* National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709;
ManTech Environmental Technology, Inc., Research Triangle Park, North Carolina 27709;
Research Triangle Institute, Research Triangle Park, North Carolina 27709;
National Cancer Institute at NIEHS, Research Triangle Park, North Carolina 27709;
¶ U.S. Environmental Protection Agency, ORD, NCEA, Washington, District of Columbia 20460; and
|| Neurotoxicology Division, NHEERL, U.S. Environmental Protection Agency, ORD, Research Triangle Park, North Carolina 27709
Received July 25, 2001; accepted November 6, 2001
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ABSTRACT |
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Key Words: mercury vapor; elemental mercury; metallic mercury; inhalation; developmental toxicity; maternal toxicity; fetal toxicity; metallothionein; developmental neurotoxicity.
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INTRODUCTION |
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The general toxicokinetics of inhaled Hg0 have been well studied. Approximately 80% of inhaled Hg0 vapor is retained and absorbed into the blood as it passes through the pulmonary circulation (Hursh et al., 1976). Part of the dissolved Hg0 can be trapped in erythrocytes; however, some Hg0 remains dissolved in the blood long enough for it to be distributed to other tissues. Because it is highly lipid soluble, Hg0 readily diffuses through cell membranes where it is rapidly oxidized by cytosolic catalase-hydrogen peroxide to mercuric mercury (Hg2+), the reactive species for most mercury compounds (Clarkson, 1997
; IPCS, 1991
). Mercuric Hg is highly reactive, and rapidly combines with intracellular ligands such as sulfhydryls, potentially disrupting enzymes and proteins essential to normal organ function.
Considerably less is known about the toxicokinetics of Hg0 vapor in the pregnant female as it relates to fetal development and reproductive outcome. During pregnancy, a number of physiological changes occur that can alter the tissue distribution and therefore the toxicity of xenobiotics. Because it is highly lipid soluble, Hg0 readily penetrates the placental barrier (Clarkson et al., 1972; Khayat and Dencker, 1982
; Lutz et al., 1996
; Warfvinge et al., 1994
; Yoshida et al., 1990
) and is taken up by fetal tissues. As in maternal tissues, oxidation of Hg0 in the fetal tissues converts it to Hg2+, which is much less likely to re-cross the placental barrier. Thus oxidation serves to trap Hg in the tissues leading to accumulation of Hg in the fetus (Baranski and Szymczyk, 1973
). Moreover, Hg from Hg0 vapor can accumulate at higher levels in the fetus than in the mother (Goering, 1992
).
The ability of Hg0 to accumulate in the fetus has led to concerns about potential developmental toxicity resulting from in utero Hg0 exposure. A limited number of poorly documented studies (Laraview, 1956, cited in Baranski and Szymczak, 1973; Steffek et al., 1987) indicate that exposure to Hg0 vapor during pregnancy can cause adverse effects. In animal studies Baranski and Szymczak (1973) reported that rat pups exposed prenatally to Hg0 vapor (approximately 2.5 mg/m3, 6 h/day for 21 days), died within 6 days after birth. These results were due in part to maternal toxicity caused by this high dose of Hg (U.S. EPA, 1984
).
All forms of mercury administered to animals have been shown to result in developmental problems such as spontaneous abortion, stillbirths, and congenital malformations (for review: Barlow and Sullivan, 1982; Schuurs, 1999
). Studies of human exposures to methylmercury have shown that the fetus is uniquely susceptible to methylmercury poisoning (Spyker et al., 1972
). Elemental mercury has been shown to accumulate in the fetus similar to methylmercury; however, relatively little information is available on the toxicity of inhaled Hg0 vapor for the pregnant female and the fetus.
In these studies we investigated the disposition and toxicity of inhaled Hg0 vapor for the pregnant rat and the potential effects on developmental outcomes. Pregnant rats were exposed to a range of Hg0 vapor concentrations to determine whether doses that do not cause maternal toxicity can adversely affect developmental outcomes. Toxicity was characterized and correlated with the tissue dose during and after exposure.
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MATERIALS AND METHODS |
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Animals.
