Cytotoxic, Genotoxic, and Mutagenic Effects of Organotellurane RF07 in Female Swiss Mice (Mus musculus)

TL;DR

This study investigates the toxic effects of an organotellurane compound in female mice, revealing DNA damage and other harmful impacts.

Contribution

This is the first study to explore the cytotoxic, genotoxic, and mutagenic effects of RF07 in mice.

Findings

RF07 increased DNA damage and clastogenic/aneugenic effects in mice.

The compound caused cytotoxicity in bone marrow erythroid precursors and myelosuppression.

RF07 also induced hepatotoxicity and altered erythrocyte ratios.

Abstract

The organotellurane RF07 (RF07) is an organic compound containing tellurium, a rare semi‐metal, and its leishmanicidal and antimalarial activity has already been reported in previous studies. This study evaluated the toxic effects of RF07 at 0.42, 21.37, and 42.75 mg/kg using comet assay, micronucleus test, and hematological/biochemical tests in Mus musculus. Female Swiss mice received intraperitoneal RF07. Blood was collected at 24, 48, and 72 h, followed by euthanasia for bone marrow analysis. Results showed RF07 increased DNA damage, clastogenic/aneugenic effects via micronuclei formation, and cytotoxicity through apoptosis in bone marrow erythroid precursors and altering the ratio of polychromatic to normochromatic erythrocytes. RF07 also caused myelosuppression and hepatotoxicity. To our knowledge, this is the first study explore these toxic effects. However, the mechanism remains unclear, requiring further investigation.

Figures

The chemical structure of RF07.

Genotoxic effects of RF07 in mouse blood cells and the ability to repair DNA damage evaluated at different exposure times. [NC: negative control. PC: positive control (CPA at 2 mg/kg). Values are mean ± SD (n = 5). ANOVA‐One‐way and post‐test of Tukey. p < 0.05 compared to the aNC group, bCP at the same exposure time; and cdifferent when compared to 24 and 48 h at 0.42 mg/kg; ddifferent when compared to 24 and 48 h at 21.37 mg/kg; and different when compared to 24 and 48 h at 42.75 mg/kg.].

Quantitative variation of damage types (0–4) for the comet test in mouse peripheral blood cells treated with the RF07 at 24, 48, and 72 h of exposure. [NC: negative control. PC: positive control (CPA a 2 mg/kg). Values are mean ± SD (n = 5). ANOVA‐One‐way post‐test Tukey. p < 0.05 when compared to aNC, and bPC.].

Photomicrographic profile of mouse blood cells exposed to the RF07 showing the types of damage and cells in apoptosis. [A. 0.42 mg/kg; B. 21.37 mg/kg; C. 42.75 mg/kg. T2: Type 2 damage. T3: Type 3 damage. T4: Type 4 damage. Ap: Apoptosis. Magnification at 400× (Giemsa staining).].

Tables

TABLE 1. Evaluation of the percentage apoptosis in mouse blood cells caused by the RF07 at different exposure time.

Treatments	Dose	24 h	48 h	72 h
NC (0.5% DMSO)	10 mL/kg	4.0 ± 2.3	4.0 ± 1.4	3.0 ± 2.4
PC (Cyclophosphamide)	2 mg/kg	31.0 ± 2.5 a	35.7 ± 2.9 a	41.5 ± 3.8 a #≠
RF07	0.42 mg/kg	39.2 ± 6.2 ab	32.5 ± 3.6 a	13.5 ± 1.2 ab#≠
	21.37 mg/kg	41.25 ± 1.2 ab	32.2 ± 1.5 a #	24.0 ± 1.4 ab #≠
	42.75 mg/kg	49.7 ± 6.7 ab	31.2 ± 1.8 a #	22.0 ± 1.6 ab #

TABLE 2. Evaluation of the cytotoxic and mutagenic activity of RF07 in mouse bone marrow cells through micronucleus (MN) test.

