ABSTRACT
Title
Effect of aflatoxins on immune response in vitro in LPS-stimulated J774A.1 murine macrophages.
Authors
G. Biancoa, S. Marzoccoa, R. Russob, S. Velottob, L. Severinob & G. Autorea
aDepartment of Pharmaceutical and Biomedical Sciences, University of Salerno, Fisciano (SA), Italy.
bDepartment of Pathology and Animal Health - Division of Toxicology, University of Naples Federico II, Naples (NA), Italy.
aDepartment of Pharmaceutical and Biomedical Sciences, University of Salerno, Fisciano (SA), Italy.
bDepartment of Pathology and Animal Health - Division of Toxicology, University of Naples Federico II, Naples (NA), Italy.
Abstract
Food contamination by mycotoxins assumed great importance in recent years for their impact on human and animal health and because they are very frequently found in food and feed in particular in cereals such as wheat, maize, barley, oats, and rye but also in cocoa, spices, wine and many other products and processed grains. Data of FAO show that about 25% of world food production is contaminated by at least one mycotoxin (Heussner et al., 2006).
Mycotoxins are produced by secondary metabolism of fungi found primarily in cereal grains and derived products. They are heterogeneous group of chemicals that elicit different toxic effects both on human and animal health. More than 400 different mycotoxins have been isolated and chemically characterized; those of major medical and agricultural concern are aflatoxins (AFs), ochratoxins, trichothecenes, zearalenone and fumonisins. AFs group includes four major products B1, B2, G1 and G2. After metabolism of AFB1 and B2 in the mammalian body, result two metabolites AFM1 and AFM2 as hydroxylated derivatives of the parent compound. AFs have high carcinogenic potential, the most powerful carcinogens in different species of animals and humans. International Agency for Research on Cancer has classified aflatoxin B1 in Group I carcinogens (Miliţă et al., 2010). Once few studies have been performed about the effects of AFB2 on immune functions and no data are available regarding the immunotoxicity of their metabolites AFM1 and AFM2, in this study we evaluate in vitro the antiproliferative activity of AFB1, AFB2, AFM1 and AFM2, alone and in combination and their effect on NO production, a significant inflammatory and immune mediator, by LPS-activated cultured J774A.1 macrophages.
MTT assay (Mosmann, 1983) revealed that among tested mycotoxins only AFB1, alone or in combination with other mycotoxins,reduced macrophage J774A.1 cell viability after 24, 48 and 72hrs of incubation. All mycotoxins was tested in not cytotoxic concentrations (5-30 μM) in LPS-stimulated macrophage pre-incubated for 24h with mycotoxins.
Both AFB1 and AFB2 alone didn’t interfere with NO production; while their combination significantlyinhibited NO production only at the highest concentration (30 μM).
AFM1 pre-incubation determined a significant inhibition on NO release at the concentration of 30 µM (P<0.05) while AFM2didn’t interfere with NO production by macrophage. Interestingly the contemporary presence of AFM1 and AFM2 significantly improved NO inhibition respect to AFM1 alone (P<0.05 and P<0.001 vs AFM1 at 15 and 30 μM respectively).
The contemporary presence of AFM1 and AFM2 with their respective precursors AFB1 and AFB2 significantly improved their inhibitory effect on NO release.
In conclusion our study firstly reported the capability of AFM1 and AFM2to interfere with macrophage immunefunction by NO modulation. Moreover these results highlighted that the effects of aflatoxins combination are stronger than those induced by single one. These data suggest that the co-contaminationwith AFB1,AFB2, AFM1 and AFM2,frequently possible in food, may significantly affect immunoreactivity.
References
HeussnerAH et al., Toxicol. In Vitro, 2006, 20(3):332-341.
Mosmann T, J. Immunol. Methods, 1983, 65:55-63.
Miliţă NM et al., Bacteriol Virusol Parazitol Epidemiol., 2010, 55(1):19-24.
Mycotoxins are produced by secondary metabolism of fungi found primarily in cereal grains and derived products. They are heterogeneous group of chemicals that elicit different toxic effects both on human and animal health. More than 400 different mycotoxins have been isolated and chemically characterized; those of major medical and agricultural concern are aflatoxins (AFs), ochratoxins, trichothecenes, zearalenone and fumonisins. AFs group includes four major products B1, B2, G1 and G2. After metabolism of AFB1 and B2 in the mammalian body, result two metabolites AFM1 and AFM2 as hydroxylated derivatives of the parent compound. AFs have high carcinogenic potential, the most powerful carcinogens in different species of animals and humans. International Agency for Research on Cancer has classified aflatoxin B1 in Group I carcinogens (Miliţă et al., 2010). Once few studies have been performed about the effects of AFB2 on immune functions and no data are available regarding the immunotoxicity of their metabolites AFM1 and AFM2, in this study we evaluate in vitro the antiproliferative activity of AFB1, AFB2, AFM1 and AFM2, alone and in combination and their effect on NO production, a significant inflammatory and immune mediator, by LPS-activated cultured J774A.1 macrophages.
MTT assay (Mosmann, 1983) revealed that among tested mycotoxins only AFB1, alone or in combination with other mycotoxins,reduced macrophage J774A.1 cell viability after 24, 48 and 72hrs of incubation. All mycotoxins was tested in not cytotoxic concentrations (5-30 μM) in LPS-stimulated macrophage pre-incubated for 24h with mycotoxins.
Both AFB1 and AFB2 alone didn’t interfere with NO production; while their combination significantlyinhibited NO production only at the highest concentration (30 μM).
AFM1 pre-incubation determined a significant inhibition on NO release at the concentration of 30 µM (P<0.05) while AFM2didn’t interfere with NO production by macrophage. Interestingly the contemporary presence of AFM1 and AFM2 significantly improved NO inhibition respect to AFM1 alone (P<0.05 and P<0.001 vs AFM1 at 15 and 30 μM respectively).
The contemporary presence of AFM1 and AFM2 with their respective precursors AFB1 and AFB2 significantly improved their inhibitory effect on NO release.
In conclusion our study firstly reported the capability of AFM1 and AFM2to interfere with macrophage immunefunction by NO modulation. Moreover these results highlighted that the effects of aflatoxins combination are stronger than those induced by single one. These data suggest that the co-contaminationwith AFB1,AFB2, AFM1 and AFM2,frequently possible in food, may significantly affect immunoreactivity.
References
HeussnerAH et al., Toxicol. In Vitro, 2006, 20(3):332-341.
Mosmann T, J. Immunol. Methods, 1983, 65:55-63.
Miliţă NM et al., Bacteriol Virusol Parazitol Epidemiol., 2010, 55(1):19-24.