Time-pregnant Long-Evans rats (225 to 250 g; Charles River Breeding Laboratories, Portage, MI) were received on gestation day (GD) 2, where GD 0 is defined as the day mating was confirmed (presence of vaginal plug). Rats were individually housed and food (NIH-07) and water (deionized, filtered tap water) provided ad libitum except during exposure. Feed contained less than 0.025 ppm total Hg. On GD 4 rats were weighed and randomized into control and treatment groups. Body weights of rats were recorded daily beginning on GD 5 through postnatal day (PND) 1. PND 0 was defined as the day of birth (between 0700 and 1600 h), and PND 1 was defined as the day following birth.
This study was conducted under federal guidelines for the use and care of laboratory animals and was approved by the NIEHS and U.S. EPA Animal Care and Use Committees. Animals were housed in a humidity- and temperature-controlled, HEPA-filtered, mass air displacement room in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Animal rooms were maintained with a light-dark cycle of 12 h (light from 0700 to 1900 h). Sentinel animals housed in the animal facility as part of an ongoing surveillance program for parasitic, bacterial, and viral infections were pathogen-free throughout the study.
Mercury Vapor Exposure System
Generation and monitoring.
Elemental mercury vapor was generated by passing conditioned air (HEPA filtered, charcoal-scrubbed, temperature and humidity controlled) through a flask containing 1020 g of Hg0. The flask containing Hg0 was immersed in a temperature-controlled water bath maintained at approximately 2°C above ambient. The resulting Hg0 vapor was diluted and delivered to the exposure system at a controlled rate using mass flow controllers.
Control animals were exposed to conditioned air in a stainless steel, 52-port nose-only exposure system (Lab Products, Rockville, MD). A second nose-only exposure system was used for Hg0 vapor exposures to 1, 2, 4, or 8 mg/m3. The airflow through both systems was maintained at approximately 12 l/min. Each experiment consisted of a group of animals exposed to one Hg0 vapor concentration and a concurrent air-exposed control group. Exposure concentrations were measured from the nose-only system once every 1530 min and air samples from the room, scrubber, and the exposure system enclosure were analyzed once every hour. Air samples were analyzed using a Jerome Model 431-X Mercury analyzer (Arizona Instruments, Phoenix, AR) that is specific for elemental mercury vapor.
Animal exposures.
Pregnant rats were exposed for 10 consecutive days from GD 6 through GD 15 to maximize the exposure of the developing fetus to Hg0. The pregnant rats were exposed by nose-only to avoid contamination of the fur and subsequent oral and dermal exposure to Hg0 vapor. Exposures were limited to 2 h a day to minimize stress caused by restraint during nose-only exposure. Each experiment consisted of one Hg0 concentration group (25 rats) and a concurrent air-exposed control group (25 rats). Pregnant rats were exposed to either Hg0 (1, 2, 4, or 8 mg/m3) or conditioned air (controls).
Maternal body and organ weights.
Five control and 5 Hg0-exposed rats from each exposure cohort were euthanized (CO2 asphyxiation) immediately after Hg0 exposure for 1 (GD 6), 5 (GD 10), and 10 (GD 15) exposures and 1 week after the last exposure (PND 1). Lung, liver, kidney, brain, uterus, and placenta were weighed and stored at 20°C for Hg analyses.
Urinalysis.
Following initial studies examining developmental outcomes, urinalyses were conducted on separate groups of pregnant rats to evaluate potential nephrotoxicity occurring during the 10-day exposures. Five rats per Hg0 exposure concentration were placed in individual metabolism cages with food and water on the day prior to Hg0 exposure (GD 5; controls) and after 1 (GD 6), 5 (GD 10), and 10 (GD 15) exposures. Urine was collected on ice overnight. In the prior study group, exposure to 8 mg/m3 Hg0 resulted in morbidity and early termination of some animals therefore this exposure concentration was not included in the urinalysis study. Samples were analyzed for alkaline phosphatase (AP), N-acetyl glucosaminidase (NAG), glucose, aspartate aminotransferase (AST), total protein, and creatinine using an automated analyzer (Monarch System 2000, Instrumentation Laboratory, Lexington, MA) and commercially available reagents.
