Environment International 37 (2011) 136141

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Environment International
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Genotoxicity of mercury: Contributing for the analysis of Amazonian populations

Maria Elena Crespo-Lpez a,, Gisele L. Macdo a, Gabriela P.F. Arrifano a, Maria da Conceio N. Pinheiro b, Jos Luiz M. do Nascimento c, Anderson M. Herculano d
a

Laboratrio de Farmacologia Molecular, Instituto de Cincias Biolgicas, Universidade Federal do Par (UFPA), Belm, Brazil Ncleo de Medicina Tropical; Universidade Federal do Par (UFPA), Belm, Brazil Laboratrio de Neuroqumica Molecular e Celular, Instituto de Cincias Biolgicas, Universidade Federal do Par (UFPA), Belm, Brazil d Laboratrio de Neuroendocrinologia, Instituto de Cincias Biolgicas, Universidade Federal do Par (UFPA), Belm, Brazil
b c

a r t i c l e

i n f o

a b s t r a c t
Mercury is an important source of environmental contamination affecting human beings throughout the world and especially in the Amazon. Riverside populations have been chronically exposed to relatively high levels of methylmercury for many years. Long-term effects of mercury exposure are not well known, but human genotoxicity was already showed in both in vitro and epidemiological studies. However, to date, only two studies were carried out in Amazonian populations with conicting results and without comparing to a non-exposed population. Aiming to highlight this question and avoid interference factors, this work analyzed in vitro genotoxicity of mercury in blood lymphocytes of Amazonian individuals by two methods (micronucleus and chromosomal aberrations). Deleterious effects of low levels (1500 g/l or 0,004 2 M) of methylmercury were only detected with the method to detect chromosomal aberrations. Mitotic index (proportion of cells in metaphase) was the parameter most sensible. Thus, this technique was applied for the analysis of an Amazonian non-exposed population (Panacauera) with similar socialeconomical characteristics of the exposed populations studied elsewhere. The mean of the mitotic index for Panacauera population was 0.0814 0.0097. Inter-individual variability of this index had no relation with sex or age. This value was above those registered for some groups of exposed populations. This fact points to mercury as the main responsible for inhibiting the cell cycle and/or the loss of proliferative capacity of the cells. These results already support mitotic index as an essential parameter for the early diagnose of mercury genotoxicity in humans, and especially in Amazonian populations. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 8 June 2010 Accepted 12 August 2010 Available online 9 September 2010 Keywords: Mercury Amazon Genotoxicity Chromosomal aberrations Micronucleus Human Methylmercury Mitotic index

1. Introduction Mercury is a major source of environmental pollution in various parts of the world and especially in the Amazon (Passos and Mergler, 2008; Berzas-Nevado et al., 2010). This fact is closely related to its intense use by the traditional mining activity because of the ability of mercury to bind to other metals, especially those of economic interest, such as gold. The release of mercury to the environment occurs when the mixture formed is heated to recover the gold. By the force of gravity, mercury drops into the riverbed by mixing the sediments. Then, mercury is transformed into organic forms by methanogenic bacteria and it is concentrated as it moves from one trophic level to the next (Gochfeld, 2003; Baird and Cann, 2004). Thus, the aquatic biota is the major route of transfer of mercury from a contaminated environment to humans, especially when the sh is part

Corresponding author. Laboratrio de Farmacologia Molecular, Instituto de Cincias Biolgicas, Universidade Federal do Par (UFPA), Rua Augusto Corra 01, Campus do Guam, 66075-110 Belm-PA, Brazil. Tel.: +55 91 32018212; fax: +55 91 32011601. E-mail address: ecrespo@ufpa.br (M.E. Crespo-Lpez). 0160-4120/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2010.08.009

