In Vitro, Oral Acute, and Repeated 28-Day Oral Dose Toxicity of a Mixed-Valence Polyoxovanadate Cluster

Abstract

Polyoxovanadates (POV) are a subgroup of polyoxometalates (POM), which are nanosized clusters with reported biological activities. This manuscript describes the first toxicity evaluation of a mixed-valence polyoxovanadate, pentadecavanadate, (Me₄N)₆[V₁₅O₃₆Cl], abbreviated as V₁₅. Cytotoxicity experiments using peripheral blood mononuclear cells (PBMC), larvae of Artemia salina Leach, and in vivo oral acute and repeated 28-day doses in mice was carried out. The LC₅₀ values in PBMC cells and A. salina were 17.5 ± 5.8 μmol L⁻¹, and 17.9 µg L⁻¹, respectively, which indicates high cytotoxic activity. The toxicity in mice was not observed upon acute exposure in a single dose, however, the V₁₅ repeated 28-day oral administration demonstrated high toxicity using 25 mg/kg, 50 mg/kg and, 300 mg/kg doses. The biochemical and hematological analyses during the 28-day administration of V₁₅ showed significant alteration of the metabolic parameters related to the kidney and liver, suggesting moderate toxicity. The V₁₅ toxicity was attributed to the oxidative stress and lipid peroxidation, once thiobarbituric acid (TBAR) levels significantly increased in both males and females treated with high doses of the POV and also in males treated with a lower dose of the POV. This is the first study reporting a treatment-related mortality in animals acutely administrated with a mixed-valence POV, contrasting with the well-known, less toxic decavanadate. These results document the toxicity of this mixed-valence POV, which may not be suitable for biomedical applications.

1. Introduction

Polyoxovanadates (POVs) belong to a subclass of polyanionic molecules made up by group 5 and 6 metal-oxo clusters referred to as polyoxometalates or polyoxidometalates (POM) [1,2,3,4]. Several studies describing POVs have demonstrated anticancer, antimicrobial, and antiviral applications [5,6,7,8,9,10]. Moreover, POVs trigger many biochemical effects such as lipoperoxidation and oxidative stress, affecting metabolic pathways and cell cycle arrest. It interferes with ions transport systems and apoptosis, induces cell morphology changes, inhibits phosphorylases and redox enzymes, and activates or deactivates cell signaling [11,12,13]. Regarding its future clinical use, POV’s toxic effects on various biological systems remain a topic in need of more experimental data. In this work, we followed the guidelines from the Organization of Economic Co-operation and Development (OECD) for measuring the acute oral toxicity and long-term toxicity of chemicals [14]. This study deviates from biological studies showing inhibition of diseased cells or simply measuring inhibition of growth or other biological activities [15]. There are currently few studies in the literature using the OECD guidelines for POVs or other vanadium compounds on healthy cells, which is the focus of this work [16,17,18].

Among POVs, decavanadate ([H_nV₁₀O₂₈⁽⁶⁻ⁿ⁾⁻], abbreviated V₁₀) is the most commonly investigated POV regarding its chemical, physical, and biological properties. It is found to be stable at acidic pH (pH 3–6) and has shown biological effects, such as inducing oxidative stress processes, stimulating enzymes, and interfering in lipid structures and cellular function [19,20,21,22]. It also has effects as an antidiabetic and anticancer agent [23]. Recently, the transition-metal-monosubstituted decavanadates V₉Mo and V₉Pt were found to be effective in inhibiting the growth of Mycobacterium smegmatis [24] and Chinese Hamster Ovary (CHO) cells [12]. In both studies, the V₁₀ cluster was reported to be a more effective growth inhibitor; however, in bacteria, the cluster is bound more tightly and hence more cell associated than in the mammalian system, where V₉Mo and V₉Pt are readily washed off [12,24].

Mixed-valence polyoxovanadates (MVPs) have contributed to an increase in the number of potential therapeutic agents for metabolic diseases and cancer [25,26,27,28]. The MVP [(Me₄N)₆[V₁₅O₃₆Cl] is a polyoxovanadate (abbreviated V₁₅, see Figure 1) that contains six Me₄N⁺ counterions, and the vanadium-oxygen cage of V₁₅ consists of one central chlorine atom, eight vanadium(IV) and seven vanadium(V) atoms, and 36 oxygen atoms (Figure 1). V₁₅ was effective against the deleterious effect of the deoxyribonucleic acid (DNA) plasmid pUC19 alkylating agents diethyl sulphate and dimethyl sulphate [29]. A chemoprotective effect of 30–40% against diethyl sulphate was additionally shown when using a more complex model of Escherichia coli DH5α cell cultures [30]. V₁₅ has also demonstrated inhibitory growth effects on microorganisms including Mycobacterium smegmatis [31]. Furthermore, the luteinizing hormone receptor (LHR), a G-protein-coupled receptor (GPCR), was used to evaluate V₁₅ and other POVs in their interaction with the cell membrane lipid interface of CHO cells [9]. Cell responses for all MVPs were greater than those seen for cells treated with the human chorionic gonadotropin (hCG) hormone, which was used in reference [9]. Despite the promising biological effects described for V₁₅, to the best of our knowledge, no evidence of toxicity (in vivo assays) was reported [2,3,29,30,31,32].

Although several POV compounds have demonstrated favorable biological properties, many aspects of the toxicity remain to be further investigated [33]. The toxicological effects of vanadium compounds in biological systems are of great concern and have been well documented in in vitro assays [34,35,36,37]. However, less information on vanadium compounds and POV toxicity is available for in vivo systems, which is critical for any potential biomedical applications [26,27].

Some animal studies have demonstrated that toxicological effects of vanadium compounds are related to the oxidovanadate ion, complex nature, dose, and administration route [35,36,38]. Cellular assays suggest some levels of toxicity are associated with the vanadium compounds due to their commonly observed redox active properties and hence potential for causing oxidative tissue damage, which can be produced by both reactive oxygen species (ROS) and reactive nitrogen species (RNS) [39]. Excess ROS and RNS species result in extensive damage to tissues, nucleic acids, lipids, and proteins through the depletion of sulfhydryl groups, lipids, and reactive parts of biomolecules and their metabolites [39,40].

In this study, toxicological evaluations in both in vitro and in vivo models have been performed to determine any potential human health risk of this multivalent POV cluster, herein abbreviated V₁₅. This is the first report of oral single-dose toxicity of a mixed-valence POV in mice.

2. Results

2.1. Preparation and Characterization of the Pentadecavanadate V₁₅

The synthesis of [(CH₃)₄N]₆[V^IV₈V^V₇O₃₆(Cl)] (V₁₅) was first described by Muller [41]. More recently, a methodology that uses cheaper and commercially available reactants was developed by Nunes and coworkers [29]. Aiming to produce enough V₁₅ to perform in vivo studies, some modifications were made to the previously described procedure to increase scale. Special care must be taken with the control of temperature, because the fresnoite type oxide (NH₄)₂VO₃ is the only product obtained when the reaction takes place at 70 °C [29], and V₁₅ can be produced as the major product under reflux.

The material was characterized in both solid and solution states, with the latter studies being performed in aqueous solution and under the conditions of biological studies. Solid-state characterization includes simulated and experimental powder X-ray diffraction studies. The pattern in powder X-ray diffraction experiments of V₁₅ was compared to previously reported single-crystal X-ray diffraction structure, showing comparable results (Figure S1, Supplementary Materials). In addition, the FTIR (infrared) spectrum of V₁₅ was recorded and showed characteristic bands of the polyoxoanion [42] (Figure S2, Supplementary Materials). The EPR (electronic paramagnetic resonance) spectrum of a pulverized sample of V₁₅ was collected at 77 K and found to show the expected broad line centered at 339 mT (g = 1.963 and with Δ_p−p of 35 mT, assigned to a ΔMS = ±1 transition (Figure S3, Supplementary Materials). This pattern confirms a partial reduction of the V(V) to V(IV) and is compatible with the intra- and intermolecular exchange interactions between vanadium(IV) centers present in the [V^IV₈V^V₇O₃₆(Cl)]⁶⁻ polyoxoanion [29,43].

