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Maternal–fetal Distribution of Manganese in the Rat Following Inhalation Exposure to Manganese Sulfate
NeuroToxicology 26 (2005) 625–632
Maternal–fetal Distribution of Manganese in the Rat Following Inhalation Exposure to Manganese Sulfate David C. Dorman*, Anna M. McElveen, Marianne W. Marshall, Carl U. Parkinson, R. Arden James, Melanie F. Struve, Brian A. Wong CIIT Centers for Health Research, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, NC 27709-2137, USA Received 11 June 2004; accepted 9 August 2004 Available online 29 September 2004
Abstract Studies examining the pharmacokinetics of manganese during pregnancy have largely focused on the oral route of exposure and have shown that the amount of manganese that crosses the rodent placenta is low. However, limited information exists regarding the distribution of manganese in fetal tissues following inhalation. The objective of this study was to determine manganese body burden in CD rats and fetuses following inhalation of a MnSO4 aerosol during pregnancy. Animals were evaluated following pre-breeding (2 weeks), mating (up to 14 days) and gestational (from gestation day (GD) 0 though 20) exposure to air or MnSO4 (0.05, 0.5, or 1 mg Mn/m3) for 6 h/day, 7 days/week. The following maternal samples were collected for manganese analysis: whole blood, lung, pancreas, liver, brain, femur, and placenta. Fetal tissues were examined on GD 20 and included whole blood, lung, liver, brain, and skull cap. Maternal lung manganese concentrations were increased following exposure to MnSO4 at 0.05 mg Mn/m3. Maternal brain and placenta manganese concentrations were increased following exposure of pregnant rats to MnSO4 at 0.5 mg Mn/m3. Increased fetal liver manganese concentrations were observed following in utero exposure to MnSO4 at 0.5 mg Mn/m3. Manganese concentrations within all other fetal tissues were not different from air-exposed controls. The results of this study demonstrate that the placenta partially sequesters inhaled manganese, thereby limiting exposure to the fetus.
# 2004 Elsevier Inc. All rights reserved. Keywords: Manganese; Inhalation; Rat; Pregnancy
INTRODUCTION Manganese has been shown to be an essential nutrient in several animal species particularly during gestation and early infancy. Manganese is required for normal amino acid, lipid, protein, and carbohydrate metabolism. Manganese-dependent enzyme families include oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Manganese metalloenzymes include arginase, glutamine synthetase, phos-
* Corresponding author. Tel.: +1 919 558 1350; fax: +1 919 558 1300. E-mail address: [email protected] (D.C. Dorman).
phoenolpyruvate decarboxylase, and manganese superoxide dismutase. No formal recommended dietary allowance (RDA) for manganese has been established, but the U.S. National Research Council has established an estimated safe and adequate dietary intake (ESADI) of 2–5 mg/day for adults (Greger, 1998). Lactation and gestation are thought to increase the manganese requirement (NAS, 2001). Pregnant women and infants often have elevated blood manganese concentrations (Spencer, 1999). Studies examining whole blood concentrations of manganese during pregnancy show an increase throughout gestation, and iron supplementation of pregnant women with normal iron status does not significantly change blood manganese concentra-
0161-813X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2004.08.004
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tions despite the sharing of some transport proteins by manganese and iron (Tholin et al., 1995). In excess, manganese can accumulate within the human striatum and globus pallidus and produce damage to dopaminergic neurons within these sites (Malecki et al., 1999; Nelson et al., 1993; Pal et al., 1999). Manganese neurotoxicity most commonly results from the chronic inhalation exposure of high (>1 mg/m3) manganese concentrations, as may occur during certain mining operations. Whether chronic exposure to much lower airborne manganese concentrations can contribute to the incidence of human neurological disease is the subject of discussion within the scientific community. Most manganese inhalation pharmacokinetic studies have been conducted in adult animals and have typically exposed rodents to high concentrations of manganese oxides (for a review, see ATSDR, 1992). Manganese pharmacokinetic studies involving potentially sensitive subpopulations (e.g., developing rodents) have been less frequently performed. Rodent studies using oral gestational exposure have demonstrated that the amount of manganese that crosses the placenta is low (Kontur and Fechter, 1985). Despite this apparent barrier, fetotoxicity (reduced fetal body weight or morphological defects) in the absence of maternal toxicity has been reported to occur in mice exposed orally to high levels of manganese (Colomina et al., 1996; Sanchez et al., 1993). To our knowledge, pharmacokinetic data on inhaled manganese in pregnant animals are not available. The objective of this study was to determine manganese body burden in CD rats following MnSO4 inhalation during pregnancy.
