Human Biomonitoring of Mercury Exposure

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1 Human Biomonitoring of Mercury Exposure The main objective of this workshop is to familiarize the participants with the choice and measurement of biomarkers for different chemical forms of mercury exposure. The workshop targets participants who are involved in exposure assessment, environmental monitoring, and public health. Mercury is a metal that has a long history of use due to its unique physical and chemical properties. As a result, mercury has resulted in many human poisonings. The main chemical forms of mercury exposures include elemental mercury (Hg 0 ), divalent mercury (Hg 2+ ) and organic mercury (mainly as methylmercury, MeHg). Exposure sources, target organs, toxicity and metabolism differ with each chemical form. For example, most MeHg exposure comes from consuming fish and seafood. MeHg is then easily absorbed by the digestive tract, entering the central nervous system (CNS) after passing the blood-brain barrier (BBB). The result is permanent injury to the CNS, particularly in the developing fetus (WHO 1990). Elemental mercury exposure to the general public mainly results from dental amalgam restorations. Additionally, workers at artisanal small scale gold mining (ASGM) sites can experience extremely high exposures to elemental mercury. Miners and gold shops workers heat amalgam to separate the mercury from the gold-mercury mixture with little or no personal protection, resulting in the evaporation of mercury and exposures through inhalation. Target organs here include the CNS, lungs and kidneys (WHO 1991). In this workshop, we will provide an overview on mercury metabolism, toxicity and exposure assessment. Exposure levels can be estimated by measuring biomarkers such as mercury concentrations in human tissues (i.e. blood, hair, urine, cord tissue, and nails). We will discuss the current state of knowledge on dose-response after exposure to the various forms of mercury and the use of biomarkers for risk management. In the main body of the workshop, we will explain 1) The appropriate selection of biomarkers for assessing different mercury exposures; 2) How to collect and preserve biomarkers; 3) Useful biomarkers for estimating fetal exposure to MeHg; 4) The strength and weaknesses of biomarkers; 5) When and how to measure total mercury and MeHg in biomarkers; 6) A demonstration of mercury analysis at National Institute for Minamata Disease booth. 1

2 Metabolism and toxicity of mercury depend on its chemical forms Human absorption of liquid Hg 0 is minimal, and acute toxicity does not occur even when the liquid mercury used in thermometers is accidentally ingested. The problem arises when liquid mercury is heated and bursts into the gaseous phase, which causes acute interstitial pneumonia when inhaled at a high concentration. Approximately 80% of inhaled gaseous Hg 0 is absorbed into the blood and easily passes through the blood-brain barrier in its un-oxidized form, thereby reaching the brain and damaging the central nervous system. With time, gaseous Hg 0 in the body is oxidized to Hg 2+, which accumulates in the kidneys and causes damage there (WHO 1991). The biological half-life of mercury absorbed from vapor into the blood is approximately 2-4 days when 90% is excreted primarily through urine, and to a lesser extent through exhaled breath and feces. Peak urine levels can lag behind peak blood levels by a few days to a few weeks, and urinary mercury levels decline with a half-life of approximately 1-3 months. Absorption of Hg 2+ through the digestive tract is comparatively low. However, a large intake of Hg 2+, such as in accidental or suicidal ingestion, causes digestive tract and kidney disorders resulting in death (WHO, 1991). MeHg is readily absorbed by the digestive tract and enters the central nervous system after passing the blood-brain barrier, thereby causing degeneration and dysfunction of nerve cells (WHO, 1991). MeHg transport into tissues appears to be mediated by the formation of a MeHg-cysteine conjugate, which is transported into cells via a neutral amino acid carrier protein (NRC, 2000; WHO, 1990). The brain of the developing fetus is very sensitive to MeHg. In addition, the MeHg concentration in fetal blood reaches approximately 2-fold higher than that of the mother, because of active MeHg transport across the placenta (NRC, 2000; WHO, 1990). Thus, fetuses are recognized as the highest-risk group for MeHg. Exposure assessment 1) The appropriate selection of biomarkers for assessing different mercury exposures MeHg exposure The preferred biomarker reflects the MeHg concentration in the brain, since the brain is the major target organ. In humans, MeHg has an average biological half-life of approximately 70 days (whole body) (WHO, 1990). Generally, the amount retained in the body becomes stable under constant MeHg exposure, and depends on dietary intake. Animal experiments indicate that the ratio of the Hg concentration in the blood to that in the brain becomes fixed under steady state conditions. 2

