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The biological monitoring of prenatal exposure to methylmercury
NeuroToxicology 28 (2007) 1015–1022
The biological monitoring of prenatal exposure to methylmercury E. Cernichiari a, G.J. Myers b, N. Ballatori a, G. Zareba a, J. Vyas c, T. Clarkson a,* b
a Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York, United States Department of Pediatrics and of Neurology, University of Rochester School of Medicine, Rochester, New York, United States c Industrial Hygiene Department, National Institute of Occupational Health, Ahmedabad, India
Received 16 January 2007; accepted 7 February 2007 Available online 23 February 2007
Abstract Several biological media have been used as indicators of the fetal body burden of methylmercury and the levels in the primary target tissue, the developing brain. These media include maternal hair and blood. The relative merits of these media will be considered both with regard to current knowledge of the physiology of mercury disposition in the body and also the practicality of field application with respect to sample, collection, transport, storage and processing. # 2007 Elsevier Inc. All rights reserved. Keywords: Methylmercury; Biological monitoring; Hair; Whole blood
Several studies have now been published on the potential adverse effects on child development of prenatal exposure to methylmercury (for a review, see Clarkson and Magos, 2006). These studies essentially test for statistical associations between the dependent variable (measures of child development) and the independent variable (the prenatal dose of methylmercury). Whereas measures of child development follow well-established methods, measures of the prenatal dose are more controversial. The relevant prenatal dose is the dose to the developing fetal brain as this tissue is the exclusive target for the toxic action of methylmercury. This dose can only be estimated by indirect measures. Such measures, usually described as ‘‘biological monitoring’’, make use of measures of mercury in surrogate tissues or biological fluids as an index of the concentration of methylmercury in the fetal brain. The rationale for the choice of the appropriate biological marker must come from an understanding of the disposition of mercury in the body, and more specifically in the maternal-fetal unit.
1. Disposition of methylmercury Methylmercury distributes rapidly to all tissues in the body (Fig. 1). Methylmercury consumed in fish is almost completely * Corresponding author at: P.O. Box 2590, Alachua, FL 32616, United States. Tel.: +1 386 418 0894. E-mail address: [email protected] (T. Clarkson). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.02.009
absorbed into the blood stream. Distribution to body tissues and organs is completed in some 30–40 h after a single meal of fish (Kershaw et al., 1980). It is eliminated from the body with a half time of about 70 days corresponding to an excretion rate of 1% of the body burden per 24 h (Miettinen, 1973). Given a whole body half time of 70 days, it will take about 1 year (equivalent to five half times) of regular intake of methylmercury to attain a steady state balance between uptake and excretion of methylmercury. In all epidemiological studies of populations consuming methylmercury in fish it has generally been assumed that a steady state distribution had been established. In the blood stream, it preferentially accumulates in the red blood cells resulting in a red cell to plasma concentration ratio of approximately 20:1 (Fig. 2; Kershaw et al., 1980). The brain to whole blood concentration ratio in adults is in the range of 5:1–10:1. However, this ratio has been measured only in a few volunteer subjects dosed with radioactive methylmercury so little information is available on the variability of this ratio (Miettinen, 1973). Animal data indicate that levels in the fetal brain may be higher than in the corresponding maternal brain (Inouye et al., 1986). In fact, methylmercury is well transported across the placenta although the actual rates of transport are unknown. As in the adult, methylmercury is taken up by the red blood cells. Indeed, in cord blood the proportion found in the red cells is even greater than in adults (Kuhnert et al., 1981). This consideration is also relevant to the role of cord blood as a biological monitoring medium, as discussed further below. Of
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Fig. 1. The disposition of methylmercury in maternal and fetal tissues. Methylmercury is efficiently absorbed from the diet and enters the portal circulation. As it passes through the liver a fraction of the absorbed dose is secreted into the bile and part is reabsorbed back into the blood compartment. Some of that remaining in the GI tract is converted by microflora to inorganic mercury and excreted in the feces. Methylmercury readily enters all tissues and organs and is transported readily across the blood–brain barrier. Its transport is bidirectional except in the case of its avid accumulation into scalp hair where it remains in the hair strand. It crosses the placenta and distributes throughout fetal tissues including the brain. Several fluids and tissues have been used as biological indicators of methylmercury in the fetal brain including fetal cord blood, placental tissue and maternal scalp hair.
the methylmercury in blood plasma, about 1% is complexed with cysteine (CH3Hg-Cys; Fig. 2), and some is also complexed with reduced glutathione (GSH). Other ligands for methylmercury include albumins, hemoglobins, keratins, and tubulins. Scalp hair accumulates methylmercury to a remarkable degree (Fig. 2). On average, the hair to whole blood concentration ratio is 250:1 and the corresponding ratio for plasma mercury is some ten times greater, about 2500:1. Once
incorporated into the hair strand, the concentration of methylmercury remains stable, and thus allows the recapitulation of past exposure from a single sample of hair. Hair grows at an average rate of just over 1 cm per month, such that 1 cm segments can recapitulate past exposure for many months or even years depending on the length of the hair sample (Phelps et al., 1980). As will be discussed later, the ability of hair to concentrate methylmercury and to maintain stable concentrations is an important consideration in evaluating hair as a biological monitoring medium. In terms of the best medium for estimating fetal methylmercury brain levels, a glance at Fig. 1 would suggest that cord blood should be the preferred as it is fetal blood and has direct contact with the fetal brain. Perhaps next in order of preference would be maternal blood as it exchanges methylmercury across the placenta to the fetus. Last in preference might be maternal scalp hair as it is more remote from the fetal brain. However when one considers the known methylmercury transport mechanisms, these conclusions will change and perhaps even reverse. 2. Mechanisms of disposition
Fig. 2. The relative concentrations of methylmercury in whole blood and scalp hair with the concentration in plasma set at unity. Approximately 95% of the methylmercury in whole blood is bound to red blood cells. The brain level is approximately 50 times and the hair level 2500 times greater than the concentration in plasma after methylmercury has attained a steady state of distribution in the body. Methylmercury complexed with the amino acid cysteine, (CH3Hg-Cys) is the species transported into tissues. This complex accounts for only a small fraction of methylmercury in plasma and most of it is bound to mercaptalbumin. Other proteins known to bind methylmercury are the keratins, tubulins and hemoglobin.
The methylmercury cation has a high chemical affinity for thiol ligands (Rabenstein et al., 1982). The reaction depicted in the equation below is rapid and reversible: CH3 Hgþ þ HSR @ CH3 HgSR In view of its high chemical affinity for thiol groups, probably greater than for any other ligand in the cell, it is assumed that
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Fig. 3. Cellular methylmercury (MeHg) uptake and export mechanisms. A major mechanism of MeHg uptake into cells involves neutral amino acid transporters, including LAT1 and LAT2. These proteins have been shown to mediate the uptake of the MeHg-L-cysteine complex (MeHg-Cys) in exchange for an intracellular amino acid. In terms of MeHg export from cells, two major mechanisms have been described. One involves amino acid transporters and the other is mediated by members of the multidrug resistance-associated (MRP) family of proteins. As indicated above, neutral amino acid carriers function as exchangers, and thus can also mediate export of MeHg-Cys, in exchange for an extracellular amino acid. Export of anions such as the MeHg-glutathione complex (MeHg-SG) is mediated by certain members of the MRP family of proteins, in particular MRP1 and MRP2. These membrane proteins mediate the ATP-driven pumping of these anions from the cell into the extracellular space (for a detailed review, see Ballatori, 2002).
methylmercury binds exclusively to thiol-containing molecules.1 In fact all compounds of methylmercury so far identified in tissues are complexes with endogenous thiol containing molecules. The rapid reversibility of the reaction allows the methylmercury cation to move readily from one thiol to another depending, among other things, on local concentrations and differing affinities for the different thiol ligands. The complex of methylmercury with the thiol containing amino acid cysteine plays a key if not exclusive role in the transport of methylmercury into mammalian cells. The structure of the methylmercury-cysteine complex is sufficiently similar to that of the large neutral amino acid, methionine, that it is transported across the cell membrane into the cell on the large neutral amino acid carriers (Fig. 3). The transport carriers are so selective that the complex with the L-optical isomer of cysteine is transported, but not the D-optical isomer (Mokrzan et al., 1995). Appreciable transport also takes place for the complex with the close structural analogue, homocysteine. This process was first discovered in studies of the transport of methylmercury across the blood–brain barrier (Kerper et al., 1992). Since then, transport of methylmercury-cysteine has been shown to occur across the placenta (Kajiwara et al., 1996) and when such amino acid carriers have been experimentally introduced into other recipient cells (Simmons-Willis et al., 2002).
