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. 2022 Sep 9;5(10):907–918. doi: 10.1021/acsptsci.2c00093

In Vitro Characterization of a Threonine-Ligated Molybdenyl–Sulfide Cluster as a Putative Cyanide Poisoning Antidote; Intracellular Distribution, Effects on Organic Osmolyte Homeostasis, and Induction of Cell Death

Johanna M Gretarsdottir †, Ian H Lambert ‡, Stefan Sturup §, Sigridur G Suman †,*
PMCID: PMC9578141  PMID: 36268119

Abstract

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Binuclear molybdenum sulfur complexes are effective for the catalytic conversion of cyanide into thiocyanate. The complexes themselves exhibit low toxicity and high aqueous solubility, which render them suitable as antidotes for cyanide poisoning. The binuclear molybdenum sulfur complex [(thr)Mo2O2(μ-S)2(S2)]− (thr - threonine) was subjected to biological studies to evaluate its cellular accumulation and mechanism of action. The cellular uptake and intracellular distribution in human alveolar (A549) cells, quantified by inductively coupled plasma mass spectrometry (ICP-MS) and cell fractionation methods, revealed the presence of the compound in cytosol, nucleus, and mitochondria. The complex exhibited limited binding to DNA, and using the expression of specific protein markers for cell fate indicated no effect on the expression of stress-sensitive channel components involved in cell volume regulation, weak inhibition of cell proliferation, no increase in apoptosis, and even a reduction in autophagy. The complex is anionic, and the sodium complex had higher solubility compared to the potassium. As the molybdenum complex possibly enters the mitochondria, it is considered as a promising remedy to limit mitochondrial cyanide poisoning following, e.g., smoke inhalation injuries.

Keywords: molybdenum, ICP-MS, LRRC8A, cell uptake, cell fractionation, A549

Cyanide has received increasing attention as a toxic chemical in inhalation injuries,1−3 and studies of smoke inhalation injuries have prompted renewed interest in the search for a safe, easily handled, and rapidly administrable detoxifying agent.4,5 The largest risk of cyanide poisoning for the general public is exposure to smoke inhalation from residential and industrial fires,6,7 thereby placing the availability of an appropriate treatment as a public health concern where timely treatment of multiple poisoning victims is still a challenge.8

Cyanide enters the bloodstream quickly and dissipates easily into tissue, where it is subsequently found as HCN under physiological conditions (pH 7.4).9,10 Cyanide is an excellent π-acid, with a strong affinity for metal ions with high oxidation states. Although cyanide interacts with metalloenzymes, having an accessible ligand position (hemoglobin, catalases, cytochromes, peroxidases, oxidases),11 its lethal action is by noncompetitive inhibition of cytochrome c oxidase (CcO), halting cellular respiration, and causing histotoxic anoxia.1 Cyanide binds noncompetitively to form a cyanoferric cytochrome oxidase complex (CcO–CN).12,13 As lethality of cyanide is primarily related to the difficulty in reversing its binding to CcO, efforts in developing antidotes have focused on preventing the formation of the CcO–CN complex.1 Treatments for cyanide poisoning most often aim to coordinate cyanide, for example, with cobalt compounds14−18 or to convert it to thiocyanate with a sulfur donor such as thiosulfate.19,20In vivo the mitochondrial rhodanese enzyme protects aerobic respiration through sulfur transfer from thiosulfate to cyanide, leading to the formation of the less toxic thiocyanate.20 Sodium thiosulfate is a natural substrate of the enzyme rhodanese (Scheme 1).4 It is often co-administered with other antidotes, e.g., as in the Cyanide Antidote Kit (CAK), or to prevent cyanide toxicity during the metabolism of sodium nitroprusside (Na2[Fe(CN)5NO]) vasodilator.21,22

Scheme 1. Reaction of Cyanide and a Sulfur Donor Catalyzed by Rhodanese.

Scheme 1

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Cytotoxicity in molybdenum complexes has been reported over a large range of inhibitory concentration (IC50) values. Organometallic molybdenum complexes have been reported with comparable or lower IC50 values than the chemotherapeutic drug cisplatin in a number of cell lines.1,23−25 On the other hand, complexes and γ-octamolybdates (clusters), with biocompatible amino acid, peptide, or biomimetic ligands, were reported noncytotoxic, and a simple MoS2 nanosheet is noncytotoxic in up to 200 μM concentration in A549 cells.26−28

Salts of the binuclear molybdenum sulfur complex ([(thr)Mo2O2(μ-S)2(S2)]−) (Figure 1), investigated in the present study, exhibit low cytotoxicity and stability in air, water, and acidic solutions.29,30 The Mo–S bonds react with cyanide forming thiocyanate,31−37 making these compounds attractive candidates for biological studies and hence evaluation as potential treatment for cyanide poisoning. The design of the selected study complex strived to maximize water solubility,30 biocompatibility, and, as will be demonstrated in the present paper, low cytotoxicity in the human alveolar (A549) cancer cells (Figure 1). The compound formula is that of a monoanionic complex ion and its counter cation. Alkali metal counter cation offers high aqueous solubility compared to organic cations or less polar cations. The complexes exhibit high aqueous solubility as alkali metal salts, air stability, and relatively low molecular weight. These properties sparked interest in further studies on the biological activity of the complexes for the purpose of achieving better insight into their mechanism of action. For investigation of biological/physiological effects of molybdenum compounds, the human alveolar carcinoma cell line (A549) was chosen as smoke inhalation is the most likely source of cyanide poisoning for the general public,2 and lung epithelial cells consequently a key point of entrance for cyanide into the body. Complexes K[(thr)Mo2O2(μ-S)2(S2)] (1) and Na[(thr)Mo2O2(μ-S)2(S2)] (2) were chosen for this study where the two complexes differ in their physical properties influenced by their counter cation, such as in aqueous solubility. For a cyanide poisoning antidote, it is beneficial if the antidote enters cells because HCN does dissipate rapidly from the bloodstream into tissue.1 The antidotal efficacy is then not limited to intercepting HCN in the bloodstream.

