Evaluation of immunosuppression induced by metronidazole in Balb/c mice and human peripheral blood lymphocytes

Abstract

The immunomodulatory effect of metronidazole (MTZ), a nitroimidazole drug used as an antiprotozoal and antibacterial agent, was investigated using Balb/c mice and human peripheral blood lymphocytes. For in vivo studies, mice were divided into six groups, six animals per group, group I received vehicle alone while the other groups (II–VI) received intraperitoneal injections of MTZ (14, 28, 42, 57, and 114 mg/kg) respectively. For in vitro studies different concentrations of MTZ (5, 10, 50, and 200 μg/ml) were used. MTZ showed a significant decrease in the percentage of circulating neutrophils and monocytes and an increase in the percentage of circulating lymphocytes. The relative weights of spleen as well as the relative body weight gain also decreased. Detectable changes were seen in the histology of spleen and thymus. Splenic plaque-forming cells (PFC), hemagglutination (HA) titer to sheep red blood cells (SRBC), spleenocytes and human peripheral blood lymphocytes proliferation (MLR) were markedly suppressed by MTZ treatment as compared to control group. MTZ also induced a significant decrease in delayed-type hypersensitivity (DTH) reaction, phagocytic activity (assessed by phagocytic capacity and phagocytic index) as well as TNF-α secretion by peritoneal macrophages. These observations indicate that MTZ significantly induced immunosuppression in mice and in human peripheral blood lymphocytes.

Introduction

Nitroheterocyclic chemicals have a wide variety of applications, ranging from food preservatives to antibiotics. 5-nitroimidazoles are a well-established group of antiprotozoan and antibacterial agents. They have a heterocyclic structure consisting of an imidazole-based nucleus with a nitro group, NO₂, in position 5. Metronidazole (MTZ) [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole)] is one of these chemotherapeutic agents that have been used in treatment of ailments caused by anaerobic bacteria and certain anaerobic protozoa, such as Trichomonas vaginalis, Entamoeba histolytica, and Giardia lamblia. Also, 5-nitroimidazoles are generally administered before various surgery operations [1].

All investigational new drugs should be evaluated for the potential to produce immunosuppression [2]. This is generally accomplished in repeat-dose toxicology studies using standard clinical and anatomic pathology methods. According to the American National Toxicology Program (NTP) and the Chemical Industry Institute of Toxicology (USA), a flexible approach to immunotoxicity assessment was designed. The assays would be applied to chemicals and drugs that demonstrate suspicious structure-activity relations and pharmacokinetic properties that suggest potential immunotoxicity. Among the indicators of immunotoxicity is the induction of neoplasia [3], [4].

Since the introduction of MTZ into clinical applications, its effectiveness in the treatment of various diseases was counterbalanced by the increasing evidence of its mutagenicity, carcinogenicity, and embryolethality. MTZ induced a reversible bone marrow depression and effects on male fertility [4], [5]. Some of its metabolites have been shown to be mutagenic in certain bacterial test systems. In addition, it has been observed that MTZ induces DNA single-strand breaks in the lymphocytes of patients on standard doses of the drug [6], [7], [8].

Toxicological investigations following long-term administration of high doses of some 5-nitroimidazole derivatives to rats and mice showed induction of various tumors. Therefore, the potential immunosuppressive effects of nitroheterocyclic compounds should be considered when these agents are evaluated in the laboratory or used in the clinic [9], [10]. On the other hand, heavy research have been started in order to study the significance of using antibiotics for their immunosuppressive side effect [11], [12].

MTZ is marketed by various generic manufacturers. In Jordan, it was found that MTZ was widely used by the Jordanian population during the year 2004, and it was estimated that only 43% of MTZ units sold was prescribed properly during the year 2004 [13]. MTZ is highly and repeatedly prescribed because of reinfections, improper prescriptions and automedication. In Jordan, patients as well as specialists reported MTZ-induced side effects such as gastritis, nausea, and vomiting (unpublished observations) but documented immunotoxic and neurotoxic effects have also been reported [14], [15], [16], [17].

In the present study we investigated the effect of MTZ on immune system cells and tissues in two models. Functional assays included screening for changes in percent body weight gain, organ body weight ratio, IgM plaque-forming cell (PFC) and human and mouse lymphocyte proliferation including mixed lymphocyte response (MLR), thymus, lymph nodes and spleen histopathology, delayed-type hypersensitivity (DTH) and peripheral blood cells parameters. The non specific immunity was also assessed by in vitro peritoneal macrophage phagocytosis as well as TNF-α production.