See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/43131971

Identification and distribution of mercury

species in rat tissues following administration
of Thimerosal or methylmercury
Article in Archives of Toxicology April 2010
DOI: 10.1007/s00204-010-0538-4 Source: PubMed

CITATIONS

READS

41

35

5 authors, including:
Jairo Rodrigues

Bruno Lemos Batista

Universidade Federal dos Vales do Jequitin

Universidade Federal do ABC (UFABC)

34 PUBLICATIONS 765 CITATIONS

59 PUBLICATIONS 751 CITATIONS

SEE PROFILE

SEE PROFILE

Fernando Barbosa
University of So Paulo
243 PUBLICATIONS 3,891 CITATIONS
SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate,

letting you access and read them immediately.

Available from: Bruno Lemos Batista

Retrieved on: 08 September 2016

Environmental Research 134 (2014) 218227

Contents lists available at ScienceDirect

Environmental Research
journal homepage: www.elsevier.com/locate/envres

A systematic study of the disposition and metabolism

of mercury species in mice after exposure to low levels
of thimerosal (ethylmercury)$
Maria Fernanda Hornos Carneiro a, Juliana Maria Oliveira Souza a, Denise Grotto a,b,
Bruno Lemos Batista a,c, Vanessa Cristina de Oliveira Souza a, Fernando Barbosa Jr.a,n
a
Laboratrio de Toxicologia e Essencialidade de Metais, Faculdade de Cincias Farmacuticas de Ribeiro Preto USP, Avenida do Caf, s/n, Monte Alegre,
CEP 14040-903 Ribeiro Preto, SP, Brazil
b
Programa de Ps-Graduao em Cincias Farmacuticas, Universidade de Sorocaba, Rodovia Raposo Tavares km 92.5, CEP 18023-000 Sorocaba, SP, Brazil
c
Centro de Cincias Naturais e Humanas, Universidade Federal do ABC, Bloco B, Avenida dos Estados 5001, CEP 0910-170 Santo Andr, SP, Brazil

art ic l e i nf o

a b s t r a c t

Article history:
Received 7 June 2014
Received in revised form
15 July 2014
Accepted 17 July 2014

Thimerosal (TM) is an ethylmercury (etHg)-containing preservative used in some vaccines despite very
limited knowledge on the kinetics and direct interaction/effects in mammals' tissues after exposure.
Thus, this study aimed to evaluate the kinetics of Hg species in mice in a time course analysis after
intramuscular injection of TM, by estimating Hg half-lives in blood and tissues. Mice were exposed to
one single intramuscular dose of 20 mg of Hg as TM. Blood, brain, heart, kidney and liver were collected at
0.5 hour (h), 1 h, 8 h, 16 h, 144 h, 720 h and 1980 h after TM exposure (n 4). Hg species in animal tissues
were identied and quantied by speciation analysis via liquid chromatography hyphenated with
inductively coupled mass spectrometry (LCICP-MS). It was found that the transport of etHg from
muscle to tissues and its conversion to inorganic Hg (inoHg) occur rapidly. Moreover, the conversion
extent is modulated in part by the partitioning between EtHg in plasma and in whole blood, since etHg is
rapidly converted in red cells but not in a plasma compartment. Furthermore, the dealkylation
mechanism in red cells appears to be mediated by the Fenton reaction (hydroxyl radical formation).
Interestingly, after 0.5 h of TM exposure, the highest levels of both etHg and inoHg were found in
kidneys (accounting for more than 70% of the total Hg in the animal body), whereas the brain
contributed least to the Hg body burden (accounts for o 1.0% of total body Hg). Thirty days after TM
exposure, most Hg had been excreted while the liver presented the majority of the remaining Hg.
Estimated half-lives (in days) were 8.8 for blood, 10.7 for brain, 7.8 for heart, 7.7 for liver and 45.2 for
kidney. Taken together, our ndings demonstrated that TM (etHg) kinetics more closely approximates
Hg2 than methylmercury (meHg) while the kidney must be considered a potential target for etHg
toxicity.
& 2014 Elsevier Inc. All rights reserved.

Keywords:
Thimerosal
Ethylmercury
Half-life
Distribution
Dealkylation

1. Introduction
Thimerosal (TM), which contains ethylmercury (etHg), has
been widely used as a preservative in a number of drug products,
including vaccines, to help prevent life-threatening contamination

Funding sources supporting the work: Fundao de Amparo Pesquisa do Estado

de So Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientco e
Tecnolgico (CNPq).
n
Corresponding author. Fax: 55 16 3602 4725.
E-mail addresses: mafehoca@fcfrp.usp.br (M.F.H. Carneiro),
souza.jmo@gmail.com (J.M. Oliveira Souza), denise.grotto@prof.uniso.br (D. Grotto),
bruno.lemos@ufabc.edu.br (B.L. Batista),
vcosouza@fcfrp.usp.br (V.C. de Oliveira Souza),
fbarbosa@fcfrp.usp.br (F. Barbosa Jr.).

http://dx.doi.org/10.1016/j.envres.2014.07.009
0013-9351/& 2014 Elsevier Inc. All rights reserved.

with microbes (Tan and Parkin, 2000). However, the potential

neurotoxic effects of organomercurial compounds, even at low
exposures (Lebel et al., 1998; Berman et al., 2008; Delong, 2011;
Bose et al., 2012; Petroni et al., 2012; Ida-Eto et al., 2013), have
provoked concerns about the use of thimerosal in vaccines and
other products (Clements et al., 2000; Ball et al., 2001).
The toxic properties of Hg compounds are directly related to
the chemical form of the element. In general, exposure to organic
forms of Hg is associated with nervous system damage, while
inorganic forms are closely connected to renal damage (Clarkson
and Magos, 2006). However, the toxicokinetics and potential toxic
properties of TM (etHg) are mostly unknown (WHO, 2012).
Due to the lack of information about the behavior of TM in the
mammalian body, the initial risk assessments for etHg were based
on studies of oral methylmercury (meHg) toxicity. However, recent

M.F.H. Carneiro et al. / Environmental Research 134 (2014) 218227

data indicate that the kinetics of tissue disposition and metabolism differ substantially between these two forms of organic Hg
(Clarkson, 2002; Magos, 2003; Burbacher et al., 2005), indicating
that meHg is not a suitable reference for risk assessment from
exposure to TM-derived Hg. Therefore, the knowledge of the
toxicokinetics of TM is mandatory to afford a meaningful assessment of the developmental effects of TM-containing vaccines.
Thus, the present study aimed to investigate the body burden
disposition of TM in mice after a single low-dose exposure. Two
forms of Hg found after the exposure (etHg and the dealkylated
form (inoHg)) were measured in blood and animal tissues by using
hyphenation of High Performance Liquid Chromatography to
Inductively Coupled Plasma Mass Spectrometry (HPLCICP-MS)
in a time course analysis to estimate Hg half-lives. It was also
demonstrated that the mechanism of etHg dealkylation appears to
be mediated by the Fenton reaction (hydroxyl radical formation).