pregnant female rats (sperm positive = gestation day (GD) 0) were exposed to air or MnSO4 from GD 0 through GD 20. Immediately after the end of the 6-h exposure on GD 20, presumed pregnant rats were killed (n = 7 pregnant rats/exposure concentration were evaluated) and maternal and fetal tissue manganese concentrations determined. Manganese concentrations were determined for two randomly selected fetuses/ litter while tissues from one additional randomly selected fetus/litter were retained. Chemicals Manganese(II) sulfate monohydrate (MnSO4H2O) was obtained from Aldrich Chemical Company Inc. (Milwaukee, WI) with a certified purity of >98%. The material is a relatively water-soluble, white to pale pink crystalline powder that contains 32% Mn by weight. Animals This study was conducted under federal guidelines for the care and use of laboratory animals (National Research Council, 1996) and was approved by the CIIT Institutional Animal Care and Use Committee. Sixweek old male and female Crl:CD1(SD) BR rats were purchased from Charles River Laboratories Inc. (Raleigh, NC). Randomization of animals to treatment groups occurred prior to the start of the inhalation exposure and was based upon a weight randomization procedure. Animals were acclimated for approximately 2 weeks prior to the start of the inhalation exposure. Animal Husbandry
MATERIALS AND METHODS Experimental Design Rats were used to evaluate maternal and fetal tissue manganese concentrations following inhalation exposure throughout the majority of gestation. This experiment consisted of 10 male and 10 female CD rats per exposure group (F0 male and female rats) that were exposed to air or MnSO4 beginning 28 days prior to breeding and for up to 14 days during the mating period. Animals were exposed to either air or MnSO4 (nominal concentrations were 0.05, 0.5, or 1 mg Mn/ m3) for 6 h/day, 7 days/week. Male F0 rats were killed by carbon dioxide (CO2) asphyxiation shortly after the completion of the 2-week breeding period and tissues were not collected from these animals. Presumed
Animal rooms were maintained at daily temperatures of 17–23 8C, relative humidity of 30–70%, and an air flow rate sufficient to provide 10–15 air changes per hour. To the extent possible, the exposure chambers were maintained at daily temperatures 18–26 8C, relative humidity of 30–70%, and an air flow rate sufficient to provide at least 12 air changes per hour. Fluorescent lighting was controlled by automatic controls (lights on approximately 0700–1900). All animals were housed in CIIT’s animal facility which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Except during inhalation exposure and breeding periods, male and female F0 rats were individually housed in polycarbonate cages containing cellulose fiber chip bedding (ALPHA-driTM; Shepherd Specialty Papers, Kalama-
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zoo, MI). Animal breedings (typically 1 male:1 female) were conducted overnight in polycarbonate cages containing cellulose fiber chip bedding. A pelleted, semi-purified AIN-93G certified diet from Bio-Serv (Frenchtown, NJ) formulated to contain approximately 10 ppm manganese and 35 ppm iron was given throughout the study. Food was available to all animals ad libitum except during inhalation exposures. Reverse osmosis purified water containing <0.222–0.546 mg Mn/L and 1.31–13.01 mg Fe/L was available ad libitum. Detailed individual animal clinical examinations were conducted and recorded at least once weekly throughout the course of the study beginning at the start of the inhalation exposures. Adult male (F0) body weights were measured and recorded at least weekly. Maternal body weights were measured and recorded at least weekly and on GD 0, 7, 14, 18. All animals had individual body weights determined at necropsy. Animals were mated at night (female added to male’s cage after each daily exposure, then removed for the next exposure day) for a period of up to 14 days, with no change in mating partners. Females were examined daily after the cohabitation period for the presence of plug/sperm in the vaginal tract. The observation of a copulation plug or sperm-positive smear was considered evidence of successful mating (sperm positive), and the day was designated GD 0. The sperm-positive female and male from that mating pair were then individually housed, and gestational exposures begun. Manganese Exposure Exposures were conducted for 6 h/day, 7 days/week. Rats were exposed in stainless steel wire cage units contained in four 8-m3 Hinners-style, stainless steel and glass inhalation exposure chambers (Lab Products, Maywood, NJ). Air flow through the chambers was adjusted to assure at least 12 air changes per hour (approximately 1600–2600 L/min). Temperature and relative humidity inside the 8-m3 chamber were monitored continuously and recorded every 30 min during each exposure. The average mean daily chamber temperature was maintained at 22 4 8C and relative humidity was maintained at 30–70%. Animal positions within the exposure chambers were rotated during the experiment to minimize experimental error due to any undetected differences in the environment or the manganese aerosol concentration. Exposure atmospheres were generated by aerosolizing MnSO4 using a dry powder generator (Wright Dust Feeder, Model
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WDF-II, BGI Inc., Waltham, MA). Separate generation systems were used for each target exposure concentration. The MnSO4 was packed in a dry powder generation cup under a pressure of 1500–3000 psi. The MnSO4 particles were carried from the dry powder generator through a single, 38-L mixing/settling chamber or through two 38-L mixing/settling chambers. An 85Kr discharging unit was suspended in the primary mixing/settling chamber for each generation system. Exposure atmospheres were measured with optical particle sensors (Real-Time Aerosol Sensors, Model RAM-S, MIE Inc., Billerica, MA). An optical particle sizing spectrometer (Aerodynamic Particle Sizer, Model 3320, TSI Inc., St. Paul, MN) was used to measure particle size distribution. Gravimetric filter samples were used to verify the optical particle sensor readings. Oxygen was monitored in each chamber one time during the study with an MDA Oxygen Sensor (Model 3300, MDA Scientific, Lincolnshire, IL). Necropsy Procedure Presumed pregnant rats were euthanized on GD 20 via CO2 asphyxiation and fetuses were killed by decapitation. Maternal (1–2 ml) blood was collected from the thoracic aorta or heart, and fetal trunk blood (>100 ml) was collected immediately after euthanasia. Fetal blood samples were pooled in order to provide an adequate sample size for manganese analysis. An aliquot of the heparinized maternal (500 ml) or fetal (100 ml) blood was placed into a plastic tube for manganese analysis. The following maternal samples were collected for manganese analysis: whole blood, lung, pancreas, liver, brain, femur, and placenta. The following fetal (n = 2 fetuses/litter) samples were collected for manganese analysis: whole blood, lung, liver, brain, and skull cap. Samples were transferred to plastic vials, frozen in liquid nitrogen, and stored at approximately 80 8C until manganese analysis. Tissue samples were thawed and weighed prior to transfer to 3-ml digestion vessels. Samples (10–30 mg) were digested in 16 M nitric acid prior to manganese analysis using a CEM MARS5 Microwave Accelerated Reaction System (CEM, Matthews, NC). Tissue manganese concentrations were determined by graphite furnace atomic absorption spectrometry (GFAAS) with a Perkin-Elmer AAnalyst 800 Atomic Absorption spectrometer equipped with AA WinLab software (version 4.1 SP). The analyte was identified using a manganese-specific hollow cathode lamp by the presence
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of an absorbance signal at the manganese analytical wavelength of 279.5 nm. Quantitation of the analyte (mg Mn/g tissue wet weight) was carried out using an external calibration curve and the AA WinLab software. Palladium nitrate and magnesium nitrate were used as matrix modifiers for all samples. The performance evaluation sample for this study was Bovine Liver Reference Standard, containing 0.1 mg Mn/mL (High Purity Standards, Charleston, SC) with a certified analysis for manganese. An additional manganese standard (calibration check standard) was prepared at a mid-level of the calibration curve using Manganese Reference Standard, 1000 mg/mL in 2% HNO3 (High Purity Standards, Charleston, SC) to evaluate the accuracy of the calibration curve. All samples were analyzed at least twice by the GFAAS. Statistics The data for quantitative, continuous variables were compared for the exposure and control groups by tests for homogeneity of variance (Levene’s test), analysis of variance (ANOVA), and Dunnett’s multiple comparison procedure for significant ANOVA. In the event the Levene’s test was significant, then the data were transformed using a natural log (ln) transformation. If the Levene’s test was significant following transformation, then the original data were analyzed by nonparametric statistics (Wilcoxon or Kruskal–Wallis). Individual data that appeared to be outliers were critically evaluated using a Dixon-type test for discordancy for an upper outlier (Barnett and Lewis, 1984). Statistical analyses were performed using JMP software from SAS Institute Inc. (Cary, NC). A probability value of 0.01 was used for Levene’s test, while p < 0.05 was used as the critical level of significance for all other statistical tests. Unless otherwise noted, data presented are mean values standard error of the mean (S.E.M.).
