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Neurobehavioral effects and oxidative biochemistry of chronic exposure to ethanol and methylmercury in the adolescence period in female rats View project
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Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs
Review
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
∗ 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) 212–220 213
1. Introduction: mercury applications and human In addition, organometallic mercury compounds, as those
intoxication present in thimerosal (thiomersal, merthiolate, sodium ethylmer-
cury thiosalicylate), an ethyl mercury-containing antibacterial and
Mercury is one of the oldest chemical elements used in human antifungal agent, have also been used as antiseptic and preservative
applications. In its elemental state, mercury is a silver-white liq- in various formulations including paints, topical medications, con-
uid, being also known as metallic mercury (Hg◦ ). However, mercury tact lens cleaners and cosmetics since the 1930s. Concern has been
may also be present in two oxidized forms [mercurous ion (Hg2 +2 ) raised by the use of thimerosal in more than 30 vaccines licensed in
and mercuric ion (Hg+2 )] and as different organometallic species the United States, with the consequence that majority of vaccines
(alkyl mercury, alkoxy mercury and phenylmercury), being the are now free of thimerosal [15–17].
short chain alkyl mercury species, as methylmercury (CH3 Hg) and Thus, the exposure to mercury (especially in cases of acute intox-
dimethylmercury ((CH3 )2 Hg), the most dangerous compounds in ication, as in the accidents of Minamata and Irak) has been arousing
terms of their toxicological effects. These organometallic com- the concern of international organisms, such as the World Health
pounds have a higher solubility in lipids when compared to Organization [18], because of its serious effects on exposed popula-
inorganic species, making it easier to diffuse through the lipidic tions. It is known that the central nervous system (CNS) is the main
matrix of the cellular membrane, therefore increasing its toxicity target of acute intoxication caused by the most dangerous mercury
potential [1–3]. compounds, as methylmercury [3,5,7,19]. This type of intoxication
Mercury versatility as a metal explains its numerous applica- is characterized mainly by ataxy, disartry, paresthesia, constraints
tions in areas as different as industry, odontology, pharmacology, in the visual field, hearing loss and disturbances in children’s ner-
primary gold mining and agriculture. Many of these applications vous system development [7–9,11,19,20]. Another typical symptom
are based on the unusual ability of mercury to bind other met- of chronic cases of intoxication is erethism that manifests itself
als making amalgams. In odontology, for example, mercury–silver through behavior and personality disorders, and may evolve into
amalgams are used to fill dental cavities. Whenever the filling depressive dysphoria [9,20].
is involved in the chewing of food, a tiny amount of mercury is Meanwhile, consequences of these intoxication outbreaks are
vaporized reaching olfactory bulbs and brain [3]. Thus, mercury not yet fully known or understood, especially if we consider long-
levels in brain may be proportional to the number of mercury- term effects of chronic exposure to relatively low levels of mercury
containing amalgams present in tooth fillings. In fact, the study [7,9,14,19,21]. Nevertheless, in the past few years several studies
of Kingman et al. [4] carried out among a military population have been published supporting the idea that relatively low con-
from United States demonstrated that individuals with amalgams centrations of mercury may be at the origin of genotoxic processes
show four–five-fold higher concentrations of mercury in urine and [7,19,22–24]. For this reason, we decided to focus this review on
blood. studies about genotoxicity of mercury compounds, which have
In the last decades, the growing use of this metal has resulted been performed in vitro as well as in vivo, and/or have included
in a significant increase of environmental contamination (predom- epidemiologic studies using human populations.
