Public perception of the safety and efficacy of childhood
vaccines has a direct impact on immunization rates (Biroscak
et al. 2003; Thomas et al. 2004). The current debate linking
the use of thimerosal in vaccines to autism and other developmental
disorders [Institute of Medicine (IOM) 2001, 2004] has led
many families to question whether the potential risks associated
with early childhood immunizations may outweigh the benefits
(Blaxill et al. 2004; SafeMinds 2005). Thimerosal is an effective
preservative that has been used in the manufacturing of vaccines
since the 1930s. Thimerosal consists of 49.6% mercury by weight
and breaks down in the body to ethylmercury and thiosalicylate
(Tan and Parkin 2000). Recent reports have indicated that some
infants can receive ethylmercury (in the form of thimerosal)
at or above the U.S. Environmental Protection Agency (EPA)
guidelines for methylmercury exposure (U.S. EPA 2005), depending
on the exact vaccinations, schedule, and size of the infant
(Ball et al. 2001). Clements et al. (2000) calculated that
children receive 187.5 µg of ethylmercury from thimerosal-containing
vaccines given over the first 14 weeks of life. According to
the authors, this amount approaches or, in some cases, exceeds
the U.S. EPA guidelines for MeHg exposure during pregnancy
(0.1 µg/kg/day). Other estimates (Halsey 1999) have indicated
that the schedule could provide repeated doses of ethylmercury
from approximately 5 to 20 µg/kg over the first 6 months
of life. Studies in preterm infants indicate that blood levels
of Hg after just one vaccination (hepatitis B) increase by > 10-fold
to levels above the U.S. EPA guidelines (Stajich et al. 2000).
The U.S. EPA guidelines for MeHg (U.S. EPA 2005) are based
on several decades of studies of humans and animal models of
developmental toxicity (Burbacher et al. 1990a; National Research
Council 2000). Because few data exist for ethylmercury, the
use of the MeHg guidelines would seem appropriate if the two
compounds have similar toxicokinetic profiles and neurodevelopmental
effects. The results from the few studies that have provided
a direct comparison of these two compounds have been reviewed
recently by Magos (2003), who concluded that a) Hg clears
from the body faster after the administration of ethylmercury
than after the administration of MeHg; b) the brain-to-blood
Hg concentration ratio established for MeHg will overestimate
Hg in the brain after exposure to ethylmercury; and c)
because ethylmercury decomposes faster than MeHg, the risk
of brain damage is less for ethylmercury than for MeHg. These
conclusions are based on only a few studies, none of which
included measurements of both blood and brain Hg levels in
infant subjects.
We initiated the present study in order to directly compare
the blood and brain levels of Hg in infant nonhuman primates
exposed orally to MeHg or via intramuscular (im) injections
of vaccines containing thimerosal. Nonhuman primates have been
used extensively in previous studies of MeHg toxicokinetics
and developmental neurotoxicity (Burbacher et al. 1986, 1990b;
Gunderson et al. 1986, 1988; Rice and Gilbert 1982, 1990, 1995;
Stinson et al. 1989; Vahter et al. 1994, 1995). The routes
of administration (oral for MeHg and im injection for thimerosal-containing
vaccines) were chosen to mimic the two routes of Hg exposure
for humans. The dosages and schedule of administration of Hg
were chosen to be comparable with the current immunization
schedule for human newborns, taking into consideration the
faster growth (~ 4 to 1) of the macaque infant (Gunderson and
Sackett 1984). The results of the present study provide important
new information regarding the comparative toxicokinetics of
these two compounds in newborns and infants.
Subjects. Forty-one infant Macaca fascicularis born
at the Washington National Primate Research Center’s
Infant Primate Research Laboratory were used in the study.
The birth weights of the infant monkeys were within the normal
range for this species; the average birth weight was 341 g
(range, 255-420 g). Infants were weighed daily throughout
the study, and any clinical problems were recorded.
Table 1
|
Mercury dosing schedule. The Hg dosing schedule
is shown in Table 1. Infants were assigned to one of three
exposure groups at birth. Seventeen infant monkeys assigned
to the thimerosal group were given the typical schedule of
vaccines for human infants (Table 1). Thimerosal (Omicron
Quimica S.A., Barcelona, Spain), dissolved in saline, was mixed
with
thimerosal-free vaccines to yield a final concentration of
4, 8, or 20 µg/mL Hg, depending on the vaccine and the
age of the infant. The total dose of Hg administered via the
vaccines was 20 µg/kg on day 0 and at 7, 14, and 21 days
of age. A dose of 20 µg/kg was chosen based on the
range of estimated doses received by human infants receiving
vaccines
during the first 6 months of life.