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Once inside the cell, methylmercury is believed to attach in part to the thiol ligand of GSH in view of the high intracellular concentrations of this thiol-containing peptide (Fig. 3). In fact, methylmercury is secreted from liver cells into bile as a complex with GSH on the glutathione carriers (Ballatori and Clarkson, 1982). Although this transport process has not yet been demonstrated for other cells, it is highly likely that it will be universal to all mammalian cells in view of the presence of high levels of intracellular GSH and GSH membrane carriers (Ballatori, 2002). The processes involved in the excretion of methylmercury provide an example of the important role of these thiol complexes in the membrane transport and excretion of methylmercury. Fecal excretion predominates, accounting for about 90% of the total elimination from the body. The process originates with the secretion of methylmercury from liver cells to bile as a complex with GSH (Fig. 1). As the methylmercury moves down the biliary tree and enters the gallbladder, GSH is hydrolyzed to its constituent amino acids by the membranebound ectoenzymes gamma-glutamyltranspeptidase and dipeptidase, resulting in the release of methylmercury as a complex with cysteine (Dutczak et al., 1991). The latter can be absorbed into the portal circulation both in the gallbladder and in the intestines to be returned to the liver to complete the enterohepatic cycle. However some of the methylmercury secreted into the GI tract comes into contact with the microflora that are capable of breaking the carbon mercury bond to release inorganic mercury (Rowland et al., 1978). The latter is poorly absorbed (less then 10%, Rahola et al., 1973) and is therefore mainly excreted in the feces (Fig. 1). Urinary excretion of methylmercury makes a small contribution to total excretion, less than 10%, probably due to the fact that the mobile form of mercury, methylmercurycysteine, is itself readily reabsorbed in the kidney tubules after filtration through the glomerulus. Methylmercury that is directly taken up by the kidney from the peritubular blood is excreted into the tubular fluid as the complex with GSH. As in the biliary tree, the GSH complex is enzymically broken down within the renal tubule, with the release of methylmercury complexed to cysteine that is reabsorbed back into the renal tubular cells (Ballatori et al., 1998). In summary, two major mobile species of methylmercury have been identified. The GSH complex is the intracellular species responsible for transport out of the cell and the cysteine complex is the extra cellular species responsible for entry into the cell. The latter is especially important for biological monitoring as it will determine entry into tissues including the target tissue, the central nervous system, and into tissues responsible for the overall body burden of methylmercury, namely the cells in the enterohepatic system. 3. Indicator media
The selenide ligand Se= or R-Se may have a higher affinity for methylmercury. However the only mercury-selenium compounds identified to date in tissues involve the inorganic mercury cation, Hg2+, in the form of mercuric selenide, HgSe. 1
The majority of studies of prenatal exposure to methylmercury have made use of hair and blood as the biological indicator media of choice. However, there is not universal agreement on the relative merits of these two media. Specifically the question
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arises as to which medium will be more likely to give a better index of levels of methylmercury in the fetal brain, given our current understanding of the disposition of methylmercury in maternal and fetal tissues. 3.1. Whole blood The half time of methylmercury in whole blood in adults has been determined to be 44 days on average (Smith et al., 1994). The half time in brain roughly determined from the decline in radioactivity in the head region in volunteers taking radioactive methylmercury was found to be about 70 days (Aberg et al., 1969). This difference in half-times implies that the brain to blood concentration ratios will be constantly changing until a steady state body burden of methylmercury is attained after approximately 1 year of regular intake of methylmercury. The assumption has been made in all epidemiological studies of fish eating populations that the mother is in steady state with respect to her body burden of methylmercury. Under these conditions, the ratio of methylmercury concentration of blood to brain should be constant. However, this conclusion may not necessarily apply to fetal tissues. The half time in the fetal brain is unknown. Indeed the kinetics of deposition will be complicated by the fact that this organ will be increasing in size at varying rates during gestation. Nevertheless, despite these uncertainties, methylmercury levels in autopsy brains of very young infants do correlate with levels in maternal blood taken following the death of the infant, and with autopsy samples of the infant’s blood (Cernichiari et al., 1995). Unfortunately, no similar data are available for cord blood samples. Measures of methylmercury concentrations in whole blood are affected by the hematocrit and by the formation of microclots as most of the mercury is bound to red blood cells. The delivery room is a busy place and the emphasis is on the health of the mother and infant. Collection of cord blood takes a secondary place and is done by the obstetrician after caring for the mother and infant or by someone not actively helping the patients (i.e. secondary personnel). The timing of cord sampling following delivery varies depending upon the medical situation and is not uniform. The timing may affect the formation of microclots even before the blood is taken. Microclots present at the time of collection or related to timing of processing the blood after collection may affect the measurement of Hg present in the sample. Collection of cord blood can be done by simply taking the top off the tube, inserting the placental end of the umbilical cord into the tube and loosening the clamp. This is often the most rapid and easiest for the obstetrician since minimal equipment is needed. Alternatively, it can be done using a needle and syringe and aspirating from the cord. Aspiration can be from the cord itself, usually on the placental side of the clamps or from vessels on the surface of the placenta itself. The samples are usually collected directly into the tube that is sent to the laboratory. The time interval between collection, transport of the sample to the laboratory, and processing the sample in the laboratory may vary. In the laboratory, care must be exercised
that the aliquot for analysis is representative of the sample. Substantial changes in hematocrit may occur when samples are allowed to sit or microclots have formed and these can affect the mercury measurement. Whole blood contains methylmercury in different chemical complexes (Fig. 2). Approximately 95% of methylmercury in human whole blood is found inside the red blood cells, and most is bound to thiol ligands of hemoglobin. Of the 5% in blood plasma, most is bound to the thiol ligand of mercaptalbumin and only about 1% is bound to cysteine (Ancora et al., 2002; Yasutake et al., 1989). Thus the transportable species of methylmercury accounts for a very small fraction of total methylmercury in whole blood. If whole blood is a measure of the transport species of methylmercury, one must assume that this species is a constant fraction of the total mercury in whole blood. Given the high mobility of methylmercury and its rapidly reversible binding to thiol ligands, this assumption may be true especially for the same individual. However, as discussed above, hematocrits can vary. In addition, binding to hemoglobin might be affected by genetically different homologues of this protein and dietary intake of protein might well affect levels of L-cysteine and other thiols in plasma, as demonstrated experimentally in mice (Adachi et al., 1994). With respect to hemoglobin binding, the brain to blood ratio of methylmercury in different animal species varies by two orders of magnitude (Table 1), due in part to differences in the affinities of the methylmercury cation for the thiol ligands of different hemoglobin proteins. 3.2. Human head hair Studies on uptake into animal fur indicate that methylmercury enters via the follicle and is only accumulated when the follicle is in the growing phase (Shi and Clarkson, 1990). These findings were confirmed in studies of human hair grafted onto athymic nude mice (Zareba et al., submitted). Autoradiographic observations in these same studies demonstrated that radioactive methylmercury was taken up by the keratinocytes and eventually deposited in the high sulfur keratin proteins. Although transport of the methylmercury-cysteine complex on the large neutral amino acid carriers has not been demonstrated specifically for the hair follicle, it is highly likely that this carrier-mediated mechanism is the route of entry. Supporting this hypothesis is the ubiquitous presence of these carriers in mammalian cells and the high demand for amino acids for synthesis of the keratin proteins in hair. In fact it is this active uptake of amino acids in growing hair that accounts for Table 1 Species differences in brain to blood concentration ratiosa Species
Methylmercury
Rat Mouse Squirrel monkey Human
0.06 1.2 6 6
a The concentration ratio is the brain level divided by the corresponding blood level. The data are taken from Suzuki et al. (1963/1973) and Ulfvarson (1962).