2. Material and methods

2.1. Reagents
All reagents used were of analytical grade and the solutions
were prepared using high purity water with a resistivity of
18.2 M cm (Milli-Q Plus, Millipore, Bedford, MA, USA). A solution
of 37% hydrochloric acid (Merck, Darmstadt, Germany) was doubly
distilled in a quartz sub-boiling apparatus (Krner Analysentechnik, Rosenheim, Germany). A clean laboratory and laminar-ow
hood capable of producing class 100 were used for preparing
solutions and samples for the HPLCICP-MS technique. All solutions used were stored in high-density polyethylene bottles.
Materials were cleaned by soaking in 10% v/v HNO3 for 24 h,
rinsing ve times with ultrapure water and dried in a class 100
laminar ow hood before use. All operations were performed on a
clean bench. A 10 mg/l standard solution of inoHg was obtained
from Perkin-Elmer (PerkinElmer, Norwalk, CT). A 1000 mg/l standard solution of meHg chloride (CH3HgCl) and 1000 mg/l standard
solution of etHg chloride (CH3CH2HgCl) in water were obtained
from Alfa Aesar (MA, USA). Analytical calibration standards of Hg
species were prepared daily over the range of 0.020.0 mg/l for the
HPLCICP-MS by suitable serial dilutions of the stock solution.
Additional reagents used for the speciation studies included
methanol (99.9% v/v, HPLC-grade, Merck, Germany), mercaptoethanol (Sigma-Aldrich, USA) and L-cysteine (Fluka, Japan).
Ammonium acetate (99.99%) was obtained from Aldrich Chemical
Company (Milwaukee, USA) and formic acid, dimethyl sulfoxide
(DMSO) and hydrogen peroxide (H2O2) were purchased from
Sigma-Aldrich (USA).
2.2. Instrumentation and method
All measurements were carried out with a HPLC (Perkin-Elmer
model L-200, a six-port injector Rheodyne 9725 and a reversephase column C8 Brownlee 3 mm, 33 mm  4.6 mm) hyphenated
to an ICP-MS (Elan DRCII PerkinElmer, Norwalk, CT, USA). Argon
with a purity of 99.999% (White Martins, So Paulo, Brazil) was
used throughout. A Vibracell VC 100 ultrasonic processor with
a titanium probe controlled by USS-100 (Sonics & Materials,
Danbury, CT, USA) was employed for Hg extraction from samples.
The ultrasonic probe was set at an amplitude of 80%, power of
50 W and a frequency of 20 kHz. Samples volume injection was
100 ml. All separations were performed at room temperature under
isocratic conditions. The isocratic mobile phase was 3% v/v
methanol 97% v/v (0.5% v/v 2-mercaptoethanol0.05% v/v
formic acid). The ow rate was 1.2 ml/min. Data were evaluated
using the software Chromeras (supplied with the instrument) and

219

quantied by external calibration using a peak area. The complete

description of experimental conditions for both ICP-MS and HPLC
were provided by Souza et al. (2013). Data were validated by the
analysis of Standard Reference Materials (SRM) 966 Toxic Metals in
Bovine Blood from the National Institute of Standards and Technology (NIST, Gaithersburg, MD) and Certied Reference Materials
(CRMs) DOLT-3, DORM-3 and TORT-2 from the National Research
Council Canada (NRCC). Found values were in adequate agreement
with target values according to a t test at the 95% condence level.
2.3. Experimental design
2.3.1. In vivo study
Animals were handled and treated according to the guidelines
of the Committee on the Care and Use of Experimental Animal
Resources of the University of So Paulo, Brazil (Opinion number
12.1.1158.53.1, issued on the 14th of December of 2012). Male Swiss
mice weighing approximately 25 g (sixth week of life) were
obtained from the central animal facility. Animals were subjected
to 12 h light/12 h dark cycles in an air-conditioned room
(2225 1C) with free access to food and water.
The animals (n 28) were exposed to 20 mg of Hg in the form of
TM (sodium ethylmercury thiosalicylate, Sigma Chemical, St Louis,
MO) through IM injection, which corresponded to approximately
0.8 mg Hg/kg. The dose was adapted to the mouse body weight
and corresponded to a dose 20 times higher than that a child of
3.5 months of age receives in terms of Hg, only from vaccines
(Clements et al., 2000). Animals were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg) to collect
blood and organs (brain, heart, kidney and liver) after 0.5 hour
(h), 1 h, 8 h, 16 h, 144 h (or 6 days (d)), 720 h (or 30 d) and 1980 h
(or 80 d) after TM exposure (4 animals per time of study). The
blood and organs were placed in metal-free Eppendorf tubes and
kept at  80 1C until analysis. Plasma samples were not included in
the present study due to the limitation to collect the necessary
volumes from mice for Hg speciation.
The total mass of each Hg species accumulated in each sample
was calculated, dividing the concentration found (mg/ml or mg/g)
by the total volume of blood in the animal body (mice of 25 g,
approximately 1.6 ml) or the mean mass obtained of the respective
organ (in grams)previously weighed in 3 control animals at
approximately 25 g. This mass balance was performed due to the
fact that Hg concentrations (as mg/ml or mg/g) do not reect
directly the absolute amount of Hg accumulated in tissues, i.e.,
tissues with smaller sizes and lower Hg concentration may contain
more Hg than tissues with higher Hg concentrations.
Moreover, along the time course, the total amount of Hg
obtained at each momenti.e., the sum of Hg levels measured in
all monitored tissueswas considered to be 100% (since most of
the Hg is concentrated in these compartments). This assumption
was made in order to better illustrate the contribution of each
organ to Hg accumulation over time. Then, the percentages of
inoHg and etHg were calculated in each organ at one time point
assuming the whole amount of the exposure dose (20 mg) to
be 100%.
2.3.2. In vitro study
An in vitro experiment was conducted to evaluate the stability
of TM and etHg in whole blood, plasma and erythrocytes. This part
of the study was conducted in three different experiments to:
(i) evaluate the stability of TM and etHg in whole blood, plasma
and erythrocytes samples; (ii) and (iii) to elucidate the mechanism
behind the dealkylation of etHg in blood. These experiments
were performed on samples collected from two healthy human
volunteers.

220

M.F.H. Carneiro et al. / Environmental Research 134 (2014) 218227

The procedure to obtain the samples was identical in all

experiments. Human blood samples (30 ml) were collected from
the cubital median vein in metal-free tubes containing heparin.
After homogenization, 15 ml of blood was centrifuged at 3000 rpm
for 10 min to obtain plasma and erythrocytes. After plasma
collection and buffy coat removal, erythrocytes were washed three
times in cold isotonic phosphate buffered saline (PBS) solution
according to the procedure described by Harisa et al. (2012). All
solutions used had been freshly prepared.
In a preliminary in vitro experiment, aliquots of whole blood,
plasma and erythrocytes were kept in a water bath at 37 1C. Then,
to these aliquots, TM or etHg solution was added resulting in a
nal Hg concentration of 3 mg/l. 24 h after the TM/etHg incubation, samples were analyzed for Hg species determination (n 4).
Since there was no statistical difference in the levels of Hg species
after incubation of samples with TM or etHg, in the following
in vitro studies, we decided to incubate the samples with
only etHg.
Subsequent in vitro experiments were carried out by the same
procedure followed in the preliminary experiment. Briey,
aliquots of whole blood, plasma and erythrocytes were maintained
in a water bath at 37 1C. Then, to these aliquots, solutions of etHg
and iron (Fe) chloride (FeCl3) were added resulting in nal
concentrations of 3 mg/l of Hg and 400 mg/dl of Fe. This Fe
concentration is about twice the typical concentration found in
human plasma. 24 h after the incubation with etHg or etHg/Fe,
samples were analyzed for Hg species determination (n 4).
In the last in vitro experiment, the analysis was focused only on
plasma samples. Human plasma aliquots were kept in a water bath
at 37 1C after collection and centrifugation. Then, to each aliquot,
solutions of etHg, H2O2 and DMSO were added to obtain plasma
samples with the following concentrations:
(a) etHg (3 mg/l); (b) etHg (3 mg/l) plus H2O2 (10 mM); (c) etHg
(3 mg/l) plus H2O2 (10 mM) plus DMSO (100 mM). Then, 24 h later,
samples were analyzed for Hg species determination (n 4).