Solution studies included both ⁵¹V NMR (nuclear magnetic resonance) and EPR studies, as well as UV-Vis spectroscopy. The multivalence of the V₁₅ anion affects the ⁵¹V NMR spectra, since the intact V₁₅ is not observed because of the unpaired electrons of the V^IV-atoms in the cluster. The ⁵¹V NMR and EPR spectra were recorded in aqueous solutions by dissolving the cluster in water and then adding phosphate-buffered saline (PBS pH 7.4) containing 1.0 mmol L⁻¹ CaCl₂ and 0.50 mmol L⁻¹ MgCl₂. The spectroscopic signature of V₁₅ was monitored in a time-dependent study up to 34 h [19] and showed that some V₁₅ was hydrolyzed in an aqueous solution and in media to give a mixture of the intact and oxidized V₁₅, other oxidovanadium(V) species (H₂VO₄⁻, H₂V₂O₇²⁻, V₄O₁₂⁴⁻, V₅O₁₅⁵⁻), and the oxidovanadium(IV) complex [VO₂(OH₂)₄]⁺. Other speciation studies were carried out in Luria–Bertani (LB) broth in the presence of other additives at lower pH values (down to 6.0) [44].

The electronic absorption UV-Vis spectra of a 0.025 mmol L⁻¹ aqueous solution of V₁₅ showed a ligand-to-metal charge transfer (LMCT, p(O)→d(V)) band below 400 nm, and a broad band that extended from 600 nm to the near-infrared was attributed to an intervalence charge transfer transition (IVCT, V^IV → V^V) in the POV anion. The spectrum did not change over a period of 24 h, which is consistent with V₁₅ remaining stable in solution (Figure S4, Supplementary Materials).

2.2. In Vitro Toxicity

V₁₅ with the formula (Me₄N)₆[V₁₅O₃₆Cl] was screened for its cytotoxicity using peripheral blood mononuclear cells (PMBC) using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cytotoxicity assay and a brine shrimp bioassay. The PMBC lethality assay measured in vitro toxicity of V₁₅, as shown in Figure 2A at 50 mol·L⁻¹. The inhibition stabilized, reaching 50%. In this assay, PBMCs showed decreasing viability at all concentrations tested (Figure 2A). Mean values of viability were 70.43 ± 3.4% at a concentration of 10 µM; 48.61 ± 3.3% at 25 µM; 41.79 ± 5.1% at 50 µM; and 28.06 ± 3.6% at 100 µM, with a dose-dependent reduction in viability. The IC₅₀ value of V₁₅ was 12.95 µM (10.99–14.91 μmol·L⁻¹, R² = 0.9440), indicating a significant inhibition of cell viability (Figure 2A).

The percentage of mortality in the A. salina nauplii response increased as the POV concentration increased (Figure 2B). The V₁₅ showed a high cytotoxic activity against A. salina, with an IC₅₀ value of 17.84 (15.75–20.21 μg·mL⁻¹, R² = 0.9857), causing 100% mortality in brine shrimp nauplii at a concentration of 125 μg mL⁻¹. See below for discussion of the results shown in Figure 2C,D.

2.3. Acute Oral Toxicity of V₁₅

In this study, the potential for mortality by acute treatment with a POV in animals was evaluated. The animals were examined for abnormal clinical signs and macroscopic findings, all of which were not observed. V₁₅ induced a transitory behavioral alteration such as piloerection and lethargy immediately after dosing in the treated animals. The body weight gain, food, and organ mass were not adversely affected by a single oral V₁₅ dose. As shown in

Table 1. Effects of the acute oral administration of V₁₅ on total body weight (g), food intake (mL), and fluid intake (mL) after 14 days in different female mice groups.

Parameters	Control	V15–50	V15–300	V15–2000
Total body weight (g)	470.5 ± 11.7 a	484.3 ± 15.8 a	463.0 ± 14.8 a	503.0 ± 20.0 a
Total food intake (g)	96.5 ± 3.8 a	89.7 ± 4.1 a	102.7 ± 4.2 a	103.3 ± 1.9 a
Total fluid intake (mL)	194.0 ± 9.9 a	189.3 ± 3.7 a	234.0 ± 4.5 b	235.0 ± 9.5 b

The data are presented as the mean ± SEM (n = 3). The values with different superscript letters are statistically significant at p < 0.05 compared to control group, using one-way ANOVA.

, minor differences were observed, and the fluid intake increased by 20% (p < 0.05) in the V₁₅-treated mice at 300 and 2000 mg/kg treatment levels. The lung weight increased by 25% (p < 0.05) for the 300 and 2000 mg/kg levels in the V₁₅-treated groups, and liver weight increased by ~10% (p < 0.05) only at a higher treatment dose compared to the untreated control group (

Table 2. Effects of the acute oral administration of V₁₅ on relative organ weight (mg/100 g of body weight) after 14 days in different female mice groups.

Organ	Control	V15–50	V15–300	V15–2000
Heart	0.56 ± 0.13 a	0.69 ± 0.26 a	0.54 ± 0.06 a	0.57 ± 0.02 a
Lung	0.54 ± 0.01 a	0.58 ± 0.07 a	0.72 ± 0.05 b	0.68 ± 0.07 b
Liver	5.93 ± 0.19 a	5.15 ± 0.15 a	5.94 ± 0.18 a	6.70 ± 0.29 b
Kidney	1.46 ± 0.04 a	1.29 ± 0.03 a	1.38 ± 0.036 a	1.38 ± 0.04 a
Spleen	0.54 ± 0.02 a	0.46 ± 0.04 a	0.64 ± 0.03 a	0.66 ± 0.04 a
Tibial anterior	0.13 ± 0.01 a	0.15 ± 0.02 a	0.12 ± 0.01 a	0.13 ± 0.01 a
Soleus	0.024 ± 0.002 a	0.021 ± 0.001 a	0.016 ± 0.002 a	0.018 ± 0.001 a

The data are presented as the mean ± SEM (n = 3). The values with different superscript letters are statistically significant at p < 0.05 compared to control group, using one-way ANOVA.

). The biochemical analysis of the biological assay is shown in

Table 3. Effects of the acute oral administration of V₁₅ on serum biochemical profile after 14 days in different female mice groups.

Parameters	Control	V15–50	V15–300	V15–2000
AST (mmol/L)	58.5 ± 1.1 a	87.0 ± 3.1 b	117.9 ± 15.4 c	139.7 ± 19.4 c
ALT (mmol/L)	66.1 ± 5.3 a	59.9 ± 0.6 a	141.6 ± 8.9 b	165.5 ± 14.4 b
Total protein (mg/dL)	45.6 ± 4.2 a	41.0 ± 1.1 a	49.5 ± 2.5 a	48.0 ± 3.6 a
Albumin (μmol/L)	314.0 ± 29.0 a	357.4 ± 4.8 a	236.8 ± 11.3 b	218.7 ± 5.4 c
BUN (mg/dL)	11.2 ± 0.6 a	11.2 ± 0.6 a	11.2 ± 0.7 a	10.5 ± 0.4 a

The data are presented as the mean ± SEM (n = 5). Values with different superscript letters are statistically significant at p < 0.05 compared to control group, using one-way ANOVA. Abbreviations used are: ALT—alanine aminotransferase; AST—aspartate aminotransferase; BUN—blood urea nitrogen.

