Keywords
Mercury; GSTM1 polymorphism; GSTT1 polymorphism; Environmental Health; Amazon
Abstract
Mercury (Hg) is considered one of the most dangerous toxic metals to the environment and to human. High Hg level may induce different types of toxic effects on human health and the main target is the Central Nervous System (CNS). One of the main mechanisms involved in neurotoxicity caused by Hg is the oxidative stress and its effect on antioxidant systems. Currently it is known that deforestation is one of the main reasons for the high levels of Hg in the aquatic ecosystems in Amazon. However, to the best of our knowledge, there are no recent investigations that evaluate the Hg exposure in a human population of the Low Amazon region. The present work aims to fill the gap of the lack of information related to how the urban and floodplain populations of Santarém city (Pará state, Brazilian Amazon) is affected by Hg exposure, also investigating the GSTM1 and GSTT1 polymorphisms for the population studied. Our results show that the individuals studied are environmentally exposed to Hg due to the fish intake. Additionally, the analysis of our data suggests that fish intake, age, and sex are more relevant for prediction of Hg level in blood than the polymorphisms of GSTM1 and GSTT1 genes.
Introduction
Mercury (Hg) is one of the most dangerous heavy metal due to its high toxicity, bioaccumulative properties and genotoxic potential, with harmful effects for the environment and for human health [1]. Hg is released in the environment from natural sources and/or anthropic activities and circulates between the atmosphere and aquatic and terrestrial ecosystems, potentially reaching humans [2].
In the Tapajós River, the human exposition to the Hg has been strongly related to the consumption of contaminated fishes [3]. The presence of Hg in fishes and humans was firstly reported in the Amazon in the early 1990 [4]. Since then, many of the data on the cumulative Hg levels in fish and human populations in this region focus on gold digging activity (garimpo) as the main responsible for the Hg environmental contamination [5], although less than 3% of the Hg deposited in Amazonian soils comes from the garimpo [6].
Currently, a major contributor to the maintenance of high levels of Hg in the Tapajós River comes from the illegal deforestation caused by the expansion of the agricultural border [7]. The conversion of forest soil in pasture soil through the burning increases the remobilization of Hg accumulated [8] and intensifies the erosion process that accelerates the transport of Hg for aquatic environments [9]. Once there, it can be biotransformed into methylmercury (MeHg), the most toxic Hg compound to humans. The MeHg is neurotoxic and crosses the placental and the bloodbrain barriers and may result in different types of fetal abnormalities (neurological and motor) [10]. Furthermore, low levels of MeHg are sufficient to cause adverse toxic effects in adults and infants [11].
One of the key mechanisms involved in neurotoxicity caused by MeHg is oxidative stress and its effect on antioxidant systems [12] especially the Glutathione (GSH) [13], an intracellular antioxidant that reacts with MeHg forming the MeHg-glutathione complex, an essential step in Hg detoxification. Glutathione S-Transferase (GST) is a family of highly polymorphic enzymes that catalyze the MeHgglutathione complex conjugation reaction or may act the transporters of MeHg-glutathione complexes [14].
Recent epidemiological studies link polymorphism alteration of GST genes to Hg levels [15-18]. GSTM1 and GSTT1 deletions were associated with high levels of Hg in Amazon riverine [19] and indigenous [20] populations. Thus, the aim of the present work is to investigate the environmental exposure to Hg in the urban and floodplain populations of Santarém city (Low Amazon region) evaluating the total Hg level, in association to factors such as age, sex, and the frequency of fish intake, as well as the polymorphisms of oxidative stress-related genes (GSTM1 and GSTT1).
Methodology
An observational, descriptive and quantitative study was conducted. The study enrolled 98 subjects of both sexes, aged between 18 and 81 years living in the Santarém urban area (n = 63), located at the confluence of Tapajos and Amazonas rivers and in the Santarém várzea area (n=35), located in the floodplain on opposite banks of the Amazon river. Both groups are formed by residents of the Santarém municipality region, which belongs to the middle region of the low Amazon.
The study was approved by the Ethics Committee of Pará State University (UEPA) (technical report no. 1,127,108). All participants were previously informed about the objectives of the study and a health/lifestyle questionnaire after the signature of a consent term. Blood samples were collected in tubes containing EDTA and total Hg blood concentration was determined by flameless atomic absorption spectrometry in DMA-80 Direct Mercury Analyzer (Milestone) equipment, according to the manufacturer’s instructions, and for each sample, the analyses were made in duplicates. According to WHO, those individuals who have Hg levels above 10μg/L were considered exposed. Genomic DNA was extracted from blood samples of all subjects using the QIAamp DNA Blood Mini Kit (Qiagen), according to the manufacturer’s instructions and stored at -20 ° C until analysis. Genotyping was performed by PCR multiplex for GSTM1 and GSTT1 genes.