Histopathology.
Maternal lung, liver, and kidney were collected on GD 6, 10, 15, and PND 1 for histopathological evaluation of potential Hg0 toxicity. A 23 mm thick section of the left lateral and median lobes of liver were collected and placed in cassettes in formalin. Kidneys were trimmed of peri-renal fat, then the right kidney was bisected crosswise and the left kidney lengthwise through the renal pelvis. One-half of each kidney was fixed in formalin. Lungs were trimmed and perfused with fixative via the trachea. Fixed tissues were mounted in paraffin blocks, sectioned, stained with hematoxylin and eosin, and evaluated by light microscopy.
Developmental toxicity endpoints.
Groups of 5 pregnant rats/concentration were euthanized (CO2 asphyxiation) immediately after air or Hg0 exposure for 1 (GD 6), 5 (GD 10), and 10 (GD 15) exposures. At each time point, the uterus was removed and the numbers of implantation sites and resorptions recorded. PND 0 was defined as the day of birth (between 0700 and 1600 h), and PND 1 was defined as the day following birth. On PND 1, pup body and organ weights and numbers of live and dead pups were recorded.
Fetal and neonatal organ and body weights.
Fetal weights were recorded on GD 10 and 15. Fetal brains and carcasses (carcass without brain) were collected and weighed on GD 15 (up to 10 fetuses/litter). Neonatal brain, kidneys, and liver were collected and weighed on PND 1.
Tissue collection for Hg analyses.
After exposure on GD 6, 10, and 15, and PND 1, 5 pregnant rats/concentration were euthanized by CO2 asphyxiation. Blood samples (23 ml) were collected by cardiac puncture and then separated into plasma and packed cells for Hg analysis. Brain, lungs, liver, kidney, fat, and uterus were collected from treated rats at all time points. Initial experiments demonstrated that tissue Hg levels in control rats did not change significantly throughout the study; therefore, Hg analyses were conducted only on blood and tissues collected from control rats on GD 15.
The following fetal tissues (up to 10 fetuses/litter) were collected and frozen at 20°C for Hg analyses: GD 6 fetuses with attached placentas (pooled 10/litter), GD 10 fetuses, GD 15 fetal brains, carcasses, and placentas (collected separately). Neonates from each of 5 litters/exposure group were randomly selected and euthanized on PND 1. Neonatal brain, liver, and kidney were collected and stored at 20°C until analyzed for total Hg.
Prior to collection of each tissue sample, stainless steel dissection instruments were rinsed in 10% hydrochloric acid and deionized water to reduce cross-contamination with Hg. Tissues were placed in glass, acid-washed vials prepared in a class 100 clean room environment and stored at 20°C until processed for Hg analysis.
Hg analyses.
Tissue samples were weighed, and homogenized in deionized water using a Polytron with a stainless steel probe. The mixing probe was cleaned with deionized water before and after each sample to avoid cross-contamination. Homogenates were digested overnight at 70°C in sealed vials. The next morning, samples were cooled, diluted with concentrated hydrochloric acid (trace metal grade), and neutralized. Digested samples were diluted to volume with deionized water and analyzed for total mercury by cold vapor atomic fluorescence spectrometry (CVAFS) by the method of Stockwell and Corns (1993). Sample preparation methods were developed and validated for each tissue type. The percent recovery from Hg-spiked samples was determined for each tissue. Detection limits varied for each tissue type. All Hg species present in tissues were converted to divalent Hg2+ during sample processing, thus Hg content is expressed as total Hg.
Metallothionein analyses.
Tissue metallothionein was measured in order to investigate its potential roles in Hg distribution and protection from Hg toxicity. Because exposure to 8 mg/m3 resulted in morbidity of dams, and lower concentrations (1 or 2 mg/m3) had no observable effects, metallothionein was measured only in tissues from animals exposed to 4 mg/m3 Hg0 vapor and air controls. Maternal kidney, lung, brain, liver, and uterus were collected on GD 6, 10, 15, PND 1, and PND 15. Placenta was collected on GD 10 and GD 15. Neonatal kidney, lung, brain, and liver were collected on PND 1, 7, 14, and 21. Tissues were weighed, and homogenized, in cold Tris-HCl buffer (pH 7.4, 4°C). The tissue cytosols were prepared by centrifugation at 20,000 x g for 20 min. Total metallothionein content in the cytosols was determined by the Cd/Hemoglobin radioassay of Onasaka et al. (1978) as modified by Eaton and Toal (1982).
