Genotoxicity of Mercury and Its Derivatives Demonstrated In Vitro and In Vivo in Human Populations Studies. Systematic Review

Beside partial coverage in three reviews so far (1994, 2009, 2019), there is no review on genotoxic studies dealing with mercury (Hg) and human exposure using the most usual genotoxic assays: sister chromatid exchanges (SCE), chromosomal aberrations (CA), cytochalasin B blocked micronucleus assay (CBMN), and single-cell gel electrophoresis (SCGE or alkaline comet assay). Fifty years from the first Hg genotoxicity study and with the Minamata Convention in force, the genotoxic potential of Hg and its derivatives is still controversial. Considering these antecedents, we present this first systematic literature overview of genotoxic studies dealing with Hg and human exposure that used the standard genotoxic assays. To date, there is not sufficient evidence for Hg human carcinogen classification, so the new data collections can be of great help. A review was made of the studies available (those published before the end of October 2021 on PubMed or Web of Science in English or Spanish language) in the scientific literature dealing with genotoxic assays and human sample exposure ex vivo, in vivo, and in vitro. Results from a total of 66 articles selected are presented. Organic (o)Hg compounds were more toxic than inorganic and/or elemental ones, without ruling out that all represent a risk. The most studied inorganic (i)Hg compounds in populations exposed accidentally, occupationally, or iatrogenically, and/or in human cells, were Hg chloride and Hg nitrate and of the organic compounds, were methylmercury, thimerosal, methylmercury chloride, phenylmercuric acetate, and methylmercury hydroxide.


Introduction
Mercury (Hg) is a highly dangerous environmental pollutant, and many studies have evaluated the activity of Hg compounds in different test systems with a wide variety of biomarkers. Nevertheless, one that is striking is its possible genotoxic effect in human populations, even at low concentrations [1][2][3][4][5][6]. Some studies recognized mutagenic and teratogenic effects and reported that it can also induce cancer, with very scarce and controversial information about the mechanisms by which it induces such effects [7,8].
Hg can be found in air, water, and soil. Environmental Hg pollution is caused by natural phenomena (erosion, volcanic eruptions) and anthropogenic activities (metal smelt-allowable Hg amount and/or MeHg in fish to be sold on the market, with alimentary guideline levels from 0.2 mg MeHg/kg in non-predatory fish to 1 mg MeHg/kg in predatory fish [9,14]. Preliminary critical limits to prevent ecological effects due to Hg in organic soils have been set at 0.07-0.3 mg/kg for the total Hg soil content [14]. EPA's reference MeHg dose is 0.1 µg/kg body weight per day according to the EPA's Water Quality Criterion for the Protection of Human Health: Methylmercury [14]. Also, the Joint the Food and Agriculture Organization of the United Nations (FAO)/The World Health Organization (WHO) Expert Committee on Food Additives (JECFA) have established a MeHg provisional tolerable weekly intake (PTWI) of 3.3 µg/kg body weight per week [14]. Legislation also exists for limiting/prohibiting Hg in cosmetic products, stipulating that Hg and its compounds may not be present as ingredients in cosmetics, including soaps, lotions, shampoos, skin bleaching products etc. (except for phenyl mercuric salts for eye make-up conservation and eye-make-up removal products in concentrations not exceeding 0.007 percent weight to weight) [14].
Although systematic research on heavy metal exposure and effects started centuries ago, new discoveries, and large gaps in understanding the mechanisms and the right assessment of the health effects caused by environmental and occupational human exposure are still pointing out the necessity for new data collection and further research. Beside partial coverage in three reviews so far (1994,2009,2019) [1,7,22], there is no literature review of the genotoxic studies dealing with Hg, its derivatives and human exposure using the most usual genotoxic assays: sister chromatid exchanges (SCE), chromosomal aberrations (CA), cytochalasin B blocked (CBMN), and single-cell gel electrophoresis (SCGE or alkaline comet) assays, making this review paper a valuable contribution to this field.

