Metallothionein alone or in combination with Prussian blue attenuates acute thallium systemic toxicity in rats.

Background: Acute Thallium (Tl) toxicosis is still a health problem, worldwide. Oral administration of Prussian blue (PB) is the antidotal treatment of election. On the other hand, metallothionein (MT) is a low-molecular-weight protein, with high content of cysteines (25–30%). MT is able to chelate metals as an ecient endogenous mechanism of detoxication. It is also a potent antioxidant. Methods: In this study, we tested the ability of MT at two doses (100 and 600 µg/rat), administered alone or in combination with Prussian blue (PB) (50 mg/kg) to decrease thallium (Tl) toxicity. A sublethal dose of Tl (16mg/kg) was injected i.p. to male Wistar rats. Antidotes were administered twice-daily, starting 24h after Tl injection, for 4 days. Tl concentrations were analyzed in body organs and brain regions, 5 days after Tl injection. Results: Results showed a diminution (p<0.05) of Tl concentrations in all organs by effect of PB alone or in combination with MT-100 and MT-600, whereas MT-100 only decreased Tl concentrations in testis, spleen, lung and liver. Likewise, Tl in brain regions was also diminished (p<0.05) by effect of PB and both MT-100 alone or in combination in most of the regions analyzed (p<0.05). The greatest diminution of Tl was achieved when the antidotes were combined. Plasma markers of renal damage, increased after Tl administration. Both PB and MT, either alone or in combination, prevented the raise of renal markers of Tl Toxicity. Conclusions: Our ndings demonstrate that the combined treatment of PB + MT is a good antidotal option against thallotoxicosis.

Once ingested, Tl is transported into the cells by means of the active mechanism of the Na + /K + -ATPase and passively through K + channels, due to the similarity of charge and atomic radius to this monovalent cation [19,20]. It is distributed throughout the organism crossing the blood-brain barrier (BBB) and the placenta barrier [21,22]. Preclinical studies with rats have supported that the half-life of Tl in blood is 72 hours and its highest concentration has been measured in the kidney, testicles and heart [23]. Even when the brain is the organ that reports the lower concentration of Tl, it is considered a target organ of the acute thallotoxicosis [2].
The mechanism of thallium toxicity is based upon the inhibition of enzymes speci c of the glycolysis, Krebs cycle and oxidative phosphorylation. Those inhibitions produce a depletion of the levels of ATP in the cells [24]. Tl has a high a nity for the sulfhydryl groups of amino acids such as cysteine and methionine. The binding a nity of thallium for potassium sites has also been reported, and that binding has been implicated in the alterations of the cellular metabolic pathways dependent on this ion [25][26][27]. Tl + also increases reactive oxygen species (ROS) formation, which in turn play an important role in brain and liver tissue damage and organ dysfunctions by lipid peroxidation (LP) [28][29][30].
Potassium ferri-cyanoferrate II (KFe[Fe(CN) 6 ]), commonly known as Prussian blue (PB), is the antidote of choice against the human thallotoxicosis [31,32]. This chelator agent is administered by oral route, decreasing the absorption of Tl to the enterohepatic circulation by 60-70% and therefore increasing elimination of Tl into feces [33].
Despite its effectiveness as antidote, in severe cases of human thallotoxicosis it´s administration is still ine cient, causing neurological and peripheral aftermath, including renal and hepatic lesions. In order to get a new therapy against Tl poisoning and to increase the e cacy of PB, other chelating agents have been administered alone or in combination with PB, such as; sodium diethyldithiocarbamate, dimercaprol (British Anti-Lewisite), D-penicillamine and the thiol amino acid L-cysteine, as antidotes. However, rat studies have shown that when those chelating agents are administered alone, they cause removal of the metal from the deposit tissues, such as bone, muscle and others, redistributing it to the brain, which aggravates the symptomatology of the intoxication [34][35][36]. On the other hand, the administration of an endogenous metalloprotein, such as metallothionein I (MT-I), has shown a chelating and antioxidant effect (35%) on the liver of rats administrated with 32 mg/kg of thallium acetate [30]. Those in vivo studies suggest the need for a better characterization of the effects on other tissues and peripheral organs, using different doses of MT. In the present study, we characterized the effect of MT-I + MT-II administration to rats treated with a sublethal dose of thallium acetate (16 mg/Kg), either alone or in combination with PB.