	Mutagenicity e cytotoxicity
Treatments	Doses	Micronuclei	EPC/(PCE+ENC)
NC (DMSO)	0.5%	1.95 ± 0.60	0.90 ± 0.02
PC (Cyclophosphamide)	2 mg/kg	33.25 ± 2.63 a	0.43 ± 0.02 a
	0.42 mg/kg	3.75 ± 1.93 b	0.86 ± 0.01 b
RF07	21.37 mg/kg	26.37 ± 1.88 a	0.64 ± 0.01 [Link] , b
	42.75 mg/kg	30.87 ± 1.26 a	0.60 ± 0.02 [Link] , b

TABLE 3. Hematological and biochemical parameters of mice treated with RF07.

Parameters	NC	PC	RF07 0.42 mg/kg	RF07 21.37 mg/kg	RF07 42.75 mg/kg
Hematological
Leukocytes Total	4552.5 ± 405.0	2392.5 ± 478.7 a	3025 ± 818.5 a , b	2550 ± 556.0 a	2675 ± 292.0 a
Neu	55.3 ± 1.9	24.0 ± 0.9 a	7.5 ± 1.8 a , a	4.1 ± 2.7 a , a	5.8 ± 8.9 a , a
Lin	36.8 ± 1.4	71.5 ± 5.3 a	67.5 ±3.1 a	74.1 ± 4.2 a	72.5 ± 10.3 a
Mon	6.1 ± 0.9	2.2 ± 4.5	23.4 ±2.8 a	20.4 ± 2.8 a	20.3 ± 1.7 a
Eos	0.4 ± 0.1	1.1 ± 0.1	0.5 ± 0.1	0.3 ± 0.1	0.5 ± 0.5
Bas	1.1 ± 0.2	1.0 ± 0.0	1.0 ± 0.1	0.9 ± 0.1	1.0 ± 0.6
RBC	8.1 ±0.9	7.1 ± 1.2	10.2 ± 2.9	4.80 ± 2.6	6.8 ± 0.8
Hb	13.1 ±1.2	12.8 ±0.8	11.2 ± 3.5	5.4 ± 2.6 a	7.3 ± 0.3
Ht	45.1 ± 5.4	36.8 ± 6.1	50.9 ± 14.4	22.9 ± 12.1 a	32.9 ± 0.9 a
MCV	56.0 ±0.9	50.8 ± 0.9	49.2 ± 2.2	47.2 ± 1.9	48.5 ± 2.5
MHC	17.0 ±0.3	17.4 ± 0.6	11.0 ± 1.3	11.0 ± 1.0	11.0 ± 0.7
MCHC	31.8 ±1.8	32.9 ± 1.2	22.3 ± 1.6	23.1 ± 1.4	22.7 ± 2.3
RDW	13.9 ±0.3	13.7 ± 1.6	19.6 ± 2.6	21.0 ± 1.4	21.3 ± 0.2
PLT	787.7 ±149.6	691.7 ± 138.0	937.5 ± 428.2	861.7 ± 398.9	1186.5 ± 37.1 a , a

Full text

Introduction

1

Tellurium (Te) is a rare non‐radioactive semi‐metal that belongs to group 16 in the periodic table and is chemically related to selenium and sulfur. It is regarded as a non‐essential trace element. Because of its properties, Te is well‐known as a component of metal alloys. It is often used as an additive to steel. Moreover, it is alloyed with aluminum, copper, lead, or tin. In addition, it is used in solar panels and some types of magnetic disks [1, 2, 3]. Based on their oxidation states, Te‐derived compounds are divided into two groups: (i) divalent derivatives, represented by compounds analogous to alcohols and thiols, (ii) hypervalent compounds with oxidation states of +4 and +6, as in inorganic or organic telluranes and pertelluranes, respectively [1].

