Development of a Cumulative Exposure Index (CEI) for Manganese and Comparison with Bone Manganese and Other Biomarkers of Manganese Exposure.

Manganese (Mn) exposure can result in parkinsonism. However, understanding of manganese neurotoxicity has been limited by the lack of a cumulative Mn biomarker. Therefore, the current goal was to develop Mn cumulative exposure indices (MnCEI), an established method to estimate cumulative exposure, and determine associations of MnCEI with blood Mn (BMn), fingernail Mn (FMn), and bone Mn (BnMn). We completed a cross-sectional study of 60 male Chinese workers. Self-reported occupational history was used to create two MnCEIs reflecting the previous 16 years (MnCEI16) and total work history (MnCEITOT). An in vivo neutron activation analysis system was used to quantify BnMn. BMn and FMn were measured using ICP-MS. Mean (standard deviation) MnCEITOT and MnCEI16 were 37.5 (22.0) and 25.0 (11.3), respectively. Median (interquartile range) BMn, FMn, and BnMn were 14.1 (4.0) &mu;g/L, 13.5 (58.5) &mu;g/g, and 2.6 (7.2) &mu;g/g dry bone, respectively. MnCEI16 was significantly correlated with FMn (Spearman&rsquo;s &rho; = 0.44; p = 0.02), BnMn (&rho; = 0.44; p < 0.01), and MnCEITOT (&rho; = 0.44; p < 0.01). In adjusted regression models, MnCEI16 was significantly associated with BnMn (&beta; = 0.03; 95% confidence interval = 0.001, 0.05); no other biomarkers were associated with MnCEI. This suggests BnMn may be a useful biomarker of the previous 16 years of Mn exposure, but larger studies are recommended.

1. Introduction

Manganese (Mn) is a widely-used metal in modern industries, including mining, welding, smelting, ore processing, ferroalloy steel production, dry-cell battery manufacturing, and pesticide manufacturing. Occupational overexposure to Mn can result in postural tremor [1], decreased visuomotor coordination [2], impaired memory [3,4], deficits in information processing and working memory [3], olfactory impairment [5], and decreased motor function [3,6]. Severe Mn intoxication results in manganism with the symptoms similar, but not identical to, idiopathic Parkinson’s disease [7,8].

Cumulative exposure indices (CEIs) have been widely used to estimate long-term exposure to metals, including manganese, particularly in occupational settings. Prior studies on occupational manganese exposure have used CEIs based on a combination of work history questionnaire data (job titles, years of employment) [4,5,9,10,11,12], which can be supplemented with Mn air sampling [4,5,9,10,11,12]. Advantages of CEIs are that they can represent flexible time periods, may incorporate exposures from multiple routes of exposure, and can be applied in an individual basis; a disadvantage is that they are often reliant on self-reported work histories, which can be affected by recall bias.

Existing biomarkers for Mn include blood, urine, hair, and toenails. Blood and urine Mn concentrations have been shown to reflect exposure over the past several days [6,13,14,15,16]. Blood Mn (BMn) was found to be significantly elevated in a group of Italian ferroalloy workers [17] and in a cross-sectional study of 96 Russian welders [18] when compared to workers without known Mn exposure. In a study of San Francisco Bay bridge welders, active welders had significantly higher BMn concentrations than those who were no longer welding [5]. In both a study of 14 Chinese male welders and 9 smelters [19], as well as in a study of gas transmission pipeline welders [20], significantly elevated urine Mn was seen in occupationally exposed workers compared to controls.

Mn levels in toenails and hair have also been suggested as potential biomarkers of Mn exposure [14,21,22]. Two recent studies report that toenail Mn is reflective of external Mn exposure in the prior 7–12 months among welders [14,23]. Hair Mn can also be reflective of several months of Mn exposure [21,22]. In a group of 47 welding school students, sampled from a longitudinal cohort study, hair Mn was associated with external air Mn concentrations [22]. In a cross-sectional study of Mn-exposed dry cell battery workers, participants with high Mn exposure had significantly higher hair Mn concentrations when compared to both low Mn-exposed and non-exposed workers [24]. However, the uses of both hair and nail Mn are limited due to a large variation among individuals [24] and the potential for external contamination.

Despite these positive correlations with Mn exposure, existing Mn biomarkers have several limitations, including a short half-life and high variability. BMn, for example, is only reflective of short-term Mn exposure due to the short half-life and high individual variability of Mn in the blood (~2 h–40 days) [16,25]. In the same study of dry cell battery workers conducted by Bader et al., urine Mn was unable to distinguish between Mn-exposed workers and controls [24]. Urine Mn also has inconsistency in studies that assess the association between the urinary Mn biomarker and external air Mn concentrations [14,26] since only 1% of absorbed Mn is found in urine [27].

