Gold-induced reactive oxygen species (ROS) do not mediate suppression of monocytic mitochondrial or secretory function

Abstract

The toxicity of anti-rheumatic gold compounds has limited their use and development, yet both the toxicological and therapeutic actions of these compounds remain unclear. In the current study, we tested the hypothesis that intracellular reactive oxygen species (ROS) induced by Au(I) or Au(III) compounds mediate their ability to suppress mitochondrial activity.

Methods

Human THP1 monocytes were exposed to HAuCl₄ · 3H₂O (Au(III)), or the anti-rheumatic compounds auranofin (AF) or gold sodium thiomalate (GSTM) for 6–72 h, after which mitochondrial activity (succinate dehydrogenase) was measured. To assess the role of cellular redox status as a mediator of mitochondrial suppression, monocytes were pre-treated with a pro-oxidant (t-butyl hydroquinone, t-BHQ) or antioxidant (N-acetyl cysteine, NAC ). ROS levels were measured 0–24 h post-gold addition to determine their role as mediators of mitochondrial activity suppression.

Results

AF was the most potent inhibitor of mitochondrial activity, followed by Au(III) and GSTM. Only Au(III) induced intracellular ROS; no ROS formation was observed in response to AF or GSTM exposure. Although anti- and pro-oxidants had some effects on mitochondrial suppression of Au compounds, collectively the data do not support redox effects or ROS formation as major mediators of Au-compound mitochondrial suppression.

Conclusions

Our results do not indicate that ROS and redox effects play major roles in mediating the cytotoxicity of AF, GSTM or Au(III).

Introduction

Gold compounds have been used therapeutically for over a century to treat a variety of diseases, but most commonly to treat rheumatoid and several other arthrides (Parish, 1992, Fricker, 1996, Simon, 2000). The most common gold-based anti-arthritic drugs today are gold sodium thiomalate (GSTM, injected intramuscularly) and auranofin (AF, oral), both of which contain Au(I) bound to organic ligands (Fig. 1, Schmidbaur, 1992, Simon, 2000). The therapeutic value of these compounds in limiting the progression of rheumatoid arthritis is unquestioned, but they have serious toxic side effects including diarrhea, skin rashes, blood dyscrasias, and proteinurea (Kean et al., 1997, Eisler, 2003). These side effects often are life-threatening and force patients to stop therapy despite a significant reduction of pain and joint destruction (Simon, 2000). In contrast, Au(III) compounds (Fig. 1, Messori et al., 2000) are not used clinically because of the reactivity and cytotoxicity of the Au(III) cation, but numerous new Au(III) compounds have been proposed for the treatment of arthritis, cancer, or other diseases (Novelli et al., 1999, Messori et al., 2000, Tiekink, 2002, Che et al., 2003, Bendek, 2004).

In spite of their long history of medicinal use, the mechanisms by which gold compounds are therapeutic or toxic are not known. This ignorance has severely limited the full clinical utility of Au(I) compounds, the development of new Au(I) or Au(III) compounds as drugs, and strategies to limit side effects (Simon, 2000, Wong et al., 2003). Historically, GSTM and AF have been considered immunosuppressive agents, thought to limit the activation of monocytes and other blood cells. These compounds have been reported to limit secretion of pro-inflammatory mediators such as IL-1β, IL6, or TNFα (Drakes et al., 1987, Remvig et al., 1988, Schmidt and Abdulla, 1988, Barrerra et al., 1996, Bondeson, 1997, Yoshida et al., 1999). By ‘suppressing’ activation of inflammatory cells, these compounds purportedly limit synovial hyperplasia, B- and T-cell activation, and erosion of cartilage and bone (Lipsky, 1994). However, recent evidence indicates that gold compounds are not simply suppressors of immune function, but complex modulators of cytokine secretion (Barrerra et al., 1996, Bondeson, 1997, Lampa et al., 2002, Seitz et al., 2003). Au-induced modulation may result in up- or down-regulation of cytokine secretion, particularly when the gold compounds are present in conjunction with other cellular activators such as TNFα or lipopolysaccharide (LPS). Similar results have been reported for Au(III) chloride (Stern et al., 2005). The mechanisms by which gold-based compounds induce these complex, cytokine-specific responses also are under-explored and unclear. Yet understanding these mechanisms is essential to optimizing the use of existing compounds and developing new ones.

Several lines of evidence suggest that Au-based compounds act, at least in part, by modifying cellular redox balance. In general, metal ions are thought to elevate levels of intracellular reactive oxygen species (ROS) (Kasprzak, 1995, Lloyd and Phillips, 1999, Kudrin, 2000). Elevated ROS levels are suspected triggers of several transcription factors such as AP1 and NFκB (Byun et al., 2002, Li and Stark, 2002) that mediate the secretion of many inflammatory cytokines and factors (Barnes and Karin, 1997). Other evidence suggests that gold compounds (e.g., AF) may activate Nrf2, a transcription factor that recruits an antioxidative cellular response (Katoaka et al., 2001). Furthermore, Au-induced oxidative stress may cause a buildup of oxidized thioredoxin, a key intracellular redox protein that mediates transcription factor activation, nuclear translocation, and DNA binding (Handel, 1997).

The ability of Au-based compounds and Au ions to elevate or modulate intracellular ROS formation at sublethal levels has not been reported in spite of the potential importance of this mechanism to the therapeutic and toxicological profile of these compounds. In the current study, we test the hypothesis that Au compounds generate reactive oxygen species (ROS) that mediate cellular effects. We have focused here on the ability of Au-based compound to suppress mitochondrial activity.