Cunha, Rodrigo L.O.R. et al. [4] demonstrated that telluranes act as potent inhibitors of cathepsin B. This inhibitory effect was further verified for cathepsins L, S, and K [5]. Cathepsin B plays a crucial role in tumor invasion and metastasis, and is frequently over‐expressed in various cancer cell types [6] Thus, organotelluranes exhibit a promising alternative in the treatment of cancer [4, 5] Similarly, the small nontoxic tellurium compound AS‐101 (a) exhibits immunomodulatory activity by inhibiting the function of some subtypes of specific lymphocytes and leukocyte integrins, (b) induces the production of interleukin (IL) ‐2, and (c) further promotes the restoration of regulatory cells of the immune system. In addition to their anticancer activities, organotellurium compounds exhibit anti‐inflammatory and antimicrobial properties [7, 8, 9], as well as a selective inhibitory effect on cysteine protease enzymes [4, 5, 10, 11]. Among them, the organotellurane RF07 is an organic compound that contains tellurium (Te) in its molecular structure (Figure 1).

The chemical structure of RF07.

Scientific reports suggest that RF07 has interesting and diverse biological activities, such as leishmanicidal and anti‐malarial activities [12, 13, 14]. However, the toxicological profile of this particular compound has only been explored in golden hamsters [12]. Acute intoxication with elemental tellurium or its inorganic derivatives has been found to cause headache, drowsiness, changes in heart rate, nausea, strong‐smelling breath, and urination with a strong odor [1, 3]. In the +4 oxidation state, tellurium compounds interact with reactive sulfhydryl groups in purified enzymes [4, 5, 15]. In isolated mitochondria, organotelluranes target inner membrane proteins, inducing the opening of the mitochondrial permeability transition pore [16, 17], and also react with cytoplasmic proteases [14]. Based on incomplete data for the toxicity of tellurium, we evaluated the toxic, cytotoxic, genotoxic, and mutagenic effects of RF07 using the comet assay, micronucleus test, and determination of hematological and biochemical parameters in Mus musculus.

MATERIALS AND METHODS

2

Chemicals and Instrumental

2.1

Cyclophosphamide was purchased from Sigma AldrichTM (St. Louis, MO, USA). All chemicals employed in this study were of analytical grade and used as indicated by their manufacturers.

For the analysis were used the Optical Microscope (BIOPTIKA) for most of the assays, Automatic Hematology Analyzer Mindray BC‐2800 Model (USA) for the hematological assays and Bioplus Biochemical Semiautomatic Analyzer BIO‐2000 IL Model (BR) for the biochemical ones.

Synthesis and Determination of Test Doses of RF07

2.2

Synthesis and characterization of RF07 were accomplished at the Center for Natural and Human Sciences (Universidade Federal do ABC, Santo André, São Paulo). Briefly, RF07 was synthesized by the reaction of p‐methoxyphenyltellurium trichloride with 1‐ethynyl‐1‐cyclohexanol under reflux in benzene, as described by [18]. Desired product RF07 was initially purified by fractional crystallization, and then by re‐crystallization. RF07 was characterized by spectroscopic techniques such as infrared, ^1^H, ^13^C, and ^125^Te NMR spectra, and by elemental analysis (C, H, and N). These techniques confirmed both the identity and the purity of the synthesized compound. On the other hand, RF07 doses (0.42; 21.37, and 42.75 mg/kg), prepared in DMSO and diluted in aqueous buffer, used in this study were selected according to [19].

Treatment of the Animals

2.3

Test protocol (CEEA 039/17) was approved by the Animal Experimentation Ethics Committe of the Universidade Federal do Piauí (UFPI). A total of 25 adult 2‐month old female Swiss mice (Mus musculus) (25–30 g) were obtained from the Central Animal House of the UFPI. Animals were kept under standard monitored conditions of temperature 24°C ± 1°C, with free access to pellets (Purina) and water, and kept in a 12 h light/dark cycle. Animals were divided into 5 groups with 5 animals each, and were subjected to different acute doses of RF07 (0.42, 21.37, and 42.75 mg/kg, intraperitoneal [i.p.]). DMSO was used as a negative control, as it is an organic compound widely used as a solvent, and was applied at a concentration of 0.5%. Cyclophosphamide (CPA), used as an alkylating and antineoplastic agent in cancer treatment, was administered at a dose of 2 mg/kg (i.p.) and used as a positive control. The exposure times adopted were 24, 48, and 72 h, with only one administration on the first day [20].

