*Article* **Phenanthroline Complexation Enhances the Cytotoxic Activity of the VO-Chrysin System**

**Agustin Actis Dato 1, Luciana G. Naso 1, Marilin Rey 2, Pablo J. Gonzalez 2, Evelina G. Ferrer <sup>1</sup> and Patricia A. M. Williams 1,\***


**Abstract:** Metal complexation in general improves the biological properties of ligands. We have previously measured the anticancer effects of the oxidovanadium(IV) cation with chrysin complex, VO(chrys)2. In the present study, we synthesized and characterized a new complex generated by the replacement of one chrysin ligand by phenanthroline (phen), VO(chrys)phenCl, to confer high planarity for DNA chain intercalation and more lipophilicity, giving rise to a better cellular uptake. In effect, the uptake of vanadium has been increased in the complex with phen and the cytotoxic effect of this complex proved higher in the human lung cancer A549 cell line, being involved in its mechanisms of action, the production of cellular reactive oxygen species (ROS), the decrease of the natural antioxidant compound glutathione (GSH) and the ratio GSH/GSSG (GSSG, oxidized GSH), and mitochondrial membrane damage. Cytotoxic activity studies using the non-tumorigenic HEK293 cell line showed that [VO(chrys)phenCl] exhibits selectivity action towards A549 cells after 24 h incubation. The interaction with bovine serum albumin (BSA) by fluorometric determinations showed that the complex could be carried by the protein and that the binding of the complex to BSA occurs through H-bond and van der Waals interactions.

**Keywords:** oxidovanadium(IV) phenantrholine chrysin; vanadium cellular uptake; anticancer; albumin interaction

#### **1. Introduction**

Chrysin (Scheme 1) is a natural polyphenol with several biological activities, such as antioxidant, anticancer, antiviral, and neuroprotective. It has low solubility at physiological conditions, low bioavailability, quick metabolism, and rapid excretion, limiting its utilization as a chemotherapeutic agent [1]. Hence, its structure has been modified (by functionalization or metal complexation) in order to improve its bioactivities. Metal-based drug development is a promising strategy for the enhancement of the pharmacological action of drugs. In particular, vanadium complexes have been recognized to display biological activities for the treatment of various diseases, such as diabetes, cancer, tuberculosis, and leishmaniasis [2]. Cancer is one of the primary causes of mortality. Moreover, lung cancer is the main cause of cancer deaths. Recently, with the knowledge of the molecular mechanisms related to this disease, it has been found that angiogenesis is one of the causes of its bad prognosis [3,4].

**Citation:** Actis Dato, A.; Naso, L.G.; Rey, M.; Gonzalez, P.J.; Ferrer, E.G.; Williams, P.A.M. Phenanthroline Complexation Enhances the Cytotoxic Activity of the VO-Chrysin System. *Inorganics* **2022**, *10*, 4. https://doi.org/10.3390/ inorganics10010004

Academic Editor: Dinorah Gambino

Received: 9 December 2021 Accepted: 23 December 2021 Published: 28 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Scheme 1.** Draw of the structure of chrysin.

We have previously studied the anticancer behavior of chrysin and chrysin oxidovanadium(IV) metal complex on osteoblast like cells [5], breast cancer cells [6], and human lung A549 cancer cells [7] and showed that complexation improved the biological effects of the polyphenol. The selection of the heterocyclic base phenanthroline (phen) included as a second ligand is related to the fact that planar ligands coordinated to metals could bind DNA through intercalation to base pairs, improving the anticancer action of binary complexes, as well as confer lipophilicity to the compounds [8]. Only a few ternary chrysin metal complexes were reported and the Ga(III), Cu(II), and Ru(II)-chrysin-ancillary aromatic chelator systems proved more cytotoxic than free chrysin in different cancer cell lines [9–11].

In the current work, we design the heteroleptic [VO(chrys)phenCl] complex aiming to enhance the biological behavior of the VO(chrys)2 complex. It was characterized in the solid state and in solution. The biological activity of the complex as an anticancer drug was studied in the human lung cancer cell line A549 and toxicity was evaluated in the cell line derived from human embryonic kidney HEK293. The mechanism of action was studied by means of cellular reactive oxygen species (ROS) generation, natural antioxidant level depletion (glutathione, GSH), mitochondrial membrane damage, and vanadium cellular uptake. The interaction of the complex with BSA was also determined.

#### **2. Results**

#### *2.1. Synthesis of [VO(chrys)phenCl]*

The complex was prepared by the replacement of one ligand chrysin in the binary [VO(chrys)2EtOH]2 complex by phen. The molar conductance of the complex measured in DMSO, Λ<sup>m</sup> = 11 (Ω−<sup>1</sup> cm<sup>2</sup> mol−1), suggested a non-electrolyte compound. The thermogravimetric analysis (oxygen atmosphere, 50 mL/min) showed that the complex is stable up to 260 ◦C, indicating that no solvation or coordination solvent molecules are present in the complex (Figure 1). The compound degraded in a series of two consecutive TG steps observed at 295 and 400 ◦C. Weight constancy is attained at 525 ◦C and the weight of the remaining solid residue, collected at 700 ◦C, was 17.2%, in good agreement with the expected value of 17.0%. The presence of V2O5 in the residue was confirmed by FTIR spectroscopy. More details can be found in Section 4.

**Figure 1.** Thermogravimetric analysis (TGA) curve for the decomposition of [VO(chrys)phenCl].

#### *2.2. FTIR Spectrum*

Δ

The assignment of the main absorption bands of the FTIR spectrum of the complex was performed in comparison with the binary VO(chrys)2 system [5]. The spectral pattern remained similar to the binary compound regarding the modifications of the vibrational bands of C=O stretching (1635 cm−1) and O-H bendings (1596, 1351, and 1247 cm−1), indicating the coordination of chyrsin to the metal center (Figure 2). The vibrational modes of phen in the 1600–1400 cm−<sup>1</sup> range (medium intensities) are associated with C=C and C=N stretching modes. These bands appeared to be overlapped with those of chrysin. However, it can be seen that the C=N stretching band of phen at ca. 1646 cm−<sup>1</sup> shifted to 1635 cm−<sup>1</sup> upon coordination, but it is masked by the C=O stretching mode (strong intensity) of the ligand chrysin. Main bands of motion of ring hydrogen atoms in phase and out of phase for phen at 852 and 738 cm−1, respectively, shifted to 847 and 725 cm−1, showing that phen is also interacting to the oxidovanadium(IV) cation [12].

**Figure 2.** FTIR spectra of [VO(chrys)phenCl] and [VO(chrys)2EtOH]2.

Moreover, the replacement of one chrysin ligand by one phen may produce a decrease of π electron donation to the V=O moiety. The shift of the V=O stretching band from 968 to 957 cm−<sup>1</sup> is indicative of a decrease of the bond order and an increase of the bond length in agreement with the ligand replacement. These results suggest that the metal ion is interacting with chrysin through C=O and the deprotonated C(5)–O group and with the N atoms of phen.

#### *2.3. EPR Measurements*

The powder EPR spectrum of the complex obtained at 120K is shown in Figure 3. A very similar spectrum was obtained at room temperature (Figure S1A). The EPR spectrum of a polycrystalline powder of [VO(chrys)phenCl] gave a unique EPR line, which does not show the typical eight line hyperfine splitting pattern of 51V nucleus (I = 7/2), suggesting the presence of extended spin–spin interactions between neighboring oxidovanadium(IV) ions in the solid complex, which collapse the hyperfine interaction into a single line [13]. Similar behavior was observed for oxidovanadium(IV) complexes of apigenin, naringenin, and quercetin [14–16] and it is characteristic of magnetically extended systems of the cation.

**Figure 3.** EPR spectrum of [VO(chrys)phenCl] recorded at 120 K. (**A**) Powder sample and (**B**) Frozen DMSO solution. EPR spectra were recorded in a Bruker EMX-Plus spectrometer equipped with a rectangular cavity. Experimental conditions: 100 kHz modulation, 4 Gpp modulation amplitude, 2 mW microwave power.

