Abstract
Background:
Bromine is a naturally occurring element that is widely present in the human environment in various chemical forms primarily as flame retardants, pesticides, and water treatments.
Objective:
In this exploratory study, we investigated the association of brain bromine concentrations on Alzheimer’s disease (AD) neuropathology, cerebral infarcts, and Lewy bodies.
Methods:
The study was conducted in 215 deceased participants of the Memory and Aging Project, a clinical-pathologic cohort study. Brain bromine levels were measured using instrumental neutron activation analysis. Multiple brain regions were assessed for diffuse and neuritic plaques, neurofibrillary tangles, cerebral macro-and microinfarcts, and Lewy bodies. Standardized measures of AD pathology (Braak, CERAD, NIA-Reagan, global AD pathology) were computed.
Results:
In linear regression models, the higher brain bromine levels were associated with more AD neuropathology (Braak (p trend = 0.01); CERAD (p trend = 0.02); NIA-Reagan (p trend = 0.02).
Conclusion:
Bromine accumulation in the brain is associated with higher level of AD neuropathology. The potential deleterious effects of this element on AD need further exploration.
INTRODUCTION
Bromine is a naturally occurring element in the environment. Human exposure to bromine may occur through various manufacturing compounds and chemicals used as flame retardants (in carpets, furniture, cushions, home insulation, textiles, electronics, construction materials, etc.), pesticides, or for water treatments. Bromine flame-retardants are considered one of the major contributors of bromine exposure and various in vivo and in vitro experiments over the years reported the neurotoxic effects of these compounds on the brain. Animal studies report detrimental effects of prenatal or neonatal exposure to bromine flame retardants among rats [1–3]. In human studies, children exposed prenatally to bromine as assessed in cord blood or in maternal and child serum had decreased levels of attention, fine motor coordination, and cognitive performance [4–6]. Only two studies examined brain levels of bromine in humans and found the concentrations to be higher in brains of Alzheimer’s disease (AD) cases than in non-demented controls. However, it is unknown if brain deposition of bromine over a lifespan is associated with AD neuropathology or cerebral infarcts in the brain. In this exploratory analysis, we examined the associations of brain bromine levels with brain neuropathologies of dementia in 215 deceased participants of a community cohort study.
MATERIAL AND METHODS
Study population
Brain cases are of deceased participants of the Rush Memory and Aging Project (MAP), an ongoing clinical-neuropathological cohort study of older adults residing in retirement communities and subsidized housing in Chicagoland. At the time of enrollment, participants were without known dementia and agreed to annual clinical neurological assessments and brain donation at death. The sample includes participants who died between 2004 and 2013, and had completed at least one dietary assessment in the years before death. All study participants gave informed consent, and the institutional review board of Rush University approved the study.
Brain neuropathology
Methods for brain autopsy and pathological evaluations were described in detail previously [7]. Briefly, brain metal analyses were performed on tissue collected from cerebral hemispheres stored at –80°C. Analyses of brain neuropathologies were conducted on tissue samples from different brain regions of the contralateral hemisphere that were fixed in 4% paraformaldehyde and then dissected and embedded in paraffin blocks, cut into 6-micron sections, and mounted onto slides. AD neuropathologies, including diffuse and neuritic amyloid plaques, and neurofibrillary tangles were identified using modified Bielschowsky silver-staining in multiple cortical regions. The raw scores (greatest number in 1 mm2 area) were standardized in each region and then averaged across the regions to create three summary scores which were then averaged to obtain the global measure of AD pathology. We also analyzed semi-quantitative measures of AD pathology including, CERAD (Consortium to Establish a Registry for Alzheimer Disease) neuritic plaques [8], Braak neurofibrillary tangle staging [9], and National Institute on Aging (NIA) - Reagan level of Alzheimer’s Disease brain neuropathology [10]. The CERAD score ranged from 1 to 4 (no neuritic plaques to frequent neuritic plaques). Braak stage scoring ranged from 0 to 6 (few neurofibrillary tangles in few regions to frequent neurofibrillary tangles spread across multiple regions). NIA-Reagan scores ranged from 1, low AD pathology to 4, high AD pathology.
Cerebral infarction counts were identified through gross inspection (presence of one or more chronic macroscopic infarcts was considered as infarcts present) and microscopically (minimum of nine regions in one hemisphere were examined for microinfarcts on 6 μm paraffin-embedded sections stained with hematoxylin/ eosin, presence of micro infarcts in any region was considered “present”) as previously described [11]. Lewy body staging was examined in six brain regions through staining with antibodies to α-synuclein [7, 12]. Absence of any Lewy body staining in different regions was considered as “not present”; otherwise it was marked as “present”.