Comet Assay by Using Peripheral Blood of the Animals

2.4

After administration of RF07, 0.5 mL caudal peripheral blood from each animal was collected at 24, 48, and 72 h. The comet assay was performed according to the method of [20, 21]. Briefly, aliquots containing 10 µL of the blood samples were mixed with a thin layer of “low melting” agarose 0.75% (90 µL) and placed on pre‐coated slides with “normal melting” agarose (1.5%). The slides were covered with coverslips (24 × 60 mm) and kept at room temperature (29°C) until solidified. The coverslips were removed and the slides were subjected to the lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X‐100, 10% DMSO, pH 10) at 4°C and protected from light for the next 72 h. Then, electrophoresis was performed (1 mM EDTA, 300 mM NaOH, pH >13) at a temperature of 4°C, and conducted at 25 V and 300 mA for 20 min. Subsequently, the slides were neutralized with 0.4 M Tris solution at pH 7.5 for 5 min and then exposed for 10 min to the fixative solution (trichloroacetic acid 15%, zinc sulphate heptahydrate 5%, and glycerol 5%). After drying, the slides were stained with 10% Giemsa solution and washed 3 times with distilled water, and dried at room temperature.

For each group (n = 5), 100 cells were photographed per slide, having two slides per animal. Evaluation was carried out from analysis of the degradation size and intensity, and the dragging of genetic material, in which 5 damage classes were identified: class 0, genetic material without damage or intact; class 1, 2, and 3 are between intact and maximum damage, whereas class 4 represents the maximum damage. Finally, the damage index (DI) was determined, with values ranging from 0 to 400, according to the formula: DI = Σ (0 × C0 + 1 × C1 + 2 × C2 + 3 × C3 + 4 × C4), where C0, C1, C2, C3, and C4 correspond to the number of nuclei assigned to classes 0, 1, 2, 3, and 4, respectively, out of 100 nuclei evaluated. The frequency of damage (FD) was also calculated according to the formula: FD (%) = 100%—Class 0 damage (C0); numbers varied from 0% to 100%. For the evaluation of apoptotic cells, 200 cells per animal were counted and results were expressed in percentage [20, 21].

Micronucleus Test in Mouse Bone Marrow Cells

2.5

The micronucleus (MN) test was conducted by employing mouse bone marrow cells, according to the method described by [22]. After euthanasia by an overdose of anesthetic, the two femoral bones of each rodent were removed and washed internally with fetal bovine serum (20%). Then, 25 µL of the cell suspension was placed on glass slides for fixation and staining with Giemsa 10%. To determine the frequency of micronucleated polychromatic erythrocytes (MNPCE), 2000 polychromatic erythrocytes (PCE) per animal were observed, totaling 10,000 cells per treatment. To determine cytotoxicity, a total of 400 erythrocytes (PCE + ENC) per animal (200 / slide) and 2000 cells per treatment were counted, two slides per animal. In this case, the erythrocyte ratio was calculated by [PCE/(PCE + ENC)]. For this analysis an optical microscope with 100× magnification was used.

Hematological and Biochemical Analyses

2.6

For hematological analysis, samples were collected in EDTA anti‐coagulant tubes to evaluate the following parameters: hemoglobin, hematocrit, hematimetric index, leukocytes, and platelets. Samples were stored for up to 48 h at −4°C. Assays were performed with the aid of the Automatic Hematology Analyzer Mindray BC‐2800 Model (USA).

For biochemical analysis, whole blood samples were collected and stored in test tubes and then centrifuged at 3500 rpm for 10 min on room temperature. The following parameters were analyzed: urea, creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Samples were stored for up to 48 h at −4°C. Assays were achieved by means of a Bioplus Biochemical Semiautomatic Analyzer BIO‐2000 IL Model (BR).