Statistical analyses.
Analysis of variance procedures were used to assess the significance of differences (p < 0.5) among gestational days, treatment effects, and the interactions between these factors (Snedecor and Cochran, 1980). Pairwise comparisons were made by Fisher's least significant difference test (Miller, 1966
).
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RESULTS |
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Maternal Tissue and Blood Hg Levels
Kidney.
Of the tissues evaluated, the highest Hg levels were detected in the kidneys of exposed rats (Fig. 2). Kidney Hg concentrations increased in a concentration and time dependent manner and attained levels up to 180 µg/g tissue in the 8 mg/m3 exposure group. At PND 1, 1 week after the last exposure, kidney Hg levels in all dose groups decreased by approximately 30% from peak levels measured on GD 15. The mean kidney Hg concentration from GD 15 control animals was 0.023 µg Hg/g tissue. The minimum detection limit (MDL) was 6.6 ng/g.
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Brain.
Hg levels in maternal brains increased with exposure duration and were proportional to exposure concentration except at the 8 mg/m3 dose group where brain levels were slightly higher than anticipated if a linear relationship existed (Fig. 2). At PND 1, 1 week after the last exposure, brain Hg levels in all dose groups decreased by about 20% from peak levels measured on GD 15. The mean maternal brain Hg concentration from GD 15 control animals was 2.14 ng/g tissue (MDL = 0.432 ng/g).
Liver.
The concentration of Hg in liver of dams exposed to Hg0 vapor from GD 6 to GD 15 increased in a dose- and time-dependent manner (Fig. 2). Liver Hg levels increased in proportion to exposure concentration at all time points. By 1 week after the last exposure (PND 1), liver mercury levels in all dose groups decreased by about 50% from peak levels measured on GD 15. The mean liver Hg concentration from GD 15 control animals was 5.01 ng/g tissue (MDL = 2.56 ng/g).
Uterus.
Hg levels (ng/g) in the uterus increased in a linear manner with increasing Hg0 exposure concentration and duration (Fig. 2). At PND 1 uterine Hg levels in all dose groups decreased by about 64% from peak levels measured on GD 15. The mean uterine Hg concentration in GD 15 control animals was 2.15 ng/g tissue (MDL = 0.44 ng/g).
Adipose.
Total Hg in adipose tissue increased with exposure concentration and days of exposure (Fig. 2). At GD 15, adipose from rats exposed to 8 mg/m3 contained approximately 4-fold more Hg than adipose from rats exposed to 4 mg/m3. At PND 1, 1 week after the last exposure, adipose Hg levels in all dose groups decreased by approximately 64% from peak levels measured on GD 15. The mean adipose Hg concentration from GD 15 control dams was 1.18 ng/g tissue (MDL = 0.59 ng/g).
Packed RBCs.
Total Hg levels in RBCs increased with exposure concentration and duration; however, a maximum level of Hg (about 600 ng/g) was attained after 10 days exposure (GD 15) to 4 mg/m3 and 5 days (GD 10) exposure to 8 mg/m3 (Fig. 2). By 1 week after the last exposure, approximately 80% of Hg was eliminated from the RBCs. The mean Hg concentration in RBCs from GD 15 control dams was 13.5 ng/g packed cells (MDL = 7.0 ng/g).
Plasma.
Blood was separated into plasma and packed erythrocytes (RBCs) before Hg analysis. Plasma contained the lowest Hg levels of all tissues evaluated (Fig. 2). Exposure time- and concentration-dependent increases in Hg levels were observed in plasma. At PND 1, 1 week after the last exposure, plasma mercury levels in all dose groups decreased by about 86% from peak levels measured on GD 15. The mean Hg concentration in plasma from control dams on GD 15 was less than the MDL of 0.133 ng Hg/g.
Placenta.
