Journal of Occupational & Environmental Medicine:
Cognitive Disorders and Tau-Protein Expression Among Retired Aluminum Smelting Workers
Lu, Xiaoting PhD; Liang, Ruifeng PhD; Jia, Zhijian; Wang, Hao; Pan, Baolong; Zhang, Qinli PhD; Niu, Qiao MD, PhD
From the Department of Occupational Health, Shanxi Medical University, Taiyuan, China.
Address correspondence to: Qiao Niu, PhD, Department of Occupational Health, Shanxi Medical University, 56 Xin Jian Nan Lu, Taiyuan 030001, China (firstname.lastname@example.org).
This study was supported by grants 81001241 and 3091010300 from the Natural Science Foundation of China.
The authors declare no conflicts of interest.
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Objectives: To analyze cognitive functions and tau-protein expression in peripheral blood lymphocytes of retired aluminum (Al)-exposed workers.
Methods: A total of 66 retired Al potroom workers and 70 unexposed controls were investigated. The cognitive functions were assessed with the Mini-Mental State Examination. The tau-protein expression in peripheral blood lymphocyte was analyzed with Western blot.
Results: The cognitive functions of the exposed group were significantly decreased. Twelve mild cognitive impairment cases in the exposed group and four mild cognitive impairment cases in the control group were diagnosed. Significantly higher p-tau181 and p-tau231 levels were detected in the Al-exposed workers than in the control group.
Conclusions: The study suggests that long-term exposure to Al may cause cognitive disorders and that p-tau181 and p-tau231 might be useful indicators for monitoring cognitive decline in Al-exposed workers.
Aluminum (Al) has been clearly shown to be neurotoxic in experimental animals and human beings and is believed to be a neurotoxic agent with deleterious effects on cognitive processes. Excessive Al exposure may occur in the workplace. Epidemiological studies have shown poor performance on cognitive tests and a higher abundance of neurological symptoms in workers occupationally exposed to Al.1 Longstreth et al2 studied three patients with a progressive neurological disorder who had worked for more than 12 years in the same potroom of an Al smelting plant. Two of the three patients exhibited cognitive deficits. The finding of mildly impaired cognitive function was reported in a study of workers who inhaled Al dust.3 A report on 25 potroom workers from an Al smelting plant found that 21 (84%) reported memory loss.4 A study from Canada reported cognitive and other neurological deficits among workers occupationally exposed to dust containing a high content of Al.5 Neurotoxic effects of Al among foundry workers were studied by Polizzi et al,6 and early neurotoxic effects were detected at a preclinical stage of Alzheimer disease (AD). Buchta et al7 reported the neurotoxicity of long-term occupational exposure to Al-containing welding fumes in terms of delay in overall reaction time of the exposed workers. Fifty Al welders underwent a neuropsychological test, which indicated that Al exposure leads to cognitive changes.8 A study on workers exposed to Al welding fumes revealed disturbances of cognitive processes and memory.9 The authors conducted and summarized a meta-analysis of the data on the effects of occupational Al exposure on cognitive and motor performance and found that cognitive performance was negatively related to U–Al.10 Substantial evidence supports the hypothesis that occupational exposure to Al has an adverse effect on cognitive function. The major feature of mild cognitive impairment (MCI) is a decline in cognitive ability. Mild cognitive impairment is a syndrome characterized by cognitive decline that is not sufficient to meet the criteria for a specific dementia.11 Although occupational Al exposure induces cognitive changes, it can cause, accelerate, or aggravate MCI. The mechanism and the relationship between MCI and Al exposure are poorly understood.
