Short-term moderate aflatoxin B₁ exposure has only minor effects on the gut-associated lymphoid tissue of Brown Norway rats

Abstract

Aflatoxin B₁ (AFB₁) is toxic to the systemic immune system in various animal species, whereas little is known about its effect on the gut-associated lymphoid tissue (GALT). It may be hypothesized that the toxicity of AFB₁ and its locally generated metabolites in the intestinal tissue may result in a disturbed intestinal integrity and, subsequently, in an impaired immune response towards dietary proteins. The objective of our study was to investigate the toxic effect of short-term moderate AFB₁ exposure on the intestinal epithelium and on the immune cells associated with the intestinal tract. The toxicological potential of AFB₁ and its metabolites to the intestinal epithelium was determined by measuring viability and genotoxic damage in isolated jejunal epithelial cells (comet assay) after 30 min incubation in vitro. In vivo toxicology studies were carried out with Brown Norway (BN) rats, which were exposed orally once a week with AFB₁ (1×100 μg/kg body weight (b.w.)/week) for 5 consecutive weeks. Viability and genotoxicity were measured in explanted jejunal epithelial cells. For studying the effectiveness of AFB₁ on immunological parameters BN rats were treated with a high (study 1: 1×1 mg/kg b.w./week) or a low (study 2: 1×100 μg/kg b.w./week) AFB₁ dose for 5 consecutive weeks with or without ovalbumin (OVA). Mesenteric lymphocytes were isolated and proliferative responsiveness, secretion of interferon-γ, and changes in lymphocyte subpopulations as well as mucosal mast cell specific protease and anti-OVA specific antibody concentrations were measured. In vitro, AFB₁ (>30 μM) induced genotoxicity in rat jejunal epithelial cells. The oral administration of AFB₁ (1×100 μg/kg b.w./week) did not induce DNA damage in jejunal epithelial cells. The high AFB₁ dose increased the number of CD8+ and CD8/CD71+ cells in mesenteric lymph nodes. The immune response towards OVA was not affected. The low AFB₁ dose only reduced the proliferative responsiveness of mesenteric lymphocytes (P<0.05). Serum concentrations of anti-OVA specific IgE antibody, of RMCPII, and the capacity of mesenteric lymphocytes to produce interferon-γ were not impaired by AFB₁. In conclusion, exposure to moderate doses of AFB₁ does not damage the intestinal epithelium and has only minor effects on the GALT. The low exposure, as it may predominantly occur in western countries, does not appear to increase the risk for sensitization to dietary antigens.

Introduction

Food contaminants may be potential factors contributing to the increased prevalence of allergies (Madsen, 1997). The primary target of ingested food contaminants are the epithelial cells of the gastrointestinal tract and the cells of the gut-associated lymphoid tissue (GALT). Exposure of the intestinal tract to food contaminants may alter the integrity of the intestinal barrier and may impair the local immune response to food allergens. In contrast to the inhalation-associated allergies (Rusznak et al., 1994), however, experimental evidence for contaminant-potentiated allergic reactions in the gastrointestinal tract are largely lacking.

Contamination of food with mycotoxins is a world-wide health problem, although the consequences for the immune system have not been well studied. In the European Community (EC) about 20% of the produced food grains and the cereal products made thereof are contaminated with mycotoxins (Smith et al., 1994). Although the average intake of mycotoxins in the EC is very low, high-level exposure may occur sporadically through the consumption of contaminated food.

AFB₁ is a mycotoxin occurring in a wide range of food and feed commodities. The daily intake of AFB₁ in children living in a country with low-level AFB₁ exposure such as Canada is 1–2 ng/kg body weight (b.w.)/day (Kuiper-Goodman, 1995). Recently, AFB₁ exposure in the UK was estimated on the basis of AFB₁-albumin adducts to be ∼3 μg/day (∼43 ng/kg b.w./day for a person of 70 kg) (Turner et al., 1998). In countries with high AFB₁ exposure, a daily exposure of 1.7 μg/kg b.w. was estimated but it could exceed 1 mg/day at certain times of the year (Yeh et al., 1989, Eaton and Groopman, 1994). The health effects of varying levels of ingestion are insufficiently understood (Smith et al., 1994).

