Equilibrium characterization of the As(III)–cysteine and the As(III)–glutathione systems in aqueous solution

Abstract

Some arsenic compounds were the first antimicrobial agents specifically synthesized for the treatment of infectious diseases such as syphilis and trypanosomiasis. More recently, arsenic trioxide has been shown to be efficient in the treatment of acute promyelocytic leukemia. The exact mechanism of action has not been elucidated yet, but it seems to be related to arsenic binding to vicinal thiol groups of regulatory proteins. Glutathione is the major intracellular thiol and plays important roles in the cellular defense and metabolism. This paper reports on a study of the interactions between arsenic(III) and either cysteine or glutathione in aqueous solution.

The behavior observed for the As(III)–glutathione system is very similar to that of As(III)–cysteine. In both cases, the formation of two complexes in aqueous solution was evidenced by NMR and electronic spectroscopies and by potentiometry.

The formation constants of the cysteine complexes [As(H₋₁Cys)₃], logK=29.84(6), and [As(H₋₂Cys)(OH)₂]⁻, logK=12.01(9), and of the glutathione complexes [As(H₋₂GS)₃]³⁻, logK=32.0(6), and [As(H₋₃GS)(OH)₂]²⁻, logK=10(3) were calculated from potentiometric and spectroscopic data.

In both cases, the [As(HL)₃] species, in which the amine groups are protonated, predominate from acidic to neutral media, and the [As(L)(OH)₂] species appear in basic medium (the charges were omitted for the sake of simplicity). Spectroscopic data clearly show that the arsenite-binding site in both complexes is the sulfur atom of cysteine. In the [As(L)(OH)₂] species, the coordination sphere is completed by two hydroxyl groups. In both cases, arsenic probably adopts a trigonal pyramidal geometry. Above pH 10, the formation of [As(OH)₂O]⁻ excludes the thiolates from arsenic coordination sites. At physiological pH, almost 80% of the ligand is present as [As(HL)₃].

Introduction

The discovery of an organoarsenic compound by Ehrlich and co-workers in 1909, salvarsan (arsphenamine), proven to be effective in the treatment of syphilis, stimulated the development of a wide range of arsenicals for the treatment of infectious diseases [1]. More recently, As₂O₃ has been reported to induce complete remission in patients with acute promyelocytic leukemia [2]. Like other antitumoral drugs, arsenic has been used in the clinical practice before its mechanism of action being completely elucidated. Arsenic trioxide was shown to inhibit cell growth and induce apoptosis in several cell lines [3], [4], [5], [6]. However, the mechanism of arsenic-induced apoptosis in tumor cells remains unclear. Arsenite induces the breaking of DNA strands, stimulates poly(ADP-ribosylation), induces an increase in cellular levels of nitric oxide and superoxide, and affects protein phosphorylation by binding to vicinal thiols [7].

The effects of arsenic can be modulated by combining it with other compounds, such as thiol-containing molecules. For example, Watson et al. [8] found that some thiols protect cells from the toxic effects of arsenite. Accordingly, Dai and co-workers [4] observed that the As₂O₃-induced apoptosis was inversely related to intracellular glutathione (GSH) concentration. In contrast, Gurr et al. [7] found that dithiothreitol enhances arsenic trioxide-induced apoptosis. The authors proposed that arsenite can complex dithiothreitol producing a new compound that has a higher potency in inducing apoptosis. Stýblo et al. [9] have shown that arsenothiols were more potent inhibitors of the glutathione reductase than arsenicals.

The biometabolism of arsenic involves a redox cycle between As(V) and As(III) with subsequent methylation of As(III), giving rise to the mono, di- and trimethylated derivatives. The reduction of arsenate to arsenite can be promoted nonenzymatically by glutathione, however, in physiological conditions, the reduction by As(V) reductases may predominate. Arsenic(III) is methylated in the liver during its hepato-enteric circulation by methyltransferases [10]. Arsenic is mostly excreted in urine in the form of methylated metabolites, being dimethylated arsenic the major form [11]. Because of this fact, biotransformation was during a long time considered a detoxification mechanism. But recently, it was proposed that, in fact, methylation is a pathway for arsenic activation because methylated forms can persist in tissues and are more toxic than inorganic arsenic(III) [12].

It is well known that arsenic(III) has a high affinity for sulfur containing molecules such as dithiothreitol [13] and glutathione [14]. Scott et al. [15] isolated and characterized an arsenite complex of glutathione as As(SG)₃ by mass spectrometry. Delnomdedieu et al. [16] found that glutathione reduces arsenate to arsenite and forms a (glutathione)₃–arsenite complex.

Another point of interest when considering the clinical use of arsenicals is the resistance to metalloid salts found in bacteria, fungi, parasites and animals. In bacteria, the resistance system responsible for detoxification of metalloids transports arsenite out of the cell. This arsenite-efflux system can be conferred by a carrier protein (ArsB) or an anion-translocating ATP-ase (ArsAB) [17]. If arsenic is present as arsenate, it must be reduced to arsenite prior to extrusion. The reduction is catalized by thiol-linked reductases that use glutaredoxin, glutathione or thioredoxin as reductants [18]. In eukaryotes, another transmembrane protein, MRP1, functions as an efflux pump. It has been shown that tumor cells overexpressing MRP1 exhibit cross-resistance to arsenite [19]. Resistance mediated by MRP1 requires intracellular GSH [20], suggesting that either GSH is a co-transported substrate or the transported substrate is an arsenite complex of GSH. There is a controversy in current literature about the formation of a complex between arsenic and GSH at physiological pH values. The formation of an As(glutathione)₃ complex has already been evidenced, but there is a lack of information about the stability of this species. So there is a need for a better quantitative understanding of the equilibrium between As(III) and glutathione. Once the stability constants of all the relevant complexes formed have been evaluated, one could simulate species distribution under physiological pH and GSH concentrations.