Statistical Analysis

2.7

Data obtained were subjected to one‐way and two‐way analysis of variance (ANOVA), and results are expressed as the mean ± standard deviation (SD). Statistical analysis was performed with the aid of Tukey post‐hoc test, using the GraphPad Prism program (version: 7.00 for Windows, GraphPad Software, San Diego California U.S.A.); differences were considered significant at p ≤ 0.05 at 95% confidence interval.

RESULTS

3

Genotoxicity Evaluation of RF07

3.1

The results of our study on the genotoxic effect of RF07 are shown in Figure 2. Our findings revealed that treatment of animals with RF07 caused significant genotoxicity with an increase in DI (Figure 2A) and FD (Figure 2B) at all test doses within the exposure times (ET) of 24, 48, and 72 h when compared with the negative control (NC) group. In the first 24 h of exposure, RF07 exerted a substantial genotoxic effect as shown by the increase in DI and FD in comparison with the positive control (CPA). However, at 48 h, DI was only statistically higher at the highest dose (42.75 mg/kg), whereas for the last exposure time (72 h), RF07 was statistically less genotoxic than CPA group (Figure 2). This could be due to the natural DNA repair capacity of the test system at 72 h. However, the genotoxic effect of RF07 at 72 h was still higher than that of the NC group.

Genotoxic effects of RF07 in mouse blood cells and the ability to repair DNA damage evaluated at different exposure times. [NC: negative control. PC: positive control (CPA at 2 mg/kg). Values are mean ± SD (n = 5). ANOVA‐One‐way and post‐test of Tukey. p < 0.05 compared to the aNC group, bCP at the same exposure time; and cdifferent when compared to 24 and 48 h at 0.42 mg/kg; ddifferent when compared to 24 and 48 h at 21.37 mg/kg; and different when compared to 24 and 48 h at 42.75 mg/kg.].

Quantitative variation of the types of damage was also evaluated, where RF07 induced significant damage of type 2, 3, and 4 in the mouse cells in comparison to the NC group at the three doses used. However, when compared with the CPA group (positive control), the test substance caused a higher incidence of type 3 damage at the lowest (0.42 mg/kg) and intermediate (21.37 mg/kg) doses, and type 4 damage at the highest dose (42.75 mg/kg) after 24 h of exposure. For ET at 48 and 72 h, RF07 caused more damage (type 4), to a lesser extent than what was observed at 24 h, when compared to CPA group as shown in Figure 3B,C.

Quantitative variation of damage types (0–4) for the comet test in mouse peripheral blood cells treated with the RF07 at 24, 48, and 72 h of exposure. [NC: negative control. PC: positive control (CPA a 2 mg/kg). Values are mean ± SD (n = 5). ANOVA‐One‐way post‐test Tukey. p < 0.05 when compared to aNC, and bPC.].

Table 1 and Figure 4 show the results of apoptosis in cells treated with RF07 at all tested doses and exposure times. These findings, revealed by photomicrographic analysis, are compared with the NC group. In addition, in comparison to CPA group, the tested substance also induced apoptotic cell death at 24 and 72 h. However, the apoptosis index at 24 h was found to be higher than that of 48 and 72 h of exposure to RF07 (Table 1 and Figure 3).

Photomicrographic profile of mouse blood cells exposed to the RF07 showing the types of damage and cells in apoptosis. [A. 0.42 mg/kg; B. 21.37 mg/kg; C. 42.75 mg/kg. T2: Type 2 damage. T3: Type 3 damage. T4: Type 4 damage. Ap: Apoptosis. Magnification at 400× (Giemsa staining).].

Cytotoxicity and Mutagenicity of RF07

3.2

We employed mouse bone marrow cells in the micronucleus (MN) test to evaluate the cytotoxicity and mutagenicity of RF07. Our results demonstrated that, among the analyzed doses, treatment with 21.37 and 42.75 mg/kg significantly (p < 0.05) reduced the PCE/(PCE + ENC) ratio in mouse bone marrow cells compared with the negative control group. In addition, there was a statistically significant difference when compared to the CPA‐treated group, which demonstrates the cytotoxic effect of this ogano‐tellurium compound. RF07 at the two highest concentrations (21.37 and 42.75 mg/kg) induced significant (p < 0.05) mutagenicity in comparison to the NC group as shown in Table 2.