Patients with MCI are at high risk for AD, with conversion rates of 15% to 53% over a 3-year period.12 Early identification of MCI and taking sound measures to prevent MCI from progressing to dementia are important. Phosphorylated tau (p-tau) is a reliable diagnostic biomarker of MCI and a prognostic biomarker for progression of MCI.13 Tau is largely an axonal protein that normally functions as a microtubule-associated protein, presumably by stabilizing the microtubules that serve as tracks for cytoplasmic transport. Hyperphosphorylated tau is the major component of the neurofibrillary tangles (NFTs) of AD.14 A hallmark feature of AD pathology is the presence of NFTs. Neurofibrillary tangles mainly consist of clusters of intracellular aggregates of conformationally abnormal and hyperphosphorylated tau protein. The presence of NFTs is associated with impairment of cognitive function, supporting a damaging effect of p-tau on the central nervous system.15 Phosphorylated tau may be a useful indicator in the evaluation of cognitive function deterioration by predicting cognitive decline.16
Aluminum has been suggested to play a role in the hyperphosphorylation of tau. Previous studies have shown that Al alters the phosphorylation state of tau and causes aggregation of tau in experimental animals and cultured neurons.17,18 We explored whether Al leads to cognitive disorders through hyperphosphorylated tau. In this study, we assessed cognitive deficits by using the Mini-Mental State Examination (MMSE), determined the expression of total tau (t-tau) and p-tau, and explored the association between cognitive function and the expression of the tau protein in a population of retired Al potroom workers.
All the study participants were retirees from the same region in Taiyuan, China. From the participants, the subjects were selected according to their previous occupational history. The study group of exposed workers was composed of 66 retired potroom workers with long-term exposure to Al in an Al smelting plant, and the control group was composed of 70 demographically similar subjects retired from a flour mill and all had no previous Al exposure. The two groups were matched for age, economic status, educational level, lifestyle, and health. All the participants gave their written informed consent, and the study was approved by the ethics and human subject committees of Shanxi Medical University.
Investigation With Questionnaire
We designed the study questionnaire, which was delivered to all the subjects by a professional investigator through face-to-face interviews to gather general information, including age, educational level, smoking and drinking habits, personal occupational history, and individual and family medical histories. Smoking was defined as currently smoking no less than 10 cigarettes per day over the last year, and drinking was defined as currently drinking wine, beer, or spirits no less than three times a week for the last 6 months.
The subjects were selected on the basis of the occupational history obtained from the questionnaire. The exclusion criteria were medication with drugs containing Al (such as antiacids) or drugs affecting the central nervous system, renal failure, past head trauma, and psychiatric, somatic, or neurological disorders. In addition, the subjects involved in this study spoke Chinese, exhibited normal eyesight and hearing, and were thought to be capable of undergoing standard psychometric testing.
Determination of Serum Al Concentration
Two 2-mL samples of venous blood were obtained from each subject in a fasting state in the morning of the day on which the clinical and cognitive function tests were performed. One blood sample was used to separate to serum, and the other was used to extract to peripheral blood lymphocytes (PBLs). The serum was separated from the 2-mL sample of venous blood on the same day. An aliquot of 0.4 mL was removed, mixed with nitric acid (1% volume per volume, 1.6 mL), and analyzed by inductively coupled plasma mass spectrometry (7500A; Agilent Technologies, Santa Clara, CA).19 The instrument was calibrated after every 10th sample, using the Al Standard Solution (Analytical Instruments, Beijing, China). Specific efforts were made to avoid contamination in all steps of the procedure.
Measurement of Cognitive Function and Screening of MCI
All the subjects were evaluated by standardized assessment procedures of the MMSE, which is a widely used tool for measuring cognitive function and screening MCI cases.20,21 The MMSE, introduced by Folstein and colleagues22 in 1975, has become a standard tool for cognitive assessment in clinical settings. The MMSE questionnaire includes orientation in time and place, immediate memory, short-term memory, calculation ability, and attention and language skills. It is scored from 0 to 30. In accordance with the utilization instructions of the MMSE, the investigators were strictly trained and the test was performed with precision.23
We screened patients with MCI defined according to the following criteria: (1) memory complaints, (2) normative activities of daily living, (3) exclusion of dementia, and (4) mild quantitative impairment of cognitive function measured by the MMSE testing, with cutoff points for MMSE scores adjusted for age and educational levels. The cutoff points were set at one standard deviation less than the mean for the control group matched with age and educational level.24,25
Determination of T-Tau and P-Tau Level
The expression of t-tau and p-tau in human PBLs was analyzed with Western blot analysis, which includes t-tau (tau5), p-tau396, p-tau262, p-tau231, and p-tau181. Peripheral blood lymphocytes were removed from the 2-mL sample of venous blood and isolated by the Ficoll–Hypaque density gradient centrifugation.26
The isolated PBLs were lysed (5 × 106 cells/100 μL) for 20 minutes at 4°C, using a commercial buffer supplemented with protease and phosphatase inhibitors, and centrifuged at 16,000g for 10 minutes. The supernatant was collected as the soluble fraction, and the protein content was assayed using the bicinchoninic acid protein assay (CoWin Biotech Co, Beijing, China). The protein samples were stored at −80°C until subsequent Western blot analysis.
