Project Human Resources
Title of the project: The role of ochratoxin A in triggering of the balcanic endemic nephropathy by using the swine as animal model
Tip of the project: Projects of Research for Stimulation of the Establishment of Young Independent Research Team
Project code: PN-II-RU-TE-2012-3-0086
Contract number: TE 4
Financing unit: UEFISCDI
The project investigate the involvement of ochratoxin A in balcanic endemic nephropathy (BEN). The project will evaluate the effect of OTA at cellular and molecular level concerning the cell growth and division, oxidative stress and inflammatory response and will represent an original approach of this subject in Romania. The results will constitute the scientific basis for the changes induced by OTA in kidneys and at the level of the whole organism, they could be correlated with clinical observations of BEN, proving or not the involvement of OTA in this disease. Beside the national benefits, of the risk analyses realized during the project, this project will contribute to the consolidation of the European database concerning the toxic cellular and molecular effects of OTA in animals; this will allow the consolidation of the UE legislation concerning the food and feed security and elaboration of new UE reglementation concerning the limits of tolerance of OTA in feed, knowing that until now there is only a recommendation of UE (CE Recommendation 576/2006) concerning the tolerance limits of OTA in fodders. The regulation concerning the maximum admitted levels for OTA will eliminate from the consummation the food and feed with concentrations higher than the maximum level admitted. In the same time, the increase of the food and feed safety and security will improve the health status of animals and implicitly of human population.
The general objective of this project is based on two important and original arguments i) necessity to go deeper with the involvement of OTA in BEN through the investigation of the cellular and molecular mechanisms at renal level. The kidney is the target organ of OTA and the principal characteristic of BEN is the formation of bilateral multiple tumors in the upper part of unirary tract. As known, the tumoral cells suffer changements of the gene expression associated to important metabolic processes (proliferation and cell differentiation, apoptosis, etc). OTA was little studied as concerning the effects on the signalization (NF-kB and MAPKs). ii) lack of scientific and clinical in Romania. This is the reason for that Romania appears very rarely in the EU reports (as SCOOP), that presents periodically the level of contamination and the incidence of the contamination with mycotoxins in the UE countries; there are few data concerning OTA contamination and concerning the BEN incidence come from Romania. Additionally, the temperate climate from Romania, with hot summers, especially in the last years, favors the development of molds of Aspergillus genra and mycotoxins synthesis. The project will use the swine as animal model and will combine studies in vitro (swine epithelial cell cultures and blood cells), with in vivo studies (studies on swine) in order to understand the mechanism of action of OTA at cellular and molecular level. In a first stage will be evaluated the changements produced by OTA in vitro on MAPKinase expression (ERK1/2, p38, JNK) and NFkb (p65), cellular signaling pathways with an important role for the progress of vital physiological processes as proliferation, apoptosis, inflamation, etc. This set of informations will be correlated with: (i) cell proliferation, necrosis and apoptosis (ii) oxidative stress, (iii) inflammatory process. In vivo investigations will start from the hypothesis that ingestion of the feed contaminated with OTA will modify the animal metabolism in generally and renal physiology in special, that will lead to immunosuppression. The consequence is a higher sensibility to infections. The use of the modern methods of in vitro investigation, using some cell lines eg : LLC-PK and in vivo models together with the expertise of the research team , will allow a better understanding of the way that OTA act at local (kidney) and systemic level (blood).
Objectives / Activities foreseen for the project:
|1. Investigation of the effects of the toxic stress induced by OTA on growth and development.||Determination of the activity of mithocondrial succinat dehydrogenase in active metabolic cells|
|Determinarea alterarii expresiei proteinelor implicate in cresterea celulara, diferentiere si apoptoza (ERK1/2, c-Jun N-terminal kinases, p38)|
|1. Estimation of the toxicity of OTA in induction of the oxidative stress.||Investigation concerning the induction of the oxidative stress in vitro|
|Investigation concerning the induction of the oxidative stress in vivo (Experimental intoxication in vivo with OTA)|
|1. Evaluation of the amplitude of the inflammatory stress induced by OTA.||The study of the expression of some markers of inflammation: synthesis of proinflammatory cytokines (IL1-b, IL-8, TNF-alpha) and anti-inflammatory (IL4, IL-10)|
|Spectrophotometrical assesement of biochemical profile|
|Determination of seric markers of inflammation|
Project will have as a result data concerning the OTA effects at cellular and molecular level, concerning cell proliferation and division, oxidative stress and inflammatory response, representing on original approach of this subject in Romania. Results will provide a scientific base for the changes induced by OTA in kidney at the level of entire organism. These results will be correlated with clinical observation concerning BEN, proving or not involvement of OTA in this disease. Beside the national benefits, risk analyses realized during the project will contribute to the consolidation of the UE data base concerning the toxic effects of OTA in animals. This will allow the consolidation of UE legislation concerning food security and will allow the establishment of a UE regulation concerning the maximum limits of OTA in animals feed, knowing that until present there is an EU recommendation (CE 576 / 2006) concerning the OTA limits in feed. This regulation will lead to the the elimination of the cereals and of other raw materials contaminated with high concentrations of OTA that that admitted to the consummation. In same time, the increase of the food and feed safety and security will improve the animal health and implicitly of the human population.