2.4. Statistical analysis

Descriptive analysis was used to elucidate some ndings of the
present study. For the in vivo study, a t test was employed to
compare the mean Hg levels (inoHg and etHg) 7standard deviation (SD) obtained in blood and organs at each time point in the
study. Hg half-lives were estimated using the pharmacokinetic
modeling software SAAM II (SAAM Institute, Seattle, WA, USA). In
the kidneys, a one-compartment model failed to provide a
satisfactory t of the mean Hg concentrationstime data. After
log-scale transformation, the half-lives for the mono/biexponential
decline of Hg concentration were estimated by standard formulas
(Bonate, 2006; Gibaldi and Perrier, 1982; Wolfsegger, 2006). The
estimation of initial and terminal half-lives was made using the
biexponential model: y a1 nexp(  b1 n) a2 nexp(  b2 n) with a
parameterization to ensure b1 4b2 40 tted by the least squares
criteria with optimization of the package function based on the
method of NelderMead. Curve peeling (Foss, 1969) was employed
to obtain starting values for nonlinear model tting. When no
adequate starting values were determined by curve peeling, a
single exponential model was tted with starting values obtained
from an Ordinary Least Squares regression on log-transformed
values with a parameterization to ensure a slope 40.
In the in vitro study, One-way ANOVA followed by Tukey's test,
when needed, was used to compare the mean concentrations of
Hg species obtained in whole blood, plasma and human erythrocytes. The inuence of Fe/H2O2/H2O2 DMSO upon etHg conversion was tested by means of the same combination of
statistical tests.

The statistical signicance was set at 5%. Statistical procedures

and gures preparation were performed using GraphPad Prism
version 5 (GraphPad Software, Inc. CA, USA).

3. Results and discussion

3.1. Hg distribution in tissues of mice exposed to thimerosal
Table 1 shows the Hg (etHg and InoHg) amounts in mass units
accumulated in the organs and blood of mice at different time
points after TM exposure. In each compartment, Hg amounts were
expressed in mg, obtained by dividing the concentration found
(mg/ml or mg/g) by the total volume of blood in the animal body
(mice of 25 g, about 1.6 ml) or the mean mass of the respective
organ (in grams)previously weighed in 3 control animals with a
body weight of 25 g. This mass balance was performed due to the
fact that Hg concentrations (as mg/ml or mg/g) do not reect
directly the absolute amount of Hg accumulated in tissues, i.e.,
tissues with smaller sizes and lower Hg concentration may contain
more Hg than tissues with higher Hg concentrations.
The total amount of Hg measured 0.5 h after TM exposure (sum
measured in organs and blood) was 17.370.8 mg (Table 1). The
remaining quantity of Hg (considering the dose administered of
20 mg) had been distributed to other organs or excreted. However,
it is reasonable to consider the sum of Hg measured in the animal
blood, brain, heart, liver and kidney as approximating 100% of the
total Hg in the body, since the contribution of other compartments
is much less relevant. Taking this in account, the contribution of
each organ and blood to the total amount is also represented as a
percentage (Table 1).
It was apparent that there is no excretion of Hg from 0.5 h to
16 h after the TM exposure, since the sum of Hg levels obtained
in the monitored organs changed slightly (ranging from
17.31 70.8 mg (0.5 h) to 17.17 70.36 mg (16 h)). Moreover, during
this same period there was no signicant change in the contribution of blood and organs in terms of percentage of the Hg body
burdenconsidering each time point individually (Table 1). The
kidney was the organ with the greatest contribution to Hg body
burden (around 7075%) followed by liver (2024%). A major cause
of accumulation of Hg in the renal tissue is likely to be related to
its physiological function of ltration. The large renal blood ow of
about 25% of the cardiac output is another important feature
contributing to uptake of toxic chemicals, since the kidney is full of
essential transport systems (Berndt et al., 1985).
After 0.5 h of TM exposure, the amount of Hg in the blood
compartment corresponded to approximately 7.6% of the injected
dose. This is consistent with the results obtained for Hg levels in
blood from infants after receiving 12.5 mg of Hg as TM (from 4.2%
to 5.1% of the injected dose) (Stajich et al., 2000; Aschner and
Ceccatelli, 2010). On the other hand, the brain accounted for less
than 1% of the total Hg in animal body and was the organ with less
Hg accumulated after TM exposure and the tissue that has the
highest loss of the accumulated Hg in percentage (from 0.5 h to
6 d 94.1%, see Table 1). The brain-to-blood Hg ratio was rather
lower than 1 (ranging from 0.1 to 0.4Table 1). This rate is in close
agreement with other studies (Harry et al., 2004; Orct et al., 2006;
Zareba et al., 2007; Rodrigues et al., 2010; Blanua et al., 2012).
Moreover, from 0.5 h to 16 h, Hg was found in brain tissue as both
inoHg and etHg (approximately 50/50), indicating that despite the
greater molecular size compared with meHg, etHg can cross the
bloodbrain-barrier. In fact, the uptake of meHg and etHg complexes with cysteine into glioma cells was shown to be mediated,
at least in part, through the L-type neutral amino acid carrier
transport system (Zimmermann et al., 2013), which corroborates
this result. However, at 6 d after exposure, no etHg was detected in

M.F.H. Carneiro et al. / Environmental Research 134 (2014) 218227

221

Table 1
Hg levels (g) determined in mice organs after thimerosal exposure and the percentage of it considering the sum of Hg measured in the animal blood, brain, heart, liver and
kidney as 100% in each time point.