Evaluation of the Biochemical and Hematological Parameters

3.3

Toxic effects caused by RF07 in mice were assessed by analyzing the plasma levels of urea, creatinine, AST, and ALT in comparison to biochemical parameters and plasma levels related to erythrogram, leukogram, and plaquetogram of hematological tests. Regarding the hematological parameters, results showed that leukopenia was significant (p < 0.05), accompanied by an increase in neutropenia, lymphocytosis, and monocytosis, at the three doses used when compared to the NC group. However, with respect to the other hematological parameters analyzed, there were no significant alterations by this test substance. For the biochemical parameters, significant alterations were only observed for AST (TGO) at the highest concentrations of 21.37 and 42.75 mg/kg used as given in Table 3.

DISCUSSION

4

Although there is an increasing use of tellurium‐containing organic compounds in chemistry and biochemistry, studies of the toxicity of these substances are still scarce in the literature. Along this line, it is known that elemental Te and its inorganic salts or organic compounds, such as organtellurans, tend to be quite toxic, however, the intensity of this toxicity depends on both the structure of the compound, the dose/concentration used, and the types of test system [23]. Thus, in the present study, we used an in vivo experimental model to evaluate the toxic, cytotoxic, genotoxic, and mutagenic effects of the tellurium‐derived RF07.

Genotoxicity observed at the three evaluated doses of RF07 in the comet assay and MN test agrees with the findings of Meinerz and coworkers [24]. These authors used diphenyl ditelluride (PhTe)2, which showed a genotoxic effect at 500 µmol/kg using the comet assay. To date, the proposed mechanism explaining the toxicity of tellurium compounds involves their ability to oxidize sulfhydryl groups, which leads to enzyme inactivation and/or a decrease in the concentration of non‐protein sulfhydryl molecules, such as reduced glutathione (GSH) [25, 26]. Indeed, it has been demonstrated that some divalent organotellurium compounds inhibit sulfhydryl‐containing enzymes, including δ‐aminolevulinate dehydratase, [26, 27] squalene monooxygenase, and Na^+^/K^+^‐ATPase [26, 27].

In addition, doses close to LD_50_, of (S)‐dimethyl 2‐(3‐(phenylalkyl) propanamide) succinate produced mutagenic effects in adult male mice, as shown by increased MN frequency and significant genotoxic effects produced on the leukocytes of mice. These effects may be related to the pro‐oxidant activity exhibited by high concentration/doses of organotellurium compounds. However, the molecular mechanism involved is still unknown, but may be related to the Te reactivity and its propensity to be oxidized to Te (IV), which may potentially interact with nucleophilic thiols in biomolecules. In this context, research findings have recently demonstrated that exposure to diphenyl diselenide causes DNA damage and GSH depletion in different tissues of mice [24, 29]. Knowledge of the relative toxicity of organic compounds containing tellurium and selenium is important due to the use of organochalcogenides as possible components of a variety of pharmacological agents [1, 29].

In a similar fashion, treatment of adult rats with diphenyl ditelluride (PhTe)2 at doses of 10 and 50 µmol/kg for 7 consecutive days induced noticeable signs of toxicity, including body weight loss, behavioral alterations, and increased lipid peroxidation in the brain. PhTe_2_ also reduced the activity of several key antioxidant enzymes in the brain, including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx), and thioredoxin reductase (TrxR) [30]. In addition, work published by different research groups [31, 32] indicated that tellurium compounds are capable of inhibiting thioredoxin reductase (TxrR) and cause cytotoxicity (in vitro), which contributes to the results of apoptosis and erythrocyte ratio observed in this present study; (PhTe)2 oxidizes dithiothreitol (DTT). Organotellurium compounds inhibit the activity of the sulfhydryl δ‐ALA‐D enzyme, which is considered one of their main targets, since it contains two cysteine residues in its active site that are easily oxidized both in vitro and in vivo [26, 33].