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 2. Mercury and human genotoxicity
directly related to the intensive use of mercury in the mining
activity [7–9], because of its ability to bind to other metals and, 2.1. Teratogenesis and carcinogenesis of mercury
particularly, to those with economical value, like gold. The forma-
tion of mercury–gold amalgams makes easier the separation of Genotoxic consequences of human exposure to chemical com-
both metals from river and soil’s sediments [3,9]. The release of pounds generate changes in the genetic material mainly by two
mercury into the environment occurs when the amalgam is heated processes: teratogenesis and carcinogenesis. The first one may
in order to recover the gold particles. By gravity, mercury precipi- express itself in progeny in the form of congenital mal-formations,
tates into the riverbed, getting mixed with the sediments. It then while the second consists in the direct development of tumours on
suffers a biotransformation process, through which methanogenic individuals exposed.
bacteria transform inorganic mercury into methylmercury [3,6,10]. Some mercury compounds, known as teratogenic agents, affect
Following this biotransformation, another process, called biomag- specifically the normal development of the central nervous system
nification, takes place, which refers to the tendency of mercury (and [17,20,25–27]. Among those compounds, methylmercury is directly
other pollutants) to concentrate as it moves from one trophic level transferred to the fetus through the placenta, while inorganic mer-
to the next. Consequently, the aquatic biota becomes the main way cury is retained in the amniotic liquid, possibly because ions have a
of mercury transference from the contaminated environment to lower solubility in lipids [25–27]. A higher incorporation of mer-
human beings, especially when fish is part of populations’ food diet cury was found in animal embryos exposed to mercury organic
[6–9,11]. compounds when compared to those exposed to the inorganic form
As expected, piscivorous and carnivorous fish from gold min- [25]. Nevertheless, inorganic mercury can accumulate in the breasts
ing areas in the Amazon showed higher mercury concentrations, and be secreted in breast milk, generating serious damages on the
followed by fish from lower trophic levels such as omnivorous, developing nervous system [28]. Development of major language
detritivorous, and herbivorous species (reviewed by [12]). In the and motor functions, attention, visual-spatial abilities and verbal
Tapajós River basin (main tributary of the Amazon River), for exam- memory are also considerably affected by low-dose prenatal mer-
ple, the highest mercury concentrations (above 500 ppb) were cury exposure [2].
found in carnivorous species such as the Sciaenidae family (Plag- However, the connection between mercury exposure and car-
isocion squamosissimus, “pescada branca”), Pseudoplatystoma sp. cinogenesis (one of the most dangerous consequences DNA induced
(“surubim”), the Pimelodidae family (Brachyplatystoma filamento- damages) remains controversial, because there are studies that
sum, “filhote”; B. fravicans, “dourada”) and the Cichlidae family seem to demonstrate the existence of genotoxic activity associated
(Cichla sp., “tucunaré”), among others [12,13]. Therefore, riverside to mercury while others have not confirmed such damaging effects
communities located downstream to the gold mining areas suffer on DNA [2,20,28].
a chronic exposure to relatively high levels of methylmercury due For example, in a retrospective cohort study including 334
to their fish-rich diet [8,9,11,14]. 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 cury could also have carcinogenic consequences [34,35,36]. Thus,
by cancer. However, these patients were matched for sex, age and increased mortality (standardized mortality ratio of 1.2–2.8) by
year of death with “control” individuals, supposedly exposed to low lung and liver cancer was observed in 6784 males and 265 females
levels, that died in the same location. In addition, data were not ana- working at mines and mills from Spain, Slovenia, Italy and Ukraine
lyzed by type of cancer. For a better evaluation, the same authors exposed to inorganic mercury [34]. However, authors discussed that
broadened the work by increasing the number of individuals partic- we have to be cautious with any conclusion because exposure to
ipating in the study and by comparing residents in Minamata city crystalline silica would have contributed to increased number of
to residents in two seaside districts, Fukuro and Tsukinoura [30]. death by lung cancer. These results were also confirmed in recent
Again, to match with intoxicated individuals, it was assumed that publications [35,36].
residents in the city were exposed to lower levels of methylmercury Despite the incidence of cancer in populations from the pre-
(by lower intake of local seafood), without a complete analysis of viously quoted studies, it is still not clear which genotoxic
mercury concentrations. Although no significant increase in num- mechanisms may be associated to the development of cancer. Cur-
ber of deaths by cancer was found in the individuals supposedly rently, there are some parameters which are used in the research
exposed to high levels of mercury intoxication (residents in the of genotoxicity, such as identification of chromosomal abnormali-
two seaside districts), an increased rate of deaths by liver cancer ties, sister chromatids exchange, presence of micronuclei bearing
was revealed among males. Unfortunately, these conclusions are DNA fragments or chromosomes and molecular biology techniques
limited by the high incidence of alcohol consumption and hepatitis [37–39].
