Influence of bifentrin, a pyrethriod pesticide, on human colorectal HCT-116 cells attributed to alterations in oxidative stress involving mitochondrial apoptotic processes.

The widespread use of pesticides is beneficial for food production; however, there are numerous adverse consequences reported in the ecosystem and humans associated with exposure to these contaminants. The pyrethriod bifenthrin (BIF) is utilized for (1) maintenance, growth, and storage of agricultural products; (2) control of internal and external parasites of farm animals; and (3) eradication of insects threatening public health. Numerous data are available regarding environmental and ecological impact of pyrethriods on the central and peripheral nervous systems; however few studies focused on non-target tissues especially in humans. Therefore, the aim of this investigation was to determine the potential cytotoxic effects of BIF on a non-target tissue using human colorectal HCT-116 cells as a model. Data demonstrated that BIF reduced cell viability and disrupted mitochondrial functions which were accompanied by increased reactive oxygen species (ROS) levels indicating the presence of oxidative stress. BIF produced a significant elevation in levels of malondialdehyde (MDA) supporting the role of oxidative stress in pesticide-mediated toxicity. Concomitantly, a fall of mitochondrial transmembrane potential (Δψ), consequently producing perturbation of fluidity as well as excitability of cellular membranes was noted. Our results also indicated that BIF induced a rise in DNA damage as evidenced by the comet assay. An increase in mitogen-activated protein kinases (MAPKs), JNK (N-terminal Kinase), p38, and ERK (extracellular-signal-regulated kinase) suggested an apoptotic effect. Data thus indicated that BIF-induced cytotoxicity in human colorectal HCT-116 cells was associated with oxidative stress, mitochondrial dysfunction, and apoptosis.

Eugenol protects against citrinin-induced cytotoxicity and oxidative damages in cultured human colorectal HCT116 cells.

This study aimed to investigate the protective effects of Eugenol (EUG), an effective antioxidant phenolic compound with a radical scavenging activity against citrinin (CTN)-induced toxicity in vitro using HCT116 cells. CTN is a well-known mycotoxin found in different constituents of the food chain. This environmental contaminant produces free radicals which interacts with cellular macromolecules and produces oxidation of protein, lipid, and DNA. The cytotoxic effects were monitored by measuring cell viability, reactive oxygen species (ROS) generation, antioxidant enzyme activities, malondialdehyde (MDA) production, protein oxidation, and DNA fragmentation. Our results have shown that the pretreatment of HCT116 cells with EUG, 2 h prior to citrinin (CTN) exposure, significantly decreased CTN-induced cell death, inhibited ROS generation, modulated activities of both catalase (CAT) and superoxide dismutase (SOD), and reduced MDA production. Level of protein-bound sulfhydryls and DNA fragmentation were also declined as compared with CTN-treated cells. These findings suggest that EUG would be an effective protective agent against CTN-induced oxidative stress, and thereby, it may complement and add to the functions of antioxidant vitamins and enzymes as a protection against the cytotoxicity of this mycotoxin.

Di (2-ethylhexyl) phthalate induces cardiac disorders in BALB/c mice.

Because of the extensive use of phthalates for domestic, medical, and industrial applications, the evaluation of their toxic effects is of major concern to public health. The aim of the present study was to assess the propensity of di (2-ethylhexyl) phthalate (DEHP), one of the most used phthalates, to cause oxidative cardiac damage in mice. DEHP was administered intraperitoneally at doses of 5, 50, and 200 mg/kg body weight for 30 consecutive days in BALB/c mice. We assessed the effect of DEHP on cardiac injury using biochemical profile (such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatinine phosphokinase (CPK), total cholesterol (T-CHOL), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C)), parameters related to myocardiac oxidative stress, such as malondialdehyde (MDA) level, protein carbonyl (PC) concentration, and DNA fragmentation. In addition, we evaluated antioxidant status; enzymatic (catalase (CAT) and superoxide dismutase (SOD) activities) and non-enzymatic (protein-bound sulfhydryl concentration (PSH)) antioxidants. Acetylcholinesterase (AChE) activity and histopathological changes were also assessed in heart mice treated with DEHP. Our results showed that DEHP induced an elevation of serum marker enzymes and perturbated the lipid profile. In addition, this phthalate increased lipid peroxidation, protein carbonyl levels, and DNA fragmentation in the heart in a dose-dependent manner. Antioxidant status was also perturbated by the increase of the CAT and SOD activities and the decrease of the protein-bound sulfhydryl concentration. AChE activity was also inhibited in the heart following the treatment with DEHP. These biochemical alterations were also confirmed by histopathological changes. Increased free radical production at various doses of DEHP would result in impairment of the redox status leading to an enhanced dose-dependent cardiotoxicity.