The EPR spectrum of the DMSO frozen solution measured at 120 K displays the typical eight-line pattern spectrum for axial-V(IV) systems, as shown in Figure 3. The simulation (see Figure S1B) predicted that the observed signal was consistent with the oxidovanadium(IV) ion in a nearly axial or pseudoaxial ligand field. The spin Hamiltonian parameters and the hyperfine coupling constants were gII = 1.941; AII = 162.2 × <sup>10</sup>−<sup>4</sup> cm<sup>−</sup>1; <sup>g</sup><sup>⊥</sup> = 1.977; A<sup>⊥</sup> = 59.5 <sup>×</sup> <sup>10</sup>−<sup>4</sup> cm−1. These parameters fit well in the corresponding gII vs. AII diagram for a 2N,2O coordination sphere [17]. Because the parallel component of the hyperfine coupling constant is sensitive to the type of donor atoms on the equatorial positions of the coordination sphere, the empirical relationship Az = ∑niAz,i is frequently used to determine the identity of the equatorial ligands in V(IV) complexes (ni, number of equatorial ligands of type i and Az,i, the contribution to the parallel hyperfine coupling from each of them) [17]. Considering the contributions to the parallel hyperfine coupling constant of the different coordination modes (CO = 44.7 × <sup>10</sup><sup>−</sup>4, ArO<sup>−</sup> = 38.6 × 10–4) [14], and N(phen) × 2 = 40.4 × <sup>2</sup> × <sup>10</sup>−<sup>4</sup> [18], the calculated value for AII of 164.1 × <sup>10</sup>−<sup>4</sup> cm−<sup>1</sup> agrees with the experimental value. From the EPR parameters, we conclude that the coordination sphere would correspond to a binding mode of (CO, O−, 2N) in the equatorial plane. The chloride ion that also linked to the metal center (see below) may be located in trans-position. Besides, it is observed that g <g<sup>⊥</sup> < ge = 2.0023 and |A|>|A⊥|, in line with an octahedral site with tetragonal compression and the dxy orbital being the ground state of the V4+ (3d1) ion. Moreover, the <sup>Δ</sup>g/Δg<sup>⊥</sup> ratio ([g-ge]/[g⊥-ge]) proved to be 2.37, showing an octahedral tetragonal distortion.

The hyperfine coupling constants were related to the dipolar hyperfine coupling parameter P, that represents the dipole–dipole interaction of the electronic and nuclear moments, through the relations of Kivelson and Lee [19]: <sup>A</sup> <sup>=</sup> −P [k + 4/7 − <sup>Δ</sup>g − 3/7 <sup>Δ</sup>g⊥] and A<sup>⊥</sup> <sup>=</sup> <sup>−</sup>P [k <sup>−</sup> 2/7 <sup>−</sup> 11/4 <sup>Δ</sup>g⊥]. *<sup>p</sup>*-value ranges from 100 to 160 <sup>×</sup> <sup>10</sup>−<sup>4</sup> cm−<sup>1</sup> in oxidovanadium(IV) compounds [20] and is calculated as *P* = gegNμBμN‹r−3›, where gN is the nuclear g-factor, ge is the g-factor of the free electron, μ<sup>N</sup> the nuclear magneton, and ‹r−3› can be calculated for the vanadium 3d orbitals. The parameter *k* (between 0.6 to 0.9) is the dimensionless Fermi contact interaction constant [21], is very sensitive to deformations of the metal orbitals, and indicates the isotropic Fermi contact contribution to the hyperfine coupling. The calculated value of *<sup>P</sup>* = 119.6 × <sup>10</sup>−<sup>4</sup> cm−<sup>1</sup> is considerably reduced when compared to the value of the free ion (160 × <sup>10</sup>−<sup>4</sup> cm−1) and indicates a considerable amount of covalent bonding in the [VO(chrys)phenCl] complex. The value of *k* = 0.71 indicates a moderate contribution to the hyperfine constant by the unpaired s-electron. Moreover, the product *<sup>P</sup>* × *<sup>k</sup>* = 85.1 × <sup>10</sup>−<sup>4</sup> cm−<sup>1</sup> represents the anomalous contribution of s-electrons to the A and A<sup>⊥</sup> components (the rest being the contribution of 3dxy electrons) [22].

#### *2.4. Stability Measurements*

The electronic spectral band for the d-d transition of [VO(chrys)phenCl] dissolved in DMSO is shifted to blue regarding the precursor complex (VO(chrys)2), in agreement with the changes in the coordination sphere (2O and 2N atoms) vs. (4O atoms) (766 nm for [VO(chrys)phenCl] vs. 796 nm for the binary compound) [5]. The position of the electronic absorption band proved similar to that of [VO(SO4)(phen)2] in DMSO (ca. 765 nm) [23]). Electronic spectra of a DMSO solution of [VO(chrys)phenCl] (t) and conductivity measurements did not show any significant change during 4 h. Figure S2B shows the spectral changes for a solution of the complex in DMSO/H2O 1/99 (1 × <sup>10</sup>−<sup>2</sup> M), during 4 h. The complex proved less stable in water solutions, but at least during 15 min (manipulation time for the cellular studies) the complex remained stable in both solutions. It is known that once the complex is added to living cells, it could undergo several chemical interactions with the oxidant and antioxidant cellular systems, including ligand release, but the differences in the anticancer effect between the free ligands and the vanadium complex could demonstrate the efficacy of the vanadium compound.

#### *2.5. Cytotoxic Assays*

The cytotoxic effect [VO(chrys)phenCl] on the human lung cancer cell line A549 was determined by the MTT assay, at 24, 48, and 72 h incubation (Table 1). The effects were compared with those of the oxidovanadium(IV) cation [16–24], chrysin [25], VO(chyrs)2 [7], and phen [8], as previously reported.

**Table 1.** Half maximal inhibitory concentration, IC50, values of VO(chrys)phenCl and its components (oxidovanadium(IV) cation (VO), chrysin, phen) on A549 cell line at 24, 48 and 72 h incubation and on HEK cell line for VO(chrys)phenCl (24 h). The IC50 values for the binary complex of VO and chrysin was added for comparisons. The results represent the mean ± the standard error of the mean (SEM) from three separate experiments.


<sup>a</sup> [16], <sup>b</sup> [24], <sup>c</sup> [25], <sup>d</sup> [8], <sup>e</sup> [7].

The replacement of one chyrsin molecule by phen in the coordination sphere of the binary complex greatly enhanced the anticancer effects at 24 and 48 h incubation. As can be seen, cell incubation time had an impact on cell viability in a dose–response manner. Higher incubation times (72 h) result in low IC50 values, and most of the cells that are responding at early times after treatment were lost at the time the assay was performed, owing to the disintegration of cells into particulate debris altering the ratio between the live and dead cell populations present at the time of analysis. Therefore, it is difficult to make cell viability comparisons after 72 h incubation and this is the reason why the mechanistic studies have been carried out in the present study at 24 h incubation [26].

It is important to emphasize that the oxidovanadium(IV) complexes may hydrolyze and/or oxidize in aqueous solution. At 24 h incubation, the VIVO cation showed a high IC50 value without affecting cell viability up to 100 μM (Table 1), and V(V) showed a decrease of cell viability of 10–20 % at 50 and 100 μM [27]. The anticancer effect of VO(chyrs)phenCl at 24 h incubation proved higher than that exerted by the oxidovanadium(IV) cation, its oxidized species, the binary complex, and the ligands. Hence, we are able to discard that the deleterious effect the complex was due to the metal ion, phen, and/or chrysin ligands generated after decomposition processes (or at least we can assume that these processes were slow at 24 h incubation).

The percentage of non-tumorigenic HEK293 cell viability vs. [VO(chrys)phenCl] concentrations is shown in Figure S3. The complex does not reduce the cellular viability in the range of tested concentrations (0–100 μM), indicating that the toxicity of the compound shows a good correlation in terms of selectivity toward A549 cancer cells within 24 h of tincubation.

#### *2.6. ROS and GSH/GSSG Cellular Levels*

Cellular oxidative stress is considered to be inducer of carcinogenesis. Cancer cells show high ROS levels and are more vulnerable to ROS. Hence, ROS generating compounds kill cancer cells selectively. Non-transformed cells have a low basal intracellular ROS level and have a full antioxidant capacity, being less vulnerable to the oxidative stress induced by different compounds in cancer cells. To study the role of the oxidative stress in the cytotoxicity induced by the complex, we measured ROS production in A549 cells at 24 h incubation. From Figure 4, it can be seen that the oxidovanadium(IV) cation did not increase cellular ROS levels, chrysin generated a very low increase of ROS, while phen and the complex elevated the amount of intracellular ROS so that they selectively damage cancer cells. The pro-oxidant nature of the complex and phen improved the anticancer effects of the flavonoid.

