Cytotoxicity of doxorubicin‐loaded Con A‐liposomes

The present study investigated the potential of Concanavalin A lectin (Con A) conjugated to liposomes (Con A‐liposomes) for targeting doxorubicin (DOX) to cells. The physicochemical properties and the cytotoxicity of DOX‐loaded Con A‐liposomes were evaluated. DOX‐loaded Con A‐liposomes were prepared by incubation of DOX‐loaded liposomes with a Con A‐SATA derivative. Lectin biological activity was monitored before and after conjugation by a hemagglutinating assay. The cytotoxicity of DOX‐loaded Con A‐liposomes was evaluated in terms of the inhibition of NCI‐H299 and HEp‐2 cell proliferation using the MTT method. The affinity of lectinized liposomes with these cells was thus assessed by evaluating the cytotoxic effect of the DOX released into cells. Stable DOX‐loaded Con A‐liposomes were obtained and their high affinity for cells was corroborated. The encapsulation of DOX into Con A‐liposomes produced an inhibition of roughly 70% of Hep‐2 cell proliferation and 50% of cell inhibition was verified on HCI‐H292. DOX in solution was able to inhibit only 20% of cell proliferation for both cell lines. Unloaded Con A‐liposomes were not cytotoxic. The encapsulation of DOX into Con A‐liposomes improves drug penetration into cells, thereby enhancing its cytotoxicity, especially in Hep‐2 cells. Drug Dev. Res. 67:430–437, 2006. © 2006 Wiley‐Liss, Inc.     

Retinoids inhibit measles virus in vitro via nuclear retinoid receptor signaling pathways

Measles virus (MV) infects 30 million children every year, resulting in more than half a million deaths. Vitamin A (retinol) treatment of acute measles can reduce measles-associated mortality by 50-80%. We sought to determine whether or not retinoids can act directly to limit MV output from infected cells. Physiologic concentrations of retinol were found to inhibit MV output in PBMC and a range of cell lines of epithelial and endothelial origin (40-50%). Near complete inhibition of viral output was achieved in some cells/lines treated with all-trans retinoic acid (ATRA) and 9-cis RA (9cRA). Important attenuation of the anti-MV effect of retinoids in R4 cells, a subclone of a retinoid-responsive cell line (NB4) deficient in RAR signaling, demonstrates that this effect is mediated at least in part by nuclear retinoid receptor signaling pathways. Inhibition of MV replication could not be fully explained as a result of retinoid effects on cell differentiation, proliferation or viability, particularly at low retinoid concentrations (1-10nM). These data provide the first evidence that retinoids can directly inhibit MV in vitro, and raise the possibility that retinoids may have similar actions in vivo.     

Surface Modification of Pharmaceutical Nanocarriers with Ascorbate Residues Improves their Tumor-Cell Association and Killing and the Cytotoxic Action of Encapsulated Paclitaxel In Vitro

Purpose  To evaluate the potential of ascorbate as a novel ligand in the preparation of pharmaceutical nanocarriers with enhanced tumor-cell specific binding and cytotoxicity. Methods  Palmitoyl ascorbate was incorporated into liposomes at varying concentrations. A stable formulation was selected based on size and zeta potential measurements. A co-culture of cancer cells with GFP expressing non-cancer cells was used to determine the specificity of palmitoyl ascorbate liposome binding. Liposomes were fluorescently labeled to facilitate analysis by flow cytometry and fluorescence microscopy. The cytotoxic action of palmitoyl ascorbate liposomes against a variety of cell types was assayed using a standard metabolic assay. The cytotoxic effect of a low dose of paclitaxel incorporated in palmitoyl ascorbate liposomes on various cell lines was also determined. Results  Palmitoyl ascorbate liposomes associated preferentially with various cancer cells compared to non-cancer cells in a co-culture model. Palmitoyl ascorbate liposomes exhibited anti-cancer toxicity in numerous cancer cell lines. Furthermore, ascorbate liposomes enhanced the effectiveness of encapsulated paclitaxel compared to paclitaxel encapsulated in ‘plain’ liposomes. Conclusions  Surface modification of liposomes with ascorbate residues represents a novel way to target and kill certain types of tumor cells and additionally can potentiate the effect of paclitaxel delivered by the liposomes.     