mean diameter (GMD) and 1.52 geometric standard deviation (GSD), 1.05 mm GMD (GSD = 1.53), and 1.07 mm GMD (GSD = 1.55) for the target concentrations of 0.150, 1.53, and 3.10 mg MnSO4/m3, respectively. Control groups were exposed to HEPA-filtered air only. The particle size distribution for the control chamber was 0.79 mm GMD (GSD = 1.52) and most likely reflects dander, feed, and other particulate sources. Clinical Observations, Pathology, and Organ Weights Inhalation exposure to MnSO4 during gestation did not affect maternal body weight gain (Fig. 1) or terminal maternal or fetal body weight (Table 1) or mean number of fetuses per litter (Table 1). The majority of all F0 male and female rats had no reported clinical signs. Most of the clinical signs that were observed in the young male and female rats were of minimal veterinary concern (e.g., hair loss, transient weight loss), are often seen in rodent studies (Everitt and Dorman, 2001), and were not related to MnSO4 inhalation. The majority (24/28) of all F0 female rats had no macroscopic lesions observed during visual inspection at necropsy. Lesions observed in the F0 female rats did not demonstrate any dose or exposure-duration association and were deemed to be unrelated to MnSO4 inhalation. Manganese exposure did not affect maternal brain, lung, pancreas, femur, placenta, or liver weights. Fetal brain, lung, skull cap, and liver weights were unaffected by MnSO4 exposure.
RESULTS Manganese Test Atmospheres The overall means (S.D.) for the chamber concentrations based on daily optical particle sensor data were 0.001 0.000, 0.157 0.011, 1.50 0.10, and 3.03 0.18 mg/m3 for the target exposure concentrations of 0, 0.15, 1.53, and 3.10 mg MnSO4/m3, corresponding to 0, 0.05, 0.5, and 1 mg Mn/m3, respectively. The particle size distribution was 1.03 mm geometric
Fig. 1. Mean (S.E.M.) maternal body weight gain during pregnancy following in utero exposure to either air or MnSO4 (mg Mn/m3). For clarity, error bars have only been shown for the control and high dose groups (n = 7 pregnant rats/exposure concentration).
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Table 1 Mean (S.E.M.) terminal (GD 20) maternal and fetal body weights and number of fetuses per litter following in utero exposure to either air or MnSO4 (mg Mn/m3) Nominal MnSO4 concentration (mg Mn/m3)
Endpoint Terminal body weight (g) Fetal body weight (g)a No. of live fetuses a
a
0
0.05
0.5
1
358.0 16.3 3.41 0.10 13.6 1.1
353.8 10.3 3.71 0.16 14.4 0.8
326.5 22.0 3.57 0.19 13.9 1.1
344.7 19.7 3.62 0.09 13.7 0.9
n = 7 dams/exposure group.
Tissue Manganese Concentrations Mean (S.E.M.) tissue manganese concentrations for the F0 female rats (dams) are presented in Fig. 2. Increased maternal lung manganese concentrations were observed following exposure to MnSO4 at 0.05 mg Mn/m3 during pregnancy. Increased maternal placental and brain manganese concentra-
tions were observed following exposure to MnSO4 at 0.5 mg Mn/m3 during pregnancy. Elevated maternal femur and liver manganese concentrations were observed following gestational exposure to MnSO4 at 0.5 and 1 mg Mn/m3; respectively; however these increases were not statistically significant (p = 0.93–0.10). Mean (S.E.M.) tissue manganese concentrations for GD 20 fetuses are presented in Fig. 3. Increased fetal liver manganese concentrations were observed following in utero exposure to MnSO4 at 0.5 mg Mn/m3. Elevated fetal skull manganese concentrations were observed following in utero exposure to MnSO4 at 1 mg Mn/m3; however this increase was not statistically significant (p = 0.129).
DISCUSSION
Fig. 2. Mean (S.E.M.) GD 20 maternal (n = 7 pregnant rats/exposure concentration) tissue manganese concentrations (mg Mn/g) following gestational exposure to either air or MnSO4 (mg Mn/m3) (*p < 0.05).
Fig. 3. Mean (S.E.M.) GD 20 fetal (n = 2 pups/litter and 7 litters/exposure concentration) tissue manganese concentrations (mg Mn/g) following in utero exposure to either air or MnSO4 (mg Mn/m3) (*p < 0.05).