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the high hair to plasma concentration ratio in the range to 2500:1 (Fig. 2). The fact that hair is essentially dried tissue may also play a role in the high hair to blood concentration ratio. In summary, the process of uptake of methylmercury into human head hair most likely starts with the transport of the methylmercury-cysteine complex into the keratinocytes, the differentiation and death of these cells during the production of the hair strand, with the ultimate deposition of methylmercury attached to protein thiol ligands. Once incorporated into the hair strand, the mercury level remains stable. In this context, the levels of mercury along the length of the hair strand should provide an historical record of the levels of the transportable species in plasma. It is not surprising therefore that hair levels closely follow whole blood levels in the same individual and over a wide range of concentrations (Fig. 4A and B). We would expect that those factors affecting the distribution of methylmercury in whole blood would be unchanged for the same person whether ingested in pregnancy (Fig. 4A) or in adult males (Fig. 4B). The concentration ratio of hair to whole blood may be expected to show variation from one individual to another. For reasons already discussed, the transportable species of methylmercury may not constitute the same fraction of total mercury in whole blood due to genetic and dietary differences. For example Budtz-Jorgensen et al. (2004) have reported that the ratio of hair to whole blood levels changes with age. These data were obtained from individuals of different ages and may well represent the influence of diet if not genetic differences as well. Such differences in hair to blood ratios have been used as an argument against the use of hair as a biological monitor for fetal brain levels. In contrast, anything that changes this ratio suggests the opposite conclusion, namely that the variance in the distribution of mercury in whole blood binding sites is the most likely explanation for why hair to blood concentration ratios may differ between individuals. The true test of hair as an appropriate biological indicator comes from an examination of the degree of correlation with levels of mercury in the target tissue, the brain. Fig. 5 presents the relationships between levels of mercury in maternal hair versus levels in four anatomical regions of autopsy brains from infants dying shortly after birth. A linear association is seen with correlation coefficients in the range of 0.8. Given the variance that inevitably arises from measures of autopsy tissues as well as the long distance transport and lengthy storage of some of these samples, these correlations are quite striking, and clearly support the use of maternal hair as an indicator of fetal brain levels. The uptake of mercury into hair is apparently selective to methylmercury and does not occur with inorganic mercury2 (Table 2). In several individuals who were highly exposed to inorganic mercury, hair levels were slightly elevated over background, but were one to two orders of magnitude lower than those that would be expected from the same blood levels of
2 The closely related chemical congener, ethylmercury, may also be taken up by hair (Zareba et al., in press).
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Fig. 4. There is a close parallel between the concentration of methylmercury in the diet and mercury in scalp hair in exposed individuals. (A) Mercury levels in a female including the period of her pregnancy represented by the shaded area. She was exposed to dietary mercury derived from homemade bread prepared from seed grain that had been treated with a methylmercury fungicide. Her hair sample was collected in 1973 and was of sufficient length to allow recapitulation of mercury levels back to 1971, a time long before her exposure to MeHg. The peak levels in her hair occurred at admission to hospital when exposure to methylmercury ended. Blood samples were collected in hospital and compared to hair levels (adapted from Amin-Zaki et al., 1976). (B) The concentration of mercury in samples of whole blood and 8 mm segments of scalp hair taken from a volunteer who consumed methylmercury in fish (halibut). The fish consumption took place over a 100-day period. Hair samples were cut close to the scalp, divided into 8 mm segments and plotted according to the growth rate of hair (assumed to be 11.8 mm per month). (The figure is based on data published by Hislop et al., 1983.)
methylmercury. This is a useful attribute of hair as some inorganic mercury may be present in the blood stream of fish eating populations. As discussed previously, some methylmercury is converted to inorganic mercury in the GI tract, whereupon some absorption back into the blood stream may occur. Methylmercury is slowly converted to inorganic mercury in various body tissues and this might also contribute to blood levels of inorganic mercury (Suda et al., 1993). Maternal amalgam tooth fillings also elevate levels of inorganic mercury in both maternal and cord blood (Bjornberg et al., 2003). Thus, the data in Table 2 further support the use of hair as a biological indicator of brain methylmercury levels. Little uptake of inorganic mercury took place into hair. Inorganic
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Fig. 5. The concentrations of mercury in four anatomical areas of the infant brain plotted against the concentration in maternal hair. The infants died soon after birth from various natural causes unrelated to their methylmercury exposure. The maternal hair concentration was measured in the first centimeter next to the scalp collected at the time of the infant’s death. Their exposure to methylmercury was via maternal consumption of ocean fish. The linear regression lines were forced through zero as it was assumed that if maternal levels of methylmercury were zero the levels in the newborn infant must also be zero. Actually when the regression lines were drawn without this restriction, the intercepts were not significantly different than zero. The linear regression coefficients fell in the range of 0.6–0.8 (adapted from data published by Cernichiari et al., 1995).
mercury is known to be poorly transported across the blood– brain barrier (Friberg and Mottet, 1989). In fact, blood levels of methylmercury that correspond to the observed levels of inorganic mercury would have been expected to result in severe brain damage. The fact that these subjects had no neurological signs or symptoms is consistent with findings on the disposition of radioactive inorganic mercury where no radioactivity was detected in the head region (Rahola et al., 1973). Thus it appears that uptake into hair mimics uptake into brain for both organic and inorganic species of mercury. Table 2 The concentrations of mercury indicator media in five individuals after exposure to inorganic mercury and comparison with expected levels if exposure were to methylmercury Subject and exposurea
Blood (ng Hg/ml)
Urine (ng Hg/ml)
Normal values
<3
<2
(1) (2) (3) (4) (5)
400 335 284 54 854
1512 1320 234 210 472
400
25
Metallic Hg Metallic Hg Mercury vapor Mercury vapor Mercuric chloride
Methylmercury a
Hair (mg Hg/g) <1 3 2.4 2.8 2.5 2.7 100
Subjects (1 and 2) exposed to metallic mercury were due to insuflation into the lungs of liquid metallic mercury from the accidental rupture of a Miller– Abbott tube during esophageal dilitation. Subjects 3 and 4 were exposed to mercury vapor by inhalation subject 5) was exposed to mercuric chloride due to a suicide attempt. The data are taken from Myers et al. (submitted) [?]. The data for illustrating what would be expected with methylmercury are based on the published pharmacokinetic parameters for the disposition of methylmercury in adult subjects (WHO, 1976).