In treated and control rats, the placental weight increased by about 10-fold between GD 10 and GD 15 (Table 3). In treated rats, Hg levels in the placenta (ng/g) increased in a dose- and time-dependent manner during exposure. Placental Hg concentrations (ng/g tissue) decreased between GD 10 and GD 15; however, because the placental weight increased approximately 10-fold during this time period, the amount of Hg in the placenta (ng Hg/total placenta) also increased approximately 10-fold. The mean placenta Hg concentration from GD 15 control animals was 2.65 ng/g tissue (MDL = 0.255 ng/g).
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Neonatal Tissue Hg Content
Mercury levels (ng total Hg/g tissue) were measured in neonatal brain, liver, and kidney at PND 1 (Fig. 5). In neonatal brain, total Hg increased with increasing exposure concentration to a maximum of about 30 ng/g in pups exposed in utero to 8 mg/m3 Hg0. Total Hg in neonatal kidney increased in a dose-dependent manner attaining about 65 ng/g in the 4 and 8 mg/m3 dose groups. Similarly, in the neonatal liver Hg levels were exposure concentration related and attained a maximum of about 130 ng/g in pups from dams exposed to 8 mg/m3 Hg0.
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DISCUSSION |
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Inhaled Hg0 vapor was distributed throughout the body of pregnant rats and accumulated in a concentration- and time-dependent manner in all tissues evaluated. The distribution of Hg is determined by tissue-specific factors such as perfusion, concentrations of Hg-binding ligands, as well as transport and elimination mechanisms. In addition, during pregnancy, a number of physiological changes occur that can alter the tissue distribution and therefore the toxicity of Hg and other xenobiotics. Changes in pulmonary function, cardiac output, blood distribution, and the rapid growth of the uterus, placenta, and fetus can thus alter the susceptibility of the pregnant female as well as affect pregnancy outcome (Mattison et al., 1991).
The maternal kidneys accumulated the highest Hg levels at all exposure concentrations and time points followed by lung > brain > liver > uterus > adipose > RBCs > plasma. There were no histopathological changes indicative of Hg toxicity in any of the tissues evaluated. However, elevated levels of alkaline phosphatase and protein in the urine of exposed rats suggested the presence of mild nephrotoxicity in these animals. The kidney is the primary depository of Hg after inhalation of Hg0 vapor and nephrotoxicity is a common sign of human Hg intoxication (IPCS, 1991).
Relatively high Hg concentrations were found in the brains of exposed pregnant rats. These high levels of Hg in the maternal brain may in part be attributed to the direct uptake of mercury via the nasal mucosa and retrograde transport transynaptically into the brain (Tjalve and Henriksson, 1999). Studies on the oxidation of Hg0 in blood indicate that because of the short transit time from the lungs to the brain, almost all the Hg0 vapor arrives at the brain unoxidized (Hursh et al., 1980
). The Hg0 readily crosses the blood-brain barrier and is oxidized in the brain to Hg2+, a form that does not easily re-cross the blood-brain membrane. Subsequent intracellular reactions of Hg2+ with sulfhydryl compounds and other ligands lead to an accumulation of Hg in the brain (IPCS, 1991
). Even though there were no overt histopathological lesions, binding of Hg2+ to essential sulfhydryls could disrupt neurological function.
The brain and kidney are the most commonly recognized target organs for Hg0 toxicity. Not only are high concentrations of Hg0 delivered to these tissues, but Hg also appears to be eliminated much more slowly from brain and kidney than from other tissues (Yoshida et al., 1999). By one week after the last exposure, only about 20% of the accumulated Hg was eliminated from the brain and kidney compared to 7080% elimination from other maternal tissues. Previous studies have reported that the half-life for a fraction of the inorganic Hg in the brain is longer than in other organs (Clarkson et al., 1988
; Hursh et al., 1980
; Lind et al., 1988
). Similarly, in the kidney, a fraction of the Hg pool reportedly has a long half-life (Hursh et al., 1976
, 1980
; Kosta et al., 1975
).
