TOXICOLOGICAL SCIENCES 93(1), 114–124 (2006)
doi:10.1093/toxsci/kfl028
Advance Access publication June 1, 2006
Brain Accumulation and Toxicity of Mn(II) and Mn(III) Exposures
Stephen H. Reaney,*,1 Graham Bench,† and Donald R. Smith‡
*Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064; †Center for Accelerator Mass Spectrometry,
Lawrence Livermore National Laboratory, Livermore, California 94550; and ‡Department of Environmental Toxicology,
University of California, Santa Cruz, California 95064
Received March 21, 2006; accepted May 27, 2006
Concern over the neurotoxic effects of chronic moderate
exposures to manganese has arisen due to increased awareness of
occupational exposures and to the use of methylcyclopentadienyl
manganese tricarbonyl, a manganese-containing gasoline antiknock additive. Little data exist on how the oxidation state of
manganese exposure affects toxicity. The objective of this study
was to better understand how the oxidation state of manganese
exposure affects accumulation and subsequent toxicity of manganese. This study utilized a rat model of manganese neurotoxicity
to investigate how ip exposure to Mn(II)-chloride or Mn(III)pyrophosphate at total cumulative doses of 0, 30, or 90 mg Mn/kg
body weight affected the brain region distribution and neurotoxicity
of manganese. Results indicate that Mn(III) exposures produced
significantly higher blood manganese levels than equimolar exposures to Mn(II). Brain manganese concentrations increased in
a dose-dependent manner, with Mn(III) exposures producing
significantly higher ( > 25%) levels than exposures to Mn(II) but
with no measurable differences in the accumulation of manganese
across different brain regions. Gamma amino butyric acid concentrations were increased in the globus pallidus (GP) with manganese
exposure. Dopamine (DA) levels were altered in the GP, with the
highest Mn(II) and Mn(III) exposures producing significantly
different DA levels. In addition, transferrin receptor and H-ferritin
protein expression increased in the GP with manganese exposure.
These data substantiate the heightened susceptibility of the GP to
manganese, and they indicate that the oxidation state of manganese exposure may be an important determinant of tissue toxicodynamics and subsequent neurotoxicity.
Key Words: GABA; dopamine; oxidation state; neurotoxicity;
PIXE; brain region.
Metal speciation (i.e., oxidation state and ligand environment) is important in determining the functionality and toxicity
of trace metals in biological systems (Finney and O’Halloran,
2003; Thompson and Orvig, 2003). This may be particularly
true for manganese, which is an essential trace element in
biology (Finley and Davis, 1999; Underwood, 1977; Yocum
1
To whom correspondence should be addressed at Department of Environmental Toxicology, University of California, 1156 High Street, Santa Cruz, CA
95064. Fax: (831) 459-3524. E-mail: [email protected].
and Pecoraro, 1999) that exhibits complex reduction-oxidation
(redox) chemistry (Klewicki and Morgan, 1998; Silva and
Williams, 1991). Concern over possible neurotoxic effects associated with chronic exposure to moderate levels of manganese in mixed oxidation states may be justified by recent studies
reporting that workers in the manganese alloy industry are
exposed to manganese aerosols of mixed oxidation states
(Mn(0), Mn(II), Mn(III), and Mn(IV)) (Thomassen et al.,
2001) and by the growing use of methylcyclopentadienyl
manganese tricarbonyl, a manganese-containing antiknock
additive in gasoline currently in use in a number of developed
countries (e.g., Canada, Australia) and proposed for use in the
United States (Cooper, 1984; Ressler et al., 2000).
Elevated occupational exposures to manganese are known to
cause significant neurotoxicity (Cook, 1974). Furthermore,
epidemiologic studies have suggested a relationship between
elevated environmental manganese exposure and an increased
risk for parkinsonian disturbances (Barbeau, 1984; Corrigan
et al., 1998; Gorell et al., 1997; Rybicki et al., 1993; Yamada
et al., 1986), an association that has also been supported by
numerous laboratory studies (Brown and Taylor, 1999; Gavin
et al., 1999; Gwiazda et al., 2002; Olanow et al., 1996; Witholt
et al., 2000). While the exact mechanisms underlying the
neurotoxic effects of manganese remain unclear, these studies
collectively suggest that elevated environmental exposures to
manganese may be sufficient to exacerbate the emergence of
neurological diseases (Gwiazda et al., 2002; Witholt et al.,
2000). In light of these observations, there is a need to further
examine the role of manganese oxidation state and its ability
to mediate the uptake, retention, and resulting toxicity of
manganese.
The mechanisms of manganese neurotoxicity may vary with
exposure dose and speciation (Roels et al., 1997); examples of
the former exist in the literature. The postmortem tissues of
workers occupationally exposed to manganese exhibit decreases in dopamine (DA) in the basal ganglia (Shinotoh
et al., 1997; Wolters et al., 1989; Yamada et al., 1986), and
some of these observations have been reproduced in nonhuman
primate models (Bird et al., 1984; Eriksson et al., 1987; Neff
et al., 1969; Olanow et al., 1996; Shinotoh et al., 1995). Based
on these and other studies, DA metabolism in the basal ganglia
The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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MANGANESE OXIDATION STATE AND TOXICITY
(e.g., striatum [Str] and globus pallidus [GP]) has emerged as
an outcome of major emphasis in mechanistic studies of
manganese neurotoxicity. However, studies have reported
seemingly conflicting results on the dopaminergic effects of
manganese (see Gwiazda et al., 2006, for review), including
decreases, increases, or no change in striatal DA concentrations
in manganese-treated animals, possibly reflecting effects of
the different exposure regimens on dopaminergic outcomes
(Bonilla and Prasad, 1984; Chandra and Shukla, 1981; Eriksson
et al., 1987; Gwiazda et al., 2002; Subhash and Padmashree,
1991). Moreover, it has recently been suggested that at lower
doses of manganese, other systems such as the GABAergic
pathways may be affected (Gwiazda et al., 2002; Zheng et al.,
2003). These inconsistent observations may well reflect differences in exposure route, magnitude, duration, etc., between
studies, though they also demonstrate the complexity of
manganese toxicity and suggest that the factors contributing
to its toxicity are not well understood.