Seventeen infant monkeys assigned to the MeHg group were
given MeHg hydroxide (MeHgOH, 97% pure; Alfa Aesar, Johnson
Matthey Co., Ward Hill, MA) dissolved in water to a concentration
of 20 µg Hg/mL. MeHg was administered to infant monkeys
via oral gavage at a dose of 20 µg/kg on their day of
birth (day 0) and at 7, 14, and 21 days of age.
Seven infant monkeys were assigned to a control group. These
monkeys did not receive any gavages or im injections. Infants
were assigned to the three groups on a semirandom basis, in
order to balance sex ratios and average birth weights across
groups.
Blood draw schedule. Blood was drawn from the
saphenous vein of all infant monkeys at birth (before any Hg
exposure). Blood was also drawn 2, 4, and 7 days after the
initial Hg exposure (day 0) and after subsequent exposures
on days 7 and 14. Depending on the sacrifice group, blood was
drawn up to 28 days after the final exposure on day 21 to further
characterize the washout kinetics of Hg (Table 1).
Sacrifice schedule. Infants were sacrificed
2, 4, 7, or 28 days after their last Hg exposure on day 21
(Table 1). Infants were sedated with an im injection of ketamine
(10 mg/kg) and atropine (0.4 mg/kg) and then given an intravenous
overdose of Nembutal (20 mg/kg; Abbott Labs, North Chicago,
IL). Autopsy personnel from the primate center drew blood and
removed the brain and other organs for analysis. The autopsy
typically lasted approximately 1 hr.
The numbers of monkeys at each sacrifice day for both the
MeHg and thimerosal groups were as follows: day 23 (2 days
after most recent dose), n = 4; day 25 (4 days after
most recent dose), n = 4; day 28 (7 days after most
recent dose), n = 5; and day 49 (28 days after most
recent dose), n = 4. The seven control monkeys were
assigned sacrifice days as follows: day 23, n = 3; day
25, n = 1; day 28, n = 2; and day 49, n =
1 (Table 1). Monkeys were assigned to sacrifice groups at birth
on a semirandom basis that balanced sex ratios and average
birth weights across groups.
Blood and brain Hg measurements. Blood
samples were prepared for Hg analysis by diluting them with
an equal volume of 1% wt/vol NaCl solution. Aliquots were removed
for Hg determination without digestion. One drop of antifoam
reagent was added to the aliquot at the time of the analysis.
Half brain samples were fixed in formaldehyde before analysis.
Samples of the fixative were analyzed to check for Hg content.
The tissue was removed from the jar and blotted dry. A homogenate
of the brain in 1% NaCl was prepared using a Polytron homogenyzer
PT 10-35 (Brinkmann Instruments, Westbury, NY) while keeping
the sample in an ice slurry. An aliquot of the homogenate was
digested with 1 mL 1% wt/vol cysteine and 2 mL 45% NaOH by
heating at 95°C for 10-15 min. Digest was allowed
to cool and then diluted to volume by addition of 7 mL 1% wt/vol
NaCl. The digests were kept in an ice slurry until analysis.
Aliquots were removed for Hg determination. One drop of antifoam
was added to the aliquot at the time of the analysis.
Total Hg concentrations in blood and total and inorganic
Hg concentrations in brain were measured using a procedure
adapted from Greenwood et al. (1977). The method determines
total Hg and its inorganic fraction (Magos and Clarkson 1972).
Cadmium chloride in the presence of stannous chloride at high
pH breaks the Hg-carbon bond with the subsequent reduction
of Hg2+ to Hg0; the latter is then measured
by cold vapor atomic absorption at 254 nm with a Hg monitor
(Laboratory Data Control, model 1235; Thermo Separation Products,
Waltham, MA). Inorganic Hg is determined by the addition of
SnCl2 in the absence of cadmium chloride. Concentration
of organic Hg was calculated from the difference between the
measured total and inorganic Hg concentrations. The original
concentration of SnCl2 used for the Magos method
(Magos and Clarkson 1972) was modified to prevent the decomposition
of the ethylmercury during assay (Magos et al. 1985). To measure
Hg in aqueous solution of thimerosal, the amount of SnCl2 was
reduced from 100 µg to 50 µg/aliquot analyzed.
For tissue homogenate samples, 500 µg SnCl2 was
added to each aliquot. All reagents used for preparation and
analysis of the samples were of analytical grade.