Figure 1.

Figure 1

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Design of study complex 1.

A recent approach to determine metal drug content in cells exploits inductively coupled plasma mass spectrometry (ICP-MS) to determine cellular uptake.38 This has been successfully performed for [(η6-p-cym)RuCl(κ2-N,C-l)] (1-bytyl-2-phenyl-benzimidazole carboxylate), a complex developed for cancer treatment.39 The applied protocol for the fractionation of cells into a nuclear, mitochondrial, and cytosolic fraction was originally developed for mouse Ehrlich Lettré Ascites (ELA) cells.40 Analysis of the compound concentration in each fraction using ICP-MS analysis provides information on the distribution of a compound within subcellular compartments. Specific subcellular compartment protein markers were used to verify the success of the fractionation process, i.e., the purity of the fraction. Cross-contamination should not be observed for pure fractions.40 This technique was applied to study the ability of the compound to enter specific subcellular compartments where a new fractionation protocol was developed for human alveolar carcinoma cells (A549), which have not been fractionated using this method before.

The cells were subsequently subjected to studies that give insight into cell function in the presence of the complexes. Cell volume maintenance/cell volume restoration, following osmotic perturbations, gives insight into how the complexes could induce toxic effects as failure to regulate the volume is known to instigate cell cycle arrest and/or death by apoptosis and autophagy.41,42 DNA binding studies were performed to study whether putative toxic effects were due to binding to cell DNA. These in vitro studies were hoped to give insight into the mechanism of action of the complexes in vivo and guide future efforts in the synthesis of highly effective and nontoxic complexes. The research question was to determine if the binuclear molybdenum sulfur complexes were accumulated in the cells and whether they were distributed into cellular compartments. If positive, they could remove cyanide from the cytosol and other subcellular compartments intercepting cyanide and prevent its inhibition of the mitochondrial cytochrome c oxidase.

Results and Discussion

Complex Stability and Solubility

The stability of complexes 1 and 2 under the planned experimental conditions was evaluated, and their cytotoxicity was studied in the A549 cells to determine if these cells are viable in the planned treatments. As shown in Figure 1, [(thr)Mo2O2(μ-S)2(S2)]− has a single threonine ligand with a hydroxyl side chain group. The complex was synthesized according to the published procedure.30 The amino acid ligand is highly biocompatible and naturally nontoxic in vivo. The disulfide ligand reacts with cyanide and forms thiocyanate, as shown in Scheme 1, thereby forming an active catalyst for the transformation of cyanide to thiocyanate with an external sulfur donor.43 The bimetallic core [Mo2O2(μ-S)2]2+ has been shown stable in HCl, and the antiferromagnetically coupled binuclear Mo(V) center is stable toward redox reactions.44 The stability of the binuclear molybdenum sulfur complex under the biological experimental conditions was investigated by monitoring the electronic spectrum of complex 1 dissolved in water, and in phosphate-buffered saline (PBS) for at least 72 h. Figure 2 shows the electronic spectra of 1 in water and the decay profile. The pH of an aqueous solution of the complex was found to be 4.20. Water displays solvatochromic effects on the charge transfer (CT) band in the electronic spectrum of the complex, broadening and bathochromically shifting the peak at 274 to 300 nm. Therefore, a band at 350 nm, that is less impacted, was used as a reference. According to the 350 nm band, the absorbance varies 5 to 7% within the experimental timeframe with no obvious trend indicating a stable complex. A solution that was ∼0.1 mM in complex 1 in 1 mM NaOH was 75% hydrolyzed in 24 h based on loss of absorbance of its CT band at 350 nm. The compounds participate in a stoichiometric reaction with cyanide according to eq 1.

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The thiocyanate formed is quantitated using FeSCN2+ calibration curve.43 This reaction has been shown to give reliable results for 1 and 2 purity determination. A direct infusion of a solution of the bulk compound was subjected to electrospray ionization (ESI) MS analysis. The ion peaks give a characteristic isotope pattern that was simulated with the complex formula. Variation in observed compared to calculated isotope patterns, for observed m/z peaks, of less than 2 ppm is acceptable as a verification of purity. The colorimetric analysis details and observed and simulated ESI MS spectra of 1 and 2 are given in the experimental section and the Supporting Information. NMR data confirmed the absence of organic impurities. The solubility of the sodium and potassium salts was quantified in water using successive dilutions of saturated solution. The sodium and potassium salts exhibited a remarkable difference in aqueous solubility. The sodium salt (2) was found to have a solubility of 210 mM, while the potassium salt (1) was found to have 70 mM. In comparison, cobinamide sulfite and hydroxocobalamin (Cyanokit) were reported with solubilities of 350 and 70 mM, respectively.45 These results indicate adequate stability and solubility in aqueous solution to be applied for cyanide poisoning treatment in practice.

Figure 2.

Figure 2

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Electronic spectrum of 1 in water shows a slow loss of absorbance over time.

Toxicity of Complexes 1 and 2 in A549 Cells

Preliminary cytotoxicity experiments of complex 1 and similar complexes suggested that complexes 1 and 2 are nontoxic.29 This is an important property for emergency treatment, and it is important to test the complex cytotoxicity in the cell line intended for the biological experiments, the human alveolar carcinoma cells A549. Cytotoxicity experiments for both the potassium and sodium salts of [(thr)Mo2O2(μ-S)2(S2)]−, 1 and 2, respectively, were performed employing MTT assays. The IC50 values were calculated at 48 h and at 72 h. The results are shown in Table 1.