A common hypothesis regarding the differential susceptibility of brain regions to manganese is that elevated exposures
lead to brain regional differences in the accumulation of manganese and other metals (e.g., iron). It has been reported that
rats exposed to very elevated manganese levels via drinking
water from an early age displayed increased brain manganese
levels and altered copper and iron levels in the Str (Lai et al.,
1999). Both animal (Newland et al., 1989) and human studies
(Dietz et al., 2001; Krieger et al., 1995) have shown indications
of increased manganese concentrations in the basal ganglia as
measured by T1-weighted MRI imaging and postmortem
analysis, further suggesting that selective manganese accumulation in the basal ganglia may be a determinant of toxicity. In
light of these studies, there is a clear need to investigate the role
of the oxidation state of manganese exposure on brain regional
uptake and toxicity in intact vertebrate organisms.
This need is underscored by several recent cell-based studies
showing that Mn(II) and Mn(III) exposures produced different
toxic effects on cellular function (Reaney and Smith, 2005).
Mn(III) exposure produced significantly greater reductions in
intracellular serotonin (5-hydroxytryptophan [5-HT]) concentrations than did Mn(II), as well as greater effects on measures
of cellular iron homeostasis, as measured by H-ferritin and
transferrin receptor (TfR) protein levels. These oxidation state
differences in manganese toxicity in intact cells substantiated
earlier in vitro studies showing that Mn(III) added to cell
lysates was significantly more effective than Mn(II) at inhibiting total aconitase activity in PC12 and primary astrocyte cell
lysates (Reaney et al., 2002).
Here we utilized a well-characterized rodent model of manganese exposure and proton-induced x-ray emission (PIXE)
spectroscopy to investigate the role of the oxidation state of
manganese exposure on (1) tissue manganese levels, including
the in situ concentrations and regional distribution of manganese and other essential trace metals (iron, copper, and zinc) in
the Str, GP, thalamus (Th), and cerebral cortex, and (2) the
115
relative neurotoxicity of manganese, as measured by altered
brain regional neurotransmitter levels, H-ferritin protein levels,
and TfR protein levels. These data will contribute to a better
understanding of the potential relative risks of environmental
exposures to manganese in different oxidation states.
MATERIALS AND METHODS
Reagents. Anti-human mouse TfR antibody was from Zymed Laboratories
(Carlsbad, CA), anti-mouse HRP secondary antibody was from Santa Cruz
Biotechnology (Santa Cruz, CA), 10% Bis-Tris precast gels was from
Invitrogen (Carlsbad, CA), and HPLC-grade solvents (methanol and acetonitrile) were from Fisher Scientific (Fairlawn, NJ). All other reagents were from
Sigma Aldrich (St Louis, MO).
Study design and animals. This study used a 3 3 2 (Mn dose 3 oxidation
state) factorial design. Thirty-six adult female retired breeder Long Evans rats
(~ 8 months old) were obtained from Charles River Laboratories (Wilmington,
MA) and randomly assigned to treatment groups following a 1-week acclimation (n ¼ 6/treatment group). Animals were treated with Mn(II) or Mn(III) at
nominal doses of 0, 2, or 6 mg Mn/kg body weight/dose via ip injection,
delivered three times a week for a duration of 5 weeks (total cumulative doses
of 0, 30, or 90 mg Mn/kg body weight). Mn(II)-exposed treatment groups were
injected with saline (control, isotonic with Mn(II)-chloride solution) or Mn(II)chloride solution (32mM), while Mn(III)-exposed groups were injected with
100mM sodium pyrophosphate (control, vehicle) or a Mn(III)-pyrophosphate
solution (33mM). Manganese solutions were prepared as previously reported
(Reaney et al., 2002). An ip exposure route was used here for several
reasons. The internal dose is accurately known, thereby avoiding the uncertainty of dose associated with other exposure routes (oral, inhalation) that
would confound our ability to achieve the objectives of the study. The stability
and purity of the Mn(III)-pyrophosphate solution have been well characterized
(Reaney et al., 2002), allowing the oxidation state delivered to be clearly
known. Finally, a large number of rodent studies have evaluated the toxicity of
Mn(II)-chloride using an injection exposure route, thereby facilitating interstudy comparison.
Animals were divided into three cohorts of 12 animals, with each cohort
balanced by treatment and body weight. Sacrifices occurred 3 days after the last
manganese dose for each cohort. Animals were sacrificed by decapitation
without anesthesia, and the brain was immediately removed and coronally
sectioned on ice to reveal GP, Str, frontal cortex, and Th brain regions. The right
hemisphere was collected for elemental determination by PIXE, and the left
hemisphere was collected for Western blot and neurotransmitter analysis.
Tissues were frozen immediately on dry ice and stored at 70C until further
processing. Concurrently, the liver was removed and stored and whole blood
collected in heparin-containing vacutainers. The liver was stored at 70C and
blood at 20C.
Liver and blood manganese concentrations. Liver (1 g) or blood (1 ml)
was aliquoted into 15-ml Teflon digestion vials and processed for manganese
analyses by graphite furnace atomic absorption spectrometry using a Perkin
Elmer (Boston, MA) 4100ZL Zeeman Spectrometer. Details of sample preparation and analyses have been previously described (Witholt et al., 2000).
PIXE analysis: brain Mn, Fe, Cu, and Zn. PIXE spectroscopy is an x-ray
fluorescence technique capable of the simultaneous measurement of many
elements below a 1-lg/g limit of quantification and micrometer-scale spatial
resolution of a sample and thus is well suited for measuring brain regional
differences in the accumulation of manganese and other elements in situ
(Mauthe et al., 1998). To prepare samples for PIXE analysis, 1- to 2-mm coronal slices containing the brain region of interest were mounted on a sectioning
chuck at 20C. Utilizing a cryostat, successive 20-lm slices were taken until
the brain region of interest was revealed. Brain regions were analyzed at
the same level for each animal and defined by discrete morphological
116
REANEY, BENCH, AND SMITH
characteristics, using the rat brain atlas of Paxinos and Watson (1986) as
a guide. The section chosen (bregma 1.88 mm) contained all brain regions of
interest. At this level the Str is clearly defined by unique tissue striata and
clearly bordered by the corpus callosum. The level at which the GP was delineated showed the internal capsule clearly separating the GP from the thalamic
nuclei. For a control brain region, the somatosensory cortex was analyzed at the
same level. Brain slices of interest were collected onto a nylon foil for PIXE
analysis. This sample was then freeze-dried until further analysis (details below).
