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Perinatal and Lifetime Exposure to Methylmercury in the Mouse: Blood and Brain Concentrations of Mercury to 26 Months of Age
NeuroToxicology 22 (2001) 467±477
Perinatal and Lifetime Exposure to Methylmercury in the Mouse: Blood and Brain Concentrations of Mercury to 26 Months of Age Sander Stern1, Christopher Cox2, Elsa Cernichiari1, Marlene Balys1, Bernard Weiss1,* 1
Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA 2 Department of Biostatistics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA Received 21 September 2000; accepted 22 May 2001
Abstract Chronic, low-level exposures to environmental toxicants, because they often begin prenatally and then persist throughout the individual's lifetime, pose challenging issues to risk assessment. Exposure to low levels of methylmercury through the diet, based largely on consumption of ®sh and sea mammals, follows this pattern. Early development is considered to be a period of heightened vulnerability during which even low-level exposures may produce undetected, ``silent'', damage that is revealed only under conditions that challenge the functional capacities of the individual. Aging, with its diminished functional capacities and compensatory reserves provides such a challenge, but, to explore this possibility, requires basic information about blood and brain levels under conditions of chronic lifetime exposure. The current research was undertaken to provide such information. One hundred female B6C3F1/HSD mice were assigned to one of three dose groups, 0, 1, or 3 ppm methylmercury chloride administered in a 5 nM sodium carbonate drinking solution. They were bred with male CBA/J HSD mice to produce the trihybrid offspring B6C3F1/ HSD CBA/J HSD. Dosing of the females began 4 weeks prior to breeding and continued for the two methylmercuryexposed groups throughout breeding and gestation. The methylmercury-treated litters were split into two subgroups, one exposed throughout its lifetime (set at 26 months) to the original dose, the other exposed through postnatal day 13 (PND 13). Brain and blood concentrations were assayed by cold-vapor atomic absorption. Samples were obtained on PND 4 and 21, and then at the end of months 14 and 26. On PND 4, brain and blood levels closely re¯ected maternal dosing. In all groups, concentrations fell sharply from PND 4 to 21, but to a greater extent in the perinatal groups. Blood levels in the 1 ppm lifetime group remained unchanged between months 14 and 26, but brain levels rose modestly. In the 3 ppm lifetime group, both brain and blood levels rose signi®cantly between months 14 and 26, suggesting an interaction between dose and age. # 2001 Published by Elsevier Science Inc.
Keywords: Methylmercury; Mice; Aging; Prenatal
INTRODUCTION One of the more challenging issues in current risk assessment is how to evaluate the outcome of chronic, low-level exposures to environmental toxicants. Such *
Corresponding author. Tel.: 1-716-275-1736; fax: 1-716-256-2591. E-mail address: [email protected] (B. Weiss).
exposures often begin prenatally and then continue through the lifetime. Methylmercury exposure follows this pattern, especially in populations who depend on ®sh or sea mammals for food. Initial exposures occur in utero, followed by subsequent transfer from the mother by breast-feeding, and then by dietary intake. The present research was designed to generally model this pattern. Neurotoxicity is an established hazard of methylmercury exposure (Clarkson, 1992; Watanabe and
0161-813X/01/$ ± see front matter # 2001 Published by Elsevier Science Inc. PII: S 0 1 6 1 - 8 1 3 X ( 0 1 ) 0 0 0 4 7 - X
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Satoh, 1996; Lebel et al., 1998). Prenatal exposure provokes especially grave concerns, even at relatively low exposure levels, because it can induce persistent effects that have been studied in humans following accidental occupational exposures (Inskip and Piotrowski, 1985), ingestion of seed grain treated with a methylmercury-based fungicide (Amin-Zaki et al., 1974; Marsh et al., 1987), or in connection with consumption of contaminated ®sh due to point sources (Igata, 1993). Other investigations have targeted populations who consume large quantities of ®sh or sea mammals whose methylmercury exposure is a product of broader, even global dispersion patterns (McKeown et al., 1983; Davidson et al., 1998; Grandjean et al., 1998; Crump et al., 1998, 2000). Investigations of neurobehavioral function in these populations have yielded an inconsistent picture of outcomes based on the prevailing exposures. Methylmercury neurotoxicity has received extensive study in laboratory animals (e.g. Spyker, 1975; Burbacher et al., 1990; Reuhl and Chang, 1979; Chang and Reuhl, 1983; Eccles and Annau, 1982; Newland et al., 1994; Bornhausen et al., 1980; Watanabe and Satoh, 1996). Enhanced susceptibility during the period of early development is a consistent feature of this literature. Most developmental inquiries, however, have tended to emphasize the consequences of early exposures alone. Furthermore, laboratory research has typically examined the outcome of a single or a few, relatively high, administered doses. As others have noted (e.g. Berlin and Kacew, 1997), to fully grasp the risks stemming from early developmental exposure requires lifetime observations. One source of concern is the possibility that exposures early in life may produce undetected, latent (Issacson, 1975), or ``silent'' damage (Grant, 1973; Reuhl and Chang, 1979), that emerges only when the functional capacities of the nervous system are challenged by other conditions, such as aging (e.g. Weiss and Simon, 1975; Weiss, 1996; Calne et al., 1986), drugs (e.g. Eccles and Annau, 1982), or complex behavioral situations (e.g. Newland et al., 1994). Delayed neurotoxicity (Weiss and Reuhl, 1994), which refers to an effect absent upon initial exposure but that appears later, has been observed following environmental methylmercury exposures in humans; e.g., ``Minamata disease'' in Japan (Igata, 1993), following consumption of contaminated grain in Iraq (Amin-Zaki et al., 1976; Cox et al., 1989), or experimentally in mice (Spyker, 1975) and in monkeys (Rice, 1996). The current research was provoked by these issues. The overall scheme, of which this was the ®rst phase,
was designed to examine the long-term behavioral consequences of exposure to methylmercury, and to relate those outcomes to both perinatal and lifetime tissue mercury values. Mice were exposed to one of two drinking water concentrations of methylmercury, 1 or 3 ppm, either exclusively during the perinatal period or beginning perinatally and then continuing to 26 months of age. Drinking water provided the medium of exposure, ®rst for the females prior to mating, then continuing through pregnancy and lactation, and ®nally, for half of the exposed offspring, throughout their experimental lifetimes. Mice were chosen as subjects because their brain/blood ratios, close to 1.0 in preliminary studies, are closer to the 3.0:10.0 ratios seen in primates (Rice, 1989; Evans et al., 1977) than the ratios seen in rats, estimated by Magos (1987) as 0.06. The differences between the two rodent species arise from the tendency of rats to sequester metals in red cells. In the rat study by Newland and Reile (1999), brain/blood ratios after perinatal exposure remained well below 1.0. To provide indexes of the attained dose, ®rst during neonatal exposure periods, and then at times that coincided with the behavioral tests administered later, mercury levels in the brain and blood of the offspring were measured on postnatal day 4 (PND 4), at weaning (PND 21), and ®nally at months 14 and 26. Dam blood was also assayed at weaning. Brain and blood levels are reported here. Behavioral assessments will be reported separately. METHODS Subjects One hundred female B6C3F1/HSD mice (source: HSD-Ind 202A; Harlan Sprague±Dawley, IN, USA) were received at age 7±8 weeks. The B6C3F1 mouse was selected because of its extensive use in both aging and toxicology research, and because it is one the strains maintained for the National Institute on Aging. They were split into two groups of 50 mice each. After the ®rst 50 subjects were assigned randomly to 50 different cages, the second 50 were then assigned randomly to the same cages. The 50 pairs of females then were randomly assigned to one of the ®ve dose groups: 0 (0 ppm, control); 1±0 (1 ppm, perinatal only); 1±1 (1 ppm, lifetime); 3±0 (3 ppm, perinatal only); 3±3 (3 ppm, lifetime). Fifty male CBA/J HSD mice (source HSD-FR208; Harlan Sprague±Dawley, IN, USA), obtained as retired breeders, were received 3
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weeks later. This mating produced the trihybrid offspring B6C3F1/HSD CBA/J HSD, which, in the experience of the mouse colony at the University of Rochester, yielded robust, healthy animals. In addition, trihybrid offspring provide greater strain generality than single strain offspring. All housing and experimental procedures were conducted in the University of Rochester Medical Center Vivarium, an AAALACcerti®ed facility, and were approved by the University Committee on Animal Research. Breeding To facilitate breeding, 2 days before being placed with the male assigned to a matched cage number, each pair of females was transferred to the cage of their designated male while the male was transferred to their cage; the cages had not been cleaned for 4 days prior to the switch. These transfers helped establish the odor of the male as a pheromone for entraining the estrous cycle in the female. Drinking solutions were also switched to maintain the methylmercury dosing of the females. On the day scheduled for initial pairing, the male was returned to its original cage, which still housed the assigned females. The drinking solutions were kept on the cages so that the males consumed the same concentration of methylmercury as the females during the brief breeding period. Each morning thereafter, between 07.00 and 08.00 h, each female was inspected for the presence of a vaginal plug, which was indicative of successful mating. That day was designated as gestation day 0 (GD 0). At that time, the female was removed, housed individually in a clean cage, and dosing continued as scheduled. Postnatal day 1 (PND 1) was the ®rst morning (08.30 h) a cleaned litter, i.e. one born the previous night, was observed. Litters were weaned on PND 21 with four (or more) males from each being saved for the behavioral testing. The offspring were initially held in pairs for approximately 25 days, and then each was housed individually. Offspring Selection, Numbering, and Identification Offspring were the trihybrid: B6C3F1/HSD CBA/ J HSD. A hybrid strain was chosen to provide wider generality for the results and because hybrids tend to be more robust than inbred strains, an important consideration for lifetime studies. Litters were culled on PND 4 as follows. First, offspring that weighed less than 75% of the mean weight for that litter were removed. A