Table 1. IC50 Values of Complexes 1 and 2 in A549 Cells.

compound 48 h 72 h
complex 1 600 μM 190 μM
complex 2 490 μM 100 μM
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The results show that complexes 1 and 2 are relatively nontoxic in A549 cells compared to the well-known and cytotoxic cisplatin.46 The observed cytotoxicity increases with time. In vivo, molybdenum has been reported as excreting most often within one day and without bioaccumulation.47 At 48 h, the IC50 of cisplatin in A549 cells is 26 μM, which is about 19 times lower than that found for 2. These results correlate with other molybdenum complexes and clusters that show aqueous solubility and have biocompatible ligands that were reported noncytotoxic.26−28,48,49 An observable trend is that biocompatible ligands are expected to display lower cytotoxicity (higher IC50 values)50 although not without exceptions.51 Interestingly, the organometallic Cp2MoCl2 is noncytotoxic, and replacing the chloride ligands with malonate or maltolato ligands also results in noncytotoxic compounds.48,49 The compounds MoO3, MoS2, and MoS42– have been shown to be relatively nontoxic in vitro and well tolerated in vivo, respectively.26,52,53 MoO3 nanoplates were proposed as a selective potential breast cancer treatment with cytotoxicity reported at 275 μg/mL (1.91 mM) toward invasive breast cancer cells (iMCF-7).52 A study of the mechanism of action showed that the compound induces generation of reactive oxygen species (ROS) based on the redox chemistry of Mo(VI).52 MoS42– has been reported noncytotoxic at concentrations of 30 μM54 and at oral doses up to 200 mg/kg per day that yielded up to 30 μM Mo concentrations in plasma.55

Complexes 1 and 2 were therefore deemed suitable for further biological studies in A549 cells. Further experiments were mostly performed with the potassium salt, K[(thr)Mo2O2(μ-S)2(S2)], complex 1, as it exhibits lower cytotoxicity.

Cellular Influx and Accumulation

To obtain insight into the mechanism of action of complex 1, the time frames for cellular accumulation and more importantly for the intracellular distribution of the compounds in A549 cells were investigated. Cell viability tests give information on the relative toxicity of the selected candidate. Protein expression gives insight into the mechanism of action. Uptake and intracellular distribution of a compound provide insight into the mechanism of action in vivo. It was the hope that the compound would be able to enter cells, distribute effectively, and potentially remove cyanide from the cytosol and other subcellular compartments, before it reached the mitochondrial cytochrome c oxidase, halting cellular respiration.

Influx

The relative influx of complex 1 was studied to determine whether it is able to enter A549 cells. The A549 cells were treated with 100 or 200 μM at several different time points. The compound concentrations were chosen based on the IC50 data. The cells were isolated and analyzed for molybdenum content using ICP-MS. The results are shown in Figure 3.

Figure 3.

Figure 3

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Cellular accumulation of 1 determined as ng Mo/mg protein in the whole cells over time. The time traces for 100/200 μM are representative of 5/4 experiments.

It was observed that within the first 5 h, the cellular content (ng per mg protein) of complex 1 increased linearly with time. The experiments were repeated on three different days. Figure 3 illustrates accumulation for 100/200 μM complex 1 as obtained for one day. Overall, a similar pattern with increasing accumulation over time and concentration was seen on all days, but the actual uptake varied substantially between days with average influx rates of 51 ± 54 ng Mo/mg protein per hour for 100 μM (n = 5) and 145 ± 53 ng Mo/mg protein per hour for 200 μM (n = 4).

This variation is most likely due to biological variation of the cells as the experiments span over 2 months and several passages of the A549 cells. The overall conclusion is that the binuclear molybdenum sulfur complex is taken up by the human alveolar cells and that accumulation is time- and concentration-dependent, although no exact relationship between concentration and absolute uptake was established in the present work. Cellular uptake studies reported for organometallic Mo(II) compounds indicated that aqueous solubility and ability to pass through biological membranes were the most important factors in achieving significant cellular uptake.24 In the present study, an overall molybdenum uptake in the range of 0.1–1.3% of the administrated molybdenum was found, which is in agreement with the 0.5% cellular uptake previously reported for an organometallic molybdenum complex.24,49

Cellular Distribution

Intracellular distribution of molybdocene dichloride employing X-ray fluorescence techniques showed nonspecific localization within the cell, although reported methods did not allow relative organelle quantification.56 ICP-MS allows quantification of a metal such as molybdenum that has close to zero background in dilute physiological, low volume, samples. If cells are fractionated into their compartments, then the subcellular distribution of the metal may be determined. Expressions of the stress-sensitive biologically relevant proteins LRRC8A (leucine-rich repeat containing protein), p21 (cyclin-dependent kinase inhibitor 1 protein), and Pp53 (tumor suppressor protein) were selected to gain insight into whether and how the complexes could induce cell death. A shift in the expression of the LRRC8A protein, i.e., an essential component of volume-sensitive transporters for organic/inorganic osmolytes, indicates an impact on the cells’ ability to control cell volume and hence basic physiological processes.57−59 Upregulation in the expression of p21 indicates cell cycle arrest,60 whereas upregulation in the expression of phosphorylated p53 (Pp53) indicates DNA damage and initiation of apoptosis.60 Intracellular distribution of K[(thr)Mo2O2(μ-S)2(S2)] in A549 cells was investigated to obtain insight into the putative mechanism of action. The cells were incubated for 24 h with a 400 μM complex solution and subsequently fractionated into a nuclear, mitochondrial, and cytosolic fraction by applying an optimized version of the protocol, which was previously developed for the ELA cell line61 and now adapted to A549 cells as outlined in the protocol shown in Figure 4. The distribution of complex 1 in cells was determined by analyzing the molybdenum concentration in each fraction using ICP-MS analysis. The fractionation was performed on seven different passages of the A549 cells. The average content found in the cytosol was 875 ± 318 ng Mo (n = 7), which is approximately 19 times higher than the background level of 46 ng Mo (n = 2). On average, 55 ng of Mo was found in the mitochondrial fraction and 120 ng of Mo in the nucleus fraction.