Citrinin induces apoptosis in human HCT116 colon cancer cells through endoplasmic reticulum stress.

The mycotoxin citrinin (CTN) is a natural contaminant of various human foods that may produce serious adverse health problems. Several studies demonstrated that citrinin exerts cytotoxic and genotoxic effects in both in vivo and in vitro systems. However, the precise mechanisms of action (MOA), particularly in intestinal cells remain unclear. The aim of the present study was to examine the precise MOA of citrinin in vitro. Data demonstrated that CTN significantly decreased the number of viable human intestinal HCT116 cells and induced apoptotic events including (1) decrease in ΔÑ°m indicative of mitochondrial membrane permeabilization, (2) activation of caspase 3, (3) elevated production of reactive oxygen species (ROS) and (4) relative persistence of plasma membrane integrity. Further, the genetic deficiency of the pro-apoptotic protein Bax protected cells against CTN-induced apoptosis, indicating that Bax is required for CTN-mediated toxicity. It was also found that CTN triggered endoplasmic reticulum (ER) stress and activated different arms of the unfolded protein response (UPR) as demonstrated by increase in expression of GRP78 (glucose-regulated protein-78), GRP94 (glucose-regulated protein-94), GADD34 (growth arrest and DNA damage-inducible protein-34), the protein disulfide isomerase associated 6 (PDIA6), CHOP (C/EBP-homologous protein) and the splicing of XBP1 (X-Box Binding Protein 1). Pretreatment of cells with the chemical chaperone 4-phenylbutyrate (PBA), known to alleviate ER stress, prevented significantly the apoptotic process triggered by CTN. Taken together, these results suggest that CTN exerts its cytotoxic effects in HCT116 cells by inducing apoptosis, at least in part, through induction of ER stress.

Cell death in relation to DNA damage after exposure to the jellyfish Pelagia noctiluca nematocysts.

Studies on the toxicity of Mediterranean jellyfish have gained attention owing to their weak toxic properties. Our research has been mainly performed on the Scyphomedusae. Pelagia noctiluca is a scyphozoan jellyfish which causes a danger to sea bathers and fishery damages in the Mediterranean Sea. To check whether the cytotoxicity of Pelagia noctiluca nematocysts was associated to DNA lesions, we have looked for DNA fragmentation by means of the Comet and chromosome aberration assays. To specify cell death pathway, we have investigated caspase-3 activation. Our results have shown that nematocysts reduced cell viability and induced DNA fragmentation in a concentration-dependent manner with a maximum effect at 150 000 nematocysts mL(-1). The high percentage of chromosome aberrations also emphasized the genotoxic character of Pelagia noctiluca nematocysts in Vero cells. This fragmentation was correlated to apoptosis induction which was confirmed by caspase-3 activation. In conclusion, the present report has suggested that Pelagia noctiluca nematocysts were able to promote apoptosis in Vero cells and therefore may be useful in cancer therapy.

Identification of proteins related to early changes observed in Human hepatocellular carcinoma cells after treatment with the mycotoxin Zearalenone.

Zearalenone (ZEA) is a mycotoxin produced by some Fusarium species. ZEA often occur as a contaminant in cereal grains and animal feeds. Human exposure occurs by ingestion of mycotoxin-contaminated products and can cause serious health problems. It was established that this mycotoxin have an hepato, haemato, immuno and genotoxic properties (Maaroufi et al., 1996; Lioi et al., 2004). While most ZEA toxic effects have been quite well investigated, more studies are required to elucidate its mechanisms of toxicity. In order to better understand the molecular mechanisms involved in ZEA toxicity, we used a proteomic approach, to assess the early changes in protein expression initiated by ZEA in HepG2 cells. Our results showed that, after 8h of exposure, cells were still viable and showed a significant change in a number of proteins involved in diverse cellular processes. These changes may provide the early affected functions and yield further insight into mechanisms underlying the involvement of mycotoxin-induced diseases.