The protein samples were resolved on a loading buffer (0.125 M Tri/HCl, pH 6.8, 4.6% sodium dodecyl sulfate, 20% glycerin, 10% β-mercaptoethanol, and 0.1% bromophenol blue) and denatured at 95°C for 10 minutes. The protein sample of 20 to 30 μg was loaded in each well and separated in 10% sodium dodecyl sulfate–polyacrylamide eletrophoresis gels. After running, the proteins were transferred onto nitrocellulose membranes, which were saturated and blocked with 5% fat-free milk at 37°C for 1 hour, and incubated with the first antibody at 4°C overnight. The following first antibodies were used: t-tau (tau 5; Santa Cruz Biotechnology, Dallas, TX), p-tau396 (Invitrogen, Carlsbad, CA), p-tau262 (Invitrogen), p-tau231 (Abcom, Cambridge, UK), and p-tau181 (Abcom). Anti–glyseraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (CoWin Biotech Co) was used for loading control. After extensive washing, mouse anti-tau5, rabbit anti–p-tau396, rabbit anti–p-tau262, rabbit anti–p-tau231, rabbit anti–p-tau181, and mouse anti-GAPDH secondary antibodies (CoWin Biotech Co) were added, and the membranes were incubated for 45 minutes followed by extensive washes (1 to 2 hours). The specific antibody–antigen complexes were detected using the enhanced chemiluminescence Western blot detection kit (CoWin Biotech Co). Graphs of the blots were obtained in the linear range of detection and quantified for the level of specific induction by scanning laser densitometry. All the experiments were independently performed three times, and the average was used for analysis. All the protein samples were normalized to the GAPDH levels and the results (mean ± standard error of the mean) are expressed as the protein to GAPDH ratio used to evaluate the relative expression of all the proteins between groups.
All the data were input into a personal computer with Epidata 3.1 software (program design: Jens M. Lauritsen and Michael Bruus, Odense, Denmark) and processed with SPSS 13.0 for windows (SPSS, Inc, Chicago, IL). The t test was used to analyze the differences between the serum Al concentration, cognitive function, and tau-protein expression between the exposed group and the control group, as well as the MCI patients and non-MCI subjects. The chi-squared test was used to analyze on significant difference in MCI between the Al-exposed group and the control group. All the results are expressed as mean ± standard error of the mean. The difference was considered significant at P < 0.05 (two-sided).
Demographic Information on the Subjects
There were no significant differences between the two groups regarding age, education level, and occupational history, as well as years of work other than Al exposure and smoking and drinking habits. The average age of the workers in the exposed group was 62 years, and that of the workers in the control group was 61 years. These results are shown in Table 1.
Serum Al Concentration
The mean concentration of serum Al in the Al-exposed workers (25.18 ± 2.65 μg/L) was significantly higher than that of the controls (9.97 ± 2.83 μg/L) (P < 0.01).
Cognitive Function and Rate of MCI in the Two Groups
The total score of the MMSE test of the Al-exposed group (26.13 ± 2.57) is significantly lower than that of the controls (27.89 ± 1.91) (P < 0.01). Figure 1 shows that the scores for orientation in time and place, short-term memory, and calculation ability significantly decreased in the exposed group compared with the control group (P < 0.01). The actual scores of the MMSE test obtained by both groups were listed in Table 2. There were 12 MCI cases (18.2%) in the exposed group and 4 MCI cases (5.7%) in the control group, and the rate of MCI in the Al-exposed group was significantly higher than that of the control group (P < 0.05).
T-Tau and P-Tau Levels
Compared with the non-MCI group, the expressions of tau5, p-tau181, p-tau231, and p-tau396 were significantly increased in the MCI cases (P < 0.05). Significantly higher expressions of p-tau231 and p-tau181 were found in the Al-exposed workers than in the controls (see Fig. 2).
This study aimed to investigate cognitive impairment and the expression of tau protein following Al exposure in retired Al potroom workers. This population has rarely been studied. Our subjects were tested for cognitive function and serum Al.