The longer half-life of Hg in kidney and brain may be due in part to the relatively high levels of MT in these tissues (Yoshida et al., 1999). Relative to other tissues, kidney and brain contained high levels of inducible MT. The cysteine-rich MT has a high affinity for Hg2+ and does not easily release it. By sequestering Hg2+, MT protects essential cellular macromolecules in the brain and kidney and at the same time increases the tissue Hg levels.
As with the other maternal tissues examined, Hg levels in the uterus increased with exposure concentration and duration. The uterus is a highly perfused organ and accumulated Hg at concentrations similar to those found in liver, but less than the amounts found in kidney, lung, and brain. Unlike most other maternal tissues evaluated, the uterus increased in size during gestation to accommodate the growing fetuses. Uterine weights doubled between GD 10 and GD 15. Because the organ mass rapidly increased during the 10-day Hg exposure, the concentration of Hg in the uterus (ng/g tissue) was expected to decrease because of dilution by the greater organ mass. However, uterine Hg concentrations continued to increase during organ growth indicating that the rate of Hg accumulation was increasing. During pregnancy the blood flow per unit weight of uterus has been shown to remain relatively constant (Beck, 1981). Therefore, it is likely that as the uterine mass increased, perfusion and the amount of Hg reaching the uterus increased proportionately.
The placenta also increases in mass during gestation in order to provide increasing amounts of nutrients and respiratory gases to the developing fetuses. In addition to supplying nutrients, the placenta can also remove toxic compounds before they reach the fetus (Goyer and Cherian, 1992). Mercuric Hg2+ has been shown to accumulate in the placenta, thereby limiting the amount reaching the fetus (Clarkson et al., 1972
; Khayat and Dencker, 1982
). As observed with the uterus, placental growth was accompanied by a proportional increase in placental Hg levels. Exposure to Hg0 had no significant effect on placental weights; this suggests there was no overt toxicity for this tissue. Placental growth includes increased expression of two key proteins: glutathione (Mover and Ar, 1997
) and metallothionein (Itoh et al., 1996
). These proteins bind mercury and other metals and may play a protective role to the fetal compartment (Aschner, 2000
; Aschner and Clarkson, 1988
; Itoh et al., 1996
). Of the maternal tissues analyzed, the placenta contained the highest MT levels. The presence and abundance of these proteins may explain the relatively high mercury tissue levels in placenta.
Developmental toxicity of Hg0 vapor was observed only at the exposure concentration that caused significant maternal toxicity (8 mg/m3). Exposure of pregnant rats to 8 mg/m3 Hg0 caused an increased incidence of resorptions, decreased litter size, and decreased birth weight. Although the total numbers of implantations were not affected by Hg0 exposure, the numbers of resorptions were significantly increased. Resorptions occurred late in gestation and were detected only after the last exposure (GD 15).
The developmental toxicity observed in the high dose group may in part be attributed to maternal toxicity. The significantly reduced weight gain of exposed rats could indirectly result in resorptions and reduced litter size. In addition, impairment of placental function could have contributed to the fetotoxicity. The placenta has many functions that are critical to the fetus, such as maintaining an adequate supply of nutrients and oxygen, elimination of wastes, and elaboration of hormones. Although effects of Hg on placental function have not been demonstrated, disruption of any of these functions could adversely affect pregnancy outcome.
In addition, Hg may directly affect fetal survival. Significant amounts of Hg passed through the placental "barrier" as indicated by the exposure time- and concentration-related increases in fetal Hg content. Fetal body weights were not affected by maternal exposure to the highest Hg0 concentration (data not shown), even though the Hg concentrations in GD 6 and GD 10 fetuses were 2- to 4-fold greater than in maternal plasma.
The concentration of Hg in fetal brain (ng/g tissue) decreased during the week between the last inhalation exposure (GD 15) and birth (PND 1). The decreased fetal brain Hg concentration could be a result of decreasing Hg levels in maternal blood, and/or dilution of the tissue-bound Hg by the increasing mass of the fetal brain. Fetal brain weights increased 10-fold from GD 15 to PND 1. To account for the increasing mass of the fetal brain, the Hg concentration data were also expressed as ng Hg in the whole fetal brain. Interestingly, when expressed as ng/brain, the amount of Hg remained constant from GD 15 to PND 1 indicating that while the fetal brain mass increased 10-fold, the amount of Hg in the fetal brain also increased 10-fold. Because the dams were no longer inhaling Hg vapor, it is likely that Hg was mobilized from maternal tissues and redistributed to the fetuses. During this same time period (GD 15 to PND 1), about 70% of Hg was being eliminated from maternal tissues.