Figure 4.

Figure 4

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Protocol for fractionation of A549 cells.

In this work, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed to evaluate the purity of the obtained fractions, and hence the success of the fractionation. Tests for the presence of MDH2 (mitochondria), MEK1/2 (cytosol), and histone H3 (nuclei) markers were performed for each cell fraction. The results of the western blotting are shown in Figure 5.

Figure 5.

Figure 5

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Western blot of whole cells and cellular fractions: left panel with mitochondrial marker MDH2 (lower 35 kDa band); right with cytosol and nucleus markers MEK1/2 and Histone, respectively. Fractions are total cell homogenate (Tot), mitochondria (Mit), nucleus (Nuc), and cytosol (Cyt). Protein markers (M) are indicated.

The Western blots show that the mitochondrial fraction indeed contains mitochondria (left panel, MDH2 protein) although it seems to express cytosolic contamination (right panel, MEK1/2). It is also seen that some cytosolic contamination appears in the nuclear fraction (right panel; MEK1/2). Hence, although a significant amount of molybdenum was confirmed and quantified in the cytosol, an optimized protocol for purification of the mitochondrial/nuclear fractions is needed to obtain accurate quantitative data for mitochondrial and nuclear accumulation of the molybdenum. The results nevertheless indicate that [(thr)Mo2O2(μ-S)2(S2)]− enters the cytosol, nucleus, and possibly the mitochondria.

DNA Binding of [(thr)Mo2O2(μ-S)2(S2)]−

As a follow-up experiment to obtain a more accurate estimate for the molybdenum content in the nucleus, the DNA of the cells was isolated and the molybdenum content of the DNA fraction was determined. Although [(thr)Mo2O2(μ-S)2(S2)]− is relatively nontoxic compared to known cytotoxic complexes such as cisplatin, it shows increased toxicity over an extended period, as it demonstrates increased toxicity after 72 h compared to 48 h in IC50 tests (Table 1). In the hope to gain better insight into the cause of the slight increase in toxicity, it was studied whether the complex binds to DNA of A549 cells. The cells were treated with [(thr)Mo2O2(μ-S)2(S2)]− for 24 and 48 h at two different concentration levels (200 and 400 μM), the DNA was isolated, and molybdenum content was determined using ICP-MS. The results are shown in Figure 6.

Figure 6.

Figure 6

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Molybdenum content in DNA isolated from A549WT cells treated with complex 1. The error bars indicate ± standard deviation (SD), n = 8.

The molybdenum values detected in DNA samples did not increase with time (24 or 48 h) indicating a fast uptake leading to a steady state. The molybdenum bound to DNA increased with concentration (t-test, p = 0,003). The data in Figure 6 demonstrate that DNA binding of complex 1 roughly increases by a factor of 2 (about 50 ng Mo/mg DNA), when the concentrations were doubled (200 to 400 μM). It can hence be concluded that DNA binding takes place and is concentration-dependent and also that cytotoxicity is low as indicated by the relatively high IC50 values (Table 1). A significantly higher DNA binding range of 40–90 μg platinum/mg DNA was found in mouse cancer cell lines (Ehrlich ascites tumor cells/ Ehrlich Lettré cells) treated with 10 μM cisplatin for 18 h.62 The ligands employed play a role in DNA interactions. A DNA binding constant of mononuclear Mo–salophen complexes with calf thymus DNA (CT-DNA) was reported as high as 104 M–1,63 whereas for MoO2(acac)2, no interaction was found with DNA.64 The interaction of Cp2MoCl2 with DNA fragments employing 31P NMR concluded that the two chloride ligands dissociated and the tetrahedral Mo(IV) center coordinated to a phosphate oxygen atom and to a nitrogen of a DNA base, both in the major and minor grooves.65 Steric hindrance of the Cp ligands prevents binding to two DNA base atoms. Additionally, molybdocene-DNA adducts were found to be unstable at pD higher than 6.0.66 Similar behavior of increased Mo content per mass of DNA for increased complex concentrations was reported for organometallic compounds, accompanied by likely intercalation of DNA for compounds with aromatic ligands,24,28 in good agreement with data reported for the salophen ligand with molybdenum.63 Complexes 1 and 2 do not have easily hydrolyzable or aromatic ligands, but they could interact with an easily accessible phosphate oxygen atom in a nondestructive manner.

Effect of [(thr)Mo2O2(μ-S)2(S2)]− on Cell Volume Regulation

Cell volume regulation under isotonic conditions and cell volume restoration following osmotic perturbations is important for cellular functions, such as metabolism, migration, and instigation of cell death by, e.g., apoptosis.41,42 Cells maintain a steady volume by regulating the intracellular concentration of ions and organic osmolytes.67 Taurine is an inert and quantitatively important organic osmolyte in mammalian cells, i.e., taurine release is increased rapidly following osmotic cell swelling but release becomes insignificant as soon as cells regulate their cellular content of osmolytes and recover normal cell volume. As taurine release can be monitored easily by tracer technique the release is often used to verify the impact of chemical drugs on the ability to control cell volume and hence basic physiological function. Taurine efflux via the volume-sensitive organic ion channel (VSOAC) from A549 cells grown in the presence of complex 1 and 2 for 20 h was determined by quantifying labeled taurine released from the cells following hypotonic perturbation.41,68 The taurine release was studied under isotonic and hypotonic conditions in an effort to indirectly determine the cells’ ability to maintain a steady volume in the presence of the compounds. Taurine release from control cells not treated with a compound was determined in parallel. The results of the taurine efflux experiments are shown in Figure 7.

Figure 7.