In the context of the fieldwork involved in epidemiological studies, the hair sample offers appreciable practical advantages. The collection is non-invasive, transport and storage can be achieved in a postal envelope and no refrigeration is required. A single hair sample can achieve recapitulation of mercury levels over months or even years depending in the length of the hair strands. Non-destructive physical methods such as X-ray fluorescent spectrometry are now available that can measure mercury in single strands of hair. The difference in mercury levels between each strand has been shown to be less than the analytical variance (Toribara, 2001). Some experimental studies have indicated that certain hair treatments might remove mercury or possibly add mercury as a contaminant (Nuttall, 2006; Wakisaka et al., 1990). However, a recent study on mercury levels in samples of hair collected from a large population found no evidence of an effect of hair treatment under actual conditions of use (McDowell et al., 2004). Another argument that has been presented against the use of hair as a biological monitor is that a higher variance was found in maternal hair samples than in the corresponding cord blood samples (Grandjean et al., 2004). As discussed above, hair levels should be an index of the mobile species of mercury in blood. This species will be variable given the factors that influence mercury distribution in blood. The variance in hair levels might well represent the actual changes taking place in the transport species. Variance therefore might actually be interpreted in favor of using hair as the preferred biomarker depending on what assumptions are made. Two of the largest studies of prenatal exposure to methylmercury came to apparently opposite conclusions.
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One study based in the Faroe Islands claimed to find adverse effects on child development (Grandjean et al., 1997) whereas the other study based in the Seychelles Islands found no adverse effects and indeed found evidence that fish consumption might be beneficial despite the concomitant methylmercury intake (Davidson et al., 2006; Myers et al., 2003). It has been claimed that problems with hair measurement such as the higher variance or changes in hair to blood ratio might introduce errors in the independent variable that masks an adverse effect in the Seychelles population. This explanation is unlikely. Both studies have been intensively reviewed by expert committees, who concluded that both were well conducted with state-of-the-art methods. The different outcomes are most likely due to differences in the characteristics of the two populations, the study designs, and the analyses and their interpretations. For example, the average concentration of methylmercury in whale meat, the major source of exposure in the Faroes, is 1.6 mg Hg/g wet weight, whereas the average concentration in the ocean fish consumed in the Seychelles population is 0.31 mg Hg/g wet weight. These different average concentrations indicate that the amount of nutrient food accompanying any given intake of methylmercury is some five times higher in the Seychelles than in the Faroes. In conclusion, the use of maternal hair as an index of fetal brain levels is well justified based on the fact that it offers superior practical advantages and has a firmer physiological base. Acknowledgments This work was supported in part by grants from the National Institute of Environmental Health Sciences, Grant #RO1 ES10219 and in part by Center Grant #P30 ES01247 References Aberg B, Ekman L, Falk R, Greitz U, Persson G, Snihs JO. Metabolism of methyl mercury (203Hg) compounds in man. Arch Environ Health 1969;19(4):478–84. Adachi T, Yasutake A, Hirayama K. Influence of dietary levels of protein and sulfur amino acids on the fate of methylmercury in mice. Toxicology 1994;93(2–3):225–34. Amin-Zaki L, Elhassani S, Majeed MA, Clarkson TW, Doherty RA, Greenwood MR, et al. Perinatal methylmercury poisoning in Iraq. Am J Dis Child 1976;130(10):1070–6. Ancora S, Rossi R, Simplicio PD, Lusini L, Leonzio C. In vitro study of methylmercury in blood of bottlenose dolphins (Turslops truncates). Arch Environ Contam Toxicol 2002;42(3):348–53. Ballatori N. Transport of toxic metals by molecular mimicry. Environ Health Perspect 2002;110(Suppl. 5):689–94. Ballatori N, Clarkson TW. Developmental changes in the biliary excretion of methylmercury and glutathione. Science 1982;2l6:61–3. Ballatori N, Wang W, Lieberman MW. Accelerated methylmercury elimination in gamma-glutamyl transpeptidase-deficient mice. Am J Pathol 1998;152(4):1049–55. Bjornberg KA, Vahter M, Petersson-Grawe K, Glynn A, Cnattingius S, Darnerud PO, et al. Methyl mercury and inorganic mercury in Swedish pregnant women and in cord blood: influence of fish consumption. Environ Health Perspect 2003;111(4):637–41. Budtz-Jorgensen E, Grandjean P, Jorgensen PJ, Weihe P, Keiding N. Association between mercury concentrations in blood and hair in methylmercury-
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exposed subjects at different ages. Environ Res 2004;95(3):385–93 [Erratum in: Environ. Res. 2006;102(2):249]. Cernichiari E, Toribar TY, Liang L, Marsh DO, Berlin M, Myers GJ, et al. The biological monitoring of mercury in the Seychelles study. Neurotoxicology 1995;16:613–62. Clarkson TW, Magos L. The toxicology of mercury and its chemical compounds. Crit Rev Toxicol 2006;36:609–62. Davidson PW, Myers GJ, Cox C, Wilding GE, Shamlaye CF, Huang LS, et al. Methylmercury and neurodevelopment: longitudinal analysis of the Seychelles child development cohort. Neurotoxicol Teratol 2006;28(5):529–35. Dutczak WK, Clarkson TW, Ballatori N. Biliary-hepatic recycling of a xenobiotic: gallbladder absorption of methylmercury. Am J Physiol 1991;G873– 80. Friberg L, Mottet NK. Accumulation of methylmercury and inorganic mercury in the brain. Biol Trace Elem Res 1989;21:201–6. Grandjean P, Budtz-Jorgensen E, Keiding N, Weihe P. Under estimation of risk due to exposure misclassification. Int J Occup Med Environ Health 2004;17:131–6. Grandjean P, Weihe P, White RF, Debes F, Araki S, YokoyamaK. et al. Cognitive deficit in 7 year-old children with prenatal exposure to methylmercury. Neurotoxicol Teratol 1997;19:417–28. Hislop JS, Collier TR, White GF, Khathing T, French E. The use of keratinized tissues to monitor the detailed exposure of man to methylmercury in fish. In: Brown SS, Savory J, editors. Chemical toxicology and clinical chemistry of metals. New York: Academic Press; 1983. p. 145–8. Inouye M, Kajiwara Y, Hirayama K. Dose- and sex-dependent alterations in mercury distribution in fetal mice following methylmercury exposure. J Toxicol Environ Health 1986;19(3):425–35. Kajiwara Y, Yasutake A, Adachi T, Hirayama K. Methylmercury transport across the placenta via neutral amino acid carrier. Arch Toxicol 1996;70(5):310–4. Kerper LE, Ballatori N, Clarkson TW. Methylmercury transport across the blood–brain barrier by an amino acid carrier. Am J Physiol 1992;262(5 Pt 2):R761–5. Kershaw TC, Clarkson TW, Dhahir P. The relationship between blood levels and dose of methyl mercury in man. Arch Environ Health 1980;35:28–36. Kuhnert PM, Kuhnert BR, Erhard P. Comparison of mercury levels in maternal blood, fetal cord blood, and placental tissues. Am J Obstet Gynecol 1981;139(2):209–13. McDowell MA, Dillon CF, Osterloh Bolger PM, Pellizzari E, Fernando R, Montes de Oca R, et al. Hair mercury levels in U.S. Children and Women of Childbearing Age: reference range data from NHANES 1999–2000. Environ Health Perspect 2004;112:1165–71. Miettinen JK. Absorption and elimination of dietary mercury (Hg2+) and methyl mercury in man. In: Miller MW, Clarkson TW, editors. Mercury mercurials and mercaptans. Springfield, IL: Charles C. Thomas Publishers; 1973. p. 233–40. Mokrzan EM, Kerper LE, Ballatori N, Clarkson TW. Methylmercury-thiol uptake into cultured brain capillary endothelial cells on amino acid system L. J Pharmacol Exp Ther 1995;272(3):1277–84. Myers GJ, Watson G, Cernichiari E, Clarkson TW. The uptake into human scalp hair of different chemical and physical species of mercury; submitted for publication. Myers GJ, Davidson PW, Cox C, Shamlaye CF, Palumbo D, Cernichiari E, et al. Prenatal methylmercury exposure from ocean fish consumption in the Seychelles child development study. Lancet 2003;361(9370):1686–92. Nuttall KL. Interpreting hair mercury levels in individual patients. Ann Clin Lab Sci 2006;36(3):248–61. Phelps RW, Clarkson TW, Kershaw TG, Wheatley B. Interrelationships of blood and hair mercury concentrations in a North American population exposed to methylmercury. Arch Environ Health 1980;35(3):161–8. Rabenstein DL, Isab AA, Reid RS. A proton nuclear magnetic resonance study of the binding of methylmercury in human erythrocytes. Biochim Biophys Acta 1982;720(1):53–64. Rahola T, Hattula T, Korolainen A, Miettinen JK. Elimination of free and protein-bound ionic mercury (20Hg2+) in man. Ann Clin Res 1973;5(4): 214–9. Rowland IR, Davies MJ, Grasso P. Metabolism of methylmercuric chloride by the gastro-intestinal flora of the rat. Xenobiotica 1978;8(1):37–43.