Quality control was assured by analysis of reference samples
before each assay run. Fisher Mercury Reference Standard Solution
(SM114-100, certified 1,000 ppm ± 1%; Fisher Scientific,
Hampton, NH) was used as a stock solution. Working standards
of 30 and 10 ng Hg/mL were made daily from appropriate dilutions
of the stock solution. In addition, the following certified
reference materials were analyzed daily before analysis of
the samples: trace elements in whole blood (Seronorm Trace
Elements, Certified Reference Material 201605, 6.8-8.5 µg/L;
Accurate Chemical & Scientific Corp., Westburg, NY), and
trace elements in human hair (Certified Reference Material
397, 12 µg/g ± 0.5; Commission of the European
Communities, Geel, Belgium). The detection limit of the instrument
was estimated to be 0.75 ng Hg per aliquot used for analysis.
Data analysis. The mean total blood Hg concentration
data from both the oral MeHg and im thimerosal groups (n =
17 in each) were analyzed using the compartmental module of
the pharmacokinetic modeling software SAAM II (SAAM Institute,
Seattle, WA).
The accumulation and washout of total blood Hg concentration-time
data from the MeHg monkeys were well described by a one-compartment
model featuring a first-order absorption process. Regression
fit of the data to the model yielded estimates of the absorption
rate constant (ka), elimination rate constant
(K), and an apparent volume of distribution (V/F; F is
the implicit bioavailability term). Half-lives (T1/2)
corresponding to each of the rate constants were calculated
by dividing ln 2 by the rate constant estimate. Blood clearance
(Cl/F) was derived from the product of K and V/F.
A one-compartment model failed to provide a satisfactory
fit of the mean total blood Hg concentration-time data
from the thimerosal monkeys. The model overpredicted the blood
concentration during accumulation; at the same time, it underpredicted
the blood concentration during washout rate (i.e., overpredicted
washout rate). Further examination of a scatter plot of the
individual monkey data suggested a biphasic pattern in the
washout of Hg from the blood after the last dose. Accordingly,
we attempted a regression fit of the mean total blood Hg concentration
data with a two-compartment model. This yielded a much better
visual fit of the data, with minimal change in the objective
function and Akaike information criterion. The two-compartment
parameter estimates from the regression analysis included the
absorption rate constant (ka), rate constants
for Hg transfer from the central to the peripheral compartment
(k12) and the return from the peripheral
to the central compartment (k21), the elimination
rate constant from the central compartment (k10),
and the apparent volume of the central compartment (Vc/F).
From these primary parameters, we further estimated the apparent
distribution volume at steady state (Vss/F)
and the peripheral volume referenced to blood concentration
(i.e., Vp = Vss - Vc).
The initial and terminal rate constants and half-lives (T1/2, and T1/2,β)
for the biexponential decline of total blood Hg concentration
were estimated by standard formulas (Gibaldi and Perrier 1982).
Blood clearance was computed by the product of Vc and k10.
For both the MeHg and thimerosal model fits, a fractional SD
of 0.1 was used as the weighting scheme.
The washout half-life of total and organic Hg in the brain
of both the oral MeHg and im thimerosal groups was estimated
by regression fit to a monoexponential model using WinNonlin
software (Pharsight Corp., Mountain View, CA). One of the day
28 brain samples from the MeHg exposure group had a spuriously
high total Hg concentration, that is, a concentration of 151
ng/g, which is more than 50% higher than the other samples
obtained on day 28 (71-90 ng/mL) and higher than those
observed at the earliest sacrifice time at day 2 (75-129
ng/g). The unreasonably high concentration is most likely due
to contamination of the sample. Therefore, data from this brain
and its corresponding blood were excluded from the regression
analysis. The average brain-to-blood concentration ratio was
also calculated using data from the earliest sacrifice duration
(2 days). Because of different washout half-lives in blood
and the brain, brain-to-blood concentration ratio is expected
to vary with the duration of washout. Samples at day 2 offered
the best measure of the extent of uptake of Hg species into
the brain that are least confounded by differences in their
clearance rate.
Between-group statistical comparisons of the rate of washout
of total Hg in blood, as well as total and organic concentrations
in the brain, were accomplished through multiple regression
analysis as implemented in the PROC GLM subroutine in SAS (version
9.1; SAS Institute, Cary, NC). PROC GLM performs multiple regression
within the framework of general linear models and can accommodate
missing data or sparse sampling and confounding from correlations
between repeated measures. Hence, it is able to provide tests
of hypotheses for the effects of time and group using blood
and brain data obtained from sacrifice of individual animals
at varying times during washout. Log-transformed blood or brain
Hg concentrations in animals from both the MeHg and thimerosal
groups were entered as the dependent variable. The independent
variables consisted of sampling time, group (MeHg = 0, thimerosal
= 1), and a time-by-group interaction. Once the overall significance
of the regression model was verified, the significant sources
of variation (i.e., time, group, and time by group) were identified.
