ABSENCE OF GENOTOXIC EFFECT OF MERCURY VAPOR IN LYMPHOCYTES OF OCCUPATIONALLY EXPOSED INDIVIDUALS

Objective : to evaluate the genotoxic effects caused by exposure to Hg vapor in individuals with occupational exposure in a municipality in northern Brazil. Methods : Blood samples were collected from 70 individuals (38 exposed and 32 unexposed controls) in heparinized tubes. The comet assay and the micronucleus test were used to detect the occurrence of genotoxic effects. The results were statistically analyzed using the Shapiro-Wilk normality test, Student's T test and ANOVA. Non-parametric data were analyzed using the Mann-Whitney and Kruskal-Wallis tests. Spearman's test was used to assess the correlation between dependent variables. Mean, geometric mean and standard deviation (SD) were calculated. Results and Conclusions : The evaluation of genotoxicity revealed no difference in the frequency of DNA damage when the two groups, control and exposed, were compared, even considering confounding variables such as smoking and alcoholism, gender, age, time of exposure. In conclusion, our results indicate that exposure to Hg vapor (elemental form) did not produce detectable genotoxic changes in blood cells in vivo. Research Implications : Although no genotoxic effects were detected in blood cells from individuals exposed to mercury vapor, studies with cells that are in more direct contact with this source of contamination, such as oral and nasal epithelial cells, should be performed to confirm whether elemental form of Hg has no genotoxic effect.


INTRODUCTION
Mercury (Hg) is considered a pollutant on a global scale, and although its best-known effects are related to its neurotoxic properties, there is evidence that this metal also causes damage to genetic material (HACON et al., 2020). This property is known as genotoxicity (SOUZA et al., 2022).
Due to the great versatility of this metal, provided by its physical-chemical properties, it is applied in several productive activities. Among those that involve Hg directly and indirectly, gold mining is the one that causes great worldwide concern regarding epidemiology and environmental health, because a massive amount of this metal is used to recover the gold present in the rock and, later, it is released into the environment during the gold purification (MASON et al., 2012).
Considering the existence of gold mining activity in the Amazon region, largely by illegal enterprises, there are many individuals at risk, as they are exposed throughout the gold extraction process, including the end of the beneficiation chain, the gold purchase and sale stores (CRESPO-LOPEZ et al., 2021). In this sense, it is important to analyze possible genotoxic effects caused by the level of exposure to Hg vapor to which these individuals are exposed.

THEORETICAL REFERENCE
Mercury (Hg) is among the most deleterious contaminant in the aquatic environment, representing a serious risk both to humans and ecosystems (VIEIRA et al., 2009;TELAHIGUE et al., 2020). Human intoxication can occur in several ways: occupational exposure, inhalation of Hg 0 vapor or through the consumption of fish contaminated by MeHg, which is formed in river sediments and passes to the food chain through bioaccumulation and biomagnification (DELAI & TAKAHASHI, 2008;HACON et al., 2020). It can be found in all environmental compartments, in three main chemical forms: elemental mercury (Hg 0 ), oxidized mercury (Hg2+) and methylmercury (MeHg) (GWOREK et al., 2017). It is released into the environment from natural and anthropogenic processes. Due to its physical-chemical characteristics, it has several applications, including in medicine, dentistry and industrial sectors, such as lamps, batteries, chips, thermometers and measuring devices, among others. However, due to its ability to form metallic alloys with other metals, one of the main uses of Hg is in gold mining (MASON et al., 2012).
Despite many studies on the Hg cycle in the environment and its toxic manifestations in humans, there are still little information about events underlying the clinical manifestation of exposure to this metal (AZEVEDO et al., 2012;De FLORA et al., 1994;ELTAIB et al., 2019;KHOURY et al., 2013;OBRIST et al., 2018;ELTAIB et al., 2019). It is known that mercury compounds are considered genotoxic (De FLORA et al., 1994;SOUZA et al., 2022), and this property may be related to four main processes: generation of free radicals and oxidative stress, action on microtubules, influence on DNA repair mechanisms and direct interaction with DNA molecules (CRESPO-LOPEZ et al., 2009, 2016SOUZA et al., 2022).
Since the 1970s, the presence of gold mines using Hg as an extractive method is a common feature in the Amazon region (CRESPO-LOPEZ et al., 2021). Considering that one of the forms of chronic contamination with Hg is occupational, it is possible that exposed individuals present a higher occurrence of DNA damage, which is usually related to carcinogenic processes (LADEIRA AND SMAJDOVA, 2017). Thus, this study aimed to evaluate possible genotoxic changes caused by exposure to Hg vapor in workers from gold trading stores in the municipality in Northern Brazil.