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litter was not saved if it consisted of fewer than four males, or if the total offspring numbered less than seven. Then, if the litter contained at least eight pups, males were selected ®rst. Up to six males, and then females were selected to establish a litter size of eight offspring. If a litter contained more than eight offspring, two were saved for mercury assays in brain and blood. Additional female offspring were saved if cross-fostering was required for other litters on that day. In a few instances, when the litter consisted of at least four males, but the total litter size was seven, and if excess females of the same age were available that day from the same dose group, they were added to the litter to maintain a litter size of eight. As a result, in the 3 ppm group only, four females, each from a different litter, were cross-fostered to two perinatal and two lifetime exposure litters. Except for the rules just noted, all selections were random. For the subsequent behavioral studies, only the males were retained because of limited experimental resources, and because males have been shown to be more sensitive than females to methylmercury (Watanabe and Satoh, 1996). For those studies, the four members of a litter were assigned to different tests to avoid litter confounding. At the time of weaning, subjects were identi®ed by small ear markings. When the subjects were later housed individually, they were randomly assigned a pup number. Each of the offspring mice, then, had a unique identi®cation number coded according to treatment, litter number, and pup number. An Implantable Micro-Identi®cation transponder (BioMedic Data Systems, Maywood, NJ), 2 mm diameter 12 mm long, consisting of a biocompatible glass encapsulated, microchip coded with an unalterable 10-digit hexadecimal number, was implanted subcutaneously with a trocar provided with the transponders. No anesthetic was required for the implantation. A BioMedic Data Systems Model DAS 4004 Pocket Scanner was used to read the code; it was programmed to simultaneously read out an additional subject data ®le number assigned to the subject at the time it was selected for one of the two operant behavioral procedures. Each subject's identi®cation was con®rmed prior to each experimental session. Maintenance The mice were housed in polycarbonate cages with wood chip bedding, a wire top holding the food and water bottle, and, above that, a ®lter top. The cages were located in rooms assigned exclusively to this
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experiment. The rooms were maintained at a temperature of 24 2 C, humidity of approximately 50%, and a 12/12 light/dark cycle beginning at 06.00 h. (Prior to weaning, a 14/10 light/dark cycle was scheduled beginning at 05.00 h). One room was used for housing all mice prior to behavioral testing. To facilitate weaning, the food (Purina 5001) was placed in the cage itself beginning on PND 17 and for several days immediately following weaning. While in the holding facility, the body weights were held constant at approximately 32 g by feeding a ®xed amount of food. Such a regimen was designed to prevent obesity and the higher levels of mortality that would be expected in mice permitted free access to food. Just prior to testing, the mice within an age group were moved, under barrier conditions, to an isolation facility consisting of two cubicles and an ante-room. The mice were housed in one of two enclosed cubicles; the behavioral apparatus was located in the adjacent cubicle. Sentinel mice were maintained in both home cage rooms throughout the experiment to ensure the integrity of the facilities, and to assist in evaluating potential sources of health problems should any have emerged within the test±subject populations. Serum samples from the sentinels, collected quarterly by staff from the Division of Laboratory Animal Medicine, were submitted for ELISA for antibodies to mouse hepatitis virus (MHV); the ELISA was always negative. Dosing Females were exposed to the methylmercury drinking solution beginning 2 days after arrival, and continuing for 4 weeks prior to breeding. Drinking ¯uid rather than feed was chosen as the exposure medium for several reasons. First, exposure through diet requires grinding and mixing, which contaminates equipment and presents an environmental hazard for personnel. In addition, drinking solutions can be freshly prepared weekly and the glassware properly cleaned. The methylmercury-treated litters were split into two subgroups, one to be exposed throughout its lifetime to the original dose, the other to be exposed until the morning of PND 13. By PND 13, postnatal mouse brain development, based upon structural and functional criteria, is nearly comparable to the human brain at birth (Rice and Barone, 2000). In addition, because the methylmercury was administered only in the drinking solution on the cage, exposure of the pups during this period would have occurred only by consuming maternal milk until the pups began to consume the
drinking solution itself, which commonly occurs at about PND 14. For dosing, a 2 l, 10 ppm stock solution was prepared weekly. Crystalline methylmercury(II) chloride (CH3HgCl, 95% purity, AESAR/Johnson Matthey Company, Catalog #37123) was added to a 5 nM sodium carbonate buffering solution (Na2CO3, sodium carbonate anhydrous ACS reagent, Sigma #S-1641), which was heated, stirred, and then brought to room temperature. The stock solution was then diluted to produce the requisite quantities of the 1 and 3 ppm dosing solutions. The sodium carbonate solution, prepared each week by adding the sodium carbonate to distilled, ®ltered water, was always used for the dilutions. It also served as the drinking solution for both the 0 ppm control group throughout the entire experiment and the perinatal exposure groups after they were no longer exposed to the methylmercury solution. Plastic bottles with stainless steel caps and extruded spouts were used to administer the methylmercury drinking solutions to the mice. Earlier tests had revealed that bottles with rubber stoppers, which are frequently used for providing drinking solutions to animals, were not satisfactory here because the stoppers became contaminated, and altered the integrity of the solution. A standard cleaning protocol ensured minimal mercury contamination of washed bottles. Mercury Concentration Assays All mercury assays were conducted by the mercury laboratories of the Shared Analytic Facility of the Environmental Health Sciences Center at the University of Rochester. Assays for the current study were based on atomic absorption spectroscopy and provided indices of total mercury. Inorganic and organic species were not analytically separated. Methylmercury Stock Solution A diluted sample of the solution was submitted each week for determining total mercury by a cold-vapor atomic absorption method (Cernichiari et al., 1995). The solutions were usually assayed only for total mercury content. Since additional analyses indicated that the concentration of inorganic mercury in the stock solution remained low over the course of the experiment, this approach was adopted to limit the cost and effort required in conducting the assays. The stock solution was prepared 146 times during the 26 months of exposure. The mean concentration was 10.02 mg/ml (S:D: 0:758 mg/ml), i.e. 10.02 ppm. The minimum concentration was 8.04 mg/ml. On one
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occasion when the offspring were approximately 90 days old, an unacceptably high concentration of 14.66 mg/ml was discovered 2 days after providing the mice with fresh solutions; all methylmercury drinking solutions were replaced immediately with a new one. The maximum concentration seen otherwise was 11.275 mg/ml. Stability of the methylmercury solution over the approximately 1 week period it remained in the bottle on the mouse cage was evaluated in two tests. Total mercury content remained constant, while the amount of inorganic mercury declined from about 2 to less than 1%. Other occasional checks during the experiment con®rmed that the inorganic mercury content of the 10 ppm stock solution was approximately 2% of the total mercury. In a test for residual methylmercury after the standard bottle-washing procedure, a bottle that had contained a 3 ppm solution was ®lled with the sodium carbonate solution and left standing for 10 days. Total mercury, obtained from a sample of the solution was 2.4 ng/ml, or 0.002 ppm, indicating that residual mercury should not have produced signi®cant contamination of the drinking solutions. Blood and Brain Blood and brain samples were obtained from females on PND 4 from a subset of the offspring culled from the litters, and on PND 21 at the time of weaning from a subset of the pups not selected for behavioral testing. The assay samples were taken only from female brains to preserve enough males for subsequent testing. Analyses of a small number of male brains, in litters large enough to provide surplus males, indicated no sex differences at those ages. Brain samples consisting of half brains from individual offspring were pooled, both within and, if necessary, between litters on PND 4 in order to obtain a mass suf®cient for the analysis (0.5 g). Brain weights among dose groups did not differ. For those PND 4 brain samples, no litter contributed to more than one sample. Between-litter pooling occurred twice for the 0 ppm group and once for the 3 ppm, lifetime group. Samples were not obtained on PND 21 from the litters that had been adjusted in size through cross-fostering on PND 4. Blood samples only were obtained on PND 21 from a subset of the dams, which by then had been exposed to the methylmercury solutions for approximately 75 days. Additional samples were obtained at month 14 or 26 from the male mice immediately after they had completed their long-term behavioral testing.
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RESULTS Breeding Outcome Table 1 summarizes the outcome of the breeding and the litter structure. The pregnant females had gained 16±18 g by GD 16. Parturition usually occurred on GD 19. Mean litter size fell between eight and nine pups. Detailed inspection of the data from PND 4 (not shown) indicated that exposure to methylmercury affected neither litter size (7±12 pups), nor body weights and gender distribution. The decrease between the initial number of dams and the ®nal number of litters shown for PND 21 re¯ects the outcome that, for some litters, total offspring number and gender distribution did not meet the earlier speci®ed criteria for inclusion in the study. The 3 ppm dose, for both the perinatal and lifetime groups, produced 14 low-weight (<75% of the litter mean weight) pups across 12 litters as determined on PND 21. Low-weight pups were not seen in the other groups. None of those 12 l were donors or recipients in the few instances of cross-fostering of females that occurred on PND 4. Dam weight gains and pup weights across groups were fairly uniform, indicating that their ¯uid consumption was adequate. Mercury Burden Brain and blood samples were obtained from a total of 56 litters across the four sample days. When the results of the sample fell below the mercury detection limits of the assay, a value equal to the detection limit multiplied by a factor of 0.5 was used to estimate ``missing values'', a common procedure when a value of zero is unreasonable or is unsuitable for log transformations. The detection limits ranged between 0.007 and 0.008 mg/g for brain and 0.002±0.007 mg/g for blood. For statistical analyses, the data were log transformed to normalize the distributions. Geometric means and their geometric standard errors were used to summarize the data. The tissue-concentration data were used to address three questions: (1) How did levels in the offspring of the different treatment groups vary over the duration of the experiment? (2) What was the relation between dam blood concentration and brain and blood concentrations of her pups on PND 21? (3) What was the relation between the blood and brain concentrations of the offspring at the selected assay times? Table 2 and Fig. 1 present the geometric means (S.E.M. in Fig. 1) for brain and blood concentrations
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Table 1 Breeding and litter summary Dose group Controla
1 ppm perinatal
1 ppm lifetimeb
3 ppm perinatal
3 ppm lifetime
Dams
15
15
16
18
18
Dam weight (g) (S.D.) GD 0 GD 16 Gain
22.41 (1.37) 38.53 (4.00) 16.12 (3.19)
23.49 (1.21) 42.16 (2.89) 18.67 (2.19)
23.21 (1.81) 40.42 (2.85) 17.22 (2.30)
22.65 (1.72) 40.13 (3.09) 17.48 (1.95)
23.96 (1.47) 40.22 (2.97) 16.25 (3.06)
Gestation period (days) (S.D.)
