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.

Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential.

Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marrow, but the number of cells obtained is limited. Here, we compared the MSC-like cell populations, obtained from alternative sources for MSC: adipose tissue and skin, with the standard phenotype of human bone marrow MSC (BM-MSCs). MSC from human adipose tissue (human adipose stromal cells (hATSCs)) and human skin (human adult skin stromal cells, (hASSCs) and human new-born skin stromal cells (hNSSCs)) grew readily in culture and the growth rate was highest in hNSSCs and lowest in hATSCs. Compared with phenotype of hBM-MSC, all cell populations were CD34(-), CD45(-), CD14(-), CD31(-), HLA-DR(-), CD13(+), CD29(+), CD44(+), CD73(+), CD90(+),and CD105(+). When exposed to in vitro differentiation, hATSCs, hASSCs and hNSSCs exhibited quantitative differences in their ability to differentiate into adipocytes and to osteoblastic cells. Using a microarray-based approach we have unveiled a common MSC molecular signature composed of 33 CD markers including known MSC markers and several novel markers e.g. CD165, CD276, and CD82. However, significant differences in the molecular phenotype between these different stromal cell populations were observed suggesting ontological and functional differences. In conclusion, MSC populations obtained from different tissues exhibit significant differences in their proliferation, differentiation and molecular phenotype, which should be taken into consideration when planning their use in clinical protocols.

Cytotoxicity effects of amiodarone on cultured cells.

Amiodarone is a potent anti-arrhythmic drug used for the treatment of cardiac arrhythmias. Although, the effects of amiodarone are well characterized on post-ischemic heart and cardiomyocytes, its toxicity on extra-cardiac tissues is still poorly understood. To this aim, we have monitored the cytotoxicity effects of this drug on three cultured cell lines including hepatocytes (HepG2), epithelial cells (EAhy 926) and renal cells (Vero). We have investigated the effects of amiodarone on (i) cell viabilities, (ii) heat shock protein expressions (Hsp 70) as a parameter of protective and adaptive response and (iii) oxidative damage.Our results clearly showed that amiodarone inhibits cell proliferation, induces an over-expression of Hsp 70 and generates significant amount of reactive oxygen species as measured by lipid peroxidation occurrence. However, toxicity of amiodarone was significantly higher in renal and epithelial cells than in hepatocytes. Vitamin E supplement restores the major part of cell mortalities induced by amiodarone showing that oxidative damage is the predominant toxic effect of the drug.Except its toxicity for the cardiac system, our findings demonstrated that amiodarone can target other tissues. Therefore, kidneys present a high sensibility to this drug which may limit its use with subjects suffering from renal disorders.

Cytotoxicity and genotoxicity induced by aflatoxin B1, ochratoxin A, and their combination in cultured Vero cells.

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are important food-borne mycotoxins that have been implicated in human health. In this study, independent and combinative toxicities of AFB1 and OTA were tested in cultured monkey kidney Vero cells. The experiments reported here were conducted to evaluate the effect of these toxins on cell viability followed by the determination of cell death pathways, using the quantification of DNA fragmentation and the expression of p53 and bcl-2 protein levels. Our results showed that AFB1 and OTA caused a marked decrease of cell viability in a dose-dependent manner. Under the same conditions, these mycotoxins increased fragmented DNA levels. In addition, p53 was activated in response to DNA damage and the expression of the antiapoptotic factor bcl-2 decreased significantly. According to these data, AFB1 and OTA seemed to be involved in an apoptotic process. Moreover, combined AFB1 and OTA induced all the toxicities observed with the mycotoxins separately. Therefore, this combination was classified as an additive response of the two mycotoxins.

Individual and combined effects of ochratoxin A and citrinin on viability and DNA fragmentation in cultured Vero cells and on chromosome aberrations in mice bone marrow cells.

Ochratoxin A (OTA) and citrinin (CTN) are two common contaminant mycotoxins which can occur jointly in a wide range of food commodities. Both mycotoxins have several toxic effects but share a significant nephrotoxic and carcinogenic potential since OTA and CTN were reported to be responsible for naturally occurring human and animal kidney diseases and tumors. Considering the concomitant production of OTA and CTN, it is very likely that humans and animals are always exposed to the mixture rather than to individual compounds. Therefore, the aim of the present study was to investigate, in vivo and in vitro, whether DNA damage is enhanced by combination of both mycotoxins as compared to their effect separately. To this end, we have assessed their effects individually or combined on cell proliferation and DNA fragmentation in cultured Vero cells and in vivo by monitoring the induction of chromosome aberrations. Our results clearly showed that cultured renal cells respond to OTA and CTN exposure by a moderate and weak inhibition of cell proliferation, respectively. However, when combined, they exert a significant increase in inhibition of cell viability. Similar results were found for the investigated genotoxicity endpoints (DNA fragmentation and chromosome aberrations). Altogether, our study showed that OTA and CTN combination effects are clearly synergistic. The synergistic induction of DNA damage observed with OTA and CTN taken concomitantly could be relevant to explain the molecular basis of the renal diseases and tumorogenesis induced by naturally occurring mycotoxins.