B prevalence, as confounding factors. Monitoring human populations in situations of exposure to
Perhaps one of the larger studies carried out with Minamata genotoxic agents is generally performed by combining the for-
disease patients [31] was a retrospective cohort including 1351 merly mentioned genetic evidences. For example, the chromosomal
intoxicated and 5667 control individuals. Interestingly, although abnormalities test is becoming an increasingly valuable param-
no increase in relative risk was detected for all cancer deaths in eter in the detection of genetic changes [38,39]. The analysis of
patients, the risk for death by leukemia was eight-fold higher than chromosomal abnormalities identifies two types of mutagenic sub-
in control group. stances: the ones that generate structural abnormalities (like breaks
To eliminate the confounding factors, others studies tried to or gaps) designated clastogenic, and those that produce numerical
compare mercury content in hair of cancer patients with those abnormalities, designated aneugenic (due to an interference in the
found in healthy individuals. Traditionally, mercury concentrations formation of the mitotic spindle, which induces changes in the chro-
found in hair have been considered as a historical biomarker of mosome distribution during cellular division). Additionally, the test
mercury exposure. Thus, an interesting case–control study carried of sister chromatids exchange allows analyzing manifestations of
out with individuals exposed to fungicides containing mercury breaks occurring in the same chromatid locus of a chromosome,
[32] showed that exposed individuals developing leukemia, pos- followed by interchange and repair [39]. Besides those mentioned
sessed higher mercury content (1.24 ppm) in hair than healthy above, another important test is the one that examines micronu-
individuals (0.49 ppm). Moreover, mercury levels were still signif- clei, detecting the loss of chromatin as a consequence of structural
icantly higher in those leukemia patients sharing the same house chromosome damage or damage in the mitotic apparatus [37–39].
than healthy individuals. Taking into account the different types of All these detection tools are extremely useful in studies related
leukemia, only diagnosis of acute leukemia significantly correlated to mercury and its compounds’ genotoxic effects in individuals
with higher levels of mercury. exposed to this contaminating agent, either by occupational or
Thus, considering studies above, the Commission on Life environmental exposure.
Sciences of the National Research Council [2] declared that epi-
demiological research was still needed to evaluate the prevalence 2.2. Mercury and biomarkers of genotoxicity: studies with human
of chromosomal aberrations and cancer, especially leukemia and tissues and populations
renal tumors, among populations suffering from chronic exposure
to mercury compounds such as methylmercury. For this reason, the The first in vivo studies performed with the aim of understanding
International Agency for Research on Cancer and the Environmental the mercury genotoxic effects were reported by Skerfving and co-
Protection Agency classified methylmercury as a “potential” human workers in 1970 [epub 40]. After that, some studies were carried
carcinogen [1] and recommended that the ability of methylmercury out during the 1970s and 1980s in order to clarify if genotoxic-
to cause chromosomal damage and promote tumor growth should ity biomarkers, such as chromosomal aberrations, micronuclei and
be taken into consideration when establishing exposure guidelines. sister chromatid exchanges, could reveal DNA damages induced by
However, in spite of the National Research Council and United States mercury exposure (reviewed by [40]). Most of these works, and
Environment Protection Agency recommendations, few epidemio- especially those that included chromosomal aberrations tests, did
logic studies have been carried out on the relation between mercury not detect differences between controls and individuals exposed
exposure and incidence of death by cancer. to mercury compounds by accident, occupation or diet. However,
Recently, the most large study (245,066 exposed individuals) the number of subjects included in those studies was relatively low
carried out in Minamata region [33] indicated that methylmercury and that may have affected the final results. For example, Skerfving
intoxication (originated by intake of contaminated fish) was prob- and co-workers, in 1970, showed that the frequency of chromo-
ably the most responsible for the increased leukemia ASMR (age somal aberrations was not significantly different in exposed and
standardized mortality ratio) found in exposed individuals. Inter- control groups. However, in 1974, when they broadened the work
estingly, the subgroup of more heavily exposed individuals (living by increasing the number of individuals participating in the study,
on the east side of Shiranui sea that was contaminated for 30 years) the frequency was slightly higher in exposed subjects [40].