19.0 (0.4)
18.9 (0.4)
18.7 (0.4)
18.9 (0.2)
18.9 (0.2)
PND 4: number of offspring (S.D.) Male Female
3.27 (1.28) 5.07 (1.94)
4.71 (1.70) 4.54 (1.31)
4.09 (1.46) 4.82 (1.31)
3.97 (1.56) 4.81 (1.45)
4.11 (1.45) 4.72 (1.41)
3.00 (0.10) 2.97 (0.11)
2.90 (0.05) 2.86 (0.04)
2.91 (0.04) 2.87 (0.04)
2.87 (0.03) 2.83 (0.03)
2.81 (0.04) 2.78 (0.03)
7.47 (0.16) 7.51 (0.17)
7.56 (0.09) 7.56 (0.08)
7.37 (0.08) 7.38 (0.09)
7.38 (0.08) 7.36 (0.08)
7.55 (0.08) 7.51 (0.08)
11.62 (0.20) 11.28 (0.24)
11.44 (0.15) 11.00 (0.17)
11.02 (0.16) 10.68 (0.18)
10.77 (0.11) 10.45 (0.16)
10.90 (0.15) 10.67 (0.14)
Low weights (0.75 mean) PND 4 PND 13 PND 21: pups/litters PND 21: males/females
0 0 0 0
1 female 0 1 0/1
0 0 0 0
0 1 litter 8/7 4c/4
0 0 6/5 3/3
PND 21 summary Litters with four or more males Total litters available Mortality: males/females
6 10 1/1
10 14 0/0
11 16 0/1
11 15 0/1
12 18 2/1
Offspring weight (g) (S.E.M.) PND 4 Male Female PND 13 Male Female PND 21 (excluding low weight) Male Female
a
One dam died on GD 18. One dam died on PND 17. c Two dams died on PND 22. b
and, in Table 2, brain/blood ratios of all methylmercury-exposed offspring for sample days PND 4 and 21. For months 14 and 26, these values are given only for the lifetime exposure groups. Brain/blood ratios are not intrinsically important, but are given here as a pharmacokinetic index showing the relative rates of decline in the two tissues. Rice (1989) found blood levels in monkeys to fall more quickly than brain levels. Such a difference was seen here between PND 4 and 21. Mercury concentrations fell below the detection limit in the 0 ppm group and for the perinatal groups at months 14 and 26. Mercury concentration in dam blood on PND 21 is also shown in Table 2. The general linear models procedure (McCullagh and Nelder, 1989) was used to examine these data. Separate ANOVAs were conducted for brain and blood values. Each analysis yielded P < 0:001 for dose
groups, sample day, and dose group sample day interaction. Data from the different sampling times can be summarized as follows. PND 4 and 21 Brain mercury concentrations averaged across the 3 ppm groups (5.64 mg/g) were 3.52 times higher than those of the 1 ppm groups (1.60 mg/g) on PND 4. The mean blood mercury concentrations of the 3 ppm groups (3.69 mg/g) were 3.41 times higher than those of the 1 ppm groups (1.08 mg/g), so, like brain, were almost linearly related to the drinking water concentrations. Both brain and blood concentrations of all groups declined between PND 4 and 21. The decline was greater in the perinatal than the lifetime groups, an outcome arising from continued access to the drinking solution by the latter offspring after PND 13 as well as continued
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Table 2 Mercury concentration (mg/g)a Day sample
Dose group 1 ppm perinatal
1 ppm lifetime
3 ppm perinatal
3 ppm lifetime
PND 4 Brain Blood Brain/blood ratio
1.63 (7) 1.04 (7) 1.56 (7)
1.58 (4) 1.12 (4) 1.41 (4)
5.62 (6) 3.75 (5) 1.71 (4)
5.65 (3) 3.62 (4) 1.58 (3)
PND 21 Brain Blood Brain/blood ratio
0.19 (4) 0.11 (4) 1.65 (4)
0.46 (4) 0.74 (4) 0.63 (4)
0.45 (4) 0.31 (4) 1.44 (4)
0.86 (4) 1.38 (4) 0.63 (4)
Dam (75) blood
1.50 (4)
6.26 (4)
3.91 (4)
16.03 (4)
Month 14 Brain Blood Brain/blood ratio
0.004 (6) 0.002 (6)
1.20 (8) 2.25 (8) 0.54 (8)
0.005 (8) 0.005 (8)
3.66 (9) 5.77 (9) 0.63 (9)
Month 26 Brain Blood Brain/blood ratio
0.005 (6) 0.003 (6)
1.62 (8) 2.42 (8) 0.67 (8)
0.006 (8) 0.003 (8)
6.59 (9) 9.18 (8) 0.68 (8)
a Geometric means: blood, brain, and brain/blood ratio for offspring across four sample days, and for dam blood on PND 21. Methylmercury exposure of the dams started 75 days preceding PND 21. Sample size is shown in parenthesis. The brain/blood ratio is not shown for the adult perinatal exposure conditions, since most of those mercury concentrations fell at, or below, the limits of detectability.
dosing of their dams, while exposure for the perinatal groups ceased on that day, except for residual dosing via milk from the dam. On PND 4, brain levels were higher than corresponding blood levels, as shown by the brain/ blood ratio. By PND 21, however, the brain/blood ratio had fallen below 1.0 for the lifetime exposure groups and remained in that range for the duration of the study.
months of age, blood levels in the 3 ppm group had risen by nearly 60% and brain levels by almost 100%. These values suggest that, at this age, the mechanisms underlying mercury elimination may have been unable to cope with the higher dose.
Months 14 and 26 Mercury concentrations for most (72%) of the blood and brain samples from the perinatally-exposed offspring fell below detection limits. Between months 14 and 26, as shown in Table 2, mercury levels for both brain and blood increased substantially for the 3 ppm lifetime group. Brain levels increased modestly although signi®cantly for the 1 ppm group, but blood levels changed only slightly. Although, with advancing age, methylmercury might have been mobilized from other tissues, when sampled at month 14, brain concentrations in the two lifetime groups differed almost in proportion to drinking water levels (Fig. 1), indicating that current exposures determined tissue burdens. By month 26, brain levels in the 3 ppm group had risen to four times the levels in the 1 ppm group. Blood level comparisons at 14 months gave a 3 ppm/1 ppm ratio of 2.5. By 26 months, the ratio had increased to 3.8, comparable to the brain ratios. Between 14 and 26
For PND 21 samples, separate analyses of covariance were conducted for the methylmercury-exposed subjects to examine the relation between mercury concentrations in dam blood compared to the concentrations in the brain and blood of her offspring. These data are shown in Fig. 2, which also plots the brain/ blood ratios in offspring.
Dam Blood Influence
Brain Mercury concentration in dam blood was not a signi®cant predictor of brain level in her offspring compared to offspring from other dams administered the same dose (P 0:63). Across doses, however, levels in pup brains increased in correspondence with maternal dose. We therefore conducted an analysis of covariance of dam blood concentration on the groupmean brain concentration of the pups, which was signi®cant (P < 0:001; R2 0:904). The regression line shows the predicted function.
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Fig. 1. Brain and blood concentrations of methylmercury (geometric mean) at different ages and corresponding brain/blood ratios. Error bars indicate geometric S.E.M. Brain/blood ratios are not plotted for months 14 and 26 for the perinatal exposure groups because most of the samples fell below detection limits.
Blood Dam blood mercury concentration was a signi®cant predictor of the concentration in the blood of her pups compared to offspring from other litters (P 0:02; R2 0:96), but there were no adjusted treatment differences (P 0:10); i.e. no departures from parallelism, indicating that the prediction is characterized by a single regression line, as shown in the ®gure. Brain/Blood Ratio Dam blood mercury concentration was a signi®cant predictor of the brain/blood concentration ratio of her pups (P < 0:001; R2 0:86). Although the correlation
Fig. 2. Relationship between methylmercury concentrations in dam blood and blood and brain levels in offspring on PND 21. Brain/blood ratios are also shown. The regression lines plot the predicted function.
between dam blood and offspring brain did not reach signi®cance, the signi®cant relationship between dam and offspring blood could underlie the present ®nding. In addition, there were signi®cant treatment differences. For the ratio, the dam blood concentration was a signi®cant predictor for the ratio versus the period of the dosing (P 0:03; R2 0:86), but not for the ratio versus the level of the dose (P 0:47), as shown by the two regression lines having a common slope. Offspring Brain versus Blood Fig. 3 shows the brain versus blood mercury concentrations for individual pups on PND 21. Within dose
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475
Fig. 3. Brain vs. blood concentrations for individual offspring on PND 21. The regression line plots the predicted function.