of the diet (Gochfeld, 2003; Baird and Cann, 2004). In the Tapajs River Basin, one of the main tributaries of the Amazon river (Fig. 1), the highest concentration of methylmercury (up to 500 ppb) were found in carnivorous sh as the Sciaenidae family (Plagisocion squamosissimus, pescada branca), the Pimelodidae family (Pseudoplatystoma sp., surubim; Brachyplatystoma lamentosum, lhote; B. fravicans, dourada) and the Cichlidae family (Cichla sp., tucunar) among others (see Berzas-Nevado et al., 2010 for a review). These species are the main protein source of the riverside communities living downstream to the gold-mining areas, who usually consumed sh ve or more meals per week (Pinheiro et al., 2008). The human poisoning with mercury in the Tapajs River region has been studied by analysis of hair samples from different populations of riverine communities (Pinheiro et al., 2006, 2007, 2008; Berzas-Nevado et al., 2010). Studies in some of these communities (e.g., Brasilia Legal, Barreiras and So Luis do Tapajos) located near the great mineral reserve of gold in the Tapajs area have shown exposure levels for mercury levels in hair above 10 ppm (reviewed by Berzas-Nevado et al., 2010). Thus, this region is suffering a chronic exposure to moderately elevated levels of mercury since the 70's.

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Fig. 1. Map of Brazil showing the states with their capitals indicated by black stars (left). The insert represents the map of the State of Par with the locations of some riverine populations (small white circles), as well as the approximate location of the main gold-mining area of the Tapajs River basin (large grey circle). Maps were obtained from Instituto Brasileiro de Geologia e Estatstica (IBGE, Brazil).

Exposure to mercury (especially the episodes of acute intoxication, such as accidents at Minamata and Iraq) has aroused the concern of international organizations such as the World Health Organization (WHO, 1990), about the severity of its effects. However, the long-term effects, especially those caused by chronic intoxication, are not so well known. In recent years, several studies support the idea that chronic exposure to relatively low concentrations of mercury could be starting genotoxic processes in humans (Amorim et al., 2000; Westphal et al., 2003; Crespo-Lpez et al., 2005, 2007, and 2009). Potential genotoxicity of mercury compounds would cause changes in genetic material, eventually resulting in the development of carcinogenic, teratogenic and/or mutagenic processes in human populations exposed (see Crespo-Lpez et al., 2009 for a review). Monitoring of human populations exposed to genotoxic agents is usually accomplished by combining information of some tests as chromosomal aberrations and micronuclei detection (Fenech, 2000; Bonassi et al., 2005; Mateuca et al., 2006). The differences in sensitivity of methods to detect the genotoxicity and the difculty to avoid other factors that may interfere with the epidemiological ndings, have been one of the main difculties in analyzing the results of studies in humans, and especially the Amazon populations. Migration of populations from different continents and the existence of many different indigenous people make that Amazonian populations show very specic physical characteristics. These characteristics may provide a differential sensitivity to genotoxic agents, such as solar radiation (Marrot et al., 2005) or others. So, the rst part of this work aimed the comparison of traditional techniques (micronuclei and chromosomal aberrations) of detection of in vitro mercury genotoxicity in blood lymphocytes of volunteers from the Amazon region and determining which method would be the most appropriate technique for detecting genotoxicity in Amazonian populations. Subsequently, to eliminate interferences and highlight the conicting results of genotoxic studies that have been carried out in the Amazon, a non-exposed population was selected and analyzed as control for genotoxicity in this type of populations. 2. Materials and methods

