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Mercury and human genotoxicity: Critical considerations and possible

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 Pharmacological Research 60 (2009) 212–220

Contents lists available at ScienceDirect

Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs

Review

Mercury and human genotoxicity: Critical considerations

and possible molecular mechanisms
Maria Elena Crespo-López a,∗ , Gisele L. Macêdo a , Susana I.D. Pereira b , Gabriela P.F. Arrifano a ,
Domingos L.W. Picanço-Diniz c , José Luiz M. do Nascimento d , Anderson M. Herculano c,d
a
Laboratório de Farmacologia Molecular, Brazil
b
Departamento de Química, Universidade de Aveiro, Aveiro, Portugal
c
Laboratório de Neuroendocrinologia, Brazil
d
Laboratório de Neuroquímica Molecular e Celular, Instituto de Ciências Biológicas, Universidade Federal do Pará (UFPA), Belém, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Mercury compounds versatility explains their numerous applications in diverse areas of industry. The
Received 27 October 2008 growing use of this metal has resulted in a significant increase of environment contamination and episodes
Received in revised form 13 February 2009 of human intoxication, arousing the concern of international organisms. Meanwhile, consequences of
Accepted 27 February 2009
these intoxication outbreaks are still not fully understood, especially if we consider long-term effects
of chronic exposure to relatively low levels of mercury compounds. In the present manuscript, studies
Keywords:
about the genotoxicity of mercury compounds, performed in vitro, in vivo, and/or including epidemio-
Mercury
logic studies of human populations were reviewed. Some mercury compounds are known as teratogenic
Methylmercury
Cancer
agents, especially affecting the normal development of the central nervous system; however, the connec-
Teratogenesis tion between mercury exposure and carcinogenesis remains controversial. Since 1990s, epidemiological
Genotoxicity studies have begun to include an increasing number of human subjects, making the results more reliable:
Oxidative stress thus, increased genotoxicity was demonstrated in human populations exposed to mercury through diet,
Human occupation or by carrying dental fillings. In fact, concentrations of methylmercury causing significant
Microtubule genotoxic alterations in vitro below both safety limit and concentration were associated with delayed
DNA psychomotor development with minimal signs of methylmercury poisoning. Based on mercury’s known
ability to bind sulfhydryl groups, several hypotheses were raised about potential molecular mechanisms
for the metal genotoxicity. Mercury may be involved in four main processes that lead to genotoxicity: gen-
eration of free radicals and oxidative stress, action on microtubules, influence on DNA repair mechanisms
and direct interaction with DNA molecules. All data reviewed here contributed to a better knowledge of
the widespread concern about the safety limits of mercury exposure.
© 2009 Elsevier Ltd. All rights reserved.

Contents

1. Introduction: mercury applications and human intoxication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

2. Mercury and human genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
2.1. Teratogenesis and carcinogenesis of mercury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
2.2. Mercury and biomarkers of genotoxicity: studies with human tissues and populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
3. Molecular mechanisms of mercury genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
3.1. Mercury and oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
3.2. Mercury action on microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
3.3. Influence of mercury on the DNA repair mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
3.4. Direct interaction of mercury with DNA molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
4. Final considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

∗ Corresponding author at: Laboratório de Farmacologia Molecular, Instituto de Ciências Biológicas, Universidade Federal do Pará (UFPA), Rua Augusto Corrêa 01,
Campus do Guamá, 66075-110 Belém-PA, Brazil. Tel.: +55 91 32018212; fax: +55 91 32011601.
E-mail address: mecl30@yahoo.es (M.E. Crespo-López).

