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Genotoxicity and cytotoxicity evaluation of two thallium compounds using the Drosophila wing somatic mutation and recombination test

Thallium (Tl) is a heavy and toxic metal and a byproduct of several human activities, such as cement production, mining, and coal combustion. Thallium is found in fruits, vegetables, and animal fodder with high Tl contamination; therefore, it is an environmental pollution issue and a toxicological contamination problem for human beings and other organisms when exposed to it. The mutagenic potential of Tl and its compounds is controversial, and there are few in vivo studies on its effects. We conducted the animal bioassay Drosophila wing somatic mutation and recombination test (SMART) to test for genotoxicity and assessed the genotoxic effects of Tl acetate (TlCH3COO) and Tl sulfate (Tl2SO4) on Drosophila melanogaster. Third instar larvae from the SMART standard cross (ST) were fed Tl acetate [0.2, 2, 20, 200, 600 and 1200 μM] and Tl sulfate [0.2, 2, 20, 200, and 600 μM]. Hexavalent chromium [CrO3, 500 μM] served as the positive control, and Milli-Q water served as the negative control. Only the high Tl2SO4 [600 μM] concentration resulted in genotoxicity with 87.6% somatic recombination, and both salts disrupted cell division of wing imaginal disc cells, showing the expected cytotoxic effects. Genotoxic risks due to high metal levels by bioaccumulation of Tl+1 or its compounds require further evaluation with other in vivo and in vitro assays.


Introduction
Thallium (Tl) is a rare, very reactive, highly toxic heavy metal that is not found free in the environment because it forms mono-and trivalent compounds. Tl is mainly found in Asia, Europe, and North America [1,2] in the form of sulfides in minerals and rocks. It is a polluting metal and a byproduct of coal combustion [3] and a high variety of industries, such as cement, fireworks, electronic devices, mercury lamps, low-temperature thermometers, special glasses, mixed crystals, radioactive isotopes, mining, steel and sulfuric acid production [2]. Some Tl salts have been used as epilators and to treat venereal diseases, such as typhus, tuberculosis, malaria and ringworm. Tl sulfate (Tl 2 SO 4 ) has been included as an active compound in insecticides and rodenticides (2.5%) since 1920, and its use was prohibited in the USA in 1972 [4]; nevertheless, it is still used as a rodenticide in some countries given its low price [2]. Recently, it has been reported that the inks used in tattoos contain this metal [5].
The concentration of Tl in wastewater can reach 3,000 μg/L [1], and frequently, Tl contaminants generated by human activities end up in the trophic chain due to their absorption and accumulation in plants [6,7]. The Tl concentration in foods, such as fruits and vegetables [6], increase by bioaccumulation, which depends on the ground concentrations, ground pH and cultivated species and varieties. In humans, an 8 mg/kg concentration may be indicative of poisoning, and a 10-15 mg/kg concentration is lethal [2]. Tl tends to form the þ3 and þ1 oxidation states, and it has been suggested that the majority of Tl þ3 is converted to Tl þ1 [8]. Its salts, such as sulfate, carbonate and acetate, are very soluble in water, but there are other salts, such as sulfide and iodide, that are slightly soluble in water. Tl þ1 and cation compounds, such as Tl acetate (TlCH 3 COO) and Tl sulfate (Tl 2 SO 4 ) (Figure 1), are more stable in water solution at neutral pH compared with Tl þ3 compounds.
In general, greater than 90% Tl is absorbed through the skin and oral mucosa, but it is also acquired by inhalation. Tl accumulates mainly in the kidney and reaches the brain after systemic administration [9]. Galv an-Arzate et al. [10] induced significant lipoperoxidation (LPO) in the brains of rats treated with daily sublethal doses of Tl [0.8 mg/kg]; at [1.6 mg/kg], all brain regions studied presented a significant increase in LPO compared to the control, suggesting that oxidative stress is involved in Tl toxicity. Thalliotoxicosis diagnosis and treatment are important, although alopecia is the distinctive symptom of Tl poisoning [4,11]. The addition of 10-15 nmol of Tl þ1 per mg/mitochondrial protein inhibits K þ1 translocation without affecting oxidative phosphorylation [12]; when Tl þ1 enters the cell, it induces oxidative stress and affects glutathione (GSH) metabolism, K þ1 -regulated homeostasis and cellular and mitochondrial membrane integrity [13]. In PC12 cells incubated for 1-72 h at a single dose of Tl þ1 or Tl þ3 [10-250 μM], H 2 O 2 increased in the mitochondria, which resulted in higher levels of reactive oxygen species (ROS) in the cytoplasm [14]. Tl þ1 [25-200 μM] in hepatocyte mitochondria induced ROS and decreased ATP, which triggered cytochrome c release and the activation of apoptosis [15]. Korotkov et al. [16] assumed that Tl þ1 might deform complex I in "Ca þ2 -loaded" mitochondria and that Tl þ1 to Tl þ3 oxidation is involved in this event. Pourahmad et al. [17] studied rat hepatocytes and associated Tl þ1 , Tl þ3 , GSH and CYP2E1 with the interruption of the electron transport chain in mitochondria and the mutual damage between mitochondria and lysosomes that resulted from an increase in ROS. The metabolism and toxicity of Tl þ1 are positively related to the induction of oxidative stress, apoptosis and LPO. Tl and its compounds are not classifiable as carcinogens to humans based on "inadequate information to assess its carcinogenic potential" [4]. Despite all the toxic effects described above, which mainly involve ROS increases related to DNA damage [18], there is a lack of data on the mutagenic effects of thallium compounds in humans [13]. Furthermore, very few studies evaluate Tl compound genotoxicity in vitro and in vivo [19]. We assessed the in vitro genotoxic effect of TlCH 3 COO and Tl 2 SO 4 in human leucocytes cultured at [0.5, 1, 5, 10, 50 and 100 μg/mL] based on structural and numerical chromosome aberrations, sister chromatid exchange and comet assays (pH > 13 and 12.1) and showed that both salts have effects on the mitotic index, exert cytotoxic, cytostatic, and clastogenic activity and cause DNA damage [20,21]. Given these previous results, the aim of this study is to gain insight into the possible in vivo genotoxicity of TlCH 3 COO and Tl 2 SO 4 through an alternative animal test that uses the insect Drosophila melanogaster to realize the wing somatic mutation and recombination test (SMART) standard cross (ST) with Cyp450 basal levels [22] by pairwise comparison with negative dissolvent control results. This bioassay detects mutations, deletions, aneuploidies, and recombinations in wing imaginal disc cells and allows cytotoxicity to be determined based on alterations in cellular division through cumulative frequency analysis of mwh clones [23,24].

