Cytotoxicity mechanisms of sodium hypochlorite in cultured human dermal fibroblasts and its bactericidal effectiveness

Abstract

We investigated the therapeutic efficacy of the topical antiseptic sodium hypochlorite (NaOCl) for antibacterial activity and in parallel the cytotoxicity mechanisms by which hypochlorite and the chloramines generated therefrom induce oxidative tissue damage, which further influences the wound-healing process. Human dermal fibroblasts were exposed to increasing concentrations of reagent NaOCl (0.00005–0.1%) at exposure times varying between 2 and 24 h and the protective effects of fetal calf serum (FCS) determined. Antibacterial power was studied by testing a wide range of hypochlorite concentrations (0.00025–0.5%) against four isolated bacterial species. Total bactericidal effects were observed only for 0.5%; concentration range 0.25–0.025% produced partial antimicrobial activity. The early NaOCl-produced cytotoxic action on cultured fibroblasts was cell ATP depletion which occurred at 0.00005% (with FCS 2%) followed by dose- and time-dependent decreases, reaching levels below 5% of control values. Using the 3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid metabolic assay to evaluate cell death, we observed that NaOCl concentrations greater than 0.05% provoked null fibroblast survival at all exposure times assayed. Hypochlorous acid proved to exert a rapid inhibitory effect on DNA synthesis, consistent with its primary role in bacterial killing by phagocytes. Cytotoxicity produced by increasing NaOCl concentrations and assessed by measuring both mitochondrial function and cell DNA synthesis was reduced with the greatest presence of FCS (10%) in culture media.

Introduction

Sodium hypochlorite (NaOCl) is a highly and rapidly effective agent against a wide range of microbes which has been used as a universal disinfectant for more than 100 years [1]. As an antiseptic, NaOCl is also applied in clinical practice together with other chloro-releasing agents (N-chloro compounds), thanks to its antimicrobial spectrum which covers bacteria, mycobacteria, spores, viruses, algae and even protozoa. Diluted NaOCl solutions are used for bladder and urethra irrigations, vaginal washing, control of certain topical mycoses or as prophylaxis in burn infections [2] and, because of its bactericidal or antifungal efficacy, in dental therapy as a root canal irrigant [3], [4], [5], [6]. Furthermore, NaOCl has been considered the most efficient disinfectant for chemical inactivation of human immunodeficiency virus (HIV) in vitro [7].

In living organisms, hypochlorite is a useful biomolecule synthesized from hydrogen peroxide and chlorine ions in a chemical reaction catalyzed by the enzyme myeloperoxidase (MPO) secreted by activated phagocytes in zones of inflammation. HOCl is a key microbicidal agent, used as a natural defense owing to its great potency as a nucleophilic non-radical oxidant and its efficacy lies in the fact that neither bacteria nor mammalian cells can counteract its toxic effect since they lack the enzymes required for its catalytic detoxification [8]. Hypochlorous acid (HOCl) is known to react with primary amines and other N-compounds to rapidly yield chloramines and nitrogen–chlorine derivatives [9].

Reactions of HOCl and chloramines with different biological molecules can cause injury to target cells and tissue damage; at low concentrations (10–20 μM) HOCl damages proteins on cell membranes and induces cell lysis [10]. Treatment of a wide range of cells with the reagent HOCl has been shown to provoke necrotic cell death [11], [12], [13], [14], [15]. Therefore, the possible cytotoxic, and thus deleterious, effects of hypochlorite on wound healing should be considered to ascertain whether NaOCl reduces or prolongs the normal tissue regeneration process. Concentrated hypochlorite solutions are known to produce infraclinical damage on intact skin [16], and with diluted solutions (NaOCl 0.5%) applied directly to cultured fibroblasts, total cell destruction with 15-min exposure has been reported [17]. The effects of 0.025–0.0025% NaOCl solutions on neutrophil, fibroblast and endothelial cell viability in vitro caused cytoplasmic vacuolation, swollen mitochondria and dilated endoplasmic reticulum in fibroblasts and endothelial cells and 90% inhibition of neutrophil migration, but no cell death [18]. The ability of intermediate concentrations of HOCl to induce apoptotic cell death has more recently been described [19].

The role of antimicrobial topical agents is to limit bacterial proliferation to such an extent that invasion of viable underlying tissue does not occur and thus permit normal tissue regeneration; consequently, the parallel study of NaOCl cytotoxicity on cells and its bactericidal power would appear to be of interest. Lineaweaver et al. [17] conducted a study using only Staphyloccus aureus to test the bactericidal efficacy of hypochlorite and evaluated cell toxicity using the trypane blue technique that only differentiates viable from non-viable cells. In a later work, the number of bacterial species was increased to five to assess antiseptic effectiveness but without performing a parallel cytotoxicity study [20]. The toxic effects of a reduced range of NaOCl concentrations on wound healing, at short exposure times have also been studied [21], although the results were limited to acute toxicity assessment.

Cell cultures have advantages over animal experimentation since they afford highly defined culture conditions, thereby avoiding the complex homeostatic mechanisms that occur in vivo. In vitro studies may be a suitable tool in cellular toxicology research and, additionally, would offer information on the sequence and mechanisms of repair response and on how they can be modulated. Therefore, determined cytotoxicity-modulating factors in vitro, such as different fetal calf serum (FCS) concentrations should be taken into account [22], [23], since their presence may closely mimic what really happens in vivo. Investigation into the cytotoxicity of commonly used antiseptics appear to be of interest, especially since effects on host cells, compared with those on pathogenic micro-organisms, have often been underestimated. Dermal fibroblasts are the main cell type involved in matrix production for the wound healing process, and therefore were chosen for this cytotoxicity study. The aims of our work were to conduct a parallel study on cytotoxicity-related antibacterial power by applying a wide range of known NaOCl concentrations to cultured human fibroblasts at exposure times varying between 2 and 24 h; the possible detoxifying or protective actions of FCS were also studied. A further objective was to establish the sub-cellular targets of hypochlorite tissue toxicity. Furthermore, since hypochlorite solutions exert rapid antiseptic effects and are chemically unstable, we aimed to study the early and sequential toxic events to ascertain the doses and exposure times of HOCl at which cell growth arrest is transient.