1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phrs.2009.02.011
 M.E. Crespo-López et al. / Pharmacological Research 60 (2009) 212–220
1. Introduction: mercury applications and human
intoxication
Mercury is one of the oldest chemical elements used in human
applications. In its elemental state, mercury is a silver-white liq-
uid, being also known as metallic mercury (Hg◦ ). However, mercury
may also be present in two oxidized forms [mercurous ion (Hg2 +2 )
and mercuric ion (Hg+2 )] and as different organometallic species
(alkyl mercury, alkoxy mercury and phenylmercury), being the
short chain alkyl mercury species, as methylmercury (CH3 Hg) and
dimethylmercury ((CH3 )2 Hg), the most dangerous compounds in
terms of their toxicological effects. These organometallic com-
pounds have a higher solubility in lipids when compared to
inorganic species, making it easier to diffuse through the lipidic
matrix of the cellular membrane, therefore increasing its toxicity
potential [1–3].
Mercury versatility as a metal explains its numerous applica-
tions in areas as different as industry, odontology, pharmacology,
primary gold mining and agriculture. Many of these applications
are based on the unusual ability of mercury to bind other met-
als making amalgams. In odontology, for example, mercury–silver
amalgams are used to fill dental cavities. Whenever the filling
is involved in the chewing of food, a tiny amount of mercury is
vaporized reaching olfactory bulbs and brain [3]. Thus, mercury
levels in brain may be proportional to the number of mercury-
containing amalgams present in tooth fillings. In fact, the study
of Kingman et al. [4] carried out among a military population
from United States demonstrated that individuals with amalgams
show four–five-fold higher concentrations of mercury in urine and
blood.
In the last decades, the growing use of this metal has resulted
in a significant increase of environmental contamination (predom-
inantly of water and food) and episodes of human intoxication
[2,5–7]. For example, the Amazon region is currently one of the
chronically contaminated points in the world where exposure is
directly related to the intensive use of mercury in the mining
activity [7–9], because of its ability to bind to other metals and,
particularly, to those with economical value, like gold. The forma-
tion of mercury–gold amalgams makes easier the separation of
both metals from river and soil’s sediments [3,9]. The release of
mercury into the environment occurs when the amalgam is heated
in order to recover the gold particles. By gravity, mercury precipi-
tates into the riverbed, getting mixed with the sediments. It then
suffers a biotransformation process, through which methanogenic
bacteria transform inorganic mercury into methylmercury [3,6,10].
Following this biotransformation, another process, called biomag-
nification, takes place, which refers to the tendency of mercury (and
other pollutants) to concentrate as it moves from one trophic level
to the next. Consequently, the aquatic biota becomes the main way
of mercury transference from the contaminated environment to
human beings, especially when fish is part of populations’ food diet
[6–9,11].
As expected, piscivorous and carnivorous fish from gold min-
ing areas in the Amazon showed higher mercury concentrations,
followed by fish from lower trophic levels such as omnivorous,
detritivorous, and herbivorous species (reviewed by [12]). In the
Tapajós River basin (main tributary of the Amazon River), for exam-
ple, the highest mercury concentrations (above 500 ppb) were
found in carnivorous species such as the Sciaenidae family (Plag-
isocion squamosissimus, “pescada branca”), Pseudoplatystoma sp.
(“surubim”), the Pimelodidae family (Brachyplatystoma filamento-
sum, “filhote”; B. fravicans, “dourada”) and the Cichlidae family
(Cichla sp., “tucunaré”), among others [12,13]. Therefore, riverside
communities located downstream to the gold mining areas suffer
a chronic exposure to relatively high levels of methylmercury due
to their fish-rich diet [8,9,11,14].
213
In addition, organometallic mercury compounds, as those
present in thimerosal (thiomersal, merthiolate, sodium ethylmer-
cury thiosalicylate), an ethyl mercury-containing antibacterial and
antifungal agent, have also been used as antiseptic and preservative
in various formulations including paints, topical medications, con-
tact lens cleaners and cosmetics since the 1930s. Concern has been
raised by the use of thimerosal in more than 30 vaccines licensed in
the United States, with the consequence that majority of vaccines
are now free of thimerosal [15–17].
Thus, the exposure to mercury (especially in cases of acute intox-
ication, as in the accidents of Minamata and Irak) has been arousing
the concern of international organisms, such as the World Health
Organization [18], because of its serious effects on exposed popula-
tions. It is known that the central nervous system (CNS) is the main
target of acute intoxication caused by the most dangerous mercury
compounds, as methylmercury [3,5,7,19]. This type of intoxication
is characterized mainly by ataxy, disartry, paresthesia, constraints
in the visual field, hearing loss and disturbances in children’s ner-
vous system development [7–9,11,19,20]. Another typical symptom
of chronic cases of intoxication is erethism that manifests itself
through behavior and personality disorders, and may evolve into
depressive dysphoria [9,20].
Meanwhile, consequences of these intoxication outbreaks are
not yet fully known or understood, especially if we consider long-
term effects of chronic exposure to relatively low levels of mercury
[7,9,14,19,21]. Nevertheless, in the past few years several studies
have been published supporting the idea that relatively low con-
centrations of mercury may be at the origin of genotoxic processes
[7,19,22–24]. For this reason, we decided to focus this review on
studies about genotoxicity of mercury compounds, which have
been performed in vitro as well as in vivo, and/or have included
epidemiologic studies using human populations.
2. Mercury and human genotoxicity
2.1. Teratogenesis and carcinogenesis of mercury
Genotoxic consequences of human exposure to chemical com-
pounds generate changes in the genetic material mainly by two
processes: teratogenesis and carcinogenesis. The first one may
express itself in progeny in the form of congenital mal-formations,
while the second consists in the direct development of tumours on
individuals exposed.
Some mercury compounds, known as teratogenic agents, affect
specifically the normal development of the central nervous system
[17,20,25–27]. Among those compounds, methylmercury is directly
transferred to the fetus through the placenta, while inorganic mer-
cury is retained in the amniotic liquid, possibly because ions have a
lower solubility in lipids [25–27]. A higher incorporation of mer-
cury was found in animal embryos exposed to mercury organic
compounds when compared to those exposed to the inorganic form
[25]. Nevertheless, inorganic mercury can accumulate in the breasts
and be secreted in breast milk, generating serious damages on the
developing nervous system [28]. Development of major language
and motor functions, attention, visual-spatial abilities and verbal
memory are also considerably affected by low-dose prenatal mer-
cury exposure [2].
However, the connection between mercury exposure and car-
cinogenesis (one of the most dangerous consequences DNA induced
damages) remains controversial, because there are studies that
seem to demonstrate the existence of genotoxic activity associated
to mercury while others have not confirmed such damaging effects
on DNA [2,20,28].
For example, in a retrospective cohort study including 334
patients of Minamata outbreak [29], no significant correlation was
214 M.E. Crespo-López et al. / Pharmacological Research 60 (2009) 212–220
found between methylmercury intoxication and death frequency
by cancer. However, these patients were matched for sex, age and