bovine serum (FBS) and phytohaemagglutinin was obtained from Cultilab (So Paulo, Brazil). 2.2. Ethical aspects All participants were informed about the study and they gave written consent for enjoying the research. This study was performed according to ethical principles for human research dened by Resolution 196/96 of the National Health Council, Ministry of Health, Brazil. In addition, this study was approved by the National Council for Ethics in Research (CONEP, Brazil) with the document n 023/2007-CEP/NMT. 2.3. Populations Blood samples used to compare the two techniques of genotoxicity (micronuclei and chromosomal aberrations detection) were obtained from ve healthy donors aged 1840 years-old living at Belm. Blood samples used to establish reference values of genotoxicity for Amazonian populations were obtained from thirty individuals (about 10% of the total population) living at Panacauera, aged between 1860 years (22 females and 8 males). This population shows a life style very similar to that of the riverine communities of the Tapajs River area: among other characteristics, the diet includes a large number of sh meals per week and shing and local agriculture represents the major activities. For all participants in the study, exclusion criteria included smokers, alcohol drinkers (more than 200 ml per day) and individuals with long-term treatment with drugs, drug dependency and/or occupational exposure to any other genotoxic compound. 2.4. Collection of blood samples and culture of blood lymphocytes Approximately 3 ml of blood were collected, by venipuncture, into syringes containing heparin as anticoagulant. Immediately, 500 l were added to 5 ml of RPMI 1640 medium supplemented with 20% FBS, antibiotics and 1.5% of phytohaemagglutinin. Cultures were incubated for 72 h at 37 C and 5% CO2 (Moorhead et al., 1960). 2.5. Exposure to methylmercury chloride

2.1. Chemicals Methylmercury chloride, antibiotics (penicillin and streptomycin), glutamine, demecolcine and cytochalasin B were purchased from Sigma-Aldrich (St Louis, MO, USA). RPMI 1640 medium was obtained from GIBCO Company (Paisley, UK). Methanol, acetic acid, potassium chloride, formaldehyde and Giemsa were purchased from Merck (Darmstadt, Germany). Heparin was obtained from Roche. Fetal Methylmercury chloride was previously diluted in distilled water and completed with RPMI 1640 to obtain the nal concentrations of 1500 g/l (or 0.0042 M). Forty eight hours after the beginning of the cultures of blood lymphocytes (belonging to the healthy volunteers from Belm), they were incubated with the different concentrations of methylmercury for 24 h at 37 C.