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Shi C-Y, Clarkson TW. Uptake of mercury by the hair of methylmercury-treated newborn mice. Environ Res 1990;51:170–81. Simmons-Willis TA, Koh AS, Clarkson TW, Ballatori N. Transport of a neurotoxicant by molecular mimicry: the methylmercury-L-cysteine complex is a substrate for human LAT1 and LAT2 amino acid carriers. Biochem J 2002;367:239–46. Smith JC, Allen PV, Turner MD, Most B, Fisher HL, Hall LL. The kinetics of intravenously administered methyl mercury in man. Toxicol Appl Pharmacol 1994;128(2):251–6. Suda I, Suda M, Hirayama K. Phagocytic cells as a contributor to in vivo degradation of alkyl mercury. Bull Environ Contam Toxicol 1993;51(3): 394–400. Suzuki T, Miyama T, Katsunuma H. Comparative study of bodily distribution of mercury in mice after subcutaneous administration of methyl, ethyl and Npropyl mercury acetates. Jpn J Exp Med 1963;33:277–82. Suzuki T, Takemoto T, Kashiwazaki H, Miyama T. Metabolic fate of ethylmercury salts in man and animal. In: Miller MN, Clarkson TW, editors. Mercury, mercurials and mercaptans. Springfield, IL: Charles C. Thomas Publishers; 1973. p. 2009–40.
Toribara TY. Analysis of single hair by XRF discloses mercury intake. Hum Exp Toxicol 2001;20:185–8. Ulfvarson U. Distribution and excretion of some mercury compounds after long term exposure. Int Arch Gewerbepathol Gewerbehyg 1962;19:412– 22. Wakisaka I, Yanagihashi T, Sato M, Nakano A. Factors contributing to the difference of hair mercury concentrations between the sexes. Nippon Eiseigaku Zasshi 1990;45(2):654–64. World Health Organization. Environmental health criteria. 1. Mercury. International program on chemical safety. Geneva: World Health Organization; 1976. Yasutake A, Hirayama K, Inoue M. Mechanism of urinary excretion of methylmercury in mice. Arch Toxicol 1989;63(6):479–83. Zareba G, Cernichiari E, Hojo R, McNitt S, Weiss B, Mumtaz MM, Jones DE, Clarkson TW. Thimerosal distribution and metabolism in neonatal mice; comparison with methyl mercury; in press. Zareba G, Cernichiari E, Goldsmith LA, Clarkson TW. Validity of methyl mercury hair analysis: mercury monitoring in human scalp/nude mice model; submitted for publication.
The biological monitoring of prenatal exposure to methylmercury E. Cernichiari a, G.J. Myers b, N. Ballatori a, G. Zareba a, J. Vyas c, T. Clarkson a,* b
a Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York, United States Department of Pediatrics and of Neurology, University of Rochester School of Medicine, Rochester, New York, United States c Industrial Hygiene Department, National Institute of Occupational Health, Ahmedabad, India
Received 16 January 2007; accepted 7 February 2007 Available online 23 February 2007
Abstract Several biological media have been used as indicators of the fetal body burden of methylmercury and the levels in the primary target tissue, the developing brain. These media include maternal hair and blood. The relative merits of these media will be considered both with regard to current knowledge of the physiology of mercury disposition in the body and also the practicality of field application with respect to sample, collection, transport, storage and processing. # 2007 Elsevier Inc. All rights reserved. Keywords: Methylmercury; Biological monitoring; Hair; Whole blood
Several studies have now been published on the potential adverse effects on child development of prenatal exposure to methylmercury (for a review, see Clarkson and Magos, 2006). These studies essentially test for statistical associations between the dependent variable (measures of child development) and the independent variable (the prenatal dose of methylmercury). Whereas measures of child development follow well-established methods, measures of the prenatal dose are more controversial. The relevant prenatal dose is the dose to the developing fetal brain as this tissue is the exclusive target for the toxic action of methylmercury. This dose can only be estimated by indirect measures. Such measures, usually described as ‘‘biological monitoring’’, make use of measures of mercury in surrogate tissues or biological fluids as an index of the concentration of methylmercury in the fetal brain. The rationale for the choice of the appropriate biological marker must come from an understanding of the disposition of mercury in the body, and more specifically in the maternal-fetal unit.
1. Disposition of methylmercury Methylmercury distributes rapidly to all tissues in the body (Fig. 1). Methylmercury consumed in fish is almost completely * Corresponding author at: P.O. Box 2590, Alachua, FL 32616, United States. Tel.: +1 386 418 0894. E-mail address: [email protected] (T. Clarkson). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.02.009
absorbed into the blood stream. Distribution to body tissues and organs is completed in some 30–40 h after a single meal of fish (Kershaw et al., 1980). It is eliminated from the body with a half time of about 70 days corresponding to an excretion rate of 1% of the body burden per 24 h (Miettinen, 1973). Given a whole body half time of 70 days, it will take about 1 year (equivalent to five half times) of regular intake of methylmercury to attain a steady state balance between uptake and excretion of methylmercury. In all epidemiological studies of populations consuming methylmercury in fish it has generally been assumed that a steady state distribution had been established. In the blood stream, it preferentially accumulates in the red blood cells resulting in a red cell to plasma concentration ratio of approximately 20:1 (Fig. 2; Kershaw et al., 1980). The brain to whole blood concentration ratio in adults is in the range of 5:1–10:1. However, this ratio has been measured only in a few volunteer subjects dosed with radioactive methylmercury so little information is available on the variability of this ratio (Miettinen, 1973). Animal data indicate that levels in the fetal brain may be higher than in the corresponding maternal brain (Inouye et al., 1986). In fact, methylmercury is well transported across the placenta although the actual rates of transport are unknown. As in the adult, methylmercury is taken up by the red blood cells. Indeed, in cord blood the proportion found in the red cells is even greater than in adults (Kuhnert et al., 1981). This consideration is also relevant to the role of cord blood as a biological monitoring medium, as discussed further below. Of
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Fig. 1. The disposition of methylmercury in maternal and fetal tissues. Methylmercury is efficiently absorbed from the diet and enters the portal circulation. As it passes through the liver a fraction of the absorbed dose is secreted into the bile and part is reabsorbed back into the blood compartment. Some of that remaining in the GI tract is converted by microflora to inorganic mercury and excreted in the feces. Methylmercury readily enters all tissues and organs and is transported readily across the blood–brain barrier. Its transport is bidirectional except in the case of its avid accumulation into scalp hair where it remains in the hair strand. It crosses the placenta and distributes throughout fetal tissues including the brain. Several fluids and tissues have been used as biological indicators of methylmercury in the fetal brain including fetal cord blood, placental tissue and maternal scalp hair.