The effacing slice was mounted on a gelatin-coated microscope slide for cresyl
violet Nissl staining to make neuronal morphological features more apparent to
assure that the proper brain regions were probed during PIXE analyses.
Brain regions of interest ( GP, Str, Th, cortex [Cx]) within freeze-dried tissue
sections were analyzed for manganese, iron, zinc, and copper using PIXE and
scanning transmission ion microscopy (STIM) at the nuclear microprobe
facility of Lawrence Livermore National Laboratory (Roberts et al., 1999).
PIXE and STIM data were obtained using incident 3 MeV proton microbeams.
Regions of interest were delineated on the associated freeze-dried section using
a 340 optical microscope within the microprobe specimen chamber. An
absolute encoder-controlled sample stage was used to position the feature of
interest in the beam path with a precision better than 10 lm. The beam spot size
was maximized to interrogate as much of the feature of interest while avoiding
interrogation of surrounding material. Beam spot sizes were square or
rectangular and varied between 0.1 3 0.1 mm and 1 3 1 mm. For PIXE
analysis, x-rays were detected with an energy-dispersive x-ray detector that
subtended a solid angle of ~ 100 msr to the specimen. The detector was located
at an angle of 135 with respect to the incident beam. Charge was collected in
a biased Faraday cup located behind the sample. Regions of interest were
examined with beam currents of up to 10 nA, for doses of up to 15 lC. X-ray
spectra were stored on a computer and analyzed offline.
X-ray spectra were analyzed using the computational iterative PIXE
spectrum fitting code PIXEF to extract characteristic x-ray peak areas (or
yields) for elements of interest (Antolak and Bench, 1994). The incident and
residual proton energies were used to convert x-ray yields to element
concentrations using PIXEF assuming the composition of the biological
material to be C5H9O2N (Bench, 1991; Lefevre et al., 1987, 1991). To verify
the system calibration for metal quantitation, thin-film organic matrix standards
containing metals at appropriate concentrations were analyzed under the same
conditions used for analysis of tissue sections each day the tissues were
analyzed. Measured manganese contents from both standards were always
within 7% of the known manganese contents. Analysis of the standards
revealed that a minimum limit of quantification for manganese in an organic
matrix was approximately 1 mg/kg.
Tissue preparation for neurotransmitters and Western blots. Twomillimeter slices of the left hemisphere of the brain were dissected to collect
the region of interest (Str, GP, Th, Cx). Brain regions were weighed, and nine
times their weight of an ice-cold 10mM HEPES, pH 7, solution was added
in a tube containing a homogenization resin and the sample homogenized for
1 min on ice per the manufacturer’s recommendations (Amersham Biosciences,
Piscataway, NJ sample grinding kit). The homogenate was further disrupted
with sonication (3 3 1 s pulses at 25 Hz, 3-mm probe), vortexed, and
centrifuged at 1000 3 g for 10 s. The supernatant was diluted in 23 RIPA
buffer for Western blots or in 53 perchlorate buffer for neurotransmitter
determination (see below).
Biochemical determinations. For DA determination, four parts of tissue
homogenate in 30mM HEPES (pH 7.4) were added to one part of a degassed
53 solution of perchlorate for a final concentration containing 400mM
perchlorate, 2mM Na2EDTA, and 2 lM 3,4-dihydroxybenzylamine (DHBA)
as internal standard. External standards were diluted with the perchlorate
solution in the same proportion as the samples. Supernatant fractions were
separated via centrifugation at 16,000 3 g for 5 min at 4C. The sample was
injected onto an HPLC (Beckman with Gold Software) fitted with a C18, 3-lm
reversed-phase column (100 mm) for separation of DA and metabolites and
measured using electrochemical detection (BAS), according to procedures
outlined by Freeman et al. (1993). Quantified compounds included DA and its
metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) and 5-HT.
Quantification was performed using DHBA-spiked external standards prepared
during sample processing. Data were expressed relative to tissue wet weight.
Tissue homogenates were also analyzed for gamma amino butyric acid
(GABA) and the excitatory amino acids aspartate and glutamate by HPLC-EC
after derivatization with o-phthalaldehyde. Derivatization was accomplished by
combining a 20-ll volume of the homogenate supernatant obtained during
processing for DA measurements (see above) with 208 ll of b-amino butyric
acid-spiked (internal standard BABA) 0.1M borate buffer (pH 9.5) and 32 ll of
derivatizing reagent (0.1M borate buffer, 0.05M sodium sulfite, 5% [vol/vol]
ethanol, and 0.02M o-phthalaldehyde). External standards were prepared at the
time of sample derivatization and derivatized using the same BABA-spiked
buffer used with the samples. The derivatized mixtures were analyzed via C18,
3-lm reversed-phase HPLC (150-mm column) with electrochemical detection.
Data were expressed relative to wet tissue weight.
Cellular abundance of the proteins TfR, H-ferritin, and tubulin was
determined with gel electrophoresis and Western blotting using previously
described methods with slight modifications (Kwik-Uribe et al., 2003). Briefly,
aliquots of the tissue homogenate in HEPES, pH 7, were further disrupted on ice
in RIPA lysis buffer (phosphate-buffered saline, containing 1% Igepal-640,
0.5% sodium deoxycholate, 0.1% sodium lauryl sulfate, 1mM sodium
orthovanadate, 1mM phenylmethylsulfonyl fluoride, 10 lg/ml pepstatin, and
10 lg/ml leupeptin). The lysate was then centrifuged at 2000 3 g for 10 min at
4C, an aliquot was taken for protein determination (by the Bradford method),
and the remaining supernatant was mixed with 43 NuPAGE sample-loading
buffer and stored at 70C until analysis. Prior to gel loading, all samples were
diluted to equal protein concentrations using NuPAGE sample-loading buffer.
Lysates in NuPAGE sample-loading buffer were reduced with 10% (vol/vol)
0.75M dithiothreitol and loaded onto NuPAGE 10% Bis-Tris gels (9 lg of
protein loaded per lane). Following separation, the proteins were transferred to
PVDF membranes and probed with a mouse monoclonal TfR (Zymed
Laboratories), H-ferritin (a kind gift from Dr J. Connor, Hershey Medical
Center, Pennsylvania State University, Hershey, PA), or rabbit b-tubulin (Santa
Cruz Biotechnology). Following immunoblotting with the appropriate HRPconjugated secondary antibodies (Santa Cruz Biotechnology), the protein bands
were visualized using an ECL PLUS detection kit (Amersham Pharmacia,
Piscataway, NJ). Band intensity was imaged and quantified using a Typhoon
scanner equipped with Image Quant software (Amersham Biosciences).
