Evaluation of Fumonisin B₁ and its metabolites absorption and toxicity on intestinal cells line Caco-2

Abstract

The aim of the present paper is to investigate intestinal absorption and toxicity of Fumonisin B₁ (FB₁) and its partially (PHFB₁ and PHFB₂) and totally hydrolyzed (HFB₁) metabolites, using the human intestinal cell line Caco-2, a very well known in vitro model of intestinal epithelium for absorption and metabolism studies. Caco-2 cells were treated for 48 h with several toxin concentrations (in the range of 1–138 μM). At the end of exposure period, no significant variation on cell viability has been observed with all chemicals tested, either in undifferentiated cells or in differentiated ones, suggesting a poor toxicity of these mycotoxins for intestinal cells. In any case, FB₁ appears the most active in this respect. For which concerns the cellular absorption, FB₁, PHFB₁ and PHFB₂ are never detected into Caco-2 cells. On the contrary, a dose-dependent absorption of HFB₁ has been observed in differentiated cells, which express enzymatic and metabolic characteristics of mature enterocytes. Thus HFB₁, losing the tricarballylic acid chain, is more bioavailable than FB₁ on intestinal cell, supporting the hypothesis that in risk evaluation of fumonisins exposure its metabolites are also relevant.

Introduction

Fumonisins are a group of mycotoxins produced by several Fusarium moulds. Fumonisin B₁ (FB₁) has been found at significant levels in corn (Sydenham et al., 1991, Doko et al., 1996). Human consumption of fumonisin-contaminated corn is linked to the esophageal cancer in an area of Southern Africa (Transkey) and China (Marasas et al., 1981, Li et al., 1980, Yang, 1980, Dutton, 1996). FB₁ is at present considered a possible carcinogen to humans and classified as class 2B (IARC, 1993). More recently, Bath et al. (1997) have found, according to an epidemiological survey on African population, that FB₁ contaminated maize was responsible for toxicity at the gastrointestinal level, causing severe abdominal pain and diarrhea. Fumonisins are also known as the causative agent of leukoencephalomalacia (ELEM) in horses (Marasas et al., 1981, Kellerman et al., 1990), pulmonary edema (PPE) in pigs (Harrison et al., 1990), hepatocarcinoma in rats (Voss et al., 1990) and brain hemorrhage in rabbits (Bucci et al., 1996).

FB₁ and FB₂ are diesters of tricarballylic acid. The backbones are polyhydric alcohols that are very similar in structure to sphingosine. This similarity explains the competitive role of this molecule for ceramide synthase and its capability to interfere with the sphingolipids metabolism. Three metabolites from FB₁ are known (Rice and Ross, 1994, Shephard et al., 1994): aminopoliol 1 (PHFB₁), aminopoliol 2 (PHFB₂), both as results of a partial hydrolysis, and the totally hydrolyzed metabolite, aminopentol (HFB₁). In particular, HFB₁ is produced during nixtamalization, a traditional treatment of corn with calcium hydroxide and heat and has been detected in commercial masa, tortilla chips, and canned sweet corn (Hendrich et al., 1993, Hopmans and Murphy, 1993). In vivo studies demonstrated that HFB₁ exerts the same toxicological effects of the parent compound (Hendrich et al., 1993, Voss et al., 1996).

FB₁, and to a lesser extent HFB₁, have been also tested on several in vitro cellular systems of different origin. As a general effect, FB₁ gave cytotoxicity (inhibition of cell proliferation, LDH release) on proliferating human keratinocytes (Tolleson et al., 1996), rat hepatocytes (Abel and Gelderblom, 1998), renal cells (MDCK and LLC-PK; Yoo et al., 1996, Abbas et al., 1995), and glioma cells (Mobio et al., 2000) with an IC₅₀ in the range of 50–100 μM. In addition to these basic effects, FB₁ can induce lipid peroxidation, cellular macromolecule inhibition (Abado-Becognee et al., 1998), accumulation of sphingoid bases (Shmelz et al., 1998), apoptosis, DNA fragmentation and hypermethylation (Mobio et al., 2000). HFB₁ is less potent in vitro than FB₁, as reported, for example, on HT-29 intestinal cells (Shmelz et al., 1998) and on rat hepatocytes (Van der Westhuizen et al., 1998).

As the gastrointestinal tract represents the first barrier for ingested chemicals, as drugs, food contaminants, natural toxins, we decided to investigate FB₁, PHFB₁, PHFB₂ and HFB₁ toxicity and absorption on Caco-2 cell line, that represent a very well characterized in vitro model of epithelial barrier for intestinal absorption and metabolism studies. This human cell line derives from a colon adenocarcinoma and, despite its origin, is able to spontaneously differentiate to small intestine enterocytes (Fogh et al., 1977, Pinto et al., 1983), according to several enzymatic, physiological and structural characteristics (Grasset et al., 1984), and to express several biotransforming activities (Baker and Baker, 1992, Zucco et al., 1994).