groups, brain levels remained nearly constant across the range seen for blood. An analysis of covariance indicated that, within a dose group, pup blood mercury concentration was not a signi®cant predictor of brain concentration (P 0:222), but, over groups, brain concentration depended on dose (P 0:003). DISCUSSION This phase of a larger study still under analysis sought to provide dosing and tissue data for mouse models of methylmercury toxicity both early and late during the lifetime. Epidemiological studies are not equipped to supply such data for humans but mice are a useful species for this purpose because, unlike rats, they do not sequester large amounts of methylmercury in red cells. Magos (1987) calculated the brain/blood ratio in rats to be about 0.06. For adult mice, he gives values of 1.2 and 1.3. The longitudinal design adopted here, comparing continuous and early developmental exposure, was deemed essential for three reasons: (a) methylmercury exposure occurs over a lifetime in populations consuming large amounts of ®sh or sea mammals; (b) exposure con®ned to the period of early
development may exert effects long after exposure has ceased even during senescence (Calne et al., 1986); (c) methylmercury may exhibit different toxicokinetic properties during advanced age than during earlier periods of life, as demonstrated repeatedly by idiosyncratic responses to drugs in the elderly (Roberts and Turner, 1988). The earliest assays of brain and blood concentrations, conducted on PND 4, predominantly re¯ect the contribution of in utero exposure, with a minor proportion of the total coming from lactation. The wide differences between PND 4 and 21 in the perinatal groups testify to the diminished role of lactation compared to gestation as a source of exposure. These results partially con®rm those of Nielsen (1992) and Nielsen and Andersen (1991), whose data from a crossfostering study indicated that mercury from gestational exposure exceeded the amount of mercury deposited by lactational exposure. The authors also concluded that transplacentally delivered mercury undergoes minimal excretion in the neonatal mouse. Their study, however, drew its conclusions from whole-body counting of 203 Hg administered as methylmercury, which can distort interpretations for two reasons. First, in neonates, one pathway of excretion is hair,
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so whole-body counting is inappropriate for de®ning pharmacokinetic variables. Second, the 203 Hg may re¯ect either the inorganic or organic species, and a large proportion of the mercury in maternal milk is in the inorganic form (Amin-Zaki et al., 1981). By PND 21, mercury levels in both the perinatal and lifetime exposure groups had fallen steeply from PND 4 levels, but the latter group exhibited higher values. PND 4 levels mostly re¯ect transplacental exposure; at that age, mice possess little capacity for excretion. By PND 21, in addition to excretion, lowered blood and brain levels re¯ect both dilution by the increase in body mass and the diminished importance of maternal milk as an exposure source. Differences between the perinatal and lifetime groups are attributable to two factors: (1) dosing of the perinatal dams ceased on PND 13, so that levels in their milk would have declined from those of the lifetime dams; (2) between PND 13 and 21, the lifetime offspring were exposed through both lactation and the drinking solution. Such an exposure pattern is similar to that for humans exposed chronically through diet. In mice exposed initially to methylmercury only by lactation, Nielsen and Andersen (1991) observed close to a four-fold increase in total mercury levels shortly after the mice began to consume a methylmercury drinking solution following weaning. The values we measured in the offspring on PND 21 were observed just prior to separating the offspring from the dams. Once these mice had access only to the methylmercury solution as their sole source of ¯uid, their total body levels could well have increased to the levels measured at month 14, close to the four-fold increase determined by Nielsen and Andersen (1991). Mercury brain/blood ratios for both lifetime groups remained comparable at PND 21 and month 26, averaging 0.63. Although our value is lower than the 1.2± 1.3 cited by Magos (1987) in exposed adults, major differences in experimental design preclude identifying the basis for the discrepancy. Two variables, among the many that would most likely affect the ratio are (1) the schedule of dosing, which was chronic in the current study, and (2) the time between administering the ®nal dose and obtaining the sample, which in our case would be on the scale of hours. We would expect that both variables in the current study would act to maintain relatively higher levels in the blood, thereby reducing the brain/blood ratio. For the 1 ppm lifetime group, mercury concentrations in blood remained fairly constant during months 14±26, but brain levels rose slightly and signi®cantly. In the 3 ppm group, both brain and blood values increased substantially from months 14 to 26. Such
an increase suggests an interaction between exposure level and age, but more detailed pharmacokinetic studies are required to con®rm these observations, and to evaluate their potential signi®cance. Newland and Reile (1999) also attempted, in rats, to trace brain±blood relationships arising from perinatal exposure. Female rats received 0, 0.5, or 6 ppm Hg (as methylmercuric chloride, 10 rats per group) in drinking water beginning either 4 or 7 weeks before mating and continuing to PND 16. The experimenters measured blood and whole-brain mercury concentrations on PND 0 and 21 and also recorded daily maternal water consumption during gestation and lactation. Both blood and brain mercury levels in offspring were correlated with ¯uid consumption during gestation, but not during lactation. As in the present study, brain mercury in the neonates decreased sharply between birth and weaning. In our study, brain:blood ratios averaged 1.64 on PND 4 and 1.54 on PND 21 for the two perinatal groups, despite the fall in both brain and blood to roughly 10% of the PND 4 values. In rats, brain:blood ratios averaged 0.14 at birth and 0.24 at weaning, another indication of how strongly mercury is bound by erythrocytes in rat blood. These experimental data con®rm Magos' calculations (1987) about the pharmacokinetic differences between the two rodent species. The primary index of methylmercury neurotoxicity, however, is described by concentrations in the target tissue, the brain. ACKNOWLEDGEMENTS Supported in part by Grants ES-01247 and ES-05433 from the National Institute of Environmental Health Sciences. We thank Dr. Allen H. Gates, who supervised the Inbred Mouse Unit of the Laboratory Animal Services Core of the Environmental Health Sciences Center, for his assistance in selecting the strains of mice used in this study. REFERENCES Amin-Zaki L, Elhassani S, Majeeed MA, Clarkson TW, Doherty RA, Greenwood MR. Intra-uterine methylmercury poisoning in Iraq. Pediatrics 1974;54:587±95. Amin-Zaki L, Elhassani S, Majeeed MA, Clarkson TW. Perinatal methylmercury poisoning in Iraq. Am J Dis Child 1976;130: 1070±6. Amin-Zaki L, Majeed MA, Greenwood MR, Elhassani SB, Clarkson TW, Doherty RA. Methylmercury poisoning in the Iraqi suckling infant: a longitudinal study over 5 years. J Appl Toxicol 1981;1:210±4.
S. Stern et al. / NeuroToxicology 22 (2001) 467±477 Berlin Jr CM, Kacew S. Environmental chemicals in human milk. In: Kacew S, Lambert GH, editors. Environmental toxicology and pharmacology of human development. Washington (DC): Taylor & Francis, 1997. p. 67±93. Bornhausen M, Musch HR, Greim H. Operant behavior performance changes in rats after prenatal methylmercury exposure. Toxicol Appl Pharmacol 1980;56:305±10. Burbacher TM, Rodier PM, Weiss B. Methylmercury developmental neurotoxicity: a comparison of effects in humans and animals. Neurotoxicol Teratol 1990;12:191±202. Calne DB, Eisen A, McGeer E, Spencer P. Alzheimer's disease, Parkinson's disease, and motoneurone disease: abiotrophic interaction between ageing and environment? Lancet 1986;8515(2):1067±70. Cernichiari E, Toribara TY, Liang L, Marsh DO, Berlin M, Meyers GJ, Cox C, Shamlaye CF, Choisy O, Davidson P, Clarkson TW. The biological monitoring of mercury in the Seychelles study. NeuroToxicology 1995;12:613±28. Chang, LW, Reuhl K. Mercury in human and animal health. In: Rose J, editor. Trace elements in health: a review of current issues. Boston: Butterworths, 1983. p. 132±49. Clarkson TW. Mercury: major issues in environmental health. Environ Health Perspect 1992;100:31±8. Cox C, Clarkson TW, Marsh DO, Amin-Zaki L. Dose±response analysis of infants prenatally exposed to methylmercury: an application of a single compartment model to single-strand hair analysis. Environ Res 1989;49:318±32. Crump KS, Kjellstrom T, Shipp AM, Silvers A, Stewart A. Influence of prenatal mercury exposure upon scholastic and psychological test performance: benchmark analysis of a New Zealand cohort. Risk Anal 1998;18:701±13. Crump KS, Van Landingham C, Shamlaye C, Cox C, Davidson PW, Myers GJ, Clarkson TW. Benchmark concentrations for methylmercury obtained from the Seychelles Child Development Study. Environ Health Perspect 2000;108:257±63. Davidson PW, Myers GJ, Cox C, Axtell C, Shamlaye C, SloaneReeves J, Cernichiari E, Needham L, Choi A, Wang Y, Berlin M, Clarkson TW. Effects of prenatal and postnatal methylmercury exposure from fish consumption on neurodevelopment: outcomes at 66 months of age in the Seychelles Child Development Study. JAMA 1998;280:701±7. Eccles CU, Annau Z. Prenatal methyl mercury exposure. II. Alterations in learning and psychotropic drug sensitivity in adult offspring. Neurobehav Toxicol Teratol 1982;4:377±82. Evans HL, Garman RH, Weiss B. Methylmercury: exposure duration and regional distribution as determinants of neurotoxicity in nonhuman primates. Toxicol Appl Pharmacol 1977;41:15±33. Grandjean P, Weihe P, White RF, Debes F. Cognitive performance of children prenatally exposed to ``safe'' levels of methylmercury. Environ Res 1998;77:165±72. Grant CA. Pathology of experimental methylmercury intoxication: some problems of exposure and response. In: Miller MW, Clarkson TW, editors. Mercury, mercurials, and mercaptans. Springfield (IL): Charles C. Thomas, 1973. p. 294±312. Igata A. Epidemiological and clinical features of Minamata disease. Environ Res 1993;63:157±69. Inskip MJ, Piotrowski JK. Review of the health effects of methylmercury. J Appl Toxicol 1985;5:113±33. Issacson RL. The myth of recovery from early brain damage. In:
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Ellis NR, editor. Aberrant development in infancy: human and animal studies. Hillsdale (NJ): Erlbaum, 1975. p. 1±25. Lebel J, Mergler D, Branches F, Lucotte M, Amorim M, Larribe F, Dolbec J. Neurotoxic effects of low-level methylmercury contamination in the Amazonian Basin. Environ Res 1998;79:20±32. Magos L. The absorption, distribution, and excretion of methyl mercury. In: Eccles CU, Annau Z, editors. The toxicity of methyl mercury. Baltimore: The Johns Hopkins University Press, 1987. p. 24±44. Marsh DO, Clarkson TW, Cox C, Myers GJ, Amin-Zaki L, AlTikriti S. Fetal methylmercury poisoning. Relationship between concentration in single strands of maternal hair and child effects. Arch Neurol 1987;44:1017±22. McCullagh P, Nelder JA. Generalized linear models, 2nd ed. London: Chapman & Hall, 1989. McKeown EGE, Ruedy J, Neims A. Methyl mercury exposure in northern Quebec. II. Neurologic findings in children. Am J Epidemiol 1983;118:470±9. Newland MC, Reile PA. Blood and brain mercury levels after chronic gestational exposure to methylmercury in rats. Toxicol Sci 1999;50:106±16. Newland MC, Yezhou S, Logdberg B, Berlin M. Prolonged behavioral effects of in utero exposure to lead or methyl mercury: reduced sensitivity to changes in reinforcement contingencies during behavioral transitions and in steady state. Toxicol Appl Pharmacol 1994;126:6±15. Nielsen JB. The toxicokinetics of mercuric chloride and methylmercuric chloride in mice. J Toxicol Environ Health 1992;37:85±122. Nielsen JB, Andersen O. Methyl mercuric chloride toxicokinetics in mice. I. Effects of strain, sex, route of administration and dose. Pharmacol Toxicol 1991;68:201±7. Reuhl KR, Chang LW. Effects of methylmercury on the development of the nervous system. NeuroToxicology 1979;1:21±55. Rice DC. Brain and tissue levels of mercury after chronic methylmercury exposure in the monkey. J Toxicol Environ Health 1989;27:189±98. Rice DC. Evidence for delayed neurotoxicity produced by methylmercury. NeuroToxicology 1996;17:583±96. Rice D, Barone Jr, S. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect 2000;108(Suppl 3):511±33. Roberts J, Turner N. Pharmacodynamic basis for altered drug action in the elderly. Clin Geriatr Med 1988;4:127±49. Spyker JM. Behavioral teratology and toxicology. In: Weiss B, Laties VG, editors. Behavioral toxicology. New York: Plenum Press, 1975. p. 311±44. Watanabe C, Satoh H. Evolution of our understanding of methylmercury as a health threat. Environ Health Perspect 1996;104:367±79. Weiss B. Long ago and far away: a retrospective on the implications of Minamata. NeuroToxicology 1996;17:257±63. Weiss B, Reuhl K. Delayed neurotoxicity: a silent toxicity. In: Chang LW, editor. Principles of neurotoxicology. New York: Marcel Dekker, 1994. p. 765±84. Weiss B, Simon W. Quantitative perspectives on the long-term toxicity of methylmercury and similar poisons. In: Weiss B, Laties VG, editors. Behavioral toxicology. New York: Plenum Press, 1975. p. 429±35.