Time elapsed

0.5 h

1h

8h

16 h

144 h (6 d)

720 h (30 d)

1980 h (80 d)

Organ

Total Hg (lg)7 SD

inoHg (%)

etHg (%)

% of Hg considering the
sum per time point

Hg [organ]/[blood]

Heart
Liver
Kidney
Brain
Blood
Total
Heart
Liver
Kidney
Brain
Blood
Total
Heart
Liver
Kidney
Brain
Blood
Total
Heart
Liver
Kidney
Brain
Blood
Total
Heart
Liver
Kidney
Brain
Blood
Total
Heart
Liver
Kidney
Brain
Blood
Total
Heart
Liver
Kidney
Brain
Blood
Total

0.117 0.02
3.26 70.31
12.447 3.34
0.177 0.04
1.3370.32
17.317 0.8
0.43 70.05
3.667 0.44
12.95 71.91
0.077 0.01
1.197 0.10
18.30 7 2.44
0.26 70.02
3.46 70.33
13.767 1.48
0.047 0.001
0.377 0.02
17.89 7 0.37
0.247 0.02
4.277 0.76
12.107 0.82
0.067 0.0001
0.517 0.19
17.177 0.36
0.047 0.01
1.197 0.26
3.5 71.29
0.017 0.0001
0.09 70.03
4.83 70.26
0.017 0.001
0.19 7 0.03
0.10 7 0.02
0.02 70.001
0.047 0.02
0.36 70.01
ND
ND
0.02
ND
ND
0.02

100
53
60
52
46

0
47
40
48
54

0.65
18.8
71.9
0.97
7.66

0.1
2.5
9.4
0.1
1.0

94
48
65
56
71

6
52
35
44
29

2.35
20
70.8
0.39
6.48

0.4
3.1
10.9
0.1
1.0

95
42
67
53
75

5
58
33
47
25

1.46
19.33
76.9
0.2
2.06

0.7
9.4
37.4
0.1
1.0

87
43
80
55
83

13
57
20
45
27

1.42
24.9
70.4
0.32
2.95

0.5
8.4
23.9
0.1
1.0

77
67
92
100
100

23
33
8
0
0

0.88
24.7
72.5
0.21
1.76

0.5
14.0
41.2
0.1
1.0

100
83
94
100
100

0
17
6
0
0

1.90
54
27.9
4.63
11.6

0.2
4.7
2.4
0.4
1.0

100

100

ND: not detected.

this tissue (inoHg: 100%). Two main scenarios may explain the
presence of inoHg in the brain: (i) etHg crosses the bloodbrain
barrier and, once inside the brain, is converted to inoHg, as
observed for meHg by Vahter et al. (1995) or; (ii) due to direct
uptake of inoHg from blood, since studies have reported inoHg in
brain after exposure to inoHg (Arvidson, 1992; Pamphlett and
Waley, 1996; Pamphlett and Jew, 2013). However, both mechanisms can be occurring simultaneously. Despite the low Hg levels
found in the brain of mice after TM exposure, and the predominance of inoHg, toxic risk cannot be excluded, since tissues
respond differently to Hg. Yasutake et al. (2010) found in mice
an increased rate of movement in the open eld test associated
with brain Hg levels of 0.4 mg/g, 3 weeks after inoHg exposure by
an intraventricular injection. This concentration is close to the
inoHg levels we found in the present study after TM exposure
(around 0.2 mg/g at 0.5 h and 0.1 mg/g at 1 hFig. 1). Therefore, as
stated by Blair et al. (1975), any Hg level in brain, either organic or
inorganic, should be viewed as potentially hazardous.
It is also interesting to observe that from 16 h to 6 d after TM
exposure there was a considerable drop in the total Hg measured
as the sum of blood and tissue levels (from 17.2 mg to 4.83 mg). At
the same time, the kidney Hg level dropped from 12.1 mg to 3.5 mg.

Thus, the contribution of the kidney to the total reduction in the

Hg levels was 69% (8.5 mg), meaning that a signicant part of Hg
was excreted by urine. On the other hand, in the same period, liver
Hg levels account for less than 25% of the total Hg in the rodent
body, meaning that the liver-biliary system is not the main route
of Hg excretion. We did not measure Hg levels in urine or feces in
the present study; however, it is reasonable to deduce that a
signicant part of the Hg excreted is eliminated by urine, since
most of the Hg found in kidney was found in the inorganic form.
This nding is contrary to the observations of Pichichero et al.
(2002), who reported that the Hg levels determined in stool and
urine samples collected from children given vaccines containing
TM indicated a substantial excretion of Hg taking place via the
fecal route. However, in their study Pichichero et al. (2002)
collected spot urine samples (at 1 time interval after vaccination)
and reported the Hg levels in concentration and not in absolute
amount of Hg excreted (i.e. without volume correction).
At 80 d after TM exposure, Hg was still detected in the kidney
but not in other compartments. It is also important to point out
that elevated kidney Hg levels were also observed in an etHg
poisoning episode in Iraq (Hilmy et al., 1976). Suzuki et al. (1973)
reported levels of total and inoHg in the tissues of a 13-year-old

222

M.F.H. Carneiro et al. / Environmental Research 134 (2014) 218227

Fig. 1. Mean levels of inorganic mercury (inoHg) (circles) and ethyl Hg (etHg) (triangules) 7 standard deviation obtained in mice blood, heart, brain, kidney and liver after
intramuscular injection of 20 mg of Hg in the form of thimerosal (TM) (n 4).

boy who died 5 d after receiving an infusion of plasma containing

high content of TM (284540 mg Hg). Considerable Hg levels were
found in the kidneys.
It has been assumed that the major effect from chronic
exposure to inoHg is nephropathy induced mainly by apoptosis
and necrosis (Sharma et al., 2007; Gewin et al., 2012). Although
TM is supposed to be decomposed rapidly into inoHg after TM
injection, the effects and mechanisms behind both etHg and inoHg
levels should be deeply explored in this organ. Considering inoHg
toxicity upon kidneys, the literature shows that even at low levels
of exposure to inoHg (approximately four times higher than the
Hg concentration we administered in the present study) rats
exhibited an impaired glomerular ltration rate (Zalups, 1997).
Moreover, the currently available literature reports renal failure,
amongst other organ injuries, in a patient who ingested 83 mg/kg
of TM (Pfab et al., 1996). However, little information on toxic
effects in kidneys after administration of TM-containing vaccines
is available. Moreover, to date, very little is known about the
mechanisms and molecular interactions underlying toxicity of
etHg/thimerosal in the kidney (Carneiro et al., 2013, 2014).

Due to an immature or impaired renal system, newborns and

renal patients, respectively, may be at high risk of toxicity after TM
exposure through vaccines. Therefore, the kidney appears to be an
important post-TM target organ, despite the little attention that
has been given in the literature to the possible harmful renal
effects of Hg after TM exposure.
Table 1 also shows the fraction of the total Hg found as etHg or
inoHg in each compartment analyzed. In this table, a considerable
change is evident in the proportion between inoHg and etHg in
the blood compartment from 0.5 to 1 h after TM exposure,
indicating a rapid dealkylation of etHg in this uid or transference
of etHg from blood to tissues. However, no considerable changes in
the amount of etHg was observed in the tissues in the same time
course, which indicates that etHg is rapidly converted to inoHg in
blood compartment when it reaches the bloodstream. Moreover,
during the time course of the exposure, the inorganic fraction
increases in all compartments, which conrms that the conversion
of etHg to inoHg after TM exposure is a dynamic process.
Table 2 presents a comparison between Hg levels found in
blood and tissues after TM exposure under different experimental

223

conditions and relatively similar exposure doses. As can be noted,

in general Hg levels in blood and tissues of the present study was
within the range of concentrations reported in other studies on
different animal species, with the exception of Harry et al. (2004).

Burbacher et al.
(2005)

Blanua et al.
(2012)
Rodrigues et al.
(2010)
Blair et al. (1975)

Orct et al. (2006)

Present study

Reference

Harry et al. (2004)

Zareba et al. (2007)

M.F.H. Carneiro et al. / Environmental Research 134 (2014) 218227

Most commonly employed time points among the references cited (some previous studies presented other time points). ND, not detected. IM, intramuscular. SC, subcutaneous. IN, intranasal.