A difference in the rate of washout of Hg in blood or brain
between groups was indicated by a significant regression coefficient
for time-by-group interaction. If there was no evidence for
interaction, a significant decline in blood or brain Hg concentration
over time for each group was assessed by the t-statistic
associated with the estimated regression coefficient for time.
The following statistical comparisons of the washout rate
of Hg were also undertaken: total Hg in blood versus total
Hg in the brain, total Hg in blood versus organic Hg in the
brain, and total Hg versus organic Hg concentration in the
brain. The difference between the pair of log-transformed Hg
concentrations for each animal sacrificed at the various times
was calculated. Individual difference values in both groups
were then entered as the dependent variable in the regression
model. The independent variables were time, group, and time-by-group
interaction. A significant regression coefficient for the time
variable indicates that the paired-log concentration difference
(or the concentration ratio) varied with time; that is, the
two concentration measures (e.g., blood and brain) did not
decline in parallel with time.
Figure 1. Weight gain of infant monkeys during
study. Error bars indicate SE.
|
Table
2
|
Figure 2. Comparison of predicted
and observed mean blood total Hg concentrations during and
after four
weekly oral doses (20 µg/kg) of MeHg. Error bars indicate
SD. |
Table 3
|
Figure 3. A semilogarithmic
plot of washout of total Hg in blood and the brain after
four weekly oral doses
(20 µg/kg) of MeHg. The data were collected from groups
of infant monkeys sacrificed 2, 4, 7, and 28 days after the
last dose. The lines represent nonlinear regression fit of
the data to a monoexponential model; the regression estimate
(± SE) of T1/2 is T1/2 =
19.1 ± 5.1 days (r = 0.81) for blood and T1/2 =
59.5 ± 24.1 days (r = 0.59) for brain. |
Figure 4. A semilogarithmic
plot of the washout of organic and inorganic Hg in the brain
after four weekly
oral doses (20 µg/kg) of MeHg. The data were collected
from groups of infant monkeys sacrificed at 2, 4, 7, and
28 days after the last dose. The lines represent nonlinear
regression fit of the data to a monoexponential model. The
regression estimate (± SE) for organic Hg is T1/2 =
58.4 ± 25.0 days (r = 0.57). The half-life
of inorganic Hg is too long (> 120 days) to be accurately
estimated from the present data (i.e., r is not
significantly different from 0). |
Table 4
|
Figure 5. Comparison of predicted and observed
mean blood total Hg concentration during and after four weekly
im injections of vaccine containing thimerosal (20 µg/kg
Hg). Error bars indicate SD. |
Figure 6. A semilogarithmic
plot of washout of total Hg in blood and the brain after
four weekly im injections
of vaccine thimerosal (20 µg/kg Hg). The data were
collected from groups of infant monkeys sacrificed at 2,
4, 7, 10, 17, and 21 days after the last dose. The lines
represent nonlinear regression fit of the data to a monoexponential
model. The regression estimate (± SE) of T1/2 is
24.2 ± 7.4 days (r = 0.74) for brain and 6.9 ± 1.7
days (r = 0.82) for blood. |
Figure 7. A semilogarithmic
plot of washout of organic and inorganic Hg in the brain
after four weekly im
injection of vaccines containing thimerosal (20 µg/kg
Hg). The data were collected from groups of infant monkeys
sacrificed at 2, 4, 7, and 28 days after the last dose. The
lines represent nonlinear regression fit of the data to a
monoexponential model. The regression estimate (± SE)
of T1/2 for organic Hg is T1/2 =
14.2 ± 5.2 days (r = 0.76). The half-life of
inorganic Hg is too long (> 120 days) to be accurately
estimated from the present data (i.e., r is not
significantly different from 0). |
Growth and health status. The weights of infant
monkeys during the study are shown in Figure 1. We found no
significant differences in the weight gain across the three
groups (
p > 0.10, all comparisons); the average
weight gain during the first 23 days of life was 135 g. The
brain
weights at sacrifice and brain-to-body weight ratios are
shown in Table 2; we found no significant differences in
brain weights
or brain-to-body weight ratios across the three groups (
p > 0.10,
all comparisons). Also, no serious medical complications
were observed in any of the monkeys.
Oral MeHg kinetics. The total blood Hg concentrations
at 2 days (observed peak) after the first dose ranged from
8 to 18 ng/mL across the monkeys, that is, a 2-fold variation.
