Comparison of in vitro metabolism and cytotoxicity of capsaicin and dihydrocapsaicin

Highlights

• We compare in vitro metabolism and cytotoxicity of capsaicin and dihydrocapsaicin.

• Novel in vitro metabolites were identified for both capsaicinoids by LC–MS/MS.

• Cytotoxic responses were more pronounced for dihydrocapsaicin than capsaicin.

Abstract

Capsaicin and dihydrocapsaicin are the major active components in pepper spray products, which are widely used for law enforcement and self-protection. The use of pepper sprays, due to their irreversible and other health effects has been under a strong debate. In this study, we compared metabolism and cytotoxicity of capsaicin and dihydrocapsaicin using human and pig liver cell fractions and human lung carcinoma cell line (A549) in vitro. Metabolites were screened and identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Using liver cell fractions, a novel aliphatic hydroxylated metabolite (m/z 322) was detected to dihydrocapsaicin but no structure was found corresponding to capsaicin. Instead, a novel phase I metabolite of capsaicin, corresponding to the structure of aliphatic demethylation and dehydrogenation (m/z 294) was identified. In addition, two novel conjugates, glycine conjugates (m/z 363 and m/z 365) and bi-glutathione (GSH) conjugates (m/z 902 and m/z 904), were identified for both capsaicin and dihydrocapsaicin. The medium of the exposed A549 cells contained ω-hydroxylated (m/z 322) and alkyl dehydrogenated (m/z 304) forms, as well as a glycine conjugate of capsaicin. As to dihydrocapsaicin, an alkyl dehydrogenated (m/z 306) form, a novel alkyl hydroxylated form, and a novel glycine conjugate were found. In A549 cells, dihydrocapsaicin evoked vacuolization and decreased cell viability more efficiently than capsaicin. Furthermore, both compounds induced p53 protein and G1 phase cell cycle arrest. Usefulness of the found metabolites as biomarkers for capsaicinoid exposures will need further investigations with additional toxicity endpoints.

Introduction

The use of various chemicals in riot controls and peacekeeping operations are nowadays general part of public protection. These chemicals have immediate incapacitating effects on humans and in most cases they are considered to be safe when handled correctly with precautions. Pepper spray is one of the most used tools for law enforcement and is widely applied by civilians for self-protection. However, potential misuse and unfavorable effects may occur, due to their wide range of usage and lack of proper knowledge. The regulation of the use and surveillance of pepper spray products vary greatly between countries and there are concerns of their use. These include particularly medical issues i.e., toxicity and safety of long-term use.

Pepper sprays (called also as “OC spray” or “pepper–gas”) contain an oily extract of chili pepper, oleoresin capsicum (OC). These OC extracts are complex mixtures resulting of varying extraction conditions and maturity of the chili peppers. The active chemicals in OC extracts are capsaicinoids, of which capsaicin (8-methyl-N-vanillyl-6-nonenamide, CAP) and dihydrocapsaicin (N-(4-hydroxy-3-methoxybenzyl)-8-methylnonanamide, DHC) constitute the major pharmacologically active and toxic ingredients of the extract, at relative proportions of 45–55% and 40–50%, respectively [1] (Table 1).

Exposure to pepper spray falls usually on the face area, producing burning pain in the skin and mucous membranes and temporary loss of vision, resulting in incapacity to act for a variable time period [2]. The major adverse responses to an exposure of pepper spray capsaicinoids have been seen in the pulmonary system. Low concentration of capsaicinoids induces irritation and coughing, while high doses can lead to bronchoconstriction and dyspnea [3], [4]. Inhalation studies with animals have shown marked acute inflammation, epithelial cell dysplasia and necrosis in the upper and lower respiratory tract. The doses causing the lesions were estimated to be about the same as human could receive during 5–10 s exposure to pepper spray [5].

The mechanisms involved in capsaicinoids action are multifaceted at cellular level and can end up to apoptosis or necrosis. The mechanisms are associated with the transient potential receptor family (TRPV), particularly the subtype 1 receptor (TRPV1). This receptor is a nonselective cation-channel that is expressed particularly in the primary sensory neurons and known to be involved in sensation of pane, burning and localized inflammation [6], [7]. Receptor activation by capsaicin has been shown to trigger cytokine production, inflammation and toxicity in bronchial epithelial cells [5], [8], [9]. Capsaicinoids have previously been associated with various cellular effects e.g., disruption of arachidonic acid metabolism, increase of lipid peroxidation, change of p53 protein level (increase or decrease), and cell cycle arrest [10], [11], [12]. The p53-protein is a critical response to various acute cellular stress conditions, particularly to DNA-damage. Accumulation of the p53 protein is known to alter expression of a number of target genes involved in DNA-repair, cell cycle arrest, autophagy or elimination of damaged cells via apoptosis [13], [14]. Due to lipophility, capsaicinoids are readily absorbed from the mucous membranes and shown to be metabolized extensively in the liver but less efficiently in the respiratory system [8], [15]. Previous studies have shown that capsaicinoids undergo phase I and phase II metabolic reactions involving both oxidative and non-oxidative pathways. Various cytochrome P450 (CYP) mediated metabolites and glutathione (GSH) conjugates from capsaicin have been characterized by liquid chromatography–tandem-mass spectrometry (LC–MS/MS) and liquid chromatography–nuclear magnetic resonance spectroscopy (LC–NMR). The main targets have been shown to be in aliphatic chain, amide bond, or aromatic ring [8], [15], [16], [17].

In this study, metabolism and cytotoxicity of the two most abundant components of the pepper spray, capsaicin and dihydrocapsaicin, were investigated and compared. The goal was to broaden the knowledge of metabolism and cytotoxicity of these compounds and identify new potential biomarkers of the exposure. Furthermore, the effect of capsaicinoid on p53 protein was studied to get information of potential mechanism for reduced cell viability after the treatment. In vitro experiments were conducted using human and pig liver subcellular fractions and the human lung carcinoma cell line (A549). Chemical analyses were performed using various LC–MS/MS methods.