Heparinized whole blood was centrifuged at 1000 3 g and the separated
plasma fraction removed and stored at 20C. Plasma prolactin concentrations
were measured by a radioimmunoassay (RIA) with a kit from Diagnostic
Systems Laboratories (Webster, TX) with RIA reagents from the National
Institute of Diabetes and Digestive and Kidney (NIDDK) Diseases Hormone
Distribution Program (Thordarson et al., 2001).
Statistics. Treatment and pairwise comparisons were performed using
two-way analysis of variance and Tukey’s post hoc comparison tests, with
manganese exposure and manganese oxidation state as the main factors. The p
values less than 0.05 were considered statistically significant for all tests. All
analyses were conducted using SYSTAT (SPSS Inc., 10th ed., 2000).
RESULTS
Rats were exposed to Mn(II)-chloride or Mn(III)-pyrophosphate at cumulative doses of 0, 30, and 90 mg Mn/kg body
weight over a 5-week period. These relatively low dose ranges
were selected based on previous studies with Mn(II) (Gwiazda
et al., 2002; Witholt et al., 2000), showing effects on GABA
concentrations in the Str and neuromotor deficits at doses
(72 mg Mn/kg body weight) comparable to those used here.
These dose ranges are also comparable, though somewhat higher,
117
MANGANESE OXIDATION STATE AND TOXICITY
Mn(III) exposures produced significantly higher blood manganese levels than comparable exposures to Mn(II) (Fig. 1a).
This is reflected by the significant effect of manganese dose
(F2,29 ¼ 567, p < 0.001) and oxidation state (F1,29 ¼ 29.9, p <
0.001) on blood manganese levels, and the significant dose 3
oxidation state interaction (F2,29 ¼ 7.40, p ¼ 0.002).
The highest Mn(III) exposure produced greater manganese
concentrations in the liver than the corresponding Mn(II)
exposure, as reflected by the overall dose (F2,29 ¼ 9.94, p ¼
0.001) and oxidation state effect (F1,29 ¼ 2.81, p ¼ 0.104;
interaction F2,29 ¼ 5.57, p ¼ 0.009) on total manganese
concentrations in the liver (Fig. 1b). The above interaction
reflects the ~ 56% increase versus control in liver manganese
concentrations of rats exposed to the highest dose of Mn(III).
There was only a ~ 5% nonsignificant increase in liver concentrations in Mn(II)-treated rats.
Manganese Concentrations in the Brain
Brain manganese levels increased significantly in all brain
regions with increasing manganese dose and oxidation state,
though within a dose or oxidation state there was no measurable difference among the four different brain regions measured (Str, GP, Th, and Cx; pBrainRegion all > 0.1) (Figs. 2a and
2b, Table 1). Other metals (Fe, Cu, Zn) did not measurably
change with manganese exposure (dose or oxidation state), and
the relative concentrations of these elements did not vary
among different brain regions (Table 1).
There was a clear effect of increasing manganese dose and
oxidation state on increasing manganese concentrations in all
four brain regions measured (GP, Str, Th, Cx), with Mn(III)
exposure producing higher brain manganese concentrations
than Mn(II) exposures (GP Mn dose F2,30 ¼ 112, p < 0.001, Mn
ox F1,30 ¼ 13.9, p ¼ 0.001, interaction p > 0.05; Str Mn dose
F2,30 ¼ 113, p < 0.001, Mn ox F1,30 ¼ 10.3, p ¼ 0.003,
interaction F2,30 ¼ 4.23, p ¼ 0.024; Th Mn dose F2,30 ¼ 114,
p < 0.001, Mn ox F1,30 ¼ 11.9, p ¼ 0.002, interaction F2,30 ¼
6.81, p ¼ 0.004; Cx Mn dose F2,30 ¼ 49.3, p < 0.001, Mn ox
F1,30 ¼ 5.12, p ¼ 0.031, interaction > 0.05). Because there
were no measurable differences between brain regions in the
accumulation of manganese, the brain regional manganese
values were treated as replicates, values were averaged together
for each individual animal, and the data were statistically
reanalyzed. This reanalysis further showed an effect of both
dose (F2,30 ¼ 207, p < 0.001) and oxidation state (F1,30 ¼ 18.5,
p < 0.001; interaction F2,30 ¼ 10.8, p < 0.001) on increasing
brain manganese concentrations (Fig. 2c).
Blood [Mn], ppb
Manganese Concentrations in Blood and Liver
A
E
500
400
D
300
C
200
100
B
A
A
0
30 mg/kg
0 mg/kg
90 mg/kg
Cumulative Mn Dose
B
14
12
Liver [Mn], ppm
to the lowest cumulative dose (10 mg/kg body weight) where
adverse neurological effects have been observed in humans
occupationally exposed to manganese (Roels et al., 1992).
Because we wanted to test the effects of manganese exposure
on aged animals, female retired breeders were utilized.
*
Mn Dose p < 0.001
Mn Ox. State p < 0.001
10
8
6
4
2
0
0 mg/kg
30 mg/kg
90 mg/kg
Cumulative Mn Dose
FIG. 1. (a) Mean blood manganese concentrations (ng/ml, ppb) and (b)
liver manganese concentrations (lg/g dry weight, ppm) for Mn(II)-exposed
(gray) or Mn(III)-exposed (black) rats (± SE, n ¼ 5 for Mn(III) 0 mg/kg dose,
n ¼ 6 for all others). Dose (x axis) shown as cumulative amount of manganese
given over the 5-week exposure period per kilogram of body weight. Different
letters (e.g., A, B) denote a significant difference between treatments based on
a Tukey post hoc analysis.