Dr. Daniela Marin (Project coordinator); Dr. Ionelia Taranu, Dr. Gina Pistol, Monica Motiu, Dr. Gras Mihai, Dr. Veronica Chedea, Dr. Lazar Cristina, Dr. Rodica Pelmus, Dr. Elena Ghita.
Project advancement and results aquired:
Phase 1. Investigation of the effects of the toxic stress induced by OTA on growth and development (May 2013- December 2013)
In this phase the cytotoxic effect of OTA on cell proliferation and the mechanisms involved in the cell proliferation and differentiation were evaluated using an epithelial renal cell line (LLC-pK).The results showed that OTA has different effects on the expression of genes involved in the signalization pathways of MAPKs, with important implications for cell growth, differentiation and apoptosis.
Phase 2: Estimation of OTA toxicity in the induction of the oxidative stress (January-December 2014)
- In vivo experimental intoxication with OTA
- Analysis of different markers of the oxidative stress (nitric oxide synthesis, estimation of the antioxidant status)
Inflammationand oxidative stress are tightly correlated, the pathway that generate the inflammatory mediators being induced by the oxidative stress. Despite this, few studies had investigated the role of the oxidative stress and inflammation in triggering the balcanic endemic nephropathy (BEN). In this phase we had investigated the effect of OTA on oxidative stress and inflammation using two models, an in vitro model epithelial line IPEC and one in vivo piglets after weaning. The results obtained in this phase have shown that OTA can interfere with the organism defense capacity and with the immune response at the intestinal level, with important implications for the pathology of BEN.
Phase 3: Evaluation of the amplitude of the inflammatory response induced by OTA
- The study of the expression of some important markers of inflammations
The food industry has important economic losses due to the mycotoxin intoxication. The pigs in particular, are very sensitive to the intoxication wit mycotoxins and represents in particularly a good model for mycotoxin intoxication in humans.In this phase, we have evaluated the inflammatory effect of OTA in the liver and kidney of weanling piglets, experimentally intoxicated with toxin. Our results showed that the presence of OTA in concentration of 50 ppb, as UE recommended value for weanling piglets, administered for 33 days induce hepatotoxicity as resulting from the increase of serum triglyceride concentration, of the ALAT transaminase activity, and of liver superoxide-dismutase together with a decrease of the plasma concentration of total proteins and albumin. These alteration are associated with a significant decrease of IL-6 synthesis and of nitric oxide in liver and a significant increase of IL-10 synthesis in kidney, as important markersof inflammation.
Publications and Conferences:
- Daniela MARIN, Gina PISTOL, Mihai GRAS, Monica MOTIU, Ionelia TARANU. 2015. Studies concerning the nephrotoxic effects of ochratoxin a using pig as a model. Lucrari Stiintifice - Seria Zootehnie, Iasi, p333-337.
- Daniela E. MARIN, Ionelia TARANU. 2015. Ochratoxin A anditseffects on immunity. Toxinreviews. 34, 1, 11-20.
- D.E. MARIN, M. Gras, G. Pistol, I. Taranu. 2014. Effect of the mycotoxin ochratoxin A on the expression of some genes involved in the MAPKs pathway. Proceedings of the Nutrition Society (in press).
- Daniela MARIN, Monica MOTIU, IoneliaTARANU. Effect of Ochratoxin A and Aristolochic Acid on an Epithelial Intestinal Cell Line. Bulletin USAVM-CN Animal Science and Biotechnologies 71(2)/2014.
- D.E. MARIN, I. TARANU, G. PISTOL. 2014. Metode de decontaminare a micotoxinelor (Methods for mycotoxins decontamination iin Romanian). Editura Inovativ Media.