Figure 7

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(A) Rate constant (min–1) for taurine release caused by the 50 μM complex 2 sodium (black circle) salt as a function of time compared with control (white circle). The arrow indicates the time of exposure to hypotonic conditions. Values are given as mean rate constants for taurine release ± SD for 8 (control) and 4 (50 μM complex 2) sets of experiments. (B) Maximum rate constant for taurine release for complexes 1 and 2 as a function of concentration. Maximum values for the rate constants in cells exposed to Na salt or K salt, taken as the rate constant 6 min after hypotonic exposure, are given relative to the max. value for control cells. Data represent three sets of experiments; * indicates significant reduction (ANOVA test, Student–Newman–Keuls method) compared to control cells.

When cells under isotonic conditions are exposed to hypotonic conditions, they swell because of osmotic pressure. Due to the swelling, the VSOAC opens and taurine as well as KCl are released to the extracellular compartment to restore cell volume. Eventually, the cell volume reaches a new steady state volume at which point the taurine flux ceases. Figure 7A shows activation of VSOAC following exposure to hypotonic conditions (time 10 min), maximal VSOAC activity 6 min following hypotonic exposure, and subsequent inactivation of VSOAC for cells that were not treated by a compound (white circles). Good cell regulation is expected for compounds that have low cytotoxicity. Treating A549 cells with variable concentrations of complexes 1 or 2 shows that the VSOAC activation is less compared to untreated cells and that inactivation is more efficient. A reduced activation and improved inactivation could be taken to indicate that VSOAC activation and/or VSOAC inactivation is accelerated in treated cells compared to nontreated cells; in other words, cell volume restoration is improved by the molybdenum sulfur complex swelling. Direct cell volume registration was not performed in the present paper. The graph in Figure 7B shows that there was no significant difference in volume regulation of cells treated with the potassium or sodium salt of [(thr)Mo2O2(μ-S)2(S2)]−, but that cell regulation is, if anything, improved with increased concentration.

Effect of the Sodium Salt (2) on Cell Fate, i.e., LRRC8A, p21, and Pp53 Expression

Expressions of three biologically relevant proteins were studied in A549 cells treated with [(thr)Mo2O2(μ-S)2(S2)]− to further study its effect on cell fate. The expression of LRRC8A was studied in cells treated with 2 to further understand observed cell volume regulation. It has previously been demonstrated that LRRC8A significantly contributes to VSOAC activity57−59 and the protein expression should therefore correlate with the ability to volume regulate.69 Downregulation in LRRC8A expression indicates little VSOAC activity, whereas upregulation indicates high activity due to cell swelling. As failure in volume regulation can lead to cell cycle arrest and apoptosis, the expressions of the cyclin-dependent kinase inhibitor 1 protein (p21) and phosphorylation of the tumor suppressor protein (p53) were studied. The expressions of p21 and Pp53 in cells can give information on whether the cells are undergoing DNA damage, cell cycle arrest, and apoptosis. The expressions of LRRC8A, p21, and Pp53 in cells treated with complex 2 under isotonic conditions for 24 and 48 h were studied using Western blot. Actin and p150 were used as control markers. Representative Western blots are given in Figure 8.

Figure 8.

Figure 8

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LRRC8A, pp53, and p21 expression as a function of time and concentration (50–200 μM and 24 or 48 h). Blots represent 10 (LRRC8A), 4 (pp53), and 9 (p21) sets of experiments.

The results indicate that the expression of the VSOAC protein LRRC8A is unaffected in cells treated with 2 which supports the hypothesis that the compound seems to affect the activation/inactivation of VSOAC, i.e., the compound improves the ability to control cell volume under hypotonic conditions. There is a slight overall upregulation in the expressions of p21 (most obvious for 200 μM for 24 h) and Pp53 (most obvious for 100 μM for 24 h), which are the proteins upregulated when, DNA damage, cell cycle arrest, and apoptosis have been elicited. However, this upregulation of cell fate markers is relatively low compared to what is previously reported for cells treated with e.g., cisplatin.69 High upregulation in p21 and Pp53 was not expected since p150 and actin expression with large solid bands was seen for the control and all treatments indicated limited apoptosis. Using expression of the LC3 protein as an indicator of autophagy following 24 h exposure to 100 μM of complex 2 indicated if anything a reduction in cell death caused by autophagy (LC3 expression relative to control was 80 ± 10% compared to control, t-test: 0.05, data not shown). Hence, [(thr)Mo2O2(μ-S)2(S2)]− induce very low cytotoxicity in A549 cells.

Conclusions

The potassium and sodium salts of the threonine molybdenum sulfur complex, 1 and 2, were subjected to various biological studies in human alveolar carcinoma cells A549 to gain insight into the mechanism of action in vivo. Cytotoxicity studies with 1 and 2 demonstrated that they have relatively low cytotoxicity in vitroi.e., relatively high IC50 values, and thus were deemed suitable for further biological studies. Cellular uptake studies demonstrated that the complexes are able to enter cells and distribute between subcellular compartments, with the majority of Mo found in the cytosol, nucleus, and possibly mitochondria. In addition, Mo was found in the cell DNA where the content was dependent on complex concentration, but not time (within 48 h).

The ability to control the expression and activity of volume-sensitive transporters was not affected by 1 and 2, indicating low toxicity toward basal physiological functions. Likewise, only a slight upregulation of the expression of proteins p21 and Pp53 was observed, indicating only a minor DNA damage and upregulation of cell cycle arrest and apoptosis with increasing concentration. The observed upregulation is relatively low compared to what is observed for cells treated with cisplatin and in agreement with the observed IC50 values.

The results indicate that the salts of the binuclear molybdenum sulfur complex anion, 1 and 2, are good candidates for further development of a novel treatment for cyanide poisoning in vivo, as they do not demonstrate severe and toxic biological effects.