year of death with “control” individuals, supposedly exposed to low
levels, that died in the same location. In addition, data were not ana-
lyzed by type of cancer. For a better evaluation, the same authors
broadened the work by increasing the number of individuals partic-
ipating in the study and by comparing residents in Minamata city
to residents in two seaside districts, Fukuro and Tsukinoura [30].
Again, to match with intoxicated individuals, it was assumed that
residents in the city were exposed to lower levels of methylmercury
(by lower intake of local seafood), without a complete analysis of
mercury concentrations. Although no significant increase in num-
ber of deaths by cancer was found in the individuals supposedly
exposed to high levels of mercury intoxication (residents in the
two seaside districts), an increased rate of deaths by liver cancer
was revealed among males. Unfortunately, these conclusions are
limited by the high incidence of alcohol consumption and hepatitis
B prevalence, as confounding factors.
Perhaps one of the larger studies carried out with Minamata
disease patients [31] was a retrospective cohort including 1351
intoxicated and 5667 control individuals. Interestingly, although
no increase in relative risk was detected for all cancer deaths in
patients, the risk for death by leukemia was eight-fold higher than
in control group.
To eliminate the confounding factors, others studies tried to
compare mercury content in hair of cancer patients with those
found in healthy individuals. Traditionally, mercury concentrations
found in hair have been considered as a historical biomarker of
mercury exposure. Thus, an interesting case–control study carried
out with individuals exposed to fungicides containing mercury
[32] showed that exposed individuals developing leukemia, pos-
sessed higher mercury content (1.24 ppm) in hair than healthy
individuals (0.49 ppm). Moreover, mercury levels were still signif-
icantly higher in those leukemia patients sharing the same house
than healthy individuals. Taking into account the different types of
leukemia, only diagnosis of acute leukemia significantly correlated
with higher levels of mercury.
Thus, considering studies above, the Commission on Life
Sciences of the National Research Council [2] declared that epi-
demiological research was still needed to evaluate the prevalence
of chromosomal aberrations and cancer, especially leukemia and
renal tumors, among populations suffering from chronic exposure
to mercury compounds such as methylmercury. For this reason, the
International Agency for Research on Cancer and the Environmental
Protection Agency classified methylmercury as a “potential” human
carcinogen [1] and recommended that the ability of methylmercury
to cause chromosomal damage and promote tumor growth should
be taken into consideration when establishing exposure guidelines.
However, in spite of the National Research Council and United States
Environment Protection Agency recommendations, few epidemio-
logic studies have been carried out on the relation between mercury
exposure and incidence of death by cancer.
Recently, the most large study (245,066 exposed individuals)
carried out in Minamata region [33] indicated that methylmercury
intoxication (originated by intake of contaminated fish) was prob-
ably the most responsible for the increased leukemia ASMR (age
standardized mortality ratio) found in exposed individuals. Inter-
estingly, the subgroup of more heavily exposed individuals (living
on the east side of Shiranui sea that was contaminated for 30 years)
always showed a high ASMR for leukemia (around 1.5) from 1961 to
1997. By other side, the subgroup with lower exposure (living on the
west side of Shiranui sea, contaminated for 10 years) demonstrated
that ASMR increased gradually during the same period (from 0.7 to
1.5, approximately).
Not only exposure to organic compounds of mercury seems to be
correlated with incidence of cancer, but exposure to inorganic mer-
cury could also have carcinogenic consequences [34,35,36]. Thus,
increased mortality (standardized mortality ratio of 1.2–2.8) by
lung and liver cancer was observed in 6784 males and 265 females
working at mines and mills from Spain, Slovenia, Italy and Ukraine
exposed to inorganic mercury [34]. However, authors discussed that
we have to be cautious with any conclusion because exposure to
crystalline silica would have contributed to increased number of
death by lung cancer. These results were also confirmed in recent
publications [35,36].
Despite the incidence of cancer in populations from the pre-
viously quoted studies, it is still not clear which genotoxic
mechanisms may be associated to the development of cancer. Cur-
rently, there are some parameters which are used in the research
of genotoxicity, such as identification of chromosomal abnormali-
ties, sister chromatids exchange, presence of micronuclei bearing
DNA fragments or chromosomes and molecular biology techniques
[37–39].
Monitoring human populations in situations of exposure to
genotoxic agents is generally performed by combining the for-
merly mentioned genetic evidences. For example, the chromosomal
abnormalities test is becoming an increasingly valuable param-
eter in the detection of genetic changes [38,39]. The analysis of
chromosomal abnormalities identifies two types of mutagenic sub-
stances: the ones that generate structural abnormalities (like breaks
or gaps) designated clastogenic, and those that produce numerical
abnormalities, designated aneugenic (due to an interference in the
formation of the mitotic spindle, which induces changes in the chro-
mosome distribution during cellular division). Additionally, the test
of sister chromatids exchange allows analyzing manifestations of
breaks occurring in the same chromatid locus of a chromosome,
followed by interchange and repair [39]. Besides those mentioned
above, another important test is the one that examines micronu-
clei, detecting the loss of chromatin as a consequence of structural
chromosome damage or damage in the mitotic apparatus [37–39].
All these detection tools are extremely useful in studies related
to mercury and its compounds’ genotoxic effects in individuals
exposed to this contaminating agent, either by occupational or
environmental exposure.
2.2. Mercury and biomarkers of genotoxicity: studies with human
tissues and populations
The first in vivo studies performed with the aim of understanding
the mercury genotoxic effects were reported by Skerfving and co-
workers in 1970 [epub 40]. After that, some studies were carried
out during the 1970s and 1980s in order to clarify if genotoxic-
ity biomarkers, such as chromosomal aberrations, micronuclei and
sister chromatid exchanges, could reveal DNA damages induced by
mercury exposure (reviewed by [40]). Most of these works, and
especially those that included chromosomal aberrations tests, did
not detect differences between controls and individuals exposed
to mercury compounds by accident, occupation or diet. However,
the number of subjects included in those studies was relatively low
and that may have affected the final results. For example, Skerfving
and co-workers, in 1970, showed that the frequency of chromo-
somal aberrations was not significantly different in exposed and
control groups. However, in 1974, when they broadened the work
by increasing the number of individuals participating in the study,
the frequency was slightly higher in exposed subjects [40].
After that, in the 1990s, epidemiological studies started to
include a higher number of subjects, increasing the reliability
of results. In 1994, for example, a study was carried out involv-
ing a group of 51 fishermen intoxicated with mercury-containing
seafood from the northern Tyrrhenian Sea [41]. A statistical correla-
tion was found between the frequency of micronuclei in peripheral
lymphocytes and mercury concentration in blood, as well as the
 M.E. Crespo-López et al. / Pharmacological Research 60 (2009) 212–220 215