Furthermore, our results showed that RF07 exhibits significant cytotoxicity in the comet assay, as demonstrated by the high percentage of apoptosis and by the marked reduction in the erythrocyte ratio observed after treatment with different doses of this compound. These findings indicate that RF07 may interfere with cellular homeostasis, leading to DNA damage and cell death. Similar cytotoxic and pro‐apoptotic effects have been reported for other organotellurium compounds. Sailer et al. [34] demonstrated that 2,2′‐dimethoxydiphenyl ditelluride and 2,2′‐diamino‐3,3′,5,5′‐tetramethyldiphenyl ditelluride induced apoptotic cell death in HL‐60 cells in a dose‐dependent manner, causing cell‐cycle arrest at the G1/S transition. Likewise, Abondanza et al. [7] reported that the organotellurium compound RT‐04 triggered apoptosis through modulation of Bcl‐2 expression in human leukemia cells, reinforcing the role of tellurium derivatives as redox‐active agents capable of disrupting mitochondrial balance. In addition, other studies described that hypervalent tellurium compounds irreversibly inhibit cysteine proteases and may promote cytotoxicity through thiol oxidation, leading to apoptotic pathways [1, 5]. These studies support our findings and suggest that RF07 may act through similar mechanisms, although structural differences could account for the variations observed in genotoxic and cytotoxic intensity.

The mechanism by which Te‐containing compounds induce apoptotic cell death is unknown. However, based on the chemical similarities between Te and Se, the intracellular mechanism of action may be comparable to that proposed for Se‐containing compounds. It is believed that inorganic and organic forms of Se can function as redox switches in numerous biochemical pathways, leading to rupture of intracellular chemistry and apoptotic cell death (35). Our results are in agreement with studies related to diphenyl ditelluride, 3,3’‐diaminodiphenyl ditelluride, and 4,4’‐di‐isopropildifenil ditelluride on HL‐60 cells; these compounds induced apoptotic cell death in a time‐ and dose‐dependent manner. This apoptosis was verified by three different analytical methods: fluorescence microscopy, gel electrophoresis, and flow cytometry. Additionally, apoptotic cell death was evident at 2 h after treatment with concentrations of 1×10^−6^ M [35].

On the other hand, evaluation of the biochemical parameters revealed hepatotoxicity at the highest concentration of RF07 (100 µg/mL). Hepatotoxicity and renal toxicities ex vivo have been already observed for some organic compounds of Te. In this context, the most common markers of hepatotoxicity are plasma activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which increase when there is damage caused by the Te‐containing compounds [19, 35]. Small doses of (PhTe)2 in rats (0.5 µmol/kg) showed an increase in the activity of ALT and AST, indicating that (PhTe)2 induces changes in normal liver and kidney functions of the animals (19). Results from our study agree with the findings of Lacerda et al. [36]. These researchers treated mice with an intraperitoneal (i.p.) injection of an organocalcogen (3‐methyl‐1‐phenyl‐2‐ (phenylmalene) oct‐2‐en‐1‐one) at doses of 125, 250, and 500 mg/kg, and after 60 min, blood analysis was performed to determine biochemical and hematological parameters; changes in ALT activity at doses of 250 and 500 mg/kg along with an increase in neutrophil count at a dose of 125 mg/kg were observed. These changes are particularly evident when compared with the hematological parameters previously reported for healthy female Swiss mice by Moura et al. (2024) [37]. The authors reported mean values of red blood cell count (RBC), hemoglobin concentration (HGB), and hematocrit (HCT) of 7.5 ± 0.63 × 10^6^/µL, 14.12 ± 0.67 g/dL, and 37.7 ± 2.95%, respectively. The total leukocyte count averaged 5.0 ± 1.89 × 10^3^/µL, with 9.0 ± 1.87% neutrophils, 83.0 ± 2.23% lymphocytes, and 8.0 ± 1.87% monocytes, while eosinophils were absent (0%). The platelet count was within the expected range for the species, with a mean value of 6672.0 ± 693.88 × 10^9^/µL [38]. These data reinforce the hematological alterations observed in our study. In other studies, human leukocytes were exposed to 5 and 50 µM of ebselen, (PhSe)2 and (PhTe)2. Results indicated that all of these compounds are cytotoxic at the highest concentration tested. In a similar fashion, it has been observed that (PhTe)2 can cause cytotoxicity and genotoxicity in leukocytes. These effects were indicated by a decrease in cell viability in the Trypan blue exclusion test and an increase in DI in the comet assay [37].