Liposome mediated dissipation of valinomycin-imposed potassium potential across erythrocytes membrane

Influence of liposomes made of phosphatidylcholine (PC) on the valinomycin-imposed potassium potential across erythrocyte membrane was examined by measuring the fluorescence change of the potential-sensitive cyanine dye. We concluded that the liposomes modulate ion selectivity of the membrane embedded valinomycin, on the basis of the following lines of evidence. (i) The valinomycin-imposed potassium potential across erythrocyte membrane (interior negative) was dissipated in the presence of PC-liposomes. (ii) When PC-liposomes were added to the cell suspension before the valinomycin, a membrane potential could not be imposed. (iii) Liposomes containing only the PC of saturated fatty acids were inactive in the potential dissipation, whereas the liposomes containing PC of unsaturated fatty acids were fully active. (iv) Liposome-mediated dissipation of the imposed-membrane potential was similarly observed in the resealed erythrocyte ghosts. (v) The liposomes did not show a detectable effect on the gramicidin-mediated proton potential. (vi) The effect of liposome was somewhat analogous to the nigericin-mediated dissipation of the valinomycin-imposed potassium potential.     

Preparation,characterization, transfection efficiency,and cytotoxicity of liposomes containing oligoamine-modified cholesterols as nanocarriers to Neuro2A cells

In the present study, three derivatives of cholesterol that contain an amino head group with a different spacer arm were synthesized. A novel gene delivery system was developed using liposomes composed of DOTAP and oligoamines-modified cholesterol. These multilamellar vesicle (MLV) liposomes were extruded through 400- and 100-nm polycarbonate filters to produce large unilamellar vesicles (LUVs). Transfection efficiency in Neuro2A cells was tested using plasmid DNA encoding Renilla luciferase, and the cytotoxicity of the prepared lipoplexes was also evaluated. LUV liposomes containing derivative III showed the highest transfection efficiency among other synthesized derivatives, but among MLV liposomes, cationic liposomes with derivative I had the best transfection activity. In all cases, transfection increased by increasing the ratio of cationic lipid to DNA. Almost all formulations were not cytotoxic on Neuro2A cells. The results indicated that the designed systems are able to transfer the DNA into Neuro2A cells with low or no toxicity.From the Clinical EditorThis study describes the ideal parameters for preparing large unilamellar vesicles as nanocarriers containing oligoamine-modified cholesterols. The results indicated that the designed systems are able to transfer the DNA into Neuro2A cells with low or no toxicity.     

Stimulation of vitamin A(1) acid signaling by the HIV protease inhibitor indinavir

HIV protease inhibitors (PIs) are effective drugs for the treatment of AIDS. However, PI therapy is sometimes associated with side-effects including increased plasma lipids and altered body fat distribution, although fat redistribution may occur in some patients not treated with PIs. Overdosage with vitamin A(1) acid (all-trans-retinoic acid, ATRA) or its metabolites may cause similar changes in lipid metabolism. Moreover, the PI indinavir and retinoids have been associated with nail, skin, and hair defects, suggesting that indinavir and retinoids may exert their effects through similar molecular mechanisms. This hypothesis was tested by examining the effects of PIs on retinoid signaling in vitro. Mesenchymal stem cells (C3H10T1/2) were cultured in the presence of various PIs (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) and synthetic retinoids, and the metabolic response was assessed by measuring the activity of a retinoid-regulated protein, alkaline phosphatase (ALP). Of the PIs tested, only indinavir stimulated ATRA-dependent ALP activity and altered stem cell morphology; the effects of indinavir occurred in the presence of ATRA, but not in its absence. Moreover, indinavir increased the effects of ATRA on lipid accumulation during fat cell differentiation. AGN 193109 (4-[[5,6-dihydro-5, 5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethynyl]-benzoic acid), a retinoic acid receptor (RAR) antagonist, inhibited the synergistic effects of indinavir and ATRA, indicating that indinavir increases RAR signaling. However, indinavir did not potentiate ALP activity in the presence of the RAR agonist CH55 (3,5-di-tert-butylchalcone 4'-carboxylic acid). Unlike ATRA, CH55 does not bind to cytosolic retinoic acid binding protein (CRABP), suggesting that CRABP may regulate the effects of indinavir on RAR signaling. These observations support the proposal that altered retinoid signaling promotes some of the adverse reactions associated with indinavir therapy, such as altered lipid metabolism.     