age of the individuals, pointing to the presence of micronuclei as a
useful tool for early detection of DNA damage.
Another interesting study on chronic exposure to mercury-
containing diets was performed in the Tapajós River basin, major
tributary of the Amazon River [22]. To our knowledge, this is
the only in vivo study performed on the analysis of genotoxicity
biomarkers in riverside populations of the Amazon. As in the case of
Franchi et al. [41], this study also supports the hypothesis of a geno-
toxic activity associated to mercury compounds. Major changes
observed consisted in polyploidy, chromatid breaks and a decrease
in the mitotic index [22].
In addition to studies on chronic intoxication by diet, very few
human epidemiological studies were carried out on genotoxicity
generated by other forms of mercury exposure since the publication
of De Flora et al. review in 1994 [40]. A more recent study deter-
mined the potential genotoxicity of dental restorative compounds
in young people (18–27 years) using a new tool, the single cell gel
electrophoresis assay (SCGE), which detects DNA fragmentation of
individual cells [42]. With this sensitive method, they found that
lymphocytes of subjects carrying dental fillings showed parame-
ters of DNA fragmentation (tail length, percentage of DNA in the tail
and tail moment) two-fold higher than lymphocytes of unexposed
controls. This work demonstrated, for the first time, the adverse
effect on human health of dental filling constituents (amalgams
and methacrylates).
The genotoxicity caused by occupational exposure to relatively
low concentrations of mercury was also the aim of other two
epidemiological studies [24,43]. Queiroz et al. [43] found a signif-
icant increase in the percentage of micronuclei in lymphocytes of
workers chronically exposed to levels considered to be biologically
safe for populations. The second study, performed by Cebulska-
Wasileska et al. [24], monitorized twenty-five workers that used
mercury electrodes for electrolysis processes in a chlorine produc-
tion company located in Tarnów city, in the vicinity of Kraków,
Poland. Although the occupational exposure did not generate differ-
ences with respect to control individuals when the sister chromatid
exchange assay was used, chromosomal damage detected by SCGE
was significantly increased in lymphocytes of exposed workers.
The differences observed in the sensibility of different methods
used to measure mercury genotoxicity and the difficulty to avoid
the effect of other factors influencing the epidemiological studies
results support the interest of performing in vitro studies under
controlled conditions and the study of human tissues exposed to
mercury compounds. The human tissues more currently used for
this purpose are primary cultures of blood lymphocytes [23,44–49],
probably because it constitutes a peripheral tissue very easy to
collect and cultivate.
Since the 1990s, many authors tried to determine the levels of
mercury compounds required to produce DNA damages in lym-
phocyte cultures with different techniques (see Table 1). Effects of
trace concentrations of different mercury compounds on the gen-
eration of significant in vitro genotoxic modifications were very
diverse, revealing, once again, the distinct sensibilities of the dif-
ferent techniques. Thus, one of the more important conclusions
of Table 1 is that detection of chromosomal aberrations seems
to be the more adequate assay for detecting genotoxicity of mer-
cury compounds because of its higher sensibility when compared
with other assays as detection of micronuclei or sister chromatid
exchanges. For example, Ogura et al. [45] clearly showed that chro-
mosomal aberrations were registered with lower concentrations
(2 and 10 ␮M) of both organic and inorganic mercury compounds,
respectively, than those producing micronuclei and increased 8-
hydroxydeoxyguanosine levels (Table 1).
Data in Table 1 also demonstrate that exposure to low con-
centrations of mercury organic compounds (as low as 0.6 ␮M
[44]) induced statistically significant genotoxic modifications (like
micronuclei and chromosomal aberrations, among others), mean-
ing that the genotoxic potential of these compounds may be higher
compared to inorganic compounds.
Besides all these studies summarized in Table 1, it is hard to
establish the definitive minimal level of mercury compounds gen-
erating genotoxicity in human lymphocytes, due to the diverse
experimental conditions used in the studies. For example, although
Rao et al. [47] showed sister chromatid exchanges with exposure to
1.052 ␮M of inorganic mercury, Lee et al. [46] did not detect any of
them with exposure below 30 ␮M (Table 1). This discrepancy could
be due to the use of different times of exposure (44 h in the study
of Lee et al. [46] and not described in the study of Rao et al. [47]),
different anions (mercury nitrate in the study of Lee et al. [46] and
mercury chloride in the study of Rao et al. [47]), etc.