. V₁₅ treatment caused an increase in the aspartate aminotransferase (AST) by 50% (p < 0.05) in the 50 mg/kg group and at 2.0-fold (p < 0.05) in the 300 and 2000 mg/kg groups. The alanine aminotransferase (ALT) levels were increased 2.5-fold (p < 0.05) for the 300 and 2000 mg/kg groups compared to untreated mice. On the other hand, V₁₅ reduced albumin content by ~25% (p < 0.05) in mice treated with higher doses of V₁₅ compared with the control group, without any alteration in blood urea nitrogen (BUN) and total protein levels.

The V₁₅ cluster has an acute oral lethal dose (LD₅₀) for female mice estimated above the 2000 mg/kg body weight based on an analysis of the dose–mortality relationship. Thus, this POV has a low order of acute toxicity by oral administration in mice, making it a category 5 of acute toxicity material [45].

2.4. Repeated-Dose 28-Day Oral Toxicity Study in Rodents

The effects of repeated doses were studied during the 28-day treatment. During the entire experimental period, mice were placed under observation, and signs of toxicity were recorded. No treatment-related changes in the external appearance or general condition were observed in either male or female mice treated with a 25 or a 50 mg/kg b.w. daily dose. The results are displayed in a Kaplan–Meier curve of both Swiss female (Figure 2C) and male mice (Figure 2D). All animals dosed at 300 mg/kg of V₁₅ did not die until the 12th day of the treatment. Furthermore, 20% of female mice did not die at the 50 mg/kg level of V₁₅ until the 22nd day (Figure 2C). At the high dose, the female mice only survived for 4 days. The survival for the female mice was 100% until the mice were treated with 100 μmol L⁻¹; the survival for the male mice was 100% until the mice were treated with 50 or 100 μmol L⁻¹, at which point the survival rate was 20 or 8 days. The most common symptoms were diarrhea, lethargy, and weight loss. Body weight and fluid intake of all mice treated with 25 and 50 mg/kg of V₁₅ did not show any alteration compared to the untreated control group (Figure 3). However, male and female mice treated with 50 mg/kg had fluctuations in food intake during dosing (Figure 3), but the total food intake in this group was slightly less than that of the V_15–25 and control groups (

Table 4. Accumulative effects of the oral repeated administration of V₁₅ on total body weight (g), food intake (g), and fluid intake (mL) in different male and female mice groups for 28-day toxicity evaluation.

Male
Parameters	Control	V15–25	V15–50
Body weight (g)	1071.80 ± 21.8 a	1.08000 ± 12.1 a	1.07000 ± 29.2 a
Food intake (g)	177.0 ± 6.6 a	170.8 ± 2.8 a	155.6 ± 5.4 b
Fluid intake (mL)	316.0 ± 5.89 a	327.5 ± 14.2 a	308.40 ± 10.5 a
Female
Parameters	Control	V15–25	V15–50
Body weight (g)	941.20 ± 28.7 a	928.80 ± 15.4 a	912.70 ± 15.5 a
Food intake (g)	146.8 ± 6.9 a	154.2 ± 5.7 a	136.3 ± 5.4 b
Fluid intake (mL)	303.6 ± 9.0 a	303.2 ± 18.1 a	322.7 ± 3.5 a

The data are presented as the mean ± SEM (n = 5). The values with different superscript letters are statistically significant at p < 0.05 compared to control group, using one-way ANOVA.

).

The necropsy did not reveal any gross pathological changes compared to the corresponding control groups. In addition, male mice did not show any effects on relative organ weight after V₁₅ treatment, except for a slight reduction in teste (20%, p < 0.05) and kidney (15%, p < 0.05) mass at 50 mg/kg. In female mice, a reduction in tibial (~40%, p < 0.05) and reproductive organ mass (uterus and ovaries ~30%, p < 0.05) was observed at both doses evaluated (

Table 5. Toxicity evaluation of the oral repeated administration of V₁₅ on relative organ weight (mg/100 g of body weight) in different male and female mice groups after 28 days.

Male Mice
Organ (mg/100 g of b.w.)	Control	V15–25	V15–50
Brain	1.13 ± 0.03 a	1.10 ± 0.06 a	1.05 ± 0.03 a
Heart	0.41 ± 0.01 a	0.51 ± 0.11 a	0.45 ± 0.01 a
Lung	0.62 ± 0.02 a	0.63 ± 0.05 a	0.52 ± 0.06 a
Liver	4.69 ± 0.24 a	5.41 ± 0.10 a	4.99 ± 0.66 a
Kidney	1.41 ± 0.05 a	1.44 ± 0.08 a	1.22 ± 0.08 b
Spleen	0.35 ± 0.04 a	0.42 ± 0.07 a	0.39 ± 0.02 a
Tibial anterior	0.15 ± 0.01 a	0.14 ± 0.01 a	0.13 ± 0.01 a
Gastrocnemius	0.36 ± 0.01 a	0.36 ± 0.02 a	0.34 ± 0.02 a
Teste	0.74 ± 0.08 a	0.64 ± 0.08 b	0.60 ± 0.01 b
Female mice
Organ (mg/100 g of b.w.)	Control	V15–25	V15–50
Brain	1.24 ± 0.09 a	1.23 ± 0.04 a	1.25 ± 0.03 a
Heart	0.48 ± 0.04 a	0.41 ± 0.01 a	0.39 ± 0.02 a
Lung	0.67 ± 0.09 a	0.64 ± 0.05 a	0.70 ± 0.05 a
Liver	5.03 ± 0.22 a	5.30 ± 0.23 a	5.78 ± 0.05 a
Kidney	1.34 ± 0.06 a	1.16 ± 0.04 a	1.14 ± 0.05 a
Spleen	0.47 ± 0.04 a	0.48 ± 0.04 a	0.50 ± 0.05 a
Tibial anterior	0.24 ± 0.06 a	0.14 ± 0.01 b	0.13 ± 0.01 b
Gastrocnemius	0.25 ± 0.01 a	0.28 ± 0.01 a	0.31 ± 0.03 a
Ovaries and uterus	1.32 ± 0.07 a	0.94 ± 0.04 b	0.92 ± 0.07 b

The data are presented as the mean ± SEM (n = 5 for both male and female mice). Values with different superscript letters are statistically significant at p < 0.05 compared to control group, using one-way ANOVA.

).

Hematological values of mice treated orally with V₁₅ for 28 days are shown in

Table 6. Evaluation of the effects of the repeated administration of V₁₅ on hematological profile in different male and female mice groups after 28 days.

Male Mice
Parameters	Control	V15–25	V15–50
RBC (×103/μ)	6.80 ± 0.50 a	6.90 ± 0.50 a	5.50 ± 0.70 a
Hemoglobin (%)	15.20 ± 1.30 a	12.50 ± 0.50 b	13.30 ± 0.60 b
MCV (μm3)	58.50 ± 2.70 a	46.70 ± 0.60 b	47.70 ± 0.40 b
MCH (pg)	20.60 ± 0.70 a	18.70 ± 0.70 a	17.40 ± 3.20 b
MCHC (g/dL)	35.50 ± 1.80 a	38.60 ± 0.10 a	37.40 ± 6.80 a
HCT (%)	43.40 ± 4.70 a	32.20 ± 1.90 b	26.40 ± 3.20 c
Platelet count (×103/μ)	531.00 ± 86.94 a	778.00 ± 23.35 b	942.75 ± 17.33 c
WBC (×103/μ)	14.00 ± 0.70 a	13.30 ± 0.10 a	14.50 ± 0.40 a
Monocytes (%)	6.90 ± 1.00 a	7.70 ± 0.30 a	7.60 ± 2.50 a
Lymphocyte (%)	89.50 ± 2.10 a	89.00 ± 0.30 a	87.40 ± 4.00 a
Female mice
Parameters	Control	V15–25	V15–50
RBC (×103/μ)	14.80 ± 0.50 a	13.90 ± 0.20 a	15.2 ± 0.20 a
Hemoglobin (%)	13.80 ± 0.60 a	11.20 ± 0.80 b	11.40 ± 0.70 b
MCV (μm3)	56.80 ± 5.40 a	47.30 ± 0.80 b	42.40 ± 0.40 c
MCH (pg)	20.90 ± 1.10 a	15.10 ± 0.40 b	15.30 ± 7.00 c
MCHC (g/dL)	37.30 ± 1.60 a	35.30 ± 2.40 a	33.50 ± 17.50 a
HCT (%)	37.30 ± 2.30 a	31.80 ± 0.40 a	11.4 ± 5.80 c
Platelet count (×103/μ)	438.82 ± 13.82 a	499.00 ± 14.47 b	911.66 ± 16.23 b
WBC (×103/μ)	6.70 ± 0.60 a	7.40 ± 0.60 a	3.60 ± 0.90 b
Monocytes (%)	3.50 ±0.60 a	4.90 ± 0.60 a	6.10 ± 1.30 b
Lymphocyte (%)	88.30 ± 4.70 a	91.1 ± 0.60 a	90.40 ± 2.40 a