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2.6. Cytokinesis-block micronucleus assay For micronuclei analysis, cytochalasin B (6 g/ml) was added to the cultures 24 h before completing 72 h of incubation (Fenech and Morley, 1985; Crespo-Lpez et al., 2007). After centrifugation (1000 rpm for 5 min), cells were treated with 0.075 M KCl and 40% formaldehyde for 5 min. Then they were xed with acetic acid: methanol (1:5) solution and stained for 5 min with 5% Giemsa. A minimum of 500 binucleated cells or 500 cells (mononucleated, binucleated and metaphases) were counted for each group to analyze each parameter. Micronuclei and nucleoplasmic bridges were identied according to established criteria (Fenech, 2000; Fenech et al., 2003; Thomas et al., 2003). Total number of cells (N), number of binucleated cells (B), number of cells with one, two, three and four micronuclei, number of cells in metaphase (M) and number of cells with nucleoplasmic bridges were scored. Results were expressed as binucleation index (B/N), frequency of micronucleated cells, number of micronuclei per micronucleated cell (micronucleation index), fraction of cells containing nucleoplasmic bridges (index of nucleoplasmic bridges) and proportion of metaphases (M/N, mitotic index) (Crespo-Lpez et al., 2007). 2.7. Detection of chromosomal aberrations For detection of chromosomal aberrations, demecolcine (10 g/ml) was added to the lymphocytes cultures, 2 h before completing 72 h of incubation (Sram et al., 1998; Lazutka et al., 1999). Then, cells were harvested by centrifugation at 1000 rpm for 5 min, and treated for 10 min with KCl 0.075 M. After that, they were xed with acetic acid: methanol solution (1:3) and stained for 5 min with 5% Giemsa. One hundred metaphases per culture were observed for analysis of chromosomal abnormalities (gaps, breaks and other deformations). In addition, ve hundred cells were scored to analyze the proportion of metaphases (mitotic index, M/N). 2.8. Statistical analysis A one-way analysis of variance (ANOVA), followed by a Student NewmanKeuls post hoc test when appropriated, was used to compare average values between groups. A P value below 0.05 was considered to be statistically signicant. Pearson's correlation was also used to verify the association between variables.
3. Results 3.1. Genotoxic effects of in vitro exposure of human lymphocytes to methylmercury detected with the cytokinesis-block micronucleus assay The presence of micronuclei was detected when human lymphocytes were contaminated with 1, 10 and 50 g/l of methylmercury, but the number of micronucleated cells was very low (frequency of micronucleated cells was below 0.3 in all cases). The micronucleation index was 1 for these cultures, meaning that those micronucleated cells only contained one micronucleus. Furthermore, we detected cells with nucleoplasmic bridges in two donors, but, again, the number of cells with these changes was relatively low (as low as 0.001) and it was not correlated with the methylmercury concentration (p N 0.05). The values of binucleation index (BI) were variable between different donors: for example, in lymphocytes cultures of donors 1 and 2, the highest BIs (up to 0.19) were detected when cells were exposed to 1 g/l of methylmercury; however, for donor 3, the highest BI (0.113) was registered with 500 g/l. BI results showed no statistically signicant difference between the different concentrations of methylmercury (Fig. 2). 3.2. Genotoxic effects of in vitro exposure of human lymphocytes to methylmercury detected with the chromosomal aberrations assay No chromosomal aberrations were detected, however, results of mitotic index (MI) showed that exposure to concentrations N 1 g/l of methylmercury increased signicantly MI values except when 10 g/l was used (Fig. 3). This signicant increase in the MI may indicate changes in the process of cell division, when compared with the control, enhancing cellular proliferation. 3.3. Selection of the control population for mercury exposure in the Amazon area and application of the technique of detection of chromosomal aberrations In vitro experiments with lymphocytes treated with methylmercury showed that the most sensitive technique was the detection of chromosomal aberrations as previously discussed. So, this method was selected to be applied in an Amazonian population that will serve as control for epidemiological studies of mercury genotoxicity. The population selected was the community of Panacauera (Fig. 1), having very similar characteristics to exposed populations of the Tapajos River, but without mercury exposure. We carried out an expedition to that community where blood samples were collected from 30 volunteer donors. Distribution according to age and sex and results of mitotic index obtained for each individual are shown in Fig. 4. No chromosomal aberrations were detected in any of the cultures of these donors. Mean mitotic index and SEM for the entire population were 0.0814 0.0097. Due to the relatively higher proportion of people over 35 years (Fig. 4), we tested the Pearson correlation between age and mitotic index for this population and a nonsignicant correlation was observed (r = 0.0872; p = 0.685). Therefore, age showed no inuence in the MI values.

Fig. 2. Binucleation index (number of binucleated cells/total number of cells) of cultures of human blood lymphocytes exposed to methylmercury for 24 hours. Results, found with cytokinesis-block micronucleus assay, are expressed as mean SEM (n = 5).

To avoid any inuence of other factors that were not due to mercury intoxication, Pearsons correlation was also tested for the age of each donor and the MI values. Nonsignicant correlations were found between age and MI for all treatments (r = 0.466 to 0.379; p = 0.429 to 0.529). Therefore, age can be considered as a factor that did not directly inuence the MI values in our work.

4. Discussion Except at mining sites, where occupational exposure occurs by direct inhalation of mercury vapor, the type of mercury exposure more common in the Amazon is through the diet by eating sh contaminated with methylmercury (Passos and Mergler, 2008; Berzas-Nevado et al., 2010). In the Tapajs River basin (Fig. 1), for example, such exposure caused high levels of intoxication in riverside

Fig. 3. Mitotic index of cultures of human lymphocytes exposed to methylmercury found with the chromosomal aberrations assay. Data are expressed as mean SEM (n = 5). **P b 0.01 and ***P b 0.001 vs control group.