Antioxidative and antigenotoxic effect of vitamin E against patulin cytotoxicity and genotoxicity in HepG2 cells.

Patulin (PAT) is a mycotoxin produced in fruits, mainly in apples, by certain species of Penicillium, Aspergillus, and Byssochlamys. It has been shown that PAT is cytotoxic, genotoxic, and mutagenic in different cell types. Several studies incriminate the oxidative stress as a mechanism of PAT-mediated toxicity. In this context, our aim was to investigate the protective role of Vitamin E (Vit E), an antioxidant agent, against PAT induced cytotoxicity and genotoxicity in cultured HepG2 cells. The obtained results showed that addition of Vit E in cells treated with PAT significantly reduce cell mortality induced by this toxin. In the same conditions, Vit E decreased the intracellular level of ROS, reduced PAT induced p53 expression, and reversed PAT induced DNA damage. In addition, Vit E prevented significantly the percentage of chromosome aberrations induced by PAT in HepG2 cells in a concentration dependant manner. These results suggest that Vit E, an exogenous antioxidant agent, plays an important role in defense against PAT-induced cytotoxicity and genotoxicity, which confirms the involvement of oxidative stress in the induction of DNA damage by PAT in HepG2 cells.

The in vitro effects of zearalenone and T-2 toxins on Vero cells.

The aim of the present study was to investigate in vitro, whether cytolethality and oxidative damage is enhanced by combination of both mycotoxins as compared to their individual effect. In our paper, we applied a tiered in vitro experimental approach in order to predict the possible health risk effects of two interactive fusarial toxins. Considering the concomitant production of zearalenone (ZEN) and T-2 toxin, it is very likely that humans and animals are always exposed to the mixture rather than to individual compounds. Our results clearly showed that cultured renal cells respond to individual (ZEN) or T-2 toxin exposure by a moderate inhibition of cell proliferation, respectively. However, when combined, they exert a more significant decrease in cell viability. Similar results were found for the investigated oxidative status endpoints. When combined, ZEN and T-2 toxin increased ROS production and heat shock protein (Hsp) 70 expression as compared to the effect of each mycotoxin taken alone. We can conclude that the mixture of ZEN and T-2 toxin increased their toxic effects. The health risk is heightened by the interactions between co-occurring mycotoxins.

Protective effect of cactus cladode extract against cisplatin induced oxidative stress, genotoxicity and apoptosis in balb/c mice: combination with phytochemical composition.

BACKGROUND: Cis-Platinum (II) (cis-diammine dichloroplatinum; CDDP) is a potent antitumor compound widely used for the treatment of many malignancies. An important side-effect of CDDP is nephrotoxicity. The cytotoxic action of this drug is often thought to induce oxidative stress and be associated with its ability to bind DNA to form CDDP-DNA adducts and apoptosis in kidney cells. In this study, the protective effect of cactus cladode extract (CCE) against CDDP-induced oxidative stress and genotoxicity were investigated in mice. We also looked for levels of malondialdehyde (MDA), catalase activity, superoxide dismutase (SOD) activity, chromosome aberrations (CA) test, SOS Chromotest, expressions of p53, bax and bcl2 in kidney and we also analyzed several parameters of renal function markers toxicity such as serum biochemical analysis. METHODS: Adult, healthy balb/c (20-25 g) male mice aged of 4-5 weeks were pre-treated by intraperitonial administration of CCE (50 mg/Kg.b.w) for 2 weeks. Control animals were treated 3 days a week for 4 weeks by intraperitonial administration of 100 µg/Kg.b.w CDDP. Animals which treated by CDDP and CCE were divided into two groups: the first group was administrated CCE 2 hours before each treatment with CDDP 3 days a week for 4 weeks. The second group was administrated without pre-treatment with CCE but this extract was administrated 24 hours after each treatment with CDDP 3 days a week for 4 weeks. RESULTS: Our results showed that CDDP induced significant alterations in all tested oxidative stress markers. In addition it induced CA in bone morrow cells, increased the expression of pro-apoptotic proteins p53 and bax and decreased the expression of anti-apoptotic protein bcl2 in kidney. On the other hand, CDDP significantly increased the levels of urea and creatinine and decreased the levels of albumin and total protein.The treatment of CCE before or after treatment with CDDP showed, (i) a total reduction of CDDP induced oxidative damage for all tested markers, (ii) an anti-genotoxic effect resulting in an efficient prevention of chromosomal aberrations compared to the group treated with CDDP alone (iii) restriction of the effect of CDDP by differential modulation of the expression of p53 which is decreased as well as its associated genes such as bax and bcl2, (iiii) restriction of serums levels of creatinine, urea, albumin and total protein resuming its values towards near normal levels of control. CONCLUSION: We concluded that CCE is beneficial in CDDP-induced kidney dysfunction in mice via its anti-oxidant anti-genotoxic and anti-apoptotic properties against CDDP.