IM
35 mg Hg divided in
4 administrations (birth, 7, 14
and 21)

Oral

IN
207 mg Hglow dose

Adult squirrel monkeys

(both gender)
Infant monkeys M.
fascicularis (both
gender)

0.006

0.41/0.05
0.073/0.007

0.012/0.020
0.012/0.004

0.84/ND

1.7
2.1
9.5/0.38
1.5
1.2
3.0/0.5
0.08
0.08
0.16/0.035

24 h
Total
6d
12 h/120 h: blood (Total Hg) 5 d:
tissues (inoHg/etHg)
6 months of exposure inoHg/organic
fraction
7d
Total
SC

Total
SC

Suckling Wistar rats (male) 0.81 mmol Hg/kg (17 mg Hg),

3 times
Suckling Wistar rats (male) 0.81 mmol Hg/kg (17 mg Hg),
3 times
Adult Wistar rats (male)
0.5 mg Hg/kg (100 mg Hg)

IM
0.8 mg Hg/kg (20 mg Hg)

8h
16 h
6d
72 h

IM
IM
70 mg Hg/kg (1.75 mg Hg)
1.4 mg Hg/kg (35 mg)

Adult CD-1 mice (male)

Neonatal ICR mice (both
gender)
Adult Swiss mice (male)

1.2
0.5
0.4/0.05

21.15/10.12
21.33/5.6
10.7/0.9
2
1.24/4.08
1.04/2.83
0.72/0.35
1.37
2.25/0.12
2.1/0.32
0.33/0.09

0.04/0.04
0.07/0.05
0.03/ND
0.108
0.2/0.06
0.3/0.06
0.08/ND
0.85

97
0.7/2.5

0.9/2.3

Total
inoHg/organic
fraction
inoHg/etHg
24 h
24 h

22.5
0.8

2.6
0.04/0.2

Liver
Heart
Brain
Blood

Hg
determined
Elapsed time after
exposurea
Route of
exposure
Experimental design dose
of Hg as TM (approximate)
Species prole

Table 2
Comparisons of Hg levels in blood and tissues after TM exposure in experimental studies.

Hg levels (approximate) in mg/l or lg/g

Kidney

3.2. Biological half-lives of etHg in blood and tissues of mice

As far as we know, this study is the rst to estimate the half-life
of TM in the blood, brain, kidney, liver and heart of mice after a
low-dose exposure. Fig. 1 shows the mean levels of inoHg and
etHg 7SD obtained in the blood, heart, brain, kidney and liver of
mice at 0.5 h, 1 h, 8 h, 16 h, 6 d, 30 d and 80 d after the IM
injection of 20 mg of Hg as TM. These data were employed to
estimate the biological half-lives in blood and tissues discussed
throughout the text.
Table 3 displays the estimated half-lives derived from one or
two-compartment analysis of log-transformed Hg levels obtained
from TM exposed mice (see Section 2 for details). The time course
of Hg levels in blood, brain, heart and liver was better described by
a one compartment model, whereas kidney levels are best
described by a two compartment model with a rapid initial phase
followed by a slower terminal phase of clearance.
The half-life of Hg in blood was estimated in 8.8 d, a value
very close to the previous reports on those in blood obtained
from humans and other animal species exposed to TM (Table 4)
but much shorter than those observed after meHg exposure
(Skerfving, 1974; Yaginuma-Sakurai et al., 2012).
Burbacher et al. (2005) reported a terminal half-life of 8.62 d in
the blood of infant monkeys after TM injection, whereas Barregrd
et al. (2011) and Pichichero et al. (2002, 2009) reported values of
5.6 d, 7 d and 6.3 d, for adults and newborn infants. Therefore,
although the biological interaction of Hg may differ between
humans and other animals (USEPA, 1997), our results on Hg halflife in blood of mice after TM exposure strongly indicate similarities between the two categories. This fact consistently stresses
the contribution of the present work to the establishment of a
toxicological prole for TM. Moreover, since the clearance of Hg
from blood after TM exposure is much faster than after meHg
exposure (Burbacher et al., 2005), the latter is not a suitable
reference for risk assessment of TM exposure. Moreover, given the
Table 3
Estimated half-lives for blood and tissues derived from one and two-compartment
analysis of the log-transformed Hg levels obtained from thimerosal exposed mice.
Organ/tissue

T1/2 (days)

Blood
Heart
Liver
Brain

8.8
7.8
7.7
10.7

Kidney

T1/2 (days)
2.7

T1/2 (days)
45.2

Table 4
Estimated blood half-lives after TM administration in different animal species.
Animal species

Blood T1/2 (days)

Reference

Mice
Monkeys

8.8
Initial: 2.1
Terminal: 8.6

Present work
Burbacher et al. (2005)

5.6
7
6.3

Barregrd et al. (2011)

Pichichero et al. (2002)
Pichichero et al. (2009)

Humans
Adults
Newborns
Newborns

224

M.F.H. Carneiro et al. / Environmental Research 134 (2014) 218227

low Hg levels in blood and shorter half-life, a risk for toxicity

seems to be lower than that after meHg exposure.
For the heart and liver, half-lives were estimated at 7.8 d and
7.7 d (Table 3), versus 10.7 d for the brain, which is much shorter
than the 26 d half-life reported for brain after meHg exposure
(Magos and Butler, 1976). On the other hand, the initial kidney
half-life was about 2.7 d and the terminal, 45.2 d (Table 3), another
indicative that kidney is the organ where Hg persists for a longer
duration after TM exposure. As a comparison, in rabbits exposed to
meHg, the total Hg half-life in liver and kidneys (0.125 mmol/kg,
twice a week for 9 weeks, by intravenous injection) was estimated
at 28 d (Petersson et al., 1991). This period is about 4 times longer
than the Hg half-life we observed in the liver but lower than the
Hg half-life in the kidneys of TM-exposed mice. Accordingly, the
kidney Hg half-life is closer to the 58 d (ranging from 35 to 90 d)
reported after inhalation of Hg vapor (Clarkson and Magos, 2006).

3.3. Retention of initial Hg dose by each tissue along time course

Fig. 2 shows a schematic representation of Hg distribution after
TM exposure where the size of the organs represents a rough
approximation of the percentage of the initial dose (20 mg of Hg as
TM) retained in each organ/tissue along a time course. To draw the
gure we used the amount of total Hg in mg in each tissue at each
time point to calculate the percentage regarding the initial dose of
20 mg Hg as TM as 100%.
Blood Hg levels did not exceed 7% of the initial dose of 20 mg of
Hg throughout the time course. The maximum and minimum
values were observed at 0.5 h and 30 d, respectively (6.63% and
0.21%). In the heart, Hg levels peaked at 2.15% of the initial Hg dose
1 h after the exposure then decreased during the follow-up until