Comparing the brain manganese concentrations (averaged
across brain regions) for each dose within oxidation state to the
blood manganese concentrations within animals showed a notable curvilinear relationship (Fig. 3). The best-fit curves for
Mn(II)- and Mn(III)-exposed animals were produced using
Michaelis-Menton–type equations of the general form: y ¼
Bmax 3 x/(Kd þ x), where Bmax¼ 4.4 (95% CI ¼ 3.9–4.8) and
5.3 (95% CI ¼ 4.6–6.0) for Mn(II)- and Mn(III)-exposed
animals, respectively. The Bmax term reflects the asymptote
of the best-fit curve, which in this case represents the saturation brain manganese concentration for each oxidation state.
The Bmax terms for the Mn(II)- versus Mn(III)-exposed
animals were marginally not significantly different (F1,31 ¼
3.96, p ¼ 0.055), suggesting that Mn(III) exposures produce
higher brain manganese concentrations at saturation than do
Mn(II) exposures. The Kd Mn(II) ¼ 22.4 and Kd Mn(III) ¼ 37.7
were not statistically different (F1,31 ¼ 1.42, p ¼ 0.242).
Brain regional GABA, DA, and 5-HT Concentrations
GABA levels increased significantly with manganese exposure in the GP and tended to increase in the Str (Fig. 4a, Table 2).
Manganese exposure increased GABA levels in the GP
118
REANEY, BENCH, AND SMITH
B
Brain [Mn], ppm
A
7
6
5
4
3
2
1
0
7
6
5
4
3
2
1
0
Brain Reg. p = 0.923
Mn Dose p < 0.001
0 mg/kg
30 mg/kg
90 mg/kg
Brain Reg. p = 0.987
Mn Dose p < 0.001
0 mg/kg
Cumulative Mn(II) Dose
C
30 mg/kg
90 mg/kg
Cumulative Mn(III) Dose
7
6
Avg. [Mn], ppm
GP
Str
Th
Cx
Mn Dose p < 0.001
E
Mn Ox. State p < 0.001
5
D
C
4
B
3
2
A
A
1
0
0 mg/kg
30 mg/kg
90 mg/kg
Cumulative Mn Dose
FIG. 2. Manganese concentrations (lg/g dry weight, ppm) in the GP (open), Str (gray), Th (black), and Cx (dark gray) of Mn(II)-exposed (a) and Mn(III)exposed (b) rats. (c) Shows average brain manganese concentrations of Mn(II)-exposed (gray) or Mn(III)-exposed (black) rats calculated using the average of all
brain regions within each animal. Data are means (± SE, n ¼ 6 rats per treatment for each Mn(II) and Mn(III) exposure group, see text). The x axis shows the
cumulative manganese dose per kilogram body weight administered over the 5-week exposure period. Brain manganese concentrations were measured by PIXE
(see text). Different letters (e.g., A, B) in (c) denote a significant difference between treatments based on a Tukey post hoc analysis.
by ~ 15–30% over controls (dose F2,29 ¼ 3.69, p ¼ 0.037; ox
F1,29 ¼ 0.047, p ¼ 0.83; interaction p > 0.05). There were
similar, though marginally nonsignificant, GABA increases of
15–45% in the Str (15–45%) (dose F2,29 ¼ 2.92, p ¼ 0.07; ox
F1,29 ¼ 5.92, p ¼ 0.021; interaction p > 0.05) but not the Th
(dose F2,29 ¼ 0.308, p ¼ 0.740; ox F1,29 ¼ 1.09, p ¼ 0.310;
interaction p > 0.05) (Table 2). It is worth noting that
differences in the effect of oxidation state were apparent if
the data were grouped by non–manganese exposed versus
manganese exposed (30 and 90 mg/kg combined) within
oxidation state. Mn(III) exposure led to a significant increase
of ~ 41% (p ¼ 0.010) in Str GABA levels, while Mn(II)
exposure resulted in no measurable change (~ 4%, p > 0.1)
compared to control.
Brain DA concentrations in the GP were altered by
manganese dose and oxidation state, though this was most
evident by the significant difference between the Mn(II) versus
Mn(III) effect at the highest exposure dose (dose F2,29 ¼ 0.304,
p ¼ 0.741; ox F1,29 ¼ 1.42, p ¼ 0.243; interaction F2,29 ¼ 4.52,
p ¼ 0.020) (Table 2, Fig. 4b). There was no manganese effect
on DA levels in the Str (dose F2,29 ¼ 0.375, p ¼ 0.690; ox
F1,29 ¼ 0.052, p ¼ 0.820; interaction p > 0.05) (Table 2).
The significant treatment interaction in the GP reflects the
significantly different effects of the highest Mn(II) and
Mn(III) exposures on DA concentrations, with a trending decrease
in DA with Mn(II) exposure and significantly higher DA in
Mn(III)-exposed animals (Fig. 4b). DA and 5-HT levels in
the Th were below the limit of quantification for the method.
Brain 5-HT concentrations (Table 2) were not measurably
altered by manganese exposure, though levels trended up
with Mn(III) exposure: GP Mn dose F2,29 ¼ 0.40, p ¼ 0.67; Mn
ox F1,29 ¼ 2.18, p ¼ 0.15; interaction p > 0.05; Str Mn dose
F2,29 ¼ 2.65. Interestingly, similar to GABA levels in the Str,
there was a significant effect of manganese oxidation state on
Str 5-HT concentrations when the data were grouped by manganese exposed (30 and 90 mg/kg combined) versus not manganese exposed within oxidation state. Mn(III) exposure led to
a significant increase of ~ 31% (p ¼ 0.028) in Str 5-HT levels,
while Mn(II) exposure resulted in no change (~ 9%, p > 0.1).
TfR and H-ferritin Protein
TfR protein abundance trended toward an increase with
increasing manganese dose in all brain regions measured (GP,
Str, Th), though the manganese dose effect was significant only
in the GP (GP Mn dose F2,29 ¼ 5.29, p ¼ 0.011, ox F1,29 ¼
0.534, p ¼ 0.471, interaction p > 0.05; Str dose F2,29 ¼ 2.56,
p ¼ 0.090, ox F1,29 ¼ 0.074, p ¼ 0.787, interaction p > 0.05; Th
dose F2,29 ¼ 1.37, p ¼ 0.27, ox F1,29 ¼ 0.001, p ¼ 0.97,
119
MANGANESE OXIDATION STATE AND TOXICITY
TABLE 1
Mean Concentrations of Manganese (Mn), Iron (Fe), Copper (Cu), and Zinc (Zn) in the GP, Str, Th, and Cx of Mn(II)- and
Mn(III)-Exposed Rats, as Measured by PIXE. Mean Values Are Expressed as a Percentage of Control (± SE, n ¼ 5–6).