The data are presented as the mean ± SEM (n = 5). Values with different superscript letters are statistically significant at p < 0.05 compared to control group, using one-way ANOVA. Abbreviations used are: HCT—hematocrit; MCH—mean corpuscular hemoglobin; MCV—mean corpuscular volume; MCHC—mean corpuscular hemoglobin concentration; RBC—red blood cell; WBC—white blood cells.

. In this study, a significant alteration was observed in some hematimetric indices in both male and female mice. V₁₅ treatment reduced hemoglobin (~15%, p < 0.05), MCV (~20%, p < 0.05), and HCT in a dose-dependent manner (25 and 40%, respectively) and stimulated platelet content in a dose-dependent manner (~45 and ~80%, respectively) in treated male mice groups compared to untreated mice. In female mice treated with V₁₅ daily for 28 days, a reduction in hemoglobin (~20%, p < 0.05), MCV (~15%, p < 0.05), and MCH (~15%, p < 0.05) was observed after the treatment with both doses, and a reduction in HCT (~70%, p < 0.05) was detected only at a higher treatment dose. V₁₅ treatment also stimulated platelet content in a dose-dependent manner (~1.5 and ~2-fold, respectively). In addition, V₁₅ reduced the WBC (~50%, p < 0.05) at a higher dose, and it increased monocytes as the dose increased compared to untreated female mice (Table 6). For other hematological parameters, no significant differences were detected between treated male mice and the control group.

The data shown in

Table 7. Effects of the oral administration of V₁₅ on serum biochemical profile in different male and female mice groups after 28 days in repeated-dose toxicity evaluation.

Male Mice
Parameters	Control	V15–25	V15–50
ALT (mmol/L)	39.90 ± 1.80 a	103.70 ± 5.40 b	122.50 ± 8.30 c
AST (mmol/L)	74.80 ± 2.40 a	81.90 ± 2.90 a	145.40 ± 8.30 b
Total protein (g/L)	46.60 ± 5.60 a	46.00 ± 11.90 a	51.60 ± 2.10 a
BUN (mg/dL)	12.00 ± 0.20 a	13.40 ± 0.90 a	11.60 ± 0.01 a
Creatinine (µmol/L)	29.80 ± 2.40 a	23.86 ± 2.60 a	71.90 ± 20.50 b
Uric acid (µmol/L)	122.31 ± 15.30 a	185.30 ± 17.30 b	211.80 ± 32.70 b
Total cholesterol (mmol/L)	1.60 ± 0.18 a	1.58 ± 0.41 a	1.80 ± 0.19 a
Triglycerides (mmol/L)	1.62 ± 0.11 a	1.84 ± 0.13 a	1.30 ± 0.10 b
Female mice
Parameters	Control	V15–25	V15–50
ALT (mmol/L)	35.40 ± 2.70 a	55.40 ± 3.10 b	76.30 ± 12.10 c
AST (mmol/L)	60.30 ± 3.10 a	73.20 ± 4.70 a	76.30 ± 12.10 b
Total protein (g/L)	41.80 ± 5.40 a	47.60 ± 6.00 a	50.70 ± 1.50 a
BUN (mg/dL)	11.49 ± 1.10 a	13.41 ± 0.28 a	11.74 ± 0.22 a
Creatinine (µmol/L)	37.53 ± 6.44 a	36.60 ± 3.60 a	34.50 ± 1.80 a
Uric acid (µmol/L)	114.92 ± 19.72 a	170.60 ± 42.0 b	204.10 ± 68.1 b
Total cholesterol (mmol/L)	2.03 ± 0.14 a	1.46 ± 0.17 b	1.83 ± 0.06 a
Triglycerides (mmol/L)	1.50 ± 0.14 a	1.21 ± 0.12 b	1.40 ± 0.07 a

The data are presented as the mean ± SEM (n = 5). Values with different superscript letters are statistically significant at p < 0.05 compared to control group, using one-way ANOVA. Abbreviations used are: ALT—alanine aminotransferase; AST—aspartate aminotransferase; BUN—blood urea nitrogen.

indicate that V₁₅ caused an increase in AST levels in male (~30%, p < 0.05) and female (95%, p < 0.05) mice. Creatinine was increased only in male mice treated with 50 mg/kg of V₁₅ (2.5-fold p < 0.05), whereas uric acid levels increased in both male and female mice treated with 25 mg/kg of V₁₅ (50%, p < 0.05) and male (70%, p < 0.05) and female (~80%, p < 0.05) mice groups treated with 50 mg/kg of V₁₅ compared with the untreated control group. Considering the lipid profile, V₁₅ reduced triglyceride (TG) levels in male mice at a higher V₁₅ treatment dose (~20%, p < 0.05), and there was a reduction in total cholesterol (TC, ~30%, p < 0.05) and TG (~20%, p < 0.05) in treated female mice compared with the control group.

2.5. Oxidative Stress

The oxidative stress parameters evaluated were reduced glutathione (GSH), oxidized glutathione (pGSSG), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT). The effect of the V₁₅ compound on the generation of ROS species in the liver of male and female mice treated for 28 consecutive days is shown in

Table 8. Effects of the repeated oral administration of V₁₅ on oxidative stress parameters in different male and female mice groups and after 28 days.

Male Mice
Parameters	Control	V15–25	V15–50
GSH (nmol/mg ptn)	10.2 ± 0.3 a	10.4 ± 0.4 a	10.1 ± 0.6 a
GSSG (nmol/mg ptn)	7.4 ± 0.4 a	7.4 ± 0.4 a	7.3 ± 0.6 a
GSH/GSSG (nmol/mg ptn)	1.4 ± 0.1 a	1.4 ± 0.1 a	1.4 ± 0.05 a
MDA (U/mg ptn)	1.3 ± 0.1 a	5.3 ± 0.3 b	4.8 ± 0.5 b
SOD (U/mg ptn)	19.9 ± 3.0 a	14.0 ± 2.2 b	15.7 ± 1.3 b
CAT (U/mg ptn)	0.023 ± 0.005 a	0.027 ± 0.010 a	0.028 ± 0.010 a
Female mice
Parameters	Control	V15–25	V15–50
GSH (nmol/mg ptn)	6.6 ± 0.2 a	7.2 ± 0.2 a	6.9 ± 0.2 a
GSSG (nmol/mg ptn)	2.4 ± 0.4 a	5.0 ± 0.3 b	4.2 ± 0.1 b
GSH/GSSG (nmol/mg ptn)	2.8 ± 0.5 a	1.5 ± 0.1 b	1.6 ± 0.1 c
MDA (U/mg ptn)	0.9 ± 0.1 a	1.0 ± 0.2 a	1.3 ± 0.1 b
SOD (U/mg ptn)	16.6 ± 3.6 a	33.7 ± 3.8 b	24.3 ± 1.9 c
CAT (U/mg ptn)	0.011 ± 0.003 a	0.029 ± 0.008 b	0.026 ± 0.004 b

The data are presented as the mean ± SEM (n = 5). The values with different superscript letters are statistically significant at p < 0.05 compared to control group, using one-way ANOVA. Abbreviations used are: GSH—glutathione reduced; GSSG—glutathione oxidized; GSH/GSSG—ratio between GSH and GSSG; MDA—malondialdehyde; SOD—superoxide dismutase; CAT—catalase.