LC 50
The LC 50 of each Tl salt was determined by feeding until pupation D. melanogaster third instar larvae (72 AE 4 h) of the flare strain with 0.5 g of DIM plus 2 mL of fresh solution at eight concentrations of Tl 2 SO 4 or TlCH 3 COO [1.9, 7.8, 31.2, 125, 500, 1000, 2000 and 4000 μM] running three independent experiments with five replicates for each compound at 25 AE 2 C and 65% relative humidity (RH) until emergence. One-factor ANOVA (P < 0.5) was performed to compare results among experiments, and the LC 50 was determined by regression.

SMART
To develop the Drosophila wing SMART, the ST cross was performed by mating virgin females of the flare strain (flr 3 /TM3, Bd S ) to males of the mwh strain (mwh/mwh) originally donated by Dr. Ulrich Graf (ETH, Zurich, Switzerland). Eggs collected from fresh yeast media from the cross were incubated at 25 AE 2 C and 65% relative humidity (RH); equal batches of third instar larvae (72 AE 4 h) were chronically exposed by feeding until pupation with 0.5 g of DIM plus 2 mL fresh solution of TlCH 3 COO [0.2, 2, 20, 200, 600 and 1200 μM] or Tl 2 SO 4 [0.2, 2, 20, 200 and 600 μM] dissolved in Milli-Q water, which was also used as a negative control. Hexavalent chromium [CrO 3 , 500 μM] was used as a positive control [22]. Transheterozygous flies (MH, marker-heterozygous flies: mwhþ/þflr 3 : wild-type wings) were randomly selected, and wings were dissected and mounted in Entellan® and scored at 40x under a microscope (double-blind). Relative spot per fly frequencies were compared through statistical analysis using SMART v2.1. software (Frei and Würgler, unpublished), which is based on the Kastenbaum-Bowman test that contrasts the expected frequencies [25]. A Mann-Whitney-Wilcoxon U test was performed to corroborate the results [26]. When the twin spot frequency was statistically significant, we mounted and scored the BH of balancer-heterozygous flies (mwhþ/TM3, BdS) with serrated wings to determine the recombinogenic activity of the compound [22]. Similarly, the size distribution of mwh clones was analyzed using the Kolmogorov-Smirnov test [23]. P < 0.05 was considered statistically significant in all tests.