Asterisks Signify That the Value is Statistically Different Than Control
Trace metal
Brain region
(control conc.)a
Mn
GP
Str
Th
Cx
GP
Str
Th
Cx
GP
Str
Th
Cx
GP
Str
Th
Cx
Fe
Cu
Zn
a
(1.4)
(1.5)
(1.5)
(1.6)
(106)
(99)
(100)
(98)
(9.2)
(9.4)
(9.4)
(9.7)
(97)
(92)
(99)
(97)
Mn(II),
0 mg/kg
Mn(II),
30 mg/kg
Mn(II),
90 mg/kg
Mn(III),
0 mg/kg
Mn(III),
30 mg/kg
Mn(III),
90 mg/kg
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
218
176
193
201
99.1
118
117
97.3
105
104
111
93.9
105
104
103
104
309
278
243
298
102
110
119
105
102
106
103
104
101
112
100
108
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
229
252
226
203
100
104
93.9
99.1
95.2
105
110
98.8
98.3
94.3
95.4
94.1
327
401
361
261
94.9
102
96.9
104
106
114
99.3
100
101
109
89.2
91.5
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
7.8
7.5
7.1
5.1
16
14
15
14
4.5
11
11
9.2
5.2
9.2
7.4
5.9
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
21*
27*
23*
36*
5.3
4.5
12
6.7
12
13
9.3
8.7
3.5
6.2
8.0
3.7
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
12*
26*
22*
23*
10
7.1
8.2
9.9
16
7.9
9.1
11
2.9
4.2
9.0
6.7
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
8.6
8.3
8.6
5.4
5.6
6.6
6.6
7.4
12
5.7
6.7
8.2
6.2
5.7
6.5
4.3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
18*
18*
22*
10*
4.9
4.9
2.0
14
4.2
4.2
17
10
3.3
3.3
7.3
4.5
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
32*
29*
22*
37*
10
12
9.1
10
5.6
16
3.6
10
4.4
5.6
7.2
5.3
Values in parentheses are average trace metal concentrations in brain region of control animals in units of micrograms per gram dry weight (ppm).
interaction p > 0.05) (Fig. 5a). Overall, however, brain TfR
levels were significantly increased with manganese exposure
when the changes, relative to controls, were averaged across
brain regions for each individual animal (Mn dose F2,29 ¼ 6.92,
7
Avg. Brain [Mn], ppm
6
5
4
3
2
1
0
0
100
200
300
400
500
600
Blood [Mn], ppb
FIG. 3. Blood manganese (ng/ml, ppb) versus brain manganese (lg/g dry
weight, ppm) concentrations for Mn(II)-exposed (gray inverted triangles) and
Mn(III)-exposed (squares) rats. The manganese concentrations of all brain
regions analyzed were averaged to calculate the brain manganese concentration
within each animal. Also shown are best-fit lines for the Mn(II)-exposed (solid
line) and Mn(III)-exposed (dashed line) rats, determined using a MichaelisMenton–type function with the form: y ¼ Bmax 3 x/(Kd þ x), where Bmax¼ 4.4
(95% CI ¼ 3.9–4.8) and 5.3 (95% CI ¼ 4.6–6.0) for Mn(II)- and Mn(III)exposed animals, respectively. The Kd for Mn(II)- and Mn(III)-exposed groups
were 22.4 and 37.7, respectively. The Bmax values were marginally not
significantly different between Mn(II) and Mn(III) groups ( p ¼ 0.055). The
Kd values were not statistically different ( p ¼ 0.242, see text).
p ¼ 0.003, Mn ox F1,29 ¼ 0.258, p ¼ 0.615, interaction p >
0.05) (Fig. 5b). For these latter analyses, each brain region was
treated as a replicate measure and all brain regions averaged
within an animal; average brain values for each animal were
averaged within treatment group.
There was a trending increase in H-ferritin protein levels in
the GP and Str with increasing manganese dose. Insufficient
sample volume was available for measurement of H-ferritin in
the Th. There was an effect of manganese dose to increase Hferritin in the GP (Fig. 6) and an effect of manganese oxidation
state in the Str, but no effects were observed on the average Hferritin levels across these brain regions (GP dose F2,29 ¼ 5.05,
p ¼ 0.013, ox F1,29 ¼ 0.797, p ¼ 0.379; interaction p > 0.05;
Str dose F2,29 ¼ 1.29, p ¼ 0.291, ox F1,29 ¼ 7.40, p ¼ 0.011,
interaction F2,29 ¼ 3.68, p ¼ 0.038; average of GP and Str
within animal, Mn dose F2,29 ¼ 2.99, p ¼ 0.067, ox F1,29 ¼
2.14, p ¼ 0.155, interaction p > 0.05). b-Tubulin levels did not
change in any of the brain regions or treatment groups measured (GP Mn dose F2,29 ¼ 0.186, p ¼ 0.831, ox F1,29 ¼ 0.017,
p ¼ 0.897; Th Mn dose F2,29 ¼ 1.235, p ¼ 0.305, ox F1,29 ¼
0.409, p ¼ 0.528; Str Mn dose F2,29 ¼ 0.615, p ¼ 0.547, ox
F1,29 ¼ 0.532, p ¼ 0.472).
Plasma Prolactin Concentrations
Plasma prolactin levels significantly decreased with increasing manganese dose but not manganese oxidation state
(Mn dose F2,29 ¼ 3.9, p ¼ 0.032; Mn ox F1,29 ¼ 0.003, p ¼
0.958; interaction p > 0.05). When data across oxidation state
treatment were averaged and reanalyzed by manganese dose,
120
GP GABA (% Control)
A
REANEY, BENCH, AND SMITH
there remained a significant manganese dose effect to decrease
plasma prolactin levels (p ¼ 0.022) (Fig. 7).