Progressive accumulation of total blood Hg was observed over
the three subsequent doses of MeHg, such that the peak total
blood Hg concentrations after the fourth dose were about 3-fold
higher (30-46 ng/mL). The interanimal variation in blood
Hg concentrations remained at about 2-fold during accumulation.
Blood Hg persisted through the entire period of washout and
was readily measurable in all four monkeys in the 28 day sacrifice
group (16-21 ng/mL). This is consistent with previous
reports of an elimination T1/2 > 20 days
for MeHg in adult M.fascicularis (Stinson et
al. 1989; Vahter et al. 1994, 1995) and explains the minimal
decline (< 20%) in blood Hg concentrations during the weekly
intervals between MeHg doses.
The time course of total blood Hg was fitted to a one-compartment
model. Figure 2 shows the excellent regression fit of the mean
blood concentration-time data. Table 3 presents parameter
estimates from the one-compartment model fit of the mean blood
Hg concentration-time data. The distribution volume of
total Hg after MeHg administration is estimated to be 1.7 L/kg,
or about 20 times the blood volume (~ 8%). This means that
only 1/20th of the body burden of Hg is confined to the vascular
space. This is consistent with the known extensive extravascular
distribution of Hg after MeHg exposure in primates and agrees
with previous estimates of Hg distribution volume in adult M.fascicularis (Stinson
et al. 1989). The elimination T1/2 of total
blood Hg is 21.5 days, which agrees with reported estimates
in adult M.fascicularis (Stinson et al. 1989;
Vahter et al. 1994, 1995). The blood clearance is estimated
at 46.1 mL/day/kg, well within the range of clearance values
observed earlier in adult M.fascicularis (Stinson
et al. 1989). It appears that the systemic disposition kinetics
of MeHg are the same between infant and adult M.fascicularis,
that is, no change during development.
A plot of the blood and brain total Hg concentration data
from the monkeys sacrificed at various times during the washout
period is shown in Figure 3. There was a significant decrease
in total Hg from the blood during the washout period (p < 0.01).
The apparent T1/2 for total Hg in blood is
19.1 ± 5.1 days (± SE of regression estimate).
The decrease in total Hg in the brain over time was marginally
significant (p < 0.07), with an apparent T1/2 of
59.5 ± 24.1 days. The T1/2 for total
Hg in brain was significantly longer than the T1/2 for
total Hg in blood (p = 0.05) for the MeHg-exposed monkeys.
The T1/2 for total Hg in brain is also longer
than the previously reported washout T1/2 from
the brain for adult M. fascicularis (37 days; Vahter
et al. 1994, 1995). It should be noted that the relatively
high SE of the half-life estimates for the brain reflects the
large interanimal variation in Hg concentrations at each sampling
time, limited number of data points, and the short duration
of sacrifice relative to the washout half-life. The concentration
of total Hg in the brain is 1.7- to 3-fold higher than in the
blood (mean ± SE of 2.5 ± 0.3) 2 days after the
last MeHg dose. This brain-to-blood concentration ratio increased
as the duration between the last dose and the sacrifice lengthened.
The ratio ranged from 3.9 to 7.4 at 28 days after the last
exposure. The time dependence for the brain-to-blood ratio
(p = 0.06) is primarily due to the difference in the
washout T1/2 between total Hg in the blood
and brain. The average brain-to-blood ratio for these infant
monkeys at day 2 after the last MeHg dose (2.5 ± 0.3)
is slightly lower than previously reported values (3-5)
for adult macaque and squirrel monkeys over various durations
of washout (Berlin et al. 1975; Stinson et al. 1989; Vahter
et al. 1994). Although the cited differences in brain uptake
and clearance of MeHg between adult and infant monkeys may
be attributed to the effects of postnatal brain growth and
development, it may also be related to variation in exposure
regimen between studies.
A plot of the organic and inorganic Hg concentrations in
the brain of MeHg-exposed monkeys sacrificed at various times
during the washout period is shown in Figure 4. The decrease
in organic Hg in the brain over time was not statistically
significant (p = 0.17). The apparent T1/2 for
the washout of organic Hg from the brain was 58.4 ± 25.0
days, close to the T1/2 for total Hg. The
concentration of inorganic Hg in the brain samples was below
the quantifiable limit of the assay (7 ng/mL) in 8 of 17 MeHg-exposed
monkeys. The average concentration of inorganic Hg for those
monkeys with values above the detection limit (n = 10)
did not change significantly over 28 days of washout and was
approximately 7-8 ng/mL (Figure 4). Inorganic Hg represented
only 6-10% of total Hg in the brain. These values are
consistent with previously reported data in adult M.fascicularis (Vahter
et al. 1994, 1995).