Retinoids, apoptosis and cancer

Retinoids are a class of natural and synthetic vitamin A analogs structurally related to all-trans-retinoic acid (ATRA). Natural retinoids are involved in the physiology of vision and as morphogenic agents during embryonic development; they are also known to play a major role in regulating growth and differentiation of a wide variety of normal and malignant cell types, and, indeed, they can in various ways inhibit cell proliferation, induce differentiation and cell death by apoptosis. The development of new active retinoids and the identification of two distinct families of retinoid receptors has led to an increased understanding of the cellular effects of activation of these receptors and of mechanisms involved in the retinoid-induced apoptosis. In this review a brief summary of cellular pathways relevant to programmed cell death is given together with therapeutic potentialities of retinoids having apoptotic activity. Structure-activity relationship studies concerning the importance of different stereochemistry at the C9 double bond of retinoids in conferring apoptotic activity will be described. It will be also described the preparation and the potent cytotoxic and apoptotic activity of a novel class of heterocycle-bridged arotinoids.     

Influence of isomerisation on the growth inhibitory effects and cellular activity of 13-cis and all-trans retinoic acid in neuroblastoma cells

Treatment with 13-cis retinoic acid (13-cis RA) has been shown to significantly improve the clinical outcome of children with high-risk neuroblastoma. Despite the large number of studies investigating the cellular effects of retinoids in neuroblastoma cells, the influence of RA isomerisation and the factors that determine the extent of RA isomerisation and uptake are unknown. The aim of this study was to establish the extent of extra- and intracellular isomerisation of 13-cis RA and all-trans retinoic acid (ATRA) in neuroblastoma cell lines, and to investigate the influence of isomerisation on their growth inhibitory effects and on the regulation of expression of cellular retinoic acid binding protein II (CRABP II) and RAR-beta. Limited extracellular isomerisation was observed up to 72 hr after incubation of four neuroblastoma cell lines with 10 microM 13-cis RA or ATRA. The retinoic acid isomer present initially in the medium accounted for >75% of extracellular retinoid exposure. By contrast, incubation with 13-cis RA resulted in intracellular levels of ATRA comparable to those of 13-cis RA. This degree of intracellular isomerisation was not observed after ATRA incubations, with 13-cis RA accounting for <10% of total intracellular retinoids. No differences were observed in the sensitivity of three N-type neuroblastoma cell lines to either 13-cis RA (IC(50): 11.2-13.9 microM) or ATRA (IC(50): 12.9-14.4 microM), despite 10-fold differences in intracellular retinoid levels. A decrease in sensitivity to 13-cis RA (IC(50)=137 microM), as compared to ATRA (IC(50)=41 microM), was observed in the S-type cell line SH S EP. RAR-beta was induced in a dose-dependent manner in SH SY 5Y cells following incubation with ATRA, whereas a weaker and delayed induction was observed with 13-cis RA. Similarly, incubation with ATRA resulted in a greater induction of CRABP II in these cells. In summary, these results indicate either an intracellular conversion of 13-cis RA to ATRA or a selective uptake of ATRA and suggest that this may mediate the differential activity of 13-cis RA in neuroblastoma cell subtypes.     

Biodistribution and improved anticancer effect of NIK-siRNA in combination with 5-FU for hepatocellular carcinoma

Increase of NF-κB inducing kinase (NIK) is known to promote the proliferation of the hepatitis B virus-derived hepatocellular carcinoma (HCC) cells. Previously, we have reported that NIK-specific siRNA in cationic liposomes was shown to suppress the expression of NIK and the proliferation of HCC cells (Cho et al., 2009). More improved suppression of NIK, followed by the improved antiproliferative effect on Hep3B cells, was achieved when 5-FU was cotreated with siRNA. Furthermore, biodistribution study after intravenous injection of siRNA into Hep3B-bearing Balb/c nude mice revealed that siRNA was highly accumulated in liver, followed by tumor, lung, spleen, kidney and heart. When encapsulated in cationic liposomes, larger amount of siRNA was found in tumor owing to the protection of siRNA from enzymatic degradation and enhanced permeability by liposome, suggesting a possible therapeutic modality of siRNA in liver-targeting cationic liposomal formulation for the treatment of hepatitis B virus-derived HCC.     