Glutathione is an antioxidant, capable to prevent damage produced by ROS in cells, ubiquitously present in all cell types at mM concentration. In mammalian cells under physiological conditions, the GSH redox couple is known to be present with steady-state concentrations of 1–10 mM. The overall ratio of GSH to its oxidized state GSSG in a cell is usually greater than 100:1, and the redox couple GSH/GSSG is used as an indicator of changes in the redox environment and of oxidative stress in the cell. In various models of oxidative stress, this ratio has been demonstrated to decrease to values of 10:1 and even 1:1 [28].

The GSH contents in A549 cell line after incubation with the complex were measured (Figure 5A). As GSH depletion is not a major cause of cytotoxicity, the GSH/GSSG was also calculated (Figure 5B) to demonstrate that the decrease of the GSH/GSSG ratio was due not only to a decrease in the level of GSH, but also to an accumulation of GSSG. Therefore, it can be seen that the cellular damage on the A549 cell line is manifested by the increased levels of ROS that exceed the defense mechanisms inducing GSH oxidation with the resultant reduction of cellular GSH. Therefore, a stress oxidative mechanism could be assumed for the cell-killing action of [VO(chrys)phenCl].

**Figure 4.** Effect of chrysin, VO(chrys)phenCl, phen and oxidovanadium(IV) cation on H2DCFDA oxidation to DCF. A549 cells were incubated at 37 ◦C in the presence of 10 μM H2DCFDA. The values are expressed as the percentage of the control level and represent the mean ± SEM. \* *p* < 0.05, ns: not significant.

**Figure 5.** Effect of [VO(chrys)phenCl] on GSH cellular levels (**A**) and GSH/GSSG ratio (**B**) in A549 cells, 24 h incubation. Results are expressed as mean ± SEM of three independent experiments. All values are statistically significant in comparison with the control.

#### *2.7. Mitochondrial Membrane Potential*

ΔΨ

The loss of mitochondrial function and the subsequent release of cytochrome C into the intracellular space are some of the mechanisms associated with the apoptosis process [29]. The mitochondrial membrane potential (Δψ) was measured to explain the increase of cellular ROS and decrease of the GSH/GSSG ratio on the A549 cell line by incubation of the metal complex. The lipophilic cationic probe DioC6 enters the mitochondria and upon depolarization it will accumulate less dye [30]. Figure 6 shows the effect of mitochondrial dysfunction with a membrane potential loss when A549 cells were treated with increasing concentrations of the metal complex in concordance with a stress oxidative mechanism.

μ

μΜ)

μ

 μ

μ

μ

**Figure 6.** Changes of the mitochondrial membrane potential (% Δψ) in A549 cells treated with increasing concentrations of VO(chrys)phenCl for 24 h. Each point represents the mean ± S.E.M of three measurements in three independent experiments. All values are statistically significant in comparison with the control.

#### *2.8. Cellular Vanadium Uptake Experiments*

The vanadium content after 24 h treatment of the compounds at a concentration equivalent to [VO(chrys)phenCl] IC50 (28.9 μM) was determined by inductively coupled plasma-mass-spectrometry, ICP-MS (see Table 2). VO(acac)2 exhibited almost the same capacity for cellular uptake as the control. Our results are comparable with data reported for the human A2780 ovarian cancer cells [31]. For [VO(chrys)phenCl], the total amount of V up-taken is ca. five-fold higher than for the binary complex. Our data suggest that its greatest cytotoxicity might be directly correlated with the incorporation of phen into the complex structure, increasing its lipophilicity, which could improve the cellular uptake.

**Table 2.** Cellular V content (determined by ICP-MS) following cell treatments with 28.9 μM of compounds for 24 h. Results are expressed as mean ± the standard error of the mean (SEM) of two independent experiments.


#### *2.9. BSA (Bovine Serum Albumin) Interactions*

The binding behavior of drugs with albumin affected their distribution and metabolism in the body. Both human serum albumin (HSA) and BSA are commonly used to determine the binding interactions because they possess near 76% sequence homology and tertiary structure similarity [32]. To study the protein binding ability of the complex, we have selected BSA herein because of its low cost and wide availability. The intrinsic fluorescence of BSA is due to tryptophan residue when excited at 295 nm. Upon titration of BSA with increasing concentrations of the complex, the fluorescence spectra showed a decrease in the intensity or quenching (Figure S4). The Stern–Volmer quenching constant was obtained from the slope of the graph of the fluorescence intensities F0/F (in the absence and presence of the quencher, respectively, and corrected by the inner-filter effect) vs. the quencher concentration, Q, according to the equation: F0/F = 1 + Kq*τ*0[Q] = 1 + KSV[Q], where Kq is the bimolecular quenching constant and τ<sup>0</sup> is the lifetime of the fluorophore in the absence of the quencher (considered 1 × <sup>10</sup>−<sup>8</sup> s for a biopolymer) [33].

An upward curvature of the plots at different temperatures can be seen, showing a combined quenching (static and dynamic). However, at low concentrations, a linear correlation was obtained (Figure 7). The linearity at lower concentrations suggested a single quenching type. Static and dynamic quenching can be differentiated by the analysis of the temperature dependence. Using the data of the linear region, Ksv and Kq (Kq = Ksv/τ0, τ<sup>0</sup> = 10−<sup>8</sup> s) were calculated (Table 3). The Ksv values showed a decrease with increasing temperature, suggesting a static quenching mechanism. Another criterion for the type of static cooling is that the result of the bimolecular cooling constant should be greater than the maximum dynamic cooling constant for a biopolymer, assumed to be 2.0 × 1010 <sup>M</sup>−1s−1. As can be seen in Table 3, all the values of Kq obtained were greater than that value, reinforcing the assumption that a static quenching type of interaction between the complex and albumin takes place.

**Figure 7.** Stern Volmer graph for VO(chrys)phenCl at different temperatures, [BSA] = 6 μM, λex = 280 nm.

**Table 3.** Stern-Volmer constant (Ksv), bimolecular quenching constant (Kq), binding constant (Kb) and number of binding sites (n) for the interaction of VO(chrys)phenCl with BSA (6 μM) in Tris-HCl buffer (0.1 M, pH 7.4).


To determine the binding constant (Kb) and the number of binding sites n (Table 3), the Scatchard equation (log[(F0 − F)/F] = log Kb + n log [Q]) was used (Figure 8). Binding constants prove to be in the order of 105−106 <sup>M</sup>−<sup>1</sup> and decrease at higher temperature, indicating a reduction in stability of the BSA-complex compound, and confirming the involvement of a static quenching. However, the obtained values suggest that the interactions are adequate for the complex to be transported and delivered by BSA. The number of binding sites (*ca*. 1) indicated that the complex may occupy one binding site of the protein.

**Figure 8.** Plots of log [(F0 − F)/F] vs. log [Q] for the VO(chrys)phenCl-BSA system at 298 K, 303 K and 310 K, [BSA] = 6 μM, λex = 280 nm.

To determine the main forces operating during the protein binding, thermodynamic parameters were determined, using the van't Hoff equation ln Ka = −ΔH/RT + ΔS/R and ΔG = ΔH − TΔS (Table 4). From the negative values of ΔG, a spontaneous interaction can be confirmed and the decrease of ΔG at higher temperatures indicated a decrease of the binding strength of the complex–protein bond. The negative values of enthalpy and entropy changes indicate that hydrogen bond and van der Waals forces were the major forces operating during the interaction [34].

**Table 4.** Thermodynamic parameters for the interactions between [VO(chrys)phenCl] with BSA.


#### **3. Discussion**

To enhance the biological action of VO(chrys)2 complex, one chrysin ligand was replaced by the planar ligand phen in the metal coordination sphere and the new complex was characterized by common analytical techniques. FTIR studies indicated that the oxidovanadium(IV) ion interacted with C=O and O− atoms of chrysin and N atoms of phen. Conductivity studies indicated that the chloride ion, which contributed to the electroneutrality of the complex, is also bonded to the V=O moiety, in trans- position (solution EPR determinations). Conductivity and spectral determinations in DMSO and DMSO/H2O showed that the complex did not produce hydrolytic species during the manipulation time of the complex. It has to be noted that the cellular experiments consist in the addition to the cells of a fresh DMSO stock solution of the complex dissolved in the culture media (with a final DMSO concentration of 0.5%) and, therefore, the studies of the stability of the complex must be performed not only on DMSO and the culture media, but the cell components must also be considered (including natural antioxidant compounds that must also been taken under consideration in those kind of studies), as previously mentioned [35]. However, the determination of the speciation in aqueous solution of the complex inside the cell is outside the scope of this study.