Table 1
Minimal concentrations of different mercury compounds producing significant in vitro genotoxic modifications (between parentheses) in primary cultures of human
lymphocytes as described elsewhere [23,44–49].

Methylmercury Dimethylmercury Inorganic mercury Phenylmercury acetate Thimerosal

Betti et al. [44] >0.6 ␮M (CA) 43.4 ␮M (sCA)

1.73 ␮M (nCA)

Ogura et al. [45] 2 ␮M (CA) 10 ␮M (CA)

5 ␮M (MN) 20 ␮M (MN)
5 ␮M (8-OHdG) 10 ␮M (8-OHdG)

Lee et al. [46] 20 ␮M (SCE) ns below 30 ␮M (SCE) 10 ␮M (SCE)

10 ␮M (PRI) 30 ␮M (PRI) 10 ␮M (PRI)

Rao et al. [47] 1.052 ␮M (SCE)

10.524 ␮M (C-anaphases)

Westphal et al., [23] 0.05 ␮g/ml (MN)

ns below 0.5 ␮g/ml (NDI with MN assay)

Silva-Pereira et al. [48] 0.1 ␮g/l (CA) 0.1 ␮g/l (CA)

100 ␮g/l (MI) ns below 1 mg/l (MI)

Eke and Çelik [49] 0.2 ␮g/ml (SCE)

0.2 ␮g/ml (PRI)
0.4 ␮g/ml (MI)