reaching the minimum percentage observed as 0.03% of the

initial dose.
In the brain, the Hg percentage ranged from 0.82 at 0.5 h to
0.08% at 30 d, whereas in the liver it reached 21.34% at 16 h,
dropping to 0.96% at 30 d. Indeed, the percentage of initial dose
retained in the kidney ranged from 0.11% at 80 d to about 69% at
8 h after TM exposure, which corresponds to approximately 22 ng
and 14 mg of Hg, respectively.
3.4. Evaluation of possible mechanisms associated with
etHg conversion into inoHg in blood samples
With increasing size, the bond between Hg and the organic
radical becomes less stable, which results in easier degradation of
etHg into inoHg. Accordingly, the bond lengths between Hg and
carbon in meHg and etHg have been estimated by Kaupp and
Malkina (1998) as 2.115 and 2.150 , respectively. Besides this, in
preliminary experiments our group observed that etHg is highly
stable at room temperature in plasma samples, but converts to
inoHg in red blood cells (unpublished data). Thus, an in vitro
experiment was conducted to evaluate the stability of TM and
etHg in whole blood, plasma and erythrocytes and also to
elucidate the mechanism behind the dealkylation of etHg in blood.
These experiments were carried out on human blood samples.
Fig. 3 shows Hg species concentrations (mean7SD) measured
after adding etHg or TM solution (3 mg/l) in human samples of
whole blood, plasma or red blood cells and incubated for 24 h.
There was no difference between the levels of Hg species formed
after the addition of TM or etHg considering whole blood,
erythrocytes or plasma individually, which indicates that the anion
thiosalicylate presents undetectable interference in the dealkylation of etHg present in TM (Fig. 3A). On the other hand, after the

Fig. 2. Schematic representation of mercury (Hg) distribution after thimerosal (TM) exposure. The size of the organs represents a rough approximation of the percentage of
the initial dose retained in each of the organ/tissue along time.

M.F.H. Carneiro et al. / Environmental Research 134 (2014) 218227

225

Fig. 3. Mean 7 SD of mercury (Hg) species concentrations measured after 24 h of adding ethyl Hg (etHg) or thimerosal (TM) solution (3 mg/l) in whole blood, plasma or red
blood cells obtained along the rst (A), second (B) or third (C) experiments of the in vitro study. One way ANOVA followed by Tukey's test. nConcentrations of etHg and inoHg
statistically lower and higher, respectively, than those obtained in plasma and erythrocytes; #Concentrations of etHg and inoHg statistically lower and higher, respectively,
than those obtained in plasma; nnConcentrations of etHg and inoHg statistically higher and lower, respectively, than those obtained in erythrocytes without Fe, and in both
blood groups; ##Concentrations of etHg and inoHg statistically lower and higher, respectively, than those obtained in plasma without Fe, Po 0.05. nnnConcentrations of inoHg
and etHg statistically higher and lower, respectively, than those obtained in plasma etHg and plasma etHg H2O2 DMSO samples, Po 0.05.

incubation time, the etHg levels in whole blood were lower than
those found both in erythrocytes and plasma for both compounds.
Moreover, in whole blood, etHg is converted more intensively
when compared to red blood cells while no dealkylation was
observed in plasma samples (Fig. 3A). This nding shows that the
partitioning of circulating etHg between red cells determines at
least in part the extension of etHg conversion to inoHg and
might contribute to augmenting the half-life of etHg in the animal
body.
There is some evidence that the dealkylation of organomercurials is triggered by reactive oxygen species and microbial organisms; however, the mechanisms behind the conversion are still
unknown (Suda et al., 1991, 1992; Qvarnstrm et al., 2003).
Therefore, subsequent experiments were carried out to ascertain
the mechanisms associated with etHg conversion into inoHg
in blood.
It is known that Fe is a cofactor of a Fenton HaberWeiss
reaction and that plasma has a much lower Fe concentration in
comparison to whole blood and red cells. Fig. 3B shows that the
incubation of FeCl3 together with etHg resulted in a signicantly
higher inoHg concentration in plasma samples when compared to
plasma without Fe addition. Consequently, greater conversion has
occurred in the plasma possibly by the action of OHd radical
formed in the presence of a greater quantity of Fe. Furthermore,
after Fe incubation, an increase in etHg conversion to inoHg was
observed in erythrocytes (Fig. 3B) (Hg species levels were statistically the same in comparison to whole blood). Furthermore,
erythrocytes and whole blood are known to present very similar
Fe concentrations (Helmer and Emerson, 1934; Fairbanks, 1994).
Based on this information, one would expect that whole blood and
erythrocytes would convert etHg to inoHg at similar rates. However, in red blood cells, a lower conversion of etHg was observed in
comparison to whole blood (Fig. 3A and B). This suggests involvement of other mechanisms in the dealkylation of etHg beyond the
oxidizing action of OHd radical as previously reported by Suda and
Takahashi (1992) and Suda et al. (1991). Potentially, other cells,
molecules and enzymes in whole blood might be involved in this
process. For instance, polymorphonuclear leukocytes from human
blood and intraperitoneal cavity of rabbits, rats and guinea pigs, in
addition to guinea pig macrophages and eosinophils and human
monocytes were already reported to participate in the conversion

of organic Hg into inoHg (Suda and Hirayama, 1992; Suda et al.,

1992).
In order to conrm that the conversion of etHg into inoHg
occurs through OHd radical formation, a further experiment was
developed with the addition of H2O2 and DMSO. The incubation of
plasma with H2O2 enhanced the conversion of etHg to inoHg as
shown in Fig. 3C. The H2O2 is formed in the second step of a
Fenton reaction and serves as a substrate for OHd formation
(Halliwell and Chirico, 1993). Later, H2O2 was added together with
DMSO, which acts as a potential scavenger of OHd, and under this
condition no statistical difference in terms of inoHg levels was
observed when compared to the plasma samples without H2O2 or
DMSO addition (Fig. 3C). The results strongly indicate that one of
the mechanisms behind the conversion of etHg to inoHg in living
organisms is mediated by OHd radicals. Moreover, few other
plausible explanations for the non-conversion of etHg in plasma
include the absence of cells, hemoglobin and lesser content of
proteins and other compounds, which renders plasma a less
oxidative environment. This condition may also contribute to the
higher stability of etHg in plasma. However, further studies are
necessary to conrm this hypothesis.
Based on these ndings and considering that etHg in plasma is
more freely available to interact with tissues than those in red
blood cells, we hypothesized that the etHg found in tissues is
transported by plasma and not by red blood cells. Moreover, once
in plasma, etHg can be either sequestered by red blood cells and
converted into inoHg or be retained in tissues. etHg might be also
converted by oxidative reactions/specic enzymes within cells
something not yet investigatedor be returned to plasma/red
blood cells. Hence, the partition coefcient of etHg in red blood
cells/plasma regulates the transport of this Hg species within the
organism, which is inuenced primarily by the conversion rate of
etHg in red blood cells. This conversion can also have an impact
upon the toxic effects of Hg after the administration of TMcontaining vaccines.

4. Conclusion
The present study reinforces the evidence that the transport of
etHg from muscle to tissues and its conversion into inoHg are

226

M.F.H. Carneiro et al. / Environmental Research 134 (2014) 218227

rapid. Moreover, the dealkylation process occurs in red blood cells,

but not in plasma, with hydroxyl radical as a potential effector.
Thus, the partition coefcient of etHg in red blood cells and
plasma potentially denes the amount of etHg that reaches tissues
after TM exposure.
Although some tissues accumulated signicant amounts of Hg
after TM exposure, the clearance time of etHg from the animal
body is shorter than that of meHg. Moreover, since etHg is quickly
metabolized to inoHg, the kinetics of the latter might determine
the elimination of Hg after TM exposure. Despite the smaller
contribution of the brain to the body burden of Hg, the potential
neurotoxicity of TM must be continuously monitored, since any Hg
level in the brain, either organic or inorganic, should be viewed as
potentially hazardous. Additionally, the longer Hg persistence and
higher accumulation of both Hg species in kidneys make the
tissue a potential candidate for toxic effects after TM exposure.
Therefore, further studies must be carried out to evaluate the
nephrotoxicity of TM.