Perinatal and Lifetime Exposure to Methylmercury in the Mouse: Blood and Brain Concentrations of Mercury to 26 Months of Age Sander Stern1, Christopher Cox2, Elsa Cernichiari1, Marlene Balys1, Bernard Weiss1,* 1
Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA 2 Department of Biostatistics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA Received 21 September 2000; accepted 22 May 2001
Abstract Chronic, low-level exposures to environmental toxicants, because they often begin prenatally and then persist throughout the individual's lifetime, pose challenging issues to risk assessment. Exposure to low levels of methylmercury through the diet, based largely on consumption of ®sh and sea mammals, follows this pattern. Early development is considered to be a period of heightened vulnerability during which even low-level exposures may produce undetected, ``silent'', damage that is revealed only under conditions that challenge the functional capacities of the individual. Aging, with its diminished functional capacities and compensatory reserves provides such a challenge, but, to explore this possibility, requires basic information about blood and brain levels under conditions of chronic lifetime exposure. The current research was undertaken to provide such information. One hundred female B6C3F1/HSD mice were assigned to one of three dose groups, 0, 1, or 3 ppm methylmercury chloride administered in a 5 nM sodium carbonate drinking solution. They were bred with male CBA/J HSD mice to produce the trihybrid offspring B6C3F1/ HSD CBA/J HSD. Dosing of the females began 4 weeks prior to breeding and continued for the two methylmercuryexposed groups throughout breeding and gestation. The methylmercury-treated litters were split into two subgroups, one exposed throughout its lifetime (set at 26 months) to the original dose, the other exposed through postnatal day 13 (PND 13). Brain and blood concentrations were assayed by cold-vapor atomic absorption. Samples were obtained on PND 4 and 21, and then at the end of months 14 and 26. On PND 4, brain and blood levels closely re¯ected maternal dosing. In all groups, concentrations fell sharply from PND 4 to 21, but to a greater extent in the perinatal groups. Blood levels in the 1 ppm lifetime group remained unchanged between months 14 and 26, but brain levels rose modestly. In the 3 ppm lifetime group, both brain and blood levels rose signi®cantly between months 14 and 26, suggesting an interaction between dose and age. # 2001 Published by Elsevier Science Inc.
Keywords: Methylmercury; Mice; Aging; Prenatal
INTRODUCTION One of the more challenging issues in current risk assessment is how to evaluate the outcome of chronic, low-level exposures to environmental toxicants. Such *
Corresponding author. Tel.: 1-716-275-1736; fax: 1-716-256-2591. E-mail address: [email protected] (B. Weiss).
exposures often begin prenatally and then continue through the lifetime. Methylmercury exposure follows this pattern, especially in populations who depend on ®sh or sea mammals for food. Initial exposures occur in utero, followed by subsequent transfer from the mother by breast-feeding, and then by dietary intake. The present research was designed to generally model this pattern. Neurotoxicity is an established hazard of methylmercury exposure (Clarkson, 1992; Watanabe and
0161-813X/01/$ ± see front matter # 2001 Published by Elsevier Science Inc. PII: S 0 1 6 1 - 8 1 3 X ( 0 1 ) 0 0 0 4 7 - X
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Satoh, 1996; Lebel et al., 1998). Prenatal exposure provokes especially grave concerns, even at relatively low exposure levels, because it can induce persistent effects that have been studied in humans following accidental occupational exposures (Inskip and Piotrowski, 1985), ingestion of seed grain treated with a methylmercury-based fungicide (Amin-Zaki et al., 1974; Marsh et al., 1987), or in connection with consumption of contaminated ®sh due to point sources (Igata, 1993). Other investigations have targeted populations who consume large quantities of ®sh or sea mammals whose methylmercury exposure is a product of broader, even global dispersion patterns (McKeown et al., 1983; Davidson et al., 1998; Grandjean et al., 1998; Crump et al., 1998, 2000). Investigations of neurobehavioral function in these populations have yielded an inconsistent picture of outcomes based on the prevailing exposures. Methylmercury neurotoxicity has received extensive study in laboratory animals (e.g. Spyker, 1975; Burbacher et al., 1990; Reuhl and Chang, 1979; Chang and Reuhl, 1983; Eccles and Annau, 1982; Newland et al., 1994; Bornhausen et al., 1980; Watanabe and Satoh, 1996). Enhanced susceptibility during the period of early development is a consistent feature of this literature. Most developmental inquiries, however, have tended to emphasize the consequences of early exposures alone. Furthermore, laboratory research has typically examined the outcome of a single or a few, relatively high, administered doses. As others have noted (e.g. Berlin and Kacew, 1997), to fully grasp the risks stemming from early developmental exposure requires lifetime observations. One source of concern is the possibility that exposures early in life may produce undetected, latent (Issacson, 1975), or ``silent'' damage (Grant, 1973; Reuhl and Chang, 1979), that emerges only when the functional capacities of the nervous system are challenged by other conditions, such as aging (e.g. Weiss and Simon, 1975; Weiss, 1996; Calne et al., 1986), drugs (e.g. Eccles and Annau, 1982), or complex behavioral situations (e.g. Newland et al., 1994). Delayed neurotoxicity (Weiss and Reuhl, 1994), which refers to an effect absent upon initial exposure but that appears later, has been observed following environmental methylmercury exposures in humans; e.g., ``Minamata disease'' in Japan (Igata, 1993), following consumption of contaminated grain in Iraq (Amin-Zaki et al., 1976; Cox et al., 1989), or experimentally in mice (Spyker, 1975) and in monkeys (Rice, 1996). The current research was provoked by these issues. The overall scheme, of which this was the ®rst phase,
was designed to examine the long-term behavioral consequences of exposure to methylmercury, and to relate those outcomes to both perinatal and lifetime tissue mercury values. Mice were exposed to one of two drinking water concentrations of methylmercury, 1 or 3 ppm, either exclusively during the perinatal period or beginning perinatally and then continuing to 26 months of age. Drinking water provided the medium of exposure, ®rst for the females prior to mating, then continuing through pregnancy and lactation, and ®nally, for half of the exposed offspring, throughout their experimental lifetimes. Mice were chosen as subjects because their brain/blood ratios, close to 1.0 in preliminary studies, are closer to the 3.0:10.0 ratios seen in primates (Rice, 1989; Evans et al., 1977) than the ratios seen in rats, estimated by Magos (1987) as 0.06. The differences between the two rodent species arise from the tendency of rats to sequester metals in red cells. In the rat study by Newland and Reile (1999), brain/blood ratios after perinatal exposure remained well below 1.0. To provide indexes of the attained dose, ®rst during neonatal exposure periods, and then at times that coincided with the behavioral tests administered later, mercury levels in the brain and blood of the offspring were measured on postnatal day 4 (PND 4), at weaning (PND 21), and ®nally at months 14 and 26. Dam blood was also assayed at weaning. Brain and blood levels are reported here. Behavioral assessments will be reported separately. METHODS Subjects One hundred female B6C3F1/HSD mice (source: HSD-Ind 202A; Harlan Sprague±Dawley, IN, USA) were received at age 7±8 weeks. The B6C3F1 mouse was selected because of its extensive use in both aging and toxicology research, and because it is one the strains maintained for the National Institute on Aging. They were split into two groups of 50 mice each. After the ®rst 50 subjects were assigned randomly to 50 different cages, the second 50 were then assigned randomly to the same cages. The 50 pairs of females then were randomly assigned to one of the ®ve dose groups: 0 (0 ppm, control); 1±0 (1 ppm, perinatal only); 1±1 (1 ppm, lifetime); 3±0 (3 ppm, perinatal only); 3±3 (3 ppm, lifetime). Fifty male CBA/J HSD mice (source HSD-FR208; Harlan Sprague±Dawley, IN, USA), obtained as retired breeders, were received 3
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weeks later. This mating produced the trihybrid offspring B6C3F1/HSD CBA/J HSD, which, in the experience of the mouse colony at the University of Rochester, yielded robust, healthy animals. In addition, trihybrid offspring provide greater strain generality than single strain offspring. All housing and experimental procedures were conducted in the University of Rochester Medical Center Vivarium, an AAALACcerti®ed facility, and were approved by the University Committee on Animal Research. Breeding To facilitate breeding, 2 days before being placed with the male assigned to a matched cage number, each pair of females was transferred to the cage of their designated male while the male was transferred to their cage; the cages had not been cleaned for 4 days prior to the switch. These transfers helped establish the odor of the male as a pheromone for entraining the estrous cycle in the female. Drinking solutions were also switched to maintain the methylmercury dosing of the females. On the day scheduled for initial pairing, the male was returned to its original cage, which still housed the assigned females. The drinking solutions were kept on the cages so that the males consumed the same concentration of methylmercury as the females during the brief breeding period. Each morning thereafter, between 07.00 and 08.00 h, each female was inspected for the presence of a vaginal plug, which was indicative of successful mating. That day was designated as gestation day 0 (GD 0). At that time, the female was removed, housed individually in a clean cage, and dosing continued as scheduled. Postnatal day 1 (PND 1) was the ®rst morning (08.30 h) a cleaned litter, i.e. one born the previous night, was observed. Litters were weaned on PND 21 with four (or more) males from each being saved for the behavioral testing. The offspring were initially held in pairs for approximately 25 days, and then each was housed individually. Offspring Selection, Numbering, and Identification Offspring were the trihybrid: B6C3F1/HSD CBA/ J HSD. A hybrid strain was chosen to provide wider generality for the results and because hybrids tend to be more robust than inbred strains, an important consideration for lifetime studies. Litters were culled on PND 4 as follows. First, offspring that weighed less than 75% of the mean weight for that litter were removed. A