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Fig. 4. Population distributions (left) according to age (up) and sex (down) and values of individual mitotic index for each volunteer (right) of Panacauera.

communities in the past (de S et al., 2006; Berzas-Nevado et al., 2010). Currently, these levels are lower but still above the limit given by WHO (1990) (see Berzas-Nevado et al., 2010 for a review). Most recent studies carried out in these populations show levels of mercury in blood 50 g/l (and above 10 g/g in hair) as mean (Lemire et al., 2006; Passos et al., 2008; Grotto et al., 2010). Thus, this study used relatively low concentrations of methylmercury (1500 g/l or 0,0042 M) to test the methods to detect genotoxicity. The main conclusion with the cytokinesis-block micronucleus assay is that it does not seem to be sensitive enough to register any change caused by this range of methylmercury concentrations (micronuclei and nucleoplasmic bridges were found in very few cases). Also, with the chromosomal aberrations assay, chromosome abnormalities were not detected in exposed lymphocytes. However, a very signicant increase of the mitotic index (MI) was shown in exposed cells (Fig. 3). Exposure to 10 g/l of methylmercury did not signicantly alter the MI, but it produced a tendency to increase the MI when compared to the control group (Fig. 3). A higher number of donors will be necessary to conrm this tendency. Precisely, the mitotic index is a parameter commonly used to identify the genotoxic effects of mercury and its compounds (see Crespo-Lpez et al., 2009 for a review). Because its sensitivity and the simple methodology, both in vitro and epidemiological studies pointed to the MI as a key-parameter for the analysis of genotoxicity of mercury (Amorim et al., 2000; Bahia et al., 2004; Silva-Pereira et al., 2005; Crespo-Lpez et al., 2007). The value of the MI indicates changes in cell proliferation, in other words, changes during the cell division. An increased MI in human blood lymphocytes exposed to low levels of methylmercury may indicate an enhanced cell proliferation and a potential to trigger carcinogenic processes. Similar results were also found in cultures of human neuroblastoma and glioblastoma exposed to 01 M of methylmercury (Crespo-Lpez et al., 2007). Our results also support the idea that this parameter would be the most sensitive for the detection of genotoxicity caused by methylmercury and they point to the method of detecting chromosomal aberrations as the most appropriate.

These differences in the sensitivity of detection of mercury genotoxicity using different assays were also reported by other authors in the past (see Crespo-Lpez et al., 2009 for a review). In these studies, methylmercury outstands as the more genotoxic compound studied in vitro, so far (Betti et al., 1992; Ogura et al., 1996; Lee et al., 1999; Silva-Pereira et al., 2005). Among all the methods used in these latter studies, the assay for detecting chromosomal aberrations was the most sensitive, as demonstrated in our work. In spite of the variability of the results, most studies show genotoxic changes only with the exposure to higher concentrations than those used in our work. This fact would explain the lack of detection of micronuclei and chromosomal aberrations in our results. Until now, there is only other in vitro study performed with human lymphocytes exposed to such low concentrations of methylmercury (Silva-Pereira et al., 2005). Interestingly, in this latter study, only exposure to 100 g/l methylmercury caused signicant variations in mitotic index. Thus, our results seem more consistent with in vivo studies conducted in the Amazonian populations (Amorim et al., 2000; Bahia et al., 2004), where signicant variations of MI were shown, even when the detection of chromosomal aberrations was not evident. Currently, there are some hypotheses to explain the molecular mechanism of genotoxicity caused by exposure to mercury compounds. These assumptions include the direct link of mercury to DNA, the production of free radicals and oxidative stress, the inhibition of spindle formation (by the action of mercury in microtubules), and the alterations of the mechanism of DNA repair (see Crespo-Lpez et al., 2009 for a review).The four hypotheses are not mutually exclusive, so it is likely that mercury can trigger all these mechanisms (and perhaps others unknown) to produce genotoxicity in exposed populations. Earlier studies also demonstrate that the tolerance limits for human exposure to mercury should be reassessed. For this, it is essential to develop solid epidemiological studies aimed at establishing the state of genotoxicity of populations. Thus, our contribution to this problem was to determine what level of alterations in the parameters of genotoxicity would be considered as normal in the Amazonian populations to eliminate interferences and support the studies carried out in this region. The