It is important to note that the period in which the amount of Hg increased in the fetal rat brain (GD 15 to PND 1) is a period of active proliferation, migration, and differentiation analogous to the second trimester in humans (Rice and Barone, 2001). While this period is one in which significant accumulation of mercury is occurring in the brain, this accumulation is occurring prior to the major wave of proliferation of astrocytes (Wiggins, 1986). This is important because astocytes normally produce significant amounts of metallothionein (Penkowa et al., 1999
; Yeiser et al., 1999
) and the increase in total metallothionein parallels the postnatal increase of astrocytes in rat brain. Astrocytes may act as sink for mercury following exposure and provide some protection of the neurons (Aschner, 1997
), but if exposure is occurring prior to astrocyte genesis and differentiation, then neurons may not be afforded the same margin of protection.
Significant levels of Hg were still present in the brain, kidney, and liver of newborn pups, even though the inhalation exposure of the dams was terminated about 1 week earlier (GD 15). Neonatal tissue Hg levels were 5- to 10-fold higher in brain and kidney and 6- to 20-fold higher in the liver than in controls. Additional studies are needed to determine how long Hg is retained in these tissues and whether these levels of Hg cause adverse effects in the neonates.
Little is known about the adverse effects of Hg0 vapor exposure during development. Previous studies demonstrated that in utero exposure results in an accumulation of Hg in the developing CNS (Vimy et al., 1990; Warfvinge et al., 1994
; Yoshida et al., 1990
). Effects of Hg0 vapor on development have been characterized mainly by CNS or behavioral disturbances rather than gross malformations (Danielsson et al., 1993
; Newland et al., 1996
). In this study the highest exposure concentration (8 mg/m3) caused increased resorptions and decreased birth weight; however, no gross fetal abnormalities were observed. The next lower concentration (4 mg/m3) caused only mild maternal toxicity and had no effect on resorptions, litter size, or birth weight. These data suggest that Hg has a relatively steep dose-response curve and that the observed developmental effects likely occurred after protective mechanisms were overwhelmed. Elemental Hg exposure may also cause less subtle effects on neonatal behavior; however, this study was designed only to evaluate the effects of Hg0 vapor on development through parturition.
The mechanism by which elemental Hg affects development is not clear. It is known that once oxidized, Hg2+ avidly binds to protein sulfhydryls. Binding of Hg2+ to essential enzymes and proteins produced by the pregnant female or by the conceptus may interfere with development. Whereas the relatively nonspecific binding of high Hg doses by the fetus resulted in resorptions, it is possible that lower exposure concentrations may limit Hg binding to the most susceptible sites and result in more specific developmental effects.
In conclusion, these studies were conducted to characterize the maternal toxicity and adverse effects on developmental outcome of inhaled Hg0 vapor. Inhaled Hg0 vapor was widely distributed and accumulated in a concentration and time-dependent manner in all maternal tissues evaluated. Maternal toxicity occurred primarily during exposure to the highest Hg0 concentration (8 mg/m3). Significant amounts of Hg crossed the placenta and accumulated in the fetus; however, effects on developmental outcome (increased resorptions and decreased birth weight) were observed only at the highest exposure concentration that caused maternal toxicity. Significant levels of Hg accumulated in the fetuses at all exposure concentrations and were still present at birth. Elimination of Hg from the dam resulted in the transfer of Hg to the fetus even after inhalation exposure was discontinued. Additional studies are needed to evaluate potential neurochemical, neuroanatomical, and neurobehavioral effects in neonates exposed to Hg in utero at Hg0 concentrations that do not cause maternal toxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Respiratory Toxicology, Mail Stop IF-00, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. Fax: (919) 541-0356. E-mail: morgand{at}niehs.nih.gov.
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