The primary goal of this study was to evaluate tissue manganese concentrations in dams and fetuses following inhalation exposure to MnSO4 during the majority of pregnancy. Exposures were conducted prior to breeding and from GD 0 to 20. Tissue samples from dams and fetuses were collected on GD 20 and manganese concentrations determined. As expected, MnSO4 exposure resulted in increased manganese concentrations in some maternal tissues. Increased maternal lung manganese concentrations were observed following gestational exposure to MnSO4 at 0.05 mg Mn/m3. Although maternal liver manganese concentrations in animals exposed to MnSO4 at 1 mg Mn/m3 were higher than those seen in airexposed controls, this increase was not statistically significant (p = 0.10). Increased biliary excretion of manganese occurs in response to high dose manganese exposure and serves to regulate hepatic manganese concentrations (Dorman et al., 2001). Increased brain and placental manganese concentrations were observed following exposure to MnSO4 at 0.5 mg Mn/m3. In vitro studies have shown that the human placenta accumulates manganese and bidir-
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ectional transfer across the placenta is low (Miller et al., 1987). In vivo rodent studies using oral gestational exposure or intravenous administration of a 54Mn tracer have further demonstrated that the amount of manganese that crosses the placenta is low (Kontur and Fechter, 1985). Kontur and Fechter (1985) demonstrated that the rat placenta and GD 15 fetuses accumulated approximately 1.5% and 0.5%, respectively, of the total radioactivity following intravenous administration of a 54Mn tracer to the dam. The mechanism involved in manganese distribution across the placenta is unknown, however, both transferrin and divalent metal transporter 1 (DMT-1) may be involved. Data in support of this hypothesis include the finding that transferrin and DMT-1 are present in the placenta (Georgieff et al., 2000; Leazer and Klaassen, 2003; Srai et al., 2002) and that placental transferrin and DMT-1 are up-regulated in maternal iron deficiency, thus minimizing the severity of fetal anemia (Gambling et al., 2001). To our knowledge, fetal manganese concentrations have not previously been evaluated in fetal rodents exposed in utero to manganese. Our results suggest that like the adult liver, the fetal liver can accumulate manganese following high dose inhalation exposure to MnSO4. Fetal brain manganese concentrations were unaffected by high dose MnSO4 exposure. In contrast, Kontur and Fechter (1985) reported elevated fore- and hind-brain manganese concentrations in newborn (PND 1) rats whose dams were exposed to high levels of manganese chloride (MnCl2) in their drinking water (0, 5, or 10 mg Mn/L) throughout gestation. Direct comparison of our results with those published by Kontur and Fechter (1985) is constrained by methods of manganese administration and the time when brain analyses were performed since postnatal brain manganese concentrations can change rapidly in response to dietary manganese exposure. A second issue of concern for inhaled manganese relates to possible reproductive toxicity effects that could occur following exposure during pregnancy. Fetotoxicity (reduced fetal body weight or morphological defects) in the absence of maternal toxicity has been reported to occur in mice exposed orally to high levels of manganese (Colomina et al., 1996; Elbetieha et al., 2001; Sanchez et al., 1993; Webster and Valois, 1987). We did not, however, observe any evidence of fetotoxicity in our study. High dose MnSO4 exposure was not associated with decreased fetal body weight or litter size despite an approximately 4–9% decrease in maternal body weight in rats exposed to 0.5 mg Mn/
m3. Structural evaluations were not performed in our study; therefore we cannot rule out subtle teratological effects. Atmospheric sources of manganese include manmade and natural sources. The wind erosion of dusts and soils can contribute to manganese in the air. Industrial sources associated with manganese emissions include ferroalloy production, iron and steel foundries, and power plant and coke oven combustion emissions (ATSDR, 2000; Lioy, 1983). Manganese is also found in the gasoline fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT) and emission of the sulfate form of manganese occurs in automobiles equipped with catalytic converters (Molders et al., 2001; Ressler et al., 1999, 2000; Zayed et al., 1999a). Systemic delivery of manganese to the adult brain is favored following inhalation of MnSO4 and other soluble forms of manganese (Dorman et al., 2001; Normandin et al., 2004). Manganese exposure concentrations used in the study were chosen based on the results of our 90-day inhalation studies (Dorman et al., 2004). A priori we anticipated that the highest and intermediate MnSO4 exposure concentrations (0.5, and 1 mg Mn/ m3) would result in elevated manganese concentrations in maternal brain, lung, and some (but not all) other tissues. The lowest proposed MnSO4 exposure concentration (0.05 mg Mn/m3) was deemed unlikely to induce elevated tissue manganese concentrations. Our findings largely confirmed our initial assumptions. Manganese exposure concentrations used in this study are much higher than that observed in the environment. Air manganese concentrations in Canadian cities (Montreal and Toronto) where MMT has been in use for over 20 years are approximately 2000-fold lower than our lowest manganese exposure concentration (Clayton et al., 1999; Loranger and Zayed, 1997; Pellizzari et al., 1999; Zayed et al., 1999b). Our results are directly relevant for occupational exposures. Moreover, the present study examined responses in a potentially sensitive subpopulation thus our data should also prove relevant for setting environmental exposure standards as well. In conclusion, this study provides important new information about the pharmacokinetics of inhaled manganese in pregnant rats. Fetal brain manganese concentrations remained unaffected despite high dose maternal exposure to MnSO4. The results of this study should prove very useful for the development of physiologically-based pharmacokinetic (PBPK) models to predict disposition
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of manganese following inhalation exposure to manganese.
ACKNOWLEDGMENTS The authors would like to thank the staff of the CIIT Centers for Health Research animal care facility for their contributions. We also thank Drs. Mel Andersen, Susan Borghoff, and Teresa Leavens for their critical review of this manuscript. This publication is based on a study sponsored and funded by the Ethyl Corporation in satisfaction of registration requirements arising under Section 211(a) and (b) of the Clean Air Act and corresponding regulations at 40 C.F.R. Subsections 79.50 et seq.