Conict of interest statement

The authors declare that they have no conicts of interest.

Acknowledgments
The authors are grateful to the Fundao de Amparo Pesquisa
do Estado de So Paulo (FAPESP-2011/08467-0) and the Conselho
Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq147713/2010-2) for nancial support and fellowships. We also
thank Samuel Simio de Souza, Ktia de Marco and Kim-Anh Le
Cao for technical support.
References
Arvidson, B., 1992. Accumulation of inorganic mercury in lower motoneurons of
mice. Neurotoxicology 13 (1), 277280.
Aschner, M., Ceccatelli, S., 2010. Are neuropathological conditions relevant to
ethylmercury exposure? Neurotox. Res. 18 (1), 5968.
Ball, L.K., Ball, R., Pratt, R.D., 2001. An assessment of thimerosal use in childhood
vaccines. Pediatrics 107 (5), 11471154.
Barregrd, L., Rekic, D., Horvat, M., Elmberg, L., Lundh, T., Zachrisson, O., 2011.
Toxicokinetics of mercury after long-term repeated exposure to thimerosalcontaining vaccine. Toxicol. Sci. 120 (2), 499506.
Berman, R.F., Pessah, I.N., Mouton, P.R., Mav, D., Harry, J., 2008. Low-level neonatal
thimerosal exposure: further evaluation of altered neurotoxic potential in SJL
mice. Toxicol. Sci. 101 (2), 294309.
Berndt, W.O., Baggett, J.M., Blacker, A., Houser, M., 1985. Renal glutathione and
mercury uptake by kidney. Fundam. Appl. Toxicol. 5 (5), 832839.
Blair, A., Clark, B., Clarke, A.J., Wood, P., 1975. Tissue concentrations of mercury after
chronic dosing of squirrel monkeys with thiomersal. Toxicology 3 (2), 171176.
Blanua, M., Orct, T., Vihnanek Lazarus, M., Sekovanic, A., Piasek, M., 2012. Mercury
disposition in suckling rats: comparative assessment following parenteral
exposure to thiomersal and mercuric chloride. J. Biomed. Biotechnol. 2012,
256965.
Bonate, P.L., 2006. PharmacokineticPharmacodynamic Modeling and Simulation.
Springer, New York.
Bose, R., Onishchenko, N., Edoff, K., Janson, L.A.M., Ceccatelli, S., 2012. Inherited
effects of low-dose exposure to methylmercury in neural stem cells. Toxicol. Sci.
130 (2), 383390.
Burbacher, T.M., Shen, D.D., Liberato, N., Grant, K.S., Cernichiari, E., Clarkson, T.,
2005. Comparison of blood and brain mercury levels in infant monkeys
exposed to methylmercury or vaccines containing thimerosal. Environ. Health
Perspect. 113 (8), 10151021.
Carneiro, M.F.H., Morais, C., Barbosa Jr., F., Gobe, G., 2013. Thimerosal in childhood
vaccines contributes to accumulating mercury toxicity in the kidney. Toxicol.
Environ. Chem. 95, 14241447.
Carneiro, M.F.H., Morais, C., Small, D.M., Vesey, D.A., Barbosa Jr., F., Gobe, G.C., 2014.
Thimerosal induces apoptotic and brotic changes to kidney epithelial cells
in vitro. Environ. Toxicol. http://dx.doi.org/10.1002/tox.22012, in press.
Clarkson, T.W., 2002. The three modern faces of mercury. Environ. Health Perspect.
110, 1123.
Clarkson, T.W., Magos, L., 2006. The toxicology of mercury and its chemical
compounds. Crit. Rev. Toxicol. 36, 609662.