always showed a high ASMR for leukemia (around 1.5) from 1961 to After that, in the 1990s, epidemiological studies started to
1997. By other side, the subgroup with lower exposure (living on the include a higher number of subjects, increasing the reliability
west side of Shiranui sea, contaminated for 10 years) demonstrated of results. In 1994, for example, a study was carried out involv-
that ASMR increased gradually during the same period (from 0.7 to ing a group of 51 fishermen intoxicated with mercury-containing
1.5, approximately). seafood from the northern Tyrrhenian Sea [41]. A statistical correla-
Not only exposure to organic compounds of mercury seems to be tion was found between the frequency of micronuclei in peripheral
correlated with incidence of cancer, but exposure to inorganic mer- 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 the effect of other factors influencing the epidemiological studies
useful tool for early detection of DNA damage. results support the interest of performing in vitro studies under
Another interesting study on chronic exposure to mercury- controlled conditions and the study of human tissues exposed to
containing diets was performed in the Tapajós River basin, major mercury compounds. The human tissues more currently used for
tributary of the Amazon River [22]. To our knowledge, this is this purpose are primary cultures of blood lymphocytes [23,44–49],
the only in vivo study performed on the analysis of genotoxicity probably because it constitutes a peripheral tissue very easy to
biomarkers in riverside populations of the Amazon. As in the case of collect and cultivate.
Franchi et al. [41], this study also supports the hypothesis of a geno- Since the 1990s, many authors tried to determine the levels of
toxic activity associated to mercury compounds. Major changes mercury compounds required to produce DNA damages in lym-
observed consisted in polyploidy, chromatid breaks and a decrease phocyte cultures with different techniques (see Table 1). Effects of
in the mitotic index [22]. trace concentrations of different mercury compounds on the gen-
In addition to studies on chronic intoxication by diet, very few eration of significant in vitro genotoxic modifications were very
human epidemiological studies were carried out on genotoxicity diverse, revealing, once again, the distinct sensibilities of the dif-
generated by other forms of mercury exposure since the publication ferent techniques. Thus, one of the more important conclusions
of De Flora et al. review in 1994 [40]. A more recent study deter- of Table 1 is that detection of chromosomal aberrations seems
mined the potential genotoxicity of dental restorative compounds to be the more adequate assay for detecting genotoxicity of mer-
in young people (18–27 years) using a new tool, the single cell gel cury compounds because of its higher sensibility when compared
electrophoresis assay (SCGE), which detects DNA fragmentation of with other assays as detection of micronuclei or sister chromatid
individual cells [42]. With this sensitive method, they found that exchanges. For example, Ogura et al. [45] clearly showed that chro-
lymphocytes of subjects carrying dental fillings showed parame- mosomal aberrations were registered with lower concentrations
ters of DNA fragmentation (tail length, percentage of DNA in the tail (2 and 10 M) of both organic and inorganic mercury compounds,
and tail moment) two-fold higher than lymphocytes of unexposed respectively, than those producing micronuclei and increased 8-
controls. This work demonstrated, for the first time, the adverse hydroxydeoxyguanosine levels (Table 1).
effect on human health of dental filling constituents (amalgams Data in Table 1 also demonstrate that exposure to low con-
and methacrylates). centrations of mercury organic compounds (as low as 0.6 M
The genotoxicity caused by occupational exposure to relatively [44]) induced statistically significant genotoxic modifications (like
low concentrations of mercury was also the aim of other two micronuclei and chromosomal aberrations, among others), mean-
epidemiological studies [24,43]. Queiroz et al. [43] found a signif- ing that the genotoxic potential of these compounds may be higher
icant increase in the percentage of micronuclei in lymphocytes of compared to inorganic compounds.