180
160
Mn Dose p = 0.037
140
120
100
DISCUSSION
80
60
40
20
0
0 mg/kg
30 mg/kg
90 mg/kg
Cumulative Mn Dose
GP DA (% Control)
B
200
180
160
140
120
100
80
60
40
20
0
*
30 mg/kg
0 mg/kg
90 mg/kg
Cumulative Mn Dose
FIG. 4. (a) Mean cellular GABA and (b) DA concentrations in the GP of
Mn(II)-exposed (gray) or Mn(III)-exposed (black) rats, expressed as percentage
of control (± SE, n ¼ 5 for Mn(III) 0 mg/kg, n ¼ 6 for all others). The x axis
shows dose as cumulative amount of manganese given over the 5-week
exposure period per kiolgram body weight. Control rats had a mean GABA
concentration in the GP of 3 mol GABA/g tissue and a mean DA concentration
of 12.5 nmol DA/g tissue.
This study shows that the oxidation state of manganese
exposure is important in modulating manganese retention and
accumulation. Mn(III) exposures as Mn(III)-pyrophosphate
resulted in greater accumulation/retention of manganese in
the blood, liver, and brain compared to equimolar exposures to
Mn(II)-chloride. The higher brain manganese levels produced
by the Mn(III) exposures were associated with increases in
brain GABA concentrations, TfR levels, and H-ferritin levels,
particularly in the GP. These data substantiate the need to better
understand the relative effects of manganese exposures in
different oxidation states and how these exposures impact
specific brain regions.
The higher levels of tissue manganese accumulation/
retention with Mn(III) exposures (Figs. 1 and 2) evidence different toxicodynamics for the Mn(II) versus Mn(III) exposures.
If it is assumed that similar amounts of the ip administered
Mn(II) and Mn(III) entered the circulation for a given dose,
these results would then suggest that there is significantly less
efficient clearance of manganese from the Mn(III) exposures.
These observations are consistent with previous animal model
studies which showed that Mn(II) exposures in the form of
Mn(II)-chloride or Mn-protein complexes (e.g., globulins) are
more efficiently cleared from the blood by the liver than are
Mn(III) exposures as the manganic-transferrin complex (Chua
and Morgan, 1997; Gibbons et al., 1976). We hypothesize that
the higher liver concentrations observed with Mn(III) exposures (Fig. 1b) could be due to possible sequestering of manganese in the abundant ferritin stores of the liver and hence less
TABLE 2
Mean GABA, DA, and 5-HT Concentrations in the GP, Str, and Th of Control and Mn(II)- and Mn(III)-Exposed Rats.
Mean Values Are Expressed as a Percentage of Control (± SE, n ¼ 5–6)
Neurotransmitter
GABA
DA
5-HT
Brain region
(control conc.)a
GP
Str
Th
GP
Str
Th
GP
Str
Th
(3.16)
(2.71)
(1.50)
(12.6)
(33.8)
(< DL)b
(1.23)
(2.74)
(< DL)
Mn(II),
0 mg/kg
Mn(II),
30 mg/kg
Mn(II),
90 mg/kg
Mn(III),
0 mg/kg
Mn(III),
30 mg/kg
Mn(III),
90 mg/kg
100
100
100
100
100
<
100
100
<
100
115
125
106
99
<
98
118
<
118
94
111
59
105
<
99
101
<
100
100
100
100
100
<
100
100
<
95
145
96
75
98
<
126
137
<
129
137
105
158
113
<
119
125
<
± 7.5
± 7.7
± 8.3
± 23
± 6.3
DL
± 9.0
± 4.0
DL
± 6.0
± 17
± 18
± 25
± 16
DL
± 8.9
± 19
DL
± 9.4
± 9.8
± 14
± 8.8
± 17
DL
± 9.2
± 12
DL
± 14
± 5.8
± 19
± 26
± 10
DL
± 18
± 3.5
DL
± 10
± 18
± 12
± 20
± 8.0
DL
± 18
± 12
DL
± 15
± 9.4
± 10
± 30
± 15
DL
± 16
± 11
DL
a
Values in parentheses are average neurotransmitter concentrations in control brain regions of control animals in units of picomoles of neurotransmitter per
gram of wet tissue weight.
b
< DL ¼ below the analytical detection limit for the method.
121
MANGANESE OXIDATION STATE AND TOXICITY
FIG. 6. Bar graph shows mean (± SE, n ¼ 6) H-ferritin protein levels in the
GP of Mn(II)-exposed (gray) or Mn(III)-exposed (black) rats, expressed as
percentage of control levels. Also shown are representative Western blots for
Mn(III)-exposed (top) or Mn(II)-exposed (bottom) rats (9 lg protein per lane).
The x axis shows the cumulative manganese dose per kilogram body weight
administered over the 5-week exposure period.
FIG. 5. (a) Bar graph shows mean (± SE, n ¼ 6) TfR protein levels in the
GP of Mn(II)-exposed (gray) or Mn(III)-exposed (black) rats, expressed as
percentage of control levels. Representative Western blots shown for Mn(III)exposed (top) or Mn(II)-exposed (bottom) rats (9 lg protein per lane). (b) Brain
TfR protein levels of Mn(II)-exposed (gray) or Mn(III)-exposed (black) rats,
calculated using the control-normalized average of all measured GP, Str, and Th
brain regions within each animal. The x axis shows the cumulative manganese
dose per kilogram body weight administered over the 5-week exposure period.
and Chen, 1994; Herak et al., 1982; Scheuhammer and
Cherian, 1985) and with studies showing that Mn(II) and
Mn(III) utilize different transport mechanisms for cellular
uptake and transport across the blood-brain barrier; DMT-1 is
known to transport Mn(II) species, and TfR-mediated endocytosis transports Mn(III)-transferrin species (Aschner and
Gannon, 1993; Murphy et al., 1991; Zheng et al., 2003).
efficient clearance of manganese when exposures occur as
Mn(III).
The higher brain manganese concentrations with Mn(III)
exposures, compared to those produced from exposures to
Mn(II), appear to be due predominantly but not solely to the
higher concentrations of manganese in blood. The best-fit
curvilinear associations between blood and brain manganese
concentrations imply that for significantly elevated blood
manganese levels, Mn(III) exposures result in higher brain
manganese concentrations than comparable exposures to
Mn(II) (Fig. 3). If so, this suggests that the availability of
blood manganese to the brain or the retention of manganese in
the brain may differ depending in part on the oxidation state of
manganese exposure. This suggestion is consistent with studies
indicating that manganese normally exists in both Mn(II) and
Mn(III) species within the blood (Hancock et al., 1973; Harris
Prolactin (% Control)
140
120
Mn Dose p = 0.032
A
100
80
A,B
60
40
B
20
0
0 mg/kg
30 mg/kg
90 mg/kg
Cumulative Mn Dose
FIG. 7. Mean plasma prolactin concentrations of manganese-exposed rats,
expressed as percentage of control (± SE, n ¼ 12 per dose). Data from Mn(II)and Mn(III)-exposed groups were combined since there was no effect of
manganese oxidation state on serum prolactin levels (see text). Dose (x axis)
shown as cumulative amount of manganese given over the 5-week exposure
period per kilogram body weight. Control rats had a prolactin concentration of
125 ng/ml. Different letters (e.g., A, B) denote a significant difference between
treatments based on a Tukey post hoc analysis.
