Hydroxycobalamin catalyzes the oxidation of diethyldithiocarbamate and increases its cytotoxicity independently of copper ions

Abstract

It is known that some metals (Cu, Zn, Cd, Au) markedly increase the toxic effect of thiocarbamates. It was shown in the present study that hydroxycobalamin (a form of vitamin B₁₂, HOCbl), which incorporates cobalt, significantly enhances the cytotoxicity of diethyldithiocarbamate (DDC), decreasing its IC₅₀ value in tumor cells three to five times. The addition of HOCbl to aqueous DDC solutions accelerated the reduction of oxygen. No hydrogen peroxide accumulation was observed in DDC + HOCbl solutions; however, catalase slowed down the oxygen reduction rate. Catalase as well as the antioxidants N-acetylcysteine (NAC) and glutathione (GSH) partially inhibited the cytotoxic effect of DDC + HOCbl, whereas ascorbate, pyruvate, and tiron, a scavenger of superoxide anion, had no cytoprotective effect. The administration of HOCbl into DDC solutions (> 1 mM) resulted in the formation of a crystalline precipitate, which was inhibited in the presence of GSH. The data of UV and NMR spectroscopy and HPLC and Mass Spectrometry (LC/MS) indicated that the main products of the reaction of DDC with HOCbl are disulfiram (DSF) and its oxidized forms, sulfones and sulfoxides. The increase in the cytotoxicity of DDC combined with HOCbl occurred both in the presence of Cu²⁺ in culture medium and in nominally Cu-free solutions, as well as in growth medium containing the copper chelator bathocuproine disulfonate (BCS). The results indicate that HOCbl accelerates the oxidation of DDC with the formation of DSF and its oxidized forms. Presumably, the main cause of the synergistic increase in the toxic effect of DDC + HOCbl is the formation of sulfones and sulfoxides of DSF.

Keywords: Cytotoxicity; Diethyldithiocarbamate; Disulfiram; Hydroxycobalamin; Oxidative stress; Vitamin B(12).

Graphical abstract

Fig. 1

Enhancement of the cytotoxic effect of DDC by HOCbl. (А). Effect of the concentration of HOCbl and DDС on the viability of А549 cells. (B). A decrease in the IC₅₀ value for DDC in combination with 25 μM HOCbl in НЕр–2, А431 and А549 cells. (С). Cytotoxic effect of 48-h incubation of НЕр–2, А431, and А 549 cells with DDC + HOCbl. (D). The time course of the death of А549 cells in the presence of DDC + HOCbl. The components (25 μM HOCbl and 1 mM DDC) were added simultaneously 24 h after the seeding of cells. The action of DDC + HOCbl was interrupted by replacing the culture medium with a fresh growth medium. The cytotoxicity was estimated at 48 h after the addition of the components (see Materials and methods). The data are the means ± SEM of five separate experiments. Dark columns denote 1 mM DDC, and light columns denote DDC + HOCbl. *P < 0.01, **P < 0.05 compared to cells treated with 1 mM DDC.

Fig. 2

Oxidative stress induced by the combined action of DDC and HOCbl. (A). HOCbl (25 μM) catalyzes the reduction of oxygen by DDC. Oxygen concentration was measured after the addition of DDC and HOCbl to PBS in a chamber at 20 °C using an O₂ electrode. Curve 1: 10 mM DDC, 250 μM HOCbl, 500 U catalase, 150 μM H₂O₂. Сurve 2: 1 mM DDC, 25 μM HOCbl, 100 U catalase, 50 μM H₂O₂. (В). Antioxidants partially inhibit the cytotoxic effect induced by DDC + HOCbl in А549 cells. Antioxidants were added to the cells 24 h after the seeding in 96-well plates, 1 h prior to, or simultaneously with the addition of 1 mM DDC + 25 μM HOCbl. The action of DDC + HOCbl was interrupted by replacing the culture medium with a fresh growth medium. The cytotoxicity was estimated at 48 h after the addition of the components (see Materials and methods). The data are the means ± SEM of five separate experiments. * P < 0.01, compared to cells treated with DDC + HOCbl.

Fig. 3

UV and NMR spectra of a sediment (1) and commercial DSF (2) dissolved in ethanol (А), PBS (pH 7.2) (В), and acetone (С). The sediment was obtained in a solution of 10 mM DDC and 250 μM HOCbl in deionized water. The concentration of the crystalline sediment is 30 mg/l in alcohol and 6 mg/l in PBS, and the concentration of commercial DSF is 30 mg/l and 4.25 mg/l, respectively.

Fig. 4

Changes in UV spectra of aqueous solutions of 1 mM DDC (А), 1 mM DDC combined with 25 μM HOCbl (В), 0.1 mM DDC + 0.1 mM Н₂О₂ (С). (А, В). Before measurements, DDC and DDC + HOCbl solutions were diluted tenfold; only the spectra of unilluminated solutions were recorded.

Fig. 5

A mass spectrum of DDC–Cbl (A) and low-molecular-weight products of DDC oxidation recorded after 4 h of the reaction (B–F).

Fig. 6

Cytotoxic effect of the products of the reaction of DDC + HOCbl in А549 cells. (А) Viability of А549 cells in the presence of the precipitate obtained in an aqueous solution of 10 mM DDC and 250 μM HOCbl (1) and of commercial DSF (2) in А549 cells without the addition of copper ions and after the addition of 4 μM CuSO₄ (3, 4, respectively). (В) Cytotoxicity of DDC + HOCbl in Cu-free and Cu-containing media. Incubation time: 3 h with serum-free DMEM and 6–7 h with DMEM + FS and HBSS. Then, incubation media were replaced by a fresh growth medium DMEM supplemented with 10% FS. The cytotoxic effect was estimated 48 h after the addition of DDC + HOCbl. (С) Comparison of the effect of the chelator of extracellular copper BCS on the cytotoxic effect of 1 mM DDC + 25 μM HOCbl and on the toxicity of 1 mM DDC/30 μM DSF + Cu²⁺ (4 μМ) in DMEM + FS. BCS (50 μM) was added 1 h prior to the addition of DDC + HOCbl and DDC/DSF + Cu²⁺. The cytotoxicity was estimated at 48 h after the addition of the components (see Materials and methods). The data are the means ± SEM of five separate experiments. *P < 0.01 compared to cells treated with 1 mM DDC or 30 mkM DSF.

Fig. 7

A scheme of the oxidation of DDC in the presence of HOCbl. The major transformations are denoted by a black solid line arrows; the disproportionation of unstable oxidized derivatives of DSF is shown by a black dashed line arrows; oxidation of DSF and its oxidized products, catalyzed by a complex Cbl–(HO₂^-/O₂^2-), is shown by a blue line double arrows; ①, ②, ③ – the stage of DDC oxidation; ② - the stage that is sensitive to thiols present in the system. ④ – A hypothetical mechanism of the oxidation DSF derivatives by the action of a cobalamin–hydroperoxide/superoxide anion complex; Left Right Double Arrows denote the reversible stage of the reaction, and blue double arrows denote the irreversible stage of the reaction; ⑤, ⑥, ⑦, ⑧ – the structures of oxidized DSF derivatives.