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Maternal–fetal Distribution of Manganese in the Rat Following Inhalation Exposure to Manganese Sulfate David C. Dorman*, Anna M. McElveen, Marianne W. Marshall, Carl U. Parkinson, R. Arden James, Melanie F. Struve, Brian A. Wong CIIT Centers for Health Research, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, NC 27709-2137, USA Received 11 June 2004; accepted 9 August 2004 Available online 29 September 2004
Abstract Studies examining the pharmacokinetics of manganese during pregnancy have largely focused on the oral route of exposure and have shown that the amount of manganese that crosses the rodent placenta is low. However, limited information exists regarding the distribution of manganese in fetal tissues following inhalation. The objective of this study was to determine manganese body burden in CD rats and fetuses following inhalation of a MnSO4 aerosol during pregnancy. Animals were evaluated following pre-breeding (2 weeks), mating (up to 14 days) and gestational (from gestation day (GD) 0 though 20) exposure to air or MnSO4 (0.05, 0.5, or 1 mg Mn/m3) for 6 h/day, 7 days/week. The following maternal samples were collected for manganese analysis: whole blood, lung, pancreas, liver, brain, femur, and placenta. Fetal tissues were examined on GD 20 and included whole blood, lung, liver, brain, and skull cap. Maternal lung manganese concentrations were increased following exposure to MnSO4 at 0.05 mg Mn/m3. Maternal brain and placenta manganese concentrations were increased following exposure of pregnant rats to MnSO4 at 0.5 mg Mn/m3. Increased fetal liver manganese concentrations were observed following in utero exposure to MnSO4 at 0.5 mg Mn/m3. Manganese concentrations within all other fetal tissues were not different from air-exposed controls. The results of this study demonstrate that the placenta partially sequesters inhaled manganese, thereby limiting exposure to the fetus.
# 2004 Elsevier Inc. All rights reserved. Keywords: Manganese; Inhalation; Rat; Pregnancy
INTRODUCTION Manganese has been shown to be an essential nutrient in several animal species particularly during gestation and early infancy. Manganese is required for normal amino acid, lipid, protein, and carbohydrate metabolism. Manganese-dependent enzyme families include oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Manganese metalloenzymes include arginase, glutamine synthetase, phos-
* Corresponding author. Tel.: +1 919 558 1350; fax: +1 919 558 1300. E-mail address: [email protected] (D.C. Dorman).
phoenolpyruvate decarboxylase, and manganese superoxide dismutase. No formal recommended dietary allowance (RDA) for manganese has been established, but the U.S. National Research Council has established an estimated safe and adequate dietary intake (ESADI) of 2–5 mg/day for adults (Greger, 1998). Lactation and gestation are thought to increase the manganese requirement (NAS, 2001). Pregnant women and infants often have elevated blood manganese concentrations (Spencer, 1999). Studies examining whole blood concentrations of manganese during pregnancy show an increase throughout gestation, and iron supplementation of pregnant women with normal iron status does not significantly change blood manganese concentra-
0161-813X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2004.08.004
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tions despite the sharing of some transport proteins by manganese and iron (Tholin et al., 1995). In excess, manganese can accumulate within the human striatum and globus pallidus and produce damage to dopaminergic neurons within these sites (Malecki et al., 1999; Nelson et al., 1993; Pal et al., 1999). Manganese neurotoxicity most commonly results from the chronic inhalation exposure of high (>1 mg/m3) manganese concentrations, as may occur during certain mining operations. Whether chronic exposure to much lower airborne manganese concentrations can contribute to the incidence of human neurological disease is the subject of discussion within the scientific community. Most manganese inhalation pharmacokinetic studies have been conducted in adult animals and have typically exposed rodents to high concentrations of manganese oxides (for a review, see ATSDR, 1992). Manganese pharmacokinetic studies involving potentially sensitive subpopulations (e.g., developing rodents) have been less frequently performed. Rodent studies using oral gestational exposure have demonstrated that the amount of manganese that crosses the placenta is low (Kontur and Fechter, 1985). Despite this apparent barrier, fetotoxicity (reduced fetal body weight or morphological defects) in the absence of maternal toxicity has been reported to occur in mice exposed orally to high levels of manganese (Colomina et al., 1996; Sanchez et al., 1993). To our knowledge, pharmacokinetic data on inhaled manganese in pregnant animals are not available. The objective of this study was to determine manganese body burden in CD rats following MnSO4 inhalation during pregnancy.
