Among all contaminants present in different aquatic ecosystems in Canada, methylmercury (MeHg) is a major source of concern for public health. Currently, it is difficult to reliably determine the threshold of MeHg concentration at which functional changes occur. On the other hand, it is well known that chronic MeHg exposure is very harmful for the nervous system. Oxidative reactions appear to be of central importance to mercury toxicity. Therefore, it is important and urgent to determine with precision the minimal dose at which oxidative stress and neurotoxic effects can be identified since some studies suggest that MeHg toxicity can be detected at level far below the minimal exposure level proposed by the World Health Organization. The main goal of this project is to investigate the effects of mercury on sensorimotor functions in the population of Salluit. We will examine the relationship between the level of MeHg and sensorimotor performance. Afterwards, specific recommendations based on quantitative evidence will be made to the concerned populations so as to diminish long-term risk on health.
Upon signature of the consent forms, participants agreed to provide urine samples, venous blood, hair and alveolar air samples. Hair and blood samples were used to assess short term and long term exposure to mercury. Selenium was measured in plasma, whole blood, hair and urine samples.
Mercury and Selenium Exposure Assessment Blood samples will be used to assess short term and long term exposure to mercury, respectively. Hair/blood concentration ratios will be calculated. Selenium will be measured in plasma and in whole blood, because selenium accumulates in red cells when the intake increases, whereas saturation is observed in the plasma. Long term exposure to selenium will be assessed by measuring this element in hair samples. Three segments of one cm each representing the last three months of exposure, will be collected and analyzed. Again hair/blood ratios will be calculated for selenium. Speciation of selenium in urine will also allow us to determine in which chemical form selenium is excreted in Inuit receiving large doses of selenium from food. Selenium in urine, blood and plasma will be analyzed by graphite furnace atomic absorption after sample dilution or after nitric acid digestion (with hydrogen peroxide). Hair selenium analyses will be performed by ICP-MS after nitric acid digestion. Blood mercury analysis will be carried out by cold vapor atomic absorption. Urinary selenium speciation analyses will be performed by HPLC-ICP/MS, while the sample is diluted and injected in the system (TMSe+, Se + 4, Se +6 and amino acids will be analyzed). Oxidative Markers Biochemical assessment of oxidative stress markers will include three novel indices. Firstly, the ratio of coenzyme Q10-reduced form (ubiquinol-10) to oxidized coenzyme Q10 (ubiquinone-10) in plasma, which is now considered as one of the most reliable and sensitive indices of an oxidative stress in vivo (Yamashita, 1997; Lagendijk, 1996; Finckh, 1995). In contrast to the total level of coenzyme Q10, which is reported to be associated with multiple factors including gender, age, cholesterol and triglycerides levels (Kaikkonen, 1999), the ubiquinol-10/ubiquinone-10 ratio index is apparently independent of these variables and thus represents an oxidative stress index of choice. Secondly, increased level of specific F2-isoprostanes (direct oxidation metabolites of arachidonic acid) in plasma and/or urine is another index recently used to demonstrate oxidative stress in several pathological conditions involving oxygen free-radical formation (Pratico, 1999; Patrono, 1997). The most easily measurable and frequently used F2-isoprostane species as marker of oxidative stress in vivo is 8-isoprostaglandin F2-alpha (Pratico, 1999, Patrono, 1997). We shall thus also measure the levels of 8-iso-PGF2 in the plasma samples. Finally, the level of plasmatic LDL oxidation will also be assessed as a third potential marker of oxidative stress. Sensorimotor Measures: Sensorimotor tests will include measures of tremor using high-sensitivity sensors, postural sway measured by a force plate, reaction time tests, eye-hand coordination in a pointing task, rapid alternating movements, saccadic eye movements and nystagmus measured with infrared sensors as well as rhythmic hand movements. In the past ten years, we have developed tests of sensorimotor functions that optimize precision, reliability and validity. We have also developed analysis methods that optimize the specificity and sensitivity of these tests. This research field is in full expansion and in a few years these technologies should help develop the next standards in neurotoxicology assessment. Postural tremor: Static and kinetic tremor from both hands will be recorded during 30 s using laser systems. These tests have been validated and shown to be highly sensitive in populations with neurological disorders. The most informative analysis parameters of static tremor are: amplitude, amplitude fluctuations, amplitude difference, skewness, peakedness, one dimensional entropy, asymmetry, asymmetric decay of the autocorrelation function, time asymmetry, first maximum of the autocorrelation function and centre of mass harmonicity. Analysis parameters of kinetic tremor are peakedness, center of mass harmonicity, median frequency, power in the 3-4 Hz, 4-6 Hz and 7-12 Hz intervals. This test lasts about fifteen minutes. Postural sway: Postural oscillations will be recorded while the subject is standing on a platform (force plate). Forces in the x and y directions will be recorded while the subject looks at a fixed target placed two meters in front of him or her. A standardization of this test is under way. Analysis