The PCR was carried out in a total volume of 20 μL, containing 3 μL of DNA template (20-50ng), 1 μL of each primers (10mM), 10 μL of PCR Master Mix (Promega) (50 units/mL of Taq DNA Polymerase supplied in a proprietary reaction buffer (pH 8.5), 400 μM each dNTP, 3 μM MgCl2) and 1 μL of Nuclease-free water. The primers used were GSTM1 Forward 5’-GAACTCCCTGAAAAGCTAAAGC-3’ and Reverse 5’-GTTGGGCTCAAATATACGGTGG-3’ and the GSTT1 primers Forward 5’- TCACCGGATCATGGCCAGCA-3’ and Reverse 5’-TTCCTTACTGGTCCTCACATCTC-3’. As an internal control β-globin gene was co-amplified using the primers forward 5’-GAAGAGCCAAGGACAGGTAC-3’ (GH20) and Reverse 5’-CAACTTCATCCACGTTCACC-3’ (PC04). The reaction was incubated at 94 ° C for 5 min, prior to 35 cycles of denaturation of 1 min at 94 ° C, annealing of 1 min at 65 ° C, and extension of 1 min at 72 ° C, followed by a final extension of 7 min at 72 ° C. The PCR products were visualized by electrophoresis in 3% Agarose gel. The internal control fragment amplified from β-globin was 268 bp of length and the normal (presence) or deletion (absence) of GSTM1 and GSTT1 genes was detected by 215 and 459 bp fragments, respectively. The contributions of the present work are twofold.
First, the relationship between levels of Hg and frequency of fish intake was investigated using the Kruskal-Wallis test, in order to verify if individuals with higher frequency of fish intake have higher Hg levels. Second, a multiple regression analysis was performed in order to investigate the effects on Hg levels due to the variables age, fish intake, sex, and presence of GSTM1 and GSTT1 polymorphisms. In relation to fish consumption, the sample was stratified into two subgroups: the high-frequency subgroup, with a consumption of fish daily or weekly; and the low frequency subgroup with a biweekly or monthly consumption. Statistical tests were performed in STATA 7.0 software, with a 5% significance level. The graph was constructed using ORIGIN 8.0 software.
Results
The study included 98 individuals of which 63 (64.3%) live in urban area and 35 (35.7%) in floodplain area of Santarém. Among them, 69.4% were female and 30.6% male, most aging between 21 and 40 years (59.8%). The average levels of total Hg were measured in the blood of the 98 subjects studied and it was observed that 54.1% had levels above the recommended by the WHO.
Table 1: Health/Lifestyle Characteristics and Hg levels of the individuals analyzed.
Table 1 shows the descriptive statistics about Hg level in both health and lifestyle of the analyzed subgroups. It was verified the GSTM1 and GSTT1 genotypes frequencies in the 98 individuals studied (Table 2). The genotypes of 95 individuals for the GSTM1 polymorphism were obtained successfully, among them 69.5% were normal and 30.5% were deletion. Regarding the GSTT1 polymorphism, 98 genotypes were obtained successfully, of which 84.7% were normal and 15.3% were deletion.
Table 2: GSTM1 and GSTT1 polymorphisms genotypes frequencies of the individuals analyzed.
The Pearson ChiSquare statistics as well as the p-values reported in Table 2 indicate that the hypothesis of independence between the table variables cannot be rejected, therefore with no significant association between them. In order to verify the relationship between Hg levels and fish intake, the sample was stratified into two groups, namely: high frequency (n = 66), with a daily or weekly intake; and low frequency (n = 32) with a biweekly or monthly intake. Kruskal-Wallis test was used to compare the total Hg levels among subgroups of high and low frequency. The results demonstrated that individuals of the high frequency subgroup often have total Hg levels higher compared to the low frequency subgroup (Kruskal-Wallis test, χ2 = 30.167, p = 0.0001) (see Figure 1. The asterisk in the figure corresponds to an outlier for the high frequency subgroup).
Figure 1: Relationship between Hg levels and frequency of fish intake.
Tables 3 and 4 exhibit Hg levels for GSTM1 and GSTT1 polymorphisms respectively. Regarding the GSTM1 polymorphism, most individuals of both high and low frequency subgroups of fish intake have the normal gene, with mean Hg levels of 18.2 μg/L and 7.5 μg/L, respectively. The hypothesis of independence of variables cannot be rejected for the individuals studied, therefore with no significant association between GSTM1 polymorphism genotypes and Hg level in high (χ2 = 1.87, p = 0.17) and low frequency (χ2 = 0.13, p = 0.71) subgroups.
Table 3: Hg levels and GSTM1 polymorphism genotypes in the individuals analyzed.