METHODS
This is a secctional study with a set of 70 subjects (38 occupationally exposed to Hg 0 and 32 non-exposed individuals), selected through convenience method. The exposed group was selected due their work in gold trade shops means, where have contact during the workday with Hg 0 . Controls were chosen from the same area, considering individuals without registry of activities dealing with gold or Hg 0 . The individuals were matched by age, gender, exposure time, smoking and drinking habits. The population features are described in Table 01. Health condition (including medical history, diagnostics X-ray, medication, chronic diseases), lifestyle and other relevant aspects related to both their daily life and work activities were assessed by a questionnaire. None of these individuals reported excessive exposure to x-rays nor the presence of chronic diseases. All subjects were fully informed about this query and signed a inform consent form before the study. Ethical approval for this study was obtained from the Instituto Evandro Chagas Ethical Comitee, under the number 3.316.689 (May 09, 2019).

Biological Samples
Urine samples (50 ml) were collected before starting the workday, in clean polyetilene bottles, and kept frozen until the Laboratory analysis.

Quantification of Hg Content in Urine (U-Hg)
Concentration of Hg-U it was assessed with the aim of a Mercury Analyser Hg-201 Sanso Seisakusho, co., and Cold Vapor Atomic Absortion Spectrometry aparattus. The samples were processed as described by the mercury analysis manual (MINISTRY OF THE ENVIRONMENT JAPAN, 2004). Briefly, the urine was thawed and mixed, and an aliquot of 2 mL was transferred for a 50 mL long neck volumetric balloon for the digestion process, containing a solution prepared with 2 mL of nitric 65% (Sigma-Aldrich) and percloric 70% (Sigma-Aldrich) acid (1:1) and 5 mL of sulfuric ácid 98 % (GFS Gruop -Double destilled, Vycor). The digestion was completed placing the balloon in a hot plate in a temperature ranging 200 -230 °C for 30 minutes. After cooling, ballons were fulfilled with deionised water (Milli-Q Millipore).

Comet Assay
We followed standard protocol originally described by Singh et al (1988) with modifications. For each individual sample, an amount of 30 µL of whole blood was mixed with 110 µL of Low Melting Pont Agarose (LMA) 0,6%, heated to 36 °C and then placed in a frosted side slide previously pre-coated with Normal Melting Point Agarose (NMA) 1,5% layer. Afterwards, a coverslip was put on the slide to flatten and spread the material, and the slide was kept at 4 o C for 20 min in 4 °C. After gently removing the coverslip, the slide was submersed in lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, pH 10, 4°C) for 48 h. and equilibrated for 20 min in a jar containing alkaline buffer (300 mM NaOH, 1 mM EDTA, pH > 13, 4°C). Finally, slides were transferred to an electrophoresis unit with alkaline buffer and subjected to an electric field of 30 v and 300 mA, equivalent to 1 v/cm at 4°C. Following electrophoresis, the material was neutralized in 0.4 M Tris (pH 7.5), rinsed with water, dehydrated in 100% ethanol for 2 min and allowed to dry at room temperature. The DNA was stained with Pro-Long gold antifade reagent with DAPI (Invitrogen, OR, USA), which also prevented slides fading and drying out. The analysis considered 100 random nucleoids, which were photographed using a Fluorescence microscope (Imager II Zeiss) ,40X objetive.