Protective effect of erythropoietin against cisplatin-induced nephrotoxicity in rats: antigenotoxic and antiapoptotic effect.

Cisplatin (Cisp) is an active cytotoxic agent that was found efficient in the treatment of various types of solid tumors. Its nephrotoxic effect has been very well documented in clinical oncology. Erythropoietin (EPO), a renal cytokine-regulating hematopoiesis, has recently been shown to exert important cytoprotective effects in many experimental injuries. The aim of this study was to explore whether EPO would protect against Cisp-induced apoptosis in rat kidney. Adult Wistar rats were treated with saline solution as the control group, Cisp alone, EPO alone, or EPO with Cisp in different treatments: 1) EPO and Cisp simultaneously administrated to animals as a cotreatment; 2) EPO administered 24 hours before Cisp as a pretreatment; and 3) EPO administered 5 days after Cisp injection as a post-treatment. Our results have shown that Cisp induced renal failure, characterized with a significant increase in serum creatinine and blood urea nitrogen (BUN) concentrations. Cisp promoted kidney DNA fragmentation and apoptotic cell death. Apoptosis was revealed by an enhancement of proapoptotic protein (e.g., p53 and Bax) levels, decrease in antiapoptotic proteins (e.g., Bcl2 and Hsp27), and increase in caspase-3 activity. Treatments with EPO restored creatinine and BUN levels and inhibited Cisp-induced DNA damage in the kidney. Apoptosis was also reduced by the upregulation of antiapoptotic protein expressions, downregulation of proapoptotic protein levels, and reduction of caspase-3 activity.

Induction of cytotoxicity of Pelagia noctiluca venom causes reactive oxygen species generation, lipid peroxydation induction and DNA damage in human colon cancer cells.

BACKGROUND: The long-lasting and abundant blooming of Pelagia noctiluca in Tunisian coastal waters compromises both touristic and fishing activities and causes substantial economic losses. Determining their molecular mode of action is, important in order to limit or prevent the subsequent damages. Thus, the aim of the present study was to investigate the propensity of Pelagia noctiluca venom to cause oxidative damage in HCT 116 cells and its associated genotoxic effects. RESULTS: Our results indicated an overproduction of ROS, an induction of catalase activity and an increase of MDA generation. We looked for DNA fragmentation by means of the comet assay. Results indicated that venom of Pelagia noctiluca induced DNA fragmentation. SDS-PAGE analysis of Pelagia noctiluca venom revealed at least 15 protein bands of molecular weights ranging from 4 to 120 kDa. CONCLUSION: Oxidative damage may be an initiating event and contributes, in part, to the mechanism of toxicity of Pelagia noctiluca venom.

Chemopreventive effect of cactus Opuntia ficus indica on oxidative stress and genotoxicity of aflatoxin B1.