Bromine analyses
Brain bromine concentrations were measured in 100 g sections from two cortical regions affected by AD (inferior temporal and midfrontal) and the cerebellum, a region largely unaffected by AD pathology. A ceramide blade was used to cut the brain tissue to avoid metal contamination. Brain bromine analyses were conducted at the University of Missouri Research Reactor using standard comparator instrumental neutron activation analysis (INAA). Samples were weighed into quartz tubes and irradiated for 15 min using a pneumatic tube irradiation system, followed by decaying for 2.5 days and counting for 2 h using a high purity germanium detector. Au was used as a standard comparator. The limit of detection for Br in the samples was 0.06 μg g–1. For the quality control, the bromine concentration in 10 samples of the human hair certified reference material (NCS DC 73347) was found to be 0.40±0.03 and the accepted value is 0.36 μg g–1. Details of this analysis and its validation have been reported earlier [13].
Other covariates
Age at death is obtained by subtracting date of birth from date of death and dividing the difference by days per year (365.25). Sex and education (in years) was self reported at the time of enrollment into the study. Apolipoprotein (APOE) genotyping was performed by Polymorphic DNA Technologies [14]. Brain samples were simultaneously assessed by instrumental neutron activation analysis for concentrations of other metals including mercury, selenium, iron, zinc, manganese, potassium, chromium, and scandium. The clinical dementia diagnosis was done annually in the MAP cohort using structured neurological examinations, computerized cognitive tests, clinical judgement by neuropsychologist and diagnostic classification by clinician as per the standardized criteria [15], as previously described [16].
Statistical methods
We used linear regression models to investigate the association of brain bromine levels with global AD pathology, CERAD score, Braak stage, and NIA-Reagan AD diagnostic score. Logistic regression models were used to examine associations of brain bromine levels with Lewy bodies and cerebral macro- and micro-infarcts which, due to their highly skewed distributions, were modeled as present or absent. We considered both linear and non-linear associations of brain bromine levels by modeling the variable in tertiles and then also as a linear trend variable where all the participants in the tertile were coded at the median level and modeled as a categorical variable. Analyses were conducted using SAS 9.3. Models were adjusted for age at death, sex, education, and APOE ɛ4. To test for statistical interactions by age (>90 years versus younger), sex, and education (≤15 versus >15 years), we added to the adjusted models a multiplicative term between brain bromine level and the potential effect modifier. The interaction was not tested for APOE ɛ4 due to low numbers of subjects with APOE ɛ4 positive (n = 47). For all tests, the level of significance was set at p≤0.05. We analyzed brain bromine concentrations averaged over the two cortical regions and for all three regions. As the findings were similar, we present only the results with bromine concentrations averaged over all three brain regions.
RESULTS
The analytical sample was on average 89.3 years at the time of death, primarily female (67%), with a mean educational level of 14.4 years, and 22% APOE ɛ4 positive. These characteristics were comparable to the total number (n = 554) of MAP deceased participants (age at death = 89.7 years, 68% women, and 14.4 years of education). The mean postmortem time interval was 7.12 h (±4.0). Based on the last study visit assessment, overall 34% participants (n = 73) had the diagnosis of clinical dementia at the time of death. The baseline characteristics did not differ by tertile of brain bromine concentration (Table 1). However, the lowest tertile of brain bromine levels had fewer cases of clinical dementia compared to the second and third tertile (21% in T1 versus 41% in T2 and T3, Table 1). As per NIA-Reagan diagnosis criteria for AD, percent cases in the highest tertile was more than in lowest tertile (Table 1). Region-specific mean brain bromine levels of 1.79 μg/g (±1.56) for inferior temporal, 2.07 μg/g (±1.81) for midfrontal, and 2.05 μg/g (±1.70) for cerebellum were significantly correlated (r = 0.60 to 0.70, p < 0.001). The mean bromine levels for the average of all the three regions of brain was 1.97 μg/g (±1.48). The Spearman correlation between brain bromine levels and other metals (assessed from this analytical sample for the same three regions) indicated brain bromine levels had a weak positive association with manganese (r = 0.20) and chromium (r = 0.25) as well as a negative association with potassium (r = –0.23). These results are presented in Supplementary Table 1.
Characteristics of deceased MAP participants by tertile of brain bromine concentrations (n = 215)
IQR, Intra-quartile range. *NIA-Reagan diagnosis of Alzheimer’s Disease.