LC 50
To determine the test concentrations, first, we determined the LC 50 for TlCH 3 COO [919 μM] by quadratic regression and for Tl 2 SO 4 [460 μM] by linear regression (Figure 2).

SMART
Based on these data, we exposed third instar larvae from the ST cross of the SMART bioassay to six concentrations of TlCH 3 COO [0.2, 2, 20,  Tables 1 and 2 show the effect of treatments with the metal compounds and controls.
As expected, the relative frequencies of spots in the CrO 3 treatment were significantly increased for each type of spot compared to the remainder of the treatments, which confirms the mutagenic and recombinogenic effects of this positive control [22,29]. Regarding Tl, only the highest concentration of Tl 2 SO 4 [600 μM] was clearly genotoxic because all the spot per fly relative frequencies were significantly higher. Considering that the twin spots are produced exclusively by somatic recombination between the marker fir and the centromere [22,24], only yielded a high frequency in Tl 2 SO 4 [600 μM], we scored the BH balancer-heterozygous flies (mwhþ/TM3, Bd S ) with serrated wings to calculate the percentage of spots induced by somatic recombination [22,30] dividing the frequency of total mwh spots on the BH wings by that on the MH wings (0.47*100/3.8) [22] obtaining a percentage of somatic mutation of 12.4% and 87.6% of recombination. The size distribution of the mwh clones and the statistical comparison of the experimental results against the negative control allowed us to detect whether the treatment altered the cellular division from the imaginal discs from the wing. The data in Table 2 show the results of the mwh clone cumulative frequency analysis that compared the results of the maximum difference between the cumulative distributions of the negative control with respect to treatments through a Kolmogorov-Smirnov test [23,24]. With both salts, all the treatments were statistically significant, with higher means at [600 μM]; in contrast, the [2.0 μM] treatment had no effect on any of the salts. Of note, only TlCH 3 COO [0.2 μM] showed an effect, so we considered this result a false positive.