阴离子脂质体－阳离子脂质体复合物介导的基因转染

目的评价阴离子脂质体-阳离子脂质体复合物介导质粒转移至HepG2肝癌细胞中及其毒副作用。方法制备携载表达绿色荧光蛋白质粒的阳离子脂质体,与阴离子脂质体形成复合物。测定脂质体复合物的zeta电位,凝胶阻滞实验考察质粒包封情况,流式细胞仪测量各阴离子脂质体-阳离子脂质体复合物的转染效率,MTT法检测细胞毒性。结果复合物能完全包裹质粒,其zeta电位低于阳离子脂质体zeta电位;脂质体复合物介导的转染效率略低于阳离子脂质体,其细胞生存率高于阳离子脂质体。结论阴离子脂质体-阳离子脂质体复合物在降低细胞毒性的同时,可实现对HepG2细胞较高的转染效率。     

Vascular targeting of doxorubicin using cationic liposomes

Tumor vessel has been recognized as an important target for anticancer therapy. Cationic liposomes have been shown to selectively target tumor endothelial cells, thus can potentially be used as a carrier for chemotherapy agents. In this study, cationic liposomes containing 20 mol% cationic lipid dimethyl dioctadecyl ammonium bromide (DDAB) and loaded with doxorubicin (DOX) were prepared and characterized. The cationic liposomal DOX showed 10.8 and 9.1 times greater cytotoxicity than control PEGylated liposomal DOX in KB oral carcinoma and L1210 murine lymphocytic leukemia cells, and 7.7- and 6.8-fold greater cytotoxicity compared to control neutral non-PEGylated liposomal DOX, repectively, in these two cell lines. Although cationic liposomal DOX had higher tumor accumulation at 30 min after intravenous administration compared to control liposomes (p<0.05), DOX uptake of these liposomes at 24h post-injection was similar to that of PEGylated liposomal DOX (p>0.05) and approximately twice the levels of the free drug and non-PEGylated liposomes. In a murine tumor model generated using L1210 cells, increased survival rate was obtained with cationic liposomal DOX treatment compared to free DOX (p<0.01), neutral liposome control (p<0.01), as well as PEGylated liposomes (p<0.05). In conclusion, the cationic liposomal DOX formulation produced superior in vitro cytotoxicity and in vivo antitumor activity, and warrants further investigation.     

Chronic all-trans retinoic acid administration induced hyperactivity of HPA axis and behavioral changes in young rats

Although clinical reports suggest a possible relationship between excess retinoids and the development of depression, the effect of retinoids on mood-related behavior remains controversial. Hyperactivity of the hypothalamus–pituitary–adrenal (HPA) axis plays a key role in the development of affective disorders. The present study aimed to elucidate the effect of retinoid on the activity of HPA axis in rat and whether this goes together with behavioral changes. All-trans retinoic acid (ATRA) was administered to juvenile male rats by daily intraperitoneal injection for 6 weeks. ATRA treatment increased basal serum corticosterone concentration as well as the thickness of adrenal cortex in young rat. Furthermore, the mRNA expression of corticotropin release factor (CRF) and retinoic acid receptor-α (RAR-α) in the hypothalamus was both markedly increased in ATRA-treated rats compared with vehicle. Some behavioral alterations were also observed. ATRA-treated rats showed anxiety-like behavior in elevated-plus maze and decreased spontaneous exploratory activities in novel open field. However, in the sucrose preference test chronic ATRA treatment did not modify behavior in the juvenile animals. Chronic administration of ATRA did not impair physical motor ability in either the prehensile traction or the beam balance/walk test. In conclusion, long-term ATRA administration resulted in hyperactivated HPA axis which was accompanied by several behavioral changes in young rat.     