In the rat AFB₁ is rapidly absorbed via the small intestinal tract that follows first-order kinetics (Ramos and Hernendez, 1996) and transferred almost completely into the mesenteric blood and transported also to the mesenteric lymph nodes (Kumagai, 1989). These lymph nodes may be the organ tissue with the highest AFB₁ exposure. Oral AFB₁ is metabolized in the intestinal tissue and in the liver by various microsomal cytochrome P450s (primarily cytochrome P450 3A4 and 1A2), resulting in AFB₁-8,9-epoxide, which binds to DNA, forming AFB₁-guanine adducts (Neal, 1995). Further AFB₁-metabolites are AFB_2α, AFQ₁, AFP₁, AFM₁, and aflatoxicol (Hsieh and Wong, 1994). While the liver is the principal site of accumulation for AFB₁ and AFB₁-metabolites, only 20–25% of the ingested AFB₁-dose will reach the liver (Hsieh and Wong, 1994). Already during the course of absorption, AFB₁ metabolism to the epoxide takes place in the small intestinal tissue, indicating the intestinal tract as a major site of AFB₁ metabolism (Kumagai, 1989, Hsieh and Wong, 1994). The metabolites of AFB₁ that are capable of covalent interaction with protein in the intestinal mucosa are AFB₁-epoxide, AFB₁-dihydrodiol, and AFB_2α (Hsieh and Wong, 1994). Rats and humans are reported to produce serum AFB₁-albumin adducts in a similar manner and rate (Wild et al., 1996). AFB₁-metabolites which are excreted with the bile into the intestine are not reabsorbed (Hsieh and Wong, 1994).

AFB₁ is known to exert systemic toxicity to the immune system. The ‘minimal effective oral dose’ of AFB₁ resulting in suppressed immune function is approximately 25 μg/kg b.w./day (Pier, 1991). In animal studies, AFB₁ at a concentration of 30–1000 μg/kg b.w. mainly suppressed cell-mediated immunity with minor effects on humoral immunity (Pier, 1991, Pestka and Bondy, 1994). The observed systemic immunological abnormalities were induced at concentrations exceeding regulated levels. While systemic immunity was the focus of most immunotoxicological studies, it is very likely that AFB₁ may have its greatest effect on the mucosa-associated lymphoid tissue, particular in the intestinal tract, before it is transported to the liver (Pestka and Bondy, 1994). Currently it is not known, whether the ingestion of AFB₁ affects local compartments of the immune system such as the gut-associated lymphoid-tissue (GALT).

The objective of this study was to simulate the short-term, high-level exposure occurring in humans consuming mycotoxin-contaminated food. We have addressed the question whether toxic or subtoxic effects through AFB₁ and its metabolites in the intestinal tissue could be involved in the triggering of food allergies. The hypothesis was that in addition to their systemic immunosuppressing activity, AFB₁ and locally generated AFB₁-metabolites could impair the intestinal epithelial integrity, thus potentially facilitating the penetration of an allergen and impairing the local immune response in the gut towards this allergen. We have used the Brown Norway (BN) rat to investigate the impact of AFB₁ on the immune response to intragastric OVA, a common food allergen in humans. As markers for immunotoxicity of AFB₁ changes in lymphocyte subpopulations, proliferation and cytokine secretion of mesenteric lymphocytes, release of mucosal mast cell protease, and concentration of anti-OVA immunoglobulins were assessed. In order to elucidate more closely possible mechanisms of combination effects, the interactive potential of AFB₁/AFB₁-metabolites with intestinal epithelial cells was determined by assessing AFB₁-induced cytotoxicity, and DNA-damage of jejunal epithelial cells in vitro and in vivo.