May 24-27, 2011 . Murcia· SpainI    r tian l y             nTrace Elements &Health    may 2011 I murcia spain
I                                                                Poster P.5.1.        Potassium apigenin: a promising     ...
Posterstvw prostate cell lines (PNT2, TRAMP-Cl) administered beforeexposlue to different X ray doses (OGy, 4Gy, 6Gy, SGy a...
POTASSIUM APIGENIN: A PROMISING                                         RADIATION COUNTERMEASURE       ((1)Miguel         ...
Poster P.5.2. Carnosic a,cid: a promising molecul,a for           radiation protection11lMiguel Alcaraz; (4 ]ulián Castill...
Posters(RO)=Camosic acid (CA)=Oiosrrún (O}=Apigenin (AP)=O­toeopherol (E)=Carnosol (Cl)=L_acid aseorbic (C)=Amifostine(AMI...
CARNOSIC ACID: A PROMISING MOLECULE                                     FOR RADIATION PROTECTION       (1)MiguelAlcaraz; (...
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Tracel 2011 3rd international symposium on trace elements & health

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Tracel 2011 3rd international symposium on trace elements & health

  1. 1. May 24-27, 2011 . Murcia· SpainI r tian l y nTrace Elements &Health may 2011 I murcia spain
  2. 2. I Poster P.5.1. Potassium apigenin: a promising radiation countermeasure(llMiguel Alcaraz; (4.IJulián Castillo; (4J Obdulio Benavente Carda; (2.IDavid Armero; (l)Miguel Alcaraz-Saura; (5}(,IDaniel Achel; (3Vicente Vicente and (6)Manuel de las HerasDepartments of (l)Radiology and Physical Medicine, (2)Nursing and (3)PathoJogy, University of Murcia. 30100-Campus de Espinardo. Murcia. (4)Department l+D+ i u trafur S.A., Camino Viejo de Pliego s/n 3Ü320-Alcantarilla, Murcia. (5)Applied Radiation Biology Cen.he, Radiological and Medica) SciencesResearch Inslitute, Ghana Atomic Energy Commission, Legon-Acera, Ghana. (6l[)epartment of Radiotherapy. Clinic Hospital oí San Carlos. University Complutense oí Madrid. CI Prol. Mart n Lagos sI n, 28040-Mad.rid.The genoprotective and radioprotective eifects of potassiumapigenin against chromosomaJ damage induced by ionizingradiation were compared wíth those oí L-ascorbic acid,o-tocopherol, diosmin, rutin and the S-containing compounds(dimethyl sulfoxide (DMSO) and Amifostine), using themicronuc1eus test for antimutagenic activity to evaluate thereduction in the frequency oí micronuclei (MN) in cytokinesis­blocked cells of human lymphocytes befare and after irradiation;and the ceH survival using the ceH viability test with MTT in 201
  3. 3. Posterstvw prostate cell lines (PNT2, TRAMP-Cl) administered beforeexposlue to different X ray doses (OGy, 4Gy, 6Gy, SGy and 10Cy).The results pomt to the significant genoprotective capacity of thesubstances assayed before radiabon: Apigenin (AP) -~ Diosmin(D) = aAocopherol (E) = L-ascorbic acid (C) = Amifostine (AMIF)(p<O,OOl» Ruhn (R) = Dimetil sulfoxide (DMSO) (p<O,05»Irradiated Controls (CO, the maximurn. protection factor attainedbeing 50%. When the substances were administered imrnediatelyafter radiation, th re ults were different: AP= E (p>O.OO1» 0=C (p>O.05»Ci, with a maximum protection factor of 38%. Incontrast, AMIF, R and DMSO lost their genoprotective effect.Cell survival obtained with 20 ,uM and 40 ~{M potassiumapigenin adminjstered before radiatian with up to 10 Gy showeda Protection Factor oí 100%, reducing radioinduced cell death by30.8% in both celllines assayed (p<O.OOI). Apigenin showed thegreatest pre-treatment pratective effect a good post-treatmentprotection capacit}o and no toxicity towards censoFor this reason, our results indicate that apigenin may bedeveloped as a radioprotectant for humans against the potentiallydamaging effects of exposure to radiabon. Human clinical trialsexamining the effect of supplementation oí potassium apigeninon disease prevention have not been conducted, although thereis considerable potential far apigenin to be develaped as aradioprotectíve agent.Iros report was supported by a grant fram th Nationa1 SpanishR + D Programme CE IT of the Spanish Ministry of Science andTedu1010gy (acronym: SENIFOOD) and by a Felloship oí thelnternational Atomic Energy Agency (GHA 10021). 202