Materials and Methods
The Pubmed and Web of Science database (Indexed as also SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, BKCI-S, BKCI-SSH, ESCI, CCR-EXPANDED, IC.) were searched for all years, with the last date of search being 23 October 2021. The retrieved articles were searched both in English and in Spanish.
The results were reviewed and only those with data evaluating genotoxicity and related biomarkers were selected. After duplicate references were eliminated, 66 articles dealing with human samples in vitro or in vivo were selected in relation to the analysis of genotoxicity. This article aims to provide an overview of the available data in this regard, covering the results obtained with Hg and its compounds in human short-term in-vitro test systems, as well as the cytogenetic data that have emerged from the biomonitoring of exposed individuals. The Preferred Reporting Items for Systematic Reviews (PRISMA) checklist [Tables S1 and S2], and the flow diagram for the selection of studies [ Figure S1], were completed.

Results
3.1. In-Vitro Genotoxic Effects for Inorganic (i)Hg Compounds iHg compounds or salts, such as mercury sulfide (HgS), mercury oxide (HgO), and HgCl 2 , are found as powders or crystals, and the natural mineral cinnabar, with the highest Hg content in the form of Hg sulfide (HgS), can be formed by Hg 0 vapor metabolism (Hg 0 biological oxidation) or by MeHg metabolism (MeHg demethylation by the intestinal microflora activity) [23].

Mercury Chloride (HgCl 2 )
HgCl 2 was able to induce CA in peripheral blood lymphocytes (PBL) [31] and SCE in white blood cells [30]. However, negative results at sub-toxic concentrations have been previously reported with the CA test [29]. Bérces et al. (1993), using the MN assay, detected the genotoxic effects of metal ions, including Hg [32]. A linear increase in the MN frequency was observed with increasing HgCl 2 concentration. The CA in human lymphocytes and the levels of 8-hydroxy-2'-deoxyguanosine  in mononuclear cells (PBMC), exposed to different concentrations of this compound, increased in a concentration-dependent manner and were significantly higher than those found in controls [33]. However, the results of the analysis of the frequency of hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) mutants in the human lymphoblastoid thymidine kinase heterozygote (TK6) cell line, after exposure, were inconsistent with respect to their mutagenic effects, although they exhibited clear cytotoxic effects [34].
The damage produced to DNA by HgCl 2 has been evaluated in a human fetal liver cell line (WRL-68), using the comet assay [35]. The compound was able to induce single strand breaks or alkali sensitive sites. The percentage of damaged nuclei and the average comet tail length (TL) of DNA increased as the concentration and exposure time increased. Recovery time from DNA damage was 8 h after partially removing the metal with PBS-EGTA [35]. A concentration-dependent comet formation was induced in U-937 cells, with concentrations between 1 and 5 µM [36]. In human leukocyte cultures, no effects were found on cell proliferation kinetics, however, the SCE frequency, as well as the C-anaphases frequency, were significantly higher with respect to the values found in the control [37].  Genotoxicity of HgCl 2 was assessed by CA and polyploidic cells count [38]. A significant increase of gaps and breaks was found at 0.1 and 1000 µg/L, but HgCl 2 did not induce aneuploidies at any concentration tested. Rozgaj et al. (2006), in a preliminary study with the purpose of HgCl 2 genotoxicity evaluation in cultured human lymphocytes using CBMN assay, did not find significant differences between the samples analyzed after 24 h of exposure, but, after 48 h, they found a significant increase in MN frequency, although not in a concentration-dependent manner [39]. However, inconsistent results were found by the same research group using the SCGE assay, evaluating the TL, tail moment (TM) and tail intensity (TI), with HgCl 2 concentrations of 50, 100, and 200 µM [40]. At 24 h exposure to doses of 50 and 100 µM, TL values were statistically different when compared with controls, but no significant differences were found in the TM or TI. At 48 h exposure, they found significant differences in TL between the positive control and the other samples, and the control sample, the highest concentration of which was 200 µM. For TI and TM, only the positive control sample demonstrated significant difference from all other samples.
Damage to DNA in human lymphocytes and the cells of salivary gland tissue was evaluated by analyzing DNA migration due to single-strand breaks, alkali-labile sites, and incomplete excision repair using the SCGE assay [41]. Genotoxic effects were evaluated below a cytotoxic dose level, with a significant increase in dose-dependent DNA migration demonstrated after exposure to 2.5 µM HgCl 2 and above, in both test systems, when compared with the negative control. On the other hand, in TK6 cells, there was a dosedependent increase in OTM from 0.1 mM and up to 2 mM [42].