Micronucleus Test
We followed the protocol proposed by Melo et al. (2008), with modifications. Blood smears were prepared in pre-coded slides. Then, they were allowed to dry at room temperature, and fixed with Absolut Ethanol 99,9% (Merck®) for 10 minutes and stained by Giemsa (5%) for 3 minutes. Slides were washed, dried at room temperature, and analyzed through an optical light microscope with 100X objective to assess the frequency of MN. For each sample, 200 cells were registered. As quality control, experiment was performed in duplicate, and the frequency of MN was the slides were duplicate prepared and the frequency of MN was blinding analyzed.

Statistical Analyses
The data analysis was carried out using Biostat 5.3 software (Mamirauá Institute, mamiraua.org.br/downloads/programas/). The Shapiro-Wilk normality test was applied to the data. T-Student test was used for parametric data for two groups; ANOVA was used for three or more groups. Non-parametric data was analyzed by the Mann-Whitney test considering two groups, while Kruskal-Wallis test was performed for comparisons of three or more groups. Spearman Correlation was used to evaluate correlation between dependent variables. Mean, geometric mean, and standard deviation (SD) were calculated.
The comet data were analyzed using the Komet J (Open Comet Sofware, cometbio.org/download_links.html). The data generated by this softwere were originally nonparametric, then they were log 10 + 1 converted. It was added a constant qual to "one" to all MN frequency in order to avoid the effect of excess of "zero" values. The study population homogeneity was assessed by Chi-Square test (X 2 ). It was also verified the non-parametric correlation between Hg-U, % DNA breakage and MN frequency. Differences under p > 0.05 were considered statistically significant.