Intramuscular thimerosal kinetics. The initial
total Hg concentrations in the day 2 blood samples, which ranged
from 6 to 14 ng/mL, are comparable with the concentrations
observed in the oral MeHg group. These blood levels are also
similar to those reported in preterm human infants receiving
12.5 µg Hg from a hepatitis B vaccine (Stajich et al.
2000). Blood Hg concentrations declined relatively rapidly
(by > 50%) between doses. As a result, there was minimal
accumulation in blood Hg concentrations during weekly dosing.
Also, blood Hg concentrations dropped below the detection limit
of the assay in some animals by day 10 after the last vaccine
injection.
The time course of total blood Hg concentrations was best
described by a two-compartment model; that is, the disposition
kinetics are biphasic, with a rapid initial phase followed
by a slower terminal phase of clearance. Table 4 presents the
parameter estimates derived from the two-compartment model
analysis. A comparison of the model prediction and the observed
blood concentration data is shown in Figure 5. The model predicted
some accumulation in peak blood Hg concentrations and minimal
accumulation in trough concentrations. Because blood concentration
data were not available before day 2, the predicted peak concentrations
are extrapolations and should be viewed with caution. The initial
volume of distribution in the central compartment was 1.7 L/kg,
which is comparable with the overall distribution volume for
oral MeHg. The initial and terminal blood half-lives were 2.1
and 8.6 days, respectively. Mercury derived from thimerosal
is eliminated much more rapidly than MeHg. The steady-state
volume of distribution (i.e., Vss or the
fully equilibrated volume) was estimated to be 2.5 L/kg, which
is 50% larger than the initial distribution volume (i.e., Vc).
Hence, the effective peripheral compartment volume at steady
state is about 0.8 L/kg. Alternately, this means that, at steady
state, partitioning of the body burden of Hg between the tissue
regions associated with the central and peripheral compartments
is about 2:1. The blood clearance of total Hg was estimated
to be 248 mL/day/kg, which is 5.4-fold higher than the estimate
for oral MeHg.
Figure 6 presents a scatter plot of the blood and brain total
Hg concentration data for monkeys sacrificed at various times
during the washout. There was a significant decrease in total
Hg concentration in the blood during the washout period (p < 0.01).
The apparent T1/2 for total Hg in blood is
6.9 ± 1.7 days. There was also a significant decrease
in total Hg concentration in the brain over time (p < 0.01),
with an apparent T1/2 of 24.2 ± 7.4
days. The T1/2 for total Hg in brain was
significantly longer than the T1/2 for total
Hg in blood (p < 0.01) for the thimerosal-exposed
monkeys. In addition, the T1/2 for total
Hg in blood and brain for these monkeys (6.9 ± 1.7 days
and 24.2 ± 7.4 days, respectively) are significantly
shorter (p < 0.01) than the T1/2 for
total Hg in blood and brain for the MeHg monkeys (19.1 ± 5.1
days and 59.5 ± 24.1 days). The concentration of total
Hg in the brain of the thimerosal-exposed monkeys is 2.6- to
4.6-fold higher than in the blood (mean ± SE, 3.5 ± 0.5)
at 2 days after the last injection. Again, this ratio increased
as the sacrifice was performed at longer durations from the
last dose, primarily due to the difference in the half-lives
of total Hg in the blood and brain.
A plot of the organic and inorganic Hg concentrations in
the brain of thimerosal-exposed infant monkeys sacrificed at
various times during the washout period is shown in Figure
7. There was a significant decrease in organic Hg in the brain
over the washout period (p < 0.01). The apparent T1/2 for
the washout of organic Hg from the brain was 14.2 ± 5.2
days, which is significantly shorter than the T1/2 for
total Hg in brain (p < 0.01). The inorganic form
of Hg was readily measurable in the brain of the thimerosal-exposed
monkeys. The average concentration of inorganic Hg did not
change across the 28 days of washout and was approximately
16 ng/mL (Figure 7). This level of inorganic Hg represented
21-86% of the total Hg in the brain (mean ± SE,
70 ± 4%), depending on the sacrifice time. These values
are considerably higher than the inorganic fraction observed
in the brain of MeHg monkeys (6-10%).