In a previous paper, the cytotoxicity of *cis*-[VIVO(OSO3)(phen)2] at 72 h incubation in the A549 cell line was related to its decomposition in cell culture medium generating the ligands and the oxidation of vanadium [36]. Another study showed that up to 24 h incubation of the same compound on different ovarian cancer cell lines displayed different IC50 values than the free ligands and inorganic V(IV) and V(V) ions, determining that the biological effects were due to the complex, which is more active than the free ligands [23]. However, at 72 h incubation, similar IC50 values were obtained, probably due to chemical changes of the metal complex.

From the data in Table 1, we are able to discard that the cytotoxic effect was due to phen and/or chrys ligands, generated after decomposition processes at least at 24 h incubation, because there were no deleterious effects of the binary complex and chrysin, while the IC50 value measured for phen was 66 μM, corresponding to more than twice the IC50 value of the complex. Besides, we measured the effect of the 24 h cell incubation with the mixture of each component of the complex in stoichiometric quantities (sodium metavanadate, chrysin and phen, physiological pH). It produced the same inhibitory effect from 10 to 50 μM (*ca*. 50% viable cells, Figure S5) and, hence, the IC50 value could not be determined. However, we can conclude that the effect of the mixture proved different from that of the complex (that inhibited cell viability in a dose dependent manner), discarding that the cytotoxic activity of the complex was due to decomposition followed by oxidovanadium(IV) oxidation in the culture media.

It is well known that ROS overproduction is the cause of the development of a number of diseases. Excessive ROS accumulation and the depletion of natural antioxidant compounds such as GSH can cause irreversible cell damage and even cell death [37]. We determined herein that one mechanism involved in the process of cell death is the induction of oxidative stress by the metal complex accompanied by disruption of the mitochondria membrane potential. Meanwhile, the loss of mitochondrial membrane potential is related to activation of the mitochondrial apoptosis pathways. The presence of the lipophilic ligand, phen, favors the cellular uptake of the metal. Phen coordinates to oxidovanadium(IV) cation, improving its transport inside cells, hence behaving as a better cytotoxic agent via the induction of oxidative stress and mitochondrial membrane damage than the VO(chrys)2 complex.

The replacement of one chrys ligand by phen in VOchrysphenCl produced a tighter interaction to BSA than VO(chrys)2 (Kb 0.76 × <sup>10</sup><sup>5</sup> <sup>M</sup>−1) [7]. Both compounds bind to BSA in a spontaneous and enthalpy-driven manner and could be transported by albumin (Kb values in the range of 104–106 M<sup>−</sup>1).

#### **4. Materials and Methods**

#### *4.1. Materials and Instrumentation*

Chrysin (Sigma, Buenos Aires, Argentina), phenanthroline hydrochloride (Merck, Buenos Aires, Argentina), and vanadyl acetylacetonate (Fluka Munich, Germany) were used as supplied. Corning or Falcon provided tissue culture materials. Dulbecco's modified Eagle's medium (DMEM, Gibco, Gaithersburg, MD, USA), TrypleTM (Invitrogen, Buenos Aires, Argentina) and fetal bovine serum (FBS, Internegocios, Buenos Aires, Argentina) were used as provided. All other chemicals used were of analytical grade. Elemental analysis for carbon, nitrogen, and hydrogen was performed using a Carlo Erba EA1108 analyzer. Vanadium content was determined by the tungstophosphovanadic method [38]. Thermogravimetric analysis was performed with Shimadzu systems (model TG-50), working in an oxygen flow of 50 mL·min−<sup>1</sup> and at a heating rate of 10 ◦C·min−1. Sample quantities ranged between 10 and 20 mg. UV-vis and diffuse reflectance spectra were recorded on a Hewlett-Packard 8453 diode-array and a Shimadzu 2600/2700 spectrophotometer. Infrared spectra were measured with a Bruker IFS 66 FTIR spectrophotometer from 4000 to 400 cm−<sup>1</sup> using the KBr pellet technique. Fluorescence spectra were obtained with a Shimadzu RF-6000 spectrophotometer equipped with a pulsed xenon lamp. The molar conductance of the complex was measured on a Conductivity TDS Probe-850084, Sper Scientific Direct, using 10−<sup>3</sup> M DMSO solutions. Exact mass spectra were obtained using a Bruker micrOTOF-Q II mass spectrometer, equipped with an ESI source operating in positive mode.

A Bruker EMX-Plus spectrometer, equipped with a rectangular cavity with 100 kHz field modulation and with standard Oxford Instruments low-temperature devices (ESR900/ITC4), was used to record the EPR spectra of both powdered and DMSO solution spectra. The spectra, obtained at 100 K to room temperature (*ca.* 298 K) were, when necessary, baseline corrected using WinEPR Processing software (Bruker, Inc., Billerica, MA, USA).

The EasySpin 5.2.3. toolbox based on MATLAB was used to simulate g- and A- values [39], assuming an axial spin-Hamiltonian of the form:

$$H = \mu\_B \left[ \mathcal{g}\_{\parallel} \mathcal{B}\_{\overline{z}} \mathcal{S}\_{\overline{z}} + \mathcal{g}\_{\perp} \left( \mathcal{B}\_{\overline{x}} \mathcal{S}\_{\overline{x}} + \mathcal{B}\_{\overline{y}} \mathcal{S}\_{\overline{y}} \right) \right] + \left[ A\_{\parallel} \mathcal{S}\_{\overline{z}} I\_{\overline{z}} + A\_{\perp} \left( \mathcal{S}\_{\overline{x}} I\_{\overline{x}} + \mathcal{S}\_{\overline{y}} I\_{\overline{y}} \right) \right]$$

where *μ<sup>B</sup>* is the Bohr magneton, and *g*, *g*⊥, *A*, *A*<sup>⊥</sup> are the components of the axial **g** and **A** tensors, respectively. *Bx/y/z*, *Sx/y/z*, and *Ix/y/z* are the components of the magnetic field, and of the spin operators of the electron and V nucleus, respectively.

#### *4.2. Preparative [VO(chrys)phenCl]*

The binary complex [VO(chrys)2EtOH]2 (0.1 mmol) prepared as in [5] and phenanthroline hydrochloride (0.2 mmol) in acetone (25 mL) were poured into a round bottomed flask and refluxed for 3 h. The precipitate was filtered and washed three times with hot acetone. The green-yellow solid was dried in an oven at 60 ◦C. Anal calc for C27H17ClN2O5V: 535.9 g/mol; C, 60.5; H, 3.2; N, 5.2; V, 9.5%. Exp, C, 60.4; H, 3.3; N, 5.3; V, 9.6. Diffuse reflectance spectrum: 201 nm, 208 nm, 226 nm, 276 nm, 333 nm (sh), 396 nm, 782 nm. UV-Vis (DMSO): 273 nm (105,300 M<sup>−</sup>1cm−1) 288 nm (91,100 M−1cm−1) 326 nm (12,341 M−1cm−1) 392 nm (6143 M<sup>−</sup>1cm−1) 769 nm (68 M−1cm−1). Electrospray ionization mass spectrometry (ESI-MS) analyses for the complex dissolved in DMSO:acetone (1:2): ESI-MS(+) (m/z) (calc for C27H17ClN2NaO5V): 558.02 [M − Na]+, (found): 558.01, for M, [VO(chrys)phenCl] and [M − Na]+, [VO(chrys)phenCl − Na]+. The mass-to-charge ratio peak detected at (m/z) 500.05 (100%) was due to the presence of [VOchrysphen]+ species: the ligand chloride dissociates under the ESI-MS conditions and the bidentate ligands remained bonded to the metal ion (chelate effect, higher stability), as shown in Figure S6.

#### *4.3. Cell Viability Assay (MTT Assay)*

Cell viability was measured by the 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) method. Briefly, A549 cells (human lung cancer cell line) and HEK293 (human embryonic kidney) were maintained at 37 ◦C in a 5% carbon dioxide atmosphere using DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% (*v*/*v*) fetal bovine serum as the culture medium. When 70–80% confluence was reached, cells were subcultured using TrypLE ™ and free phosphate buffered saline (PBS) (11 mM KH2PO4, 26 mM Na2HPO4, 115 mM NaCl, pH 7.4). For the treatments, cells were seeded at a density of 1 × <sup>10</sup><sup>5</sup> per well in 48 well plates, grown overnight, then incubated with the complex, metal ion, and ligands in FBS free medium. After different incubation times, at 37 ◦C, 100 μg of MTT per well were added and incubated was performed in a CO2 incubator for 2 h at 37 ◦C. DMSO was then added to dissolve formazan crystals and the absorbance of each well was measured by a plate reader at a test wavelength of 560 nm. Data are presented as the percentage of cell viability (%) of the treated group with respect to the untreated cells (control), whose viability is assumed to be 100%.