. In male mice, the V₁₅ treatment increased hepatic MDA content, and the SOD activity decreased (~30%, p < 0.05). On the other hand, in V₁₅-treated female mice, the GSSG levels increased, and the GSH/GSSG ratio decreased, which is consistent with oxidative damage. In addition, at elevated MDA content at a higher treatment dose (~30%, p < 0.05), the SOD and CAT activities in female mice were increased compared with the untreated group. The V₁₅ treatment clearly impacted female and male mice differently, although evidence of oxidative stress in both male and female mice was apparent.

Alterations in mice liver and kidneys after treatment were confirmed by hematoxylin and eosin (H&E) histology analysis in V_15–25 and V_15–50 groups, as well as by comparison to the untreated mice group. The histopathological findings shown in Figure 4 and Figure 5 include vacuolization, mononuclear cell infiltrates, local macrovesicular steatosis, hypertrophy of hepatocyte nuclei, focal degeneration, hepatocyte degeneration, structural architecture damage, and hepatic remodeling in male and female mice treated with both doses. A microscopic evaluation of kidney tissue sections revealed focal tubular damage, swelling in the renal glomerulus, thickening of basement membranes, cell peeling, vacuolization, and cytoplasmic debris in male and female mice treated with both doses. Control animals of both sexes exhibited normal hepatic and kidney architecture.

3. Discussion

Polyoxometalates, including POVs, have been studied for potential biomedical applications, including antibacterial, antidiabetic, antiprotozoal, antiviral, and anticancer applications [1,2,3]. Given the wide structural and electronic range of POVs, a wide range of biological activities is apparent [46,47,48,49,50,51,52,53,54], and it is important to evaluate the possible toxicological effects. In general, short-term toxicity of vanadium administration has been observed in several species, including mammals [35,55,56] and fish [57,58]. However, the oral treatment by a single dose of a POV, and particularly a multivalent POV, has not been reported previously. The present study is the first report of an oral dose of POV repeated for 28 days in mice. These studies are important because some of the POVs have been found to have undesirable side effects [33], whereas others have been found to have beneficial effects [1,2,3]. The detailed mechanism of action of most POVs, however, is not understood and likely will vary depending on the specific POV or POM. We recently reported that V₁₅ was able to activate signal transduction [9,13] and suggested that the redox properties of V₁₅ were important to that mode of action. V₁₅ has also been found to be effective in inhibiting cell growth [31], and the mode of action involved bacterial excretion of a temperature-insensitive material. In this case, V₁₅ behaves similar to V₁₀, which has several activities that are not attributed to its redox properties [1,22].

Several methods are useful to predict the toxicity of different types of substances [59,60], and the toxicity of the V₁₅ cluster was screened against PMBC viability and a brine shrimp lethality bioassay [61,62]. Cell viability is one of the vital methods for toxicology analysis, which measures the cellular response to a toxic agent and provides information on survival and cell death. In this study, we found that V₁₅ has a high cytotoxicity effect with low IC₅₀ values in both PBMCs (17.5 μmol L⁻¹) and the brine shrimp assay (17.9 mg mL⁻¹). The studies demonstrated that molecules with IC₅₀ values less than 100 μmol L⁻¹ are considered very toxic [63]. Thus, comparable studies have shown the cytotoxicity of the vanadium compounds investigated [64,65,66]. Although the mechanism of vanadium toxicity remains to be elucidated and likely to vary depending on the compound examined, the cytotoxicity of vanadium is associated with the ability to accumulate vanadium ions and generate redox imbalance through an increase in intracellular ROS species [39].

The in vivo acute oral toxicity test represents a useful initial step in the toxicity assessment of compounds with potential biomedical applications [45]. Though the oral acute single administration of V₁₅ did not cause any mortality, it showed a potential toxic effect through transitory behavior alterations, such as piloerection and lethargy after dosing, organ coefficients in the lung and liver, and biochemical parameters that indicated metabolic damage. Importantly, the toxicity evaluation is essential to identify a dose range and, hence, differences between efficacy and toxicity. This information would be useful to assess potential biomedical applications of a compound and identify any potential clinical symptoms that might be triggered by toxic levels of vanadium compounds under investigation.

In addition, there are no previous reports concerning repeated 28-day oral administration and toxicity of POVs. The Kaplan–Meier curve demonstrated that deaths was dose- and time-dependent. Survival analysis is important when the time between exposure and events is of clinical importance. Animal deaths occurred in both male and female Swiss mice sooner at higher dose treatments. Our data from the 28-day repeated-dose toxicity evaluation showed that male and female albino Swiss mice responded to the repeated exposure to V₁₅ with observable side effects. All animals treated with 300 mg/kg of V₁₅ died before the 12th day of the experiment (Figure 2). All of them demonstrated mortality/moribundity and clinical signs of toxicity, including lethargy, diarrhea, slimming, and abnormal gait and breathing.

Animals treated with a lower dose of 50 mg/kg of V_15, on the other hand, showed only a slight reduction in food and water intake. In addition, the weight of the kidney and testis was reduced in males treated with 50 mg/kg, and the weight of the skeletal muscles and reproductive organs was reduced in females treated with 25 and 50 mg/kg treatments. The observed reduction in organ weight could indicate that kidneys, skeletal muscles, and reproductive organs are potential targets following oral and repetitive treatments with soluble vanadium compounds. Kamboj and Kar showed that after two days of a single dose of vanadium exposure (0.08 nmol/kg), necrosis and a reduction in testes weight was detected in rats [67]. Furthermore, CD-1 male mice exposed to V₂O₅ inhalation for 12 weeks exhibited changes in the testes weight and the action of the testicular cytoskeleton, which might at least in part explain the reported reprotoxic effect of some vanadium compounds [68,69]. However, there are only a few reports concerning the effects of vanadium on the female reproductive system [70,71]. It has been shown that vanadium reduces glycogen content in the ovaries and uterus tissue, as well as protein content and acid phosphatase activity in the uterus. Furthermore, a microscopic evaluation of the ovaries revealed a decreasing mature follicle diameter, ovum disintegration, disorganized and hypertrophied growing follicles, and loose and fibrotic stroma [72]. Our results suggest that reprotoxic effects induced by POV are an important parameter that should be further investigated in future investigations of potential compounds for clinical use.

Hematological parameter evaluations were used to determine the extent of the harmful effect of V₁₅ exposure. Repetitive dosing of V₁₅ for 28 days at both doses caused similar toxic effects in male and female subjects. The impact of vanadium compounds on blood parameters has been demonstrated previously [73,74,75]. Sanchez-Gonzaléz et al. reported anemia in healthy rats treated with bis(maltolate)oxidovanadium(IV) dissolved in drinking water [73]. However, other studies have reported unchanged hematological parameters with treatment by different vanadium compounds. Specifically, Dai et al. (1994) showed that bis(maltolato)oxidovanadium(IV) (BMOV), ammonium metavanadate, and vanadyl sulfate in drinking water caused no hematological damage for 12 weeks [74]. Our results suggest a moderate normocytic normochromic anemia in both male and female mice treated with V₁₅. Some vanadium compounds could affect physiological iron concentration [73] and may explain the anemia observed in animals exposed to POV. Furthermore, the thrombocytosis detected in V₁₅-treated male and female mice may be due to inflammation, which is supported by the observed leukocyte count results. Our results agree with previous reports [75]. A higher predictive value for human toxicity for the hematological system is obtained upon the extrapolation of animal data. Thus, we suggest that V₁₅ may be severely toxic to hematopoiesis.