There are notable similarities and differences in the kinetics
of Hg after oral administration of MeHg and im injection of
thimerosal in vaccines. The absorption rate and initial distribution
volume of total Hg appear to be similar between im thimerosal
and oral MeHg. This means approximately equal peak total blood
Hg levels after a single exposure to either MeHg or thimerosal
or after episodic exposures that are apart by longer than four
elimination half-lives (i.e., > 80 days for MeHg or > 28
days for thimerosal). Studies in preterm and term human infants
have reported similar results (Stajich et al. 2000). Infants
receiving 12.5 µg Hg from a single hepatitis B vaccine
had blood Hg levels at 48-72 hr, consistent with what
would be anticipated after an equivalent dose of MeHg.
Although the initial distribution volume of total Hg is similar
for the two groups, a biphasic exponential decline in total
blood Hg is observed only after im injections of thimerosal.
This suggests continual distribution into and localization
in tissue sites over time. It is relevant to note that the
kidney-to-blood concentration gradient of total Hg is much
higher in the thimerosal monkeys than in the MeHg monkeys (mean ± SE,
95.1 ± 10 vs. 5.8 ± 0.6). The second slower phase
of washout could also represent the gradual biotransformation
of ethylmercury (the presumed principal organic form of Hg
after thimerosal administration) to Hg-containing metabolites
that have a different tissue distribution or are more slowly
eliminated. Further investigations of the disposition fate
of thimerosal-derived Hg should address these issues.
Total Hg derived from im thimerosal is cleared from the infant M.
fascicularis much more quickly than MeHg. The washout T1/2 of
total blood Hg after im injections of thimerosal in vaccines
is much shorter than the T1/2 of MeHg (6.9
vs. 19.1 days). These results support the earlier conclusion
of Magos (2003) that Hg is cleared from the body faster after
the administration of ethylmercury than after the administration
of MeHg. More interestingly, the washout blood Hg T1/2 in
the thimerosal-exposed infant macaques (7 days) is remarkably
similar to the blood Hg T1/2 reported for
human infants injected with thimerosal-containing vaccines
reported by Pichichero et al. (2002).
An important consequence of the difference in blood half-lives
is the remarkable accumulation of blood Hg during repeated
exposure to MeHg. Although the initial blood Hg concentration
(at 2 days after the first dose) did not differ between the
MeHg and thimerosal groups, the peak blood Hg concentration
in the MeHg-exposed monkeys rose to a level nearly three times
higher than in the thimerosal monkeys after the fourth dose.
Furthermore, the blood clearance of total Hg is 5.4-fold higher
after im thimerosal than after oral MeHg exposure. The results
indicate that for an equivalent level of chronic exposure,
the area under the curve of total blood Hg concentrations in
human infants receiving repeated im injections of thimerosal-containing
vaccines will be
significantly lower than that in those exposed chronically to MeHg via the
oral route.
A much lower brain concentration of total Hg was observed
in the thimerosal monkeys compared with the MeHg monkeys, that
is, a 3- to 4-fold difference for an equivalent exposure of
Hg. Moreover, total Hg is cleared much more rapidly from the
brain after thimerosal than after MeHg exposure (24 vs. 60
days). It appears that the difference in brain Hg exposure
between thimerosal and MeHg is largely driven by their differences
in systemic disposition kinetics (i.e., the blood level). The
average brain-to-blood partitioning ratio of total Hg in the
thimerosal group was slightly higher than that in the MeHg
group (3.5 ± 0.5 vs. 2.5 ± 0.3, t-test, p =
0.11). Thus, the brain-to-blood Hg concentration ratio established
for MeHg will underestimate the amount of Hg in the brain after
exposure to thimerosal.
The large difference in the blood Hg half-life compared with
the brain half-life for the thimerosal-exposed monkeys (6.9
days vs. 24 days) indicates that blood Hg may not be a good
indicator of risk of adverse effects on the brain, particularly
under conditions of rapidly changing blood levels such as those
observed after vaccinations. The blood concentrations of the
thimerosal-exposed monkeys in the present study are within
the range of those reported for human infants after vaccination
(Stajich et al. 2000). Data from the present study support
the prediction that, although little accumulation of Hg in
the blood occurs over time with repeated vaccinations, accumulation
of Hg in the brain of infants will occur. Thus, conclusion
regarding the safety of thimerosal drawn from blood Hg clearance
data in human infants receiving vaccines may not be valid,
given the significantly slower half-life of Hg in the brain
as observed in the infant macaques.
There was a much higher proportion of inorganic Hg in the
brain of thimerosal monkeys than in the brains of MeHg monkeys
(up to 71% vs. 10%). Absolute inorganic Hg concentrations in
the brains of the thimerosal-exposed monkeys were approximately
twice that of the MeHg monkeys. Interestingly, the inorganic
fraction in the kidneys of the same cohort of monkeys was also
significantly higher after im thimerosal than after oral MeHg
exposure (0.71 ± 0.04 vs. 0.40 ± 0.03). This
suggests that the dealkylation of ethylmercury is much more
extensive than that of MeHg.