BACKGROUND: Aflatoxin B1 (AFB1) is potent hepatotoxic and hepatocarcinogenic agent. In aflatoxicosis, oxidative stress is a common mechanism contributing to initiation and progression of hepatic damage. The aim of this work was to evaluate the hepatoprotective effect of cactus cladode extract (CCE) on aflatoxin B1-induced liver damage in mice by measuring malondialdehyde (MDA) level, the protein carbonyls generation and the heat shock proteins Hsp 70 and Hsp 27 expressions in liver. We also looked for an eventual protective effect against AFB1-induced genotoxicity as determined by chromosome aberrations test, SOS Chromotest and DNA fragmentation assay. We further evaluated the modulation of p53, bax and bcl2 protein expressions in liver. METHODS: Adult, healthy balbC (20-25 g) male mice were pre-treated by intraperitonial administration of CCE (50 mg/Kg.b.w) for 2 weeks. Control animals were treated 3 days a week for 4 weeks by intraperitonial administration of 250 µg/Kg.b.w AFB1. Animals treated by AFB1 and CCE were divided into two groups: the first group was administrated CCE 2 hours before each treatment with AFB1 3 days a week for 4 weeks. The second group was administrated without pre-treatment with CCE but this extract was administrated 24 hours after each treatment with AFB1 3 days a week for 4 weeks. RESULTS: Our results clearly showed that AFB1 induced significant alterations in oxidative stress markers. In addition, it has a genotoxic potential and it increased the expression of pro apoptotic proteins p53 and bax and decreased the expression of bcl2. The treatment of CCE before or after treatment with AFB1, showed (i) a total reduction of AFB1 induced oxidative damage markers, (ii) an anti-genotoxic effect resulting in an efficient prevention of chromosomal aberrations and DNA fragmentation compared to the group treated with AFB1 alone (iii) restriction of the effect of AFB1 by differential modulation of the expression of p53 which decreased as well as its associated genes such as bax and bcl2. CONCLUSION: We concluded that CCE might have a hepatoprotective effect against aflatoxicosis in mice, probably acting by promoting the antioxidant defence systems.

Presence of ochratoxin A in Tunisian blood nephropathy patients. Exposure level to OTA.

Ochratoxin A (OTA) produced by Aspergillus and Penicillium genera contaminates cereals and different food compounds. OTA presents a wide range of toxic effects, especially nephrotoxicity. It is also considered to be the main causal agent of Balkan Endemic Nephropathy (BEN) which is similar to the Chronic Interstitial Nephropathy with unknown aetiology seen in Tunisia. In this study, we attempted to confirm the relationship between OTA blood levels and the development of renal pathology. Hence, serum OTA levels were measured in several groups of patients having different renal diseases: a group presenting Chronic Interstitial Nephropathy (CIN) with unknown aetiology, a group presenting Chronic Interstitial Nephropathy (CIN) with known aetiology, a group presenting Chronic Glomerular Nephropathy (CGN), and a group presenting Chronic Vascular Nephropathy (CVN). Each group was compared to a healthy control group. In the healthy group, 49% of individuals showed OTA concentrations ranging from 1.7 to 8.5 ng/ml, with a mean value of 3.3±1.5 ng/ml. However, among nephropathic patients, the group with CIN of unknown aetiology showed the highest incidence (76%), ranging from 1.8 to 65 ng/ml with a mean value of 18±7 ng/ml. Even in the healthy group, the calculated Daily Intake (DI) ranged from 5.0 to 24.9 ng/kgb.w./day when compared to the recommended DI by the scientific committee on foods of 5 ng/kgb.w./day, indicating a high degree of exposure to OTA in the Tunisian population. Our study confirms the involvement of this nephrotoxic mycotoxin, present at high blood levels in the Tunisian population, in the outcome of this particular human nephropathy (CIN with unknown aetiology) which is similar to BEN.

Molecular events involved in ochratoxin A induced mitochondrial pathway of apoptosis, modulation by Bcl-2 family members.

In this study, we looked for the role of the mitochondrion in the cytotoxicity of ochratoxin A (OTA), which is one of the most abundant food-contaminating mycotoxins in the world. In different human carcinoma cell lines, OTA triggered a mitochondria-dependent apoptotic process, which is characterized by opening of the mitochondrial permeability transition pore (PTPC), loss of mitochondrial transmembrane potential (ΔΨ(m) ), increase in O(2) [chemp](-) production, mitochondrial relocalization of Bax, release of cytochrome c, and caspase activation. However, studies performed on purified organelles suggested that OTA does not directly target the mitochondrion. In addition, we showed that mitochondrial alterations induced by this mycotoxin are favored by the proapoptotic protein Bax, but not Bak. These alterations are prevented by the antiapoptotic proteins, Bcl-2 and to a lesser degree by Bcl-X(L). Taken together, these data indicate that although mitochondria, PTPC members and proteins of Bcl-2 family play a pivotal role in OTA-induced apoptosis, they do not constitute real targets to overcome its toxicity.