Brain bromine levels and brain neuropathology
In the linear regression models adjusted for age at death, sex, education, and APOE ɛ4, higher brain bromine concentrations were significantly associated with brain AD neuropathology (Table 2). Those in the highest tertile of brain bromine levels when compared to those in the lowest tertile, were marginally associated with more Global AD pathology (p = 0.06), and significantly associated with higher CERAD score, Braak Stage as well as NIA-Reagan score (Table 2). Logistic regression models indicated no significant associations of brain bromine levels with Lewy bodies, macroinfarcts, or microinfarcts (Table 3). We further investigated if the brain bromine levels also relate with clinical diagnosis of dementia in these participants. Higher bromine levels were found to be associated with clinical dementia as well (T2 versus T1: OR = 2.1 (95% CI: 1.00–4.5); T3 versus T1: OR = 2.4 (95% CI: 1.14–5.2); p trend = 0.047). The test for statistical interaction by age at death, sex or education on the brain bromine levels and neuropathology did not reach statistical significance (all p > 0.14).
Estimated effects (β) and 95% confidence intervals of brain bromine levels on Alzheimer’s disease (AD) neuropathology in a cross-sectional analysis among 215 deceased brains*
*Linear regression models adjusted for age at death, sex, education, and APOE ɛ4.
Estimated effects of brain bromine levels on Lewy bodies, cerebral macro- and micro-Infarcts in a cross-sectional analysis among 215 autopsied brains participants of the Memory and Aging Project*
*Logistic models adjusted for age at death, sex, education, and APOE ɛ4.
DISCUSSION
To our knowledge, this is the first study to report on the relations of brain bromine concentrations to brain neuropathologies associated with dementia. Higher bromine concentrations were associated with greater burden of AD neuropathology.
The findings are supported by two small case-control studies that found greater bromine concentrations in AD brains compared with controls without dementia [17, 18]. Of interest, bromine levels for the MAP cohort sample of 1.97 μg/g (±1.48) were greater than in these previous reports using the same technique (0.40 to 0.87 μg/g in one study [17] and 0.33 to 1.31 μg/g in another [18]). Given these previous studies were much smaller (n < 20 samples), with wide age range (38–92 years) and more than two decades old, the higher brain bromine values in the MAP cohort suggest accumulation with older age and possibly higher environmental exposure than in years past.
In vitro and in vivo experiments of bromine compounds, widely used as flame-retardants, have linked the compounds to increased oxidative stress and neuronal apoptosis [19] as well as decreased cell viability and neuronal growth [20]. Some animal studies also indicate that neonatal exposure to bromine compounds impair motor function [21] and reduce learning and memory in adulthood [2, 22]. Human studies found that both prenatal and childhood bromine exposure measured in cord blood or serum were associated with lower scores on attention, fine motor coordination, cognition, and overall adverse neurodevelopment [4–6].
Out of various possible bromine exposures for humans, flame-retardants are quite well studied. Poly brominated dimethyl ethers (PBDE) were once widely used in flame retardants, but due to concerns of their effects on health, production decreased worldwide in recent years [23]. PBDE are organic lipophilic compounds that have the propensity to migrate into the external environment, and as such are ubiquitous and persistent environmental contaminants [24]. Studies have reported that some foods, including meat, fish, and dairy, continue to be high in these compounds [25–27]. Further, data from the National Health and Nutrition Examination Survey (NHANES) showed higher serum PBDE concentrations in older (>60 years) than younger (25–59 years) adults suggesting increased accumulation with age [28]. Another recent report on NHANES samples for one decade (2005-06 through 2013-14) found decreased plasma concentrations of various forms of PBDE except, 2,2’,4,4’,5,5’-hexabromobiphenyl (BB153) which was found to increase by 8.5% in older adults (≥60 years) over time [29]. However, we do not have any evidence if the environmental exposure or plasma levels of these flame-retardants may directly affect brain bromine levels in humans. Although the PBDE is banned, there are some novel polymeric flame-retardants containing bromine that are used widely. Any ultraviolet irradiation and heat exposure to these retardants results in leaching out of brominated degeneration products [30]. Thus, the flame-retardants that are used currently may still be causing the environmental exposure of bromine.
A primary limitation of the study is the cross-sectional analysis which prevents causal interpretation of the findings. In addition, the study has no data on the source of environmental exposure to bromine. Major strengths include the large number of brain cases analyzed from participants of a well-defined community cohort, assessment of multiple brain regions for bromine concentrations, and a number of brain pathologies examined using standardized measures.
In this exploratory cross-sectional analysis, we found that higher brain bromine concentrations are associated with more burden of AD neuropathology. The potential for deleterious effects of bromine exposure on brain health is intriguing and reasonable, and deserves further investigation.