Previous reports have indicated that the dealkylation of
Hg is a detoxification process that helps to protect the central
nervous system (Magos 2003; Magos et al. 1985). These reports
are largely based on histology and histochemistry studies of
adult rodents exposed to Hg for a short period of time. The
results of these studies indicated that damage to the cerebellum
was observed only in MeHg-treated animals that had much lower
levels of inorganic Hg in the brain than animals comparably
treated with ethylmercury. Moreover, the results did not indicate
the presence of inorganic Hg deposits in the area where the
cerebellar damage was localized (granular layer).
In contrast, previous studies of adult M. fascicularis monkeys
exposed chronically to MeHg have indicated that demethylation
of Hg occurs in the brain over a long period of time after
MeHg exposure and that this is not a detoxification process
(Charleston et al. 1994, 1995, 1996; Vahter et al. 1994, 1995).
Results from these studies indicated higher inorganic Hg concentrations
in the brain 6 months after MeHg exposure had ended, whereas
organic Hg had cleared from the brain. The estimated half-life
of organic Hg in the brain of these adult monkeys was consistent
across various brain regions at approximately 37 days (similar
to the brain half-life in the present infant monkeys). The
estimated half-life of inorganic Hg in the brain in the same
adult cohort varied greatly across some regions of the brain,
from 227 days to 540 days. In other regions, the concentrations
of inorganic Hg remained the same (thalamus) or doubled (pituitary)
6 months after exposure to MeHg had ended (Vahter et al. 1994,
1995). Stereologic and autometallographic studies on the brains
of these adult monkeys indicated that the persistence of inorganic
Hg in the brain was associated with a significant increase
in the number of microglia in the brain, whereas the number
of astrocytes declined. Notably, these effects were observed
6 months after exposure to MeHg ended, when inorganic Hg concentrations
were at their highest levels, or in animals solely exposed
to inorganic Hg (Charleston et al. 1994, 1995, 1996). The effects
in the adult macaques were associated with brain inorganic
Hg levels approximately five times higher than those observed
in the present group of infant macaques. The longer-term effects
(> 6 months) of inorganic Hg in the brain have not been
examined. In addition, whether similar effects are observed
at lower levels in the developing brain is not known. It is
important to note that “an active neuroinflammatory process” has
been demonstrated in brains of autistic patients, including
a marked activation of microglia (Vargas et al. 2005).
The American Academy of Pediatrics and the U.S. Public Health
Service (1999) published a joint statement that urged “all
government agencies to work rapidly toward reducing children’s
exposure to mercury from all sources.” The statement
recommended that thimerosal be removed from vaccines as soon
as possible as part of this overall process. Between 1999 and
2001, vaccines currently recommended for children ≤ 6
years of age were made available in thimerosal-free formulations
in the United States (Centers for Disease Control and Prevention
2001). Exposures to thimerosal through pediatric vaccines,
however, still occur in other countries where multiple-dose
vials are used to maintain childhood immunization programs
and the control of preventable disease (Knezevic et al. 2004).
Recent publications have proposed a direct link between the
use of thimerosal-containing vaccines and the significant rise
in the number of children being diagnosed with autism, a serious
and prevalent developmental disorder (for review, see IOM 2001).
Results from an initial IOM review of the safety of vaccines
found that there was not sufficient evidence to render an opinion
on the relationship between ethylmercury exposure and developmental
disorders in children (IOM 2001). The IOM review did, however,
note the possibility of such a relationship and recommended
further studies be conducted. A recently published second review
(IOM 2004) appears to have abandoned the earlier recommendation
as well as backed away from the American Academy of Pediatrics
goal. This approach is difficult to understand, given our current
limited knowledge of the toxicokinetics and developmental neurotoxicity
of thimerosal, a compound that has been (and will continue
to be) injected in millions of newborns and infants.
The key findings of the present study are the differences
in the disposition kinetics and demethylation rates of thimerosal
and MeHg. Consequently, MeHg is not a suitable reference for
risk assessment from exposure to thimerosal-derived Hg. Knowledge
of the biotransformation of thimerosal, the chemical identity
of the Hg-containing species in the blood and brain, and the
neurotoxic potential of intact thimerosal and its various biotransformation
products, including ethylmercury, is urgently needed to afford
a meaningful interpretation of the potential developmental
effects of immunization with thimerosal-containing vaccines
in newborns and infants. This information is critical if we
are to respond to public concerns regarding the safety of childhood
immunizations.