Mercury Nitrate
Only one study was found regarding the genotoxic potential of Hg nitrate, reporting no SCE induction in cultured human lymphocytes; however, at 30 µM, Hg nitrate could produce endoreduplication (E) [43].

In-Vitro Genotoxic Effects for Organic Compounds
Among the organic Hg compounds, one of greatest concern is MeHg (CH 3 Hg), produced in the environment mostly by microorganisms (bacteria and fungi), rather than by human activity.
CH 3 Hg is widely distributed in the body, crossing both blood-brain and placental barriers in humans [44]. The main system affected by CH 3 Hg is the central nervous system, although others, such as urinary and immune systems are also compromised. From the point of view of its genotoxic potential, it has been the most evaluated species.
Ethylmercury (C 2 H 5 HgCl) and phenylmercury (C 6 H 5 HgCl) chloride were able to induce chromosomal alterations in human HeLa S3 cell cultures [45], and phenylmercury acetate (PMA or CH 3 COOHgC 6 H 5 ) increased the SCE and E frequency in a concentrationdependent manner in cultured human lymphocytes [43].
The same situation occurred for dimethylmercury [(CH 3 ) 2 Hg], which could induce numerical and structural CA, although with a lower potency compared with the effect induced by methyl chloride in human lymphocytes [46].
A similar effect was found with thimerosal, also known as thiomersal, or with the trade names merthiolate, mertodol or metorgan. This oHg compound, o-carboxyphenyl-thio-ethylsodium salt, with a recognized antiseptic and antifungal action, is used especially in vaccines in domestic and farmed animals and in humans. The WHO considers small doses of thimerosal safe regardless of multiple/repetitive exposures to vaccines that are predominantly taken during pregnancy or infancy. In human lymphocytes a significant MN induction was observed in the CBMN test [3], and there was also s significant increase in SCE assay with/without metabolic S9 activation, together with a significant decrease in mitotic (MI) and proliferation indexes (PRI) [47]. There are no other studies performed on human samples with genotoxic assays of our interest, but since its main metabolised compound is C 2 H 5 HgCl, and exposure is usually in the early developmental stages of organism, toxicological assays were performed, and although in-vitro and in-vivo conditions did not give similar results, and, while the exact mechanisms of action are still unknown, it has been demonstrated that it causes immunotoxic and neurotoxic effects [48]. These data raise some concern about the widespread use of thimerosal in some vaccines that are still in use, mostly in developing countries.
Methylmercury (MeHg or CH 3 Hg) CH 3 Hg induces structural and numerical CAs in human lymphocytes [49]. Likewise, it increases the SCE frequency in lymphocytes from whole blood cultures [30,49], producing chromosome breaks [50] and altering their segregation [31], in the same test system. Ogura et al. (1996) reported that it induced a significant concentration-dependent increase in CA in human lymphocytes and in the levels of 8-hydroxy-2'-deoxyguanosine  in PBMC, being more potent than the inorganic methyl chloride species [33]. Similarly, the frequency of SCE and E increased to 20 µM, in the same test system [43].
Studies on this compound have demonstrated inconsistencies regarding its mutagenic capacity when analysing the hypoxanthine phosphoribosyltransferase (hprt) locus mutations frequencies and CA number in the TK6 cell line, also demonstrating cytotoxic properties and causing a marked mitosis frequency reduction, alone or in combination with HgCl 2 [34].
Its genotoxicity was demonstrated in human PBL, through CA and polyploid cells' induction; as proof of its cytotoxicity, it lowered the mitotic index (MI) [38,51].
A significant increase in the frequency of both genotoxicity biomarkers and a significant MI decrease was observed at all concentrations evaluated compared to the control, either alone or in an evident synergistic combination with HgCl 2 .
Human brain cell lines of glioblastoma (U373) and neuroblastoma (B103) were exposed to methylmercury chloride (CH 3 HgCl), for 24 (U373) or 48 (B103) hours [6]. The binucleation index, the frequency of cells with MN, as well as the metaphasic MN and nucleoplasmic bridges were determined. Statistical analysis showed significant increases and percentages in the treated cells. Each cell line was shown to be different from each genotoxic damage biomarker, which seems to indicate the existence of different toxicity mechanisms. This work demonstrated the ability of CH 3 HgCl to cause genotoxicity in cells of the nervous system, with relatively low levels of exposure. In a human lymphoblastoid cell line (TK6) an increment of OTM was induced [42].
Crespo-López et al. (2016) analysed the possible genotoxicity and alterations in the cell cycle and proliferation of a glioma line (C6) exposed to a low, non-lethal and non-apoptotic concentration of CH 3 HgCl [52]. Treatment without promoting cell death significantly increased genotoxicity markers (DNA fragmentation, MN, nucleoplasmic bridges, and nuclear buds). In the same way, it caused changes in the cell cycle, which suggests cell cycle arrest. This work demonstrated that exposure to a sublethal CH 3 HgCl concentration, considered relatively safe, according to current limits, induces genotoxicity and alterations in the proliferation of cells of glial origin.
Patnaik and Padhy (2018) compared the genotoxicity of CH 3 HgCl and methylmercury hydroxide (CH 3 HgOH) in the human neuroblastoma cell line SH-SY5Y using the comet assay and demonstrated that both compounds were capable of inducing DNA fragmentation, with CH 3 HgCl being the most toxic for this cell line [53]. Table 2 lists the studies on the in-vitro genotoxic effects of oHg compounds.