Clements, C.J., Ball, L.K., Ball, R., Pratt, D., 2000. Thiomersal in vaccines. Lancet 355
(9211), 12791280.
Delong, G., 2011. A positive association found between autism prevalence and
childhood vaccination uptake across the U.S. population. J. Toxicol. Environ.
Health A 74 (14), 903916.
Fairbanks, V.F., 1994. Iron in medicine and nutritionModern Nutrition in Health and
Disease8th ed. Lea & Febiger, Philadelphia, pp. 191202.
Foss, S.D., 1969. A method for obtaining initial estimates of the parameters in
exponential curve tting. Biometrics 25, 580584.
Gewin, L., Vadivelu, S., Neelisetty, S., Srichai, M.B., Paueksakon, P., Pozzi, A., Harris,
R.C., Zent, R., 2012. Deleting the TGF-beta receptor attenuates acute proximal
tubule injury. J. Am. Soc. Nephrol. 23 (12), 20012011.
Gibaldi, M., Perrier, D., 1982. Pharmacokinetics. Marcel Dekker, New York.
Halliwell, B., Chirico, S., 1993. Lipid peroxidation: its mechanism, measurement,
and signicance. Am. J. Clin. Nutr. 57 (5 Suppl), S715S725.
Harisa, G.I., Alanazi, F.K., El-Bassat, R.A., Malik, A., Abdallah, G.M., 2012. Protective
effect of pravastatin against mercury induced vascular cells damage:
erythrocytes as surrogate markers. Environ. Toxicol. Pharmacol. 34 (2)
428435.
Harry, G.J., Harris, M.W., Burka, L.T., 2004. Mercury concentrations in brain and
kidney following ethylmercury, methylmercury and thimerosal administration
to neonatal mice. Toxicol. Lett. 154 (3), 183189.
Helmer, O.M., Emerson, C.P., 1934. The iron content of the whole blood of normal
individuals. J. Biol. Chem. 104 (1), 157161.
Hilmy, M.I., Rahim, S.A., Abbas, A.H., 1976. Normal and lethal mercury levels in
human beings. Toxicology 6, 155159.
Ida-Eto, M., Oyabu, A., Ohkawara, T., Tashiro, Y., Narita, N., Narita, M., 2013. Prenatal
exposure to organomercury, thimerosal, persistently impairs the serotonergic
and dopaminergic systems in the rat brain: implications for association with
developmental disorders. Brain Dev. 35 (3), 261264.
Kaupp, M., Malkina, O.L., 1998. Density-functional analysis of C-13 and H-1
chemical-shifts and bonding in mercurimethanes and organomercury
hydradesthe role of scalar relativistic, spin-orbit, and substituent effects. J.
Chem. Phys. 108, 36483659.
Lebel, J., Mergler, D., Branches, F., Lucotte, M., Amorim, M., Larribe, F., Dolbec, J.,
1998. Neurotoxic effects of low-level methylmercury contamination in the
Amazonian Basin. Environ. Res. 79 (1), 2032.
Magos, L., 2003. Neurotoxic character of thimerosal and the allometric extrapolation of adult clearance half-time to infants. J. Appl. Toxicol. 23, 263269.
Magos, L., Butler, W.H., 1976. The kinetics of methylmercury administered repeatedly to rats. Arch. Toxicol. 35 (1), 2539.
Orct, T., Blanusa, M., Lazarus, M., Varnai, V.M., Kostial, K., 2006. Comparison of
organic and inorganic mercury distribution in suckling rat. J. Appl. Toxicol. 26
(6), 536539.
Pamphlett, R., Jew, S.K., 2013. Uptake of inorganic mercury by human locus ceruleus
and corticomotor neurons: implications for amyotrophic lateral sclerosis. Acta
Neuropathol. Commun. 1, 13.
Pamphlett, R., Waley, P., 1996. Uptake of inorganic mercury by the human brain.
Acta Neuropathol. 92, 525527.
Petersson, K., Dock, L., Soderling, K., Vahter, M., 1991. Distribution of mercury in
rabbits subchronically exposed to low levels of radiolabeled methyl mercury.
Pharmacol. Toxicol. 68 (6), 464468.
Petroni, D., Tsai, J., Agrawal, K., Mondal, D., George, W., 2012. Low-dose methylmercury-induced oxidative stress, cytotoxicity, and tau-hyperphosphorylation
in human neuroblastoma (SH-SY5Y) cells. Environ. Toxicol. 27 (9),
549555.
Pfab, R., Muckter, H., Roider, G., Zilker, T., 1996. Clinical course of severe poisoning
with thiomersal. J. Toxicol. Clin. Toxicol. 34 (4), 453460.
Pichichero, M.E., Cernichiari, E., Lopreiato, J., Treanor, J., 2002. Mercury concentrations and metabolism in infants receiving vaccines containing thiomersal:
a descriptive study. Lancet 360, 17371741.
Pichichero, M.E., Gentile, A., Giglio, N., Alonso, M.M., Fernandez Mentaberri, M.V.,
Zareba, G., Clarkson, T., Gotelli, C., Gotelli, M., Yan, L., Treanor, J., 2009. Mercury
levels in premature and low birth weight newborn infants after receipt of
thimerosal-containing vaccines. J. Pediatr. 155 (4), 495499.
Qvarnstrm, J., Lambertsson, L., Havarinasab, S., Hultman, P., Frech, W., 2003.
Determination of methylmercury, ethylmercury, and inorganic mercury in
mouse tissues, following administration of thimerosal, by species-specic
isotope dilution GC-inductively coupled plasma-MS. Anal. Chem. 75 (16),
41204124.
Rodrigues, J.L., Serpeloni, J.M., Batista, B.L., Souza, S.S., Barbosa Jr., F., 2010.
Identication and distribution of mercury species in rat tissues following
administration of thimerosal or methylmercury. Arch. Toxicol. 84 (11),
891896.
Sharma, M.K., Sharma, A., Kumar, A., Kumar, M., 2007. Evaluation of protective
efcacy of Spirulina fusiformis against mercury induced nephrotoxicity in
Swiss albino mice. Food Chem. Toxicol. 45 (6), 879887.
Skerfving, S., 1974. Methylmercury exposure, mercury levels in blood and hair, and
health status in Swedes consuming contaminated sh. Toxicology 2 (1), 323.
Souza, S.S., Campiglia, A.D., Barbosa Jr., F., 2013. A simple method for methylmercury, inorganic mercury and ethylmercury determination in plasma samples by
high performance liquid chromatography-cold-vapor-inductively coupled
plasma mass spectrometry. Anal. Chim. Acta 761, 1117.
Stajich, G.V., Lopez, G.P., Harry, S.W., Sexson, W.R., 2000. Iatrogenic exposure to
mercury after hepatitis B vaccination in preterm infants. J. Pediatr. 136,
679681.

M.F.H. Carneiro et al. / Environmental Research 134 (2014) 218227

Suda, I., Hirayama, K., 1992. Degradation of methyl and ethyl mercury into
inorganic mercury by hydroxyl radical produced from rat-liver microssomes.
Arch. Toxicol. 66 (6), 398402.
Suda, I., Takahashi, H., 1992. Degradation of methyl and ethyl mercury into
inorganic mercury by other reactive oxygen species besides hydroxyl radical.
Arch. Toxicol. 66 (1), 3439.
Suda, I., Totoki, S., Takahashi, H., 1991. Degradation of methyl and ethyl mercury
into inorganic mercury by oxygen free radical-producing systems: involvement
of hydroxyl radical. Arch. Toxicol. 65 (2), 129134.
Suda, I., Totoki, S., Uchida, T., Takahashi, H., 1992. Degradation of methyl and ethyl
mercury into inorganic mercury by various phagocytic cells. Arch. Toxicol. 66
(1), 4044.
Suzuki, T., Takemoto, T.-I., Kashiwazaki, H., Miyama, T., 1973. Metabolic fate of
ethylmercury salts in man and animal. In: Miller, MW, Clarkson, TW (Eds.),
Mercury, Mercurials and Mercaptans. Charles C. Thomas, Springeld, IL,
pp. 209232.
Tan, M., Parkin, J.E., 2000. Route of decomposition of thiomersal (thimerosal). Int. J.
Pharm. 208 (12), 2334.
USEPA (United States Environmental Protection Agency), 1997. Mercury Study
Report to Congress (Available from). Ofce of Air Quality Planning and
Standards and Ofce of Research and Development, Washington D.C., USA
(accessed October, 2013).
Vahter, M., Mottet, N.K., Friberg, L.T., Lind, S.B., Charleston, J.S., Burbacher, T.M.,
1995. Demethylation of methyl mercury in different brain sites of Macaca

227

fascicularis monkeys during long-term subclinical methyl mercury exposure.

Toxicol. Appl. Pharmacol. 134 (2), 273284.
Wolfsegger, M.J., 2006. The R package PK for basic pharmacokinetics. Biometrie und
Medizin 5, 6168.
WHO (World Health Organization), 2012. Children's exposure to mercury compounds. Available from http://www.who.int/ceh/publications/children_expo
sure/en/ (accessed March, 2014).
Yaginuma-Sakurai, K., Murata, K., Iwai-Shimada, M., Nakai, K., Kurokawa, N.,
Tatsuta, N., Satoh, H., 2012. Hair-to-blood ratio and biological half-life of
mercury: experimental study of methylmercury exposure through sh consumption in humans. J. Toxicol. Sci. 37 (1), 123130.
Yasutake, A., Marumoto, M., Yoshida, M., 2010. Neurotoxic action of inorganic
mercury injected in the intraventricular space of mouse cerebrum. J. Toxicol.
Sci. 35, 767771.
Zalups, R.K., 1997. Reductions in renal mass and the nephropathy induced by
mercury. Toxicol. Appl. Pharmacol. 143, 366379.
Zareba, G., Cernichiari, E., Hojo, R., Nitt, S.M., Weiss, B., Mumtaz, M.M., Jones, D.E.,
Clarkson, T.W., 2007. Thimerosal distribution and metabolism in neonatal mice:
comparison with methyl mercury. J. Appl. Toxicol. 27 (5), 511518.
Zimmermann, L.T., Santos, D.B., Naime, A.A., Leal, R.B., Drea, J.G., Barbosa Jr., F.,
Aschner, M., Rocha, J.B., Farina, M., 2013. Comparative study on methyl- and
ethylmercury-induced toxicity in C6 glioma cells and the potential role of LAT-1
in mediating mercurial-thiol complexes uptake. Neurotoxicology 38C, 18.