pregnant female rats (sperm positive = gestation day (GD) 0) were exposed to air or MnSO4 from GD 0 through GD 20. Immediately after the end of the 6-h exposure on GD 20, presumed pregnant rats were killed (n = 7 pregnant rats/exposure concentration were evaluated) and maternal and fetal tissue manganese concentrations determined. Manganese concentrations were determined for two randomly selected fetuses/ litter while tissues from one additional randomly selected fetus/litter were retained. Chemicals Manganese(II) sulfate monohydrate (MnSO4H2O) was obtained from Aldrich Chemical Company Inc. (Milwaukee, WI) with a certified purity of >98%. The material is a relatively water-soluble, white to pale pink crystalline powder that contains 32% Mn by weight. Animals This study was conducted under federal guidelines for the care and use of laboratory animals (National Research Council, 1996) and was approved by the CIIT Institutional Animal Care and Use Committee. Sixweek old male and female Crl:CD1(SD) BR rats were purchased from Charles River Laboratories Inc. (Raleigh, NC). Randomization of animals to treatment groups occurred prior to the start of the inhalation exposure and was based upon a weight randomization procedure. Animals were acclimated for approximately 2 weeks prior to the start of the inhalation exposure. Animal Husbandry
MATERIALS AND METHODS Experimental Design Rats were used to evaluate maternal and fetal tissue manganese concentrations following inhalation exposure throughout the majority of gestation. This experiment consisted of 10 male and 10 female CD rats per exposure group (F0 male and female rats) that were exposed to air or MnSO4 beginning 28 days prior to breeding and for up to 14 days during the mating period. Animals were exposed to either air or MnSO4 (nominal concentrations were 0.05, 0.5, or 1 mg Mn/ m3) for 6 h/day, 7 days/week. Male F0 rats were killed by carbon dioxide (CO2) asphyxiation shortly after the completion of the 2-week breeding period and tissues were not collected from these animals. Presumed
Animal rooms were maintained at daily temperatures of 17–23 8C, relative humidity of 30–70%, and an air flow rate sufficient to provide 10–15 air changes per hour. To the extent possible, the exposure chambers were maintained at daily temperatures 18–26 8C, relative humidity of 30–70%, and an air flow rate sufficient to provide at least 12 air changes per hour. Fluorescent lighting was controlled by automatic controls (lights on approximately 0700–1900). All animals were housed in CIIT’s animal facility which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Except during inhalation exposure and breeding periods, male and female F0 rats were individually housed in polycarbonate cages containing cellulose fiber chip bedding (ALPHA-driTM; Shepherd Specialty Papers, Kalama-
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zoo, MI). Animal breedings (typically 1 male:1 female) were conducted overnight in polycarbonate cages containing cellulose fiber chip bedding. A pelleted, semi-purified AIN-93G certified diet from Bio-Serv (Frenchtown, NJ) formulated to contain approximately 10 ppm manganese and 35 ppm iron was given throughout the study. Food was available to all animals ad libitum except during inhalation exposures. Reverse osmosis purified water containing <0.222–0.546 mg Mn/L and 1.31–13.01 mg Fe/L was available ad libitum. Detailed individual animal clinical examinations were conducted and recorded at least once weekly throughout the course of the study beginning at the start of the inhalation exposures. Adult male (F0) body weights were measured and recorded at least weekly. Maternal body weights were measured and recorded at least weekly and on GD 0, 7, 14, 18. All animals had individual body weights determined at necropsy. Animals were mated at night (female added to male’s cage after each daily exposure, then removed for the next exposure day) for a period of up to 14 days, with no change in mating partners. Females were examined daily after the cohabitation period for the presence of plug/sperm in the vaginal tract. The observation of a copulation plug or sperm-positive smear was considered evidence of successful mating (sperm positive), and the day was designated GD 0. The sperm-positive female and male from that mating pair were then individually housed, and gestational exposures begun. Manganese Exposure Exposures were conducted for 6 h/day, 7 days/week. Rats were exposed in stainless steel wire cage units contained in four 8-m3 Hinners-style, stainless steel and glass inhalation exposure chambers (Lab Products, Maywood, NJ). Air flow through the chambers was adjusted to assure at least 12 air changes per hour (approximately 1600–2600 L/min). Temperature and relative humidity inside the 8-m3 chamber were monitored continuously and recorded every 30 min during each exposure. The average mean daily chamber temperature was maintained at 22 4 8C and relative humidity was maintained at 30–70%. Animal positions within the exposure chambers were rotated during the experiment to minimize experimental error due to any undetected differences in the environment or the manganese aerosol concentration. Exposure atmospheres were generated by aerosolizing MnSO4 using a dry powder generator (Wright Dust Feeder, Model
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WDF-II, BGI Inc., Waltham, MA). Separate generation systems were used for each target exposure concentration. The MnSO4 was packed in a dry powder generation cup under a pressure of 1500–3000 psi. The MnSO4 particles were carried from the dry powder generator through a single, 38-L mixing/settling chamber or through two 38-L mixing/settling chambers. An 85Kr discharging unit was suspended in the primary mixing/settling chamber for each generation system. Exposure atmospheres were measured with optical particle sensors (Real-Time Aerosol Sensors, Model RAM-S, MIE Inc., Billerica, MA). An optical particle sizing spectrometer (Aerodynamic Particle Sizer, Model 3320, TSI Inc., St. Paul, MN) was used to measure particle size distribution. Gravimetric filter samples were used to verify the optical particle sensor readings. Oxygen was monitored in each chamber one time during the study with an MDA Oxygen Sensor (Model 3300, MDA Scientific, Lincolnshire, IL). Necropsy Procedure Presumed pregnant rats were euthanized on GD 20 via CO2 asphyxiation and fetuses were killed by decapitation. Maternal (1–2 ml) blood was collected from the thoracic aorta or heart, and fetal trunk blood (>100 ml) was collected immediately after euthanasia. Fetal blood samples were pooled in order to provide an adequate sample size for manganese analysis. An aliquot of the heparinized maternal (500 ml) or fetal (100 ml) blood was placed into a plastic tube for manganese analysis. The following maternal samples were collected for manganese analysis: whole blood, lung, pancreas, liver, brain, femur, and placenta. The following fetal (n = 2 fetuses/litter) samples were collected for manganese analysis: whole blood, lung, liver, brain, and skull cap. Samples were transferred to plastic vials, frozen in liquid nitrogen, and stored at approximately 80 8C until manganese analysis. Tissue samples were thawed and weighed prior to transfer to 3-ml digestion vessels. Samples (10–30 mg) were digested in 16 M nitric acid prior to manganese analysis using a CEM MARS5 Microwave Accelerated Reaction System (CEM, Matthews, NC). Tissue manganese concentrations were determined by graphite furnace atomic absorption spectrometry (GFAAS) with a Perkin-Elmer AAnalyst 800 Atomic Absorption spectrometer equipped with AA WinLab software (version 4.1 SP). The analyte was identified using a manganese-specific hollow cathode lamp by the presence
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of an absorbance signal at the manganese analytical wavelength of 279.5 nm. Quantitation of the analyte (mg Mn/g tissue wet weight) was carried out using an external calibration curve and the AA WinLab software. Palladium nitrate and magnesium nitrate were used as matrix modifiers for all samples. The performance evaluation sample for this study was Bovine Liver Reference Standard, containing 0.1 mg Mn/mL (High Purity Standards, Charleston, SC) with a certified analysis for manganese. An additional manganese standard (calibration check standard) was prepared at a mid-level of the calibration curve using Manganese Reference Standard, 1000 mg/mL in 2% HNO3 (High Purity Standards, Charleston, SC) to evaluate the accuracy of the calibration curve. All samples were analyzed at least twice by the GFAAS. Statistics The data for quantitative, continuous variables were compared for the exposure and control groups by tests for homogeneity of variance (Levene’s test), analysis of variance (ANOVA), and Dunnett’s multiple comparison procedure for significant ANOVA. In the event the Levene’s test was significant, then the data were transformed using a natural log (ln) transformation. If the Levene’s test was significant following transformation, then the original data were analyzed by nonparametric statistics (Wilcoxon or Kruskal–Wallis). Individual data that appeared to be outliers were critically evaluated using a Dixon-type test for discordancy for an upper outlier (Barnett and Lewis, 1984). Statistical analyses were performed using JMP software from SAS Institute Inc. (Cary, NC). A probability value of 0.01 was used for Levene’s test, while p < 0.05 was used as the critical level of significance for all other statistical tests. Unless otherwise noted, data presented are mean values standard error of the mean (S.E.M.).