Genotoxic Effects in Exposed Individuals
Human populations can be exposed to Hg 0 or its derivatives accidentally, through occupational exposure, or through food. The first in-vivo studies on the genotoxic effects induced by exposure to this metal were carried out during the 1970s and 1980s, determining the CA, MN, and SCE frequencies. The results of epidemiological studies related to human Hg exposure have been increasing since the end of the last century, which makes the results more reliable, demonstrating an increase in genotoxicity in human populations exposed to Hg through diet, the occupation or wearing of dental fillings [22]. Table 3 represents studies dealing with genotoxic effects in human populations exposed to Hg.      Ly-human lymphocytes; CA-chromosomal aberrations; MN-micronuclei; WB-whole blood; C 6 H 5 Hg-phenylmercury; C 2 H 5 Hg + -ethylmercury; L-human leukocytes; PB-peripheral blood; CBMN-cytokinesis blocked micronucleus assay including not only MN but other biomarkers; SCE-sisters chromatid exchange; 8-OHdG-8-Hydroxy-2'-deoxyguanosine; (CH 3 ) 2 Hg-dimethylmercury; iHg-inorganic mercury; mHg-metallic mercury; oHg-organic mercury; CH 3 COOHgC 6 H 5 -phenylmercury acetate; Contam water-contaminated water; SCGE-single cell gel electrophoresis or alkaline comet assay; ROS-reactive oxygen species; MI-mitotic index; BC-blood cells RBC-red BC; conc-concentration; E-exposed; C-control.

Accidental Exposures
Mudry de Pargament et al. (1987) reported the induction of SCE in lymphocytes of children exposed to PMA used in diaper disinfection [60].

Exposure from Contaminated Food
The more frequent consumption of seal meat (six times a week, an average diet Hg concentration of 62.5 µ/L) increased SCE frequency when compared to less frequent consumers (once or twice a week, an average diet Hg concentration of 22 µ/L) [65].
However, Monsalve and Chiappe (1987) did not find a differences in SCE or CA frequency between subjects exposed to this compound through the consumption of fish caught in a contaminated area and subjects who consumed fish from uncontaminated area, even though the difference in hair samples CH 3 Hg concentrations between those two groups was statistically significant and higher in the subjects who ate fish from the area [56]. Skerfving et al. (1970) compared CA frequency in two groups of individuals in relation to the consumption of fish contaminated (or not) with CH 3 Hg and found a correlation of Hg concentration in erythrocytes, with fequent structural chromosomal rearrangements, but not with polyploidy or aneuploidy [54]. Later, in an extension of their study, they found a slight increase in CA frequency [55].