An imbalance in oxidative stress is recognized as one potential cause of vanadium toxicity [76]. In addition, the great concern is the accumulation of vanadium in organs, such as the kidney or liver, which induces oxidative harm, lipid peroxidation, and toxicity [76,77,78,79]. Studies have demonstrated that some vanadium compounds may induce and exacerbate lipid peroxidation (LPO) and also affect human health. On the other hand, other vanadium compounds have protective effects on LPO [20,39]. Furthermore, vanadium compounds can sometimes generate free radicals, and thus form ROS/RNS directly, but they can also act indirectly through the effects on LPO [20,80,81,82]. Oster et al. (1993) showed that some vanadium compound treatments are correlated with TBAR production in both the liver and kidney in male rats [83]. Furthermore, Gândara et al. (2005) showed that decavanadate did not induce short-term LPO (12 h), but it later (24 h) increased LPO in the liver of H. didactylus [84]. Apart from this, rabbits exposed to vanadium pentoxide (V₂O₅) had increased LPO in the liver and kidney [85]. Our findings demonstrated an increase in LPO levels and a subsequent significant increase in liver lipid peroxidation in treated male animals at both doses and females at a higher dose of V₁₅.

The prooxidant and antioxidant response induced by V₁₅ was different in male and female mice. SOD activity was depressed in male mice, whereas it was stimulated in female mice treated with POV. In addition, the activity of the GSH antioxidant system decreased, whereas CAT activity increased only in female mice. Our data suggest that V₁₅ has a higher toxic effect in female mice compared with male mice. GSH is the largest endogenous cellular redox buffer, and thus the most significant intracellular non-enzymatic antioxidant source [86]. Therefore, it could be possible that the GSH depletion and changes in the GSH:GSSH ratio may represent the mechanism of oxidative intensity and a higher toxicity of V₁₅ in female mice. Female mice appear to be less vulnerable to oxidative stress under physiological conditions, but during stressful conditions, it is unclear whether female mice are more protected against oxidative stress than males [87]. The literature is controversial regarding the effects of vanadium compounds on GSH levels. NaVO₃ in drinking water decreased GSH content in female rats [84]; on the other hand, ammonium metavanadate in drinking water increased GSH in the liver in the same animal model [88]. In 1993, Thompson and McNeill did not detect any noticeable differences in the amount of GSH in male rat livers treated with VOSO₄ in drinking water for 12 weeks [89]. Some of these differences have been traced to specific conditions of the interaction of the vanadium compound with the GSH [90]. In conclusion, our results demonstrate that oxidative stress is associated with the toxicity of POVs, and that multiple mechanisms are likely responsible for V₁₅ oral toxicity. According to our results, decavanadate inhibits respiration and induces membrane depolarization of the mitochondria, acting as a strong inhibitor, most likely through interference with the respiratory chain, and specifically complex III of the mitochondrial inner membrane, in the heart and liver [19,21].

The assessment of kidney and liver function in response to a new drug has been supported by the measurements of serum marker enzymes and/or metabolites [91,92]. In this study, the exposure of Swiss mice (male and female) to repeated doses of V₁₅ produced several treatment-related effects, particularly in the groups treated with high doses of V₁₅. Serum levels of ALT and AST have been observed as markers of liver injury, due to a variety of etiologies, such as medication toxicity and viral hepatitis [92]. In addition, a serum increase in ALT and AST levels is indicative of cell damage and a loss of the functional integrity of the hepatocyte membrane. Our data are comparable to previous reports on the liver toxicity of vanadium compounds in fish [57,84], broilers [93], and mammals [82,94,95]. These findings are related to an increase in liver histopathology with focal inflammation, diffused swelling of hepatocytes, and marked centrilobular hepatocellular injury. Pathological changes resulted from POV-induced free radical generation and the diminished antioxidant status of the compound. Similar histological alterations in the liver and kidney were seen in rabbits treated with vanadium pentoxide (V₂O₅) [85]. In addition, mice exposed to V₂O₅ inhalation for 6 weeks demonstrated inflammatory foci in liver parenchyma [95,96]. Cardiac myocytes also showed vacuolization and granular degeneration after 44 days of metavanadate (NH₄VO₃) exposure [97]. Taken together, previous results and observations in this paper suggest that oxidative stress is at the core of this histomorphology alteration and impairment of liver function and liver toxicity.

The kidney is a common target organ for toxicity and injury because of its exposure to drugs, including metallodrugs. Thus, renal function is usually assessed by serum levels of creatinine, uric acid, and BUN. These parameters may provide information on the effects of V₁₅ on the tubular and/or glomerulus part of the kidney. V₁₅ treatment increased serum creatinine levels in male mice at higher doses, which suggests the nephrotoxicity of this POV. In addition, hyperuricemia (HUA) may result from an imbalance in UA metabolism, which can lead to hyperuricemia and represent a very important signal of kidney toxicity. Both a reduction in glomerular filtration rate and an increase in net tubular absorption may result in HUA [91]. The kidney toxicity induced by vanadium compounds detected by an increasing UC has been demonstrated previously [94]. In this study, the increasing UA exerted by V₁₅ plays an important role in inducing renal damage in both male and female mice. In addition, the microscopic appearance of kidney tissues of male and female mice dosed at 25 and 50 mg/kg of V₁₅ showed that morphological alterations are compatible with nephrotoxicity induced by metals. Similar alterations were found in H. didatylys exposed to decavanadate oligomers [58] or ammonium metavanadate in broilers [93]. It has been demonstrated that vanadium may induce morphological damage to several organs, including kidneys. CD-1 mice exposed to vanadium oxide inhalation showed histological evidence of inflammatory foci [78]. Another study showed that V₁₀ and a high-fat diet in Wistar rats caused harm to kidney cytoarchitecture [98]. Furthermore, vanadium compounds have the capacity to disturb the cell redox balance, which can result in damage to the antioxidant enzymatic and non-enzymatic systems. In general, these pathological changes in the liver and kidney resulted from POV-induced free radical generation and compromised the activity of the antioxidant status in these tissues.

4. Materials and Methods

4.1. Ethical Statement

The experimental strategy for these studies was approved by the human ethics committee of the Health Sciences Center/UFPE with the process number CAAE 04633218.6.0000.8807. Additionally, all animal care procedures were approved by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 8023, revised 1978) and followed the Ethics Committee on the Use of Animals of the Universidade Federal de Pernambuco (CEUA/UFPE, protocol 0021/2021).

4.2. Chemicals and General Methods

Ammonium metavanadate (NH₄VO₃, 99%) and D-mannitol (C₆H₁₄O₆, 98%), both from Sigma-Aldrich^® (St. Louis, MO, USA), and tetramethylammonium chloride (C₄H₁₂ClN, 98%) from Fluka^® were used without purification. Deuterium oxide (D₂O, 99.9 atom %D) was purchased from Oakwood Chemical and used as received. Ultrapure water (18.2 mΩ·cm) was used in the synthesis and characterization of V₁₅, excepted when otherwise stated. The ⁵¹V NMR data were collected on a Bruker NEO 400 MHz spectrometer with a 105.2 MHz frequency for vanadium at 298 K, using 4096 scans in the f1 domain, 8 steady-state transitions, a spectra width of 800 ppm, a transmitter frequency offset of −548 ppm, 0.01 s relaxation delay, a 0.08192 s acquisition time, and a 16 μs pulse. Fourier-transform infrared (IR) spectra (400 to 4000 cm^–1) were recorded from KBr pellets on a Bruker VERTEX-70 spectrometer with a resolution of 4 cm⁻¹. Electron paramagnetic resonance in X-band (EPR) spectra (9.5 GHz) were recorded at 77 K on an X-band (9.5 GHz) Bruker EMX-Micro spectrometer using a pulverized sample. Electronic absorption spectra of 0.250 mmol L⁻¹ aqueous solution of V₁₅ were observed from 340 to 900 nm, using an AvaLight UV-Vis/NIR light source and an AvaSpex-UL S2048 Fiber-Optic Spectrometer.