Cytotoxic effects exerted by polyarylsulfone dialyser membranes depend on different sterilization processes.

Polyarylsulfone group is one of the most important polymeric materials used in the biomedical field, due to its excellent properties, such as good thermal, chemical, and mechanical stability. There are three important polyarylsulfone polymers, all of which have excellent electrical properties: polysulfone (PSu), polyarylsulfone (PAS) and polyarylethersulfone (PAES). All these polymers have excellent creep, radiation and high temperature resistance. In this study, we aimed to determine the effect of three sterilization processes (steam, ethylene oxide and gamma rays) on cytotoxicity of polyarylsulfone dialysis membranes. Ten long-term dialysis patients and ten age-matched healthy controls were enrolled in our study. We analysed (1) serum effect on cultured endothelial cell viability using MTT assay and (2) lipid peroxidation assessed by serum malondialdehyde (MDA) formation at the beginning (T0), the middle (T2) and the end (T4) of haemodialysis (HD) session. Our results clearly showed that steam-sterilized membranes improve endothelial cell viability when compared to ethylene oxide or gamma rays-sterilized ones. Moreover, there is a increased generation of MDA in patients sera during HD session. The serum MDA concentration was about 3, 6 and 10 times higher, respectively, for steam, ethylene oxide and gamma rays sterilization procedures when compared to the MDA amount in healthy subject sera. We concluded that using steam instead of ethylene oxide or gamma rays for sterilization may improve the biocompatibility of polyarylsulfone membranes.

Ochratoxin A levels in spices and dried nuts consumed in Tunisia.

A total of 112 samples of spices (24 caraway, 20 coriander, 25 curcuma, 20 black pepper and 23 red pepper) and 110 samples of dried nuts (44 almonds, 42 peanuts and 24 pistachio) purchased from popular markets in 24 regions of Tunisia were analyzed for ochratoxin A (OTA) by fluorescence HPLC. The average levels of contamination of OTA found in spice samples were 244, 206, 290, 274 and 203 µg/kg, respectively, for caraway, coriander, curcuma, black pepper and red pepper. Concerning dried nut samples, the average levels were 61, 60 and 89 µg/kg, respectively, for almonds, peanuts and pistachio. Contamination levels were higher than the usual norms (10.0 OTA µg/kg) established by the European Commission in 2005 . This survey is the first to be carried out on the natural occurrence of OTA in the main spices and dried nuts consumed by the Tunisian population.

Sequential events of apoptosis induced by zearalenone in cultured hepatocarcinoma cells.

Zearalenone (ZEA) is a fungal metabolite that can contaminate feed and foodstuffs and can cause serious health problems for animals as well as for humans. The present investigation was conducted to determine the chronological succession of the main events that characterise ZEA-induced toxicity in human hepatocarcinoma cells. To this aim, we have monitored the effects of ZEA on (1) cell viability, (2) heat-shock protein expression, (3) oxidative damage, (4) DNA fragmentation, (5) the cell cycle and (6) the cell-death-signalling pathway. Our results demonstrated that ZEA reduced cell viability in a time- and dose-dependent manner. When we exposed HepG2 cells to 100 µM ZEA (80% of cells are viable) for different treatment times (2, 4, 8, 24, 30, 48 and 60 h), we demonstrated an induction of Hsp70 protein, an increase in reactive oxygen species (ROS) generation, DNA fragmentation and cell-cycle arrest. These events begin after only 2 h of mycotoxin exposure and are earlier than those implicated in the execution of apoptosis. However, significant apoptotic cell death was observed after at least 30 h of ZEA exposure as a consequence of increased Bax expression, decreased Bcl-2 expression and mitochondrial membrane potential (Δψm)-released cytochrome c and activated caspase-3 and caspase-9.

Pathway of deoxynivalenol-induced apoptosis in human colon carcinoma cells.