Bone Mn (BnMn) has been suggested as a viable biomarker for cumulative Mn exposure because of Mn’s extensive accumulation in human bone (40% of the total Mn body burden) [28] and for its relatively long half-life (~8–9 years) in bone [29]. BnMn can be quantified using in vivo neutron activation analysis (IVNAA) technology, a concept originally developed and later applied by researchers from McMaster University for the assessment of Mn in the hand bones of Mn-exposed workers [30]. Researchers from this team utilized an accelerator-based IVNAA system in a feasibility study to assess BnMn in a group of 29 welders and 10 controls and found that BnMn among welders was significantly higher than that of controls [31]. In the same study, these researchers showed BnMn was significantly associated with a cumulative exposure index of participants’ lifetime occupational Mn exposure. However, despite the conceptual feasibility of using IVNAA for BnMn assessment, the large equipment is immovable, which limits the application of this novel technique.

Over the past eight years, our research team has developed a compact neutron generator-based transportable IVNAA system that can be used to assess Mn in hand bones [32,33]. Our team has previously reported on BnMn in a pilot study [34] and differences in BnMn and BMn by exposure group within a cross-sectional study of Chinese workers [35]. However, these analyses lacked a rigorous measurement of cumulative Mn exposure for each individual in the study; this assessment is needed to firmly establish whether BnMn is useful as a cumulative measure of exposure. Therefore, in this analysis, we develop two cumulative exposure indices for manganese exposure and compare these with BMn, fingernail Mn (FMn), and BnMn measurements. Our hypothesis was that a cumulative exposure index of Mn would be correlated with BnMn to a greater degree than FMn or BMn.

2. Materials and Methods

2.1. Study Population

Sixty-one male workers from Zunyi, China were recruited for this cross-sectional study. Zunyi is colloquially referred to as “the manganese capital” of China and is home to numerous Mn-related industries. Thirty participants were recruited from an equipment manufacturing and installation factory (“manufacturing”), with limited use of Mn, and thirty-one participants were recruited from a ferroalloy factory, where manganese is extensively used. Individuals were excluded if they had any active neurological or psychiatric diseases, any known motor or cognitive impairment that was not directly related into Mn exposure, or if they did not complete the BnMn measurement. One ferroalloy worker was excluded from the study due to a lack of BnMn data, leaving 60 study participants for analyses. Five (N = 5) participants did not provide a sufficient quantity of fingernail sample for analyses but did complete BnMn measurements. Therefore, the population was reduced for analyses involving FMn (N = 55).

Participants were brought to Zunyi Medical College on one weekday to complete all of the study protocols; participants did not work on this day. Participants completed a questionnaire, provided blood and fingernail samples, and completed a bone Mn scan. Study protocols and questionnaire responses were translated into Mandarin then back-translated to English to check the accuracy of the data prior to use. Age and years of education attained were collected from participants’ completed questionnaires. The Purdue Biomedical Institutional Review Board and Zunyi Medical College Ethical Review Board approved this study. Participants signed an informed consent document prior to their participation.

2.2. Cumulative Exposure Index

Work history and demographic information were collected using a questionnaire. Participants reported their job title, employer, and dates of employment for current and past jobs. These data were used to create two cumulative occupational Mn exposure indices (MnCEI) using methods adapted from prior literature [36,37]. One index included the entirety of each participant’s work history (MnCEITOT). The second incorporated jobs held after 1 January 2000 (MnCEI16); this was an average of 16.1 (SD: 0.2) years prior to the study date. Prior research suggests BnMn has a half-life of 8–9 years [38]; thus the MnCEI16 was designed to reflect approximately two half-lives for BnMn.

To calculate the MnCEI, past and current jobs were grouped into similar job types, using 20 occupational groups defined by the United States Bureau of Labor Statistics [39]. Prior studies with data on air Mn concentrations for different occupations [13,40,41,42,43,44] were used to inform placement of each job into a Mn exposure rank (high/medium/low). Details of these classifications are presented in Table A1. Both the MnCEITOT and MnCEI16 were calculated as MnCEI = ∑ rank where n = job, rank = exposure rank (high = 3, medium = 2, low =1), and Y = years employed in the job. The difference in calculation of the two CEIs is that for MnCEITOT, all reported jobs were included and for MnCEI16, only jobs held after 1 January 2000 were included.

Table A1

The classification of job titles into exposure ranks used in cumulative exposure index (CEI) calculation.