3 Therefore, the mercury concentration in the blood/red blood cells is a good biomarker (WHO, 1990). The mercury concentration in the hair also reflects blood MeHg concentration during hair formation and is frequently used as a biomarker for evaluating exposure to MeHg (WHO, 1990). Generally, the Hg concentration in the hair is 250 to 300-fold higher than that in the blood, because sulfur-containing proteins rich in the hair bind to MeHg. A number of studies have employed Hg concentrations in toenails and/or fingernails as biomarkers for exposure to MeHg. In most of these studies, toenails rather than fingernails were preferred, because toenails are often less contaminated than fingernails, especially among dental personnel and gold miners who handle Hg amalgam. Inorganic mercury/mercury vapor exposure The major form of mercury in the urine is inorganic mercury, and the total mercury concentration in the urine reflects the amount of inorganic mercury accumulated in the kidney, which is a good biomarker for evaluating exposure to inorganic mercury/mercury vapor (WHO, 1995). Serum or plasma is also known as a good biomarker of recent exposure to inorganic mercury/mercury vapor (WHO, 1991), due to its relatively short half time. The use of hair as a biomarker for mercury vapour is contentious and not advised. Mercury readily attaches to the proteins in hair making it is impossible to distinguish between the internal concentration of mercury and external contamination. Several studies have attempted to pretreat the hair to remove the external contamination, but only with limited success. 2) How to collect and preserve blood, hair, urine biomarkers; Blood: Mercury concentration in blood indicates recent or current exposure to MeHg. Blood samples are commonly obtained by venipuncture and collected into heparin-na or EDTA-2K containing Vacutainer tubes. The samples must be stored at 4ºC for periods of up to 7-10 days. Freeze at -20ºC or lower for longer periods of storage. Normally, mercury distribution in red blood cells and plasma is 2:1. Therefore, the samples must be well mixed before mercury analysis. Mercury in red blood cell is also a good biomarker to MeHg exposure and most of mercury is MeHg. For the exposure to inorganic mercury/mercury vapor assessment, the plasma/serum must be separated from red blood cells avoiding haemolysis of the sample. The minimum volume required of either whole blood or serum/plasma sample is 1-2 ml. (UNEP/WHO 2008). Hair: Hair samples are useful for assessing exposure to MeHg. Hair mercury concentrations reflect the blood mercury level when the hair was formed. The total in hair is 3

4 approximately 250 to 300 times higher than that in blood. Hair samples are usually collected approximately 50 strands from the occipital region of the head close to the scalp as possible. The strands must be tied with a cotton thread to ensure the hair strands close to the scalp. Hair monitoring also has the advantage of showing the monthly exposure to MeHg by measuring the hair Hg in 1-cm segments as the average growth rate of hair is approximately 1 cm per month (Boischio and Cernichiari 1998). Although, the time lag of approximately 1 month before the newly grown hair appears from the scalp to the surface should also be considered. It may be important to rule out any potential external contamination concerns by asking the participants about their potential exposures to mercury vapour. The minimum weigh required of hair sample for each mercury analysis is 5-10 mg. (UNEP/WHO 2008). Urine: Urine samples are useful for assessing recent exposure to inorganic mercury/mercury vapor. The urine mercury concentrations reflect the mercury level accumulated in kidneys. Bacteria growth is rapid in urine samples at room temperature. Therefore, the samples must be kept frozen (to be below -20ºC) to reduce the bacterial growth, because bacteria can reduce the mercury concentrations in urine by changing the mercury compounds (mainly divalent mercury) to elemental mercury. Adding of 0.1g of sulfamic acid and 50 L of Triton X-100 solution to 50 ml of urine sample can keep the sample stable at room temperature at least one month. The creatinine correction is frequently applied to measure mercury in urine to correct for variations in urine concentrations, even when the rate of mercury excretion is constant. Urine samples should be homogenized by shaking before analysis to lessen problems of precipitates. When collecting urine samples from individuals who may be handling mercury, such as miners or gold shop workers, it is advisable to instruct participants to first wash their hands before sample collection. The minimum volume required of urine sample is 5-10 ml. (UNEP/WHO 2008) 3) Useful biomarkers for estimating fetal exposure to MeHg; The organ targeted by MeHg exposure during gestation is the fetal brain. For this reason, biomarkers reflecting the MeHg exposure level in the fetus during the gestation are very important for predicting the effects of MeHg on child development. In the Faroe Islands study, cord blood mercury concentration was the preferred biomarker for MeHg exposure. In the Seychelles study, maternal hair Hg concentration was used as the only biomarker for fetal exposure. 4

