’s work and ours might be that we included several variables, such as CVD status and MEHP, which were not considered in Weng’s study. In addition, we set a cutoff value for each test to distinguish individuals with potential cognitive impairment from healthy individuals and designed a two-stage PCA-WQSR analysis that divided the phthalate metabolites into different PCs and estimated the co-exposure correlation and contribution more robustly.
Our epidemical data showed that MBzP was the only metabolite that was positively associated with the four cognitive outcome impairments in single pollutant models, and the co-exposure pattern remained significantly positive in DSST deficit with LMW phthalates, in which MBzP was the predominant contributor. These findings were partly in accordance with a previous epidemiologic study respecting the impact of phthalates on children's cognitive functions, which reported an inverse association of MBzP with children’s full-scale IQ (Li et al., 2019). Moreover, as shown in Table S2, the concentrations of MBzP in the urinary samples were relatively low among all the phthalates, further implying the vulnerability of cognitive functions to MBzP toxicity. Apart from the increased cognitive deficit risk observed for MBzP, we also found a positive association of MEHP, a metabolite of DEHP, with IR impairment in Q4 vs. Q1 of a single phthalate analytic model. Nonetheless, the association became statistically nonsignificant in co-exposure of DEHP HWM phthalates (PC1) and WQSR models. Considering the biased estimation and inflated false caused by the collinearity of phthalates in single pollutant analysis (Pitard and Viel, 1997), the finding that MEHP was associated with cognitive impairment in elders should be interpreted with caution. However, as a typical representative of phthalates, DEHP was reported to disrupt spatial learning and memory abilities, impair blood‒brain barriers and decrease synaptic proteins in experimental animals (Ahmadpour et al., 2021; Sun et al., 2021). One possible explanation for this observation is that the concentrations of DEHP used in those experimental animals were much higher than the environmental DEHP exposure of humans in this study. In addition, we also observed negative associations of MEOHP and MNCP with AF impairment in Q2 vs. Q1 of the single pollutant model and non-DEHP HWM phthalates (PC2) with DSST impairment in Q4 vs. Q1, and with DR impairment in Q2 vs. Q1 of the PCA co-exposure model. These results should not be simply deduced as protective factors as the simple design of single pollutant model might cause potential bias, and moreover, although statistically nonsignificant, the ORs were > 1 in Q4 vs. Q1. Besides, the results of the PCA co-exposure model became nonsignificant in the WQSR validation analysis.
It is acknowledged that urinary phthalate metabolites can serve as proper internal exposure biomarkers and are suitable surrogates of parent prototypes (Reeves et al., 2021). Therefore, as mentioned above, BBP was selected as the potential cognitive-toxic candidate for the subsequent causality analysis in this study. As a nonnegligible member of the LWM phthalate family, BBP was considered a priority pollutant by the European Commission and US Environmental Protection Agency (Silano et al., 2019a; USEPA, 2014). The neurotoxicity of BBP has also gained public attention. For instance, a systematic review of evidence from birth cohorts reported that prenatal exposure to BBP was linked to decreased cognitive scores and behavior in children (Zhang et al., 2019). Moreover, given the lower water solubility of BBP than other phthalates (Xie et al., 2011), which is an important factor that is inversely associated with the neurotoxicity of phthalates (Screnci et al., 2000), it is theoretically plausible that BBP may serve as a critical neurotoxic phthalate. However, to date, regarding cognitive impairment, only one experimental study has been conducted to explore the effect of BBP on cognitive function in mice, and their findings should be interpreted with caution because the doses they applied were relatively high, which were 10 to 25 times higher than ours (Min et al., 2014). In this study, we found that BBP exposure resulted in pathological disruption of the hippocampus and a reduction in learning and memory abilities in mice at relatively low doses, further validating the essential role of BBP as a potential neurotoxic phthalate in the progression of cognitive impairment.
Hippocampal genes are considered “key modulators” in cognitive functions including, learning and memory (Li et al., 2022). To understand the potential mechanism behind the detrimental effect of BBP on cognition, which has rarely been explored previously, the expression profile of genes in the hippocampus was analyzed in control and 50 mg/kg/day BBP treated mice. Our findings of KEGG pathway analysis demonstrated that the downregulated DEGs were mainly related to the neuroactive ligand‒receptor interaction pathway, a group of membrane receptors involved in the identification of neuroactive ligands such as the classic neurotransmitters serotonin, acetylcholine and dopamine, which modulate learning and memory directly (Huang et al., 2016). Moreover GO annotation results further confirmed that the top enriched variational expression genes were related to the modulation of membrane and protein binding processes, and subsequently affected intercellular neural signal transduction, recognition and transport (Vit and Petrak, 2017). The decreasing trend of neuroactive receptors htr2c, ptger1 and chrna3 was also validated in our qPCR analysis, which were reported to identify serotonin, amplify dopamine receptors and modulate nicotinic acetylcholine, respectively (Hebras et al., 2020; Schuch et al., 2016; Tanaka et al., 2009). Thus, the disrupted functions of these DEGs may be the potential mechanism by which BBP induced cognitive decline in mice. In addition, as illustrated in Fig. 6C, most of the upregulated DEGs were enriched in cytokine-cytokine receptor interaction pathway, which regulates neuroinflammation through cytokines like TNF, IL11 and LIL12a (Ou et al., 2021). TNF signaling, by interacting with the corresponding specific receptors, modulates the strength of synaptic transmission and directly inhibits synaptic plasticity in the hippocampus (Liu et al., 2017). IL11 is a member of IL6-type cytokines family that is involved in the process of acute-phase response to infection or environmental injury, and it was found to be increased in the cerebrospinal fluid (CSF) of patients with cognitive decline (Galimberti et al., 2008). The excitement of IL12a was associated with proinflammatory reaction and contributed to reduced performance in the processing speed of elders (Lin et al., 2019). Therefore, in combination with cognitive and historical results, we deduced that BBP interfered with cognitive function by activating hippocampal inflammation and subsequently disrupting neuroactive receptors.
This research possesses several advantages. First, the epidemical data were representative of subjects recruited from the national population-based program NHANES. Second, the survey weighted logistic single pollutant model and two-stage PCA-WQSR mixed pollutant model were collectively designed to identify the potential candidate with cognitive toxicity, which estimates the joint association more robustly and evaluates the contribution more intuitively. Third, experimental were used to confirm the possible causality of BBP with cognitive impairment and to explore the potential mechanisms, which made it more reliable to interpret the results. Nonetheless, the present study also has several drawbacks. First, although several covariates were considered in both the single and joint association analyses, some undetected or unnoticed confounders might also exist. Second, this study was performed among American populations; therefore, the results may not be generalizable worldwide. Third, transcriptome analysis is a primary and exploratory method in the evaluation of potential mechanisms, and further studies of loss- or gain-of-function analysis are needed to validate the vital genes or pathways involved in BBP caused cognitive impairment.