4.2.1. Synthesis of V₁₅-(Me₄N)₆[V₁₅O₃₆(Cl)]

Mannitol (1.428 g, 7.839 mmol), tetramethylammonium chloride (0.7530 g, 6.870 mmol), and ammonium metavanadate (1.621 g, 13,86 mmol) were weighed and transferred to a round-bottom flask. Water (75 mL) was added, and the reaction mixture was stirred under reflux (which is 93 °C in Colorado) for 24 h. The solution changed from colorless to deep green in 2 h. A small amount of black powder started forming after 30 min. After 24 h, the reaction mixture was filtered to remove the black solid and kept at 4 °C to crystalize. Due to the small amount of black solid isolated, the insoluble black side product was confirmed by IR as (NH₄)₂V₃O₈ [29]. Pure V₁₅ was obtained from the mother liquor after 4 days as dark green crystals, which were filtered off, washed with 10 mL of cold water, and dried under air. The average yield was 1.36 g, 81%, and the synthesis was reproduced 16 times to generate 10.864 g of V₁₅. The dark-green crystals of V₁₅ were insoluble in common organic solvents and soluble in hot water.

4.2.2. Characterization of V₁₅ in the Solid and Solution States

The solid V₁₅, with a formula [(CH₃)₄N]₆[V^IV₈V^V₇O₃₆(Cl)], was confirmed by single-crystal X-ray diffraction analysis. One dark-green crystal of V₁₅ was subjected to analysis at 300 K on a Bruker D8 Venture diffractometer equipped with a Photon 100 CMOS detector using Mo-Kα radiation (μ(Mo-K_α) = 0.711 mm⁻¹). Crystal data: crystal system hexagonal P6_3/mmc, a = b = 13.720 Å, c = 20.020 Å, α = β = 90°, and γ = 120°. The X-ray powder diffraction (PXRD) pattern of the V₁₅ was registered at 40 kV and 30 mA on a Shimadzu XRD600 diffractometer equipped with a Cu-target tube (Cu-K_α, k = 1.5418 Å). The calculated diffractogram of V₁₅ was generated from the single-crystal crystallographic information (CIF) file available at Cambridge Crystallographic Data Centre, CCDC code 794586, using Mercury 4.0 software [99].

To verify the purity of the compound in bulk solid, powder X-ray diffraction pattern of V₁₅ was compared with the previously described single-crystal X-ray diffraction structure, showing good correspondence.

The IR spectrum of solid V₁₅ contains characteristic bands of the polyoxoanion at 560, 658, 728, and 793 cm⁻¹ assigned to ν_as(V−O−V) and 980 cm⁻¹ to ν(V=O). Me₄N⁺ bands appeared at 1288 cm⁻¹ as ρ_r(CH₃), strong δ_as, and partial reduction in the vanadium (V) and is compatible with the intra- and intermolecular exchange interaction among vanadium (IV) centers reported in the [V^IV₈V^V₇O₃₆(Cl)]⁶⁻ (V₁₅) polyoxoanion [29,43]. The EPR spectrum of the pulverized V₁₅ sample was measured at 77 K and found to be like those reported previously [29].

The electronic absorption spectra were recorded for a 0.025 mmol L⁻¹ aqueous solution of V₁₅ and showed a ligand-to-metal charge transfer (LMCT, p(O)→d(V)) band below 400 nm and a broad band that extended from 600 nm to the near-infrared assigned to an intervalence charge transfer transition (IVCT, V^IV → V^V) in the polyoxoanion. The spectra changed little over a period of 24 h, like studies by ⁵¹V NMR and EPR spectroscopy that monitored up to 34 h of bioassays with pH 7.4 [9,30]. These studies show that V₁₅ slowly decomposes in aqueous solution to give V₁₅ in the presence of species of oxovanadates (H₂VO₄⁻, H₂V₂O₇²⁻, V₄O₁₂⁴⁻, V₅O₁₅⁵⁻) and the oxidovanadium(IV) complex [VO₂(OH₂)₄]⁺. Importantly, no precipitation of black solid (NH₄)₂V₃O₈ was observed, further consistent with the lack of formation of the fresnoite type oxide (NH₄)₂VO₃ and a solution containing hydrolytically stable V₁₅.

4.3. Peripheral Blood Mononuclear Cells (PBMC) and Cytotoxicity Assay by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) of V₁₅

PBMCs were obtained from the peripheral blood of healthy volunteers and collected in heparinized tubes. For PBMC separation, the peripheral blood was centrifuged using Ficoll Paque Plus (GE Healthcare Biosciences^®, Pittsburgh, PA, USA). After separation, the cells were counted and placed in 96-well plates (5 × 10⁵ cells/100 μL/well) in RPMI-1640 medium (Gibco, ThermoFischer scientific^®, Waltham, MA, USA), supplemented with L-Glutamine, 10% fetal bovine serum (FBS) (Gibco, ThermoFischer scientific^®), 10 mmol L⁻¹ HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Gibco, Thermo Fischer scientific^®), and 200 U/mL penicillin/streptomycin (Gibco). After being isolated, cells were exposed to V₁₅ at serial concentrations of 10, 25, 50, and 100 µmol L⁻¹ and then kept for 48 h in a humid atmosphere containing 5% CO₂ at 37 °C. Cells treated with the solvent dimethyl sulfoxide (DMSO 0.1% v/v) were used as a control. The cytotoxicity was then quantified by the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to the purple reduction product formed in living cells. The absorbance was measured at 570 nm by the Elx808 (Biotek, Shoreline, WA, USA, USA apparatus^®). Thus, the cytotoxic activity of V₁₅ was quantified as the observed percentage of the control absorbance. The results are reported as the average value calculated from three separate experiments.

4.4. Toxicity against Larvae of Artemia Salina Leach

Artemia salina (Leach, 1819) (Tropical^®, Rio de Janeiro, Brazil) dry cysts were used to evaluate V₁₅-(Me₄N)₆[V₁₅O₃₆Cl] toxicity. Firstly, cysts were hydrated in artificial seawater (40 g sea salt/L, pH 8,0) and exposed to constant aeration at ambient temperature (25 ± 3 °C) for 48 h. After hatching, the larvae (nauplii) were transferred into vials (n = 10 per vial) containing seawater with 5, 10, 25, 50, 100, 125, 250, 500, and 1000 mg mL⁻¹ final concentrations of V₁₅ taken from the stock solutions of 1 mg mL⁻¹ in DMSO 5% for 24 h at 25 ± 3 °C [61]. The control wells contained 10 nauplii in artificial seawater and DMSO 5%. Two experiments were performed in triplicate, with a total of 110 specimens per treatment, and assessments of mortality and survival of nauplii were carried out by observation of mobility for live nauplii identified after 24 h under a microscope.

4.5. Oral Acute Toxicity

The evaluation of acute oral toxicity was measured in accordance with Guideline 423, which describes the acute toxic class method reported by the Organization of Economic Co-operation and Development (OECD) for measuring acute oral toxicity of chemicals [45]. Nulliparous and non-pregnant female mice (three for each group) were acclimatized in a propylene cage for five days before dosage, fasted for 3 h, and weighed before the administration of a POV dose. The V₁₅ was dissolved in NaCl 0.9% (m/v) and administered by gavage in mice in a single dose using animal feeding needles (100 μL/100 g b.w.). Animals were randomly divided into three groups with three animals each: (a) a control group treated with 0.9% (m/v) NaCl; (b) a low-dose group treated with 300 mg/kg of V₁₅ (V_15–300); and (c) a high-dose group treated with 2000 mg/kg of V₁₅ (V_15–2000).