Occupational or Environmental Exposure
Verschaeve et al. (1976) found an increase in aneuploidy frequency in subjects exposed to metallic (m)Hg, amalgams, C 6 H 5 Hg, and C 2 H 5 Hg + [58]. Exposure to the latter also showed an increase in the structural CAs frequency, PMA induced a significant increase in hyperpolyploidy frequency [59]. Subjects with Hg amalgams showed an increase in aneuploidy frequency when compared with the control group [79].
Contrary that reported in these studies, the same research group ) and Mabille et al. (1984), found no genotoxic effects in workers exposed to metallic (m) Hg [61,62]. As in the latter cases, in a group of workers exposed to mHg vapors, as well as in another group exposed to a mixture of CH 3 HgCl and C 2 H 5 HgCl, there was no significant increase in genotoxic damage, but, when combining the two groups, there was a significant increase in the frequency of acentric fragments. Popescu et al. (1979) determined the genotoxic damage due to occupational exposure to mHg or oHg [64]. Exposed workers had higher CA incidence when compared to the control group, but with no differences observed between the unexposed and exposed groups with respect to the frequency of aneuploidies and polyploidies.
A significant SCE increase was found in workers in two factories exposed to mHg and iHg [66]. Using the MN test, Barregård et al. (1991) did not find an increase in genetic damage in workers exposed to a chlor-alkali plant when compared with a control group [67]. On the other hand, fulminant Hg-exposed workers in an explosives factory presented a statistically significant increase in MN and CA frequency when compared with controls but found no correlation between this type of damage and Hg urine concentrations [73].
In Slovenian miners, a significant MN, CA, and SCE frequency increase was related to their employment seniority [74]. Similar results were found by Cruz-Esquivel et al. (2019) using the comet assay in peripheral blood lymphocytes and a micronucleus (MN) cytome assay (BMCyt) in exfoliated buccal cells of Colombian miners [78].
Higher levels of CA, MN, SCE, and HGPRT mutations were found in workers of a chloroalkali plant [69], a mercury-producing plant [70], a battery plant and dentists [71]. In contrast, workers in a chloroalkali plant exposed to Hg vapor did not show significant differences in CA and MN frequencies in peripheral lymphocytes [68], nor were MN or SCE different in harbour workers with potential exposure to river silt aerosols [72].
Cytogenetic monitoring of fishermen with environmental Hg exposure was carried out by evaluating the MN, CA, and SCE frequencies in PBL [75]. A statistical correlation was found between the MN frequency and the total Hg blood concentration, supporting the usefulness of this biomarker for early DNA damage detection. In the same way Queiroz et al. (1999) observed a significant increase in the MN percentage of people exposed to Hg compared to an unexposed group [70]. No correlation was found between the MN percentage and age, duration of exposure, or urinary Hg concentrations with the levels considered biologically safe for the exposed population. Amorim et al. (2000) examined the cytogenetic alterations in the peripheral lymphocytes of a population who lived on the banks of the Tapajós River in Brazil, with respect to contamination by CH 3 HgCl, using hair Hg as a biological indicator of exposure [2]. The results showed a clear relationship between CH 3 HgCl contamination and cytogenetic damage in lymphocytes at levels well below 50 mg/g-the values considered safe by the World Health Organization (WHO). The main changes found consisted of polyploidies, chromatid breaks and an MI decrease.
Cebulska-Wasilewska et al. (2005) carried out a population study to evaluate whether occupational Hg exposure can cause genotoxicity and affect the efficacy of DNA repair mechanisms [4]. Although the exposure did not generate differences when the SCE test was used with respect to the control individuals, the chromosomal damage detected by the comet test increased significantly in the lymphocytes of the exposed workers, in the same way, as the authors determined, that Hg causes a significant decrease of DNA repair capacity. The latter could eventually lead to a carcinogenic process [84]. In this regard, the International Agency for Research on Cancer (IARC 1993, WHO) classifies CH 3 Hg within group 2B, as a possible carcinogen, with other inorganic forms classified in group 3 ("It cannot be classified with respect to its carcinogenicity to humans") [85]. Currently there is still no evidence that either CH 3 Hg or other iHg forms can cause cancer in humans.
In their study on cytogenotoxicity in uroepithelial cells from women exposed to inorganic Hg in a mining area, Soto-Ríos et al. (2010) provided evidence that these people are at increased risk of developing not only different types of DNA damage, but, are also at increased risk of tumour development [76]. The first cellular changes that can increase the possibility of cancer risk indicate that exposure to Hg-containing mining wastes is a health risk, recommending that these cellular changes should be considered in the preliminary assessment of the health risks associated with this occupational exposure.
As a part of the BioMadrid project, derived from the concern for DNA stability in newborns and their parents who were environmentally exposed to various metals, an association was found between elevated Hg blood levels in fathers with an increase in the frequency of CBMN, which were both significantly higher when compared to the fathers who had "normal" Hg levels. The results showed a statistically significant correlation in the frequency of CBMN parameters between parents and new borns. An association was found also between the CBMN rate and elevated Hg levels in mothers and fathers, but not in newborns. This result provides information on the relationship between shared and genetic environmental effects [77].
Crespo-López et al. (2011), analysed, in vitro, the Hg blood genotoxicity in the lymphocytes of Amazonian individuals using two methods, MN and CA [51]. The induced frequencies were very low and only the cell cycle was significantly inhibited when comparing exposed versus unexposed populations.
The evaluation of genotoxic damage in artisanal mining workers exposed to Hg was carried out by Rosales-Rimanche et al. (2013) [57]. It was reported that 15% of the workers presented MN in buccal epithelium cells, also registering other nuclear abnormalities such as nucleoplasmic bridges, buds, and binucleated cells, demonstrating the genetic damage association with occupational exposure.
Castaño Arias et al. (2014) determined the magnitude of genetic damage with Hg exposure in mining workers using the comet test, finding greater damage (TI values) in the exposed group when compared with the control group [82]. Similarly, in a heavy metals-effects study in a human population of the La Mojana region, Colombia, Calao and Marrugo (2015) found significant associations between Hg presence and DNA damage in the same bioassay and using the same biomarker [63].