parameters include: mean sway (in mm), transverse sway (in mm) sagittal sway (in mm), sway area (in mm2) and sway index. This test is part of a battery commercialized by DPD Inc. and lasts about five minutes. Reaction time measures: Simple reaction time will be measured in both hands as part of the DPD battery. Reaction time to sequences of three finger movements (1-3-2) will also be measured to evaluate fronto-striatal voluntary programming functions. This test yields mean and variance measures and lasts six minutes. Eye-hand coordination: This test quantifies aiming movements performed between two target locations on a plane. Subjects are seated in front of a board and will have to execute multiple aiming movements with each hand as precisely and rapidly as possible. Informative analysis parameters of aiming movements include speed, precision of the trajectory, hesitations, tremor, transit and contact duration, and Fitts’ constant. These parameters are operationally described in a validation study. This test lasts about five minutes. A new version of this test which can record the exact trajectory of all portions of the movement will also be validated and will replace the standard aiming test in later studies (see below). Pointing movements will also be examined in unpracticed conditions to test attentional motor control. This will be done by measuring trajectories of two-dimensional pointing movements performed with a stylus on a graphics tablet. The visual feedback of the stylus seen on a computer screen will either be direct (baseline condition) or mirror-inverted (unpracticed condition). This test has been validated in populations with frontal and striatal damage and it lasts about ten minutes. Rapid alternating movements: Simultaneous alternating pronation-supination movements of the two forearms will also be recorded with a validated system involving the rotation of two soft spheres connected to optical encoders via flexible rods. Informative parameters include: duration, amplitude, speed, maximal slope, the uniformity of shape and amplitude of oscillations, the coefficient of variation, fluidity, sharpness, symmetry, coherence, and tremor. This test lasts about five minutes. Eye movements: Optokinetic nystagmus will be measured by having the subjects fixate a screen on which vertical colored bands will pass from left to right. Measures include the slope of the slow and rapid phases, the number of saccades and reaction time. Voluntary saccades to peripheral targets will also be measured and trajectory will be sampled at 1000 Hz to record target acquisition hesitations and tremors. Horizontal and vertical voluntary saccades will be recorded to single targets as well as sequences of three targets. These measures will be obtained using a computerized system based on infrared technology (Ober 2). These tests will last about twenty minutes. Rhythmic movements: The capacity to follow temporal constraints will be examined using rhythmic arm movements to a metronome at different speeds. Three types of movements will be examined: taps of the index finger, pronation-supination of the forearm and sequences of three arbitrary hands postures (fist-palm-side). Measurement parameters include maximum frequency achieved, movement lag relative to the metronome, variance and precision. These tests last about fifteen minutes. The total duration of the test protocol is about 90 min. These tests have previously been used in the Cree population of James Bay exposed to mercury (Beuter, 1998, 1999a;b,c) in workers exposed to manganese (Beuter, 1994a; Beuter, 1999d) and on a variety of patient populations with neurological disorders (Beuter, 1994b, 1999e; Lepage, Décary, 1995; 1996; Edwards, 1997; Edwards, 1998; Lambert, 1999; Richer, 1999; Richer, in press).
Laboratory analysis 1. Analytical chemistry Selenium in urine, blood and plasma will be analyzed by graphite furnace atomic absorption after sample dilution or after nitric acid digestion (with hydrogen peroxyde). Hair selenium analyses will be performed by ICP-MS after nitric acid digestion. Blood mercury analysis will be carried out by cold vapor atomic absorption. Urinary selenium speciation analyses will be performed by HPLC-ICP/MS, while the sample is diluted and injected in the system (TMSe+, Se + 4, Se +6 and amino-acids will be analyzed). 2. Antioxidant and oxidative stress markers Simultaneous assessment of ubiquinol-10, ubiquinone-10, carotenoids, and tocopherols in human plasma will be performed by high-performance liquid chromatography-coulometric electrochemical detection, according to Finckh (1995, 1999). Plasmatic levels of 8-iso-PGF2 will be measured by a reliable and sensitive enzyme immunoassay commercially available (Cayman Chemical), as described by Hoffman (1996). Oxidized LDLs in plasma will be determined by an immunoassay using a monoclonal antibody against LDLox (Mono 12E7, from Biodesign, International and custom kits from Pharmingen).
1- Cognitive Neuroscience Laboratory, Université du Québec à Montréal; 2- Research Center – CHUQ and Laval University; 3- Public Health Center – CHUQ and Laval University ; 4- Québec Toxicology Center – CHUQ
This project was submitted and funded by the Toxic Substances Research Initiative in 1999 (TSRI). The goal of the study was to determine the biological exposure to mercury and selenium in two exposed populations of fish eaters having different patterns of exposure (seasonal vs stable, high vs low selenium intake). Study populations are 1) 800 Hydro-Quebec workers located in LG2, LG3 and LG4 sectors of the hydroelectric complex in James Bay; and 2) 150 residents of Salluit who voluntarily accepted to participate in a reassessment of mercury exposure to estimate the temporal trends between 1978 and 1999.