ns = non-significant; CA = chromosomal aberrations (s = structural; n = numerical); MN = mincronuclei; 8-OHdG = increased 8-hydroxydeoxyguanosine levels; SCE = sister
chromatid exchanges; PRI = decreased proliferation rate index, as defined by: [(1 × number of first division metaphases) + (2 × number of second division
metaphases) + (3 × subsequent division metaphases)]/total number of metaphases; NDI = nuclear division index; MI = decreased mitotic index, as defined by: number of
metaphases in 1000 cells.
216 M.E. Crespo-López et al. / Pharmacological Research 60 (2009) 212–220
Anyway, supporting the hypothesis of mercury’s genotoxic
action, Silva-Pereira et al. [48] demonstrated the genotoxic effect
of 1–10 ␮M of methylmercury and inorganic mercury on cultures
of human lymphocytes exposed in vitro, highlighting a synergistic
action promoted by inorganic and organic mercury co-treatments.
According to Table 1, methylmercury stands out as the most
genotoxic compound among all mercury compounds studied to
date [23,44–49]. In fact, the lowest concentration (0.1 ␮g/l or
0.4 nM) of methylmercury necessary to cause significant genotoxic
alterations (chromosomal aberrations) in vitro was below the safety
limit established by the World Health Organization for mercury
exposure [18]. It suggests that this effect may be directly related
to cellular changes (affecting the formation of the mitotic spindle
and the occurrence of polyploidies) conducting to potential biolog-
ical effects [48], which is especially interesting if we consider that
lymphocytes are not the main target for mercury toxicity.
Recently, the results reported by our group [19] demonstrated,
for the first time, that exposure to methylmercury can in fact gen-
erate genotoxic effects in cells of the central nervous system origin.
The study was performed using two different cell lines, human neu-
roblastoma and glioblastoma, as in vitro model and the main result
obtained consisted in a high frequency of micronucleated cells,
which indicates methylmercury does interfere in the chromosome
distribution during the anaphase period, leading to the appearance
of micronulei. Considering that concentrations of 2.5–10 ␮M in
the brain have already been associated with delayed psychomotor
development in cases of exposure to minimal levels of methylmer-
cury intoxication [11,21,50,51], our results suggest that exposure to
levels ten times lower than those previously reported may lead to
genotoxic consequences on the developing central nervous system,
pointing to a need of reviewing the tolerance values published in
1990 by the World Health Organization [18].
Most part of the studies published so far used low concentra-
tions of mercury, close or equivalent to those found in nature and in
everyday life products. The results obtained contributed to a grow-
ing discussion and led some authors to question the safety levels
currently established by international committees [9,11,19], since
a chronic exposure to relatively low doses of mercury seems to
activate several cellular mechanisms that could potentially lead to
carcinogenic and/or teratogenic processes.
Based on mercury’s ability to bind sulfhydryl groups, several
hypotheses were raised on potential mechanisms responsible for
the metal’s genotoxicity. Mercury’s action over chemical groups
present in different structures of a living organism constitutes the
base of many toxicity mechanisms (e.g. binding protein’s sulfhydryl
groups can cause changes in different biochemical processes that
may result in DNA damages). Therefore, mercury and its compounds
are potentially involved in four main mechanisms that take place
in cells and may lead to genotoxicity: direct action on DNA, gen-
eration of free radicals and oxidative stress, inhibition of mitotic
spindle formation (action over the microtubules) and influence on
DNA repair mechanisms (Fig. 1).
3. Molecular mechanisms of mercury genotoxicity
3.1. Mercury and oxidative stress
One of the first molecular mechanisms described to explain pos-
sible genotoxic consequences of mercury was oxidative stress (DNA
damage due to action of free radicals generated by the metal) as
showed in Table 2.
Free radicals are highly reactive chemical species that, in addi-
tion to an important physiological role, can also cause DNA damages
to DNA and, consequently, to cells leading, eventually, to carcino-
genic processes (see [52] for a review). These transient and unstable
Fig. 1. Molecular mechanisms of mercury genotoxicity. Mercury compounds enter
the cell through plasmatic membrane or transport proteins (grey cylinder). (1) Inside
the cell, they may produce reactive oxygen species (ROS) which react directly with
DNA or, indirectly, induce conformational changes in proteins responsible for the
formation and maintenance of DNA (DNA repair enzymes, proteins of microtubules).
Mercury compounds may be also able to bind directly to: (2) DNA molecules, forming
mercury species-DNA adducts, (3) “zinc fingers” core of DNA repair enzymes (white
large arrow), affecting their activity and (4) microtubules, avoiding mitotic spindle
formation and chromosome segregation.
chemical species easily react with nearby molecules with con-
sequent energy liberation. Free radicals are also responsible for
initiating auto-catalytic reactions through which proteins, lipids
and carbon hydrates are also converted to free radicals, thus prop-
agating a chain reaction.
Reactive oxygen species (ROS) constitute the main type of free
radicals implicated in pathogenic mechanisms. The most important
species are: superoxide radical, hydrogen peroxide, hydroxyl rad-
ical, oxygen singlet, alkyl radical, peroxyl radical and nitric oxide
[53]. ROS may be playing a potential pro-cancer role by promoting
proliferation, invasiveness, angiogenesis, metastasis and suppress-
ing apoptosis [52].
Mercury compounds have the ability to induce cellular damage
through an increase of ROS levels [53] and therefore ROS produc-
tion was already proposed as a molecular mechanism involved in
mercury genotoxicity (Fig. 1 and Table 2) [42,46,47,54]. The gener-
ation of free radicals can result on a widespread variety of effects,
but two of them are especially important to classify free radicals as
genotoxic.
First of all, direct action of free radicals on nucleic acids may gen-
erate genetic mutations (Table 2) [54]. Curiously, although mercury
compounds were not mutagenic in bacterial assays, inorganic mer-
cury was able to induce mutational events in eukaryotic cell lines
with doses as low as 0.5 ␮M [54].
Secondly, free radicals may induce conformational changes in
proteins responsible for the formation and maintenance of DNA,
such as repair enzymes, DNA-polymerases, and even tubulin and
kinesine motor proteins, responsible for mitotic spindle and chro-
mosomal segregation [24,52,55,56].
To prevent these deleterious effects, cells develop antioxidant
mechanisms that work to avoid and correct an excessive forma-
tion of free radicals, transforming the oxidant species into more
stable compounds. Such defenses include low molecular weight
molecules, like reduced glutathione, vitamins C and E, melatonin,
and enzymes such as superoxide dismutase, catalase, glutathione
peroxidase and glutathione reductase. Thus, in response to mercury
exposure intracellular glutathione levels increase and may protect
cells by playing an antioxidant role and chelating mercury [54].
Table 2
Summary of experimental studies associating mercury genotoxicity to molecular mechanisms.

Authors R Mercury compound Genotoxic effect Tissue/culture Molecular mechanism Highlights

Phenylmercury acetate

M.E. Crespo-López et al. / Pharmacological Research 60 (2009) 212–220

Lee et al. (1997) [46] Methylmercury SCE; PRI Human lymphocytes Oxidative stress CAT and SOD do not protect
Inorganic mercury

Schurz et al. (2000) [54] Inorganic mercury Mutations NIH/3T3 (primary mouse Oxidative stress Induced ROS generation
embryonic fibroblast) declines in time when MT
increases

SCE Co-treatment with

Rao et al. (2001) [47] Inorganic mercury Human lymphocytes Oxidative stress
C-anaphases Vitamin C protects

Thier et al., (2003) [59] Inorganic mercury Micronuclei CREST positive V79 (hamster lung Action on microtubules Inhibition of tubulin
fibroblasts) polymerization and motility

Bonacker et al. (2004) [60] Inorganic mercury Micronuclei CREST positive V79 (hamster lung Action on microtubules Disassembly of
fibroblasts) paclitaxel-stabilised
microtubules

Stoiber et al. (2004) [56] Inorganic mercury Micronuclei V79 (hamster lung Action on microtubules Amino acetic acid-like
fibroblasts) chelating agents (EGTA, EDTA,
etc.) do not protect

CA Oxidative stress Lower efficiency of DNA repair

Cebulska-Wasilewska et al. (2005) [24] Mercury vapor Human lymphocytes
DNA strand breaks Disruption of DNA repair against X-rays challenge

Inorganic mercury
Methylmercury Direct interaction with Covalent coordination to
Li et al. (2006) [64] Mercury species-DNA adducts –
Ethylmercury DNA molecules N sites of DNA bases
Phenylmercury

Di Pietro et al. (2008) [42] Dental amalgams DNA strand breaks Human lymphocytes Oxidative stress Direct correlation with dose
and exposure time

R = number of reference; SCE = sister chromatid exchanges; PRI = decreased proliferation rate index; CAT = catalase; SOD = superoxide dismutase; ROS = reactive oxygen species; MT = metallothioneins; CA = chromosomal aberrations.

