Ridha Oueslati

Zearalenone induces immunotoxicity in mice: possible protective effects of radish extract (Raphanus sativus).

Radish (Raphanus sativus) has been extensively studied for its preventive effects against different degenerative diseases. Zearalenone (ZEN) is a mycotoxin produced by Fusarium spp and is frequently implicated in immunological disorders and occasionally in hyperoestrogenic syndromes contributing to the increased risk of cancer and other diseases. The aims of this study were, firstly, to quantitatively evaluate the Tunisian radish extract (TRE) for its total flavonoids, isothiocyanates and antioxidant activity and, secondly, to investigate the protective role of TRE against immune system disorders in Balb/c mice treated with ZEN for two weeks. The results indicated that mice treated with ZEN (40 mg kg−1) alone showed a significant decrease in lymphocytes of the total white blood cells, immunoglobulin profile (IgG and IgM), B cells, T‐cell sub‐types (CD3+, CD4+ and CD8+) and natural killer and pro‐inflammatory cytokines. Mice treated with TRE (5, 10 or 15 mg kg−1) for 7 days before, during or after ZEN treatment, however, showed a significant improvement in lymphocyte, immunoglobulin profile, T‐cell sub‐types, B cells and pro‐inflammatory cytokines. Moreover, treatment with the highest dose of TRE (15 mgkg−1) enhanced the release of tumour necrosis factor‐α and interleukin‐1β but the other parameters were comparable with those of the control. It could be concluded that TRE was effective in protecting against ZEN‐induced immunological disorders. These results supported our hypothesis that TRE contains several compounds that are able to prevent or inhibit ZEN toxicity.

Interaction of aflatoxin B1 and fumonisin B1 in mice causes immunotoxicity and oxidative stress: Possible protective role using lactic acid bacteria

Abstract Aflatoxins (AF) are important foodborne mycotoxins implicated in human health and have immunocytotoxic effects. The aims of this study were to evaluate a new aflatoxin B1 (AFB1) and fumonisin B1 (FB1)-binding/degrading micro-organism for biological detoxification, to examine its ability to degrade AFB1 and FB1 in liquid medium, and to evaluate its potential in vivo protective role against any combined effects from AFB1 and FB1 on host splenocyte caspase-3 activity (reflecting DNA damage/cell death) and mRNA levels of select inflammation-regulating cytokines. Balb/c mice were divided into groups (10/group) and treated daily for 2 weeks by oral gavage with AFB1 (80 µg/kg BW), FB1 (100 µg/kg), AFB1 + FB1, or lactic acid bacteria (Lactobacillus paracasei BEJ01, 2 × 109 CFU/L, ∼2 mg/kg) – alone or in combination with the AFB1 and/or FB1. After the exposures, spleens were collected for measures of caspase-3 activity, lipid peroxidation (LP), and glutathione (GSH) content, expression of anti-oxidation protective enzymes (GPx and SOD), and mRNA levels of inflammation-regulating cytokines (e.g. IL-10, IL-4, IFNγ, TNFα). Thymii were also removed for analysis of apoptosis. The results indicated that, in the spleen, exposure to the mycotoxins led to increased caspase-3 activity, LP, and IL-10 and IL-4 mRNA levels, but decreased GSH content and down-regulated expression of GPx and SOD, and of IFNγ and TNFα mRNA. Co-treatment using Lactic Acid Bacteria (LAB) with AFB1 or FB1 suppressed levels of DNA fragmentation, normalized splenic LP and increased GSH levels, up-regulated expression of GPx and SOD, and normalized mRNA levels of the analyzed cytokines. It is concluded that AFB1 and FB1 might have combinational (synergistic moreso than additive) toxic effects in situ. Further, it can be seen that use of LAB induced protective effects against the oxidative stress and (immuno)toxicity of these agents in part through adhesion (and so likely diminished bioavalability).

Raphanus sativus extract prevents and ameliorates zearalenone-induced peroxidative hepatic damage in Balb/c mice

Objectives Raphanus sativus (radish) is a species of crucifer, which includes widely consumed vegetables, distributed in Asia, Africa and Europe. It is a rich source of bioactive molecules including anthocyanins, glucosinolates, isothiocyanates and other flavonoids, and miscellaneous phenolic substances. We have evaluated the hepatoprotection of R. sativus extract against zearalenone, an estrogenic mycotoxin initiating hepatotoxicity in male Balb/c mice.

In-vitro free radical scavenging, antiproliferative and anti-zearalenone cytotoxic effects of 4-(methylthio)-3-butenyl isothiocyanate from Tunisian Raphanus sativus.

Objectives The aim of this study was to investigate the antiradical and antioxidant properties of 4‐(methylthio)‐3‐butenyl isothiocyanate (MTBITC) extracted from Raphanus sativus and to assess the effects of MTBITC on tumour cell growth, cytotoxicity induced by zearalenone, an oestrogenic mycotoxin, and modulation of the expression of the genes involved in these aspects of cell behaviour.