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We used male Wistar rats weighing 200 to 250 g; they were obtained from National Institute of Neurology and Neurosurgery, México. Rats were housed four per acrylic cage and maintained under standard laboratory conditions (12:12 light-dark cycles, 23 ± 2 °C) and 40% relative humidity, had free access to food and water. All animals were

Thallium analysis
The levels of Tl in kidney, testis, spleen, heart, lung, and liver (peripherals organs) and hippocampus, striatum, hypothalamus, midbrain, cerebellum and cortex (brain regions) were analyzed by atomic absorptions spectrophotometry using the analytical conditions reported previously by our research group [23]. Tissue samples were acid-digested in metal-free concentrated nitric acid (Suprapur Merck) and analyzed using an atomic absorption spectrophotometer (Perkin Elmer 3110) equipped with a graphite furnace (Perkin Elmer HGA-600) and auto sampler (AS-60). All of the material used (polypropylene tubs and tips) in Tl analysis was previously washed and immersed in a 3% nitric acid solution for 24 h and rinsed with deionized water to avoid external Tl contamination. The quanti cation of metal in biological tissues was performed using a calibration curve constructed with a thallium standard (Perkin Elmer). Results of Tl content in peripheral organs and brain regions are expressed as µg of thallium per gram of wet tissue.

Biochemical markers of liver and renal damage
To determine biochemical markers of renal and hepatic functioning, blood samples were withdrawn and stored in clean tubes. After centrifugation at 1500 g, for 10 min, serum was stored at -20° C until analysis. Creatinine, urea, alanine amino-transferase (ALT), aspartate amino-transferase (AST) and total proteins (TP) were measured using Cobas c 111 autoanalizer (Roche, USA), with commercially available kits (Roche diagnostics).

Statistics
An exploratory analysis of the data was performed to determine normal distribution (Kolmogorov-Smirnov's test) and homogeneity of variances (Levene's test). In order to obtain normal distribution of the data, a logarithmic transformation was applied and then, logarithmic values were analyzed using one-way ANOVA followed by Dunnett's Page 5/13 test. All analyses were performed using an SPSS 22.0 software (Chicago, Illinois, USA). Differences were considered statistically signi cant when p < 0.05