Finally, it is worth mentioning that toxicity is only caused if the detoxification mechanisms are supplanted by an excessive amount of Te or its compounds, such as RF07. Because it is a rare element, the danger of occupational or environmental poisoning by this element is not a great threat to human existence, but nonetheless it is still considered a toxic element according to the literature; the present study has highlighted its toxicity and genotoxic effects.

Conclusions

5

Results from this investigation revealed that RF07 exhibits genotoxicity in the animals’ peripheral blood by significantly increasing the DI and FD. Moreover, RF07 increased the formation of MN in the mouse bone marrow. In addition, RF07 exerted a dose‐ and time‐dependent cytotoxic effect in mice. The observed effects suggest that RF07 may increase intracellular oxidative stress and cause mutations in cellular macromolecules. Results also showed that RF07 induces hepatotoxicity and disorders in hematopoietic tissues, highlighting the need for further studies to determine safe concentrations in a pharmaceutical context. The effects observed were dose‐dependent, time‐dependent, and partially reversible. Furthermore, additional studies are required to better understand its potential effects and establish safe concentrations for pharmaceutical applications.

Author Contributions

Conceptualization: Felipe Emannuel Alvino de Jesus, Octávio Augusto de Carvalho Maia, and João Marcelo de Castro e Sousa. Methodology: Felipe Emannuel Alvino de Jesus, Octávio Augusto de Carvalho Maia, Rosália Maria Tôrres de Lima, Pedro Marcos de Almeida, Diego Di Felipe Ávila Alcantara, and João Marcelo de Castro e Sousa. Software, validation, formal analysis: Felipe Emannuel Alvino de Jesus and João Marcelo de Castro e Sousa. Investigation: Felipe Emannuel Alvino de Jesus, Octávio Augusto de Carvalho Maia, Rosália Maria Tôrres de Lima, Ag‐Anne Pereira Melo de Meneses, Audinei de Sousa Moura, Antonielly Campinho dos Reis, Maria Luísa Lima Barreto do Nascimento, Taline Alves Nobre, and Athanara Alves de Sousa. Resources: João Marcelo de Castro e Sousa. Data curation: Felipe Emannuel Alvino de Jesus, Octávio Augusto de Carvalho Maia, Rosália Maria Tôrres de Lima, Ag‐Anne Pereira Melo de Meneses, Audinei de Sousa Moura, Antonielly Campinho dos Reis, Maria Luísa Lima Barreto do Nascimento, Taline Alves Nobre, and Athanara Alves de Sousa. Writing – original draft preparation: Felipe Emannuel Alvino de Jesus, Octávio Augusto de Carvalho Maia, Rosália Maria Tôrres de Lima, Ag‐Anne Pereira Melo de Meneses, Audinei de Sousa Moura, Antonielly Campinho dos Reis, Maria Luísa Lima Barreto do Nascimento, Taline Alves Nobre, Athanara Alves de Sousa, Shamya Gabriella Corrêa Coelho, Glissia Lysandra dos Santos Marciel, and Isabela Ribeiro de Sá Guimarães Nolêto. Writing – review and editing: Felipe Emannuel Alvino de Jesus and João Marcelo de Castro e Sousa. Visualization, supervision: João Marcelo de Castro e Sousa. Project administration: João Marcelo de Castro e Sousa. All authors have read and agreed to the published version of the manuscript.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.