217
218 M.E. Crespo-López et al. / Pharmacological Research 60 (2009) 212–220
An interesting work performed in vivo [14] already demon-
strated that glutathione levels are also higher in human populations
exposed to methylmercury intoxication (levels of mercury con-
tent in hair of 12–15 ␮g/g) by a fish-rich diet. Furthermore, these
authors found a direct positive correlation between glutathione and
mercury levels in blood. Interestingly, another human population
from the same area (Tapajós River basin, major tributary of Ama-
zon River) previously showed cytogenetic alterations (increased
mitotic index and polyploidal aberrations) that were also associ-
ated to mercury exposure (median levels of mercury content in hair
of 10–13 ␮g/g) [22].
In fact, glutathione may act as the main cell defense line against
mercury compounds. Data from Herculano et al. [57] also suggest
that high levels of intracellular glutathione may have a neuropro-
tective function against intoxication with mercury.
In addition, other antioxidant substances, as l-ascorbic acid
(vitamin C), also showed their protective role against mercury
genotoxicity in vitro by preventing sister chromatid exchanges and
abnormal mitosis (Table 2) [47]. Moreover, a substantial inverse
relation between mercury levels in blood and consumption of trop-
ical fruits, and especially oranges (fruit with high levels of vitamin
C), was demonstrated in exposed populations of the Amazon [58],
revealing another aspect of the protective role of the antioxidant
compounds.
Nevertheless, although addition of antioxidant enzymes as cata-
lase and superoxide dismutase (75 and 150 ␮g/ml, respectively)
does not seem to protect lymphocytes against in vitro genotoxicity
of organic mercury compounds (Table 2) [46], human epidemio-
logical studies demonstrated that enzymatic activity was altered in
exposed populations [14], probably contributing to the genotoxic
alterations detected. The results obtained suggested the hypothesis
that chronic exposure to relatively low levels of mercury may inhibit
antioxidant enzymatic activity originating a progressive cell oxida-
tive stress [14]. According to the authors, this phenomenon could
represent an important peripheral marker for mercury toxicity in
exposed populations.
3.2. Mercury action on microtubules
The influence of mercury species on microtubule network is
known since the 1970s, when the main proteins constituting micro-
tubules, tubulin and kinesin, were described as preferential targets
for mercury binding. However, only recently were these alterations
of microtubule network related, for the first time, to potential mer-
cury genotoxic effects (Fig. 1 and Table 2) [56,59,60].
In the first work reported (Table 2), above 4 ␮M of mercury salts,
independently of the anion, were able to inhibit polymerization
of isolated tubulin in a dose-dependent manner [59]. In addition,
0.1 ␮M of these salts was sufficient to decrease kinesin-driven
motility (measured by gliding of taxol-stabilised microtubules
across a kinesin-coated glass surface) and to produce a significant
increase of micronuclei in V9 hamster lung fibroblasts. The most
interesting results revealed that in vitro induction of micronuclei
started at concentrations (10 nM) below those affecting the isolated
motor proteins, revealing a high sensibility to the genotoxic effect.
The relation between mercury genotoxic effects and micro-
tubule alterations apparently is high enough to prevent chelating
compounds as ethylenediaminotetraacetate or ethyleneglycol
bis(2-amino-ethyl)-tetraacetic acid, traditionally used for detoxifi-
cation of polluted areas, from reducing those toxic consequences
(Table 2) [56]. However, other chelators containing sulfhydryl
groups, such as l-cysteine and dithiothreitol, proved efficient in
preventing the mercury inhibition of spontaneous assembly of
microtubule proteins (tubulin containing microtubule associated
proteins) in a cell-free environment at physiological temperature
[56]. These results strongly suggest that mercury effects on micro-
tubule network depend on metal binding to sulfhydryl groups of
motor proteins. This binding would, thereby, prevent the binding
of guanosine triphosphate to these proteins, and thus block the
biochemical cascade responsible for cellular functioning [61].
As for inorganic mercury, 10 ␮M of it also induced disassembly
of isolated microtubule proteins and in vitro decomposition of the
microtubule network (Table 2). However, inactivation of tubulin by
inorganic mercury was not followed by protein denaturation, being
a reversible process [56,60]. Therefore, microtubule disassembly
seems to be carried out by release of tubulin dimmers from the
terminal endings and not by fragmentation [56,60].
Since cytoskeletal proteins are involved in cell movement,
mitotic spindle formation, chromosomal segregation and nuclear
division, all these evidences indicate that part of inorganic mercury
chromosomal genotoxicity could be due to functional impairment
of kinesin and/or microtubules, leading to disturbances in chro-
mosome distribution. This process would generate problems on
chromosome segregation during mitosis by preventing chromo-
some translocation, and eventually lead to micronuclei formation.
In spite of these important results, we must remain cautious
since more evidences are necessary to confirm the relation between
microtubular inhibition and genotoxicity in human cells.
3.3. Influence of mercury on the DNA repair mechanism
Another possible mechanism responsible for mercury genotoxi-
city could be its effect on DNA repair mechanisms, which constitute
the defense system designated to protect genome integrity (Fig. 1
and Table 2). A deficient defense system could eventually lead to