mean diameter (GMD) and 1.52 geometric standard deviation (GSD), 1.05 mm GMD (GSD = 1.53), and 1.07 mm GMD (GSD = 1.55) for the target concentrations of 0.150, 1.53, and 3.10 mg MnSO4/m3, respectively. Control groups were exposed to HEPA-filtered air only. The particle size distribution for the control chamber was 0.79 mm GMD (GSD = 1.52) and most likely reflects dander, feed, and other particulate sources. Clinical Observations, Pathology, and Organ Weights Inhalation exposure to MnSO4 during gestation did not affect maternal body weight gain (Fig. 1) or terminal maternal or fetal body weight (Table 1) or mean number of fetuses per litter (Table 1). The majority of all F0 male and female rats had no reported clinical signs. Most of the clinical signs that were observed in the young male and female rats were of minimal veterinary concern (e.g., hair loss, transient weight loss), are often seen in rodent studies (Everitt and Dorman, 2001), and were not related to MnSO4 inhalation. The majority (24/28) of all F0 female rats had no macroscopic lesions observed during visual inspection at necropsy. Lesions observed in the F0 female rats did not demonstrate any dose or exposure-duration association and were deemed to be unrelated to MnSO4 inhalation. Manganese exposure did not affect maternal brain, lung, pancreas, femur, placenta, or liver weights. Fetal brain, lung, skull cap, and liver weights were unaffected by MnSO4 exposure.
RESULTS Manganese Test Atmospheres The overall means (S.D.) for the chamber concentrations based on daily optical particle sensor data were 0.001 0.000, 0.157 0.011, 1.50 0.10, and 3.03 0.18 mg/m3 for the target exposure concentrations of 0, 0.15, 1.53, and 3.10 mg MnSO4/m3, corresponding to 0, 0.05, 0.5, and 1 mg Mn/m3, respectively. The particle size distribution was 1.03 mm geometric
Fig. 1. Mean (S.E.M.) maternal body weight gain during pregnancy following in utero exposure to either air or MnSO4 (mg Mn/m3). For clarity, error bars have only been shown for the control and high dose groups (n = 7 pregnant rats/exposure concentration).
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Table 1 Mean (S.E.M.) terminal (GD 20) maternal and fetal body weights and number of fetuses per litter following in utero exposure to either air or MnSO4 (mg Mn/m3) Nominal MnSO4 concentration (mg Mn/m3)
Endpoint Terminal body weight (g) Fetal body weight (g)a No. of live fetuses a
a
0
0.05
0.5
1
358.0 16.3 3.41 0.10 13.6 1.1
353.8 10.3 3.71 0.16 14.4 0.8
326.5 22.0 3.57 0.19 13.9 1.1
344.7 19.7 3.62 0.09 13.7 0.9
n = 7 dams/exposure group.
Tissue Manganese Concentrations Mean (S.E.M.) tissue manganese concentrations for the F0 female rats (dams) are presented in Fig. 2. Increased maternal lung manganese concentrations were observed following exposure to MnSO4 at 0.05 mg Mn/m3 during pregnancy. Increased maternal placental and brain manganese concentra-
tions were observed following exposure to MnSO4 at 0.5 mg Mn/m3 during pregnancy. Elevated maternal femur and liver manganese concentrations were observed following gestational exposure to MnSO4 at 0.5 and 1 mg Mn/m3; respectively; however these increases were not statistically significant (p = 0.93–0.10). Mean (S.E.M.) tissue manganese concentrations for GD 20 fetuses are presented in Fig. 3. Increased fetal liver manganese concentrations were observed following in utero exposure to MnSO4 at 0.5 mg Mn/m3. Elevated fetal skull manganese concentrations were observed following in utero exposure to MnSO4 at 1 mg Mn/m3; however this increase was not statistically significant (p = 0.129).
DISCUSSION
Fig. 2. Mean (S.E.M.) GD 20 maternal (n = 7 pregnant rats/exposure concentration) tissue manganese concentrations (mg Mn/g) following gestational exposure to either air or MnSO4 (mg Mn/m3) (*p < 0.05).
Fig. 3. Mean (S.E.M.) GD 20 fetal (n = 2 pups/litter and 7 litters/exposure concentration) tissue manganese concentrations (mg Mn/g) following in utero exposure to either air or MnSO4 (mg Mn/m3) (*p < 0.05).