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groups at Panacauera were higher than those of Brasilia Legal showing a trend that became statistically signicant for groups 4554 years and for men (Fig. 5). Thus, this mitotic index of Brasilia Legal, lower than that of Panacauera, would indicate an inhibition of cell cycle progression and/or a loss of proliferative capacity caused by mercury intoxication (Amorim et al., 2000; Silva-Pereira et al., 2005). However, a higher number of participants will be necessary to conrm this hypothesis. The latter conclusion seems to contradict previous in vitro results where we found an increase in mitotic index caused by mercury (Crespo-Lpez et al., 2007; this study). However, the doses of methylmercury used in this study and the study of Crespo-Lpez et al. (2007) were very low. Our hypothesis is that the mitotic index is one of the rst parameters to be affected by mercury, and that low doses of mercury increase cell proliferation, but relatively higher doses (or chronic intoxication for longer times) inhibit proliferation and/or produce cell death. These results support the idea that the mitotic index could serve as a key-parameter for the early diagnosis of the damage caused by mercury exposure, contributing, by this way, to the knowledge about the epidemiological consequences of chronic mercury intoxication of the Amazon populations. Funding sources This work was supported by the FAPESPA Research Grant No. 092/ 2008. JLM do Nascimento, GL Macdo and GPF Arrifano thank CNPq and CAPES for their grants. References
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Fig. 5. Mitotic index (mean SD) of populations of Panacauera (results of this study) and Brasilia Legal (results of Amorim et al., 2000) grouped by sex (up) and range of age (down). *p b 0.05 vs the same group of Brasilia Legal.

population of Panacauera (Fig. 1) was selected due to its characteristics, very similar to those of the riverside populations of Tapajos. Panacauera has been monitored for many years and it has been already used as control population in comparative studies (Pinheiro et al., 2006, 2007, 2008). Mean mercury levels in hair of inhabitants of Panacauera always were 23 g/g (Pinheiro et al., 2006, 2007, 2008). Unfortunately, there are only two in vivo studies performed in riverside populations of the Amazon about mercury genotoxicity (Amorim et al., 2000; Bahia et al., 2004). Both studies used the method to detect chromosomal aberrations in blood lymphocytes of the population of Brasilia Legal (Fig. 1) (exposed to methylmercury through diet). The rst study supports the hypothesis of a genotoxic activity associated to mercury compounds detecting polyploidy and a decrease in the mitotic index as major changes (Amorim et al., 2000). Later, the same population was analyzed again to conrm those previous results but no signicant relationship between mitotic index and total mercury levels in hair was found (Bahia et al., 2004). Although this fact was justied by a decrease in mercury levels since 1995, it could be also due to the reduced number of individuals included in the latter study (as low as 26) when compared to that of the rst one (n = 94). Nevertheless, the attempt of an association between both factors (level of mercury in hair and mitotic index in lymphocytes) may be moot since the mercury in hair refers to a relatively recent exposure and the mitotic index may reect accumulated changes over the years. So, eventually it would be possible to detect changes in cell proliferation without necessarily a correlation with the levels of mercury intoxication of the last months. This could be an alternative explanation for the results of Bahia et al. (2004). Thus, the only way to clarify whether there are changes caused by mercury is through the use of a control population with low or no mercury exposure. This is the case of community Panacauera. For a better analysis, data of mitotic index of Panacauera were grouped by sex or age (Fig. 5) and compared with the results of Brasilia Legal (Amorim et al., 2000). The mean mitotic indices of all population

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