  4. 4. POTASSIUM APIGENIN: A PROMISING RADIATION COUNTERMEASURE ((1)Miguel Alcaraz; (3)Julián Castillo; (3)Obdulio Benavente-García;(1)Miguel Alcaraz-Saura; (4)(*)Daniel Achel; (2)Vicente Vicente y (5)Manuel de las Heras Departments of Radiology and Physical Medicine(1) and (2)Pathology, Faculty of Medicine, University of Murcia. 30100-Campus de Espinardo. Murcia. (3)Department I+D+ i Nutrafur, Camino Viejo de Pliego s/n 30320-Alcantarilla, Murcia. (4)Applied Radiation Biology Centre, Radiological and Medical Sciences Research Institute, Ghana Atomic Energy Commission, Legon- Accra, Ghana. (5)Department of Radiotherapy. Clinic Hospital of San Carlos. University Complutense of Madrid. C/ Prof. Martín Lagos s/n, 28040-Madrid. INTRODUCTION cytokinesis-block micronucleus test (MN) with cytochalasin-B on This work is carried out as part of our efforts to find a non toxic irradiated human lymphocytessubstance that can be used to reduce the harmful effects induced byionizing radiation. This compound could protect against genetic damage,mutations and tetarogenic lesions that act through free radical production.Recently substances with characteristic anti-oxidative properties aregaining attention as potential radioprotective candidates. In this study weassay the antioxidant substance potassium apigenin (AP), a habitualsubstance in human diet, for its radioprotective capacity against damageinduced by the exposure to X-rays, Its modified structure serves to increaseits solubility water solubility.MATERIAL AND METHODS Fig. 1: Different types of cells in the microscopic preparations: CB:cytokinesis- blocked cell; MNCB: cytokinesis-blocked cell with micronúcleos; L: lymphocyte; erythrocyte hemolized.Chemicals: Apigenin (API) and Diosmine (D) were supplied by Nutrafur S.A. (Murcia, Spain); 99% L-ascorbicacid (AA) and 99% δ-tocopherol (E) were obtained from Sigma Co. (Madrid, Spain). Dimethylsulphoxide GENOTOXICITY(DMSO) and Rutin (R) were obtained from Merck (Darmstadt, Germany); and Amifostine (AMF) were obtainedfrom Schering-Plough, SA (Ethyol® injectable, Madrid, Spain).AP was studied by means of two routine assays commonly employed in radiological protection:Genoprotection assay: We used the cytokinesis-block micronucleus test (MN) with cytochalasin-B onirradiated human lymphocytes to evaluate the genoprotective effects of AP following the technique describedby IAEA (2001). Samples of blood were cultivated at 37°C for 72 hours in 4.5 ml of HAM F-10 medium (SigmaCo, Madrid) containing 15% fetal bovine serum, 16µg/ml phyitohemaglutinin (Sigma Co, Madrid), 100 µg/ml MN/500 CBpenicilin/streptomicin (Sigma Co, Madrid) and glutamine 1 µg/ml; cytochalasina B (6µg/ml, Sigma Co.) wasadded 44 hours after the inception of the cultivation. At the end of 72 hours the culture was terminated and thecells harvested. At harvest the culture solution was centrifuged and a hypotonic solution KCl (75μM) wasadded to the collected cells. A fixative solution of methanol:acetic acid (3:1) was added and slides prepared.The preparations were stained with May-Grünwald and Giemsa stains. Scoring was done by counting thenumber of MN per 3000 binucleated cells (CB), using a Zeiss light microscope (Oberkochem, Germany) with a CYTOTOXICITYmagnification of 400X. The test substances were added to the samples of human blood cultures to obtain afinal concentration of 25 μM, before and after the exposure to X-rays. ControlCytoprotection assay: We used the MTT cell survival assay to assess cytoprotective effect using two prostatecell lines PNT2 (normal epithelial cells) and TRAMP-C1 (tumoral cells).The cells were cultivated normally in thepresence of API (20μM and 40μM) for 24 and 48 hr after the exposure to X-rays. Briefly: the cells were (p<0,001) versus C Treatmentsincubated in 200 μl of medium supplemented with 50 μl of MMT (MTT) (8mg/ml) in 96 well microplates (4h,37ºC, 5%CO2). After