RESULTS AND DISCUSSION
Epidemiologic data from the sampled individuals are shown in Table 01 and included both Exposed and non-exposed (control) individuals. Males were the majority (76%). Ages were 32 ± 8,8 and 34 ± 4,7 years (means ± S.D.) for exposed and non-exposed, respectively. They may be considered as non-smokers since only a few individuals had related this habit. On the other hand, alcohol consumption was related as regular habit. More than a half of the exposed participants had been working for few years on that activity. The assessment of U-Hg of the study population revealed that exposed individuals were almost 7-fold above the level observed in non-exposed, considering the geometric mean, as shown in table 02. Table 02 -Descriptive assessment of U-Hg exposure between the exposed and non-exposed groups.
Group N Parameter Geometric Mean Mean S.D. Min -Max In gold trading shops, workers are continuously exposed to variable levels of Hg, even though this business represent the final step of a gold mining chain, each gold burn during the trade can still emit 2 to 5% of Hg vapor (MELO et al., 2008;WHO, 2014). According to Hg-U levels, we found concentrations 7-fold higher in the exposed individuals compared to nonexposed ones, revealing a potential health risk that this occupational scenario might inflict to them, and showing the relevance of monitoring studies.
Acute exposition to Hg vapor from heating mercury-gold amalgam is related to paroxysmal cough, dyspnea, chest pain, tachypnea, nausea, vomiting, fever and leukocytosis as initial effects, followed by air-flow obstruction and diffusing capacity of the lung for carbon monoxide (ABBAS et al., 2017). However, none of these clinical features was reported by the sampled individuals.
In accordance with ACGIH, the Biological Exposure Indice (BEI) for occupational limits is under 20 µg/g creatinine (LAUTHARTTE et al., 2018), meaning that values below this level are most unlikely to experience noticeble adverse effects. However, although the majority of participants of this study presented concentrations under this limit, a quarter of them had concentration above it.
Results concerning U-Hg values in exposed individuals are still controversial. Similar to our results, previous research had similar results in the same study area, showed a frequency of 22% individuals having U-Hg above the permissible limits (LAUTHARTTE et al., 2018). In the same ways, data from gold-mining workers in Ecuador, showed a proportion of 15,5% individuals above BEI's limit (ROSA et al., 2000). Nonetheless, studies in Asian showed that almost 98% of miners and residents near mining area exhibit U-Hg levels above the safe occupational limits. It resembles us that mining is the front-line activity in the gold chain extraction including Hg handling, and for this reason exposed individuals must be monitored in order to understand the level of intoxication in different segments of workers related to this process.
Despite the fact that Hg was related as a genotoxic agent (SOUZA et al., 2022), genotoxicity studies on Hg exposure remain scarce, and most of them are related to organic mercury (CRESPO-LOPEZ et al., 2009). In the present study, we applied both CA and MN, considering that the combination of these assays improves the capability for detecting DNA damage, as they have differences in sensitivity (ELTAIB et al., 2019). Statistical analysis of comet assay (CA) and micronucleus frequency (MN) showed no significant difference between exposed and non-exposed, according to table 03. ANOVA teste showed no statistically significant differences when Hg level was considered as a factor for increasing DNA damage percentage assessed by the comet assay, as shown in table 04. The same results were obtained when analyzing the results of MN by Kruskal-Wallis test.
Considering MN frequency and comet results in comparison with the levels of Hg exposure obtained from the exposed group, it was observed that most of them (68,4%) were categorized with medium and high exposure levels, 21% of which were beyond the maximum limit established for occupational exposure (Table 04). When the confounding variables (age, gender and exposure time) were tested to compare endpoint effects, it could be noticed that neither age, nor exposure time exhibited differences within the groups for both assays (CA and MN). In relation to gender, this variable did not represent any effect to CA endpoints in this study. Nonetheless, statistically significant differences were obtained between female gender, control males and exposed females, when MN frequency was considered (table 05). 8 20 a 30 5 93,58 ± 1,37 6,47 ± 1,34 3,31 ± 1,6 -1,5 ± 0,57 * Significant difference between genders, p < 0.05. ° Significant difference of females between non exposed and exposed group, p < 0.05.
Despite the fact that this study had simulated six categories to analyze further possible confounding variation related to alcohol and cigarette consumption -WA (Without Addiction), NED (Non-Exposed Drinkers), EXD (Exposed Drinkers), NES (Non-Exposed Smokers), EXS (Exposed Smokers) and ESD (Exposed, Smokers and Drinkers) -no significant differences were observed between these categories, revealing this result had not been critical for this approach, and details are shown in table 06.  Nonetheless, despite of this, no statistically significant differences were observed between exposed and non-exposed individuals, even considering individuals with higher U-Hg. Since we have used blood cells, a possible explanation to this scenario is the fact that mercury can be quickly removed from the blood, redistributed and sequestered into different tissues, resulting in a lack of direct correlation between blood mercury concentration and the severity of mercury poisoning (BHOWMIK AND PATRA, 2015). However, CA in individuals exposed to amalgam dental restorations (presence of Hg vapor) showed damage to the genetic material directly proportional to the variables age, quantity of restorations and also in relation to the CA parameters -percentage of DNA in the tail and Tail Moment (SILVA-PEREIRA et al., 2005).
It could be argued that the fact that our sample was processed after being kept fronzen at -80 o C may also have increased the frequency of DNA damage, thus decreasing the differences between exposed control and its statistical significance, despite studies of optimization of protocols for DNA damage by CA demonstrate that the use of frozen whole blood samples (-80oC) does not cause harm that may prove to be statistically significant ( (AL-SALMANI et al., 2011;KOPPEN et al., 2018). Thus, the non-concordance between the studies may be related to methodological and sampling differences.

CONCLUSION
The evaluation of genotoxicity revealed no evidence of DNA damage in blood cells from individuals exposed to mercury vapor (elemental form). Despite of this, further studies using cells that are more directly in contact with this source of contamination, such as buccal and nasal epithelial cells, should be performed to confirm if this inorganic form of Hg has any genotoxic potential.