Thallium levels in peripheral organs
Thallium levels in peripheral organs after antidotal treatment are shown in Fig. 1. In panel A, the mean ± one SEM of kidney thallium levels (Tl) are shown; values are expressed as µg of Tl per gram of wet tissue. As can be observed, kidney Tl in the control group (C) averaged 11.97 ± 2.21 (n = 11), while the group of Tl with PB were 5.44 ± 0.64 (n = 13), showing a 54% of diminution, as compared to group C average (p < 0.05). Likewise, the Tl plus MT-100 group averaged 10.05 ± 1.40 (n = 5), a diminution of 16% as compared to group C. Also, the mean of the group PB + MT-100 was 2.34 ± 0.25, in this group, the highest decrease was observed, 80% vs C group (n = 5) (p < 0.05). The group MT- showed the greatest decrease of Tl, as compared to group C levels (71 and 73%, respectively). All groups were different from C (p < 0.05) except treatment with MT-600.
Tl levels measured in the heart are shown in panel D. The mean value of group C was 4.72 ± 0.40, while in the PB group the value was 1.73, 63% lower than group C. Likewise, in the group MT-100, the mean was 3.91 ± 0.37, a diminution of 17% vs group C. Finally, the combination of antidotes, groups PB + MT-100 and PB + MT-600, produced the greatest decrease of Tl levels, 74% (1.24 ± 0.15) and 68% (1.53 ± 0.11) vs group C, respectively. In this organ only PB antidotes combined with MT at 100 and 600 were different when compared with group C (p < 0.05).
Panel E shows the results of lung Tl. The mean of group C was 4.30 ± 0.55, while the PB group averaged 1.61 ± 0.13; a reduction of 63% vs group C. Likewise, in the MT-100 group we observed a mean of 2.41 ± 0.35, 44% lower than group C average. Again, the combination of antidotes showed the greatest decrease as compared to the control group average, showing diminutions of 81% (PB + MT-100) and 66% (PB + MT-600), respectively. All groups were different from C (p < 0.05) except treatment with MT-600.
Liver Tl concentrations are shown in panel F. The mean value of group C was 4.18 ± 0.050, while the PB group averaged 1.21 ± 0.09, a reduction of 71% as compared to group C. MT-100 and MT-600 groups showed mean values of 2.73 ± 0.09 and 2.96 ± 0.35, respectively, diminutions of 35 and 29% both vs group C, respectively. Again, the combination of antidotes had the greatest reduction of Tl content, which was 78% and 74%, respectively, as compared to the average of group C. All groups were different from C (p < 0.05).
Thallium levels in brain regions Figure 2 shows the Tl levels in different brain regions. Panel A shows the means ± SEM of Tl in the hippocampus; the results are expressed as µg of Tl per gram of wet tissue. As observed, control group (C) Tl averaged 2.13 ± 0.20 (n = 11), while PB group the mean was 0.72 ± 0.05 (n = 13), 66% lower than group C (p < 0.05), meanwhile, the mean was 1.92 ± 0.034 (n = 5) in the group MT-100. Likewise, mean Tl of group PB + MT-100 was 0.49 ± 0.09; this group showed the lowest decrease of Tl observed, 77% (n = 5), when compared with group C (p < 0.05). Group MT-600 averaged 1.86 ± 0.020 (n = 7), nally in the group PB + MT-600, the average was 0.72 ± 0.05 (n = 5), showing a diminution of 66% vs group C (p < 0.05). Only PB antidotes combined with MT at 100 and 600 were different when compared with group C (p < 0.05).
Panel B shows the mean Tl levels in the striatum. Tl concentrations averaged 2.05 ± 0.17 in group C, while in the PB group the mean was 0.71 ± 0.04, 65% lower than that observed in group C (p < 0.05). Meanwhile, group MT-100 averaged 1.47 ± 0.05, a 28% reduction compared to group C mean (p < 0.05). The combination of antidotes PB + MT-100 and PB + MT-600 showed the highest decrease of Tl levels vs group C, with reductions of 75 and 66%, respectively (p < 0.05). All groups were different from C (p < 0.05) except treatment with MT-600.
Panel C shows the mean Tl concentrations in the hypothalamus. Tl in Group C averaged 2.19 ± 0.21, while the mean value of group PB was 0.70 ± 0.04; 68% lower than the mean of group C. On the other hand, groups MT-100 and MT-600 averaged 1.61 ± 0.21 and 1.80 ± 0.21, showing a diminution of 26 and 18%, respectively, when compared to group. Again, the combination of antidotes showed the greatest Tl reduction as compared to the mean of group C: 77% (PB + MT-100) and 67% (PB + MT-600). All groups were different from C (p < 0.05) except treatment with MT-600.
Tl levels in the midbrain are shown in panel D. Group C showed a mean of 1.71 ± 0.017, while PB group averaged 0.61 ± 0.04; 64% lower than group C. Likewise, the mean of MT-100 group was 1.18 ± 0.09; 31% lower than group C.
Panel E shows Tl levels measured in cerebellum. The mean value of group C was 1.56 ± 0. 13, while in the PB group the mean value was 0.52 ± 0.04; 66% lower than group C. Groups MT-100 and MT-600 showed mean values of 1.21 ± 0.09 and 1.23 ± 0.16, respectively, Tl diminutions of 22 and 21%, respectively, as compared to group C. Finally, the combination of antidotes showed the greatest reduction as compare to control group mean (0.40 ± 0.06 and 0.40 ± 0.03 respectively) or approximately 74% of diminution for both groups. All groups were different from C (p < 0.05).
Panel F shows Tl levels in the cortex. There is a Tl concentration of 1.63 ± 0.13 in group C, while in the PB group the average value was 0.50 ± 0.03, 69% lower than that observed in group C (p < 0.05). Groups MT-100 and MT-600 showed means of 1.27 ± 0.07 and 1.29 ± 0.15, 22 and 21% lower than group C, respectively (p < 0.05). Similarly, the combination of antidotes with the different doses of MT (PB + MT-100 and PB + MT-600) showed the highest reduction of the levels of Tl, as compared to group C. That reduction was 75 and 69%, respectively (p < 0.05). All groups were different from C (p < 0.05).

Renal and hepatic biochemical markers
The results of antidotal treatments on renal and hepatic thallium toxicity markers are shown in the Table 1. We observed a signi cant increase in serum creatinine and urea of 2.4 and 3.6-fold, respectively, by effect of Tl intoxication (Group C), as compared healthy control (HC) group. No alterations in those markers were found in all of the groups receiving treatment either with PB or MT antidotes alone or in combination, as compared to HC averages.