#### *4.4. Oxidative Stress Determinations*

Reactive oxygen species (ROS) generation in A549 cell lines was measured by oxidation of 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCFDA) to 2 ,7 -dichlorofluorescein (DCF). Briefly, 24-well plates were seeded with 5 × <sup>10</sup><sup>4</sup> cells per well and allowed to adhere overnight. Then, different concentrations of the compounds were added. After 24 h incubation, media was removed, and cells were loaded with 10 μM H2DCFDA diluted in clear media for 30 min at 37 ◦C. Media was then separated and the cell monolayers rinsed

with PBS and lysated into 1 mL 0.1% Triton-X100. The oxidized product DCF was analyzed in the cell extracts using fluorescence spectroscopy (λex, 485 nm; λem, 535 nm) [40].

Natural antioxidant levels of glutathione (GSH) and its oxidized product (GSSG) were determined in A549 cell lines in culture. Confluent cell monolayers from 24 well dishes were incubated with different concentrations of the compounds at 37 ◦C for 24 h. Then, the monolayers were washed with PBS and harvested by incubating them with 300 μL Triton 0.1% for 30 min. For GSH determinations, 100 μL aliquots were mixed with 1.8 mL of ice cold phosphate buffer (Na2HPO4 0.1 M-EDTA 0.005 M, pH 8) and 100 μL o-phthaldialdehyde (OPT) (0.1% in methanol) [41]. For the determination of GSSG, the cellular extracts were incubated with 0.04 M of N-ethylmaleimide (NEM) to avoid GSH oxidation, 100 μL aliquots were mixed with 1.8 mL NaOH 0.1 M and OPT, and the fluorescence was determined (λex, 350 nm; λem, 420 nm). Standard curves with different concentrations of GSH were processed in parallel. The protein content in each cellular extract was quantified using the Bradford assay [42]. The better marker for the cellular redox status, the GSH/GSSG ratio, was calculated as % control for all the experimental conditions.

The mitochondrial membrane potential was assessed to evaluate the mitochondrial function, using the DiOC6 (3,3 -Dihexyloxacarbocyanine Iodide) fluorescent probe. The probe was added to the wells (400 nM concentration) and incubated for 30 min at 37 ◦C. The cells were resuspended in PBS and measured using fluorescence spectroscopy (λex, 485 nm; λem, 535 nm) [43].

#### *4.5. Cellular Vanadium Uptake Experiments*

For vanadium uptake experiments, cells were grown to 80% confluence in 100 mm petri dishes. Incubations with the treatment compounds (28.9 μM of VO(chyrs)phenCl, VO(chrys)2 and VO(acac)2 in 0.5% DMSO) were performed for 24 h. Afterwards, Vcontaining media were removed, and the cell layers were washed twice with PBS. Cells were detached using TrypLE enzyme solution for ca. 15 min at 37 ◦C. The cell suspensions were collected into centrifuge tubes and pelleted at 4000× *g* for 2 min. The cell pellets were washed once with phosphate buffered saline (1.0 mL per tube) and lysed with 100 μL 0.10 M NaOH overnight at 4 ◦C. Each lysate (2 μL) was mixed with 98 μL of Bradford reagent and the absorbance at 560 nm was measured using a plate reader for the determination of protein content. Freshly prepared solutions (0–2.0 mg mL−<sup>1</sup> in 0.10 M NaOH) of bovine serum albumin were used for calibration. The rest of the lysate was diluted to 1.0 mL with 20% HNO3 and vanadium contents in the resultant solutions were determined by ICP-MS. Corresponding amounts of NaOH and HNO3 solutions were used to prepare the blank samples. The content of vanadium in the cell lysates was calculated in nmol *V* per mg protein [44].

#### *4.6. BSA Interactions*

The fluorescent technique was used for the measurement of the interactions with BSA. BSA in Tris-HCl (0.1 M, pH = 7) was kept constant at 6 μM and titrated with different concentrations of the complex ranging from 2 to 30 μM with an incubation time of 1 h. For the experiments, the quenching of the emission intensity of BSA (at 336 nm) by the complex was monitored at λex = 280 nm with excitation and emission slits of 10 nm. Three different temperatures were selected for thermodynamic determinations (298, 303, and 310 K). Because of the absorption of the BSA-complex near the excitation wavelength, the fluorescence intensities were corrected by the inner-filter effect, using: Fcorr = Fobs × <sup>e</sup>(1/2Aex+1/2 Aem) , where Fcorr and Fobs are the fluorescence intensities corrected by inner-filter effect and recorded, respectively, Aex and Aem are the electronic absorbances of the solutions at excitation and emission wavelengths, respectively [33]. Three independent replicates were performed for each sample and concentration.

#### *4.7. Statistical Analysis*

Statistical differences were analyzed using the analysis of variance method (ANOVA) followed by the test of least significant difference (Fisher). Statistical significance was defined as *p* < 0.05.

#### **5. Conclusions**

A new oxidovanadium(IV) metal complex with the flavonoid chrysin and phen has been synthesized and characterized. In vitro cytotoxicity testing showed that the compound exhibit significant cytotoxicity towards A549 cell line at different incubation times, indicating that the compound has the potential to act as an effective metal-based anticancer drug. The induction of intracellular reactive oxygen species (ROS) production, perturbation of mitochondrial membrane potential, and GSH and GSH/GSSG depletion suggested the ability of the complex to induce cell death by the initiation or progression of oxidative stress. It can be seen that while the complexation of the natural antioxidant chrysin by the oxidovanadium(IV) cation increased the anticancer effect of the flavonoid, the addition of phen in the coordination sphere produced a higher cytotoxic effect and a better vanadium cellular uptake. In this sense, we can discard a total decomposition of the complex leading to the release of the free ligands (being only one of them (phen) biologically active but with low cytotoxicity) at 24 h incubation. In addition, the metal complex did not show toxic effects against a non-tumorigenic cell line and could be stored and transported by albumin.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/inorganics10010004/s1, Figure S1. EPR spectrum of [VO(chrys)phenCl]. (A) Powder sample at 120 K (black) and 298 K (red). (B) Frozen DMSO solution EPR spectrum recorded at 120 K (black) together with simulation (red). EPR spectra of both powder and DMSO solution were recorded in a Bruker EMX-Plus spectrometer, equipped with a rectangular cavity. Experimental conditions: 100 kHz modulation field, 4 Gpp modulation amplitude and 2 mW microwave power. The spectra were baseline corrected using WinEPR Processing software (Bruker, Inc.) and simulations were performed with the Easy Spin 5.2.3. toolbox based on MATLAB assuming an axial spin-Hamiltonian. The spin Hamiltonian parameters obtained were gII = 1.941; AII = 162.2 <sup>×</sup> <sup>10</sup>−<sup>4</sup> cm−1; g<sup>⊥</sup> = 1.977; <sup>A</sup><sup>⊥</sup> = 59.5 <sup>×</sup> <sup>10</sup>−<sup>4</sup> cm−1. Figure S2. Spectral variation of a dissolution of [VO(chrys)phenCl] in (A) DMSO, (B) DMSO/H2O 1/99 (1 <sup>×</sup> <sup>10</sup>−<sup>2</sup> M), during 4 h. Figure S3. Cell viability assay at different of [VO(chrys)phenCl] concentrations after treatment for 24 h on HEK293 cells. The results are expressed as a percentage of the control level and represent the mean ± the standard error of the mean (SEM) from three separate experiments. \* indicates significant values in comparison with the control level (*p* < 0.05). Figure S4. The fluorescence spectra of BSA at various temperatures for VOchrysphen (0, 4, 6, 8,10, 30 μM). λex = 280 nm, [BSA] = 6 μM. Figure S5. Cell viability assay of a mixture of sodium metavanadate, chrysin and phen (1:1:1), physiological pH at different concentrations after treatment for 24 h on A549 cells. The results are expressed as a percentage of the control level and represent the mean ± the standard error of the mean (SEM) from three separate experiments. \* indicates significant values in comparison with the control level (*p* < 0.05). Figure S6. Electrospray ionization–mass spectrometry (ESI-MS) spectrum of [VO(chrys)phenCl].