TPGS修饰脂质体对阿霉素抗肿瘤活性的增敏作用

目的制备聚乙二醇1000维生素E琥珀酸酯(TPGS)修饰的阿霉素脂质体并考察其对阿霉素抗肿瘤活性的增敏作用。方法用阳离子树脂吸附法测定阿霉素脂质体的包封率;MTT法测定对MCF-7和MCF-7/ADR的毒性;用荧光显微镜观察阿霉素的细胞摄取,并用HPLC测定细胞内的阿霉素含量。结果 TPGS修饰的阿霉素脂质体增加了MCF-7/ADR对阿霉素的摄取,并增强了对MCF-7和MCF-7/ADR细胞的毒性。结论 TPGS修饰脂质体能显著增强MCF-7和MCF-7/ADR对阿霉素的敏感性。     

Albumin modulates docosahexaenoic acid-induced cytotoxicity in human hepatocellular carcinoma cell lines

Fish oil-containing diets rich in cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) provide protection against tumorigenesis. The mechanisms of the cytotoxic effects of DHA include the production of reactive oxygen species (ROS). Albumin has antioxidant property and binds fatty acids, it may protect the cells against the DHA-induced cytotoxicity. In this study, we compared the susceptibility of three human hepatocellular carcinoma (HCC) cell lines (HepG2, Hep3B, Huh7) to the cytotoxic effects of DHA, and examined the changes in the susceptibility following albumin overexpression using transfection vectors or albumin downregulation using small interfering RNA (siRNA). HepG2 cells were the most susceptible to DHA-induced cytotoxicity and increased oxidative activities by DHA compared to Hep3B and Huh7 cells. The cytotoxic effects of DHA were concentration-dependently abrogated by typical antioxidants, a radical scavenger, an iron chelator and incubation with exogenous albumin. Overexpression of albumin in HepG2 cells markedly attenuated DHA-induced oxidative activities and cytotoxicity. Furthermore, knockdown of albumin in both Hep3B and Huh7 cells significantly enhanced the effects of DHA. The results of our in vitro experiments indicate that the cytotoxic effects of DHA on HCC cell lines are modulated by albumin.     

Lysine-containing cationic liposomes activate the NLRP3 inflammasome: Effect of a spacer between the head group and the hydrophobic moieties of the lipids

Cationic lipids containing lysine head groups and ditetradecyl, dihexadecyl or dioctadecyl glutamate hydrophobic moieties with/without propyl, pentyl or heptyl spacers were applied for the preparation of cationic liposomes using a simple bath type-sonicator. The size distribution, zeta potential, cellular internalization, and cytotoxicity of the liposomes were characterized, and the innate immune stimulation, e.g., the NLRP3 inflammasome activation of human macrophages and THP-1 cells, was evaluated by the detection of IL-1β release. Comparatively, L3C14 and L5C14 liposomes, made from the lipids bearing lysine head groups, ditetradecyl hydrophobic chains and propyl or pentyl spacers, respectively, were the most potent to activate the NLRP3 inflammasome. The possible mechanism includes endocytosis of the cationic liposomes and subsequent lysosome rupture without significant inducement of reactive oxygen species production. In summary, we first disclosed the structural effect of cationic liposomes on the NLRP3 inflammasome activation, which gives an insight into the application of nanoparticles for improved immune response.     

Effects of all-trans-retinoic acid and all-trans-retinoyl glucuronide in two in vitro systems of distinct biological complexity

In vitro systems are widely used to evaluate the embryotoxic potential of retinoids. The effective concentrations of these retinoids, however, are not consistent in the various in vitro systems used in evaluating embryotoxicity. This may be explained by the different level of complexity for each individual system, which may lead to different concentrations of the substances in the target tissues. To verify this hypothesis we have compared two in vitro systems of distinct biological complexity: the rat whole embryo culture system, and the mouse limb bud organ culture system. The lipid soluble, teratogenic retinoid all-trans-retinoic acid (ATRA), and all-trans-retinoyl-beta-D-glucuronide (ATRAG), an endogenous, water-soluble and biologically active retinoid with limited placental transfer, were compared with regard to their embryotoxic potential in vitro. In both in vitro systems, ATRAG showed a lower degree of embryotoxicity than ATRA. In the limb bud organ culture, ATRAG revealed only slightly less toxicity than ATRA, whereas the effective concentrations of the two compounds in the whole embryo culture system differed by almost two orders of magnitude. During incubation with ATRAG, ATRA is generated by hydrolysis and is found in culture media and exposed tissues. The presence of membrane barriers around the developing embryo in the whole embryo culture system possibly prevents the transfer of ATRAG to the embryo and, therefore, its exposure to the active hydrolysis product ATRA. From these results we conclude that analysis of retinoid concentrations in the culture media and in the exposed tissues is essential for the interpretation of results obtained from in vitro toxicity testing.     