The mycotoxin, deoxynivalenol (DON), is generally detected in cereal grains and grain-based food products worldwide. Therefore, DON has numerous toxicological effects on animals and humans. The present investigation was conducted to determine the molecular aspects of DON toxicity on human colon carcinoma cells (HT 29). To this aim, we have monitored the effects of DON on (i) cell viability, (ii) Heat shock protein expressions as a parameter of protective and adaptive response, (iii) oxidative damage and (iv) cell death signalling pathway. Our results clearly showed that DON treatment inhibits cell proliferation, did not induce Hsp 70 protein expression and reactive oxygen species generation. We have also demonstrated that this toxin induced a DNA fragmentation followed by p53 and caspase-3 activations. Finally, our findings suggested that oxidative damage is not the major contributor to DON toxicity. This mycotoxin induces direct DNA lesions and could be considered by this fact as a genotoxic agent inducing cell death via an apoptotic process.

Fusarial toxin-induced toxicity in cultured cells and in isolated mitochondria involves PTPC-dependent activation of the mitochondrial pathway of apoptosis.

Mycotoxins produced by the Fusarium molds can cause a variety of human diseases and economic losses in livestock. Fusaria produce predominantly two types of mycotoxins: the nonestrogenic trichothecenes including T-2 toxin and the mycoestrogens such as zearalenone (ZEN). In a previous report, we demonstrated that the hepatotoxicity of these mycotoxins involves the mitochondrial pathway of apoptosis. Here, we observed that both fusarotoxins induced cell death by a mitochondria-dependent apoptotic process which includes opening of the mitochondrial permeability transition pore complex (PTPC), loss of mitochondrial transmembrane potential, increase in O(2)(.-) production, mitochondrial relocalization of Bax, cytochrome c release, and caspase activation. Studies performed on isolated mouse liver mitochondria showed that both ZEN and T-2 toxin might act directly on mitochondria to induce a PTPC-dependent permeabilization of mitochondrial membranes. Moreover, they may target different members of PTPC. Indeed, although the inner membrane protein adenine nucleotide translocase could be the target of T-2 toxin, ZEN seems to target the outer membrane protein voltage-dependent anion channel. Cells pretreatment with the p53 inhibitor pifithrin-alpha suggested that ZEN but not T-2 toxin triggered a p53-dependent mitochondrial apoptotic pathway. Finally, mitochondrial alterations induced by ZEN and T-2 toxin are mediated by Bcl-2 family proteins, such as Bax, and prevented by Bcl-x(L) and to a lesser extent by Bcl-2. Taken together, these data indicate that mitochondria play a pivotal role in both ZEN- and T-2 toxin-induced apoptosis and that PTPC members and proteins of Bcl-2 family should be interesting targets to overcome fusarotoxin toxicity.

Toxicities induced in cultured human hepatocarcinoma cells exposed to ochratoxin A: oxidative stress and apoptosis status.

Ochratoxin A (OTA) is a mycotoxin currently detected in stored animal and human food supplies as well as in human sera worldwide. OTA has diverse toxicological effects; however, the most prominent one is the nephrotoxicity. The present investigation was conducted to determine the molecular aspects of OTA toxicity in cultured human hepatocellular carcinoma cells. With this aim, we have monitored the effects of OTA on (i) cell viability, (ii) heat shock protein expressions as a parameter of protective and adaptive response, (iii) oxidative damage, and (iv) cell death signaling pathway. Our results clearly showed that OTA treatment inhibits cell proliferation, downregulates Hsp 70 and Hsp 27 protein and mRNA levels, and did not induce a significant reactive oxygen species generation. We have also demonstrated a decrease in mitochondrial membrane potential, a cytochrome c release, and an activation of caspase 9 and caspase 3 in response to OTA exposure. Moreover, OTA activates p53 expression, while some of its transcriptional target genes (Bax, Bak, PUMA, and p21) were found to downregulate. According to these data, we concluded that OTA may exert an inhibitory action on the transcriptional process. Besides, oxidative damage is not a major contributor to OTA toxicity. This mycotoxin induces a mitochondrial and caspase-dependent apoptotic cell death, which seems to be mediated by p53 transcriptional independent activities.