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litter was not saved if it consisted of fewer than four males, or if the total offspring numbered less than seven. Then, if the litter contained at least eight pups, males were selected ®rst. Up to six males, and then females were selected to establish a litter size of eight offspring. If a litter contained more than eight offspring, two were saved for mercury assays in brain and blood. Additional female offspring were saved if cross-fostering was required for other litters on that day. In a few instances, when the litter consisted of at least four males, but the total litter size was seven, and if excess females of the same age were available that day from the same dose group, they were added to the litter to maintain a litter size of eight. As a result, in the 3 ppm group only, four females, each from a different litter, were cross-fostered to two perinatal and two lifetime exposure litters. Except for the rules just noted, all selections were random. For the subsequent behavioral studies, only the males were retained because of limited experimental resources, and because males have been shown to be more sensitive than females to methylmercury (Watanabe and Satoh, 1996). For those studies, the four members of a litter were assigned to different tests to avoid litter confounding. At the time of weaning, subjects were identi®ed by small ear markings. When the subjects were later housed individually, they were randomly assigned a pup number. Each of the offspring mice, then, had a unique identi®cation number coded according to treatment, litter number, and pup number. An Implantable Micro-Identi®cation transponder (BioMedic Data Systems, Maywood, NJ), 2 mm diameter 12 mm long, consisting of a biocompatible glass encapsulated, microchip coded with an unalterable 10-digit hexadecimal number, was implanted subcutaneously with a trocar provided with the transponders. No anesthetic was required for the implantation. A BioMedic Data Systems Model DAS 4004 Pocket Scanner was used to read the code; it was programmed to simultaneously read out an additional subject data ®le number assigned to the subject at the time it was selected for one of the two operant behavioral procedures. Each subject's identi®cation was con®rmed prior to each experimental session. Maintenance The mice were housed in polycarbonate cages with wood chip bedding, a wire top holding the food and water bottle, and, above that, a ®lter top. The cages were located in rooms assigned exclusively to this
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experiment. The rooms were maintained at a temperature of 24 2 C, humidity of approximately 50%, and a 12/12 light/dark cycle beginning at 06.00 h. (Prior to weaning, a 14/10 light/dark cycle was scheduled beginning at 05.00 h). One room was used for housing all mice prior to behavioral testing. To facilitate weaning, the food (Purina 5001) was placed in the cage itself beginning on PND 17 and for several days immediately following weaning. While in the holding facility, the body weights were held constant at approximately 32 g by feeding a ®xed amount of food. Such a regimen was designed to prevent obesity and the higher levels of mortality that would be expected in mice permitted free access to food. Just prior to testing, the mice within an age group were moved, under barrier conditions, to an isolation facility consisting of two cubicles and an ante-room. The mice were housed in one of two enclosed cubicles; the behavioral apparatus was located in the adjacent cubicle. Sentinel mice were maintained in both home cage rooms throughout the experiment to ensure the integrity of the facilities, and to assist in evaluating potential sources of health problems should any have emerged within the test±subject populations. Serum samples from the sentinels, collected quarterly by staff from the Division of Laboratory Animal Medicine, were submitted for ELISA for antibodies to mouse hepatitis virus (MHV); the ELISA was always negative. Dosing Females were exposed to the methylmercury drinking solution beginning 2 days after arrival, and continuing for 4 weeks prior to breeding. Drinking ¯uid rather than feed was chosen as the exposure medium for several reasons. First, exposure through diet requires grinding and mixing, which contaminates equipment and presents an environmental hazard for personnel. In addition, drinking solutions can be freshly prepared weekly and the glassware properly cleaned. The methylmercury-treated litters were split into two subgroups, one to be exposed throughout its lifetime to the original dose, the other to be exposed until the morning of PND 13. By PND 13, postnatal mouse brain development, based upon structural and functional criteria, is nearly comparable to the human brain at birth (Rice and Barone, 2000). In addition, because the methylmercury was administered only in the drinking solution on the cage, exposure of the pups during this period would have occurred only by consuming maternal milk until the pups began to consume the
drinking solution itself, which commonly occurs at about PND 14. For dosing, a 2 l, 10 ppm stock solution was prepared weekly. Crystalline methylmercury(II) chloride (CH3HgCl, 95% purity, AESAR/Johnson Matthey Company, Catalog #37123) was added to a 5 nM sodium carbonate buffering solution (Na2CO3, sodium carbonate anhydrous ACS reagent, Sigma #S-1641), which was heated, stirred, and then brought to room temperature. The stock solution was then diluted to produce the requisite quantities of the 1 and 3 ppm dosing solutions. The sodium carbonate solution, prepared each week by adding the sodium carbonate to distilled, ®ltered water, was always used for the dilutions. It also served as the drinking solution for both the 0 ppm control group throughout the entire experiment and the perinatal exposure groups after they were no longer exposed to the methylmercury solution. Plastic bottles with stainless steel caps and extruded spouts were used to administer the methylmercury drinking solutions to the mice. Earlier tests had revealed that bottles with rubber stoppers, which are frequently used for providing drinking solutions to animals, were not satisfactory here because the stoppers became contaminated, and altered the integrity of the solution. A standard cleaning protocol ensured minimal mercury contamination of washed bottles. Mercury Concentration Assays All mercury assays were conducted by the mercury laboratories of the Shared Analytic Facility of the Environmental Health Sciences Center at the University of Rochester. Assays for the current study were based on atomic absorption spectroscopy and provided indices of total mercury. Inorganic and organic species were not analytically separated. Methylmercury Stock Solution A diluted sample of the solution was submitted each week for determining total mercury by a cold-vapor atomic absorption method (Cernichiari et al., 1995). The solutions were usually assayed only for total mercury content. Since additional analyses indicated that the concentration of inorganic mercury in the stock solution remained low over the course of the experiment, this approach was adopted to limit the cost and effort required in conducting the assays. The stock solution was prepared 146 times during the 26 months of exposure. The mean concentration was 10.02 mg/ml (S:D: 0:758 mg/ml), i.e. 10.02 ppm. The minimum concentration was 8.04 mg/ml. On one
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occasion when the offspring were approximately 90 days old, an unacceptably high concentration of 14.66 mg/ml was discovered 2 days after providing the mice with fresh solutions; all methylmercury drinking solutions were replaced immediately with a new one. The maximum concentration seen otherwise was 11.275 mg/ml. Stability of the methylmercury solution over the approximately 1 week period it remained in the bottle on the mouse cage was evaluated in two tests. Total mercury content remained constant, while the amount of inorganic mercury declined from about 2 to less than 1%. Other occasional checks during the experiment con®rmed that the inorganic mercury content of the 10 ppm stock solution was approximately 2% of the total mercury. In a test for residual methylmercury after the standard bottle-washing procedure, a bottle that had contained a 3 ppm solution was ®lled with the sodium carbonate solution and left standing for 10 days. Total mercury, obtained from a sample of the solution was 2.4 ng/ml, or 0.002 ppm, indicating that residual mercury should not have produced signi®cant contamination of the drinking solutions. Blood and Brain Blood and brain samples were obtained from females on PND 4 from a subset of the offspring culled from the litters, and on PND 21 at the time of weaning from a subset of the pups not selected for behavioral testing. The assay samples were taken only from female brains to preserve enough males for subsequent testing. Analyses of a small number of male brains, in litters large enough to provide surplus males, indicated no sex differences at those ages. Brain samples consisting of half brains from individual offspring were pooled, both within and, if necessary, between litters on PND 4 in order to obtain a mass suf®cient for the analysis (0.5 g). Brain weights among dose groups did not differ. For those PND 4 brain samples, no litter contributed to more than one sample. Between-litter pooling occurred twice for the 0 ppm group and once for the 3 ppm, lifetime group. Samples were not obtained on PND 21 from the litters that had been adjusted in size through cross-fostering on PND 4. Blood samples only were obtained on PND 21 from a subset of the dams, which by then had been exposed to the methylmercury solutions for approximately 75 days. Additional samples were obtained at month 14 or 26 from the male mice immediately after they had completed their long-term behavioral testing.
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RESULTS Breeding Outcome Table 1 summarizes the outcome of the breeding and the litter structure. The pregnant females had gained 16±18 g by GD 16. Parturition usually occurred on GD 19. Mean litter size fell between eight and nine pups. Detailed inspection of the data from PND 4 (not shown) indicated that exposure to methylmercury affected neither litter size (7±12 pups), nor body weights and gender distribution. The decrease between the initial number of dams and the ®nal number of litters shown for PND 21 re¯ects the outcome that, for some litters, total offspring number and gender distribution did not meet the earlier speci®ed criteria for inclusion in the study. The 3 ppm dose, for both the perinatal and lifetime groups, produced 14 low-weight (<75% of the litter mean weight) pups across 12 litters as determined on PND 21. Low-weight pups were not seen in the other groups. None of those 12 l were donors or recipients in the few instances of cross-fostering of females that occurred on PND 4. Dam weight gains and pup weights across groups were fairly uniform, indicating that their ¯uid consumption was adequate. Mercury Burden Brain and blood samples were obtained from a total of 56 litters across the four sample days. When the results of the sample fell below the mercury detection limits of the assay, a value equal to the detection limit multiplied by a factor of 0.5 was used to estimate ``missing values'', a common procedure when a value of zero is unreasonable or is unsuitable for log transformations. The detection limits ranged between 0.007 and 0.008 mg/g for brain and 0.002±0.007 mg/g for blood. For statistical analyses, the data were log transformed to normalize the distributions. Geometric means and their geometric standard errors were used to summarize the data. The tissue-concentration data were used to address three questions: (1) How did levels in the offspring of the different treatment groups vary over the duration of the experiment? (2) What was the relation between dam blood concentration and brain and blood concentrations of her pups on PND 21? (3) What was the relation between the blood and brain concentrations of the offspring at the selected assay times? Table 2 and Fig. 1 present the geometric means (S.E.M. in Fig. 1) for brain and blood concentrations
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Table 1 Breeding and litter summary Dose group Controla
1 ppm perinatal
1 ppm lifetimeb
3 ppm perinatal
3 ppm lifetime
Dams
15
15
16
18
18
Dam weight (g) (S.D.) GD 0 GD 16 Gain
22.41 (1.37) 38.53 (4.00) 16.12 (3.19)
23.49 (1.21) 42.16 (2.89) 18.67 (2.19)
23.21 (1.81) 40.42 (2.85) 17.22 (2.30)
22.65 (1.72) 40.13 (3.09) 17.48 (1.95)
23.96 (1.47) 40.22 (2.97) 16.25 (3.06)
Gestation period (days) (S.D.)