the methylmercury in blood plasma, about 1% is complexed with cysteine (CH3Hg-Cys; Fig. 2), and some is also complexed with reduced glutathione (GSH). Other ligands for methylmercury include albumins, hemoglobins, keratins, and tubulins. Scalp hair accumulates methylmercury to a remarkable degree (Fig. 2). On average, the hair to whole blood concentration ratio is 250:1 and the corresponding ratio for plasma mercury is some ten times greater, about 2500:1. Once
incorporated into the hair strand, the concentration of methylmercury remains stable, and thus allows the recapitulation of past exposure from a single sample of hair. Hair grows at an average rate of just over 1 cm per month, such that 1 cm segments can recapitulate past exposure for many months or even years depending on the length of the hair sample (Phelps et al., 1980). As will be discussed later, the ability of hair to concentrate methylmercury and to maintain stable concentrations is an important consideration in evaluating hair as a biological monitoring medium. In terms of the best medium for estimating fetal methylmercury brain levels, a glance at Fig. 1 would suggest that cord blood should be the preferred as it is fetal blood and has direct contact with the fetal brain. Perhaps next in order of preference would be maternal blood as it exchanges methylmercury across the placenta to the fetus. Last in preference might be maternal scalp hair as it is more remote from the fetal brain. However when one considers the known methylmercury transport mechanisms, these conclusions will change and perhaps even reverse. 2. Mechanisms of disposition
Fig. 2. The relative concentrations of methylmercury in whole blood and scalp hair with the concentration in plasma set at unity. Approximately 95% of the methylmercury in whole blood is bound to red blood cells. The brain level is approximately 50 times and the hair level 2500 times greater than the concentration in plasma after methylmercury has attained a steady state of distribution in the body. Methylmercury complexed with the amino acid cysteine, (CH3Hg-Cys) is the species transported into tissues. This complex accounts for only a small fraction of methylmercury in plasma and most of it is bound to mercaptalbumin. Other proteins known to bind methylmercury are the keratins, tubulins and hemoglobin.
The methylmercury cation has a high chemical affinity for thiol ligands (Rabenstein et al., 1982). The reaction depicted in the equation below is rapid and reversible: CH3 Hgþ þ HSR @ CH3 HgSR In view of its high chemical affinity for thiol groups, probably greater than for any other ligand in the cell, it is assumed that
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Fig. 3. Cellular methylmercury (MeHg) uptake and export mechanisms. A major mechanism of MeHg uptake into cells involves neutral amino acid transporters, including LAT1 and LAT2. These proteins have been shown to mediate the uptake of the MeHg-L-cysteine complex (MeHg-Cys) in exchange for an intracellular amino acid. In terms of MeHg export from cells, two major mechanisms have been described. One involves amino acid transporters and the other is mediated by members of the multidrug resistance-associated (MRP) family of proteins. As indicated above, neutral amino acid carriers function as exchangers, and thus can also mediate export of MeHg-Cys, in exchange for an extracellular amino acid. Export of anions such as the MeHg-glutathione complex (MeHg-SG) is mediated by certain members of the MRP family of proteins, in particular MRP1 and MRP2. These membrane proteins mediate the ATP-driven pumping of these anions from the cell into the extracellular space (for a detailed review, see Ballatori, 2002).
methylmercury binds exclusively to thiol-containing molecules.1 In fact all compounds of methylmercury so far identified in tissues are complexes with endogenous thiol containing molecules. The rapid reversibility of the reaction allows the methylmercury cation to move readily from one thiol to another depending, among other things, on local concentrations and differing affinities for the different thiol ligands. The complex of methylmercury with the thiol containing amino acid cysteine plays a key if not exclusive role in the transport of methylmercury into mammalian cells. The structure of the methylmercury-cysteine complex is sufficiently similar to that of the large neutral amino acid, methionine, that it is transported across the cell membrane into the cell on the large neutral amino acid carriers (Fig. 3). The transport carriers are so selective that the complex with the L-optical isomer of cysteine is transported, but not the D-optical isomer (Mokrzan et al., 1995). Appreciable transport also takes place for the complex with the close structural analogue, homocysteine. This process was first discovered in studies of the transport of methylmercury across the blood–brain barrier (Kerper et al., 1992). Since then, transport of methylmercury-cysteine has been shown to occur across the placenta (Kajiwara et al., 1996) and when such amino acid carriers have been experimentally introduced into other recipient cells (Simmons-Willis et al., 2002).
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Once inside the cell, methylmercury is believed to attach in part to the thiol ligand of GSH in view of the high intracellular concentrations of this thiol-containing peptide (Fig. 3). In fact, methylmercury is secreted from liver cells into bile as a complex with GSH on the glutathione carriers (Ballatori and Clarkson, 1982). Although this transport process has not yet been demonstrated for other cells, it is highly likely that it will be universal to all mammalian cells in view of the presence of high levels of intracellular GSH and GSH membrane carriers (Ballatori, 2002). The processes involved in the excretion of methylmercury provide an example of the important role of these thiol complexes in the membrane transport and excretion of methylmercury. Fecal excretion predominates, accounting for about 90% of the total elimination from the body. The process originates with the secretion of methylmercury from liver cells to bile as a complex with GSH (Fig. 1). As the methylmercury moves down the biliary tree and enters the gallbladder, GSH is hydrolyzed to its constituent amino acids by the membranebound ectoenzymes gamma-glutamyltranspeptidase and dipeptidase, resulting in the release of methylmercury as a complex with cysteine (Dutczak et al., 1991). The latter can be absorbed into the portal circulation both in the gallbladder and in the intestines to be returned to the liver to complete the enterohepatic cycle. However some of the methylmercury secreted into the GI tract comes into contact with the microflora that are capable of breaking the carbon mercury bond to release inorganic mercury (Rowland et al., 1978). The latter is poorly absorbed (less then 10%, Rahola et al., 1973) and is therefore mainly excreted in the feces (Fig. 1). Urinary excretion of methylmercury makes a small contribution to total excretion, less than 10%, probably due to the fact that the mobile form of mercury, methylmercurycysteine, is itself readily reabsorbed in the kidney tubules after filtration through the glomerulus. Methylmercury that is directly taken up by the kidney from the peritubular blood is excreted into the tubular fluid as the complex with GSH. As in the biliary tree, the GSH complex is enzymically broken down within the renal tubule, with the release of methylmercury complexed to cysteine that is reabsorbed back into the renal tubular cells (Ballatori et al., 1998). In summary, two major mobile species of methylmercury have been identified. The GSH complex is the intracellular species responsible for transport out of the cell and the cysteine complex is the extra cellular species responsible for entry into the cell. The latter is especially important for biological monitoring as it will determine entry into tissues including the target tissue, the central nervous system, and into tissues responsible for the overall body burden of methylmercury, namely the cells in the enterohepatic system. 3. Indicator media
The selenide ligand Se= or R-Se may have a higher affinity for methylmercury. However the only mercury-selenium compounds identified to date in tissues involve the inorganic mercury cation, Hg2+, in the form of mercuric selenide, HgSe. 1
The majority of studies of prenatal exposure to methylmercury have made use of hair and blood as the biological indicator media of choice. However, there is not universal agreement on the relative merits of these two media. Specifically the question