5 Each biomarker has its advantages and disadvantages. Cord blood circulates in the fetal body and can directly reflect the MeHg concentrations in the fetal organs, including the fetal brain at birth (NRC 2000). Segmental analysis of maternal hair is able to provide time-course information of MeHg exposure during the gestation, because the average hair growth rate is commonly assumed to be about 1 cm per month. Umbilical cord tissue has also been used to determine fetal MeHg exposure levels in some studies (Grandjean and others 2005; Nishigaki and Harada 1975). Maternal Hg levels in fingernails and toenails at parturition also showed strong correlations with those in cord blood (Sakamoto and others 2015). 4) The strength and weaknesses of biomarkers of hair, blood and urine; Hair ; Strength: Total mercury analysis of hair is useful to estimate internal exposure to MeHg. Hair sampling is simple, non-invasive and easy to preserve. Segmental hair total mercury analysis can also provide the past exposure to MeHg since hair grows approximately 1 cm per month. Weakness: Hair is sometimes contaminated with adhesion of mercury vapor in the contaminated site such as ASGM and external mercury from mercury containing cosmetics. When external contamination is suspected, mercury speciation is necessary. On the other hand, hair permanent treatment removes mercury from the hair (WKO 1990). In addition, hair Hg analysis has involves a number of variables such as hair growth rate, density, color, waving, ethnicity and age (WHO 1990). Blood; Strength: Total mercury analysis in blood is useful to estimate current internal exposure to MeHg. Blood circulates in human body and can directly reflect the MeHg concentrations in the target organ of the brain. Cord blood is also useful to estimate fetal exposure to MeHg, especially the fetal brain at birth as mentioned before. Basically, mercury concentration in blood or red blood cells is the good biomarker of exposure to MeHg. In addition, the plasma/serum separated from red blood cells can be used as biomarker for exposure to inorganic mercury/mercury vapor. Weakness: Blood sampling is invasive to the subjects and requires proper sterile equipment and infectious disease control also to prevent infection to the persons conducting 5

6 sample collection and analysis. Subject consent is necessary but providing blood will be opposed by cultural or ethical reasons. The storage and transportation of the sample is more complicated compare to hair samples. Blood samples represent recent exposures to MeHg and/or Hg 0. Urine: Strength: Urine sampling is much less invasive and generally more acceptable to the participant than blood sampling. With the use of the proper preservatives, urine is easier to transport and store than blood in field conditions. Weakness: Urine is only useful to measure mercury vapour exposure, as MeHg is not excreted in the urine. The urine mercury concentration must be corrected for urine dilution rates with a creatinine correction test, which represents an additional step and cost. 5) When and how to measure total mercury and MeHg in biomarkers; For screening of exposure to MeHg, total mercury measurement is first recommended for blood, red blood cells, cord blood and hair samples. However, when the high inorganic mercury/mercury vapor contamination was suspected, inorganic mercury or MeHg concentration speciation is necessary, especially in hair samples who are working in ASGM using elemental mercury. For screening of exposure to inorganic mercury/mercury vapor, total mercury measurement is first recommended for plasma/serum and urine. 6) A demonstration of mercury analysis technology for hair and nails at the National Institute for Minamata Disease booth; Total mercury concentrations in hair or nail will be determined by Mercury Analyzer MA-3000 (Nippon Instruments Co. Ltd., Tokyo, Japan) using the principle of thermal decomposition,gold amalgamation, and atomic absorption in accordance with USEPA A number of analytical methods are available to determine mercury concentration, and an analytical method depends on various factors (such as an analytical regulation of each country), laboratory skills, analytical equipment, etc. Whatever analytical method will be used, it is important to practice careful quality control/quality assurance of the obtained data, including simultaneous determination of suitable certified reference materials (CRMs) 6

7 (UNEP/WHO 2008). 7) Additional essential information; The questionnaire on personal data (age, address, weight et al.), food frequency (especially for fish consumption), work exposure (gold mine et al.), should be collected at the time of biomonitoring (UNIDO 2008). The details of blood, hair and urine sampling and other information were cited from Guidance for identifying populations at risk from mercury exposure issued by UNEP/WHO (2008), Protocols for environmental and health assessment of mercury released by artisanal and small-scale gold miners issued by UNIDO (2008) and WHO IPCS Environmental Health Criteria 101 Methylmercury (1990), and Inorganic Mercury 108 (1991). References; Boischio, A.A.; Cernichiari, E. Longitudinal hair mercury concentration in riverside mothers along the Upper Madeira river (Brazil). Environ Res. 77:79-83; 1998 Grandjean, P.; Budtz-Jorgensen, E.; Jorgensen, P.J.; Weihe, P. Umbilical cord mercury concentration as biomarker of prenatal exposure to methylmercury. Environ Health Perspect. 113: ; 2005 Nishigaki, S.; Harada, M. Methylmercury and selenium in umbilical cords of inhabitants of the Minamata area. Nature. 258: ; 1975 NRC. National Research Council. Toxicological Effects of Methylmercury, Academic Press, Washington, DC.; 2000 Sakamoto, M.; Chan, H.M.; Domingo, J.L.; Oliveira, R.B.; Kawakami, S.; Murata, K. Significance of fingernail and toenail mercury concentrations as biomarkers for prenatal methylmercury exposure in relation to segmental hair mercury concentrations. Environ Res. 136: ; 2015 UNEP/WHO. Guidance for identifying populations at risk from mercury exposure; 2008 UNIDO. Protocols for environmental and health assessment of mercury released by artisanal and small-scale gold miners; 2008 WHO. Methylmercury. Environmental Health Criteria 101. Geneva: World Health Organization; 1990 WHO. Inorganic mercury. Environmental Health Criteria 118. Geneva: World Health Organization.;

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