19.0 (0.4)
18.9 (0.4)
18.7 (0.4)
18.9 (0.2)
18.9 (0.2)
PND 4: number of offspring (S.D.) Male Female
3.27 (1.28) 5.07 (1.94)
4.71 (1.70) 4.54 (1.31)
4.09 (1.46) 4.82 (1.31)
3.97 (1.56) 4.81 (1.45)
4.11 (1.45) 4.72 (1.41)
3.00 (0.10) 2.97 (0.11)
2.90 (0.05) 2.86 (0.04)
2.91 (0.04) 2.87 (0.04)
2.87 (0.03) 2.83 (0.03)
2.81 (0.04) 2.78 (0.03)
7.47 (0.16) 7.51 (0.17)
7.56 (0.09) 7.56 (0.08)
7.37 (0.08) 7.38 (0.09)
7.38 (0.08) 7.36 (0.08)
7.55 (0.08) 7.51 (0.08)
11.62 (0.20) 11.28 (0.24)
11.44 (0.15) 11.00 (0.17)
11.02 (0.16) 10.68 (0.18)
10.77 (0.11) 10.45 (0.16)
10.90 (0.15) 10.67 (0.14)
Low weights (0.75 mean) PND 4 PND 13 PND 21: pups/litters PND 21: males/females
0 0 0 0
1 female 0 1 0/1
0 0 0 0
0 1 litter 8/7 4c/4
0 0 6/5 3/3
PND 21 summary Litters with four or more males Total litters available Mortality: males/females
6 10 1/1
10 14 0/0
11 16 0/1
11 15 0/1
12 18 2/1
Offspring weight (g) (S.E.M.) PND 4 Male Female PND 13 Male Female PND 21 (excluding low weight) Male Female
a
One dam died on GD 18. One dam died on PND 17. c Two dams died on PND 22. b
and, in Table 2, brain/blood ratios of all methylmercury-exposed offspring for sample days PND 4 and 21. For months 14 and 26, these values are given only for the lifetime exposure groups. Brain/blood ratios are not intrinsically important, but are given here as a pharmacokinetic index showing the relative rates of decline in the two tissues. Rice (1989) found blood levels in monkeys to fall more quickly than brain levels. Such a difference was seen here between PND 4 and 21. Mercury concentrations fell below the detection limit in the 0 ppm group and for the perinatal groups at months 14 and 26. Mercury concentration in dam blood on PND 21 is also shown in Table 2. The general linear models procedure (McCullagh and Nelder, 1989) was used to examine these data. Separate ANOVAs were conducted for brain and blood values. Each analysis yielded P < 0:001 for dose
groups, sample day, and dose group sample day interaction. Data from the different sampling times can be summarized as follows. PND 4 and 21 Brain mercury concentrations averaged across the 3 ppm groups (5.64 mg/g) were 3.52 times higher than those of the 1 ppm groups (1.60 mg/g) on PND 4. The mean blood mercury concentrations of the 3 ppm groups (3.69 mg/g) were 3.41 times higher than those of the 1 ppm groups (1.08 mg/g), so, like brain, were almost linearly related to the drinking water concentrations. Both brain and blood concentrations of all groups declined between PND 4 and 21. The decline was greater in the perinatal than the lifetime groups, an outcome arising from continued access to the drinking solution by the latter offspring after PND 13 as well as continued
S. Stern et al. / NeuroToxicology 22 (2001) 467±477
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Table 2 Mercury concentration (mg/g)a Day sample
Dose group 1 ppm perinatal
1 ppm lifetime
3 ppm perinatal
3 ppm lifetime
PND 4 Brain Blood Brain/blood ratio
1.63 (7) 1.04 (7) 1.56 (7)
1.58 (4) 1.12 (4) 1.41 (4)
5.62 (6) 3.75 (5) 1.71 (4)
5.65 (3) 3.62 (4) 1.58 (3)
PND 21 Brain Blood Brain/blood ratio
0.19 (4) 0.11 (4) 1.65 (4)
0.46 (4) 0.74 (4) 0.63 (4)
0.45 (4) 0.31 (4) 1.44 (4)
0.86 (4) 1.38 (4) 0.63 (4)
Dam (75) blood
1.50 (4)
6.26 (4)
3.91 (4)
16.03 (4)
Month 14 Brain Blood Brain/blood ratio
0.004 (6) 0.002 (6)
1.20 (8) 2.25 (8) 0.54 (8)
0.005 (8) 0.005 (8)
3.66 (9) 5.77 (9) 0.63 (9)
Month 26 Brain Blood Brain/blood ratio
0.005 (6) 0.003 (6)
1.62 (8) 2.42 (8) 0.67 (8)
0.006 (8) 0.003 (8)
6.59 (9) 9.18 (8) 0.68 (8)
a Geometric means: blood, brain, and brain/blood ratio for offspring across four sample days, and for dam blood on PND 21. Methylmercury exposure of the dams started 75 days preceding PND 21. Sample size is shown in parenthesis. The brain/blood ratio is not shown for the adult perinatal exposure conditions, since most of those mercury concentrations fell at, or below, the limits of detectability.
dosing of their dams, while exposure for the perinatal groups ceased on that day, except for residual dosing via milk from the dam. On PND 4, brain levels were higher than corresponding blood levels, as shown by the brain/ blood ratio. By PND 21, however, the brain/blood ratio had fallen below 1.0 for the lifetime exposure groups and remained in that range for the duration of the study.
months of age, blood levels in the 3 ppm group had risen by nearly 60% and brain levels by almost 100%. These values suggest that, at this age, the mechanisms underlying mercury elimination may have been unable to cope with the higher dose.
Months 14 and 26 Mercury concentrations for most (72%) of the blood and brain samples from the perinatally-exposed offspring fell below detection limits. Between months 14 and 26, as shown in Table 2, mercury levels for both brain and blood increased substantially for the 3 ppm lifetime group. Brain levels increased modestly although signi®cantly for the 1 ppm group, but blood levels changed only slightly. Although, with advancing age, methylmercury might have been mobilized from other tissues, when sampled at month 14, brain concentrations in the two lifetime groups differed almost in proportion to drinking water levels (Fig. 1), indicating that current exposures determined tissue burdens. By month 26, brain levels in the 3 ppm group had risen to four times the levels in the 1 ppm group. Blood level comparisons at 14 months gave a 3 ppm/1 ppm ratio of 2.5. By 26 months, the ratio had increased to 3.8, comparable to the brain ratios. Between 14 and 26
For PND 21 samples, separate analyses of covariance were conducted for the methylmercury-exposed subjects to examine the relation between mercury concentrations in dam blood compared to the concentrations in the brain and blood of her offspring. These data are shown in Fig. 2, which also plots the brain/ blood ratios in offspring.
Dam Blood Influence
Brain Mercury concentration in dam blood was not a signi®cant predictor of brain level in her offspring compared to offspring from other dams administered the same dose (P 0:63). Across doses, however, levels in pup brains increased in correspondence with maternal dose. We therefore conducted an analysis of covariance of dam blood concentration on the groupmean brain concentration of the pups, which was signi®cant (P < 0:001; R2 0:904). The regression line shows the predicted function.
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Fig. 1. Brain and blood concentrations of methylmercury (geometric mean) at different ages and corresponding brain/blood ratios. Error bars indicate geometric S.E.M. Brain/blood ratios are not plotted for months 14 and 26 for the perinatal exposure groups because most of the samples fell below detection limits.
Blood Dam blood mercury concentration was a signi®cant predictor of the concentration in the blood of her pups compared to offspring from other litters (P 0:02; R2 0:96), but there were no adjusted treatment differences (P 0:10); i.e. no departures from parallelism, indicating that the prediction is characterized by a single regression line, as shown in the ®gure. Brain/Blood Ratio Dam blood mercury concentration was a signi®cant predictor of the brain/blood concentration ratio of her pups (P < 0:001; R2 0:86). Although the correlation
Fig. 2. Relationship between methylmercury concentrations in dam blood and blood and brain levels in offspring on PND 21. Brain/blood ratios are also shown. The regression lines plot the predicted function.
between dam blood and offspring brain did not reach signi®cance, the signi®cant relationship between dam and offspring blood could underlie the present ®nding. In addition, there were signi®cant treatment differences. For the ratio, the dam blood concentration was a signi®cant predictor for the ratio versus the period of the dosing (P 0:03; R2 0:86), but not for the ratio versus the level of the dose (P 0:47), as shown by the two regression lines having a common slope. Offspring Brain versus Blood Fig. 3 shows the brain versus blood mercury concentrations for individual pups on PND 21. Within dose
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475
Fig. 3. Brain vs. blood concentrations for individual offspring on PND 21. The regression line plots the predicted function.