centrifugation (240g for 10 mn) to remove non metabolized MTT, 100 μl of DMSO was (p<0,001) versus  Ci        60added to each well to solubilized the MTT taken up by the live cells. Finally, MTT reduction data were o   (p<0,001) versus  Ci 55 M Magnitude of Protection (%) ) 50 agnitude of protection (%expressed as absorbance at 570nm and 690nm with a Multiskan MCC/340P spectrophotometer. Control + Apigenin 24h + 48 h 45An X-ray equipment Andrex SMART 200E machine was used (YXLON International, Hamburg, Germany) 40operating at 120 kV, 4.5 mA, DFO 36 cm and ambient temperature to administer a dose of 2 Gy for the MN 35assay and of 0 Gy, 4Gy 6Gy 8Gy and 10 Gy for the cell survival determinations. Statistical analysis consisted 30of pair wise contrast analysis of variance to compare the percentages of cell survival in the cultures with 25 20different concentrations of the compounds and contrasting their means with the method of minimum 15significant differences. The analysis was carried out with a logarithmical transformation of the data to 10accommodate the adjustments made for the ANOVA. Finally, the results were used to obtain the Magnitude of 5Protection: Magnitude of protection (%) = ((Fcontrol – Ftreated) / Fcontrol) x 100 (where Fcontrol = frequency of MN in 0 RO CA E AP AA Amif Te R D DMSirradiated blood lymphocytes and Ftreated = frequency of MN in blood lymphocytes treated pre and post-γ- PF (%) 57.7 PF (%) 57,7 50 50 50 50 50 50 38.5 38,5 38.5 38,5 23,1 23.1 23,1 23.1 23,1 23.1 16,6 16.6irradiation as described previously) and the Dose Reduction Factor (DRF) determined. Figure 2: Control+Apigenin 24h and 48h Treatments Trratamientos TreatmentsRESULTS Apigenin-K 24h Apigenin-K 24h Genoprotection assayThe results point to the significant genoprotective capacity of the substances assayedbefore radiation: Apigenin (AP) = Diosmin (D) = α-tocopherol (E) = L-ascorbic acid (C) =Amifostine (AMIF) (p<0,001)> Rutin (R) = Dimethyl sulfoxide (DMSO) (p<0,05)> IrradiatedControls (Ci); the maximum protection factor attained being 50%. When the substanceswere administered immediately after radiation, the results were different: AP= E(p>0.001)> D= C (p>0.05)>Ci, with a maximum protection factor of 38%; however, incontrast, AMIF, R and DMSO lost their genoprotective effects.Cytoprotection assayIn the PNT2 cell line assay AP showed a radioprotective effect by significantly increasingthe cell survival to 10 Gy (p <0.001) after X-ray exposure, this represens a Factor of Doses APIG 20 CONTROL APIG 40 DosesProtection of 100% and 70% at 24h according to the concentration of AP studied (40 μMand 20 μM, respectively); these values descended respectively to 61% and 50% at 48h (p Figure 3 : Cellular survival (%) with Apigenin at the 24 hours . (p<0,001 versus Ci).<0.001), we determined a (DRF) of FRD of between 3.1-3.3 according to the concentrationof AP assayed. Apigenin-K 48h Apigenin-K 48hIn the line TRAMP-C1 assay, AP also showed a radioprotective effect by increasing thecell survival significantly to 10 Gy after exposure to X-rays (p <0.001), representing aFactor of Protection of 58% at 24h and increased to 69% and 85% at 48h (p <0.001)respectively. A Dose Reduction Factor of between 2.5-3 was determined according to theconcentrations of AP assayed. CONCLUSION our results indicate that potassium-apigenin may be developed as aradioprotectant for humans against the potentially damaging effects of exposureto radiation. Human clinical trials examining the effect of supplementation of Doses APIG 20 CONTROL APIG 40 Dosespotassium apigenin on disease prevention have not been conducted, although Figure 4: Cellular survival (%) with Apigenin at the 48 hours. (p<0.001 versus Ci).there is considerable potential for apigenin to be developed as a radioprotectiveagent. This report was supported by a grant from the National Spanish R + D Programme CENIT of the Spanish Ministry of Science and Technology (acronym: SENIFOOD) and by a Fellowship of the International Atomic Energy Agency (GHA10021).