Cytotoxicity and oxidative damage in kidney cells exposed to the mycotoxins ochratoxin a and citrinin: individual and combined effects.

ABSTRACT Ochratoxin A (OTA) and citrinin (CTN) are two mycotoxins, quite common contaminants, that can occur jointly in a wide range of food commodities. Both mycotoxins have several toxic effects but both share a significant nephrotoxic potential since OTA and CTN were reported to be responsible for naturally occurring human and animal kidney diseases. Considering the concomitant production of OTA and CTN, it is very likely that humans and animals are always exposed to the mixture rather than to individual compounds. Therefore, the aim of the present study was to investigate, using kidney cell culture (Vero cells), whether cytotoxicity and essentially oxidative cell damage (a key determinant of renal diseases) are enhanced by combination of both mycotoxins as compared to their effect separately. To this end, we have assessed their effects individually or combined on cell proliferation using three different cell viability assays (MTT, Trypan Blue, and Neutral Red). In addition, the role of oxidative stress was investigated by measuring the malondialdehyde (MDA) level and the expression of the heat shock protein Hsp 70. Our results clearly showed that cultured renal cells respond to OTA and CTN exposure by a moderate and weak inhibition of cell proliferation and induction of oxidative stress, respectively. However, when combined, they exert a significant increase in inhibition of cell viability as well as the induction of MDA level and Hsp 70 expression. OTA and CTN combination effects are clearly of synergistic nature. The enhanced induction of oxidative stress observed with OTA and CTN simultaneously could be relevant to explain the molecular basis of the renal diseases induced by these mycotoxins.

Heat shock proteins (Hsp 70) response is not systematic to cell stress: case of the mycotoxin ochratoxin A.

Ochratoxin A (OTA) is a mycotoxin routinely detected in improperly stored animal and human food supplies as well as in human sera worldwide. OTA has multiple toxic effects; however, the most prominent is nephrotoxicity. Thus, OTA is involved in the pathogenesis of human nephropathy in Balkan areas. In this study, we address the question of the appropriate functioning of the basal cellular defense mechanisms, after exposure to OTA, which, up to now, has not been investigated satisfactorily. In this context, we have monitored the effect of OTA on (i) the inhibition of cell viability, (ii) the oxidative damage using the GSH depletion, (iii) the inhibition of protein synthesis through the incorporation of [(3)H] Leucine and (iv) the induction of Hsp 70 gene expression as a parameter of cytotoxicity, oxidative damage and particularly as a protective and adaptative response. This study was conducted using the Human Hep G2 hepatocytes and monkey kidney Vero cells under exposure conditions ranging from non-cytotoxic to sub-lethal. Our results clearly showed that OTA inhibits cell proliferation, strongly reduces protein synthesis and induces the decrease of GSH in concentration-dependent manner in both Hep G2 and Vero cells. However, although cytotoxicity and oxidative damage (main inducers of Hsp expression) occur, no change was observed in Hsp 70 level under the multiple tested conditions. Inhibition of protein synthesis could not explain the absence of Hsp 70 response since concentrations, which did not influence protein synthesis, also failed to display the expected Hsp 70 response. Our data are consistent with recently published reports where considerable differences were noticed in the ability of relevant toxicants to induce Hsp. These results raised doubt about the universal character of Hsp induction which seems to be more complex than originally envisioned. It could be concluded that Hsp 70 induction is not systematic to cell stress.

Induction of Hsp 70 in Vero cells in response to mycotoxins cytoprotection by sub-lethal heat shock and by Vitamin E.

This paper analysed the toxicity mechanisms of several mycotoxins using Hsp 70 expression, cytoprotection of Vero cells by sub-lethal heat shock (sub-LHS) and Vitamin E. Our aim was (i) to determine whether Citrinin (CTN), Zearalenone (ZEN) and T2 toxin (T2) could induce the expression of Hsp 70, (ii) to check whether or not elevated levels of Hsp and Vitamin E pre-treatment could provide cytoprotection from these mycotoxins, and finally (iii) to emphasize the eventual involvement of oxidative stress on mycotoxin's toxicity. Our study demonstrated that the three examined mycotoxins induced Hsp 70 expression in a dose-dependent manner. A cytoprotective effect of Hsp 70 was obtained when Vero cells were exposed to sub-lethal heat shock followed by a 12h recovery prior to mycotoxins treatment and evidenced by a reduction of their cytolethality. This cytoprotection suggested that Hsp 70 might constitute an important cellular defence mechanism. A cytoprotective action was also obtained although at lesser extent, when cells were pre-treated with an antioxidant agent, the Vitamin E before mycotoxins treatment. This Vitamin E cytoprotection evoked the involvement of oxidative stress in mycotoxins induced toxicity, which was further, confirmed by the reduction of Hsp 70 expression when cells were pre-treated with Vitamin E prior to mycotoxins. Our data clearly shows that oxidative stress is certainly involved in the toxicity of the three studied mycotoxins, Citrinin, Zearalenone and T2 toxin and may therefore constitutes a relevant part in their toxicities; however, at variable extent from one mycotoxin to another.