**Author Contributions:** Conceptualization, P.A.M.W.; validation, E.G.F., L.G.N. and P.A.M.W.; formal analysis, A.A.D. and L.G.N.; investigation, A.A.D.; measurements, P.J.G. and M.R.; resources, P.A.M.W.; writing—original draft preparation, A.A.D., E.G.F. and P.J.G.; writing—review and editing, P.A.M.W.; visualization, P.A.M.W.; supervision, P.A.M.W. and L.G.N.; project administration, P.A.M.W.; funding acquisition, P.A.M.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by ANPCyT, 2019-0945 and UNLP X871.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the Supplementary Materials.

**Acknowledgments:** This work was supported by UNLP (X871), CICPBA, and ANPCyT (PICT 2018- 0985), Argentina. LGN and EGF are members of the Research Career, CONICET, Argentina. PAMW is member of the Research Career, CICPBA. AAD is fellowship holder from CONICET.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


**Nelly López-Valdez, Marcela Rojas-Lemus and Teresa I. Fortoul \***

Departamento de Biología Celular y Tisular, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México City 04510, Mexico; nellylopezvaldez@gmail.com (N.L.-V.);

marcelarojaslemus@hotmail.com (M.R.-L.)

**\*** Correspondence: fortoul@unam.mx; Tel.: +52-55-5623-2182

**Abstract:** Lung cancer has the highest death rates. Aerosol drug delivery has been used for other lung diseases. The use of inhaled vanadium (V) as an option for lung cancer treatment is explored. Four groups of mice were studied: (1) Saline inhalation alone, (2) Single intraperitoneal (i.p.) dose of urethane, (3) V nebulization twice a week (Wk) for 8 Wk, and (4) A single dose of urethane and V nebulization for 8 Wk. Mice were sacrificed at the end of the experiment. Number and size of tumors, PCNA (proliferating cell nuclear antigen) and TUNEL (terminal deoxynucleotidyl tranferase dUTP nick-end labeling) immunohistochemistry were evaluated and compared within groups. Results: The size and number of tumors decreased in mice exposed to V-urethane and the TUNEL increased in this group; differences in the PCNA were not observed. Conclusions: Aerosol V delivery increased apoptosis and possibly the growth arrest of the tumors with no respiratory clinical changes in the mice.

**Keywords:** urethane; vanadium; aerosol delivery; lung cancer; apoptosis; antineoplastic

#### **1. Introduction**

Lung cancer is a worldwide health problem, and this tumor has the highest estimated death rates because of the delay in the diagnosis and in the beginning of its treatment [1]. Usually, the patients have been mistreated because they do not have a previous history of smoking, which is the reason why lung cancer is not the first suspected diagnosis, even though its frequency among this group of patients has steadily increased. Some of the risk factors reported among non-smokers are environmental and occupational exposures, sex hormones, and wood smoke exposure [2,3]. The main histological subtype observed in recent years is adenocarcinoma [4].

Lung cancer treatment depends on the size of the tumor, the histologic type, and the clinical variables. Surgery, radiotherapy, and chemotherapy and or its combinations are the treatment options. The 5-year relative survival rate is about 17% for all patients and all of the stages of this pathology; if the disease is detected in early stages, the survival rate increases to 54%. Nonetheless, only 15% of the cases are diagnosed at early stages [5]. In addition, the resistance to chemotherapy and the cost of the new options, oriented to specific molecular targets, is expensive [4,6,7]. On the other hand, resistance to these treatments has also increased [8]. Additionally, because of these described events, it is important to keep looking for new treatment options for lung cancer.

The urethane model for lung tumors is a chemical carcinogenesis method that has been used to study tumor progression and treatments [9–11].

Metal compounds have been tested as possible antineoplastic agents, such as platinum compounds. Vanadium (V) is a transitional element with controversial effects [12]. Vanadium compounds have emerged as possible options for therapeutic uses because of the induction of reactive oxygen species (ROS), the activation of apoptotic cell death mechanisms, autophagy, and the inhibition of cell proliferation [13–15]. As antineoplastic

**Citation:** López-Valdez, N.; Rojas-Lemus, M.; Fortoul, T.I. The Effect of Vanadium Inhalation on the Tumor Progression of Urethane-Induced Lung Adenomas in a Mice Model. *Inorganics* **2021**, *9*, 78. https://doi.org/10.3390/ inorganics9110078

Academic Editor: Dinorah Gambino

Received: 9 October 2021 Accepted: 27 October 2021 Published: 29 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

agents, organic compounds have been studied in vitro in a variety of cancer types such as pancreatic ductal carcinoma [16].

Recently other optional routes for drug delivery have been proposed for the treatment of diverse pathologies [17] such as lung cancer [11]. Hamzawy et al. reported intratracheal administration of temozolomide in lung tumors induced by urethane [18] and Roger-Parra intranasally delivered anti-collagen-V for lung cancer treatment with promising results [11].

Aerosol drug delivery has been a technique used for the treatment of a variety of diseases and recently for experimental antineoplastic therapies [11]. This route increases the bioavailability and decreases the time of action because the doses decrease the risk of side effects, and the drug reaches the lung tissue without the metabolic changes that occur in the liver [19,20]. These benefits have been observed in asthma, COPD, and some lung infections [21,22]. In this study, we explore the administration of V by inhalation as a possible alternative route for the treatment of lung cancer in the urethane-induced lung tumor mice model.

#### **2. Results**

During the whole experiment, no signs or changes in the patterns of food intake and water consumption were observed. Body weight was statistically different when comparing the beginning (T0) with the end of the experiment (8th week) in each group. Body weight at 8th week was not statistically different among group I (control), group II (urethane), and group IV (urethane-V). In group III (vanadium), weight was significantly higher compared with the control, urethane, and urethane-V mice groups (Figure 1).

**Figure 1.** Weight of the mice per group. The weight at the beginning (Time 0) compared to the end of the experiment (8th week) was different. In group III (vanadium) the weight gain was higher than in the other groups. N = 10, values are expressed as the mean of body weight ± SEM in grams (ANOVA *p* ≤ 0.05 Tukey's post hoc).\* statistically significant differences vs. T0; a: statistically significant differences versus group III 8th week.

Except in group III (vanadium) where an increase in the weight of the mice was recorded, no other changes in the physical appearance or motor behavior of the animals were observed.

#### *2.1. Lung Histology*

Panoramic photomicrographs show the presence of pulmonary adenomas in mice treated with urethane, i.e., groups II and IV (Figure 2B,D, respectively). Groups I and III did not develop lung adenomas (Figure 2A,C, respectively).

**Figure 2.** Adenoma development in mice treated with urethane. No tumors are observed in the control (**A**) and in vanadium (**C**) groups. Arrow heads (➤) indicate the adenomas in urethane (**B**) and urethane–vanadium (**D**) groups. Hematoxylin–Eosin. Bar 2 mm.

Detailed changes in the lung tissue observed in the experimental groups are shown in Figure 3. In group I (Figure 3A), bronchioles and alveolar walls with a well-preserved structure were observed; inflammatory foci as well as focal hyperplasia of the bronchiolar and alveolar epithelium, as well as solid adenomas, are clearly identified in group II (Figure 3B). In the V-exposed group (group III; Figure 3C), perivascular and peribronchiolar lymphocytic inflammatory infiltrate were observed, whereas no adenomas were identified; in the urethane-V group (group IV), focal bronchiolar epithelial hyperplasia, small and scanty adenomas, and lymphocytic infiltrate were the main observed features (Figure 3D).

The mean number of tumors in group II was 9 ± 1.13, whereas in group IV it was 2 ± 0.51; when both groups were compared, a statistically significant difference was observed (Figure 4).

**Figure 3.** Details of the changes observed in the lungs. Control group (**A**) with no changes in the lung's parenchyma, whereas in the V-exposed group (**C**) epithelial hyperplasia and inflammatory infiltrate (\*) are observed. In urethane (**B)** and urethane-V (**D**) groups, the adenomas (↑) are observed as well as the hyperplastic epithelium (\*). Hematoxylin–Eosin stain. Bar 0.5 mm.

**Figure 4.** Quantitation of developed adenomas per group. No tumors were observed in group I (control) and group III (vanadium). In group II (urethane), the tumors were larger than in group IV (urethane-V group). N = 10, values are expressed as the mean number of adenomas ±SEM (ANOVA *p* ≤ 0.05 Tukey's post hoc). \* Statistically significant differences versus group I; a: statistically significant differences between group II and IV.