Discussion
The results presented here, are in agreement with what was previously published by Montes et al. [37]. PB is an effective antidote of election for thallotoxicosis. The mechanism of Tl decorporation by PB is well-known; during its passage through the intestine, PB exchange K + by Tl + ions on the surface of the lattice of K Fe (Fe (CN) 6 ) (PB). Once exchanged, PB forms a stable compound with thallium, that is in turn excreted into the feces, accelerating Tl decorporation [38]. MT, on the other hand, has been proposed as a chelating antidote for Tl in thallotoxicosis [30]. In our hands, MT was moderately effective as an antidotal treatment against acute thallium systemic poisoning, when given alone (100 or 600 µg) or in combination with PB. MT reduced the thallium levels in tissues of metabolic importance, such as lung and liver, and also in the two brain regions containing the highest concentrations of thallium, such as hypothalamus and cerebellum. The systemic effect of MT did not produce an undesirable redistribution of Tl to the central nervous system (target organ of thallotoxicosis), as it has been observed after the administration of other chelating agents, such as D-penicillamine or endogenous thiols such as L-cysteine [35,36], as MT is unable to cross the blood-brain-barrier [39] then, it is possible that a putative Tl-MT complex may not be able to cross to the brain to produce a redistribution of the metal. Particularly, the administration of 600 µg of MT showed a systemic, rather than cerebral, increase of renal Tl concentrations produced by a redistribution of the metal to the kidney. Interestingly, this renal thallium accumulation did not induce signi cant kidney damage, as the levels of creatinine (a biomarker of kidney dysfunction) were similar to the HC group values. Tl decorporation is more evident in the kidney and hippocampus, when MT is administered simultaneously to PB (PB + MT-100 and 600 groups). This additive pharmacological effect of MT in combination with PB has been reported for others exogenous chelating agents studied experimentally in vivo [37]. Taken together, results of the present study suggest a chelating effect of MT on Tl [30,40,41]. Exogenous administration of MT has been reported to be protective against heavy metals' intoxication [42]. In this study, hepatic Tl accumulation decreased at both doses of MT (100 or 600 µg), as reported by Kılıç,and Kutlu [30] in a similar rat model of acute thallotoxicosis. It is important to remark that most of the effects of MT were not dose-dependent in body organs and brain regions, suggesting that the lower dose of MT (100 µg/rat) is achieving the maximal effect of the protein on Tl decorporation. In fact, the highest dose of MT employed (600 µg/rat), was the one that produced a redistribution effect on kidney Tl. organ concentrations of thallium without increasing the metal concentration of the brain. Because this effect of decorporation of Tl by MT was modest, it is possible that other MT-induced mechanisms are also participating to prevent Tl-induced damage. The antioxidant actions of MT are well-known and have been suggested as an advantage of the protein compared to other chelating agents [43]. As Tl is able to induce free radicals' overproduction with consequent lipid peroxidation and formation of reactive oxygen species [29], an antioxidant action of MT may be an additional protective mechanism to explain why the protein protected from the renal effects of acute thallium poisoning, in spite of the redistribution of Tl to the kidney. The increased renal Tl concentrations observed in the group of animals treated with MT-600 may also be the re ection of a higher excretion of the metal by this organ. This remains speculative until future experiments.
The present study contributes with relevant pre-clinical data to evaluate the pharmacological e cacy of MT alone or in combination with PB for the antidotal treatment of acute thallotoxicosis. For most of the organs and brain regions, both antidotes showed a summation of effects when combined, with no statistically signi cant interaction. That result indicates that PB and MT are acting by different, unrelated mechanisms. As MT is unable to cross the bloodbrain barrier e ciently, thallium is been chelated only in the periphery, without redistribution to the brain. This represents an advantage of the protein over other chelating agents that aggravate thallium toxicity when administered alone [35,39].

Conclusion
The results presented in this work reinforces the idea of a combined treatment using PB plus a chelator as an effective antidote schedule against acute thallium toxicity, as suggested by the U.S. Food and Drug Administration [31,32]. Also, our results encourage the investigation on the participation of endogenous mechanisms of protection against acute thallotoxicosis, such as those exerted by MT.

Declarations
Ethics approval and consent to participate The guidelines for animal care was approved by the biology committee, in accordance with the o cial Mexican standard, that established internationally and nationally by the Mexican O cial Standard NOM-062- ZOO-1999 (which observes technical speci cations for the production, care and use of laboratory animals) and the guidelines for Care and Use of Laboratory Animals of the National Institutes of Health (USA). Finally, the protocol was registered with the National Institute of Neurology and Neurosurgery with the number 75/12.

Consent for publication
Not applicable.

Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request. 100 or 600 µg/ rat; PB + MT-100 and PB + MT-600 groups: Combined treatments of PB and MT to 100 and 600 µg/ rat respectively. One-way ANOVA followed by the Dunnett's test. * p< 0.05 vs C group.