  5. 5. Poster P.5.2. Carnosic a,cid: a promising molecul,a for radiation protection11lMiguel Alcaraz; (4 ]ulián Castillo; (4)Obdulio Benavente García; (2)David Annero; (l)Miguel Alcaraz-Saura; (5)()Daniel Achel and (3Vicente Vicente Departments of (IJRadiology and Physical Medicine, (l)Nursing and (3) Pathology, University af Murcia. 30100-Campus de Espinarda. Murcia. (4)Department I+D+ i Nutrafur S.A., Canüno Viejo de Pliego 51 n 30320-Alcantarílla, Murcia. (5JApplied Radiaban Biology Centre, Radialagical and Medical SciencesResearch lnstitute, Ghana Atamic Energy Comrnission, Legan-Acera, Ghana.The genoprotectíve effect of carnosic acid against damage índucedby ionizing radiabon was compared with the effect of severalantioxidant compounds by means of the micronucleus test forantímutagenic activity, in which the reduchon in the frequency ofmicronuclei was evaluated in cytokinesis-blocked cells of humanlymphocytes before and after 2 Gy of Y-radiatian. Also, theradioprotective effect of the most effective compounds was thenstudied by a cell viability test (MTT) in the PNT2 (normal prostate)and B16Flü (m.elanoma) ceH lines when they are administeredbefare exposure to X ray doses (OGy,4Gy,6Gy,8Gy and lOGy).The results point to the significant genoprotective capacityoi the substances assayed before radiation: Rosmarinic acid 203
  6. 6. Posters(RO)=Camosic acid (CA)=Oiosrrún (O}=Apigenin (AP)=O­toeopherol (E)=Carnosol (Cl)=L_acid aseorbic (C)=Amifostine(AMIF) (p<O.OOl»Green Tea Exhact (GTA)-Rutín (R)=DimethyIsulfoxide (DMSO) (p<O.OS»irradiated eontrols (Ci), with amaximumradiation Protection Factorof 58%. When the substanceswere administered irnmediately after radiation, the results weredifferent: CA=AP=E=Cl (p>O.OOl»O=C (p>o.05»Ci, with amaximum protection factor oí 46%, while RO, At1IF, R and DMSOhad no protective capacity.Cel1 survival obtained with 20 ~M and 40 ~M carnosic acidadministered before radiation with up to 10 Gy showed aProtection Factor oí 100%, eliminatll1g 30.8% oí radioll1ducedcell death in both ceH Unes assayed. In the case oi normal cells(PNT2), the protective capacity followed tlús order CA=mixture(AP+CA)=mixture (AP+RO) (p<O.OOl»API=RO (p<O.05»Ci.In the tumoral cell lineo a ayed (B16FlO) on.ly CA acted aradioprotector (p<O.OOl), while the rest oí the substances showednosignificanteffectin this respect (APIG, RO, DMSO and mixtures).Carnosic acid shoved the greatest pre-exposure protectiveeffect, a good post-treahnent protection capacity and no toxicitytowards ceUs. For this reason, oUJ results indicate that camosicacid may be developed as a radioprotectant for humans againstthe potentiaUy damaging effects of exposlIre to radiation.Tbis report was supported by a grant from the National SpanishR + O Programme CENIT oí the Spanish Ministry of Science andTechnology (acronym: SENIFOOD) and by a Fellowship oí theInternational Atomic Energy Agency (GHAI0021).TOPIes: 9. Trace Elements and Human Health 204
  7. 7. CARNOSIC ACID: A PROMISING MOLECULE FOR RADIATION PROTECTION (1)MiguelAlcaraz; (4)Julián Castillo; (4)Obdulio Benavente García; (2)David Armero; (1)Miguel Alcaraz-Saura; (5)(*)Daniel Achel and (3)Vicente VicenteDepartments of (1)Radiology and Physical Medicine, (2)Nursing and (3)Pathology, University of Murcia. 30100-Campus de Espinardo. Murcia.(4)Department I+D+ i Nutrafur S.A., Camino Viejo de Pliego s/n 30320-Alcantarilla, Murcia.