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arises as to which medium will be more likely to give a better index of levels of methylmercury in the fetal brain, given our current understanding of the disposition of methylmercury in maternal and fetal tissues. 3.1. Whole blood The half time of methylmercury in whole blood in adults has been determined to be 44 days on average (Smith et al., 1994). The half time in brain roughly determined from the decline in radioactivity in the head region in volunteers taking radioactive methylmercury was found to be about 70 days (Aberg et al., 1969). This difference in half-times implies that the brain to blood concentration ratios will be constantly changing until a steady state body burden of methylmercury is attained after approximately 1 year of regular intake of methylmercury. The assumption has been made in all epidemiological studies of fish eating populations that the mother is in steady state with respect to her body burden of methylmercury. Under these conditions, the ratio of methylmercury concentration of blood to brain should be constant. However, this conclusion may not necessarily apply to fetal tissues. The half time in the fetal brain is unknown. Indeed the kinetics of deposition will be complicated by the fact that this organ will be increasing in size at varying rates during gestation. Nevertheless, despite these uncertainties, methylmercury levels in autopsy brains of very young infants do correlate with levels in maternal blood taken following the death of the infant, and with autopsy samples of the infant’s blood (Cernichiari et al., 1995). Unfortunately, no similar data are available for cord blood samples. Measures of methylmercury concentrations in whole blood are affected by the hematocrit and by the formation of microclots as most of the mercury is bound to red blood cells. The delivery room is a busy place and the emphasis is on the health of the mother and infant. Collection of cord blood takes a secondary place and is done by the obstetrician after caring for the mother and infant or by someone not actively helping the patients (i.e. secondary personnel). The timing of cord sampling following delivery varies depending upon the medical situation and is not uniform. The timing may affect the formation of microclots even before the blood is taken. Microclots present at the time of collection or related to timing of processing the blood after collection may affect the measurement of Hg present in the sample. Collection of cord blood can be done by simply taking the top off the tube, inserting the placental end of the umbilical cord into the tube and loosening the clamp. This is often the most rapid and easiest for the obstetrician since minimal equipment is needed. Alternatively, it can be done using a needle and syringe and aspirating from the cord. Aspiration can be from the cord itself, usually on the placental side of the clamps or from vessels on the surface of the placenta itself. The samples are usually collected directly into the tube that is sent to the laboratory. The time interval between collection, transport of the sample to the laboratory, and processing the sample in the laboratory may vary. In the laboratory, care must be exercised
that the aliquot for analysis is representative of the sample. Substantial changes in hematocrit may occur when samples are allowed to sit or microclots have formed and these can affect the mercury measurement. Whole blood contains methylmercury in different chemical complexes (Fig. 2). Approximately 95% of methylmercury in human whole blood is found inside the red blood cells, and most is bound to thiol ligands of hemoglobin. Of the 5% in blood plasma, most is bound to the thiol ligand of mercaptalbumin and only about 1% is bound to cysteine (Ancora et al., 2002; Yasutake et al., 1989). Thus the transportable species of methylmercury accounts for a very small fraction of total methylmercury in whole blood. If whole blood is a measure of the transport species of methylmercury, one must assume that this species is a constant fraction of the total mercury in whole blood. Given the high mobility of methylmercury and its rapidly reversible binding to thiol ligands, this assumption may be true especially for the same individual. However, as discussed above, hematocrits can vary. In addition, binding to hemoglobin might be affected by genetically different homologues of this protein and dietary intake of protein might well affect levels of L-cysteine and other thiols in plasma, as demonstrated experimentally in mice (Adachi et al., 1994). With respect to hemoglobin binding, the brain to blood ratio of methylmercury in different animal species varies by two orders of magnitude (Table 1), due in part to differences in the affinities of the methylmercury cation for the thiol ligands of different hemoglobin proteins. 3.2. Human head hair Studies on uptake into animal fur indicate that methylmercury enters via the follicle and is only accumulated when the follicle is in the growing phase (Shi and Clarkson, 1990). These findings were confirmed in studies of human hair grafted onto athymic nude mice (Zareba et al., submitted). Autoradiographic observations in these same studies demonstrated that radioactive methylmercury was taken up by the keratinocytes and eventually deposited in the high sulfur keratin proteins. Although transport of the methylmercury-cysteine complex on the large neutral amino acid carriers has not been demonstrated specifically for the hair follicle, it is highly likely that this carrier-mediated mechanism is the route of entry. Supporting this hypothesis is the ubiquitous presence of these carriers in mammalian cells and the high demand for amino acids for synthesis of the keratin proteins in hair. In fact it is this active uptake of amino acids in growing hair that accounts for Table 1 Species differences in brain to blood concentration ratiosa Species
Methylmercury
Rat Mouse Squirrel monkey Human
0.06 1.2 6 6
a The concentration ratio is the brain level divided by the corresponding blood level. The data are taken from Suzuki et al. (1963/1973) and Ulfvarson (1962).
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the high hair to plasma concentration ratio in the range to 2500:1 (Fig. 2). The fact that hair is essentially dried tissue may also play a role in the high hair to blood concentration ratio. In summary, the process of uptake of methylmercury into human head hair most likely starts with the transport of the methylmercury-cysteine complex into the keratinocytes, the differentiation and death of these cells during the production of the hair strand, with the ultimate deposition of methylmercury attached to protein thiol ligands. Once incorporated into the hair strand, the mercury level remains stable. In this context, the levels of mercury along the length of the hair strand should provide an historical record of the levels of the transportable species in plasma. It is not surprising therefore that hair levels closely follow whole blood levels in the same individual and over a wide range of concentrations (Fig. 4A and B). We would expect that those factors affecting the distribution of methylmercury in whole blood would be unchanged for the same person whether ingested in pregnancy (Fig. 4A) or in adult males (Fig. 4B). The concentration ratio of hair to whole blood may be expected to show variation from one individual to another. For reasons already discussed, the transportable species of methylmercury may not constitute the same fraction of total mercury in whole blood due to genetic and dietary differences. For example Budtz-Jorgensen et al. (2004) have reported that the ratio of hair to whole blood levels changes with age. These data were obtained from individuals of different ages and may well represent the influence of diet if not genetic differences as well. Such differences in hair to blood ratios have been used as an argument against the use of hair as a biological monitor for fetal brain levels. In contrast, anything that changes this ratio suggests the opposite conclusion, namely that the variance in the distribution of mercury in whole blood binding sites is the most likely explanation for why hair to blood concentration ratios may differ between individuals. The true test of hair as an appropriate biological indicator comes from an examination of the degree of correlation with levels of mercury in the target tissue, the brain. Fig. 5 presents the relationships between levels of mercury in maternal hair versus levels in four anatomical regions of autopsy brains from infants dying shortly after birth. A linear association is seen with correlation coefficients in the range of 0.8. Given the variance that inevitably arises from measures of autopsy tissues as well as the long distance transport and lengthy storage of some of these samples, these correlations are quite striking, and clearly support the use of maternal hair as an indicator of fetal brain levels. The uptake of mercury into hair is apparently selective to methylmercury and does not occur with inorganic mercury2 (Table 2). In several individuals who were highly exposed to inorganic mercury, hair levels were slightly elevated over background, but were one to two orders of magnitude lower than those that would be expected from the same blood levels of
2 The closely related chemical congener, ethylmercury, may also be taken up by hair (Zareba et al., in press).
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Fig. 4. There is a close parallel between the concentration of methylmercury in the diet and mercury in scalp hair in exposed individuals. (A) Mercury levels in a female including the period of her pregnancy represented by the shaded area. She was exposed to dietary mercury derived from homemade bread prepared from seed grain that had been treated with a methylmercury fungicide. Her hair sample was collected in 1973 and was of sufficient length to allow recapitulation of mercury levels back to 1971, a time long before her exposure to MeHg. The peak levels in her hair occurred at admission to hospital when exposure to methylmercury ended. Blood samples were collected in hospital and compared to hair levels (adapted from Amin-Zaki et al., 1976). (B) The concentration of mercury in samples of whole blood and 8 mm segments of scalp hair taken from a volunteer who consumed methylmercury in fish (halibut). The fish consumption took place over a 100-day period. Hair samples were cut close to the scalp, divided into 8 mm segments and plotted according to the growth rate of hair (assumed to be 11.8 mm per month). (The figure is based on data published by Hislop et al., 1983.)