The area occupied by tumors in group II was 0.6 ± 0.07 mm2, whereas in group IV it was 0.39 ± 0.05 mm2, a difference which was statistically significant (Figure 5). No qualitative differences were observed in the amount and the spread of the infiltrate in groups II and IV.

**Figure 5.** Area (mm2) occupied by lung adenomas. In group II (urethane) the adenomas were larger compared with those observed in group IV (urethane-V). N = 10, values expressed as the mean area ± SEM (two-tailed Student's *t*-test with Welch's correction, *p* ≤ 0.05). \* Statistically significant differences between group II versus group IV.

#### *2.2. Proliferative Index*

PCNA positive nuclei stain in the lung parenchyma was observed in the four groups. In group I, the positive cells were observed mainly in some bronchiolar cells as well as in group IV. In groups II and IV, the nuclei stain was in the tumor cells (Figure 6) and no statistical difference was observed in the proliferative index calculated only in the adenomas in groups II (PI 24.18%) and IV (PI 25.93%) (Figure 7).

**Figure 6.** Immune stain for PCNA in lung adenomas. Positive PCNA nuclei (ochre color) were observed in urethane (**A**) and urethane-V (**B**) with no statistical difference between them. Hematoxylin counterstain. Bar 50 μm.

**Figure 7.** Proliferation index. The index indicated that between groups II and IV, no statistical difference was observed. N = 10, values expressed as mean percentage of PCNA positive nuclei ± SEM (two-tailed Student's *t*-test with Welch's correction). No statistically significant differences were observed.

#### *2.3. TUNEL Assay*

Positive TUNEL nuclei stain in the lung parenchyma was observed in the four groups. In group I, the positive cells were scanty inflammatory cells in the parenchyma, whereas in the V-exposed group (III), the stain was in the inflammatory cells and scarce in the bronchiolar epithelia. In groups II and IV, the nuclei stain was in the tumor cells, the inflammatory foci, and in the bronchiolar epithelium (Figure 8).

**Figure 8.** TUNEL immuno-essay. Positive nuclei TUNEL stain (ochre color) were observed in urethane group (**A**) and urethane-V group (**B**) (↑). Light green counterstain. Bar 50 μm

A clear statistical difference was observed in the apoptotic index calculated only in the adenomas in groups II (AI 5.1%) and IV (AI 10%) (Figure 9).

**Figure 9.** Apoptotic index. The index was higher in group IV (urethane-V) compared with group II (urethane). N = 10, values are expressed as mean percentage of TUNEL positive nuclei ± SEM (two-tailed Student's *t*-test with Welch's correction *p* ≤ 0.05). \*\*\* Statistically significant differences between group II versus group IV.

#### **3. Discussion**

In the present study, we report that V aerosol delivery interfered with the development of lung adenomas induced by urethane. The decrease in the area and the number of tumors was the result of increasing apoptosis of the tumor cells evaluated by TUNEL; however, no effect was observed in the PCNA proliferation marker. No respiratory clinical compromise was observed in the mice exposed to aerosolized V, and only a difference in weight gain was notorious in the V-exposed groups.

Vanadium as an antineoplastic agent has been previously explored. Köpf-Maier et al. found that vanadocene dichloride (VDC) reduces cell proliferation in leukemia tumor cells [23]. In female Sprague Dawley rats, Thompson et al., in MNU-1 (1-methyl-1 nitrosurea)-induced mammary carcinogenesis, show that supplementation with vanadium sulfate reduced the incidence and the average amount of neoplasms [24]. Other studies from Köpf-Maier's group report that the antitumor activity of VDC on the Fluid Erlich ascites tumor is because of its heterochromatin accumulation [25], mitotic aberration induction, transitory mitosis suppression, and reversible cell accumulation in the late S and G2 phases [23]. Bishayee et al. [26] suggest that the antineoplastic action of VDC might be the result of the effect on the antioxidant status in the liver and the modulation of drug metabolism enzymes of phases I and II. Sankar-Ray et al. suggested the suppression of cell proliferation, induction of apoptosis, and DNA cross-links reduction as other possible antineoplastic mechanisms. In our experimental model, an increase in apoptotic cells was observed, but not in PCNA proliferation biomarkers [27].

These results suggest that apoptosis could be the mechanisms by which V is acting on the adenomas. Recently, Rozzo et al, [15] reported the effect of V compounds in the melanoma A375 cell line in which apoptosis is observed, as well as the arrest of cell cycle in two different phases, probably by different mechanisms. The findings reported here suggest that V in aerosol delivery could be acting by inducing apoptosis and possible the tumors' cell cycle arrest [28–30].

Lu et. al. demonstrated that some synthetic V complexes showed pro-apoptotic activities in MGC803 (human gastric cancer cell line) cells related to the increase in proteins such as Bax, caspases 3 and 9, as well as the decrease in Bcl2 [31]. On the other hand, Xi et al. reported the induction of apoptotic cell death in A549 and BEAS-2B lung cancer lines associated with the overexpression of caspase 3 induced by the exposure to V nanoparticles [32].

The generation of reactive oxygen species (ROS) [28] and their effect on the neoplastic cells such as: DNA damage, oxidative alterations of other cellular organelles leading to apoptosis, and different types of cell death mechanisms, could explain the antineoplastic effects of V compounds [5].

#### *Aerosol Delivery*

In a variety of respiratory diseases, the aerosol delivery of drug treatments has been used with good results [33]. The best examples are inhaled steroids for asthma, which reduce the symptoms and the systemic effects of steroids [21]. Hamzamy et al. reported the intratracheal administration of temozolomide in gold nanoparticles or liposomes as antineoplastic carriers for the treatment of urethane-induced lung adenomas in mice [18]. Gagnadoux et al., with gemcitabine delivered by the same route, reported the potential use of aerosol delivery for lung cancer treatment [20]. The aerosol delivery of chemotherapy for lung cancer treatment in patients with non-small cell lung carcinoma (NSCLC) was reported. 5-fluorouracil (5-FU) was delivered by an ultrasonic nebulizer in two different situations: one in patients prior to surgical resection, in which the authors demonstrated that the concentration of the 5-FU was 5 to 15 times higher in the tumor than in the normal lung tissue, and two, conducted in patients with unresectable tumor in which the 5-FU was also administered by aerosol delivery, two to three times a week, reporting less pulmonary or systemic side effects. Additionally, the main agent used in the therapeutic schemes for lung cancer, cisplatin, was delivered by inhalation with less systemic side effects and with promising preclinical results [34]. With the urethane model, Abdelaziz et al. reported the reduction in lung tumors in BALB/c mice by the inhalation of lactoferrin/Chondroitin-Functionalized Monoolein Nanocomposites, supporting the use of inhalation as a possible route for lung cancer treatment, stressing that by inhalation route the agent employed in the treatment reaches higher concentrations in the tumors [35].

In our study, with whole body exposure, the regression of the adenomas was almost complete, observing some areas of peribronchiolar inflammation with no clinical effects observed in the mice, only a weight increase in V-exposed groups, which could be explained by the anabolic activity reported for V [36].

Vanadium has been reported as a possible chemotherapeutic agent for different types of neoplasms. Some antineoplastic drugs have been administered by aerosol delivery with promising results [20,32]. Here, we propose V as a potential antineoplastic agent for lung cancer by aerosol delivery that will reduce the number of tumor cells and possibly the systemic side effects reported for V. Other V compounds need further analysis to find the dose and the best protocol for the administration for this element, which opens another possible treatment for NSCLC.

#### **4. Materials and Methods**

#### *4.1. Animals*

Forty CD-1 adult male mice weighing 33–35 g were housed in hanging plastic cages (10 animals per cage), kept in an animal facility (with an average temperature of 21 ◦C, 57% humidity and controlled lighting −12:12 h light/dark regime), and fed with Rodent Laboratory chow (PMI nutrition international, Brentwood, MO, USA and Agribrands Purina, Cuautitlan, Mexico) and filtered water ad libitum. Mice were obtained from the vivarium at the School of Medicine, UNAM, and managed according to the Mexican official norm NOM-062-ZOO-1999 for the production, care, and use of laboratory animals. The project was reviewed and approved by the Research and Ethical Committee from the School of Medicine (#04-2005).