122
REANEY, BENCH, AND SMITH
There was a clear effect of manganese dose and oxidation
state on a number of brain outcomes, including levels of
neurotransmitter and proteins involved in cellular iron metabolism (Table 2, Figs. 4–6). The oxidation state of manganese
exposure had a significant impact on GABA in the Str and GP,
as well as H-ferritin levels in the Str. However, these effects
must be qualified by the fact that the Mn(III) exposures
produced higher brain manganese levels—making it difficult
to distinguish brain effects arising from manganese concentration versus oxidation state of exposure. A notable exception to
this qualification is the effect of the highest Mn(III) exposure to
significantly increase DA levels by ~ 60% in the GP, compared
to an ~ 40% decrease caused by comparable Mn(II) exposures,
relative to their respective controls (Fig. 4b). The mechanistic
basis for this latter observation remains unclear, though a recent
review of manganese effects on striatal DA content in rodent
models has shown highly variable effects, from no change,
increase, or decreases in striatal DA (Gwiazda et al., 2006),
though none of these studies compared the effects of soluble
Mn(II) versus soluble Mn(III) exposures.
Previous studies have shown that both dopaminergic and
GABAergic systems of the basal ganglia are affected by
manganese (Autissier et al., 1982; Bonilla, 1978; Chandra
and Shukla, 1981; Eriksson et al., 1987; Gianutsos and Murray,
1982; Gwiazda et al., 2002; Lipe et al., 1999; Neff et al., 1969).
While reported manganese effects on brain DA levels are quite
variable, possibly due to experimental differences between
studies (Gwiazda et al., 2006), rodent studies by and large are
consistent in reporting increases in striatal GABA levels following manganese exposure (few studies have evaluated
GABA levels in the GP) (Bonilla, 1978; Bonilla and Prasad,
1984; Gianutsos and Murray, 1982; Gwiazda et al., 2002; Lipe
et al., 1999). These GABAergic effects are consistent with
histological and MRI evidence from monkeys and human
studies indicating that the GABA-rich GP is among the most
sensitive brain regions affected by manganese. Our data suggest that the apparent increased susceptibility of the GP and Str
may be due to their inherent sensitivity to manganese since
there were no measurable differences in accumulated manganese, iron, copper, or zinc across the GP, Str, Th, andCx brain
regions measured here.
Increases in TfR protein and H-ferritin expression with
increasing manganese dose were apparent in the Str and GP,
though a statistically significant dose response occurred only in
the GP. The concurrent increase in TfR and H-ferritin protein
levels evidence disruption of iron regulation since normally
these proteins respond in opposite directions to iron deficiency
or overload. An increase in TfR protein levels or mRNA resulting from exposure to Mn(II) (Kwik-Uribe et al., 2003; Zheng
and Zhao, 2001; Zheng et al., 1999) and Mn(III) (Reaney and
Smith, 2005) has been previously observed. There are clear
toxicological implications for these outcomes since disruption
of cellular iron metabolism due to elevated manganese exposure has been implicated in increased oxidative stress and
cellular dysfunction, including association with neurodegenerative diseases such as Parkinson’s disease (Dexter et al., 1991;
Gorell et al., 1997; Riederer et al., 1989; Sofic et al., 1988).
Serum prolactin levels have been evaluated as a marker for
manganese exposure in the occupational setting by several
studies (Ellingsen et al., 2003; Mutti et al., 1996). The rationale
for the use of serum prolactin as a surrogate of manganese
exposure is based on the assumption that increases in systemic
manganese results in a reduction of brain regional DA leading
to a lack of inhibition of prolactin synthesis and secretion in the
pituitary gland. Here we found that despite the lack of robust
changes in DA concentrations in the brain regions analyzed
(GP, Str, Th), serum prolactin concentrations decreased with
increasing manganese exposure. Previous animal studies have
reported varied effects of manganese exposure on serum prolactin levels, including no effect (Merritt and Brown, 1984) or
increased serum prolactin levels (Merritt and Brown, 1984).
While the basis for the apparent differences in the effect of
manganese on serum prolactin is not clear, the fact that significant alterations in serum prolactin levels exist suggests that
altered serum prolactin may be an important toxicological outcome of manganese exposure.
In summary, the cumulative dose range of manganese used
here (30–90 mg Mn/kg body weight) is at the lower range of
exposures used in studies reporting adverse effects in rodents
(Bonilla and Prasad, 1984; Chandra and Shukla, 1981;
Eriksson et al., 1987; Gwiazda et al., 2002; Subhash and
Padmashree, 1991). Exposures to Mn(III) produced higher
blood and brain manganese concentrations than equimolar exposures to Mn(II), though there were no measurable differences
in the manganese accumulation across the four brain regions
examined here. Similarly, there were no differences in the brain
region levels of iron, copper, and zinc due to manganese
treatment. However, there were effects of manganese dose and
oxidation state on a number of brain outcomes. These data
substantiate the heightened susceptibility of the GP and Str
brain regions to manganese, and they indicate that the oxidation state of manganese exposure may be an important determinant of tissue toxicodynamics and subsequent neurotoxicity.
ACKNOWLEDGMENTS
We thank R. Gwiazda, C. Kwik-Uribe, D. Crooks, G. Thordarson, and D. Di
Monte for their assistance. We also would like to thank our biostatistician,
Dr Curt Engelhard, for his assistance with the statistics. This research was
supported by NIEHS grant ES010788, the University of California Toxic
Substances Research and Teaching Program. This work was performed in part
under the auspices of the U.S. Department of Energy by University of
California, Lawrence Livermore National Laboratory, under Contract No.
W-7405-Eng-48 and a CAMS minigrant.
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