(5)Applied Radiation Biology Centre, Radiological and Medical Sciences Research Institute, Ghana Atomic Energy Commission, Legon-Accra, Ghana. INTRODUCTION There is a continued quest for the development of non-toxic compounds that can reduce the effect of exposure to GENOTOXICITY ionizing radiation. Such compounds could potentially protect humans against the genetic damage, mutation, alteration in the human system and teratogenic effects of toxic agents (including radiation) which act through the generation of free radicals. For many years, and despite their high toxicity, only sulfhydryl (SH) compounds were known to provide radioprotective effects when administered biologically before exposure to ionizing radiation and only recently were other radioprotective substances discovered whose common characteristic was their antioxidant capacity. Subsequently, other substances were found to have the same effect when administered after exposure to ionizing radiation and, more recently, some substance have been MN/500 CB described as radioprotectors only when administered after exposure to radiation. Yet other substances present a radioprotective capacity that differs according to whether they are administered pre or post radiation. Fig. 1: Different types of cells in the In this work we assayed different antioxidant compounds that show protective capacity against the chromosome damage microscopic preparations: CB:cytokinesis- induced by ionising radiation and related their effects with their chemical characteristics (Alcaraz et al., 2009) blocked cell; MNCB: cytokinesis-blocked cell with micronúcleos; L: lymphocyte; E: erythrocyte hemolized. MATERIAL AND METHODS Treatments Chemicals: 82% Carnosic acid (oil soluble, os) (CA), 86% carnosol (os) (COL), green tea extract (90% catechins) (p<0,001) versus C (water soluble, ws) (GTEA) and grape seed extract (95% procyanidins) (ws) (GSE), were supplied by Nutrafur S.A. CYTOTOXICITY (p<0,001) versus Ci (Murcia, Spain). 95% rosmarinic acid (ws) (RO), 99% L-ascorbic acid (ws) (AA) and 99% δ-tocopherol (os) (E) were o (p<0,001) versus Ci obtained from Sigma Co. (Madrid, Spain). Dimethylsulphoxyde (DMSO) was obtained from Merck (Darmstadt, 60 Germany). Dimethyl sulphoxide (DMSO) and Rutin (R) were obtained from Merck (Darmstadt, Germany); and 55 Magnitude of protection (%) Amifostine (AMF) were obtained from Schering-Plough, SA (Ethyol® injectable, Madrid, Spain). CA was studied by 50 Magnitude of Protection (%) means of two routine assays commonly employed in radiological protection: 45 Cellular survival (%) 40 Genoprotection assay: We used the cytokinesis-block micronucleus test (MN) with cytochalasin-B on irradiated 35 human lymphocytes to evaluate the genoprotective effects of these substances and CA following the technique 30 described by IAEA (2001). Samples of blood were cultivated at 37°C for 72 hours in 4.5 ml of HAM F-10 medium 25 20 (Sigma Co, Madrid) containing 15% fetal bovine serum, 16µg/ml phytohemaglutinin (Sigma Co, Madrid), 100 µg/ml 15 (penicilin/streptomycin) penicillin/streptomycin (Sigma Co, Madrid) and glutamine 1 µg/ml; cytochalasina B Apigenin-K 24h 10 (6µg/ml, Sigma Co.) was added 44 hours after the inception of the cultivation. At the end of 72 hours the culture Doses 5 0 was terminated and the cells harvested. At harvest the culture solution was centrifuged and a hypotonic solution RO CA E AP AA Amif Te R D DMS KCl (75μM) was added to the collected cells. A fixative solution of methanol:acetic acid (3:1) was added and slides PF (%) 57.7 PF (%) 57,7 50 50 50 50 50 50 38.5 38,5 38.5 38,5 23.1 23,1 23.1 23,1 23.1 23,1 16.6 16,6 prepared. The preparations were stained with May-Grünwald and Giemsa stains. Scoring was done by counting the Cellular survival (%) Trratamientos Treatments Treatments number of MN per 3000 binucleated cells (CB), using a Zeiss light microscope (Oberkochem, Germany) with a magnification of 400X. The test substances were added to the samples of human blood cultures to obtain a final concentration of 25 μM, before and after the exposure to X-rays. Cytoprotection assay : We used the MTT cell survival assay to assess cytoprotective effect using two cell lines PNT2 (normal epithelial cells) and B16F10 (melanoma) cell lines. The cells were cultivated normally in the presence of CA and other test compounds (20μM and 40μM) for 24 and 48 hr after the exposure to X-rays. Briefly: the cells Doses were incubated in 200 μl of medium supplemented with 50 μl of MMT (MTT) (8mg/ml) in 96 well microplates (4h, 37ºC, 5%CO2). After centrifugation (240g for 10 mn) to remove non metabolized MTT, 100 μl of DMSO was added to Fig. 3: Control+Carnosic acid 24h and each well to solubilized the MTT taken up by the live cells. Finally, MTT reduction data were expressed as 48h (PNT2 cell line) absorbance at 570nm and 690nm with a Multiskan MCC/340P spectrophotometer. Fig. 2: Different types of cells in the microscopic preparations: CB:cytokinesis-blocked cell; MNCB: An X-ray equipment Andrex SMART 200E was used (YXLON International, Hamburg, Germany) operating at 120 kV, cytokinesis-blocked cell with micronúcleos; L: 4.5 mA, DFO 36 cm and ambient temperature to administer a dose of 2 Gy for the MN assay and 0 Gy, 4Gy 6Gy 8Gy lymphocyte. and 10 Gy for the cell survival determinations. Statistical analysis consisted of pair wise contrast analysis of variance to compare the percentages of cell survival in the cultures with different concentrations of the compounds and contrasting their means with the method of minimum significant differences. The analysis was Cellular survival (%) Cellular survival (%) carried out with a logarithmical transformation of the data to accommodate the adjustments made for the ANOVA. Finally, the results were used to obtain the Magnitude of Protection: Magnitude of protection (%) = ((Fcontrol – Ftreated) / Fcontrol) x 100 (where Fcontrol = frequency of MN in irradiated blood lymphocytes and Ftreated = frequency of MN in blood lymphocytes treated pre and post-γ-irradiation as described previously) and the Dose Reduction Factor (DRF) determined. RESULTS Doses Doses Fig. 4: Control+Carnosic acid 24h and 48h (B16F10 melanoma cells) Genoprotection assay: The results point to a significant genoprotective capacity of the substances assayed before radiation: Rosmarinic acid (RO)=Carnosic acid (CA)=Diosmin (D)=Apigenin (AP)=δ-tocopherol (E)=Carnosol (Cl)=L_acid ascorbic (C)=Amifostine Cellular survival (%) Cellular survival (%) (AMIF) (p<0.001)>Green Tea Extract (GTA)=Rutin (R)=Dimethyl sulfoxide (DMSO) (p<0.05)>irradiated controls (Ci), with a maximum radiation Protection Factor of 58%. When the substances were administered immediately after radiation, the results were different: CA=AP=E=Cl (p>0.001)>D=C (p>0.05)>Ci, with a maximum protection factor of 46%, while RO, AMIF, R and DMSO had no protective capacity. Cytoprotection assay : Cell survival obtained with 20 µM and 40 µM carnosic acid Doses Doses administered before radiation with up to 10 Gy showed a Protection Factor of 100%, eliminating 30.8% of radioinduced cell death in both cell lines assayed. In the case of normal cells (PNT2), the protective capacity followed this order; CA=mixture (AP+CA)=mixture (AP+RO) (p<0.001)>API=RO (p<0.05)>Ci. In the tumoral cell lines Cellular survival (%) Cellular survival (%) assayed (B16F10) only CA acted as radioprotector (p<0.001), while the rest of the substances showed no significant effect in this respect (APIG, RO, DMSO and mixtures). CONCLUSION Doses Doses Carnosic acid showed the greatest pre-exposure protective effect, a good post-treatment protectioncapacity and no toxicity towards cells. For this reason, our results indicate that carnosic acid may be Figure 5: Cellular survival (%) with different mixtures of substances. (p<0.001 versus Ci).developed as a radioprotectant for humans against the potentially damaging effects of exposure toradiation. This report was supported by a grant from the National Spanish R + D Programme CENIT of the Spanish Ministry of Science and Technology (acronym: SENIFOOD) and by a Fellowship of the International Atomic Energy Agency (GHA10021).

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