Job Title 1	Occupational Category 2	Air Mn 3	Exposure Rank
Welder/arc welder	Metal ore processing	Welders: 0.01 to 2.13 mg/m3 [40]; Ferroalloy workers: 0.098 to 0.374 mg/m3 [13]	3: high (direct exposure)
Smelter
Ore combustion
Ore extraction
Manganese ore processing
Underground Mn ore mining
Manganese electrolysis
Director	Management (ferroalloy)	Ferroalloy workers (indirect exposure) = 0.01–0.11 mg/m3 [13]; Mining = 0.21 mg/m3 [41]; Die-casting foundry = up to 0.14 mg/m3 [43]	2: medium (indirect exposure)
Manager/Project manager
Ore production manager
Equipment management
Driver	Transportation and material moving (Ferroalloy)
Driver: ore transporting
Trainman
Metal product assembler	Assemblers and fabricators
Maintenance	Installation maintenance and repair (Ferroalloy)
Equipment repair
Machine repair
Cleaner	Building grounds and cleaning (Ferroalloy)
Machine operator	Non-ferrous metal production and processing
Miner	Mining
Die-casting hardware	Molding
Refinery	Metal-refining furnace operators and tenders
Lime processing	Lime product manufacturing
Factory/general worker	Other occupations (ferroalloy) 4
Wireman
Advertisement	Advertisement and marketing	Auto repair: 0.00045 mg/m3 [38]; Administrative Support: 0.001 mg/m3 [44]	1: low (low/no exposure)
Marketing
Driver	Transportation and material moving (manufacturing)
Stevedore
Secretary	Administrative support
Security guard/personnel	Protective services
Machine repair	Installation, maintenance, and repair (manufacturing)
Weeding	Building and grounds cleaning (manufacturing)
Designer	Industrial designers
Car repair	Automotive repair and maintenance
Cement factory worker	Construction
Construction worker
Road construction/roadman
Brick carrier
Manager	Management (manufacturing)
Vegetable storage manager	Other occupations (manufacturing/other employer) 4
General/other worker
Private business
Wireman

1 As reported by participants; 2 Occupational category encompassing groups of jobs with similar responsibilities, these are based on US Department of Labor definitions [39] and current factory; 3 Previously reported air Mn concentrations for jobs in a specific occupational category; 4 Job titles did not match a U.S. Department of Labor category.

2.3. Biological Samples and Determination of Mn Biomarkers

Trained clinical staff collected whole blood samples from participants during their study visit using standard protocols. Trace metal-free vacutainers (Becton-Dickinson, Franklin Lakes, NJ, USA) were used to limit external metal contamination. Samples were frozen at −20 °C and stored until analysis. Whole blood samples were analyzed for Mn at the Chinese Centers for Disease Control and Prevention in Beijing using inductively-coupled mass spectrometry (ICP-MS); these methods have been described previously [45,46]. Briefly, blood samples were diluted with 0.01% Triton-X-00 (Sigma-Aldrich, Saint Louis, MO, USA) and 0.5% ultrapure concentrated nitric acid (Merck, Darmstadt, Germany). Samples were vortexed and then analyzed using XSERIES 2 ICP-MS (Thermo Fisher, Waltham, MA, USA). The SeronormTM Trace Elements Whole Blood Control Level 1 (Sero AS, Billingstad, Norway) was used for internal quality insurance. None of the collected samples had a Mn concentration below the detection limit (DL) of 0.11 μg/L.

Although toenail Mn is more commonly used than fingernail Mn, FMn has been used in previous studies as a biomarker of exposure [47]. Fingernails were used in this study due to a concern that toenails would have a greater potential for external contamination because participants were observed to wear open-toed shoes to the factory. Participants were asked to thoroughly wash their hands with soap to remove external debris. Fingernail samples from participants’ 10 fingers were collected using a titanium dioxide nail clipper. Fingernail samples were stored in small Ziploc bags and kept at room temperature until analysis. Samples were cleaned twice using ultrasonic cleaning procedures in 1% Triton X-100 solution (Sigma-Aldrich Inc., Saint Louis, MO, USA) [48]. Following each cleaning, the nails were rinsed multiple times with deionized (DI) water and then dried at 60 °C. After the second round of cleaning, the fingernail samples were digested at 200 °C in ultrapure nitric acid (Sigma-Aldrich Inc., Saint Louis, MO, USA), then analyzed for Mn using the ELEMENT-2 mass spectrometer (ThermoFinnigan, Bremen, Germany) at Purdue University’s Campus-Wide Mass Spectrometry Center. Mn concentrations were corrected for systematic error using an internal standard run simultaneously with the samples. The DL for fingernail Mn ranged from 1.31 to 3.97 ppb. Seventeen (28.3%) FMn measurements were below the DL but still had detectable concentrations which were larger than blank samples. Replacement of values