Isothiocyanate from the Tunisian radish (Raphanus sativus) prevents genotoxicity of Zearalenone in vivo and in vitro

Zearalenone (ZEN) is a naturally occurring contaminant of animal feed that has been implicated in several mycotoxicoses in farm livestock. Recently some information has become available indicating that ZEN caused cancer or at least increased its prevalence, although the mechanism of action is unknown. Many papers mentioned that exposure to ZEN results in genotoxicity and DNA damage. Therefore, we investigated the chemo-preventive role of 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) extracted from Tunisian Raphanus sativus (radish) on the cytogenetic effect of ZEN in Balb/c mice and in in vitro cultures of mouse lymphocytes isolated from mouse spleen. We determined chromosome aberrations and micronuclei as well as the mitotic index and DNA fragmentation following ZEN treatment alone or in combination with MTBITC. This report is the first to provide evidence of a statistically significant decrease of structural chromosome aberrations and micronuclei associated with an augmentation of the mitotic index and prevention of DNA fragmentation in all mice treated with ZEN-MTBITC and in mouse lymphocyte cultures. The MTBITC alone was safe and succeeded in reducing the toxicity of ZEN by counteracting its deleterious effect, thus protecting against the genotoxicity and clastogenicity from ZEN.

Natural occurrence of aflatoxins (B1 and M1) in feed, plasma and raw milk of lactating dairy cows in Beja, Tunisia, using ELISA

Beja is an agricultural area in northwest Tunisia. It contributes to national needs by offering cereals and milk to the market for human and animal consumption. A small number of studies on mycotoxin occurrence in feedstuffs and raw milk from lactating dairy cows in this region are available. Therefore, 226 samples were collected from farms and local markets during November 2008 until April 2010. Samples consisted of 112 raw cow milk, 56 blood from lactating cows and 58 feed destined for dairy cows. Plasma and feed were analysed for aflatoxin B1 (AFB1). Milk samples were analysed for aflatoxin M1 (AFM1). All samples were treated using a simultaneous methanolic-aqueous extraction, followed by immunoaffinity column clean-ups and were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA). Recoveries were 80%–95% and 81%–92% for AFB1 and AFM1, respectively, while the limit of detection (LOD) was 0.01 µg/kg or µg/l for both mycotoxins. Results revealed the presence of AFB1 in 84.4% of the feed samples (mean 18.7 ± 1.4 µg/kg), and 39.2% of the plasma-examined samples (median 7.1 ± 1.0 µg/l) were found to be contaminated at levels higher than the Tunisian and the European Union (EU) limit for dairy animals, which are 20 and 5 µg/kg in animal feed, respectively. AFM1 was detected in 60.7% of the cow raw milk samples examined (median 13.6 ± 1.4 µg/l). Contaminated levels were higher than the EU limit of 0.05 µg/l. It was concluded that more precaution should be taken on hygiene controls in order to prevent fungal contamination.

Interaction of Lactobacillus plantarum MON03 with Tunisian Montmorillonite clay and ability of the composite to immobilize Zearalenone in vitro and counteract immunotoxicity in vivo

Background and aim: The present study was conducted to determine the abilities of the living Lactobacillus plantarum MON03 (LP) cells, Tunisian montmorillonite clay and their composites to accumulate Zearalenone (ZEA) from a liquid medium and elucidate the preventive effect of their composite in ZEA-contaminated balb/c mice showing immunotoxicity disorders. Materials and methods: In the in vitro study, LP (2 × 109 CFU/mL), TM (0.5 mg) and LP+TM were incubated with 50 µg mL−1 ZEA for 0, 12 and 24 h. For the in vivo study, the composite MT+LP was evaluated also for possible protection regarding ZEA-immunotoxicity in Balb/c mice as a sensitive model. Results: Results indicated that TM and LP+TM had a high capacity of adsorbing ZEA 87.2 ± 2.1 and 94.2 ± 2.1%, respectively. However, LP alone able to remove only 78% after 24 h of incubation. The quantity of adsorbed ZEA by LP, TM and LP+TM were 39, 43,5 and 47 µg mL−1 of PBS, respectively. The in vivo results indicated that mice orally exposed to ZEA- (40 mg/kg bw) for 2 weeks showed severe immunotoxicity typical of fusarotoxicosis regarding thymocytes and splenocytes cell viability count, IFN-γ, IL-12, TNF-α production and B-cell activation. Mice treated with LP and TM alone, and LP+MT in combination with ZEA were comparable to the control. Conclusion: Both LP and TM are safe by themselves and their composite succeeded to exert a potential prevention by counteracting ZEA-immunotoxicity and can be implicated in the biotechnology of ZEA removal in human food and animal feed.

Immunotoxicity of zearalenone in Balb/c mice in a high subchronic dosing study counteracted by Raphanus sativus extract.