#### *4.2. Experimental Protocol*

Adult male mice were randomly assigned into four groups of 10 mice each: group I (negative control) inhaled saline 0.9% during the exposures; group II (urethane alone as positive control), received a single dose (ip) of urethane 1mg/g (ethyl carbamate, 99% purity,

Sigma Aldrich, St. Louis, MO, USA) in accordance with previous studies using the urethaneinduced lung tumorigenesis model [37]; group III (vanadium) inhaled V2O5 (0.02 mol/L) (99.99%, Sigma, St. Louis, MO, USA) in saline 1h twice a week (Tuesday and Thursday) for the 8 weeks of exposure time; and group IV (urethane and V) was as in groups II (urethane alone) and III (vanadium). At the end of the 8-week exposure, mice were anesthetized with (ip) lethal dose of pentobarbital sodium (PiSa Pharmaceutical, Guadalajara, Jalisco, México) 0.3 mg/mL and perfused via aorta with saline followed by 4% paraformaldehyde, whereas the lungs were fixed intratracheally with 4% paraformaldehyde at Total Lung Capacity (TLC) [38].

#### *4.3. Vanadium Exposure and Cardiothoracic Block Dissection*

Mice of the V-exposed groups (III and IV) inhaled 1h twice a week in an acrylic box chamber measuring 45 cm × 21 cm × 35 cm (3.3 L total volume), that could house 20 mice per session. An ultra-nebulizer DeVillbiss Ultraneb 99 (DeVillbiss Healthcare, Somerset, PA, USA) system was used to nebulize the vanadium solution at a flow rate of 10 L/min; according to the manufacturer's provided information, about 80% of the aerosolized particles reaching the mice would be expected to have a mass median aerodynamic diameter (MMAD) of 0.5–5 μm. The concentration of vanadium in the chamber was quantified as follows: a filter was placed at the external outlet of the nebulizer during the entire exposure period and samples were collected at a flow rate of 10 L/min. The filter was removed and weighed after each exposure; the V on each filter was quantified as follows: six-filters per inhalation exposures were evaluated. The source of the fog was located at the top of the chamber to ensure a homogeneous exposure. Mice behavior was always observed to detect any changes. As it has been reported in earlier studies, the final concentration in the chamber was 1.56 mg V/m3 [36] and it is in the range of the World Health Organization (0.01–60 mg/m3) for V concentrations detected in occupational exposures [39]. The V concentration in the blood was analyzed by mass spectrometry of induction-coupled plasma (ICP-MS) using a Bruker equipment, model Aurora M90, with coupled autosampler (Bruker Corp., Billerica, MA, USA). The concentration of the metal was 436 ppb or nanograms of V per of dry weight tissue (ng/g).

#### *4.4. Tissue Sampling and Preparation*

Experimental and control groups were sacrificed at the end of the exposures (8 Wk). Animals were anesthetized with sodium pentobarbital aqueous solution of (PiSa Pharmaceutical, Guadalajara, Jalisco, México) 0.3 mg/mL (ip) and perfused via aorta with saline, followed by 4% paraformaldehyde (pH 7.4) in phosphate buffer. The cardiopulmonary block was removed, and then the lungs were dissected and processed for paraffin wax embedding; 5 μm thickness tissue sections were obtained and stained with hematoxylineosin, and proved for immunohistochemical evaluation with anti-PCNA antibody and TUNEL assay. Changes were assessed with a light microscope BX51 (Olympus, Miami, FL, USA). Samples were photographed with a digital camera attached to the microscope (Media Cybernetics Inc., Bethesda, MD, USA). The number and area of the tumors were measured with Motic Images 2.0 software (Motic, Kowloon Bay, Kowloon, Hong Kong).

#### *4.5. Immunohistochemistry for PCNA*

Tissue sections were placed in poly-L-lysine (SIGMA, St Louis, MO, USA) coated slides. Antigen retrieval was achieved by incubation in a citrate buffer (pH 7.4) at 103425 Pa for 3 min, after which the slides were washed in phosphate-buffered saline (PBS). Endogenous peroxidase was blocked with 3% H2O2 (J.T. Baker, Phillipsburg, NJ, USA) for 10 min. The sections were rinsed several times with PBS-Albumin, washed for 10 min in PBS, (MP Biomedicals Inc, Kuwait city, Kuwait) and incubated for 1 h at 37 ◦C in rabbit monoclonal anti-PCNA (Abcam, Cambridge, MA, USA), and diluted 1:100 in PBST- (PBS with 0.1% Tween 20 and Albumin). The sections were washed in PBS and incubated for 30 min at 37 ◦C with the biotinylated universal link secondary antibody (Dako, Carpinteria, CA,

USA), rinsed several times in PBS, and incubated for 30 min at 37 ◦C in HRP streptavidin complex (Dako, Carpinteria, CA, USA). Immunoreactivity was visualized by incubation in 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Zymed Laboratories Inc, San Francisco, CA, USA). Samples skipping primary antibody were also included as negative controls. Immunoreactivity to PCNA-exposed lungs was measured in tissue sections to calculate the proliferation index in the adenomas; the total adenomas were evaluated from the slides of the lung of each animal and the number of positive nuclei were also counted; positive nuclei were considered when an ochre color stain was observed when the developer for the reaction was diaminobenzidine. The evaluation was completed with an image analyzer, using the software Image-Pro-Plus version 6.0. (Media Cybernetics Inc., Silver Spring, MD, USA) coupled to a digital camera (Evolution MP Color, Media Cybernetics Inc., Silver Spring, MD, USA) on a light microscope Olympus BX51 (Olympus America Inc., Melville, NY, USA).

Proliferative index (PI) was determined by a relation between the number of positive nuclei per adenoma by the total number of cells in each tumor by 100.

#### *4.6. TUNEL Assay*

The enzymatic TUNEL assay (TdT-mediated dUPT-biotin nick end labeling) (Dead-End™ Colorimetric TUNEL System, Promega Corp., Madison, WI, USA) was used to evaluate the apoptotic index. The assay identifies DNA strand breaks by marking the free 3'-OH terminal, with biotinylated desoxyuridine with the enzymatic reaction with terminal deoxynucleotidyl transferase (tdT). The biotin signaling is detected by the streptavidinmarked nuclei with the HRP enzyme bound by the biotinylated nucleotides which are visualized with HRP,3 3 -diaminobenzidine (DAB). The apoptotic nuclei are observed in ochre color in the light microscope. The assay was performed according to the providers' indications. The slides were counterstained with light green to increase the visibility of the apoptotic nucleus.

The apoptotic index (AI) was calculated in 40X photomicrographs from the different groups. Five fields randomly were selected. TUNEL positive nuclei were counted within the tumors and the total nuclei from each tumor. The index was calculated with the formula: Total amount of marked nuclei/ total nuclei X 100. The total nuclei were counted in Feulgen-stained slides [40]. Apoptotic index was compared between Group II and Group IV.

#### *4.7. Statistical Analysis*

In the different experimental groups comparison of the number of the tumors was carried out with an analysis of variance (ANOVA) with Tukey's post hoc test, whereas the analysis of the tumors area, the PI and AI was carried out with Student's *t*-test with Welch's correction (GraphPad Prism Software V 6.0, La Jolla, CA, USA). Differences were considered when *p* < 0.05.

#### **5. Conclusions**

Inhaled vanadium decreases the number and size of urethane-induced lung adenomas in mice by inducing apoptosis of the tumor cells, and with no observed clinical effects.

A limitation of this study was that the effect of V was only analyzed at a single point. It would be interesting to evaluate the effects of V overtime, to establish a wider picture of its beneficial and side effects.

Further studies about the mechanisms that lead to apoptotic cell death overtime in this model would be of interest, as well as if other types of cell death could be involved in this process.

In addition, to study if V interferes with urethane's metabolism throughout the whole experiment, we would add more information about the effects observed in this study.

**Author Contributions:** Conceptualization, N.L.-V.; methodology, N.L.-V. and M.R.-L.; formal analysis, N.L.-V.; investigation, N.L.-V. and M.R.-L.; resources, N.L.-V. and T.I.F.; writing—original draft preparation, N.L.-V.; writing—review and editing, N.L.-V. and T.I.F.; project administration, T.I.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors thank Raquel Guerrero-Alquicira for the tissue processing and to Armando Zepeda-Rodríguez and Francisco Pasos-Nájera for the artwork with the figures, all from the Departamento de Biología Celular y Tisular, Facultad de Medicina, UNAM. Alejandra Núñez-Fortoul edited English of the final version of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