methylmercury. This is a useful attribute of hair as some inorganic mercury may be present in the blood stream of fish eating populations. As discussed previously, some methylmercury is converted to inorganic mercury in the GI tract, whereupon some absorption back into the blood stream may occur. Methylmercury is slowly converted to inorganic mercury in various body tissues and this might also contribute to blood levels of inorganic mercury (Suda et al., 1993). Maternal amalgam tooth fillings also elevate levels of inorganic mercury in both maternal and cord blood (Bjornberg et al., 2003). Thus, the data in Table 2 further support the use of hair as a biological indicator of brain methylmercury levels. Little uptake of inorganic mercury took place into hair. Inorganic
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Fig. 5. The concentrations of mercury in four anatomical areas of the infant brain plotted against the concentration in maternal hair. The infants died soon after birth from various natural causes unrelated to their methylmercury exposure. The maternal hair concentration was measured in the first centimeter next to the scalp collected at the time of the infant’s death. Their exposure to methylmercury was via maternal consumption of ocean fish. The linear regression lines were forced through zero as it was assumed that if maternal levels of methylmercury were zero the levels in the newborn infant must also be zero. Actually when the regression lines were drawn without this restriction, the intercepts were not significantly different than zero. The linear regression coefficients fell in the range of 0.6–0.8 (adapted from data published by Cernichiari et al., 1995).
mercury is known to be poorly transported across the blood– brain barrier (Friberg and Mottet, 1989). In fact, blood levels of methylmercury that correspond to the observed levels of inorganic mercury would have been expected to result in severe brain damage. The fact that these subjects had no neurological signs or symptoms is consistent with findings on the disposition of radioactive inorganic mercury where no radioactivity was detected in the head region (Rahola et al., 1973). Thus it appears that uptake into hair mimics uptake into brain for both organic and inorganic species of mercury. Table 2 The concentrations of mercury indicator media in five individuals after exposure to inorganic mercury and comparison with expected levels if exposure were to methylmercury Subject and exposurea
Blood (ng Hg/ml)
Urine (ng Hg/ml)
Normal values
<3
<2
(1) (2) (3) (4) (5)
400 335 284 54 854
1512 1320 234 210 472
400
25
Metallic Hg Metallic Hg Mercury vapor Mercury vapor Mercuric chloride
Methylmercury a
Hair (mg Hg/g) <1 3 2.4 2.8 2.5 2.7 100
Subjects (1 and 2) exposed to metallic mercury were due to insuflation into the lungs of liquid metallic mercury from the accidental rupture of a Miller– Abbott tube during esophageal dilitation. Subjects 3 and 4 were exposed to mercury vapor by inhalation subject 5) was exposed to mercuric chloride due to a suicide attempt. The data are taken from Myers et al. (submitted) [?]. The data for illustrating what would be expected with methylmercury are based on the published pharmacokinetic parameters for the disposition of methylmercury in adult subjects (WHO, 1976).
In the context of the fieldwork involved in epidemiological studies, the hair sample offers appreciable practical advantages. The collection is non-invasive, transport and storage can be achieved in a postal envelope and no refrigeration is required. A single hair sample can achieve recapitulation of mercury levels over months or even years depending in the length of the hair strands. Non-destructive physical methods such as X-ray fluorescent spectrometry are now available that can measure mercury in single strands of hair. The difference in mercury levels between each strand has been shown to be less than the analytical variance (Toribara, 2001). Some experimental studies have indicated that certain hair treatments might remove mercury or possibly add mercury as a contaminant (Nuttall, 2006; Wakisaka et al., 1990). However, a recent study on mercury levels in samples of hair collected from a large population found no evidence of an effect of hair treatment under actual conditions of use (McDowell et al., 2004). Another argument that has been presented against the use of hair as a biological monitor is that a higher variance was found in maternal hair samples than in the corresponding cord blood samples (Grandjean et al., 2004). As discussed above, hair levels should be an index of the mobile species of mercury in blood. This species will be variable given the factors that influence mercury distribution in blood. The variance in hair levels might well represent the actual changes taking place in the transport species. Variance therefore might actually be interpreted in favor of using hair as the preferred biomarker depending on what assumptions are made. Two of the largest studies of prenatal exposure to methylmercury came to apparently opposite conclusions.
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One study based in the Faroe Islands claimed to find adverse effects on child development (Grandjean et al., 1997) whereas the other study based in the Seychelles Islands found no adverse effects and indeed found evidence that fish consumption might be beneficial despite the concomitant methylmercury intake (Davidson et al., 2006; Myers et al., 2003). It has been claimed that problems with hair measurement such as the higher variance or changes in hair to blood ratio might introduce errors in the independent variable that masks an adverse effect in the Seychelles population. This explanation is unlikely. Both studies have been intensively reviewed by expert committees, who concluded that both were well conducted with state-of-the-art methods. The different outcomes are most likely due to differences in the characteristics of the two populations, the study designs, and the analyses and their interpretations. For example, the average concentration of methylmercury in whale meat, the major source of exposure in the Faroes, is 1.6 mg Hg/g wet weight, whereas the average concentration in the ocean fish consumed in the Seychelles population is 0.31 mg Hg/g wet weight. These different average concentrations indicate that the amount of nutrient food accompanying any given intake of methylmercury is some five times higher in the Seychelles than in the Faroes. In conclusion, the use of maternal hair as an index of fetal brain levels is well justified based on the fact that it offers superior practical advantages and has a firmer physiological base. Acknowledgments This work was supported in part by grants from the National Institute of Environmental Health Sciences, Grant #RO1 ES10219 and in part by Center Grant #P30 ES01247 References Aberg B, Ekman L, Falk R, Greitz U, Persson G, Snihs JO. Metabolism of methyl mercury (203Hg) compounds in man. Arch Environ Health 1969;19(4):478–84. Adachi T, Yasutake A, Hirayama K. Influence of dietary levels of protein and sulfur amino acids on the fate of methylmercury in mice. Toxicology 1994;93(2–3):225–34. Amin-Zaki L, Elhassani S, Majeed MA, Clarkson TW, Doherty RA, Greenwood MR, et al. Perinatal methylmercury poisoning in Iraq. Am J Dis Child 1976;130(10):1070–6. Ancora S, Rossi R, Simplicio PD, Lusini L, Leonzio C. In vitro study of methylmercury in blood of bottlenose dolphins (Turslops truncates). Arch Environ Contam Toxicol 2002;42(3):348–53. Ballatori N. Transport of toxic metals by molecular mimicry. Environ Health Perspect 2002;110(Suppl. 5):689–94. Ballatori N, Clarkson TW. Developmental changes in the biliary excretion of methylmercury and glutathione. Science 1982;2l6:61–3. Ballatori N, Wang W, Lieberman MW. Accelerated methylmercury elimination in gamma-glutamyl transpeptidase-deficient mice. Am J Pathol 1998;152(4):1049–55. Bjornberg KA, Vahter M, Petersson-Grawe K, Glynn A, Cnattingius S, Darnerud PO, et al. Methyl mercury and inorganic mercury in Swedish pregnant women and in cord blood: influence of fish consumption. Environ Health Perspect 2003;111(4):637–41. Budtz-Jorgensen E, Grandjean P, Jorgensen PJ, Weihe P, Keiding N. Association between mercury concentrations in blood and hair in methylmercury-
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