carcinogenic processes [24,52].
An interesting study including workers intoxicated by occu-
pational exposure to mercury vapor analyzed the DNA repair
efficiency in primary cultures of blood lymphocytes irradiated
with X-rays (2 Gy) or UV-C (6 J/m2 ) light (Table 2) [24]. These
workers exposure was not acute but was equivalent to the max-
imum permissible amounts (0.025 mg/m3 ) for metallic mercury.
This fact may explain the absence of differences in control individ-
uals with respect to the non-irradiated cultures using single cell gel
electrophoresis assay (SCGE, which detects DNA fragmentation of
individual cells). However, when lymphocyte cultures were irradi-
ated for two hours, cells chronically exposed to mercury underwent
a significant reduction on the repair of X-ray-damages but not
UV-light-induced DNA damages [24]. Consequently, it was hypoth-
esized that exposure to relatively low concentrations of metal
would interfere on the recombination and base excision repair
mechanisms but not on the nucleotide excision repair mechanisms
[24,62].
Besides an indirect action on the DNA repair system (inhibition
of DNA polymerase caused by increased free radicals production)
[55], mercury could also be directly binding to the “zinc fingers”
core of DNA repair enzymes, affecting their activity. These “zinc
fingers” are the specific sequence of the protein chain that binds to
DNA, and they contain an atom of zinc and four cysteines and/or
histidines. Thus, the high affinity of mercury to sulfhydryl groups
present on these cysteines may severely deform the structural
integrity and activity of the enzymes [24,63]. To our knowledge,
this elegant work is the first epidemiological study in humans
demonstrating deleterious effects on the DNA repair system with
“permissible” levels of mercury exposure.
3.4. Direct interaction of mercury with DNA molecules
All molecular mechanisms described before as potentially
responsible for mercury genotoxicity are mainly based on
mercury–protein interactions and how mercury species affect
these proteins (DNA repairs enzymes, antioxidant enzymes,
 M.E. Crespo-López et al. / Pharmacological Research 60 (2009) 212–220
cytoskeleton proteins, etc.). Interestingly, a recent report opened
a different possibility: the direct interaction between mercury
compounds and DNA molecules (Fig. 1 and Table 2) [64].
Using isolated DNA, the authors demonstrated that mer-
cury species coordinate covalently to endocyclic and exocyclic N
sites of DNA bases. Among the different mercury species tested
(methylmercury, ethylmercury, phenylmercury and inorganic mer-
cury), organomercury compounds exhibited a much higher affinity
to DNA than inorganic mercury. Moreover, methylmercury showed
the fastest binding rate, pointing to a rapid formation of sta-
ble complexes between this compound and DNA. Apparently,
every three or four base pairs on the DNA helix may bind one
organic or inorganic mercury compound, respectively. The direct
participation of the four base types in the interaction with mer-
cury species was detected, with predominance of guanine and
cytosine–methylmercury bindings, adenine and cytosine–ethyl
mercury bindings and, finally, thymine and inorganic mercury bond
formation. No special selectivity was shown in the binding of
phenylmercury to either of the four DNA bases. Curiously, despite
these interactions between DNA molecules and mercury species,
no alterations of DNA original conformation were registered, and
only the DNA secondary structure was affected.
Although formation of mercury species-DNA adducts is still
to be proved under physiological conditions, these data already
point to an additional mechanism that can explain potential geno-
toxic effects of mercury compounds. This mechanism is especially
interesting in the case of organometallic species due to their
capacity to easily cross nuclear membranes to reach the DNA
helix.
4. Final considerations
Genotoxicity of mercury compounds is a controversial theme.
Although epidemiological studies on this subject are still uncom-
mon, in vitro studies seem to clearly point to a particular
susceptibility of DNA to damages caused by mercury [19,48].
Major consequences of genotoxicity, carcinogenesis and terato-
genesis are hardly due to a unique deleterious factor, especially if
we considered environmental factors such as mercury exposure.
In spite of this, genotoxic alterations as chromosome aberrations
and micronuclei were already detected in populations chronically
exposed to mercury levels below the safety values defined by the
World Health Organization [18,22,42].
Moreover, considering that mercury brain content ranging from
2.5 to 10 ␮M has already been associated to delayed psychomotor
development in cases of exposure to minimal amounts of mercury
poisoning [11,21,50,51], it was recently suggested that exposure to
levels ten times lower could also lead to genotoxic consequences on
the developing central nervous system [19], pointing to the need of
reviewing the tolerance values published in 1990 by the WHO [18].
All data reviewed here contributed to a better knowledge of
the widespread concern on the safety limits of mercury exposure.
By establishing the exact relation between genotoxic processes
and mercury in human populations and the specific mechanisms
involved in these processes, we will be able to prevent intoxica-
tion episodes and develop pharmacological strategies to avoid toxic
consequences.
Acknowledgements
This work was supported by the FINEP research grant “Rede
Instituto Brasileiro de Neurociência (IBN-Net)” #01.06.0842-00 and
FAPESPA research grant #092/2008. GL Macêdo and JLM do Nasci-
mento thank CAPES and CNPq, respectively, for their grants.
219
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