Radish (Raphanus sativus) is a cruciferous plant, rich on flavonoids, isothiocyanates, and phenolic acids. They show anti-inflammatory and immunomodulatory activity both in vitro and in vivo. Isothiocyanates and flavonoids have been reported previously to prevent low-sub-chronic dose of zearalenone (ZEN) causing immunotoxicity. The present study focuses on the amelioration of fusarotoxicosis in Balb/c mice by feeding two concentrations of radish extract. The extract at 15 and 30 mg/kg bw, was evaluated to reduce the deleterious effects in immunological parameters of high subchronic doses of 40 and 80 mg of ZEN/kg bw on modulation of lipopolysaccharide (LPS). ZEN consuming mice showed a “dose-related” decrease in weight gain and in the immune relative weights organs. Moreover, Atrophy and lymphoid depletion were seen in the histopathology of spleen. Ingestion of ZEN at either level had a significant effect on total red blood cell numbers and on their relative number of lymphocytes. Likewise, ZEN alters the production of regulatory cytokines and antibody of LPS stimulated mice. By contrast, the additions of radish extract with a low or high dose of ZEN moderately decreased the affected mice and/or the severity of lesions, and all tested parameters were normal or at least near normal levels. In addition, the radish extract alone did not produce any significant changes in all tested parameters compared with the controls. In conclusion, radish extract was effective for the protection of high dose ZEN-immunotoxication in mice and it could contribute to a solution of the ZEN immunotoxicity in humans and in farm animals.

Ability of Lactobacillus rhamnosus GAF01 to remove AFM1 in vitro and to counteract AFM1 immunotoxicity in vivo

Aflatoxin M1 (AFM1) has been detected in many parts of the world both in raw milk and many dairy products, causing great economic losses and human disease. Unfortunately, there are few studies dealing with AFM1 immunotoxicity/interactions with lactic acid bacteria for potential application as a natural preventive agent. The aim of this study was to isolate (from dairy products) food-grade probiotic bacteria able to degrade/bind AFM1 in vitro and evaluate whether the same organism(s) could impart a protective role against AFM1-induced immunotoxicity in exposed Balb/c mice. Bacteria (Lactobacillus plantarum MON03 and L. rhamnosus GAF01) were isolated from Tunisian artisanal butter and then tested for abilities to eliminate AFM1 from phosphate-buffered saline (PBS) and reconstituted milk (containing 0.05, 0.10, and 0.20 µg AFM1/ml) after 0, 6, and 24 h at 37°C. Results showed that the selected bacteria could ‘remove’ AFM1 both in PBS and skimmed milk. The binding abilities of AFM1 by L. plantarum MON03 and L. rhamnosus GAF01 strains (at 108 CFU/ml) in PBS and reconstituted milk ranged, respectively, from 16.1–78.6% and 15.3–95.1%; overall, L. rhamnosus showed a better potential for removal than L. plantarum. ‘Removal’ appeared to be by simple binding; the bacteria/AFM1 complex was stable and only a very small proportion of mycotoxin was released back into the solution. L. rhamnosus GAF01 had the highest binding capacity and was selected for use in the in vivo study. Those results indicated that use of the organism prevented AFM1-induced effects on total white and red blood cells, and lymphocyte subtypes, after 15 days of host treatment. These studies clearly indicated that L. rhamnosus GAF01 was able to bind AFM1 in vitro and—by mechanisms that might also be related to a binding effect—counteract AFM1-induced immunotoxicity. Moreover, by itself, this bacterium was not toxic and could potentially be used as an additive in dairy products and in biotechnology for mycotoxin detoxification.

Potential preventive role of lactic acid bacteria against Aflatoxin M1 immunotoxicity and genotoxicity in mice

Abstract Aflatoxin M1 (AFM1) is a mycotoxin produced by numerous Aspergillus species in pre- or post-harvest cereals and milk. Exposure to AFM1 imparts potent economic losses in the livestock industry. Toxicologically, it also causes severe immune system problems. The aims of this study were to evaluate a new AFM1-binding/degrading microorganism for biologic detoxification, to examine its ability to degrade AFM1 in liquid medium, and to evaluate its potential for in vivo preventative effects against AFM1-induced immunotoxicity and genotoxicity in mice. Lactobacillus plantarum MON03 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to AFM1 in PBS (93%) within 24 h of incubation. Further, the LP was able to tolerate gastric acidity, bile salts, and adhere efficiently to Caco-3 cells in vitro. The in vivo study used Balb/c mice that received either vehicle (control), LP only (at 1 × 109 CFU/L, ∼1 mg/kg bw), AFM1 (100 mg/kg bw), or AFM1 + LP daily for 15 days (by gavage); two other groups received a single dose of colchicine (4 mg/kg) or mitomycin C (1 mg/kg) as positive controls for induction of micronuclei and chromosomal aberrations, respectively. The results showed that, compared to in control mice, AFM1 treatment led to significantly decreased body weight gains, and caused cytotoxic/genotoxic effects as indicated by increases in frequencies of polychromatic erythrocytes, as well as those with micronucleation (PCEMN) and chromosomal aberrations, among bone marrow cells. The concurrent administration of LP with AFM1 strongly reduced the adverse effects of AFM1 on each parameter. Mice receiving AFM1 + LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. By itself, the bacteria caused no adverse effects. Based on the data, it is concluded that the test bacteria could potentially be beneficial in the detoxification of AFM1-contaminated foods and feeds for humans and animals.