workers chronically exposed to levels considered to be biologically Besides all these studies summarized in Table 1, it is hard to
safe for populations. The second study, performed by Cebulska- establish the definitive minimal level of mercury compounds gen-
Wasileska et al. [24], monitorized twenty-five workers that used erating genotoxicity in human lymphocytes, due to the diverse
mercury electrodes for electrolysis processes in a chlorine produc- experimental conditions used in the studies. For example, although
tion company located in Tarnów city, in the vicinity of Kraków, Rao et al. [47] showed sister chromatid exchanges with exposure to
Poland. Although the occupational exposure did not generate differ- 1.052 M of inorganic mercury, Lee et al. [46] did not detect any of
ences with respect to control individuals when the sister chromatid them with exposure below 30 M (Table 1). This discrepancy could
exchange assay was used, chromosomal damage detected by SCGE be due to the use of different times of exposure (44 h in the study
was significantly increased in lymphocytes of exposed workers. of Lee et al. [46] and not described in the study of Rao et al. [47]),
The differences observed in the sensibility of different methods different anions (mercury nitrate in the study of Lee et al. [46] and
used to measure mercury genotoxicity and the difficulty to avoid 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].
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
Phenylmercury acetate
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
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
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- tubule network depend on metal binding to sulfhydryl groups of
strated that glutathione levels are also higher in human populations motor proteins. This binding would, thereby, prevent the binding
exposed to methylmercury intoxication (levels of mercury con- of guanosine triphosphate to these proteins, and thus block the
tent in hair of 12–15 g/g) by a fish-rich diet. Furthermore, these biochemical cascade responsible for cellular functioning [61].
authors found a direct positive correlation between glutathione and As for inorganic mercury, 10 M of it also induced disassembly
mercury levels in blood. Interestingly, another human population of isolated microtubule proteins and in vitro decomposition of the
from the same area (Tapajós River basin, major tributary of Ama- microtubule network (Table 2). However, inactivation of tubulin by
zon River) previously showed cytogenetic alterations (increased inorganic mercury was not followed by protein denaturation, being
mitotic index and polyploidal aberrations) that were also associ- a reversible process [56,60]. Therefore, microtubule disassembly
ated to mercury exposure (median levels of mercury content in hair seems to be carried out by release of tubulin dimmers from the
of 10–13 g/g) [22]. terminal endings and not by fragmentation [56,60].
In fact, glutathione may act as the main cell defense line against Since cytoskeletal proteins are involved in cell movement,
mercury compounds. Data from Herculano et al. [57] also suggest mitotic spindle formation, chromosomal segregation and nuclear
that high levels of intracellular glutathione may have a neuropro- division, all these evidences indicate that part of inorganic mercury
tective function against intoxication with mercury. chromosomal genotoxicity could be due to functional impairment
In addition, other antioxidant substances, as l-ascorbic acid of kinesin and/or microtubules, leading to disturbances in chro-
(vitamin C), also showed their protective role against mercury mosome distribution. This process would generate problems on
genotoxicity in vitro by preventing sister chromatid exchanges and chromosome segregation during mitosis by preventing chromo-
abnormal mitosis (Table 2) [47]. Moreover, a substantial inverse some translocation, and eventually lead to micronuclei formation.
relation between mercury levels in blood and consumption of trop- In spite of these important results, we must remain cautious
ical fruits, and especially oranges (fruit with high levels of vitamin since more evidences are necessary to confirm the relation between
C), was demonstrated in exposed populations of the Amazon [58], microtubular inhibition and genotoxicity in human cells.
revealing another aspect of the protective role of the antioxidant
compounds. 3.3. Influence of mercury on the DNA repair mechanism
Nevertheless, although addition of antioxidant enzymes as cata-
lase and superoxide dismutase (75 and 150 g/ml, respectively) Another possible mechanism responsible for mercury genotoxi-
does not seem to protect lymphocytes against in vitro genotoxicity city could be its effect on DNA repair mechanisms, which constitute
of organic mercury compounds (Table 2) [46], human epidemio- the defense system designated to protect genome integrity (Fig. 1
logical studies demonstrated that enzymatic activity was altered in and Table 2). A deficient defense system could eventually lead to
exposed populations [14], probably contributing to the genotoxic carcinogenic processes [24,52].