The retired potroom workers showed a mean level of serum Al that was almost three times as high as that of the control population. It is not easy to evaluate this difference because values of Al concentration in air samples may vary, and even a mean value calculated across hours of exposure is less reliable than biological monitoring. Urinary Al is predominantly related to current exposure, whereas serum Al may relate more to prolonged exposure.27 The report of Polizzi et al6 showed that the serum Al concentration of the occupationally exposed group that had been separated from their former work for approximately 10 years was two times higher than that of the control group. The serum Al concentration could be monitored as the body burden index after separating from occupational Al exposure.
Exposure to Al may induce long-term alterations of visual memory, working memory, and attention/concentration, as previously reported in hemodialyzed patients,13 inert gas metal welders,9 and foundry workers.6 Neurotoxicity of high levels of Al is well documented in these types of workers.28 A cohort of 3777 elderly subjects followed up for 8 years supports the hypothesis that Al concentrations in drinking water may have an effect on cognitive decline.29 From a 15-year follow-up of the cohort, the authors found that cognitive decline with time was greater in subjects with a higher daily intake of Al from drinking water or a higher geographic exposure to Al.30
We demonstrated the adverse effect of Al exposure on cognitive function. Time and place orientation, short-term memory, and calculation ability were significantly decreased in occupationally Al-exposed workers. We found that the MCI prevalence rate of the Al-exposed group was 18.2%, higher than that of the control group. The general prevalence in China is 3% to 8%.31 Our results are consistent with the study of Polizzi et al6 and other studies that showed the damaging effect of Al on cognition in the preclinical period in occupationally Al-exposed workers.
Aluminum neurotoxicity can be induced by many mechanisms,32,33 because it promotes the aggregation of the hyperphoshorylated tau protein. Crapper et al32 and Klatzo et al33 described neurofibrillary degeneration in humans and experimental animals associated with higher Al brain concentrations, suggesting a possible role in the etiology of AD. Aluminum promotes the formation and accumulation of hyperphosphorylated tau, thereby playing a major role in NFT formation. Neurofibrillary tangles are aggregations within the neuronal cytoplasm of the microtubule protein tau, which is aberrantly hyperphosphorylated. Neurofibrillary pathology is highly correlated with cognitive deterioration. Phosphorylated tau may be a useful indicator in the evaluation of cognitive function deterioration by predicting cognitive decline, and p-tau181 and p-tau231 were particularly highly sensitive in predicting cognitive decline.16,34 Bramblett and colleagues35 demonstrate that phosphorylation of Ser396 may destabilize microtubules in AD, resulting in the degeneration of the affected cells. Ser262 has a major effect on binding to microtubules.36 Mandelkow and colleagues14 found that Ser262 is uniquely phosphorylated in the brains of AD patients, and phosphorylation at Ser262 could explain the reason that AD tau fails to stabilize microtubules. The growing need for reliable and manageable disease-specific in vivo markers has prompted an extensive search for reliable biomarkers that may correlate with the expression and progression of relative diseases in peripheral tissues such as PBLs. Kvetnoy and colleagues37 identified tau-protein in the PBL of AD patients. The clear difference in its expression between healthy and sick people allows consideration to tau-protein as the most promising marker, and blood lymphocytes as a suitable extra brain sample, for the lifetime diagnosis of AD.37,38 We tried to detect tau protein and p-tau in human blood lymphocytes. Our data identified that p-tau181 and p-tau231 expressions in PBL were significantly higher in Al-exposed workers than those in the controls. Elevated expressions of t-tau and p-tau are hypothesized to reflect neuronal damage. This study results support this hypothesis by showing elevated p-tau181 and p-tau231 expressions in the lymphocytes of Al-exposed workers.
Long-term exposure to Al can cause cognitive disorders and may be a risk factor for MCI. Both p-tau181 and p-tau231 in PBLs seem to be useful prognostic biomarkers for monitoring cognitive decline in Al-exposed workers.
We thank the medical staff of the Physical Examination Center of the Chinese People's Liberation Army 264th Hospital for assistance with the blood samples. We also thank the graduates and postgraduates of Shanxi Medical University for their selfless contribution to this study. More importantly, we sincerely thank the workers who consented to participate in this study.
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