All animals were closely monitored for the first four hours to identify piloerection as well as changes in eyes, skin, and fur, toxic effects on the mucous membranes, behavior pattern disorientation, asthenia, hypoactivity, hyperventilation, lethargy, lack of sleep, tremors, salivation, diarrhea, convulsion, coma, motor activity, or death. During the study, the animals were monitored daily for 14 days. Daily monitoring included measuring body weight, food intake, and water consumption. On the 14th day, the female mice were euthanized by intravenous injection of a solution containing both xylazine (80 mg/kg, i.p.) and ketamine (10 mg/kg, i.p.). The blood was drawn, and the organs collected for biochemical and macroscopic analysis. The LD₅₀ was estimated based on mortality in each group as previously reported [100].

4.6. Toxicity Administered by the Repeated 28-Day Oral Toxicity Dose

The repeated oral dose 28-day toxicity test was carried out in accordance with Guideline 407 for rodents reported by the Organization of Economic Co-operation and Development (OECD) to evaluate chemicals for long-term oral toxicity [101]. Kaplan–Meier plots were used to assess the data on mice survival. The animals were acclimated in propylene cages for five days, at which point male, nulliparous, and non-pregnant female mice were fasted for three hours and weighed before they were given the compound dose. The mice were divided into groups of 10 (5 male and 5 female each) to be treated daily over 28 consecutive days by gavage using animal feeding needles (100 μL/100 g b.w.) The groups were divided as follows: (a) the control group was treated with 0.9% m/v NaCl; (b) the low-dose group was treated with 25 mg/kg of V₁₅ (V_15–25); (c) the high-dose group was treated with 50 mg/kg of V₁₅ (V_15–50); and (d) then treated with 300 mg/kg of V₁₅ (V_15–300). The number of deaths was recorded, and a Kaplan–Maier survival probability curve was plotted [102].

Animals were weighed once a week, and their basic morphological parameters, food and water intake, and any behavioral alterations were tracked. At the end of the 28 days of treatment, all animals were anesthetized by intraperitoneal administration of a xylazine and ketamine mixture (10 mg and 80 mg/kg, respectively) and euthanized by cervical dislocation. Blood samples were collected for biochemical and hematological examination, and the extracted liver and kidney organs were weighed and kept for histopathology.

4.7. Biochemical and Hematological Analysis

Mice fasted overnight were anesthetized as described above in the 14 days and 28 days following treatment. Blood samples were collected by the retro-orbital technique with or without heparin for hematological and serum biochemical analysis, respectively.

Hematological parameters included red blood cells (RBC), red cell volume distribution (RDW), hemoglobin concentration (HBG), hematocrit (HCT), platelet count (PLT), mean platelet volume (MPV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC), and lymphocytes (LYM). The test was performed using a multiparameter automatic hematology analyzer SDH-20, Labtest^®, Curitiba, Brazil) designed for hematology testing samples.

The serum levels of several parameters were measured, including enzyme levels of alanine (ALT) and aspartate aminotransferase (AST), as well as metabolite levels including blood urea nitrogen (BUN), creatinine (CRE), total cholesterol (TC), total protein (TP), triacylglycerol (TG), and uric acid (UA) with a colorimetric assay using commercial kits (Lab Test Diagnostic SA^®, Santa Lagoa, Brazil). Optical densities were measured by spectrophotometry using a Varioskan TM Lux multimode microplate reader on a Thermo Scientific^®, Waltham, MA, USA instrument at the wavelengths designated on the datasheet for each biochemical parameter. Baseline measurements of controls were obtained by comparing the optical densities of the samples with the measurements using the appropriate standards provided in the kits. Data are showed in terms of U/mL (ALT and AST) and mg/dL for the others [16].

4.8. Oxidative Stress Evaluation in the Liver

Oxidative stress markers and lipid peroxidation in the liver were measured using thiobarbituric acid (TBAR), which measures the malondialdehyde (MDA) formed when lipids are oxidized according to the method reported previously [103]. Glutathione peroxidase (GPx) activity was measured in an assay using glutathione reductase as reported by Paglia and Valentine (1967) [104]; glutathione-reduced (GSH) levels were measured using a fluorometrical method as described by Hissin and Hilf (1976) [105]; the antioxidant activity of superoxide dismutase (SOD) was measured by monitoring superoxide anions generated by xanthine oxidase as described by Misra and Fridovich (1972) [106]; and the catalase (CAT) activity was monitored by measuring the decomposition of H₂O₂ by absorbance at 240 nm as reported by Aebi (1984) [107].

4.9. Organ Mass and Histopathological Analysis

After the 14th and 28th days of treatment, all mice were necropsied after blood withdrawal for anatomical localization and visible examination of detectable macroscopic organ changes (color, aspect, and size). Selected organs examined included the heart, liver, kidney, lungs, testicles, uterus, ovaries, spleen, adipose tissue, stomach, and soleus and extensor digitorum longus (EDL) muscles. These organs were carefully excised and trimmed of fat and connective tissue before weighing. The organ weight values were converted to g/100 g of body weight (percentage of body weight).

Following the 28-day treatment plan, the liver and kidney were fixed in 10% formalin buffer solution for 24 h at ambient temperature before being rinsed with water for four hours. They were then dehydrated stepwise using 70% (v/v) of EtOH. Dehydrated tissues were rendered transparent by addition of xylene. Tissues were added to pre-heated paraffin for sufficient infiltration, and after cooling, paraffin blocks were formed before being cut into 5 μm sections using a microtome (Leica^® RM 2025, Heidelberger, Germany). The paraffin was removed, and specimens were dehydrated and stained with hematoxylin-eosin for observation with an inverted optical microscope (Leica^®, Heidelberger, Germany) connected to a video camera (Leica^® DFC 280, Wetzlar, Germany) and a computer monitor. An expert pathologist took pictures of the liver and kidney samples and examined them for any signs of cellular harm or morphological changes.

4.10. Statistical Analysis

Unless otherwise specified, the data were reported in triplicates as mean ± standard error of the mean (S.E.M). The data of V₁₅-treated groups and their respective control groups was analyzed by one-way analysis of variance (ANOVA) followed by the Bonferroni test. The long-rank Kaplan–Meier survival test was used to compare the survival distribution of the different doses and treatment groups. A p value less than 0.05 was considered statistically significant. Graph Pad Prism^® (GraphPad Software, San Diego, CA, USA) version 6.0 software was used for all statistical analysis.

5. Conclusions

Administration of the mixed-valence polyoxovanadate (POV) V₁₅, a nanosized anionic cluster, induces in vitro and in vivo toxicity in mice. The acute toxicity demonstrated that the LD₅₀ value is more than 2000 mg/kg; thus, this POV has a low order of acute oral toxicity in mice and is a category 5 compound. However, the repeated 28-day assessment with systematic administration demonstrated toxic effects of this POV, especially in the liver and kidney tissues, and this is more toxic than in acute studies. The multivalent V₁₅ was found to be stable under the condition resembling those used for the in vitro and in vivo studies. This suggests that the observed toxicity is mainly caused by the intact V₁₅, considering the multivalent nature of the cluster and the ease of its oxidation. The redox properties of the cluster are likely related to its mode of action. Accordingly, the toxicity is attributed to redox damage produced by V₁₅, which is consistent with induced hematological, hepatic, and renal toxicity observed in both male and female mice. Due to the oxidation of V₁₅ and the resulting oxidative damage, tissues and metabolites are also likely involved in the process. Clinically, these results suggest that future research with any type of redox-active compounds may result in toxicity. Thus, there is a need to design non-toxic vanadium compounds, particularly POVs, for biomedical applications, which will be most beneficial if the delivery vehicle involved could control the reactivity of these compounds. These results should encourage researchers to develop new strategies to reduce the toxic effects of POVs, which could involve encapsulation in liposomes, other protective delivery systems, and complex-containing bioconjugate molecules.