Regarding the GSTT1 polymorphism, in both subgroups the normal gene was more frequent, with mean levels of total Hg 17.6 μg/L in the high frequency subgroup and 7.3 μg/L, the low frequency subgroup. Again, the hypothesis of independence of variables cannot be rejected for the individuals studied, therefore with no significant association between GSTT1 polymorphism genotypes and Hg level in (χ2 = 0.0005, p = 0.98) and low frequency (χ2 = 0.012, p = 0.91) subgroups.
Table 4: Hg levels and GSTT1 polymorphism genotypes in the individuals analyzed.
A multiple linear regressions was run to predict Hg level from the variables fish intake, age, sex, presence of GSTM1 polymorphism, and presence of GSTT1 polymorphism. Table 5 exhibits both univariate and multivariate regression coefficients.
Discussion
relationship between exposure and their respective toxic effects and thus characterize the risk to human and environmental health [2]. The assessment of the risks to human health from exposure to Hg in the Amazon region has focused mainly on enviromental factors [21]. However, it has been shown that genetic factors may influence toxicokinetics and/or toxicodynamics of Hg [22].
Therefore, the present study evaluated the total Hg levels in the blood of individuals living in Santarém in order to investigate the association with the consumption of fish and/or the absence of the GSTM1 and GSTT1 polymorphisms involved in the MeHg-glutathione complex conjugation reaction and/or transporter. Different studies have been performed with riverine populations of the Tapajós River to assess whether the Hg levels are associated with frequent fish intake [21].
In the present study, the average levels of total Hg found in the blood was 13.9 μg/L, which corroborates the hypothesis that the population of Santarém has shown Hg levels above the limit recommended by the WHO [23] over the years. Our results showed that subjects from the high frequency fish intake subgroup have higher levels of Hg compared to subgroup of low frequency (χ2 = 41.68; p = 0.0001), both in urban and floodplain areas. Then, we conclude that the population of Santarém is environmentally exposed to Hg poisoning through frequent fish intake. Regarding effects on the Hg level in blood, the variables fish intake, age, sex, GSTM1 polymorphism, and GSTT1 polymorphism (Table 5) statistically significantly predicted Hg level, F(5, 88) = 10.41, p < 0.0005, R2 = 0.3717. The variables fish intake, age and sex added statistically significantly to the prediction (p < 0.05), although the presence or deletion of GSTM1 or GSTT1 polymorphisms did not (p = 0.478 and p = 0.301, respectively).
Table 5: Univariate and multivariate regression coefficients for the response variable Hg level.
Conclusion
From the blood levels assessment of total Hg in the present study, it can be concluded that urban and floodplain populations of Santarém are environmentally exposed to Hg contamination through frequent fish intake. According to the analysis of our data, the fish intake, age and sex are more relevant for the Hg level in blood than genetic factors related to GSTM1 and GSTT1 polymorphisms. In a near future, the population of Santarém will be exposed to new environmental impacts of deforestation growth and installation of complex hydropower plants at Tapajós River. We highlight the need of further studies in order to determine which environmental factors (in addition to the frequent fish intake) may influence the toxicokinetics and/or toxicodynamics of Hg in humans, as well as genetic factors, in order to minimize damage of Hg exposure to human health.
Acknowledgment
We would like to thank to all the volunteers who participated in this work and to CAPES, CNPq and FAPESPA for financial support. We also would like to thank Dr. Troy Patrick Beldini and Dr. Celson Pantoja Lima for English language editing and checking, Christian Diniz Lima e Silva and Ellen Naiany Araújo de Freitas for the technical support during the blood samples collection, as well as the reviewers for the useful comments and suggestions.
References
1. World Health Organization (WHO). Environmental Health Criteria Document 101 - Methylmercury. Geneva: World Health Organization. 1990.
2. Wright DA, Welbourn P. Environmental Toxicology. Cambridge University Press, 2002.
3. Sampaio da Silva D, Lucotte M, Roulet M, Poirier H, Mergler D, Crossa M. Mercúrio nos peixes do rio Tapajós, Amazônia Brasileira. Revista de Gestão Integrada em Saúde do Trabalho e Meio Ambiente. 2006; 1: 1-31.
23. Passos CJ, Lucotte M, Queiroz A, Mergler D, Peleja R, Goch Y, et al. Condições sócio-econômicas e exposição ao mercúrio (Hg) através do consumo de peixe: um estudo de caso em Santarém, Pará, Brasil. Revista Saúde Ambiente. 2003; 6: 3-11.
Citation
Meneses HNM, Neves AC, Costa FAA, Oliveira RB, Meneses AAM, Rodrigues LRR, et al. Mercury Levels and Glutathione S-Transferase Polymorphisms Evaluation in a Population of the Low Amazon, Brazil. SM J Public Health Epidemiol. 2016; 2(2): 1027