Experimental Section

Materials and Methods

Antibiotics (penicillin, streptomycin), Dulbecco’s modified Eagle’s medium (DMEM, with glucose (4500 mg/L) plus sodium bicarbonate, without sodium pyruvate), fetal calf serum (FBS), and trypsin/EDTA were from Invitrogen, Denmark. [1,2-3H(N)]-taurine (NET1205250UC, specific activity: 0.707 TBq/mmol) and scintillation cocktail (Ultima Gold) were from PerkinElmer, Denmark. Unless otherwise stated, chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.) or Calbiochem (Europe).

Inorganic Solutions

Phosphate-buffered saline (PBS) contained (in mM) 137 NaCl, 2.6 KCl, 6.5 Na2HPO4, and 1.5 KH2PO4. Isotonic (320 mOsm) NaCl Ringer solution contained (in mM) 152.5 NaCl, 5 KCl, 1 Na2HPO4, 0.1 MgCl2, 1 CaCl2, and 10 HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Hypotonic (200 mOsm) NaCl Ringer solution was prepared by reducing the NaCl concentration without changing the concentration of the other components. All media were adjusted to pH 7.4 with NaOH. KCl-Tris buffer (pH 7.4), used for cell fractionation, consisted of KCl (100 mM), Tris-HCl (50 mM), MgCl2 (5 mM), and Na2EDTA (1 mM). Lysis buffer, used to lyse cells and extract proteins, contained NaCl (150 mM), HEPES (20 mM), EDTA (1 mM), glycerol (10% v/v), 1% SDS, Triton X-100 (0.5% v/v), protein phosphatase inhibitor NaVO3 (1 mM), and protease inhibitor cocktail (1% v/v).

Analytical Purity of 1 and 2

Complexes 1 and 2 were synthesized and purified as verified by elemental analysis according to published procedures and their solubility was determined as described previously.30 The compounds were subjected to ESI MS spectrometry (Bruker MicroTOF) where the anion of the complex is observed in the negative scan mode. The complexes share the same anion and should show nearly identical spectra. Simulated isotope patterns compared to the found isotope pattern further verifies the compound purity. Figures S2 and S3 show the obtained and simulated spectra for the molecular anion peaks, [M-cation]−. Their purity was further assessed based on the reaction of the complex with cyanide according to the reaction shown in eq 2, where one sulfur atom reacts with the cyanide forming one equivalent of thiocyanate in a stoichiometric reaction

graphic file with name pt2c00093_0003.jpg 2

Colorimetric determination (Varian Cary 100 Bio spectrophotometer) of SCN– concentration formed as FeSCN2+ against a calibration curve gives the concentration of the complex with ±2% accuracy (Figure S1) compared to CHN elemental analysis.

Cell Culture

Wild-type, human lung adenocarcinomic alveolar epithelial cells (A549), were purchased from American Type Culture Collection (ATCC, Manassas, VA) and grown in CellStar 75 cm2 flasks (Greiner Bio-one Frickenhausen, Germany) in DMEM supplemented with FBS (10% v/v), penicillin (100 units/mL), and streptomycin (100 μg/mL). Cell cultures were subcultured every 3–4 days using 0.25% trypsin/EDTA (Invitrogen, 5 mg porcine trypsin, 2 mg EDTA/mL PBS) and kept at 37 °C, 5% CO2, and 100% humidity.

Stability Studies

Complex stability in Deionised (DI) water, 10 mM PBS, and 10 mM NaOH were determined by applying UV–visible spectroscopy (Cary 100, Varian, Palo Alto, Ca.) at ambient temperature. A solution of the compound at ∼20 μM was prepared in the respective solvent, and the electronic spectrum was monitored at regular time intervals. The spectra were plotted using GraphPad. The results are given in the main text.

Toxicity Studies/MTT Assay in A549 Cells

The MTT calorimetric assay estimates the ability of cells to convert the yellow soluble tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into a blue formazan precipitate. A549 cells were seeded in 96-well microplates at a density of 16 × 103 in 200 μL of medium and incubated (37 °C, 5% CO2) overnight. After treatment with complex 1 or 2 (400 μM) for 48 or 72 h, the growth medium was discarded and replaced with 100 μL of fresh medium. The MTT solution (5 mg/mL sterilized PBS) was added, and the plate was incubated (37 °C, 5% CO2) for 3 h. Detergent SDS-HCl solution (100 μL, 5 mL of 0.01 M HCl, 0.5 g of SDS) was added to each well and mixed to lyse the cells and solubilize the colored formazan crystals. The samples were measured at 570 nm using a FLUOstar OPTIMA 96-well microplate plate reader (BMG LabTechnologies, Offenburg, Germany). Data obtained were reported in terms of relative cell viability compared to the nonstimulated control. Each experiment was performed in triplicate.

Cellular Molybdenum Influx and Accumulation

Molybdenum influx and accumulation in A549 cells were determined as (i) the initial uptake (ng Mo/mg protein) within 0–5 h exposure of complexes 1 and (ii) total molybdenum uptake (ng Mo/106 cells) following 24/48 h complex 1 exposure. For the determination of the initial Mo uptake, A549 cells, plated in six-well BioLite, were exposed to 100/200 μM complex 1, dissolved in DMEM, for 2, 3, 4, and 5 h at 37 °C. The influx was terminated by aspiration of the DMEM followed by rapid addition/aspiration of ice-cold PBS. The cells were lysed by the addition of 96% ethanol, and the tray was left to let the alcohol evaporate. Samples were dissolved with 0.1% HCl/0.65% HNO3 before molybdenum determination by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 8800, Agilent Technologies, Santa Clara, Ca). For the quantification of data, one well in the BioLite tray was used to determine the protein content per well by the Lowry technique. Mo content (ng/well) was converted to ng per mg protein using the protein content (mg/well).