Discussion
Thallium exerts potent toxic effects on a wide variety of in vitro and in vivo biological systems through different routes of exposure [4,19]. Considered an industrial and mining polluting byproduct [31], thallium is present in the environment at subtoxic levels, but it can be found in food [32], plants [6,7], and animals [33] because of its bioaccumulation, posing threats to human health and the environment. Therefore, it was important to assess whether it poses a genotoxic risk to human health and to other organisms. Given the few in vivo reports on Tl genotoxicity, an alternative animal bioassay, the D. melanogaster wing spot test, was performed with the aim of collecting robust data using this alternative eukaryotic model organism, which shares 77% of the genes that cause disease in humans [34] and 80-90% or more of conserved functional domains in nucleotides and proteins [35]. The relative spontaneous spot frequencies obtained in the negative Milli-Q water control were compared against those of the positive control, CrO 3, a potent carcinogen that produces chromosomal alterations through oxidative stress and whose toxic, teratogenic and mutagenic effects have been confirmed in humans, birds [36] and D. melanogaster [22]. not have clear explanations for these differences, but we propose that these findings could be related to the corresponding molecule dissociation and/or the contribution of sulfur to a putative generation of genotoxic sulfur compounds [37], such as hydrogen sulfide (H 2 S) [38] or sulfur dioxide (SO 2 ) [39]. It has been demonstrated that Tl exhibit a certain affinity for guanines in the major groove of B-DNA and for thymine, which could affect the molecule. Thus, the genotoxic effect observed with Tl 2 SO 4 [600 μM] could be related to the reported adduct formation between Tl and DNA nitrogenous bases [40] but also to ROS increases that generate DNA damage [18]. In addition, Yildirim et al. [41] found significant differences in chromosome aberrations and sister chromatid exchanges three days after administration of Tl to patients exposed to this metal for a myocardial perfusion study. Decreases in the mitotic index and replicative index in vitro were demonstrated by Rodríguez-Mercado et al. [20,21], who suggested that Tl þ1 induces genetic damage and is cytotoxic. The genotoxic effects of Tl 2 SO 4 [600 μM] in this in vivo study are evidence of the potential effects of Tl 2 SO 4 that have been reported in in vitro studies at [1.8-369.8 μM] [21,42].
The differences found in this study might be attributed to the compound's absorption distribution, metabolism, and excretion (ADME) of this metal salt when administered in vivo.
The mwh clone cumulative frequency analysis showed a significant effect of both Tl salts ( Table 2); this parameter is related to cytotoxicity that induces cell division disruption, aneuploidy [24,27,28] or apoptosis in imaginal wing cells. These cytotoxic effects are consistent with published reports about Tl þ1 toxicity caused by its cellular accumulation [43]. To explain these results, we also considered putative damage derived from Tl þ1 toxicity as glutathione (GSH) depletion in the mitochondrial redox balance [44,45]. On the other hand, the production of ROS [14,15] and its known effects on mitochondria could have induced apoptosis, which would account for the cell division disruption observed ( Table 2). Hantson et al. [27] detected an increase in micronuclei in lymphocyte binucleated cells of a patient who had ingested Ti 2 SO 4 and associated it with the ability of metals to induce aneuploidy.    [25] m: minimal risk multiplication factor for the assessment of negative results. For the final statistical diagnosis of all positive (þ) and negative (-) results the nonparametric Mann-Whitney and Wilcoxon U-test with significance levels α and β ¼ 0.05 was used to exclude false positive or negative diagnoses [26]. One side binomial test, significance levels α and β: significant results: þ (α 0.05); no significant results: -(β 0.05). b ST: standard cross. c Clone frequencies per fly divided by the number of cells examined per fly (48,800) provides an estimate of formation frequencies per cell and per cell division in chronic exposure experiments [26]. d BH flies were scored to calculate the percentage of somatic mutation and recombination in this treatment [22].
The present findings in D. melanogaster are important because it has genes that are related to human diseases and conserved functional domains in nucleotides and proteins [34,35,46,47]. Additionally, using this model can demonstrate the genotoxicity of compounds [48,49] as well as alterations in metabolic pathways [50,51,52] given that enzymes related to energy metabolism are common between flies and humans [53]. Therefore, based on the aforementioned Tl effects and the data generated in this study, we consider that the genotoxic and cytotoxic risks [38] due to the high exposure by bioaccumulation of this heavy metal salt in nature, food and humans, mainly in certain regions of the world that have no control over the polluting sources, such as mining [7,54], coal power stations and cement production [31], should be further assessed using more genotoxicity and cytotoxicity studies with other assays to increase our knowledge about the types of risk related to this metal.
Finally, this study evaluated a wide range of concentrations, including the Tl concentrations found in the blood or urine of intoxicated people, which reached values of 8.3-880 μg/L and 68-42,000 μg/L or greater, respectively [19,55,56]. Additionally, we believe that high concentrations must be tested because Tl accumulates in different tissues and may be found at even higher concentrations than those detected in biological fluids.

Conclusions
Survival rates obtained with D. melanogaster precisely demonstrated that different compound effects were related to the amount of Tl þ1 in each compound. Our D. melanogaster wing SMART results showed that Tl 2 SO 4 [600 μM] was genotoxic in all types of spots and induced a high recombinogenic effect, but TlCH 3 COO had no significant genotoxic activity at any concentration tested. Cell division disruption by both salts indicates cytotoxic effects of Tl þ1 , and these findings are consistent with previous reports of the toxicity of this metal in other organisms and assays. Tl and its compounds have been proposed to be more cytotoxic than genotoxic, and the results obtained in this study support this view. However, more genotoxicity studies with other in vivo assays should be performed.

María de los
Angeles Reyes-Rodríguez: Performed the experiments; Analyzed and interpreted the data.
Luis Felipe Santos-Cruz: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data.
Dur an-Díaz: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.
Laura Castañeda-Partida:Contributed reagents, materials, analysis tools or data; Wrote the paper.
Irma Elena Dueñas-García: Conceived and designed the experiments; Performed the experiments.
María Eugenia Heres-Pulido:Conceived and designed the experiments; Performed the experiments; Wrote the paper.
Juan Jos e Rodríguez-Mercado:Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Data availability statement
Data included in article/supplementary material/referenced in article.

Declaration of interests statement
The authors declare no conflict of interest.

Additional information
No additional information is available for this paper. Table 2. Results obtained from analyzing the accumulation frequency in the mwh clones using the Kolmogorov-Smirnov test [24]. Treatments with Tl acetate (TlCH 3 COO) and Tl sulfate (Tl 2 SO 4 ). Milli-Q water and chromium trioxide (CrO 3 ) serve as negative and positive controls, respectively.