groups, brain levels remained nearly constant across the range seen for blood. An analysis of covariance indicated that, within a dose group, pup blood mercury concentration was not a signi®cant predictor of brain concentration (P 0:222), but, over groups, brain concentration depended on dose (P 0:003). DISCUSSION This phase of a larger study still under analysis sought to provide dosing and tissue data for mouse models of methylmercury toxicity both early and late during the lifetime. Epidemiological studies are not equipped to supply such data for humans but mice are a useful species for this purpose because, unlike rats, they do not sequester large amounts of methylmercury in red cells. Magos (1987) calculated the brain/blood ratio in rats to be about 0.06. For adult mice, he gives values of 1.2 and 1.3. The longitudinal design adopted here, comparing continuous and early developmental exposure, was deemed essential for three reasons: (a) methylmercury exposure occurs over a lifetime in populations consuming large amounts of ®sh or sea mammals; (b) exposure con®ned to the period of early
development may exert effects long after exposure has ceased even during senescence (Calne et al., 1986); (c) methylmercury may exhibit different toxicokinetic properties during advanced age than during earlier periods of life, as demonstrated repeatedly by idiosyncratic responses to drugs in the elderly (Roberts and Turner, 1988). The earliest assays of brain and blood concentrations, conducted on PND 4, predominantly re¯ect the contribution of in utero exposure, with a minor proportion of the total coming from lactation. The wide differences between PND 4 and 21 in the perinatal groups testify to the diminished role of lactation compared to gestation as a source of exposure. These results partially con®rm those of Nielsen (1992) and Nielsen and Andersen (1991), whose data from a crossfostering study indicated that mercury from gestational exposure exceeded the amount of mercury deposited by lactational exposure. The authors also concluded that transplacentally delivered mercury undergoes minimal excretion in the neonatal mouse. Their study, however, drew its conclusions from whole-body counting of 203 Hg administered as methylmercury, which can distort interpretations for two reasons. First, in neonates, one pathway of excretion is hair,
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so whole-body counting is inappropriate for de®ning pharmacokinetic variables. Second, the 203 Hg may re¯ect either the inorganic or organic species, and a large proportion of the mercury in maternal milk is in the inorganic form (Amin-Zaki et al., 1981). By PND 21, mercury levels in both the perinatal and lifetime exposure groups had fallen steeply from PND 4 levels, but the latter group exhibited higher values. PND 4 levels mostly re¯ect transplacental exposure; at that age, mice possess little capacity for excretion. By PND 21, in addition to excretion, lowered blood and brain levels re¯ect both dilution by the increase in body mass and the diminished importance of maternal milk as an exposure source. Differences between the perinatal and lifetime groups are attributable to two factors: (1) dosing of the perinatal dams ceased on PND 13, so that levels in their milk would have declined from those of the lifetime dams; (2) between PND 13 and 21, the lifetime offspring were exposed through both lactation and the drinking solution. Such an exposure pattern is similar to that for humans exposed chronically through diet. In mice exposed initially to methylmercury only by lactation, Nielsen and Andersen (1991) observed close to a four-fold increase in total mercury levels shortly after the mice began to consume a methylmercury drinking solution following weaning. The values we measured in the offspring on PND 21 were observed just prior to separating the offspring from the dams. Once these mice had access only to the methylmercury solution as their sole source of ¯uid, their total body levels could well have increased to the levels measured at month 14, close to the four-fold increase determined by Nielsen and Andersen (1991). Mercury brain/blood ratios for both lifetime groups remained comparable at PND 21 and month 26, averaging 0.63. Although our value is lower than the 1.2± 1.3 cited by Magos (1987) in exposed adults, major differences in experimental design preclude identifying the basis for the discrepancy. Two variables, among the many that would most likely affect the ratio are (1) the schedule of dosing, which was chronic in the current study, and (2) the time between administering the ®nal dose and obtaining the sample, which in our case would be on the scale of hours. We would expect that both variables in the current study would act to maintain relatively higher levels in the blood, thereby reducing the brain/blood ratio. For the 1 ppm lifetime group, mercury concentrations in blood remained fairly constant during months 14±26, but brain levels rose slightly and signi®cantly. In the 3 ppm group, both brain and blood values increased substantially from months 14 to 26. Such
an increase suggests an interaction between exposure level and age, but more detailed pharmacokinetic studies are required to con®rm these observations, and to evaluate their potential signi®cance. Newland and Reile (1999) also attempted, in rats, to trace brain±blood relationships arising from perinatal exposure. Female rats received 0, 0.5, or 6 ppm Hg (as methylmercuric chloride, 10 rats per group) in drinking water beginning either 4 or 7 weeks before mating and continuing to PND 16. The experimenters measured blood and whole-brain mercury concentrations on PND 0 and 21 and also recorded daily maternal water consumption during gestation and lactation. Both blood and brain mercury levels in offspring were correlated with ¯uid consumption during gestation, but not during lactation. As in the present study, brain mercury in the neonates decreased sharply between birth and weaning. In our study, brain:blood ratios averaged 1.64 on PND 4 and 1.54 on PND 21 for the two perinatal groups, despite the fall in both brain and blood to roughly 10% of the PND 4 values. In rats, brain:blood ratios averaged 0.14 at birth and 0.24 at weaning, another indication of how strongly mercury is bound by erythrocytes in rat blood. These experimental data con®rm Magos' calculations (1987) about the pharmacokinetic differences between the two rodent species. The primary index of methylmercury neurotoxicity, however, is described by concentrations in the target tissue, the brain. ACKNOWLEDGEMENTS Supported in part by Grants ES-01247 and ES-05433 from the National Institute of Environmental Health Sciences. We thank Dr. Allen H. Gates, who supervised the Inbred Mouse Unit of the Laboratory Animal Services Core of the Environmental Health Sciences Center, for his assistance in selecting the strains of mice used in this study. REFERENCES Amin-Zaki L, Elhassani S, Majeeed MA, Clarkson TW, Doherty RA, Greenwood MR. Intra-uterine methylmercury poisoning in Iraq. Pediatrics 1974;54:587±95. Amin-Zaki L, Elhassani S, Majeeed MA, Clarkson TW. Perinatal methylmercury poisoning in Iraq. Am J Dis Child 1976;130: 1070±6. Amin-Zaki L, Majeed MA, Greenwood MR, Elhassani SB, Clarkson TW, Doherty RA. Methylmercury poisoning in the Iraqi suckling infant: a longitudinal study over 5 years. J Appl Toxicol 1981;1:210±4.
S. Stern et al. / NeuroToxicology 22 (2001) 467±477 Berlin Jr CM, Kacew S. Environmental chemicals in human milk. In: Kacew S, Lambert GH, editors. Environmental toxicology and pharmacology of human development. Washington (DC): Taylor & Francis, 1997. p. 67±93. Bornhausen M, Musch HR, Greim H. Operant behavior performance changes in rats after prenatal methylmercury exposure. Toxicol Appl Pharmacol 1980;56:305±10. Burbacher TM, Rodier PM, Weiss B. Methylmercury developmental neurotoxicity: a comparison of effects in humans and animals. Neurotoxicol Teratol 1990;12:191±202. Calne DB, Eisen A, McGeer E, Spencer P. Alzheimer's disease, Parkinson's disease, and motoneurone disease: abiotrophic interaction between ageing and environment? Lancet 1986;8515(2):1067±70. Cernichiari E, Toribara TY, Liang L, Marsh DO, Berlin M, Meyers GJ, Cox C, Shamlaye CF, Choisy O, Davidson P, Clarkson TW. The biological monitoring of mercury in the Seychelles study. NeuroToxicology 1995;12:613±28. Chang, LW, Reuhl K. Mercury in human and animal health. In: Rose J, editor. Trace elements in health: a review of current issues. Boston: Butterworths, 1983. p. 132±49. Clarkson TW. Mercury: major issues in environmental health. Environ Health Perspect 1992;100:31±8. Cox C, Clarkson TW, Marsh DO, Amin-Zaki L. Dose±response analysis of infants prenatally exposed to methylmercury: an application of a single compartment model to single-strand hair analysis. Environ Res 1989;49:318±32. Crump KS, Kjellstrom T, Shipp AM, Silvers A, Stewart A. Influence of prenatal mercury exposure upon scholastic and psychological test performance: benchmark analysis of a New Zealand cohort. Risk Anal 1998;18:701±13. Crump KS, Van Landingham C, Shamlaye C, Cox C, Davidson PW, Myers GJ, Clarkson TW. Benchmark concentrations for methylmercury obtained from the Seychelles Child Development Study. Environ Health Perspect 2000;108:257±63. Davidson PW, Myers GJ, Cox C, Axtell C, Shamlaye C, SloaneReeves J, Cernichiari E, Needham L, Choi A, Wang Y, Berlin M, Clarkson TW. Effects of prenatal and postnatal methylmercury exposure from fish consumption on neurodevelopment: outcomes at 66 months of age in the Seychelles Child Development Study. JAMA 1998;280:701±7. Eccles CU, Annau Z. Prenatal methyl mercury exposure. II. Alterations in learning and psychotropic drug sensitivity in adult offspring. Neurobehav Toxicol Teratol 1982;4:377±82. Evans HL, Garman RH, Weiss B. Methylmercury: exposure duration and regional distribution as determinants of neurotoxicity in nonhuman primates. Toxicol Appl Pharmacol 1977;41:15±33. Grandjean P, Weihe P, White RF, Debes F. Cognitive performance of children prenatally exposed to ``safe'' levels of methylmercury. Environ Res 1998;77:165±72. Grant CA. Pathology of experimental methylmercury intoxication: some problems of exposure and response. In: Miller MW, Clarkson TW, editors. Mercury, mercurials, and mercaptans. Springfield (IL): Charles C. Thomas, 1973. p. 294±312. Igata A. Epidemiological and clinical features of Minamata disease. Environ Res 1993;63:157±69. Inskip MJ, Piotrowski JK. Review of the health effects of methylmercury. J Appl Toxicol 1985;5:113±33. Issacson RL. The myth of recovery from early brain damage. In:
477
Ellis NR, editor. Aberrant development in infancy: human and animal studies. Hillsdale (NJ): Erlbaum, 1975. p. 1±25. Lebel J, Mergler D, Branches F, Lucotte M, Amorim M, Larribe F, Dolbec J. Neurotoxic effects of low-level methylmercury contamination in the Amazonian Basin. Environ Res 1998;79:20±32. Magos L. The absorption, distribution, and excretion of methyl mercury. In: Eccles CU, Annau Z, editors. The toxicity of methyl mercury. Baltimore: The Johns Hopkins University Press, 1987. p. 24±44. Marsh DO, Clarkson TW, Cox C, Myers GJ, Amin-Zaki L, AlTikriti S. Fetal methylmercury poisoning. Relationship between concentration in single strands of maternal hair and child effects. Arch Neurol 1987;44:1017±22. McCullagh P, Nelder JA. Generalized linear models, 2nd ed. London: Chapman & Hall, 1989. McKeown EGE, Ruedy J, Neims A. Methyl mercury exposure in northern Quebec. II. Neurologic findings in children. Am J Epidemiol 1983;118:470±9. Newland MC, Reile PA. Blood and brain mercury levels after chronic gestational exposure to methylmercury in rats. Toxicol Sci 1999;50:106±16. Newland MC, Yezhou S, Logdberg B, Berlin M. Prolonged behavioral effects of in utero exposure to lead or methyl mercury: reduced sensitivity to changes in reinforcement contingencies during behavioral transitions and in steady state. Toxicol Appl Pharmacol 1994;126:6±15. Nielsen JB. The toxicokinetics of mercuric chloride and methylmercuric chloride in mice. J Toxicol Environ Health 1992;37:85±122. Nielsen JB, Andersen O. Methyl mercuric chloride toxicokinetics in mice. I. Effects of strain, sex, route of administration and dose. Pharmacol Toxicol 1991;68:201±7. Reuhl KR, Chang LW. Effects of methylmercury on the development of the nervous system. NeuroToxicology 1979;1:21±55. Rice DC. Brain and tissue levels of mercury after chronic methylmercury exposure in the monkey. J Toxicol Environ Health 1989;27:189±98. Rice DC. Evidence for delayed neurotoxicity produced by methylmercury. NeuroToxicology 1996;17:583±96. Rice D, Barone Jr, S. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect 2000;108(Suppl 3):511±33. Roberts J, Turner N. Pharmacodynamic basis for altered drug action in the elderly. Clin Geriatr Med 1988;4:127±49. Spyker JM. Behavioral teratology and toxicology. In: Weiss B, Laties VG, editors. Behavioral toxicology. New York: Plenum Press, 1975. p. 311±44. Watanabe C, Satoh H. Evolution of our understanding of methylmercury as a health threat. Environ Health Perspect 1996;104:367±79. Weiss B. Long ago and far away: a retrospective on the implications of Minamata. NeuroToxicology 1996;17:257±63. Weiss B, Reuhl K. Delayed neurotoxicity: a silent toxicity. In: Chang LW, editor. Principles of neurotoxicology. New York: Marcel Dekker, 1994. p. 765±84. Weiss B, Simon W. Quantitative perspectives on the long-term toxicity of methylmercury and similar poisons. In: Weiss B, Laties VG, editors. Behavioral toxicology. New York: Plenum Press, 1975. p. 429±35.