alterations detected. The results obtained suggested the hypothesis An interesting study including workers intoxicated by occu-
that chronic exposure to relatively low levels of mercury may inhibit pational exposure to mercury vapor analyzed the DNA repair
antioxidant enzymatic activity originating a progressive cell oxida- efficiency in primary cultures of blood lymphocytes irradiated
tive stress [14]. According to the authors, this phenomenon could with X-rays (2 Gy) or UV-C (6 J/m2 ) light (Table 2) [24]. These
represent an important peripheral marker for mercury toxicity in workers exposure was not acute but was equivalent to the max-
exposed populations. imum permissible amounts (0.025 mg/m3 ) for metallic mercury.
This fact may explain the absence of differences in control individ-
3.2. Mercury action on microtubules uals with respect to the non-irradiated cultures using single cell gel
electrophoresis assay (SCGE, which detects DNA fragmentation of
The influence of mercury species on microtubule network is individual cells). However, when lymphocyte cultures were irradi-
known since the 1970s, when the main proteins constituting micro- ated for two hours, cells chronically exposed to mercury underwent
tubules, tubulin and kinesin, were described as preferential targets a significant reduction on the repair of X-ray-damages but not
for mercury binding. However, only recently were these alterations UV-light-induced DNA damages [24]. Consequently, it was hypoth-
of microtubule network related, for the first time, to potential mer- esized that exposure to relatively low concentrations of metal
cury genotoxic effects (Fig. 1 and Table 2) [56,59,60]. would interfere on the recombination and base excision repair
In the first work reported (Table 2), above 4 M of mercury salts, mechanisms but not on the nucleotide excision repair mechanisms
independently of the anion, were able to inhibit polymerization [24,62].
of isolated tubulin in a dose-dependent manner [59]. In addition, Besides an indirect action on the DNA repair system (inhibition
0.1 M of these salts was sufficient to decrease kinesin-driven of DNA polymerase caused by increased free radicals production)
motility (measured by gliding of taxol-stabilised microtubules [55], mercury could also be directly binding to the “zinc fingers”
across a kinesin-coated glass surface) and to produce a significant core of DNA repair enzymes, affecting their activity. These “zinc
increase of micronuclei in V9 hamster lung fibroblasts. The most fingers” are the specific sequence of the protein chain that binds to
interesting results revealed that in vitro induction of micronuclei DNA, and they contain an atom of zinc and four cysteines and/or
started at concentrations (10 nM) below those affecting the isolated histidines. Thus, the high affinity of mercury to sulfhydryl groups
motor proteins, revealing a high sensibility to the genotoxic effect. present on these cysteines may severely deform the structural
The relation between mercury genotoxic effects and micro- integrity and activity of the enzymes [24,63]. To our knowledge,
tubule alterations apparently is high enough to prevent chelating this elegant work is the first epidemiological study in humans
compounds as ethylenediaminotetraacetate or ethyleneglycol demonstrating deleterious effects on the DNA repair system with
bis(2-amino-ethyl)-tetraacetic acid, traditionally used for detoxifi- “permissible” levels of mercury exposure.
cation of polluted areas, from reducing those toxic consequences
(Table 2) [56]. However, other chelators containing sulfhydryl 3.4. Direct interaction of mercury with DNA molecules
groups, such as l-cysteine and dithiothreitol, proved efficient in
preventing the mercury inhibition of spontaneous assembly of All molecular mechanisms described before as potentially
microtubule proteins (tubulin containing microtubule associated responsible for mercury genotoxicity are mainly based on
proteins) in a cell-free environment at physiological temperature mercury–protein interactions and how mercury species affect
[56]. These results strongly suggest that mercury effects on micro- these proteins (DNA repairs enzymes, antioxidant enzymes,
M.E. Crespo-López et al. / Pharmacological Research 60 (2009) 212–220 219
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