The primary goal of this study was to evaluate tissue manganese concentrations in dams and fetuses following inhalation exposure to MnSO4 during the majority of pregnancy. Exposures were conducted prior to breeding and from GD 0 to 20. Tissue samples from dams and fetuses were collected on GD 20 and manganese concentrations determined. As expected, MnSO4 exposure resulted in increased manganese concentrations in some maternal tissues. Increased maternal lung manganese concentrations were observed following gestational exposure to MnSO4 at 0.05 mg Mn/m3. Although maternal liver manganese concentrations in animals exposed to MnSO4 at 1 mg Mn/m3 were higher than those seen in airexposed controls, this increase was not statistically significant (p = 0.10). Increased biliary excretion of manganese occurs in response to high dose manganese exposure and serves to regulate hepatic manganese concentrations (Dorman et al., 2001). Increased brain and placental manganese concentrations were observed following exposure to MnSO4 at 0.5 mg Mn/m3. In vitro studies have shown that the human placenta accumulates manganese and bidir-
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ectional transfer across the placenta is low (Miller et al., 1987). In vivo rodent studies using oral gestational exposure or intravenous administration of a 54Mn tracer have further demonstrated that the amount of manganese that crosses the placenta is low (Kontur and Fechter, 1985). Kontur and Fechter (1985) demonstrated that the rat placenta and GD 15 fetuses accumulated approximately 1.5% and 0.5%, respectively, of the total radioactivity following intravenous administration of a 54Mn tracer to the dam. The mechanism involved in manganese distribution across the placenta is unknown, however, both transferrin and divalent metal transporter 1 (DMT-1) may be involved. Data in support of this hypothesis include the finding that transferrin and DMT-1 are present in the placenta (Georgieff et al., 2000; Leazer and Klaassen, 2003; Srai et al., 2002) and that placental transferrin and DMT-1 are up-regulated in maternal iron deficiency, thus minimizing the severity of fetal anemia (Gambling et al., 2001). To our knowledge, fetal manganese concentrations have not previously been evaluated in fetal rodents exposed in utero to manganese. Our results suggest that like the adult liver, the fetal liver can accumulate manganese following high dose inhalation exposure to MnSO4. Fetal brain manganese concentrations were unaffected by high dose MnSO4 exposure. In contrast, Kontur and Fechter (1985) reported elevated fore- and hind-brain manganese concentrations in newborn (PND 1) rats whose dams were exposed to high levels of manganese chloride (MnCl2) in their drinking water (0, 5, or 10 mg Mn/L) throughout gestation. Direct comparison of our results with those published by Kontur and Fechter (1985) is constrained by methods of manganese administration and the time when brain analyses were performed since postnatal brain manganese concentrations can change rapidly in response to dietary manganese exposure. A second issue of concern for inhaled manganese relates to possible reproductive toxicity effects that could occur following exposure during pregnancy. Fetotoxicity (reduced fetal body weight or morphological defects) in the absence of maternal toxicity has been reported to occur in mice exposed orally to high levels of manganese (Colomina et al., 1996; Elbetieha et al., 2001; Sanchez et al., 1993; Webster and Valois, 1987). We did not, however, observe any evidence of fetotoxicity in our study. High dose MnSO4 exposure was not associated with decreased fetal body weight or litter size despite an approximately 4–9% decrease in maternal body weight in rats exposed to 0.5 mg Mn/
m3. Structural evaluations were not performed in our study; therefore we cannot rule out subtle teratological effects. Atmospheric sources of manganese include manmade and natural sources. The wind erosion of dusts and soils can contribute to manganese in the air. Industrial sources associated with manganese emissions include ferroalloy production, iron and steel foundries, and power plant and coke oven combustion emissions (ATSDR, 2000; Lioy, 1983). Manganese is also found in the gasoline fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT) and emission of the sulfate form of manganese occurs in automobiles equipped with catalytic converters (Molders et al., 2001; Ressler et al., 1999, 2000; Zayed et al., 1999a). Systemic delivery of manganese to the adult brain is favored following inhalation of MnSO4 and other soluble forms of manganese (Dorman et al., 2001; Normandin et al., 2004). Manganese exposure concentrations used in the study were chosen based on the results of our 90-day inhalation studies (Dorman et al., 2004). A priori we anticipated that the highest and intermediate MnSO4 exposure concentrations (0.5, and 1 mg Mn/ m3) would result in elevated manganese concentrations in maternal brain, lung, and some (but not all) other tissues. The lowest proposed MnSO4 exposure concentration (0.05 mg Mn/m3) was deemed unlikely to induce elevated tissue manganese concentrations. Our findings largely confirmed our initial assumptions. Manganese exposure concentrations used in this study are much higher than that observed in the environment. Air manganese concentrations in Canadian cities (Montreal and Toronto) where MMT has been in use for over 20 years are approximately 2000-fold lower than our lowest manganese exposure concentration (Clayton et al., 1999; Loranger and Zayed, 1997; Pellizzari et al., 1999; Zayed et al., 1999b). Our results are directly relevant for occupational exposures. Moreover, the present study examined responses in a potentially sensitive subpopulation thus our data should also prove relevant for setting environmental exposure standards as well. In conclusion, this study provides important new information about the pharmacokinetics of inhaled manganese in pregnant rats. Fetal brain manganese concentrations remained unaffected despite high dose maternal exposure to MnSO4. The results of this study should prove very useful for the development of physiologically-based pharmacokinetic (PBPK) models to predict disposition
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of manganese following inhalation exposure to manganese.
ACKNOWLEDGMENTS The authors would like to thank the staff of the CIIT Centers for Health Research animal care facility for their contributions. We also thank Drs. Mel Andersen, Susan Borghoff, and Teresa Leavens for their critical review of this manuscript. This publication is based on a study sponsored and funded by the Ethyl Corporation in satisfaction of registration requirements arising under Section 211(a) and (b) of the Clean Air Act and corresponding regulations at 40 C.F.R. Subsections 79.50 et seq.
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