To determine Mo accumulation, A549 cells were incubated in DMEM containing 400 μM complex 1 for 24/48 h. Incubation was terminated by gentle wash with PBS, the cells were detached by trypsination, and neutralization of trypsin activity was performed by the addition of DMEM (1:10). The cells were transferred to CellStar tubes (15 mL) and washed three times in PBS by successive centrifugation (600g/3 min) before resuspension at 2 × 106 cells/mL (density determined by a Bechman Coulter particle counter Z1). Aliquots of the cell suspension were taken, and the cells precipitated by centrifugation (600g/3 min) whereafter the cell pellets were dissolved in 0.1% HCl/ 0.65% HNO3 and left to dissolve (24 h, room temperature) before Mo determination by ICP-MS.

Cellular Distribution

A549 cells, grown in four T175 flasks in DMEM containing 400 μM complex 1 for 24 h, were washed in PBS, the cells were detached by trypsination, and fresh DMEM was added to each flask to neutralize the trypsin. Cell suspensions were pooled in CellStar tubes (50 mL), and the cells were precipitated by centrifugation (600g/3 min). The cell pellet was resuspended in KCl-Tris buffer and transferred to Eppendorf tubes (1.5 mL). A KCl-Tris buffer was used as it mimicked cytosolic ion concentrations. The solution was centrifuged (1200g/5 min). The pellet was dissolved in KCl-Tris buffer, and to destroy cellular plasma membranes, a mechanical force was applied, i.e., homogenization of the pellet in the Eppendorf tube with a plastic pistil (Figure 4) that fitted tightly to the bottom of the Eppendorf tube (10 strokes–rest on ice–10 strokes). The crude homogenate, representing nuclei, mitochondria, cell debris, and cytosol, was separated by centrifugation (1500g/5 min) giving rise to a supernatant, containing mitochondria and cytosol, and a pellet, containing nuclei as well as intact cells, that had survived the homogenization. The supernatant was centrifuged (9000g/10 min) producing the cytosolic fraction (supernatant) and a “crude mitochondrial fraction” (pellet). The pellet containing nuclei/intact cells was suspended in KCl-Tris buffer and centrifuged (600 g/5 min) giving rise to a pellet, containing intact cells that was discharged, and a supernatant containing nuclei. The latter was centrifuged (1500 g/5 min), the supernatant aspirated, and the pellet was taken to represent a “crude nuclear fraction”. The crude mitochondrial and nuclear fractions were at this point “contaminated” with cytosol and consequently rinsed by centrifugation (mitochondria: 2 times 9000g/10 min; nuclei: 2 times 1500g and to time 20,000g/10 min), and each time we used KCl-Tris buffer. After the last precipitation, the mitochondrial and nuclear pellets were dissolved in lysis buffer and proceeded for (i) protein quantification using the BioRad DC Protein Assay Reagents (see above), (ii) protein identification by SDS-PAGE/western blot analysis for checking fraction purity, and (iii) Mo content by ICP-MS.

DNA Binding of K[(thr)Mo2O2(μ-S)2(S2)]

For the quantification of DNA-bound molybdenum, cellular genomic DNA from A549 cells, preexposed to 200/400 μM complex 1 for 24/48 h, was purified using a Puregene Core Kit A (Qiagen Sciences, Maryland). Following preincubation with complex 1, the cells were prepared as indicated for total Mo uptake. An amount of 2 × 106 cultured cells was washed three times in PBS (600g/3 min), lysed in cell lysis solution, provided with the kit, whereafter proteins were precipitated by centrifugation (15,000g/2min) using a protein precipitation solution, provided with the kit. The supernatant was transferred to a tube containing isopropanol, and following subsequent centrifugation (15,000g/2 min), the supernatant was discarded, and the DNA pellet was washed by the addition of 70% ethanol and precipitation by centrifugation (15,000g/2 min). The washed DNA pellet was dissolved in DNA hydration solution, provided with the kit, and incubated at 65°C (1 h) to fully dissolve the DNA. The DNA content (μg/μL) was measured the following day using a NanoDrop. The DNA samples were evaporated and treated with 0.1% HCl/ 0.65% HNO3, and the amount of molybdenum was determined by ICP-MS. DNA binding is given as ng Mo per mg DNA.

Cell Volume Regulation

The release of 3H-taurine from preloaded cells, used to determine the activity of the volume-regulated anion channel complex VSOAC, was carried out as previously published.70 Briefly, the cells were grown to 80% confluence in six-well BioLite trays (Thermo Fisher Scientific, Rochester, NY) and loaded in the DMEM for 2 h with 3H-taurine (1 μL per well, 3.7·104 Bq). The release experiment was instigated by washing the cells three times with isotonic NaCl Ringer solution to remove dead/nonattached cells as well as extracellular 3H-taurine. At 2 min intervals, the NaCl Ringer’s solutions from each well were transferred to a Snaptwist scintillation vial (Simport, Canada) and immediately substituted with fresh solution, i.e., isotonic NaCl Ringer at the time points 2, 4, 6, and 8 min and hypotonic NaCl Ringer at time points within the time frame 10–30 min. After the last samples under hypotonic conditions were collected, NaOH (1 M) was added to each well and the BioLite tray was shaken for 1 h. NaOH samples were collected in a scintillation vial, and each well was washed twice with water, which was similarly transferred to scintillation vials. Scintillation cocktail (3.5 mL, Parkard Ultima Gold) was added to each vial, and after shaking the vials, the 3H-activity was measured using a scintillation counter (PerkinElmer scintillation counter, Waltham, MA), along with a blank (scintillation fluid only).

Taurine release through the VSOAC complex is a nonsaturable process, following a first-order kinetics, i.e.,

graphic file with name pt2c00093_m001.jpg

where d[A]/dt is the change in total taurine pool per time unit, [A]o is the initial taurine pool, and k is the fractional rate constant (min–1). The correlation between the taurine pool remaining in the cells at a given time point “t” ([A]t) and the preceding time point ([A]t–2) is given by

graphic file with name pt2c00093_m002.jpg

By plotting ln ([A]t/[A]t–2) vs time, the slope between a time point “t” and the preceding time point “t – 2” indicates the −k value. In the present work, we plotted the rate constant versus time and used the maximal rate constant, obtained after 6 min hypotonic exposure, to indicate VSOAC activity.