The effects of vitamin A on cells of innate immunity in vitro

Retinoid treatment is suggested to promote development of inflammatory bowel disease, although preclinical studies are not supportive. We evaluated the effect of retinoids on cytokine response in in vitro-differentiated human dendritic cells (ivDCs) and macrophages (ivMACs) derived from healthy human donors and in cultured human THP-1 cells. Effect on human intestinal epithelial cell integrity was also assessed. Each cell type was incubated (±lipopolysaccharide [LPS]) with all-trans retinoic acid (ATRA), 13-cis-RA (isotretinoin) and 4-oxo-13-cis-RA. Cytokine analysis was performed by array analysis. Cultured human endothelial colorectal adenocarcinoma (Caco-2) cells were incubated with these retinoids and media analyzed for leakage by spectrofluorometric analysis. ATRA consistently and significantly inhibited LPS-induced release of the pro-inflammatory cytokines tumor necrosis factor, interleukin (IL)-6, macrophage inflammatory protein (MIP)-1α and MIP-1β. All retinoids tested stimulated release of the anti-inflammatory cytokines granulocyte–macrophage colony-stimulating factor and IL-10, and also monocyte chemotactic protein-1, vascular endothelial growth factor and eotaxin-1. Incubation with retinoids did not significantly alter the permeability of Caco-2 monolayers. Pre-treatment of each cell type with retinoids promoted an anti-inflammatory cytokine profile with only minimal effect on intestinal epithelial cell permeability; consistent with in vivo studies.     

Lysine-based surfactants in nanovesicle formulations: the role of cationic charge position and hydrophobicity in in vitro cytotoxicity and intracellular delivery

Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, the authors developed nanovesicles containing bioactive cationic lysine-based amphiphiles and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. Different cytotoxic responses were found among the formulations, depending on surfactant, cell line and endpoint assayed. The induction of mitochondrial dysfunction, oxidative stress and apoptosis were the general mechanisms underlying cytotoxicity. Fluorescence microscopy analysis demonstrated that nanovesicles were internalised by HeLa cells and evidenced that their ability to release endocytosed materials into cell cytoplasm depends on the structural parameters of amphiphiles. The cationic charge position and hydrophobicity of surfactants determine the nanovesicle interactions within the cell and, thus, the resulting toxicity and intracellular behaviour after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute in reducing the uncertainty surrounding their potential health hazards.     

The role of oxidative stress in zearalenone-mediated toxicity in Hep G2 cells: oxidative DNA damage, gluthatione depletion and stress proteins induction

Zearalenone (ZEN) is a fusarial mycotoxin with several adverse effects in laboratory and domestic animals including mainly estrogenicity. While most ZEN toxic effects have been quite well investigated, little is known regarding its mechanism of toxicity. Our previous investigations have shown the involvement of cytotoxicity, inhibition of macromolecules synthesis as well as genotoxicity. However, there are no available data regarding the involvement of the oxidative stress pathway in ZEN toxicity. In this context, the aim of this study was to find out whether ZEN induces oxidative cell damage. Using human hepatocytes Hep G2 cells, ZEN-induced stress response is monitored at several levels in these cells. ZEN mediated induction of oxidative DNA damage (comet assay using the repair enzymes), modulation of gluthatione (GSH), cytotoxicity (growth inhibition) and the oxidative stress responsive gene Hsp 70 and Hsp 90 were investigated with respect to concentration and time dependency. Hep G2 cells respond to ZEN exposure by loss of cell viability, induction of oxidative DNA damage, GSH depletion and Hsp 70 and Hsp 90 induction already at concentrations, which are not yet cytotoxic. The perturbation of the oxidative status was further confirmed by the significant reduction of the induced oxidative DNA damage as well as stress protein induction when cells were pre-treated with Vitamin E prior to exposure to ZEN. Our study clearly demonstrates that oxidative damage is likely to be evoked as one of the main pathway of ZEN toxicity. This oxidative damage may therefore be an initiating event and contribute, at least in part, to the mechanism of ZEN different genotoxic and cytotoxic effects.