Amalgams
Dental fillings provide significant iatrogenic exposure to xenobiotic compounds. Experimental data suggest that amalgams, which contain Hg, cause a deterioration of the pro-antioxidant cellular redox balance. A study was carried out to evaluate the potential genotoxicity of dental restorative compounds in the PBL of exposed subjects compared with a control group, using an SCGE assay [80]. The comet TL, TI, TM, and Olive tail moment (OTM) were twice as high in the exposed group with significant differences from the unexposed group. In addition, the authors demonstrated the association between the number of amalgams and the exposure time with DNA damage. The main mechanism underlying genotoxicity was attributed to the ability of implants (mercury) to trigger the generation of reactive oxygen species (ROS), capable of causing DNA damage [80].
Genotoxic damage in the oral mucosa cells of subjects with Hg-based dental restorations (amalgams) was evaluated with SCGE, MN, and a nuclear abnormalities (NA) test as markers of cell death. The results showed that amalgams can induce genetic damage by increasing MN frequency and marginally by the damage detected through the comet assay. The relevance of this study lies in the fact that subjects with Hg-based restorative materials are exposed continuously and for long periods of time to this metal [81].
Hg dental amalgam has a long history of apparently safe use despite the continuous release of mercury vapor. However, some studies suggest that it can cause DNA damage, particularly for individuals with common genetic variants [86]. This and other studies suggest that susceptibility to Hg toxicity differs between individuals based on multiple genes, so exposure levels to Hg vapor from dental amalgams can be dangerous for certain subpopulations. For this reason, efforts are being made to reduce or eliminate the use of Hg-based dental amalgam. Similar results were obtained by Mary et al. (2018) using MN test in the oral mucosa cells [83]. The analysis of the alterations was carried out in the same subject before and after dental restoration with amalgams, serving as their own controls. The mucosa samples were taken before the intervention and 10 days after. A statistically significant difference was found in MN frequencies when comparing both samples (before and after). Similarly, the damage increased as the number of restorations in the individual increased.