SDS-PAGE—Western Blotting

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blot was used to separate proteins of interest by size and to quantify their expression. Cells were grown in 6 cm2 Petri dishes in the absence/presence of complex 2 (50, 100, 200 μM) for 24/48 h. The cells were washed gently once in ice-cold PBS before being lysed in lysis buffer. Lysates were sonicated (2 times 10 s) and subsequently centrifuged (20,000g, 5 min) to precipitate soluble compounds in the extracts. The protein content in the supernatant was determined with a commercial protein assay (BioRad DC Protein Assay Reagent A/BioRad DC Protein Assay Reagent S (Hercules, California)) using bovine serum albumin as a standard. Lysates were diluted in ddH2O, mixed with NuPAGE sample buffer including the redox agent dithiothreitol (DTT, boiled 5 min, 95 °C), and subsequently proceeded for SDS-PAGE gel electrophoresis NuPAGE precast 4–12% Bis-tris gels in NuPAGE MOPS ((3-(N-morpholino)propanesulfonic acid)/MES (2-(N-morpholino)ethanesulfonic acid)) SDS running buffer (Invitrogen, MA) in NOVEX chambers. A separate lane was dedicated to an Invitrogen BenchMark Protein Ladder (Life Technologies) to indicate molecular weights. Following electrophoresis, proteins were transferred to nitrocellulose membranes using an Invitrogen NuPAGE blotting system. Successful protein transfer was verified by Ponceau-S protein staining. Nitrocellulose membranes (Invitrogen) were rinsed for Ponceau staining and to avoid unspecific binding of antibodies, the membranes were placed in a TBST blocking buffer (0.01 M Tris–HCl, 0.15 M NaCl, 0.1% Tween 20, pH 7.6) containing 5% nonfat dry milk (1 h, 37 °C). The membranes were incubated overnight at 4 °C with primary antibodies and diluted in blocking buffer. The following day, the membranes were washed in TBST before exposure to secondary antibodies, diluted in blocking buffer, for 1 h at room temperature. The following antibodies were used at the indicated dilution: Monoclonal mouse anti-LRRC8A (94 kDa; SAB1412855; 1:250), Monoclonal mouse anti-p21Waf1/Cip1 (21 kDa; P1484; 1:500), Rabbit anti-Phospho-p53 (Ser 15) (53 kDa; #9284; 1:500), Rabbit anti-LC3A/B (16 kDa LC3-I/14 kDa LC3-II, #4108; 1:200), Histone H3 (18 kDa; 1:500), MDH2 (35 kDa; 1:200), and MEK1/2 (45 kDa; 1:100). As householding/reference proteins, we used Mouse monoclonal anti-β-actin (42 kDa; No A1978; 1:1000) and Mouse anti-p150 (150 kDa; No 610474; 1:1000). Secondary AP-conjugated anti-mouse and anti-rabbit antibodies were used in a dilution of 1:5000. Antibodies were purchased from Cell Signaling (Danvers, MA), BD Biosciences, α Diagnostic (San Antonio, TX), Yorkshire Bioscience, or Sigma-Aldrich. Following the final washes in TBST, membranes were developed using NBT/BCIP (Kem-En-Tec Nordic A/S, Taastrup, DK), scanned, and quantified using UN-SCAN-IT gel, version 6.1 (Silk Scientific).

Statistics

All data were statistically tested (GraphPad Prism version 7.03) by one-way ANOVA with the Newman–Keul method and Student’s t-test. In bar plots and scatterplots, the error bars indicate standard error of the mean (SEM).

Acknowledgments

Funding from the Icelandic Centre of Research grant no. 195726 is gratefully acknowledged. This contribution is based upon collaboration from COST Action CA18202, NECTAR—Network for Equilibria and Chemical Thermodynamics Advanced Research, supported by COST (European Cooperation in Science and Technology). Thiago Oliveira is thanked for the determination of the aqueous solubility of 1 and 2. Technician Dorthe Nielsen is thanked for skilled technical assistance on the many cell experiments.

Glossary

Abbreviations

A549

human alveolar cells

acac

acetylacetonate

CAK

cyanide antidote Kit

CcO

cytochrome c oxidase

CcO-CN

cyano-cytochrome c oxidase

CT

charge transfer

Cp

cyclopentadienyl

DMF

dimethylformamide

ELA

Ehrlich Lettré ascites cells

HCN

hydrogen cyanide

HT29

colon cancer cells

IC50

half-maximized inhibitory concentration

ICP MS

inductively coupled plasma mass spectrometry

iMCF-7

invasive breast cancer cells

LRRC8A

Leucine-rich repeat containing protein

MCF-7

breast cancer cells

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

p150

phosphoinositide-3-kinase, regulatory subunit 4, p21, cyclin-dependent kinase inhibitor 1 protein

PBS

phosphate-buffered saline

Pp53

tumor suppressor protein

PT45

pancreatic cancer cells

ROS

reactive oxygen species

TLC

thin-layer chromatography

thr

threonine

VSOAC

volume-sensitive organic anion channels

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00093.

  • Analysis employed to confirm the purity of compounds 1 and 2 (Figures S1–S3) (PDF)

Author Contributions

All authors have given approval to the final version of the manuscript.

Icelandic Centre of Research, grant nr. 195726.

The authors declare the following competing financial interest(s): S. Suman is the author of patents describing molybdenum compounds as cyanide poisoning antidotes.

Supplementary Material

pt2c00093_si_001.pdf (2.4MB, pdf)

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