Induction of cytochrome P450-dependent mixed function oxidase activities and peroxisome proliferation by chloramine-T in male rat liver

Highlights

• Rats received orally chloramine-T (doses of 1.25, 2.25, 5 and 10 mg/kg bw, 6 days).

• Chloramine-T induces CYP enzymes in liver microsomes and peroxisome proliferation.

• These new data contribute to understand a new mode of action involved in the chloramine-T toxicity.

Abstract

Chloramine-T is an antimicrobial agent recognized for its disinfectant properties widely used in food industry. As an N-chloro-compound, chloramine-T contains electrophilic chlorine and in water it hydrolyses to hypochlorite. Chlorine as hypochlorous acid or hypochlorite is a very reactive chemical which can function as both an oxidant and halogenating agent. Because chloramine-T could be involved in the metabolic activation of drugs, in the present study the effects of chloramine-T on the activities of some drug metabolizing enzymes in rat liver microsomes and peroxisome proliferation were determined in vivo. Rats were treated orally with chloramine-T at doses of 1.25, 2.50, 5 and 10 mg/kg body weight (bw)/day for 6 days. The activities of CYP2E1, CYP1A1/2 CYP2B1/2, CYP3A4 and CYP4A1/2 enzymes significantly increased after treatment with 2.50, 5 and 10 mg/kg bw/day, in a dose-dependent manner as compared to control. This effect was not observed after chloramine-T treatment at dose of 1.25 mg/kg bw/day. Our results suggest that chloramine-T may potentiate the toxicity of many xenobiotics via metabolic activation and/or accumulation of reactive metabolites.

Introduction

Chloramine-T (N-chloro-para-toluenesulfonamide) (Fig. 1) is an antimicrobial agent with widespread use in a broad range of practices, including medical, dental and veterinary, food processing and agricultural. Chloramine-T has been used in Europe as a surface disinfectant or sanitation biocidal agent since the early 1900s, in a wide of industries that range from hospital to agricultural use. The aquaculture industry has become very interested in developing chloramine-T for use as a therapeutic agent against proliferative gill disease and bacterial gill disease. Since chloramine-T is able to effectively kill pathogens (bacteria [both Gram-negative and -positive], fungi, viruses, protozoa), its application as anti-infective is advisable, all the more so as it can exhibit additional beneficial properties such as destruction of toxins, degradation of biofilms and anticoagulative and anti-inflammatory activities (Ofodile, 2007, Gottardi et al., 2013). Moreover, because antibiotic resistance is growing public health crisis, the use of antibiotics has to be minimized in humans, animals and plants. Microorganisms do not develop resistances to chloramine-T as often happens with antibiotics. The potential usefulness of chloramine-T for disinfectant and anti-infective treatments requires detailed information on toxic properties.

The initial breakdown product of chloramine-T in water as it loses its chlorine atom is the compound para-toluenesulfonamide (p-TSA). The compound p-TSA is the major metabolite of chloramine-T (Fig. 1), relatively stable molecule. The European Medicinal Agency (EMA, previously named EMEA) evaluated the available toxicity studies on chloramine-T. The oral LD₅₀ value of chloramine-T is 935 and 1100 mg/kg bw in the rat and mouse, respectively. Gastric inflammation, apathy and gastric bleeding as well as intestinal hemorrhage were observed in animals which died (EMEA , 1999). In a 90-day toxicity study in Wistar rats (EMEA, 1999), animals were exposed to diets containing chloramine-T equivalent to 0, 5, 15, 50 or 150 mg/kg bw per day. Relative kidney weight was significantly increased in both sexes at dose equal or higher than 50 mg/kg bw. The NOAEL (no observed adverse effect level) was considered to be 15 mg/kg bw per day. However, in a two-generation lifetime feeding study in Sprague-Dawley rats with the related substance ortho-TSA (EMEA , 1999), the NOEL for systemic toxicity (reduced weight gain and histological alterations such as centrilobular foci of basophilic cells and peliosis in liver) was 25 mg/kg feed equivalent to approximately 1.25 mg/kg bw. No carcinogenicity studies have been performed for either chloramine-T or para-TSA. All non-GLP (Good Laboratory Practice) tests of genotoxicity of chloramine-T assayed gave negative results (EMEA , 1999). No TDI (Tolerable Daily Intake) has been identified for chloramine-T or for para-TSA due to the lack of long-term toxicity data and limited detail on the available studies (FAO/WHO, 2008).

As an N-chloro-compound, chloramine-T contains electrophilic chlorine and can be compared with the O-chlorinated sodium hypochlorite or N-chloramines. Chloramine-T is capable of inhibiting bacterial growth by two mechanisms, with both the phenylsulfonamide moiety and the electrophilic chlorine. The mode of action of chloramine-T is thought to be through oxidative processes, quickly destroying cell material or disrupting essential cellular processes. Chloramine-T contains approximately 25% available chlorine and it is considered as an oxidant by forming primarily HOCl or OCl, highly destructive to bacteria (Hegna and Clausen, 1988, Dychdala, 2001, Gottardi et al., 2013). Because chloramine-T suppress the activities of bacteria by inducing the generation of oxygen and chlorine-free radicals, the use of chlorine to minimize microbial risks has its advantages and disadvantages. Chlorine-containing biocides for food contact surfaces must be used appropriately and in accordance with manufacturer recommendations. Chloramine-T is a well-known efficient oxidant; it destroys the activities of a pathogen through the process of oxidative stress cascade. Reactive oxygen species (ROS) are generated ubiquitously in aerobic organisms. Microbicidal effects are mediated primarily by the NADPH oxidase-dependent generation of ROS and reactive nitrogen species. However, when these cytotoxic agents overwhelm the endogenous antioxidant defense system, oxidative stress and oxidative damage occur as reflected by oxidative modification of macromolecules such as lipids, proteins and DNA (Yu, 1994). Covalent modification of proteins by oxidative systems has been implicated in various pathological conditions. Serum albumin can be converted to an oxidized form in various pathophysiological states. The type and number of oxidized amino acid residues of serum albumin increase with the concentration of chloramine-T. The active oxidants in this system are HO and chloro radicals (Anraku et al., 2001). It has been demonstrated the validity of chloramine-T treatment as a model for oxidative damage in vivo (Anraku et al., 2003).

Although the cytochrome P450 (CYP) enzymes normally generate metabolites with diminished biologic activity and represent a defense for detoxifying the ROS entities O₂ and O₂², there are numerous examples where these enzymes catalyze the metabolic activation of chemically inert agents to electrophiles (Ioannides and Parke, 1990; Guengerich et al., 1991, Hinson et al., 1994). The link between P450-mediated metabolism and toxicity of several compounds has been showed (Nyarko et al., 1997, Gonzalez and Gelboin, 1994). Because chloramine-T can potentially produce mutagenic electrophilic organoclorines and the ability of chloramine-T to induce hepatic drug metabolizing enzymes has not been reported, our study was performed with the objective to establish if chloramine-T interacts with microsomal CYP system in rat liver and to determine whether peroxisomal proliferation is also co-affected by chloramine-T.