Compounds against Hg Genotoxicity
Efforts have been made to find therapeutic agents capable of reducing the genotoxicity of different natural or anthropogenic compounds. As can be seen in this review, the genotoxicity induced by Hg compounds remains controversial. However, different agents have been tested in order to assess their antigenotoxic (or protective) properties in relation to the effects induced by Hg and its derivatives [37]. Epidemiological studies have shown that enzyme activity is altered in the exposed populations, which could contribute to an increase in genotoxic damage, since it has been proposed that Hg can inhibit antioxidant enzyme activity causing stress within the cells and organism [87]. Thus, the analysis of these markers could be useful in the evaluation of compounds' toxicities [22].
Catalase (CA) and superoxide dismutase (SOD) have been used for their antigenotoxic (protective) properties against PMA effects, however, at concentrations of 75 and 150 g/mL, they were not able to present this property [43]. L-ascorbic acid (vitamin C) has demonstrated its protective capacity against CH 3 HgCl-induced damage in cultured human leukocytes, probably due to its antioxidant and nucleophilic nature [37]. Purohit and Rao. (2014) evaluated the protective effect of melatonin (MLT) and αtocopherol against Hg-induced genotoxicity in cultured human lymphocytes using the SCE test, cell proliferation and the replication index [88]. Exposure to the metal significantly increased the SCE frequency, cell proliferation kinetics inhibition and caused a decrease in the replication index, compared to controls. The addition of α-tocopherol and MLT individually and in combination showed a mitigating effect by reducing the genotoxic potential in the treated cultures. The percentage of improvement was comparatively high with both MLT and α-tocopherol, but also with their combination. Similar results were found when melatonin, curcumin, and andrographolide were evaluated against the Hg genotoxic effect in the same test system and with similar biomarkers. The results revealed a CH 3 HgCl dose-dependent increase with the SCE test after Hg treatment, while supplementation with these three antioxidant compounds effectively abrogated genotoxic damage in treated cultures and improved cell cycle kinetics. The antimutagenic activity of these compounds on Hg-induced genotoxicity was in the following order: melatonin > curcumin > andrographolide [89].
Ali (2018) evaluated the role of garlic and vitamin E in mitigating the genotoxic damage induced by CH 3 HgCl in human lung cells line WI-38 [90]. The treatments led to a significant and dramatic increase in DNA damage, evidenced by the comet TL and TM values when compared with the control samples' values. However, garlic and/or vitamin E significantly reduced DNA damage in these treated cells. Protection with garlic alone was more effective than with vitamin E alone, while the combination of both was the most effective regimen.

Mechanism of Hg Genotoxic Action
Like other metals, Hg damages DNA through multiple mechanisms, recognizing its ability to bind to sulfhydryl groups. Different hypotheses have been raised about the possible molecular mechanisms of Hg genotoxicity, involving four main processes that lead to genotoxicity: the generation of free radicals and oxidative stress, action on microtubules, influence on DNA repair mechanisms, and direct interaction with DNA molecules [7,22].
The greatest contribution to the genotoxicity of Hg and its derivatives is due to their ability to generate ROS species, accompanied by the decrease in protective glutathione reserves. The genotoxic capacities of the different species are qualitatively comparable, which may suggest a differential bioavailability and the participation of a common genotoxic entity [1]. ROS are formed when Hg enters the cell directly through the plasma membrane or through protein transporters [37,43,80].
The damage can be direct, by oxidizing nitrogenous bases, or indirect, by interacting with other biologically important molecules, such as fatty acids, DNA polymerases, and microtubules [4,77,81,85,87,91]. Glutathione levels, a potent free radical scavenger and metal chelating agent, have been reported as decreased in Hg-exposed populations [2,87].
Cebulska-Wasilewska et al. (2005) speculated that occupational exposure to low Hg concentrations, within the permitted ranges, interferes with DNA repair processes by recombination and by base-pair cleavage [4].

Conclusions
The versatility of Hg compounds explains their many applications in various areas of industry. Its growing use has resulted in a significant increase in environmental pollution and in the increased number of episodes of human intoxication, arousing the concern of international organizations. However, the consequences of these poisoning outbreaks are not yet fully understood, especially when we consider the long-term effects of chronic exposure at relatively low levels [22].
The genotoxic effect caused by Hg still generates great controversy, given the diversity of responses that different Hg compounds can produce. CH 3 Hg compounds stand out as the most genotoxic among the diversity of forms derived from this metal [5,22].
Genotoxic alterations, such as CA and MN, have been detected in populations chronically exposed to Hg levels below the safety values defined by the WHO [2,5,80], using mainly peripheral blood cultures and, in some cases, oral mucosa epithelial cells.
The findings of several studies aimed at the biomonitoring of cytogenetic effects on PBL from people exposed to Hg and its compounds from accidental, occupational, or food sources were negative, controversial, doubtful, or uncertain as to the actual role played by Hg in some positive results [1,5,22]. The discrepancies found may be due to the different potency of the iHg and oHg derivatives, as well as to the different protocols applied in terms of exposure times, bioassays, and biomarkers, due to their different sensitivities. It would be pertinent to standardize genotoxicity tests, in order to have more reliable results.